RMCP Vol. 13 Num. 3 (2022): July-September [english version] by Revista Mexicana de Ciencias Pecuarias

Edición Bilingüe Bilingual Edition

Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 3, pp. 584-845, JULIO-SEPTIEMBRE-2022

ISSN: 2448-6698

Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 3, pp. 584-845, JULIO-SEPTIEMBRE-2022

REVISTA MEXICANA DE CIENCIAS PECUARIAS Volumen 13 Numero 3, JulioSeptiembre 2022. Es una publicación trimestral de acceso abierto, revisada por pares y arbitrada, editada por el Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Avenida Progreso No. 5, Barrio de Santa Catarina, Delegación Coyoacán, C.P. 04010, Cuidad de México, www.inifap.gob.mx Distribuida por el Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, Km 15.5 Carretera México-Toluca, Colonia Palo Alto, Cuidad de México, C.P. 05110. Editor responsable: Arturo García Fraustro. Reservas de Derechos al Uso Exclusivo número 04-2022-033116571100-102. ISSN: 2448-6698, otorgados por el Instituto Nacional del Derecho de Autor (INDAUTOR). Responsable de la última actualización de este número: Arturo García Fraustro, Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad, Km. 15.5 Carretera México-Toluca, Colonia Palo Alto, Ciudad de México, C.P. 015110. http://cienciaspecuarias. inifap.gob.mx, la presente publicación tuvo su última actualización en julio de 2022. Corral de engorda intensiva de corderos Dorper x Katahdin destinados a la producción de cortes finos del Instituto de Ciencias Agrícolas de la UABC. Fotografía: Ulises Macías Cruz

DIRECTORIO EDITOR EN JEFE Arturo García Fraustro

FUNDADOR John A. Pino EDITORES ADJUNTOS Oscar L. Rodríguez Rivera Alfonso Arias Medina

EDITORES POR DISCIPLINA Dra. Yolanda Beatriz Moguel Ordóñez, INIFAP, México Dr. Ramón Molina Barrios, Instituto Tecnológico de Sonora, Dr. Alfonso Juventino Chay Canul, Universidad Autónoma de Tabasco, México Dra. Maria Cristina Schneider, Universidad de Georgetown, Estados Unidos Dr. Feliciano Milian Suazo, Universidad Autónoma de Querétaro, México Dr. Javier F. Enríquez Quiroz, INIFAP, México Dra. Martha Hortencia Martín Rivera, Universidad de Sonora URN, México Dr. Fernando Arturo Ibarra Flores, Universidad de Sonora URN, México Dr. James A. Pfister, USDA, Estados Unidos Dr. Eduardo Daniel Bolaños Aguilar, INIFAP, México Dr. Sergio Iván Román-Ponce, INIFAP, México Dr. Jesús Fernández Martín, INIA, España Dr. Maurcio A. Elzo, Universidad de Florida Dr. Sergio D. Rodríguez Camarillo, INIFAP, México Dra. Nydia Edith Reyes Rodríguez, Universidad Autónoma del Estado de Hidalgo, México Dra. Maria Salud Rubio Lozano, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México Dra. Elizabeth Loza-Rubio, INIFAP, México Dr. Juan Carlos Saiz Calahorra, Instituto Nacional de Investigaciones Agrícolas, España Dr. José Armando Partida de la Peña, INIFAP, México Dr. José Luis Romano Muñoz, INIFAP, México Dr. Jorge Alberto López García, INIFAP, México

Dr. Alejandro Plascencia Jorquera, Universidad Autónoma de Baja California, México Dr. Juan Ku Vera, Universidad Autónoma de Yucatán, México Dr. Ricardo Basurto Gutiérrez, INIFAP, México Dr. Luis Corona Gochi, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México Dr. Juan Manuel Pinos Rodríguez, Facultad de Medicina Veterinaria y Zootecnia, Universidad Veracruzana, México Dr. Carlos López Coello, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México Dr. Arturo Francisco Castellanos Ruelas, Facultad de Química. UADY Dra. Guillermina Ávila Ramírez, UNAM, México Dr. Emmanuel Camuus, CIRAD, Francia. Dr. Héctor Jiménez Severiano, INIFAP., México Dr. Juan Hebert Hernández Medrano, UNAM, México Dr. Adrian Guzmán Sánchez, Universidad Autónoma Metropolitana-Xochimilco, México Dr. Eugenio Villagómez Amezcua Manjarrez, INIFAP, CENID Salud Animal e Inocuidad, México Dr. José Juan Hernández Ledezma, Consultor privado Dr. Fernando Cervantes Escoto, Universidad Autónoma Chapingo, México Dr. Adolfo Guadalupe Álvarez Macías, Universidad Autónoma Metropolitana Xochimilco, México Dr. Alfredo Cesín Vargas, UNAM, México Dra. Marisela Leal Hernández, INIFAP, México Dr. Efrén Ramírez Bribiesca, Colegio de Postgraduados, México

TIPOGRAFÍA Y FORMATO: Oscar L. Rodríguez Rivera

Indizada en el “Journal Citation Report” Science Edition del ISI . Inscrita en el Sistema de Clasificación de Revistas Científicas y Tecnológicas de CONACyT; en EBSCO Host y la Red de Revistas Científicas de América Latina y el Caribe, España y Portugal (RedALyC) (www.redalyc.org); en la Red Iberoamericana de Revistas Científicas de Veterinaria de Libre Acceso (www.veterinaria.org/revistas/ revivec); en los Índices SCOPUS y EMBASE de Elsevier (www.elsevier. com).

REVISTA MEXICANA DE CIENCIAS PECUARIAS La Revista Mexicana de Ciencias Pecuarias es un órgano de difusión científica y técnica de acceso abierto, revisada por pares y arbitrada. Su objetivo es dar a conocer los resultados de las investigaciones realizadas por cualquier institución científica, relacionadas particularmente con las distintas disciplinas de la Medicina Veterinaria y la Zootecnia. Además de trabajos de las disciplinas indicadas en su Comité Editorial, se aceptan también para su evaluación y posible publicación, trabajos de otras disciplinas, siempre y cuando estén relacionados con la investigación pecuaria. Se publican en la revista tres categorías de trabajos: Artículos Científicos, Notas de Investigación y Revisiones Bibliográficas (consultar las Notas al autor); la responsabilidad de cada trabajo recae exclusivamente en los autores, los cuales, por la naturaleza misma de los experimentos pueden verse obligados a referirse en algunos casos a los nombres comerciales de ciertos productos, ello sin embargo, no implica preferencia por los productos citados o ignorancia respecto a los omitidos, ni tampoco significa en modo alguno respaldo publicitario hacia los productos mencionados. Todas las contribuciones serán cuidadosamente evaluadas por árbitros, considerando su calidad y relevancia académica. Queda entendido que el someter un manuscrito implica que la investigación descrita es única e inédita. La publicación de Rev. Mex. Cienc. Pecu. es trimestral en formato bilingüe Español e Inglés. El costo

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VISIT OUR SITE IN THE INTERNET Full articles from year 1963 to date and Instructions for authors can be accessed via the site http://cienciaspecuarias.inifap.gob.mx

REVISTA MEXICANA DE CIENCIAS PECUARIAS

REV. MEX. CIENC. PECU.

VOL. 13 No. 3

JULIO-SEPTIEMBRE-2022

CONTENIDO Contents ARTÍCULOS Articles

Pág.

Isolated Escherichia coli resistance genes in broiler chicken Genes de resistencia a aislados de Escherichia coli en pollos de engorda Diana López-Velandia, Edna Carvajal-Barrera, Egberto Rueda-Garrido, Martín Talavera- Rojas, María Vásquez, María Torres-Caycedo ………………………………………………………………………………….584 Detección del virus de la lengua azul en ovinos por RT- PCR en tiempo real en diferentes sistemas de producción en San Martín, Perú Detection of bluetongue virus in sheep by real-time RT-PCR in different production systems in San Martín, Perú Alicia María López Flores, Roni David Cruz Vasquez, Víctor Humberto Puicón Niño de Guzmán, Alicia Bartra Reátegui, Orlando Ríos Ramírez, Fredy Fabián Domínguez ………………………………….596 Detección del virus de la diarrea viral bovina en artiodáctilos silvestres en cautiverio en México Detection of bovine viral diarrhea virus in captive wild artiodactyls in Mexico Jocelyn Medina-Gudiño, Ninnet Gómez-Romero, José Ramírez-Lezama, Luis Padilla-Noriega, Emilio Venegas-Cureño, Francisco Javier Basurto-Alcántara ………………………………………………………….…612 Evaluación de la reacción en cadena de la polimerasa en tiempo real acoplado a separación inmunomagnética (PCRTR-IMS) como método alternativo para la detección rutinaria de Salmonella spp. en carne de res en México Evaluation of real-time polymerase chain reaction coupled to immunomagnetic separation (rtPCRIMS) as an alternative method for the routine detection of Salmonella spp. in beef in Mexico Gloria Marisol Castañeda-Ruelas, José Roberto Guzmán-Uriarte, José Benigno Valdez-Torres, Josefina León-Félix…………………………………………………………………………………………………………..…625 Prevalence of Mycobacterium avium subsp. paratuberculosis and associated risk factors in dairies under mechanical milking parlor-systems in Antioquia, Colombia Prevalencia de Mycobacterium avium subsp. paratuberculosis y factores de riesgo asociados en lecherías bajo sistemas de sala de ordeño mecánico en Antioquia, Colombia Nathalia M. Correa-Valencia, Nicolás F. Ramírez-Vásquez, Jorge A. Fernández-Silva …………………643

III

Effects of nutrition in the final third of gestation of beef cows on progeny development Efecto de la nutrición en el último tercio de la gestación de vacas de carne sobre el desarrollo de la progenie John Lenon Klein, Sander Martinho Adams, Amanda Farias de Moura, Daniele Borchate, Dari Celestino Alves Filho, Dieison Pansiera Antunes, Fabiana Moro Maidana, Gilmar dos Santos Cardoso, Ivan Luiz Brondani, Ricardo Gonçalves Gindri ……………………………………………………………………….658 Establishment of tropical forage grasses in the Cerrado biome Establecimiento de gramíneas forrajeras tropicales en el bioma del Cerrado Antonio Leandro Chaves Gurgel, Gelson dos Santos Difante, Carolina Marques Costa, João Virgínio Emerenciano Neto, Gustavo Henrique Tonhão, Luís Carlos Vinhas Ítavo, Alexandre Menezes Dias, Iuri Mesquita Moraes Vilela, Vivian Garcia de Oliveira, Pâmella Cristina da Silva Lima, Andrey William Alce Miyake ....................................................................………………………………………..674 Efectividad del clorhidrato de zilpaterol en la finalización de corderos: Patente vs. Genérico Effectiveness of zilpaterol hydrochloride in lamb finishing: Patent vs. Generic Arnulfo Vicente Pérez, Leonel Avendaño-Reyes, Juan E. Guerra-Liera, Rubén Barajas Cruz, Ricardo Vicente-Pérez, M. Ángeles López-Baca, Miguel A. Gastelum Delgado, Alfonso J. Chay-Canul, Ulises Macías-Cruz ………………………………………………………………………………………………………………………690 Forage availability in Xaraés grass pastures subjected to nitrogen sources of the slow and fast release Disponibilidad de forraje en praderas de pasto Xaraés en respuesta a fuentes de nitrógeno convencionales y tratadas con N-(n-butil) triamida tiofosfórica (NBPT) Luís Henrique Almeida Matos, Carlindo Santos Rodrigues, Douglas dos Santos Pina, Vagner Maximino Leite, Paula Aguiar Silva, Taiala Cristina de Jesus Pereira, Gleidson Giordano Pinto Carvalho ..................................................................................…………………………………….….706

REVISIONES DE LITERATURA Reviews Diagnóstico, prevención y control de enfermedades causadas por Chlamydia en pequeños rumiantes. Revisión Diagnosis, prevention and control of diseases caused by Chlamydia in small ruminants. Review Fernando De Jesús Aldama, Roberto Montes de Oca Jiménez, Jorge Antonio Varela Guerrero …..725 Comportamiento de ingestión y consumo de forraje por vacas en pastoreo en clima templado. Revisión Ingestion behavior and forage intake by grazing cows in temperate climate. Review Juan Daniel Jiménez Rosales, Ricardo Daniel Améndola Massiotti .........………………………………….743 La citometría de flujo, un universo de posibilidades en el ámbito veterinario. Revisión Flow cytometry, a universe of possibilities in the veterinary field. Review Luvia Enid Sánchez-Torres, Alejandra Espinosa-Bonilla, Fernando Diosdado-Vargas …………………763

Presencia de alcaloides pirrolizidínicos en miel y los efectos de su consumo en humanos y abejas. Revisión Presence of pyrrolizidine alkaloids in honey and the effects of their consumption on humans and honeybees. Review Laura Yavarik Alvarado-Avila, Yolanda Beatriz Moguel-Ordóñez, Claudia García-Figueroa, Francisco Javier Ramírez-Ramírez, Miguel Enrique Arechavaleta-Velasco ……………………787 Efectos de los fitoestrógenos en la fisiología reproductiva de especies productivas. Revisión Effects of phytoestrogens on the reproductive physiology of productive species. Review Miguel Morales Ramírez, Dinorah Vargas Estrada, Iván Juárez Rodríguez, Juan José Pérez-Rivero, Alonso Sierra Reséndiz, Héctor Fabián Flores González, José Luis Cerbón Gutiérrez, Sheila Irais Peña-Corona ………..…………………………………………………………..................................803

NOTAS DE INVESTIGACIÓN Technical notes Estructura genética y aptitud ambiental de poblaciones de pasto banderita [ Bouteloua curtipendula (Michx.) Torr.] en Chihuahua, México Genetic structure and environmental aptitude of sideoats grama [ Bouteloua curtipendula (Michx.) Torr.] populations in Chihuahua, Mexico Alan Álvarez-Holguín, Carlos Raúl Morales-Nieto, Raúl Corrales-Lerma, Jesús Alejandro PrietoAmparán, Ireyli Zuluami Iracheta-Lara, Nathalie Socorro Hernández-Quiroz ………………..............830

Actualización: marzo, 2020 NOTAS AL AUTOR La Revista Mexicana de Ciencias Pecuarias se edita completa en dos idiomas (español e inglés) y publica tres categorías de trabajos: Artículos científicos, Notas de investigación y Revisiones bibliográficas.

Los autores interesados en publicar en esta revista deberán ajustarse a los lineamientos que más adelante se indican, los cuales en términos generales, están de acuerdo con los elaborados por el Comité Internacional de Editores de Revistas Médicas (CIERM) Bol Oficina Sanit Panam 1989;107:422-437. 1.

Página del título Resumen en español Resumen en inglés Texto Agradecimientos y conflicto de interés Literatura citada

Sólo se aceptarán trabajos inéditos. No se admitirán si están basados en pruebas de rutina, ni datos experimentales sin estudio estadístico cuando éste sea indispensable. Tampoco se aceptarán trabajos que previamente hayan sido publicados condensados o in extenso en Memorias o Simposio de Reuniones o Congresos (a excepción de Resúmenes). Todos los trabajos estarán sujetos a revisión de un Comité Científico Editorial, conformado por Pares de la Disciplina en cuestión, quienes desconocerán el nombre e Institución de los autores proponentes. El Editor notificará al autor la fecha de recepción de su trabajo. El manuscrito deberá someterse a través del portal de la Revista en la dirección electrónica: http://cienciaspecuarias.inifap.gob.mx, consultando el “Instructivo para envío de artículos en la página de la Revista Mexicana de Ciencias Pecuarias”. Para su elaboración se utilizará el procesador de Microsoft Word, con letra Times New Roman a 12 puntos, a doble espacio. Asimismo se deberán llenar los formatos de postulación, carta de originalidad y no duplicidad y disponibles en el propio sitio oficial de la revista.

Por ser una revista con arbitraje, y para facilitar el trabajo de los revisores, todos los renglones de cada página deben estar numerados; asimismo cada página debe estar numerada, inclusive cuadros, ilustraciones y gráficas.

Los artículos tendrán una extensión máxima de 20 cuartillas a doble espacio, sin incluir páginas de Título, y cuadros o figuras (los cuales no deberán exceder de ocho y ser incluidos en el texto). Las Notas de investigación tendrán una extensión máxima de 15 cuartillas y 6 cuadros o figuras. Las Revisiones bibliográficas una extensión máxima de 30 cuartillas y 5 cuadros.

Los manuscritos de las tres categorías de trabajos que se publican en la Rev. Mex. Cienc. Pecu. deberán contener los componentes que a continuación se indican, empezando cada uno de ellos en página aparte.

Página del Título. Solamente debe contener el título del trabajo, que debe ser conciso pero informativo; así como el título traducido al idioma inglés. En el manuscrito no es necesaria información como nombres de autores, departamentos, instituciones, direcciones de correspondencia, etc., ya que estos datos tendrán que ser registrados durante el proceso de captura de la solicitud en la plataforma del OJS (http://ciencias pecuarias.inifap.gob.mx).

Resumen en español. En la segunda página se debe incluir un resumen que no pase de 250 palabras. En él se indicarán los propósitos del estudio o investigación; los procedimientos básicos y la metodología empleada; los resultados más importantes encontrados, y de ser posible, su significación estadística y las conclusiones principales. A continuación del resumen, en punto y aparte, agregue debidamente rotuladas, de 3 a 8 palabras o frases cortas clave que ayuden a los indizadores a clasificar el trabajo, las cuales se publicarán junto con el resumen.

Resumen en inglés. Anotar el título del trabajo en inglés y a continuación redactar el “abstract” con las mismas instrucciones que se señalaron para el resumen en español. Al final en punto y aparte, se deberán escribir las correspondientes palabras clave (“key words”).

10. Texto. Las tres categorías de trabajos que se publican en la Rev. Mex. Cienc. Pecu. consisten en lo siguiente: a) Artículos científicos. Deben ser informes de trabajos originales derivados de resultados parciales o finales de investigaciones. El texto del Artículo científico se divide en secciones que llevan estos encabezamientos:

Introducción Materiales y Métodos Resultados Discusión Conclusiones e implicaciones Literatura citada

referencias, aunque pueden insertarse en el texto (entre paréntesis).

Reglas básicas para la Literatura citada Nombre de los autores, con mayúsculas sólo las iniciales, empezando por el apellido paterno, luego iniciales del materno y nombre(s). En caso de apellidos compuestos se debe poner un guión entre ambos, ejemplo: Elías-Calles E. Entre las iniciales de un autor no se debe poner ningún signo de puntuación, ni separación; después de cada autor sólo se debe poner una coma, incluso después del penúltimo; después del último autor se debe poner un punto.

En los artículos largos puede ser necesario agregar subtítulos dentro de estas divisiones a fin de hacer más claro el contenido, sobre todo en las secciones de Resultados y de Discusión, las cuales también pueden presentarse como una sola sección. b) Notas de investigación. Consisten en modificaciones a técnicas, informes de casos clínicos de interés especial, preliminares de trabajos o investigaciones limitadas, descripción de nuevas variedades de pastos; así como resultados de investigación que a juicio de los editores deban así ser publicados. El texto contendrá la misma información del método experimental señalado en el inciso a), pero su redacción será corrida del principio al final del trabajo; esto no quiere decir que sólo se supriman los subtítulos, sino que se redacte en forma continua y coherente.

El título del trabajo se debe escribir completo (en su idioma original) luego el título abreviado de la revista donde se publicó, sin ningún signo de puntuación; inmediatamente después el año de la publicación, luego el número del volumen, seguido del número (entre paréntesis) de la revista y finalmente el número de páginas (esto en caso de artículo ordinario de revista). Puede incluir en la lista de referencias, los artículos aceptados aunque todavía no se publiquen; indique la revista y agregue “en prensa” (entre corchetes).

c) Revisiones bibliográficas. Consisten en el tratamiento y exposición de un tema o tópico de relevante actualidad e importancia; su finalidad es la de resumir, analizar y discutir, así como poner a disposición del lector información ya publicada sobre un tema específico. El texto se divide en: Introducción, y las secciones que correspondan al desarrollo del tema en cuestión.

En el caso de libros de un solo autor (o más de uno, pero todos responsables del contenido total del libro), después del o los nombres, se debe indicar el título del libro, el número de la edición, el país, la casa editorial y el año. Cuando se trate del capítulo de un libro de varios autores, se debe poner el nombre del autor del capítulo, luego el título del capítulo, después el nombre de los editores y el título del libro, seguido del país, la casa editorial, año y las páginas que abarca el capítulo.

11. Agradecimientos y conflicto de interés. Siempre que corresponda, se deben especificar las colaboraciones que necesitan ser reconocidas, tales como a) la ayuda técnica recibida; b) el agradecimiento por el apoyo financiero y material, especificando la índole del mismo; c) las relaciones financieras que pudieran suscitar un conflicto de intereses. Las personas que colaboraron pueden ser citadas por su nombre, añadiendo su función o tipo de colaboración; por ejemplo: “asesor científico”, “revisión crítica de la propuesta para el estudio”, “recolección de datos”, etc. Siempre que corresponda, los autores deberán mencionar si existe algún conflicto de interés. 12. Literatura citada. Numere las referencias consecutivamente en el orden en que se mencionan por primera vez en el texto. Las referencias en el texto, en los cuadros y en las ilustraciones se deben identificar mediante números arábigos entre paréntesis, sin señalar el año de la referencia. Evite hasta donde sea posible, el tener que mencionar en el texto el nombre de los autores de las referencias. Procure abstenerse de utilizar los resúmenes como referencias; las “observaciones inéditas” y las “comunicaciones personales” no deben usarse como

En el caso de tesis, se debe indicar el nombre del autor, el título del trabajo, luego entre corchetes el grado (licenciatura, maestría, doctorado), luego el nombre de la ciudad, estado y en su caso país, seguidamente el nombre de la Universidad (no el de la escuela), y finalmente el año. Emplee el estilo de los ejemplos que aparecen a continuación, los cuales están parcialmente basados en el formato que la Biblioteca Nacional de Medicina de los Estados Unidos usa en el Index Medicus. Revistas

Artículo ordinario, con volumen y número. (Incluya el nombre de todos los autores cuando sean seis o menos; si son siete o más, anote sólo el nombre de los seis primeros y agregue “et al.”).

VII

Basurto GR, Garza FJD. Efecto de la inclusión de grasa o proteína de escape ruminal en el comportamiento de toretes Brahman en engorda. Téc Pecu Méx 1998;36(1):35-48.

XI)

Sólo número sin indicar volumen. II) Stephano HA, Gay GM, Ramírez TC. Encephalomielitis, reproductive failure and corneal opacity (blue eye) in pigs associated with a paramyxovirus infection. Vet Rec 1988;(122):6-10.

XII) Cunningham EP. Genetic diversity in domestic animals: strategies for conservation and development. In: Miller RH et al. editors. Proc XX Beltsville Symposium: Biotechnology’s role in genetic improvement of farm animals. USDA. 1996:13.

III) Chupin D, Schuh H. Survey of present status ofthe use of artificial insemination in developing countries. World Anim Rev 1993;(74-75):26-35.

Tesis.

No se indica el autor.

XIII) Alvarez MJA. Inmunidad humoral en la anaplasmosis y babesiosis bovinas en becerros mantenidos en una zona endémica [tesis maestría]. México, DF: Universidad Nacional Autónoma de México; 1989.

IV) Cancer in South Africa [editorial]. S Afr Med J 1994;84:15.

Suplemento de revista.

XIV) Cairns RB. Infrared spectroscopic studies of solid oxigen [doctoral thesis]. Berkeley, California, USA: University of California; 1965.

V) Hall JB, Staigmiller RB, Short RE, Bellows RA, Bartlett SE. Body composition at puberty in beef heifers as influenced by nutrition and breed [abstract]. J Anim Sci 1998;71(Suppl 1):205.

Organización como autor. XV) NRC. National Research Council. The nutrient requirements of beef cattle. 6th ed. Washington, DC, USA: National Academy Press; 1984.

Organización, como autor. VI) The Cardiac Society of Australia and New Zealand. Clinical exercise stress testing. Safety and performance guidelines. Med J Aust 1996;(164):282-284.

XVI) SAGAR. Secretaría de Agricultura, Ganadería y Desarrollo Rural. Curso de actualización técnica para la aprobación de médicos veterinarios zootecnistas responsables de establecimientos destinados al sacrificio de animales. México. 1996.

En proceso de publicación. VII) Scifres CJ, Kothmann MM. Differential grazing use of herbicide treated area by cattle. J Range Manage [in press] 2000.

XVII) AOAC. Oficial methods of analysis. 15th ed. Arlington, VA, USA: Association of Official Analytical Chemists. 1990.

Libros y otras monografías

XVIII) SAS. SAS/STAT User’s Guide (Release 6.03). Cary NC, USA: SAS Inst. Inc. 1988.

Autor total. VIII) Steel RGD, Torrie JH. Principles and procedures of statistics: A biometrical approach. 2nd ed. New York, USA: McGraw-Hill Book Co.; 1980.

XIX) SAS. SAS User´s Guide: Statistics (version 5 ed.). Cary NC, USA: SAS Inst. Inc. 1985.

Publicaciones electrónicas

Autor de capítulo. IX)

XX) Jun Y, Ellis M. Effect of group size and feeder type on growth performance and feeding patterns in growing pigs. J Anim Sci 2001;79:803-813. http://jas.fass.org/cgi/reprint/79/4/803.pdf. Accessed Jul 30, 2003.

Roberts SJ. Equine abortion. In: Faulkner LLC editor. Abortion diseases of cattle. 1rst ed. Springfield, Illinois, USA: Thomas Books; 1968:158-179.

Memorias de reuniones. X)

Olea PR, Cuarón IJA, Ruiz LFJ, Villagómez AE. Concentración de insulina plasmática en cerdas alimentadas con melaza en la dieta durante la inducción de estro lactacional [resumen]. Reunión nacional de investigación pecuaria. Querétaro, Qro. 1998:13.

XXI) Villalobos GC, González VE, Ortega SJA. Técnicas para estimar la degradación de proteína y materia orgánica en el rumen y su importancia en rumiantes en pastoreo. Téc Pecu Méx 2000;38(2): 119-134. http://www.tecnicapecuaria.org/trabajos/20021217 5725.pdf. Consultado 30 Ago, 2003.

Loeza LR, Angeles MAA, Cisneros GF. Alimentación de cerdos. En: Zúñiga GJL, Cruz BJA editores. Tercera reunión anual del centro de investigaciones forestales y agropecuarias del estado de Veracruz. Veracruz. 1990:51-56.

VIII

XXII) Sanh MV, Wiktorsson H, Ly LV. Effect of feeding level on milk production, body weight change, feed conversion and postpartum oestrus of crossbred lactating cows in tropical conditions. Livest Prod Sci 2002;27(2-3):331-338. http://www.sciencedirect. com/science/journal/03016226. Accessed Sep 12, 2003.

ha hectárea (s) h hora (s) i.m. intramuscular (mente) i.v. intravenosa (mente) J joule (s) kg kilogramo (s) km kilómetro (s) L litro (s) log logaritmo decimal Mcal megacaloría (s) MJ megajoule (s) m metro (s) msnm metros sobre el nivel del mar µg microgramo (s) µl microlitro (s) µm micrómetro (s)(micra(s)) mg miligramo (s) ml mililitro (s) mm milímetro (s) min minuto (s) ng nanogramo (s)Pprobabilidad (estadística) p página PC proteína cruda PCR reacción en cadena de la polimerasa pp páginas ppm partes por millón % por ciento (con número) rpm revoluciones por minuto seg segundo (s) t tonelada (s) TND total de nutrientes digestibles UA unidad animal UI unidades internacionales

13. Cuadros, Gráficas e Ilustraciones. Es preferible que sean pocos, concisos, contando con los datos necesarios para que sean autosuficientes, que se entiendan por sí mismos sin necesidad de leer el texto. Para las notas al pie se deberán utilizar los símbolos convencionales. 14 Versión final. Es el documento en el cual los autores ya integraron las correcciones y modificaciones indicadas por el Comité Revisor. Los trabajos deberán ser elaborados con Microsoft Word. Las fotografías e imágenes deberán estar en formato jpg (o compatible) con al menos 300 dpi de resolución. Tanto las fotografías, imágenes, gráficas, cuadros o tablas deberán incluirse en el mismo archivo del texto. Los cuadros no deberán contener ninguna línea vertical, y las horizontales solamente las que delimitan los encabezados de columna, y la línea al final del cuadro. 15. Una vez recibida la versión final, ésta se mandará para su traducción al idioma inglés o español, según corresponda. Si los autores lo consideran conveniente podrán enviar su manuscrito final en ambos idiomas. 16. Tesis. Se publicarán como Artículo o Nota de Investigación, siempre y cuando se ajusten a las normas de esta revista. 17. Los trabajos no aceptados para su publicación se regresarán al autor, con un anexo en el que se explicarán los motivos por los que se rechaza o las modificaciones que deberán hacerse para ser reevaluados.

versus

gravedades

Cualquier otra abreviatura se pondrá entre paréntesis inmediatamente después de la(s) palabra(s) completa(s).

18. Abreviaturas de uso frecuente: cal cm °C DL50 g

caloría (s) centímetro (s) grado centígrado (s) dosis letal 50% gramo (s)

19. Los nombres científicos y otras locuciones latinas se deben escribir en cursivas.

Updated: March, 2020 INSTRUCTIONS FOR AUTHORS Revista Mexicana de Ciencias Pecuarias is a scientific journal published in a bilingual format (Spanish and English) which carries three types of papers: Research Articles, Technical Notes, and Reviews. Authors interested in publishing in this journal, should follow the belowmentioned directives which are based on those set down by the International Committee of Medical Journal Editors (ICMJE) Bol Oficina Sanit Panam 1989;107:422-437. 1.

Title page Abstract Text Acknowledgments and conflict of interest Literature cited

Only original unpublished works will be accepted. Manuscripts based on routine tests, will not be accepted. All experimental data must be subjected to statistical analysis. Papers previously published condensed or in extenso in a Congress or any other type of Meeting will not be accepted (except for Abstracts). All contributions will be peer reviewed by a scientific editorial committee, composed of experts who ignore the name of the authors. The Editor will notify the author the date of manuscript receipt. Papers will be submitted in the Web site http://cienciaspecuarias.inifap.gob.mx, according the “Guide for submit articles in the Web site of the Revista Mexicana de Ciencias Pecuarias”. Manuscripts should be prepared, typed in a 12 points font at double space (including the abstract and tables), At the time of submission a signed agreement co-author letter should enclosed as complementary file; coauthors at different institutions can mail this form independently. The corresponding author should be indicated together with his address (a post office box will not be accepted), telephone and Email.

Title page. It should only contain the title of the work, which should be concise but informative; as well as the title translated into English language. In the manuscript is not necessary information as names of authors, departments, institutions and correspondence addresses, etc.; as these data will have to be registered during the capture of the application process on the OJS platform (http://cienciaspecuarias.inifap.gob.mx).

Abstract. On the second page a summary of no more than 250 words should be included. This abstract should start with a clear statement of the objectives and must include basic procedures and methodology. The more significant results and their statistical value and the main conclusions should be elaborated briefly. At the end of the abstract, and on a separate line, a list of up to 10 key words or short phrases that best describe the nature of the research should be stated.

Text. The three categories of articles which are published in Revista Mexicana de Ciencias Pecuarias are the following:

a) Research Articles. They should originate in primary

works and may show partial or final results of research. The text of the article must include the following parts:

To facilitate peer review all pages should be numbered consecutively, including tables, illustrations and graphics, and the lines of each page should be numbered as well.

Introduction Materials and Methods Results Discussion Conclusions and implications Literature cited

Research articles will not exceed 20 double spaced pages, without including Title page and Tables and Figures (8 maximum and be included in the text). Technical notes will have a maximum extension of 15 pages and 6 Tables and Figures. Reviews should not exceed 30 pages and 5 Tables and Figures.

In lengthy articles, it may be necessary to add other sections to make the content clearer. Results and Discussion can be shown as a single section if considered appropriate.

Manuscripts of all three type of articles published in Revista Mexicana de Ciencias Pecuarias should contain the following sections, and each one should begin on a separate page.

b) Technical Notes. They should be brief and be evidence for technical changes, reports of clinical cases of special interest, complete description of a limited investigation, or research results which

should be published as a note in the opinion of the editors. The text will contain the same information presented in the sections of the research article but without section titles.

names(s), the number of the edition, the country, the printing house and the year. e. When a reference is made of a chapter of book written by several authors; the name of the author(s) of the chapter should be quoted, followed by the title of the chapter, the editors and the title of the book, the country, the printing house, the year, and the initial and final pages.

c) Reviews. The purpose of these papers is to

summarize, analyze and discuss an outstanding topic. The text of these articles should include the following sections: Introduction, and as many sections as needed that relate to the description of the topic in question.

f. In the case of a thesis, references should be made of the author’s name, the title of the research, the degree obtained, followed by the name of the City, State, and Country, the University (not the school), and finally the year.

10. Acknowledgements. Whenever appropriate, collaborations that need recognition should be specified: a) Acknowledgement of technical support; b) Financial and material support, specifying its nature; and c) Financial relationships that could be the source of a conflict of interest.

Examples The style of the following examples, which are partly based on the format the National Library of Medicine of the United States employs in its Index Medicus, should be taken as a model.

People which collaborated in the article may be named, adding their function or contribution; for example: “scientific advisor”, “critical review”, “data collection”, etc. 11. Literature cited. All references should be quoted in their original language. They should be numbered consecutively in the order in which they are first mentioned in the text. Text, tables and figure references should be identified by means of Arabic numbers. Avoid, whenever possible, mentioning in the text the name of the authors. Abstain from using abstracts as references. Also, “unpublished observations” and “personal communications” should not be used as references, although they can be inserted in the text (inside brackets).

Key rules for references a. The names of the authors should be quoted beginning with the last name spelt with initial capitals, followed by the initials of the first and middle name(s). In the presence of compound last names, add a dash between both, i.e. Elias-Calles E. Do not use any punctuation sign, nor separation between the initials of an author; separate each author with a comma, even after the last but one. b. The title of the paper should be written in full, followed by the abbreviated title of the journal without any punctuation sign; then the year of the publication, after that the number of the volume, followed by the number (in brackets) of the journal and finally the number of pages (this in the event of ordinary article). c. Accepted articles, even if still not published, can be included in the list of references, as long as the journal is specified and followed by “in press” (in brackets).

Journals

Standard journal article (List the first six authors followed by et al.) I)

Basurto GR, Garza FJD. Efecto de la inclusión de grasa o proteína de escape ruminal en el comportamiento de toretes Brahman en engorda. Téc Pecu Méx 1998;36(1):35-48.

Issue with no volume II) Stephano HA, Gay GM, Ramírez TC. Encephalomielitis, reproductive failure and corneal opacity (blue eye) in pigs associated with a paramyxovirus infection. Vet Rec 1988;(122):6-10. III) Chupin D, Schuh H. Survey of present status of the use of artificial insemination in developing countries. World Anim Rev 1993;(74-75):26-35.

No author given IV) Cancer in South Africa [editorial]. S Afr Med J 1994;84:15.

Journal supplement V) Hall JB, Staigmiller RB, Short RE, Bellows RA, Bartlett SE. Body composition at puberty in beef heifers as influenced by nutrition and breed [abstract]. J Anim Sci 1998;71(Suppl 1):205.

d. In the case of a single author’s book (or more than one, but all responsible for the book’s contents), the title of the book should be indicated after the

Organization, as author VI) The Cardiac Society of Australia and New Zealand. Clinical exercise stress testing. Safety and performance guidelines. Med J Aust 1996;(164):282284.

In press VII) Scifres CJ, Kothmann MM. Differential grazing use of herbicide-treated area by cattle. J Range Manage [in press] 2000. Books and other monographs

Author(s) VIII) Steel RGD, Torrie JH. Principles and procedures of statistics: A biometrical approach. 2nd ed. New York, USA: McGraw-Hill Book Co.; 1980.

Organization as author XV) NRC. National Research Council. The nutrient requirements of beef cattle. 6th ed. Washington, DC, USA: National Academy Press; 1984. XVI) SAGAR. Secretaría de Agricultura, Ganadería y Desarrollo Rural. Curso de actualización técnica para la aprobación de médicos veterinarios zootecnistas responsables de establecimientos destinados al sacrificio de animales. México. 1996. XVII) AOAC. Official methods of analysis. 15th ed. Arlington, VA, USA: Association of Official Analytical Chemists. 1990. XVIII) SAS. SAS/STAT User’s Guide (Release 6.03). Cary NC, USA: SAS Inst. Inc. 1988. XIX) SAS. SAS User´s Guide: Statistics (version 5 ed.). Cary NC, USA: SAS Inst. Inc. 1985.

Chapter in a book IX)

Roberts SJ. Equine abortion. In: Faulkner LLC editor. Abortion diseases of cattle. 1rst ed. Springfield, Illinois, USA: Thomas Books; 1968:158-179.

Conference paper X)

XI)

Thesis XIII) Alvarez MJA. Inmunidad humoral en la anaplasmosis y babesiosis bovinas en becerros mantenidos en una zona endémica [tesis maestría]. México, DF: Universidad Nacional Autónoma de México; 1989.

Electronic publications XX) Jun Y, Ellis M. Effect of group size and feeder type on growth performance and feeding patterns in growing pigs. J Anim Sci 2001;79:803-813. http://jas.fass.org/cgi/reprint/79/4/803.pdf. Accesed Jul 30, 2003. XXI) Villalobos GC, González VE, Ortega SJA. Técnicas para estimar la degradación de proteína y materia orgánica en el rumen y su importancia en rumiantes en pastoreo. Téc Pecu Méx 2000;38(2): 119-134. http://www.tecnicapecuaria.org/trabajos/20021217 5725.pdf. Consultado 30 Jul, 2003. XXII) Sanh MV, Wiktorsson H, Ly LV. Effect of feeding level on milk production, body weight change, feed conversion and postpartum oestrus of crossbred lactating cows in tropical conditions. Livest Prod Sci 2002;27(2-3):331-338. http://www.sciencedirect.com/science/journal/030 16226. Accesed Sep 12, 2003. 12. Tables, Graphics and Illustrations. It is preferable that they should be few, brief and having the necessary data so they could be understood without reading the text. Explanatory material should be placed in footnotes, using conventional symbols.

13. Final version. This is the document in which the authors have already integrated the corrections and modifications indicated by the Review Committee. The works will have to be elaborated with Microsoft Word. Photographs and images must be in jpg (or compatible) format with at least 300 dpi resolution. Photographs, images, graphs, charts or tables must be included in the same text file. The boxes should not contain any vertical lines, and the horizontal ones only those that delimit the column headings, and the line at the end of the box.

XIV) Cairns RB. Infrared spectroscopic studies of solid oxigen [doctoral thesis]. Berkeley, California, USA: University of California; 1965.

XII

14. Once accepted, the final version will be translated into Spanish or English, although authors should feel free to send the final version in both languages. No charges will be made for style or translation services.

MJ m µl µm mg ml mm min ng

mega joule (s) meter (s) micro liter (s) micro meter (s) milligram (s) milliliter (s) millimeter (s) minute (s) nanogram (s) P probability (statistic) p page CP crude protein PCR polymerase chain reaction pp pages ppm parts per million % percent (with number) rpm revolutions per minute sec second (s) t metric ton (s) TDN total digestible nutrients AU animal unit IU international units

15. Thesis will be published as a Research Article or as a Technical Note, according to these guidelines. 16. Manuscripts not accepted for publication will be returned to the author together with a note explaining the cause for rejection, or suggesting changes which should be made for re-assessment.

17. List of abbreviations: cal cm °C DL50 g ha h i.m. i.v. J kg km L log Mcal

calorie (s) centimeter (s) degree Celsius lethal dose 50% gram (s) hectare (s) hour (s) intramuscular (..ly) intravenous (..ly) joule (s) kilogram (s) kilometer (s) liter (s) decimal logarithm mega calorie (s)

versus

gravidity

The full term for which an abbreviation stands should precede its first use in the text. 18. Scientific names and other Latin terms should be written in italics.

XIII

https://doi.org/10.22319/rmcp.v13i3.5627 Article

Isolated Escherichia coli resistance genes in broiler chicken

Diana López-Velandia a* Edna Carvajal-Barrera b Egberto Rueda-Garrido c Martín Talavera- Rojas d María Vásquez b María Torres-Caycedo a

Universidad de Boyacá-Tunja-Colombia.

Universidad de Santander, Facultad de Ciencias Médicas y de la Salud, Grupo de investigación CliniUDES, Bucaramanga, Colombia. c

Universidad de Santander, Facultad de Ciencias Exactas, Naturales y Agropecuarias, Grupo de investigación en Ciencias Agropecuarias GICA, Bucaramanga, Colombia. d

Universidad Autónoma del Estado de México, UAEM, Toluca, Estado de México. México.

*Corresponding author: dplopez@uniboyaca.edu.co

Abstract: Poultry production due to consumer demand has increased annually, which leads to the use of additives such as antibiotics to favor rearing conditions, this increases the deficiency in the composition of production animals’ intestinal microbiota and can generate microbiological and genetic changes; this microbiota can reach humans through food chain, generating a possible horizontal transfer of genes that encode resistance to antibiotics. The objective was to identify resistance profiles and the genes that encode it. Materials and 584

Rev Mex Cienc Pecu 2022;13(3):584-595

methods: From 200 chickens, 35 strains of Escherichia coli with extended spectrum betalactamase resistance phenotype were isolated from healthy broilers, from production farms in Santander (Colombia). 83 % of the AmpC gene, 86 % of the blaCTXM gene, 54 % of the blaSHV gene and 57 % of the blaTEM gene were identified. Regarding the genes that code for resistance to quinolones, 94 % of the qnrB gene, 9 % of the qnrC gene and 0 % of the qnrA gene were identified. The coexistence of the genes that encode for resistance to antibiotics is a serious problem that requires vigilance, in view of this; control strategies must be generated to avoid the spread through the food chain, as well as strategies for the control of the use of antibiotics in the production. Key words: Poultry, Resistance, Gene, Antibiotics.

Received: 28/02/2020 Accepted: 25/11/2021

Introduction In Latin-American countries, chicken is one of most consumed foods because of its easiness for getting it, its low price, its high protein content, and low lipid content, it is the second favorite meat(1). Poultry production increases every year, that is why additives as antibiotics have been employed to encourage upbringing conditions, these additives increase deficiencies in gut microbiota composition. When chickens are born their small intestine is immature and requires morphological, biochemical, and molecular changes that occur during the first two weeks of life, as the animal grows, it is established a microbial community which is more complex through the time. Antibiotics consummation causes digestive disorders be more frequent and produces a low natural resistance to colonization by pathogen microorganisms(2,3). Antibiotics residue can reach the consumer through the food chain causing allergic reactions, bacterial resistance and microflora alteration. In different countries, there are difficulties in commercialization due to a breach in the established rules related to substances concentrations presented in the food. Likewise, several studies have been developed in which bacterial pathogens are referred, including resistant isolations, can be transmitted from chicken to humans(4). In general, antibiotics haven’t been used as growing promoters in animals’ diets around the world during decades. This fact has caused a great concern since human health can be affected when generating bacterial resistance; because antibiotics used for infections 585

Rev Mex Cienc Pecu 2022;13(3):584-595

treatments in humans are employed. Beta lactam antibiotics and fluoroquinolones are broad-spectrum agents commonly used for treating infections, the resistance to this type of antimicrobials has easily arisen. The last reports have demonstrated that resistance to this kind of antibiotics can lead several impacts, which depend on the bacterial strains(5). Some countries present a restricted use of antibiotics as growing promoters, for instance Sweden since 1986, Finland since 1995(6), the European Union since January first 2006(7); among others. In Colombia the use of antibiotics is regulated by different resolutions and decrees, however, there are not restrictions in antibiotics commercialization for veterinary use; for what in some cases, provision is empirical and with no specialized prescription(8). They can be caused by mutations in chromosomal genes and the presence of conjugative and nonconjugative plasmids in the genes(9). The objective of this article was to establish resistance profiles and the genes which codify it.

Material and methods From 200 production chickens, samples were taken with a sterile swab from different organs (trachea, intestines, deep and superficial air-abdominal sacs, pericardium, manufacturing bag, intestine, intestinal contents and pancreas), they were sown in BHI broth and incubated at 37 °C for 24 h, later it was seeded on Mac Conkey agar and incubated at 37 °C for 24 h, they were isolated 35 Escherichia coli strains with extendedspectrum beta-lactamase (ESBL) resistance phenotype of healthy broiler chickens from farms in Santander (Colombia); it was made the microbiological confirmation of gender and species by using BBL Crystal® system and sensitivity tests by means of Kirby Bauer method following CLSI guidelines (2017), using Klebsiella pneumoniae ATCC 700603 strain as positive control and Escherichia coli ATCC 25922 strain as negative control. The susceptibility disks employed were ceftriaxone (CRO 30 µg)(Oxoid ®), cefotaxime (CTX 30 µg) (Oxoid ®), cefepime (FEP 30 µg) (Oxoid ®), nalidixic acid (FEP 30 µg) (Oxoid ®), ciprofloxacin (CIP 1 µg) (Oxoid ®), norfloxacin (NOR 2 µg) (Oxoid ®), piperacillin (PRL 30 µg) (Oxoid ®), aztreonam (ATM 30 µg) (Oxoid ®) and amoxicillin/clavulanic acid (AMC 30 µg) (Oxoid ®). The strains were cultivated in Brain Heart Infusion (BHI) to 37 °C all the night in stirring to make the DNA extraction according to the manufacturer's instructions (Wizard® Genomic DNA Purification Kit), they were considered ideal strains in concentration ≥100μg/μL and DNA-proteins relation A260/280 to determine optimal purity with an OD rate between 1.8 to 2.0 (MaestroNano Micro-Volume Spectrophotometer). They were identified by endpoint PCR blaTEM genes (700 pb), blaSHV genes (700 pb), blaCTX genes (500 pb) and Amp-C genes (550 pb) with the protocole modified by López et al(10); and qnrA genes (580 pb), qnrB genes (264 pb), qnrC genes (428 pb) with Aguilar et al protocole (11). The amplified products were visualized by 586

Rev Mex Cienc Pecu 2022;13(3):584-595

electrophoresis in agarose gel to 1% with TAE to 1% and Safeview classic as a colorant. Gels were visualized by using Ultra Slim Led Illuminator.

Results Susceptibility profiles were 63 % (n=22/35) for ceftriaxone (Oxoid ®), 69 % (n= 23/35) cefepime (Oxoid ®), 77 % (n =27/35) cefotaxime (Oxoid ®), 86 % (n= 30/35) norfloxacin (Oxoid ®), 89 % (n =31/35) ciprofloxacin (Oxoid ®), 91 % (n =32/35) piperacillin (Oxoid ®), 91 % (n =32/35) aztrenam (Oxoid ®), 97 % (n =34/35) amoxicillin/clavulanic acid (Oxoid ®) and 97 % (n=34/35) nalidixic acid (Oxoid ®). Regarding antibiotics groups it was presented E.coli 70 % of resistance to cefalosporines, 90 % to quinolones and 93 % to beta lactams (Figure 1). Regarding genes, they were identified fragments of the expected size, for the genes which codify for beta lactamase resistance it was identified 83 % of AmpC gene, 86 % of blaCTXM gene, 54 % of blaSHV gene and 57 % of blaTEM gene (Figure 2). Regarding genes which codify for quinolone resistance, it was identified 94 % of qnrB gene, 9 % of qnrC gene and 0 % of qnrA gene (Figure 2 and Table 1). Figure 1: Antibiotics resistance groups

587

Rev Mex Cienc Pecu 2022;13(3):584-595

Figure 2: Gel de electroforesis de gen blaCTMX

C1= 1Kb, C2= Positive control, C3= Mx1, C4= Mx2, C5=Mx3, C6= Mx4, C7= Mx5, C8=Mx6, C9= Mx7, C10= Mx8, C11= Mx9, C12= Mx10, C13= Mx11, C14= negative control.

Table 1: Results of the amplified genes Sam ple 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Am pc P P P N P P P P P N P P P P P P P P P P P

blaCT X-M P P P N P P P P P N N P P P P N P P P P P

blaS HV P N P N P P P N N P N N P N P N N P P P P

blaTE M P P P N N P N P P N P P N P N P N N N P N

qnr A N N N N N N N N N N N N N N N N N N N N N

qnr B N N P P P P P P P P P P P P P P P P P P P

qnr S N N P P N N N N N N N N N N N N N N N P N 588

CR O R I R S R R S R R R I S I R R S S I S R R

CT X I R R I R I I R R R R S R R R S S R R R R

FE P I S R S R R S R R R I S R R I S S I R R R

AM C R R R R R R R R R R R R R R R R R R R R R

CI P R I R R R I R R R R R I R R R R R R R R R

NO R S I R R R I R R R R I R R R R R R R R R R

PR L I R R R R R R R R R R R R R R R I R R R R

AT M I R R R R R R R R R I R R R R R R R R R R

Rev Mex Cienc Pecu 2022;13(3):584-595

22 23 24 25 26 27 28 29 30 31 32 33 34 35

P P P P P N P P P N P N P N

N P P P P N P P P N P P P N

P N P P N N P N P N N P P N

N N P P P N P P P N P P P N

N N N N N N N N N N N N N N

P P P P P P P P P P P P P P

N N N N N N N N N N N N N N

S R R R R R R R R I S R R R

S R R R R R R R R R R R R R

R R R R R R R R R R R R R R

P= positive; N= negative; R= resistant; S= sensitive; I= intermediate. CRO= ceftriaxone; CTX= cefotaxime; FEP= cefepime; AMC= amoxycillin/clavulanic acid. CIP= ciprofloxacin; NOR= norfloxacin; PRL= piperacillin; ATM= aztreonam.

Discussion From susceptibility profiles, it can be noticed the strains presented multiresistence, taking into account that these ones had resistance to more than four antibiotics; 49 % presented resistance to all the antibiotics, these results generate a great concern. In South Korea, they found resistance to even eleven antibiotics, including ciprofloxacin(12). Yurong et al obtained similar results when finding resistance to more than five antimicrobial agents; in which, ciprofloxacin and levofloxacine stand out(13). Regarding antibiotic groups, it excels that 93 % presented resistance to beta-lactams, followed by quinolones in 90 % and cephalosporins in 70 % (Figure 3); antimicrobials which are employed for daily use of bacterial infections in humans. Similar reports were made in Korea with resistance to ampicilin (75 %), followed by tetracycline (69 %) and ciprofloxacin (65 %)(14). While Varga et al(15) identified resistance to beta-lactams, sulfonamides and tetracyclines in poultry. In Colombia, bacteria resistant to multiple drugs such as ceftiofur, enrofloxacin, nalidixic acid and tetracycline, were isolated from the meat of poultry from independent stores and from a distribution center of the main chain, which generates an alarm for the health entities of the country(16).

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Figure 3: Genes prevalence

Within resistance it is necessary to confirm the phenotypes of resistance by means of PCR identifying the genes which codify it, for beta-lactamases they can be found the next genes blaTEM, blaSHV , blaCTX and Amp-C(17); and for fluoroquinolones the genes are qnrA , qnrB and qnrC(11); from the profiles previously analyzed, it can be noticed similarity to what other authors reported. Researches made in Brazil, exposed that the isolations which present the genes blaCTX-M-2 or blaCMY-2 tend to accumulate resistance to a higher rate of non-betalactamic antimicrobials(4). In China, the genes which predominated in isolations were blaCTXM and blaTEM, likewise they found variants of blaCMY; as long as blaSHV was not identified(13); as well as in studies made in Pakistan(18), while for the current research, it was presented in a 57 %. Alonso et.al refer that the dissemination of blaSHV can occur by horizontal transfer, mainly caused by plasmids, which could facilitate the dissemination of this gene(19). Regarding AmpC, in the United Kingdom, they were analyzed imported chicken finding un 23 % of this gene, as well as they were identified mutations of this one(20). However, in countries like Ecuador they obtained a high prevalence of the blaCTXM gene, results that differ from those obtained in the study(21). The use of antibiotics as growing promoters in animals generates a great concern due to a spreading of resistant bacteria, since chicken has an easy commercialization. In the present study it is noticed that 26 % of the strains presented the four genes, 46 % three genes, 14 % two genes, 3 % one gene and 4 % no gene; Molecular biology techniques have a great relevance, because through them, they are confirmed the resistance phenotypes by punctual mutations in the target genes in susceptible bacteria(22).

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The qnr genes are mediated by plasmids, transmissible by conjugation that relates to their potential for circulation. The primer has been reported in 1998 and since then five types of qnr genes (qnrA, qnrB, qnrS, qnrC, and qnrD) have been reported, containing more than 30 alleles(23). The animals can act as reservoirs for a series of zoonotic infections, which can be transmitted to humans by direct contact or through the food chain(24). Kilani et al in animal samples, 17.6 % identified qnr-type genes, as well as genes for beta-lactamases, which is why it is similar to what was identified in the present study(25). Clemente et al detected in E. coli isolations the gene gyrA in food producer animals which expressed in a whole the gene blaCMY-2(26). In the research made by Montes et al they reported only 1 % of the gene gyrB and the gene gyrA 0%(11), while in Quito, 36 % of isolations of Broiler chicken in a poultry the gene qnrB. Results similar to those reported in Brazil, in which they were able to identify variants of the gyr gene in isolates from food and humans, observing a reduced susceptibility to ciprofloxacin(27). The results obtained in the present research about the presence of genes which codify for resistance to quinolines is high regarding the other researches made by other authors, different studies have found that genes qnr are highly distributed in E. coli isolated of healthy humans, domestic and farm animals(9).

Conclusions and implications The coexistence of genes which codify for antibiotics resistance is a serious problem that requires vigilance. In light of this situation they must generate control strategies to avoid spreading through food chain, since chicken is one of the most available foods within market basket. These results reflect the resistance found mainly for antimicrobials that act by inhibiting wall synthesis and protein synthesis, such as cephalosporins and gentamicin respectively, showing evidence of the theory of the production of extended-spectrum betalactamases mechanism that may be plasmid mediated, which represents an emerging resistance problem. The limitations of this study include a sampling bias, since only one farm was worked, in addition to having no stool samples. Therefore, this study could overestimate the frequency of resistance by samples coming from birds that may have already been treated with antimicrobials.

Acknowledgments

Researchers express their gratitude to University of Boyacá, Santander University, and every single person who contributed in any way to this project.

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Ethical standards compliance

Every procedure was made taking into account the institutional and national research committee and Helsinky declaration of 1964 and its subsequent ammendments or similar ethical standars. This study was approved by the local ethical committee.

Funding

This research was supported by Universidad de Boyacá, Tunja, Colombia. and Universidad de Santander (UDES), Bucaramanga, Colombia.

Conflict of interests

Authors declare there is not any conflict of interests. Literature cited: 1. Tyson GH, Nyirabahizi E, Crarey E, Kabera C, Lam C, Rice-Trujillo C, et al. Prevalence and antimicrobial resistance of Enterococci isolated from retail meats in the United States, 2002 to 2014. Appl Environ Microbiol 2018;84(1):1–9. 2. Blajman J, Zbrun M, Astesana D, Berisvil A, Romero A, Fusari M, et al. Probióticos en pollos parrilleros: una estrategia para los modelos productivos intensivos. Rev Argent Microbiol 2015;47(4):360–367. 3. Carvajal EB, Hernández WA, Torres MC, López DV, Rueda EG, Vásquez MR. Antimicrobial resistance of Escherichia coli strains isolated from the bursa of Fabricius in broilers. Rev Inv Vet Perú 2019;30(1):430–437. 4. Botelho LAB, Kraychete GB, Costa e Silva JL, Regis DVV, Picão RC, Moreira BM, et al. Widespread distribution of CTX-M and plasmid-mediated AmpC β-lactamases in Escherichia coli from Brazilian chicken meat. Mem Inst Oswaldo Cruz. 2015;110(2):249–254. 5. Redgrave L, Sutton S, Webber M, Piddock L. Fluoroquinolone resistance: mechanisms, impact on bacteria, and role in evolutionary success. Trends Microbiol 2014;22(8):438–445.

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6. Wierup M. The swedish experience of the 1986 year ban of antimicrobial growth promoters, with special reference to animal health, disease prevention, productivity, and usage of antimicrobials. Microb Drug Resist 2001;7(2):183–90. 7. Chávez GLA, López HA, Parra SJE. Inclusion of probiotic strains improves immune parameters in broilers. Rev CES Med Zootec 2015;10(2):160–169. 8. Arenas NE, Melo VM. Producción pecuaria y emergencia de antibiótico resistencia en Colombia: Revisión sistemática Livestock production and emergency antibiotic resistance in Colombia: Systematic Review Infectio 2018;22(2):110–119. 9. Armas-Freire PI, Trueba G, Proaño-Bolaños C, Levy K, Zhang L, Marrs CF, et al. Unexpected distribution of the fluoroquinolone-resistance gene qnrB in Escherichia coli isolates from different human and poultry origins in Ecuador. Int Microbiol 2015;18(2):85–90. 10. López D, Torres M, Castañeda L, Prada C. Determinación de genes que codifican la resistencia de betalactamasas de espectro extendido en bacilos Gram negativos aislados de urocultivos. Rev Investig Salud Univ Boyacá. 2017;3(2):107. 11. Aguilar-Montes de Oca S, Talavera-Rojas M, Soriano-Vargas E, Barba-León J, Vazquez-Navarrete J. Determination of extended spectrum β-lactamases/AmpC βlactamases and plasmid-mediated quinolone resistance in Escherichia coli isolates obtained from bovine carcasses in Mexico. Trop Anim Health Prod 2015;47(5):975– 981. 12. Lim JS, Choi DS, Kim YJ, Chon JW, Kim HS, Park HJ, et al. Characterization of Escherichia coli producing extended-spectrum β-lactamase (ESBL) isolated from chicken slaughterhouses in South Korea. Foodborne Pathog Dis [Internet]. 2015;12(9):741–748. 13. Li Y, Chen L, Wu X, Huo S. Molecular characterization of multidrug-resistant avian pathogenic Escherichia coli isolated from septicemic broilers. Poult Sci 2015;94(4):601–611. 14. Lee HJ, Cho SH, Shin D, Kang HS. Prevalence of antibiotic residues and antibiotic resistance in isolates of chicken meat in Korea. Korean J food Sci Anim Resour 2018;38(5):1055–1063. 15. Varga C, Guerin MT, Brash ML, Slavic D, Boerlin P, Susta L. Antimicrobial resistance in fecal Escherichia coli and Salmonella enterica isolates: A two-year prospective study of small poultry flocks in Ontario, Canada. BMC Vet Res 2019;15(1):464

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16. Donado-godoy P, Byrne BA, León M, Castellanos R, Vanegas C, Coral A, et al. Prevalence, resistance patterns, and risk factors for antimicrobial resistance in bacteria from retail chicken meat in Colombia. J Food Prot 2015;78(4):751–759. 17. López D, Torres M, Prada C. Genes de resistencia en bacilos Gram negativos: Impacto en la salud pública en Colombia. Univ y Salud. 2016;29(1):190. 18. Ahmad K, Khattak F, Ali A, Rahat S, Noor S, Mahsood N, et al. Carbapenemases and extended-spectrum β-lactamase–producing multidrug-resistant Escherichia coli isolated from retail chicken in peshawar: first report from Pakistan. J Food Prot 2018;81(8):1339–1345. 19. Alonso CA, Michael GB, Li J, Somalo S, Simón C, Wang Y, et al. Analysis of blaSHV12-carrying Escherichia coli clones and plasmids from human, animal and food sources. J Antimicrob Chemothe 2017;72(6):1589–1596. 20. Dierikx CM, van der Goot JA, Smith HE, Kant A, Mevius DJ. Presence of ESBL/AmpC -Producing Escherichia coli in the broiler production pyramid: A descriptive study. Cloeckaert A, editor. PLoS One 2013;8(11):e79005. 21. Hedman HD, Eisenberg JNS, Vasco KA, Blair CN, Trueba G, Berrocal VJ, et al. High prevalence of extended-spectrum beta-lactamase ctx-m-producing Escherichia coli in small-scale poultry farming in rural Ecuador. Am J Trop Med Hyg 2019;19;100(2):374–376. 22. Laube H, Friese A, von Salviati C, Guerra B, Rösler U. Transmission of ESBL/AmpCproducing Escherichia coli from broiler chicken farms to surrounding areas. Vet Microbiol 2014;172(3–4):519–527. 23. Martínez L, Pascual A, Jacoby G. Quinolone resistance from a transferable plasmid. Lancet 1998;351(9105):797–799. 24. Machuca J, Agüero J, Miró E, Conejo M del C, Oteo J, Bou G, et al. Prevalence of quinolone resistance mechanisms in Enterobacteriaceae producing acquired AmpC βlactamases and/or carbapenemases in Spain. Enfermedades Infecc Microbiol Clin 2017;35(8):485–490. 25. Kilani H, Ferjani S, Mansouri R, Boutiba I, Abbassi M. Occurrence of plasmidmediated quinolone resistance determinants among Escherichia coli strains isolated from animals in Tunisia: Specific pathovars acquired qnr genes. J Glob Antimicrob Resist 2020;1(20):50–55.

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26. Clemente L, Manageiro V, Jones-Dias D, Correia I, Themudo P, Albuquerque T, et al. Antimicrobial susceptibility and oxymino-β-lactam resistance mechanisms in Salmonella enterica and Escherichia coli isolates from different animal sources. Res Microbiol 2015;166(7):574–583. 27. Campioni F, Souza RA, Martins VV, Stehling EG, Bergamini AMM, Falcão JP. Prevalence of gyra mutations in nalidixic acid-resistant strains of Salmonella enteritidis isolated from humans, food, chickens, and the farm environment in Brazil. Microb Drug Resist 2017;23(4):421–428.

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https://doi.org/10.22319/rmcp.v13i3.5873 Article

Detection of bluetongue virus in sheep by real-time RT-PCR in different production systems in San Martin, Peru

Alicia María López Flores a* Roni David Cruz Vasquez a Víctor Humberto Puicón Niño de Guzmán a Alicia Bartra Reátegui a Orlando Ríos Ramírez a Fredy Fabián Domínguez a

Universidad Nacional de San Martin. Laboratorio de Sanidad Animal de la Escuela Profesional de Medicina Veterinaria, San Martin, Perú.

* Corresponding author: alicialopezflores@unsm.edu.pe

Abstract: The present study aimed to determine the prevalence of Bluetongue Virus (BTV) in sheep, by the real-time Reverse Transcription-polymerase chain reaction (RT-PCR) technique. Three hundred sixty-six sheep from the ten provinces of the Peru region were evaluated. The methodology used was the collection of blood samples from the jugular vein of the sheep, then the process of extraction and purification of RNA was carried out with the QIAmp® kit, then the reverse transcription to obtain the cDNA, and finally perform the real-time RT-PCR, for which the SuperScript III platinium One-step qRTPCR kit was used, with the primers and probes being directed to segment 10 of the NS3 gene of BTV. The results of the real-time RT-PCR test revealed two positive sheep with a value of cycle threshold (Ct) of 35.21 and 35.57, with a prevalence of 0.54 % of BTVpositive sheep in the extensive production system, with environmental conditions that favor the development of the Culicoides vector. It is concluded that, by means of the real-time RT-PCR technique, the presence of BTV in this region of Peru is confirmed, which makes future studies necessary to determine the detection of other potential

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serotypes of BTV in the Peruvian Amazon in order to improve the control strategies of the disease. Key words: RNA, Gene, Molecular diagnosis, Ruminants, Amazonia.

Received: 19/11/2020 Accepted: 08/11/2021

Introduction Bluetongue virus [BTV; in Spanish: virus de la lengua azul (VLA)], genus Orbivirus(1), without envelope, the genome has 10 segments of double-stranded RNA, seven structural and five non-structural proteins(2). VP7 is a viral capsid protein(3). VP2 of the surface of the virion(4). In addition, serotypes 9, 13, 18(5) and 27(6), with serotype 28 being recently identified(7). Bluetongue is a disease, non-contagious and transmitted through the bite of Culicoides insects(8). Of the 1,357 species of Culicoides in the world(9), only about 30 have been reported as vectors(10). Vectors, when feeding on an animal with BTV, will be infected all their lives(11), they can infect animals with variations in clinical signs(12). Sheep manifest seromucous nasal discharge, facial edema, thrombo-hemorrhagic fever, coronitis and ulceration in the lips and hard palate(13); the viremia is usually detected between 3 and 5 days after infection, reaching febrile peaks on day 7(14). However, these clinical signs (e.g., fever in sheep and cattle, salivation in cattle, facial edema in sheep) were observed in infected and non-infected herds, these signs are not indicative of the disease(15). Likewise, macroscopic lesions are pulmonary edema, cranio-ventral pulmonary consolidation, swollen and cyanotic tongue; and hemorrhagic lesions in rumen mucosa(16). Cattle, goats, buffaloes and other wild ruminants act as reservoirs of the virus, being animals that may not show clinical signs(17,18,19), however, there are reports of mortality in cattle with BTV serotype 8(20); other clinical signs such as amaurosis, which is the inability to stand and the absence of the sucking reflex in infected calves(21). Therefore, the different serotypes of BTV in small and large ruminants indicate its enzootic expansion(22). Likewise, the transmission of the virus, host-vector is complex, with a variety of ecological drivers(23). The epidemiology of BTV is listed in the OIE Terrestrial Animal Health Code(24). It has generated outbreaks in Israel in 2006, generating losses of $ 2.5 million(25), as well as an outbreak in Western Europe(26). Thus, it has had an annual cost of $ 3 billion, it has become one of the diseases of economic importance(27).

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Currently, this disease is found worldwide, except in Antarctica(28). It has been reported in Brazil in sheep(16); in Sudan, in reproductive disorders in cattle(29); in Japan(30) and India, in a transplacental transmission of BTV-1, in the middle stage of gestation in sheep(31). In Australia, BTV originated from Southeast Asia(32). While in the United Kingdom, wild animals in zoos are susceptible to arboviruses, acting as native hosts of Culicoides(33). In Peru, BTV has been reported in sheep, camelids and wild animals in tropical areas(34,35,36). Preliminary studies were conducted in 1984 and 1987, finding 88, 41 and 56 % of sheep from three regions (north, central and south)(37) that were seropositive for BTV and 21 % of seroreactor alpacas from the southern region(38). In studies in Tayassu tajacu in Madre de Dios, 7.5 % of samples with BTV-specific antibodies are reported and 29.2 % had antibodies to the BTV serogroup(34). Recently, Navarro et al(35) confirmed the presence of the virus in sheep of extensive farming, in addition to identifying the Culicoides that are vectors of the disease. However, the San Martín region, in its tropical climate, is an optimal environment for the vector and facilitates its infection routes in domestic and wild hosts; mentioned by Felippe-Bauer(39), who reported Culicoides species. In relation to the diagnosis, the seroneutralization test is specific, but its disadvantage is the low sensitivity and cost(40). However, competitive ELISA is suggested in llamas, wild ruminants(41), sheep and goats(42). Currently, the tests recommended by the OIE for BTV are RT-PCR, agar gel immunodiffusion assay and competitive ELISA(43,44). With the real-time RT-PCR being for the rapid detection of BTV directed to Seg1/VP1(45,46), Seg-2/VP2 and Seg-10/NS3(47), it is a widely used method(48), in the detection of all BTV serotypes(49), based on the TaqMan fluorescence probe(50,51,52,53) to detect BTV in samples of infected sheep(54). Therefore, the objective of the study was to detect BTV in sheep by real-time RT-PCR, considering the influence of environmental characteristics and presence of the disease.

Material and methods Place of study The study was conducted in the San Martín region, Peru. Blood samples were collected from sheep in stable health condition, in the 10 provinces of the San Martín region (Table 1), in the period from August to December 2018.

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Table 1: Number of sheep sampled by province suitable for the development of the vector No. of samples 9 11 35 41 74 41 15 24 30 86 366

Provinces Moyobamba Rioja El Dorado Lamas Bellavista Picota Mariscal Cáceres Huallaga Tocache San Martín Total

Sample collection Three hundred sixty-six blood samples were collected from the jugular vein of the randomly selected sheep, which were from 42 farms. For the collection, vacuum vacutainer needles and tubes with EDTA anticoagulant were used. The samples were transported in a refrigerated container with cooling gel for their processing in the Laboratory of Molecular Biology and Genetics of the Professional School of Agronomy - Faculty of Agricultural Sciences, National University of San Martín. According to the 2012 Census(55), the population is 7,656 sheep in the entire department of San Martín. The sample was calculated based on this population:

Where: n is the sample size; Z= is the confidence level 95%= 1.96; p= is the probability of success 50%/100= 0.5; q= is the probability of failure 50%/100= 0.5; E= is the error level 5%/100= 0.05; N= is the population size= 7656. n= (1.96)2 (0.5) (0.5) (7656) (0.05)2(7656-1) + (1.96)2(0.5) (0.5) n=366 Sample subpopulation formula.

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Nh= subpopulation or group; N= total population; n= total sample; nh= sample of the groups.

RNA extraction For the processing of the blood sample, it was centrifuged at 800 xg for 10 min, then the plasma was extracted, and sterile PBS was added, the tube was inverted several times to mix, centrifuge again at 800 xg for 10 min to separate the red blood cells from the PBS. The QIAmp® kit, Qiagen brand, was used for RNA extraction. The RNA extraction step was performed according to the procedures specified by the manufacturer. The final product obtained is RNA in Buffer, to finally continue with the real-time RT-PCR, which targets segment 10 of BTV (NS3 Gene).

Real-time Reverse transcription - Polymerase chain reaction Real-time RT-PCR is a test of choice for diagnosis. The method described here is recommended by the OIE and Hofmann et al(56), to detect segment 10 of the NS3 Gene of BTV. To obtain the cDNA by real-time reverse transcription and PCR, the SuperScript III platinium One-step qRT-PCR kit, Invitrogen brand, was used, being the sequences of the primers for the detection of segment 10 of the NS3 gene of BTV. The primary solutions of the primer were diluted to a concentration of 20 pmol/μl, the nucleotide sequences of the primers: VLA_IVI_F 5’-TGG-AYA-AAG-CRA-TGTCAA-A-3’, VLA_IVI_R 5’-ACR-TCA-TCA-CGA-AAC-GCT-TC-3’(57). The probe solution for the NS3 gene of BTV was diluted to a concentration of 5 pmol/μl, the sequence of the probe: VLA_IVI_P 5’FAM-ARG-CTG-CAT-TCG-CAT-CGT-ACGC-3’ BHQ1. Zero point five microliters of each primary primer at a concentration of 20 pmol/μL were added to each well, the plate must be kept on ice. Then 2 μL of RNA samples, both from the target sample and from the positive and negative controls, are added to the corresponding wells of the plate following the distribution. The denaturing temperature was 95 °C for 5 min, and they were kept on ice for another 3 min. A volume of the primary mixture(57) of the RT-PCR was prepared, following the manufacturer’s instructions. The probe was included in the primary mixture to obtain a final concentration of 0.2 pmol/μL per sample. Twenty microliters of primary mixture were distributed in each well of the plate located in the real-time thermocycler programmed for reverse transcription and amplification, detection by fluorescence of cDNA.

Temperature conditions The reaction was carried out in a light cycler 480 System Roche Applied Biosystems. Following the following thermal profile: Reverse transcription 48 °C for 30 min, reverse

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transcriptase inactivation or initial denaturation 95 °C for 2 min, followed by 50 cycles, amplification 95 °C for 15 sec, 56 °C for 30 sec, 72 °C for 30 sec(57).

Results and discussion Real-time RT-PCR analysis Real-time RT-PCR results indicated that 0.54 % (2/366) of the samples were BTVpositive. For the first individual, with code (E8), positive for BTV, it had a cycle threshold (Ct) value of about 35.21, with a dissociation temperature™ value of 84 °C for BTV. For the sample with code (G12) – C+VLA 1/10, it had a Ct of 28.22 and 31.63 for the sample with code (H12) – C+VLA 1/100. No amplification was observed in the negative control. The high viral load and amplification of a specific product were evidenced (Figure 1). Figure 1: Results of the real-time RT-PCR test for the first positive individual (E8), in the amplification curve, it is observed with a Ct value of 35.21. Opticon Monitor software v.3.0

For the second individual (E6) positive for BTV, it had a Ct value of about 35.57, with a dissociation temperature™ value of 84 °C. For the sample (G12)- C+VLA 1/10, it had a Ct of 28.81 and 32.72 for the sample (H12)- C+VLA 1/100. No amplification was observed in the negative control. The high viral load and amplification of a specific product were evidenced (Figure 2).

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Figure 2: Results of the real-time RT-PCR test for the second positive individual (E6), in the amplification curve, a Ct value of 35.57 is observed. Opticon Monitor Software v.3.0

A panoramic review in South America, using serological studies for the detection of antibodies conducted on cattle, goats, sheep and buffaloes, indicates varied minimum and maximum prevalences in the following countries: in Argentina (0-95 %)(58), Brazil (1.22-89.69 %)(59,60), Chile (0-19.6 %)(61,62), Colombia (51.8-56 %)(63,64), Ecuador (10 %)(65), Guyana (0-56 %)(66), Suriname (82-91 %)(66) and Venezuela (74.894.7 %)(67), in Peru, preliminary studies in 1984 and 1987 reported a seroprevalence of 87.5, 41 and 55.5 % of BTV in sheep from the north, center and south of the country’s highlands(37). In the present research, the viral genome of BTV was detected, with the prevalence being 0.54 % in sheep sera by real-time RT-PCR, which confirms the epidemiological presence of this virus in this region. The areas where the positive animals were detected are very close to water tributaries, such as the Sisa River and the Ishangayacu Ravine. With environmental sensitivities being as a key component of the capacity of the vector(68). It is also possible that BTV infection in the sampled sheep was subclinical, as the animals were under normal conditions. This is explained, according to Maclachlan et al(69), because the development of the clinical signs of the disease depends on whether the infection is endemic or not; as a consequence, animals have antibodies, but rarely show clinical signs. This is possibly because in sheep the period of viremia rarely persists for more than 14 d, unlike in cattle, whose viremia can be up to 90 to 120 d. As mentioned by Navarro(36), Peru is one of the countries that is predisposed to present BTV disease, since it has several ecosystems conducive to the development of Culicoides. It is considered necessary to determine the seroprevalence of BTV in livestock areas, identify the different serotypes, map the location of the different species of Culicoides spp., in the different geographical areas and altitudes, and determine the endemic areas.

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Environment of BTV-positive sheep An assessment of the surroundings of the farms was carried out to observe the habitat of the Culicoides vector. It was found that BTV-positive animals are near water tributaries, which contributes to the development of the vector (Table 2). On the other hand, the positive sheep were of the Pelibuey breed, however, the predisposition due to breed is not determinant. In addition, extensive farming of sheep allows their farming together with cattle, chickens, dogs and horses. In the surveys conducted, the owners of extensive, intensive and semi-intensive farms do not deworm their animals, nor do they fumigate for the presence of flies or mosquitoes. Table 2: Environment of BTV-positive sheep Province District

Bellavista

San Pablo

San Bellavista Pablo

Location of the farm Angélica Sector

Farming system

Environment of the farm

Breed

Extensive

Left bank of Pelibuey the Sisa River

Hamlet of San Extensive Ignacio

Left bank of the Pelibuey Ishangayacu Ravine

Body condition 3

2.5

Epidemiological studies indicate that BTV exists in a large area in the world, between 40° north latitude and 35° south latitude, with tropical, subtropical and temperate ecosystems(70), these characteristics coincide with the San Martín region. The first studies of the presence of the BTV-transmitting vector in this region were conducted by Felippe-Bauer et al(39), who identified five species of Culicoides that are vectors of the virus. Similarly, Navarro et al(35), of the 7,930 mosquitoes captured, 94.8 % were identified as Culicoides insignis, and the presence of BTV in sheep in the Pucallpa region is also confirmed(36). On the other hand, the Culicoides vector usually develops in areas where there are certain types of drivers such as land use, trade, animal husbandry and the presence of wild animals as a reservoir of BTV; the latter is reinforced by the work carried out by Rivera et al(34), who found 7.5 % of white-lipped peccaries (Tayassu pecari) positive for BTV in the Madre de Dios region. The present research work is the first to be developed in the entire San Martín region, where the results using real-time RT-PCR show the presence of BTV. Felippe-Bauer et al(39) mention having found the Culicoides vector of the virus. Therefore, it is required to detect other existing serotypes of BTV in domestic and wild animals of the region that are susceptible or reservoirs of the disease.

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Farming systems Regarding the farming system, most producers opt for an extensive sheep farming in the San Martín Region (Table 3). Table 3: Production systems Farming systems Provinces Moyobamba Rioja El Dorado Lamas Bellavista Picota Mariscal Cáceres Huallaga Tocache San Martín

Intensive

Extensive

0 0 0 0 0 0 0 0 0 33

9 11 35 27 74 29 15 19 20 15 Total

Semi Intensive 0 0 0 14 0 12 0 5 10 38 366

Conclusions and implications It is concluded that BTV has a low prevalence in sheep in the San Martín Region of Peru, however, future studies are needed to determine morbidity and the detection of other potential BTV serotypes in the country, to better elucidate the management of vectors and control strategies of the disease. Acknowledgements This research work received financial support from the Research and Development Institute of the UNSM-T, competition for undergraduate thesis projects, period 2018, with Resolution No. 611 -2018 – UNSM/CU – R/ NLU. Literature cited: 1.

Batten CA, Henstock MR, Steedman HM, Waddington S, Edwards L, Oura CA. Bluetongue virus serotype 26: infection kinetics, pathogenesis and possible contact transmission in goats. Vet Microbiol 2013;162(1):62–67. https://doi.org/10.1016/j.vetmic.2012.08.014.

Patel A, Roy P. The molecular biology of Bluetongue virus replication. Virus Res 2014;(182):5‐20. doi: 10.1016/j.virusres.2013.12.017.

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Rev Mex Cienc Pecu 2022;13(3):596-611

Roy P. Bluetongue virus proteins and particles and their role in virus entry, assembly, and release. Adv Virus Res 2005;64:69–123. https://doi.org/10.1111/tbed.12625.

Wilson WC, Hindson BJ, O'Hearn ES, Hall S, Tellgren-Roth C, Torres C, et al. A multiplex real-time reverse transcription polymerase chain reaction assay for detection and differentiation of Bluetongue virus and Epizootic hemorrhagic disease virus serogroups. J Vet Diagn Invest 2009;21(6):760-70. doi: 10.1177/104063870902100602. PMID: 19901276.

Verdezoto J, Breard E, Viarouge C, Quenault H, Lucas P, Sailleau C, et al. Novel serotype of bluetongue virus in South America and first report of epizootic haemorrhagic disease virus in Ecuador. Transbound Emerg Dis 2018;65(1):244247. doi: 10.1111/tbed.12625. Epub 2017 Feb 26. PMID: 28239988.

Schulz C, Bréard E, Sailleau C, Jenckel M, Viarouge C, Vitour D, et al. Bluetongue virus serotype 27: detection and characterization of two novel variants in Corsica, France. J Gen Virol 2016;97(9):2073-2083. doi: 10.1099/jgv.0.000557. Epub 2016 Jul 19. PMID: 27435041.

Bumbarov V, Golender N, Jenckel M, Wernike K, Beer M, Khinich E, Zalesky O, Erster O. Characterization of bluetongue virus serotype 28. Transbound Emerg Dis 2020;67(1):171-182. doi: 10.1111/tbed.13338. Epub 2019;31469936.

Purse BV, Brown HE, Harrup L, Mertens PP, Rogers DJ. Invasion of bluetongue and other orbivirus infections into Europe: the role of biological and climatic processes. Rev Sci Tech 2008;27(2):427-42. PMID: 18819670.

Borkent A, Willis WW. World species of biting midges (Diptera: Ceratopogonidae). Vol. 233. New York, USA: American Museum of Natural History. 1997.

10. Linley JR. Autogeny in the Ceratopogonidae: literature and notes. Fla. Entomol 1983;(66):228–234. 11. Maclachlan NJ, Zientara S, Wilson WC, Richt JA, Savini G. Bluetongue and epizootic hemorrhagic disease viruses: recent developments with these globally reemerging arboviral infections of ruminants. Curr Opin Virol 2019;34:56-62. doi: 10.1016/j.coviro.2018.12.005. Epub 2019 Jan 14. PMID: 30654271. 12. Labadie T, Sullivan E, Roy P. Multiple routes of bluetongue virus egress. eicroorganisms. 2020;27;8(7):965. doi: 10.3390/microorganisms8070965. PMID: 32605099; PMCID: PMC7409164. 13. Schwartz-Cornil I, Mertens PP, Contreras V, Hemati B, Pascale F, Bréard E, Mellor PS, MacLachlan NJ, Zientara S. Bluetongue virus: virology, pathogenesis and immunity. Vet Res 2008;39(5):46. doi: 10.1051/vetres:2008023.

605

Rev Mex Cienc Pecu 2022;13(3):596-611

14. Foster NM, Luedke AJ, Parsonson IM, Walton TE. Temporal relationships of viremia, interferon activity, and antibody responses of sheep infected with several bluetongue virus strains. Am J Vet Res 1991;52(2):192–196. https://pubmed.ncbi.nlm.nih.gov/1707246/. Accessed Feb 17, 2020. 15. Elbers ARE, Backx A, Ekker HM, van der Spek AN, van Rijn PA. Performance of clinical signs to detect bluetongue virus serotype 8 outbreaks in cattle and sheep during the 2006-epidemic in The Netherlands. Vet Microbiol;2021;129(1–2):156– 162. https://pubmed.ncbi.nlm.nih.gov/18164148/. 16. Bianchi RM, Panziera, Welden FTC, Almeida GL, Cargnelutti JF, Flores EF, Kommers GD, Fighera RA. Clinical, pathological and epidemiological aspects of outbreaks of bluetongue disease in sheep in the central region of Rio Grande do Sul. Pesq. Vet Bras 2017;37(12):1443-1452. https://doi.org/10.1590/S0100736X2017001200014. 17. Wilson AJ, Mellor PS. Bluetongue in Europe: past, present and future. Philos Trans R Soc Lond B Biol Sci 2009; 27;364(1530):2669-81. doi: 10.1098/rstb.2009.0091. PMID: 19687037; PMCID: PMC2865089. 18. Lorca-Oró C, López-Olvera JR, Ruiz-Fons F, Acevedo P, García-Bocanegra I, Oleaga Á, Gortázar C, Pujols J. Long-term dynamics of bluetongue virus in wild ruminants: relationship with outbreaks in livestock in Spain, 2006-2011. PLoS One. 2014;18;9(6):e100027. doi: 10.1371/journal.pone.0100027. PMID: 24940879; PMCID: PMC4062458. 19. Lager IA. Bluetongue virus in South America: overview of viruses, vectors, surveillance and unique features. Vet Ital 2004;40(3):89-93. PMID: 20419641. 20. Santman-Berends IM, van Schaik G, Bartels CJ, Stegeman JA, Vellema P. Mortality attributable to bluetongue virus serotype 8 infection in Dutch dairy cows. Vet Microbiol 2011;24;148(2-4):183-188. doi: 10.1016/j.vetmic.2010.09.010. Epub 2010 Sep 16. PMID: 20889271. 21. Vinomack C, Rivière J, Bréard E, Viarouge C, Postic L, Zientara S, et al. Clinical cases of Bluetongue serotype 8 in calves in France in the 2018-2019 winter. Transbound Emerg Dis 2020;67(3):1401-1405. doi: 10.1111/tbed.13466. Epub 2020 Jan 8. PMID: 31883429. 22. Maan S, Tiwari A, Chaudhary D, Dalal A, Bansal N, Kumar V, et al. A comprehensive study on seroprevalence of bluetongue virus in Haryana state of India. Vet World 2017;10(12):1464-1470. doi: 10.14202/vetworld.2017.14641470. Epub 2017 Dec 13. PMID: 29391687; PMCID: PMC5771171.

606

Rev Mex Cienc Pecu 2022;13(3):596-611

23. Mayo C, McDermott E, Kopanke J, Stenglein M, Lee J, Mathiason C, Carpenter M, Reed K, Perkins TA. Ecological dynamics impacting bluetongue virus transmission in North America. Front Vet Sci 2020;17;7:186. doi: 10.3389/fvets.2020.00186. PMID: 32426376; PMCID: PMC7212442. 24. OIE. Listed diseases, infections and infestations https://www.oie.int/es/enfermedad/lengua-azul/. Accessed Jun 13, 2021.

2021.

25. Kedmi M, Van Straten M, Ezra E, Galon N, Klement E. Assessment of the productivity effects associated with epizootic hemorrhagic disease in dairy herds. J Dairy Sci 2010;93(6):2486‐2495. doi:10.3168/jds.2009-2850. 26. Velthuis AG, Saatkamp HW, Mourits MC, de Koeijer AA, Elbers AR. Financial consequences of the Dutch bluetongue serotype 8 epidemics of 2006 and 2007. Prev Vet Med 2010;1;93(4):294-304. doi: 10.1016/j.prevetmed.2009.11.007. Epub 2009 Dec 3. PMID: 19962204. 27. Rushton J, Lyons N. Economic impact of Bluetongue: a review of the effects on production. Vet Ital 2015;51(4):401-406. doi: 10.12834/VetIt.646.3183.1. PMID: 26741252. 28. Van der Sluijs MT, de Smit AJ, Moormann RJ. Vector independent transmission of the vector-borne bluetongue virus. Crit Rev Microbiol 2016;42(1):57-64. doi: 10.3109/1040841X.2013.879850. Epub 2014 Mar 19. PMID: 24645633. 29. Elhassan AM, Babiker AM, Ahmed ME, El Hussein AM. Coinfections of Sudanese dairy cattle with bovine herpes virus 1, bovine viral diarrhea virus, bluetongue virus and bovine herpes virus 4 and their relation to reproductive disorders. J Advanced Vet Anim Res 2017;3(4):332-337. http://doi.org/10.5455/javar.2016.c169. 30. Kato T, Shirafuji H, Tanaka S, Sato M, Yamakawa M, Tsuda T, Yanase T. Bovine arboviruses in Culicoides biting midges and sentinel cattle in Southern Japan from 2003 to 2013. Transbound Emerg Dis 2016;63(6):e160-e172. doi: 10.1111/tbed.12324. Epub 2015 Jan 19. PMID: 25597441. 31. Saminathan M, Singh KP, Khorajiya JH, Dinesh M, Vineetha S, Maity M, et al. An updated review on bluetongue virus: epidemiology, pathobiology, and advances in diagnosis and control with special reference to India. Vet Q 2020;40(1):258-321. doi: 10.1080/01652176.2020.1831708. PMID: 33003985; PMCID: PMC7655031. 32. Kedmi M, Van Straten M, Ezra E, Galon N, Klement E. Assessment of the productivity effects associated with epizootic hemorrhagic disease in dairy herds. J Dairy Sci 2010;93(6):2486‐2495. doi:10.3168/jds.2009-2850.

607

Rev Mex Cienc Pecu 2022;13(3):596-611

33. England ME, Pearce-Kelly P, Brugman VA, King S, Gubbins S, Sach F, et al. Culicoides species composition and molecular identification of host blood meals at two zoos in the UK. Parasit Vectors 2020;16;13(1):139. doi: 10.1186/s13071-02004018-0. PMID: 32178710; PMCID: PMC7076997. 34. Rivera H, Cárdenas L, Ramírez M, Manchego A, More J, Zúñiga A, et al. Orbivirus infection in white-lipped peccaries (Tayassu pecari) from Madre de Dios region, Perú. Rev Inv Vet Perú 2013;24(4):544–550. https://doi.org/10.15381/rivep.v24i4.2738. 35. Navarro D. Identificación de Culicoides spp, como vectores del virus Lengua Azul en áreas de ovinos seropositivos de Pucallpa, Ucayali [tesis maestría]. Perú: Universidad Nacional Mayor de San Marcos; 2017. 36. Navarro MD, Rojas M, Jurado PJ, Manchego SA, Ramírez VM, Castillo EA, et al. Molecular detection of Bluetongue virus in Culicoides insignis and sheep of Pucallpa, Perú. Rev Investig Vet Perú 2019;30(1):465–476. http://dx.doi.org/10.15381/rivep.v30i1.15690. 37. Rosadio RH, Evermann JF, De Martini JC. A preliminary serological survey of viral antibodies in peruvian sheep. Vet Microbiol 1984;10(1):91–96. doi: 10.1016/0378-1135(84)90059-2. 38. Rivera H, Madewell BR, Ameghino E. Serologic survey of viral antibodies in the Peruvian alpaca (Lama pacos). Am J Vet Res 1987;48(2):189-191. PMID: 3826854. 39. Felippe-Bauer ML, Cáceres A, Silva CS, da Valderrama-Bazan W, Gonzales-Pérez A. Costa JM. New records of Culicoides latreille (Diptera: Ceratopogonidae) from Peruvian Amazonian region. Biota Neotropic 2008;8(2):33–38. https://doi.org/10.1590/s1676-06032008000200002. 40. Singer RS, Boyce WM, Gardner IA, Johnson WO, Fisher AS. Evaluation of bluetongue virus diagnostic tests in free-ranging bighorn sheep. Prev Vet Med 1998;35(4):265‐282. doi:10.1016/s0167-5877(98)00067-1. 41. Afshar A, Heckert RA, Dulac GC, Trotter HC, Myers DJ. Application of a competitive ELISA for the detection of bluetongue virus antibodies in llamas and wild ruminants. J Wild Dis 1995;31(3):327-30. doi: 10.7589/0090-3558-31.3.327. PMID: 8592352. 42. Elmahi MM, Karrar ARE, Elhassan AM, Hussien MO, Enan KA, Mansour MA, El Hussein ARM. Serological investigations of Bluetongue Virus (BTV) among sheep and goats in Kassala State, Eastern Sudan. Vet Med Int 2020;2020:8863971. doi: 10.1155/2020/8863971. PMID: 33062245; PMCID: PMC7547342.

608

Rev Mex Cienc Pecu 2022;13(3):596-611

43. Saminathan M, Singh KP, Vineetha S, Maity M, Biswas SK, Manjunathareddy GB, et al. Virological, immunological and pathological findings of transplacentally transmitted bluetongue virus serotype 1 in IFNAR1-blocked mice during early and mid-gestation. Sci Rep 2020;10(1):2164. doi: 10.1038/s41598-020-58268-0. 44. Díaz-Cao JM, Lorca-Oró C, Pujols J, Cano-Terriza D, de Los Ángeles Risalde M, Jiménez-Ruiz S, Caballero-Gómez J, García-Bocanegra I. Evaluation of two enzyme-linked immunosorbent assays for diagnosis of bluetongue virus in wild ruminants. Comp Immunol Microbiol Infect Dis 2020;70:101461. doi: 10.1016/j.cimid.2020.101461. Epub 2020 Feb 29. PMID: 32151837. 45. Shaw AE, Monaghan P, Alpar HO, Anthony S, Darpel KE, Batten CA, et al. Development and initial evaluation of a real-time RT- PCR assay to detect bluetongue virus genome segment 1. J Virol Meth 2007;145(2):115-126. https://doig.org/10.1016/j.jviromet.2007.05.014. 46. Toussaint JF, Sailleau C, Breard E, Zientara S, De Clercq K. Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments. J Virol Meth 2007;140(1):115- 123. https://doi.org/10.1016/j.jviromet.2006.11.007. 47. Van Rijn PA, Geurts Y, Van der Spek AN, Veldman D, Van Gennip RG. Bluetongue virus serotype 6 in Europe in 2008-emergence and disappearance of an unexpected non-virulent BTV. Vet Microbiol 2012;158(1):23-32. doi: 10.1016/j.vetmic.2012.01.022. 48. Rojas JM, Rodríguez-Martín D, Martín V, Sevilla N. Diagnosing bluetongue virus in domestic ruminants: current perspectives. Vet Med 2019;(10):17–27. https://doi.org/10.2147/VMRR.S163804. 49. Mulholland C, McMenamy MJ, Hoffmann B, Earley B, Markey B, Cassidy J, Allan G, et al. The development of a real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay using TaqMan technology for the pan detection of bluetongue virus (BTV). J Virol Meth 2017;(245):35–39. https://doi.org/10.1016/j.jviromet.2017.03.009. 50. MacLachlan NJ. Bluetongue: pathogenesis and duration of viraemia. Vet Ital 2004;40(4):462–467. PMID: 20422570. 51. Mertens PPC, Maan NS, Prasad G, Samuel, AR, Shaw AE, Potgieter AC, Anthony SJ, Maan S. Design of primers and use of RT-PCR assays for typing European bluetongue virus isolates: differentiation of field and vaccine strains. J Gen Virol 2007;88(10):2811-2823. doi: 10.1099/vir.0.83023-0.

609

Rev Mex Cienc Pecu 2022;13(3):596-611

52. Hoffmann B, Beer M, Reid SM, Mertens P, Oura CA, Van Rijn PA, et al. A review of RT-PCR technologies used in veterinary virology and disease control: sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health. Vet Microbiol 2009;139(1-2):1-23. https://doi.org/10.1016/j.vetmic.2009.04.034. 53. Maan NS, Maan S, Belaganahalli MN, Ostlund EN, Johnson DJ, Nomikou K, Mertens PP. Identification and differentiation of the twenty-six bluetongue virus serotypes by RT-PCR amplification of the serotype-specific genome segment 2. PLoS One 2012;7(2):e32601. doi: 10.1371/journal.pone.0032601. Epub 2012 Feb 28. PMID: 22389711; PMCID: PMC3289656. 54. Lakshmi I, Putty K, Raut SS, Patil SR, Rao PP, Bhagyalakshmi B, et al. Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus. Vet World 2018;11(4):452–458. https://doi.org/10.14202/vetworld.2018.452-458. 55. INEI. Resultados Definitivos. IV Censo Nacional Agropecuario [Internet]. IV Censo Nacional Agropecuario. 2012: https://sigrid.cenepred.gob.pe/sigridv3/documento/906. Consultado 21 May, 2021. 56. Hofmann MA, Renzullo S, Mader M, Chaignat V, Worwa G, Thuer B. Genetic characterization of toggenburg orbivirus, a new bluetongue virus, from goats, Switzerland. Emerg Infect Dis 2008;14(12):1855-61. doi: 10.3201/eid1412.080818. PMID: 19046507; PMCID: PMC2634640. 57. Manual Terrestre de la OIE-2014.Lengua azul: Infección por el virus de la Lengua Azul. Capítulo 2.1.3. 58. Lager IA. Bluetongue virus in South America: overview of viruses, vectors, surveillance and unique features. Vet Ital 2004;40(3):89-93. PMID: 20419641. 59. Cunha RG, de Souza DM, Texeira AC. Anticorpos precipitantes para o virus da lengua azul em soros de bovinos do estado do Rio de Janeiro. Biológico, São Paulo. 1982;48(4):99-103. 60. Melo CB, Oliveira AM, Castro RS, Lobato ZIP, Leite RC. Precipitating antibodies against the bluetongue virus in bovines from Sergipe, Brazil. Cienc Vet Trop Recife 1999;2(2):125-127. 61. Fundación Vida Silvestre Argentina (FVSA). Operativo de captura de venados de las pampas en campos del Tuyú. FVSA, Buenos Aires. 2004; vidasilvestre.org.ar/pastizales/2_6.asp. Consultado 9 Jul, 2004. 62. Tamayo R, Schoebitz R, Alonso O, Wenzel J. First report of bluetongue antibody in Chile. Progr Clin Biol Res1983;178:555-558.

610

Rev Mex Cienc Pecu 2022;13(3):596-611

63. Homan EJ, Lorbacher de Ruiz H, Donato AP, Taylor WP, Yuill TM. A preliminary survey of the epidemiology of bluetongue in Costa Rica and Northern Colombia. J Hyg Camb 1985;94:357- 363. 64. Homan EJ, Taylor WP, Lorbacher de Ruiz H, Yuill TM. Bluetongue virus and epizootic haemorrhagic disease of deer virus serotypes in northern Colombian cattle. J Hyg Camb 1985;95:165-172. 65. Lopez WA, Nicoletti P, Gibbs EPJ. Antibody to bluetongue virus in cattle in Ecuador. Trop Anim Hlth Prod 1985;17:82. 66. Gibbs J, Greiner E, Alexander F, King H, Roach C. Serological survey of ruminant livestock in some countries of the Caribbean region and South America for antibody to bluetongue virus. Vet Rec 1984;114(26):635-638. 67. Gonzalez MC, Perez N, Siger J. Serologic evidence of bluetongue virus in bovines from Aragua State, Venezuela. Rev Fac Cienc Vet UCV2000;41(1-3):3-12. 68. Purse BV, Carpenter S, Venter GJ, Bellis G, Mullens BA. Binomics of temperate and tropical Culicoides Midges: Knowledge gaps and consequences for transmission of Culicoides -Borne viruses. Annu Rev Entomol 2014;(60):373–392. http://doi.org/10.1146/annurev-ento-010814-020614. 69. Maclachlan NJ, Drew CP, Darpel KE, Worwa G. The pathology and pathogenesis of bluetongue. J Comp Pathol 2009;141(1):1-16. doi: 10.1016/j.jcpa.2009.04.003. Epub 2009 May 23. PMID: 19476953. 70. Mertens PP, Diprose J, Maan S, Singh KP, Attoui H, Samuel AR. Bluetongue virus replication, molecular and structural biology. Vet Ital 2004;40(4):426-37. PMID: 20422565.

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https://doi.org/10.22319/rmcp.v13i3.6067 Article

Detection of bovine viral diarrhea virus in captive wild artiodactyls in Mexico

Jocelyn Medina-Gudiño a Ninnet Gómez-Romero a José Ramírez-Lezama b Luis Padilla-Noriega c Emilio Venegas-Cureño d Francisco Javier Basurto-Alcántara a*

Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia, Departamento de Microbiología e Inmunología. Ciudad de México, México. b

Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia, Departamento de Patología, Ciudad de México, México. c

Universidad Nacional Autónoma de México. Facultad de Medicina. Departamento de Microbiología y Parasitología. Ciudad de México, México. d

Servicio Nacional de Sanidad, Inocuidad y Calidad Agroalimentaria. Centro Nacional de Servicios de Diagnóstico en Salud Animal, Departamento de Validación de Técnicas, Estado de México, México.

*Corresponding author: basurto@unam.mx

Abstract: Bovine viral diarrhea virus (BVDV) is a pestivirus that infects a broad range of wild and domestic artiodactyls. Pestiviruses can cause a variety of respiratory, gastrointestinal and reproductive disorders that generate substantial losses in the livestock industry. Sharing of

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water and food sources between wild and domestic populations increases the risk of interspecies pestivirus transmission. Monitoring pestivirus prevalence in both population types is vital. No data is currently available on pestivirus genetic diversity in wild artiodactyl populations in Mexico. Isolation and genetic analysis were done for BVDV from serum samples collected from 371 captive wild artiodactyls in four regions in central and eastern Mexico. Samples from two water buffaloes and one fallow deer were positive for BVDV by RT-PCR. Phylogenetic analysis of the amplified sequences placed them in BVDV subgenotype 1b. A cytopathic strain was isolated from the deer sample. This is the first report of bovine viral diarrhea virus in wild artiodactyls in Mexico and the first to identify the virus subtype. Key words: Pestivirus, BVDV 1b, Genotyping, Isolation, Wild fauna.

Received: 20/09/2021 Accepted: 21/12/2021

Introduction Bovine viral diarrhea virus (BVDV) belongs to the Pestivirus genus, in the Flaviviridae family. This genus includes bovine viral diarrhea virus 1 (BVDV1) and bovine viral diarrhea virus 2 (BVDV2), among other viruses of veterinary importance. The International Committee on Taxonomy of Viruses (ICTV) classifies BVDV1 as a Pestivirus A species and BVDV2 as a Pestivirus B species(1). BVDV1 has 21 subgenotypes (1a-1u), while BVDV2 has 4 subgenotypes (2a-2d)(2). Strains of BVDV are classified as cytopathic (CP) or noncytopathic (NCP) biotypes, according to their effect on cultured cells(3). The CP biotypes cause vacuolation and cell death, while NCP biotypes cause no visible alterations in cell culture(4-5). The BVDVs infects a wide range of animals belonging to the order Artiodactyla. Until recently, BVDV had been recognized primarily as a pathogenic agent in domestic cattle. It can cause a range of symptoms from subclinical infections to acute infections characterized by inappetence, transient fever, diarrhea, respiratory disorders and reproductive disorders such as abortions, mummifications, congenital defects, stillbirths or the birth of immunotolerant, persistently infected (PI) animals(6-7). However, it is now recognized as a causal agent of reproductive, respiratory, immunological and neurological alterations in wild artiodactyls as well(8-14).

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Vertical transmission of BVDV can occur via transplacental infection, mating or use of infected semen or embryos(15). Horizontal transmission occurs through direct or indirect contact with the oral and/or nasal secretions of infected animals(12). Factors such as shared land use and animal migration can promote the spread of pestiviruses between domestic and wild animals(9). Natural water and food sources are common points of interaction where viruses can spread to a wide variety of hosts(12). BVDV infections generate significant economic losses in the livestock industry in the form of decreased milk production, substandard reproductive performance, growth retardation, congenital defects, predisposition to concomitant diseases and increased mortality of young animals(16). Estimated losses are 46 million dollars/year in England, 44.5 million dollars/yr in New Zealand(17-18), and 20 million dollars per million births in Denmark(19). Limited data is available on the genetic diversity of circulating pestiviruses in Mexico; indeed, just one study has been done in cattle in which four BVDV genetic variants were identified (subgenotypes 1a, 1b, 1c and, 2a)(20). Considering its economic impact in the livestock industry(17-19), its ease of transmission between domestic and wild artiodactyls(9,12,21), and the presence of BVDV antibodies in white-tailed deer in Mexico, the present study objective was to detect and identify the BVDV genotypes present in wild animals in Mexico to estimate their potential prevalence in the evaluated populations.

Material and methods Blood samples

A total of 371 blood samples were collected from wild artiodactyls in captivity. The animals were 2 to 3 yr old and included water buffalo (Bubalus bubalis), fallow deer (Dama dama), white-tailed deer (Odocoileus virginianus), eland antelope (Taurotragus oryx) and wild boar (Sus scrofa). They were from Mexico City and the states of Veracruz, Querétaro and Estado de Mexico. Using Vacutainer TM tubes, from 3 to 6 ml of blood were taken from the jugular vein of each animal. Once the blood coagulated, the samples were centrifuged for 20 min at 2,000 rpm in a clinical centrifuge. The serum was transferred to microcentrifuge tubes and stored at -70 °C until analysis.

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RNA extraction

Total RNA was extracted from serum samples using TRIzol TM LS Reagent, following the manufacturer’s instructions. Briefly, 400 µl serum was mixed with 900 µl TRIzol™ LS Reagent by inverting the microfuge tube six times. The mixture was incubated for 5 min at 4 °C, 240 µl chloroform added, homogenized, and incubated for 5 minutes at 4 °C. The mixture was centrifuged for 15 min at 13,000 g and 4 °C. The supernatant (200 µl) was transferred to a nuclease-free tube, 600 µl isopropanol was added and the mixture homogenized. It was incubated at -20 °C for one hour and centrifuged again for 15 min at 13,000 xg and 4 °C. The supernatant was discarded, and the pellet was washed with 1 ml 75% ethanol and centrifuged for 5 min at 13,000 xg and 4 °C. The supernatant was discarded, and the pellet dried for 5 min at room temperature. The pellets were suspended in 20 µl nuclease-free water and stored at -70 °C until use. Total RNA was also extracted from BVDV-free MDBK cells as a negative control and from the NADL reference strain as a positive control in the RT-PCR

Reverse transcription (RT)

Using a 20 µl/reaction total volume, reverse transcription was done with M-MLV Reverse Transcriptase (Thermo-Fisher), following a manufacturer-recommended protocol. Reaction ingredients were 500 ng RNA (1-10 µl), 1 µl random primers (Invitrogen) (0.2 µg/µl), 1 µl dNTP Mix (10 mM each) and nuclease-free water (sufficient to complete 12 µl). These were homogenized by pipetting, incubated for 5 min at 65 °C, and, 4 µl 5X buffer, 2 µl 0.1 M DTT and 1 µl ribonuclease inhibitor (40 U/µl) added. The mixture was homogenized by pipetting and incubated for 2 min at 37 °C. Complementary DNA was synthesized by adding 1 µl MMLV Reverse Transcriptase (200 U/µl) and incubating for 50 min at 37 °C. The reaction was stopped by heating the mixture to 70 °C for 15 min.

Polymerase chain reaction (PCR) and sequencing

The PCR analysis of the pestivirus 5' UTR region was done using the 5UTRfwd/STAR-Trev, 324/326 and HCV90/HCV368 primers, which amplify 292 bp, 288 bp and 278 bp fragments, respectively(22-24). Each reaction was prepared in a 25 µl total volume with 2 µl cDNA, 1 µl sense primer (10 µM), 1 µl antisense primer (10 µM), 1 µl dNTP Mix (10 mM each), 0.2 µl Taq polymerase (5 U/µl), 5 µl buffer (10 X) and 14.8 µl nuclease-free water.

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The analysis was run in a thermocycler (Select Cycler II, Select BioProducts) using these parameters: initial denaturation for 4 min at 94 °C; 30 amplification cycles using denaturation for 30 sec at 94 °C, alignment for 30 sec at 56.2 °C (5UTRfwd/STAR-Trev primers), 55 °C (324/326 primers) and 50 °C (HCV90/HCV368 primers), and extension for 30 sec at 72 °C; and final extension for 10 min at 72 °C. The RT-PCR products were separated by 1% agarose gel electrophoresis, following the methodology described(15,25). Agarose gels were stained with GelRed® following the manufacturer’s protocol. The RT-PCR products were viewed with a UV transilluminator and purified with the QIAquick® Gel Extraction Kit (Qiagen GmbH) following the manufacturer's protocol. The amplicons were sequenced in duplicate in both directions with the BigDyeTM Terminator Sequencing Kit on an ABI PRISM® 3130xl Genetic Analyzer at the UNAM Biotechnology Institute.

Phylogenetic analysis

Phylogenetic reconstruction of the 5' UTR region sequences of the detected viruses was done using the MEGA 10 program, with the maximum verisimilitude method and the Kimura 2 substitution method with 1,000 bootstraps(26-28).

Viral isolation

Viral isolation was done following standard cell culture techniques(29), and using 25 cm2 cell culture dishes with a 70% confluence of MDBK cells (BVDV-free) in MEM supplemented with 5% horse serum. Samples (500 μl) of serum identified as positive by RT-PCR were deposited in the culture dishes and identification of infected MDBK cell cultures done by observing cytopathic effects (vacuolation and lysis) 48 h after incubation, RT-PCR, and sequencing. The isolation titer was measured using the Reed-Muench method(30). Culture supernatant (100 μl) containing the isolated virus and 900 μl MEM supplemented with 5% horse serum were placed in each well of a 96-well microplate. Decimal log dilutions were done in triplicate from 10-1 to 10-8, and the microplates incubated for 48 h at 37 ºC.

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Results The RT-PCR analysis with the 324/326 primers identified BVDV RNA in the serum sample of a two-year-old female fallow deer identified as 7916 (Mexico City). The 5UTRfwd/STARTrev and 324/326 primers identified BVDV RNA in serum samples from two six-mo-old water buffaloes identified as BUMA 6 and BUMA 15 (Mil Aguas, Veracruz). Phylogenetic analysis of the 5' UTR amplicons to identify BVDV species and subgenotypes in the sequences of the three positive samples grouped them in BVDV subgenotype 1b (Figure 1). Figure 1: Phylogenetic analysis of isolated pestivirus based on a fragment of the 5' UTR region

Reconstruction of the phylogenetic tree rooted in the 5' UTR region built with the maximum verisimilitude method using the MEGA 10 program with a Kimura 2 substitution model and 1,000 bootstraps. Reference virus sequences are identified by their GenBank Access numbers, and viral sequences identified in the present study are in bold with an asterisk.

Phylogenetic analysis of the 5' UTR region indicated that the BUMA 6 (GenBank #: MN811650) and BUMA 15 (GenBank #: MN811651) sequences exhibited 99.8 % identity with the NY reference strain of BVDV, which belongs to subgenotype 1b. The 7916 (GenBank #: MN811649) sequence exhibited 99.8 % identity with the Osloss reference strain of BVDV, which also belongs to subgenotype 1b.

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Use of BVDV-free MDBK cells allowed isolation of a CP biotype virus from the fallow deer (7916) serum; the titer of the isolated BVDV 1b was 106 CCID50/ml. Virus isolation was unsuccessful in the water buffalo (BUMA 6 and BUMA 15) serum samples.

Discussion The BVDV subgenotype 1b is considered the most widely distributed worldwide in cattle, followed by subgenotypes 1a and 1c(2). Subgenotype 1b has been detected in domestic artiodactyls in various countries (including Mexico) on all five continents(2,20). In wild animals, subgenotype 1b has been detected in countries such as Canada (bison)(31), China (yak)(32), Germany (bongo)(33) and the United States (alpaca and white-tailed deer)(34-35). To date, only one study has addressed bovine viral diarrhea prevalence in wild animals in Mexico(36). Serum samples were collected from white-tailed deer from three states in northeastern Mexico (Coahuila, Nuevo León, and Tamaulipas) and a 63.5 % seroprevalence identified. However, this study only identified antibodies, and did not involve direct identification nor genetic characterization of BVDV. The present study is therefore the first to report on isolation and direct detection of BVDV in wild animals in Mexico, and the first report of BVDV prevalence in wild animals in central and eastern Mexico. The primers used in the present analyses allow detection of various pestivirus species (Pestivirus A-E, G-H)(22-24), but only BVDV1 subgenotype 1b RNA (Pestivirus A) was detected. This was found in approximately 1% of the analyzed wild artiodactyl samples. This proof of BVDV1 subgenotype 1b in wild animals in Mexico suggests that it may transmit between wild and domestic artiodactyl populations. The present results may contribute to design of effective disease control programs that include monitoring of domestic and wild artiodactyls, in addition to vaccination to confer specific protection against the BVDV subgenotypes currently circulating in Mexico. Infections with the CP or NCP biotypes of BVDV have different implications for disease severity(37-38). The NCP biotype is commonly detected in samples associated with respiratory disorders, while the CP biotype is usually detected in samples from animals with reproductive, enteric, or systemic disorders(38). Infections with the CP biotype can cause disorders in embryonic development, such as mummifications, hydrocephalus, retinal dysplasia, arthrogryphosis, and abortions(39). Intrauterine infections with the NCP biotype that occur between d 42 and 125 of pregnancy can cause persistent infections in the fetus and, consequently, the birth of persistently infected animals that are immunotolerant to BVDV, remain seronegative, and disseminate the virus throughout their lives(6,40).

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Simultaneous infection with the CP and NCP biotypes in PI artiodactyls results in mucosal disease, which is lethal. Therefore, presence of the CP and NCP biotypes in the same farm or location is important because it increases the possibility of co-infections with both biotypes(41). PI bulls can shed between 104 and 107 CCID50/ml BVDV in semen during their lifetime(42), a titer similar to the 106 CCID50/ml isolated from a clinically healthy female fallow deer (7916) in the present study. This titer suggests that the doe may have been in an early stage of viremia during an acute, but subclinical, infection which would allow virus detection in the serum sample. It is also similar to the 105.8 DICC50/ml in serum, and 105.8 DICC50/ml to 106.3 DICC50/ml in nasal secretions reported in white-tailed deer(12). Unlike in previous studies, the BVDV CP isolated in the present study was from an animal free of clinical signs. It remains unclear if the viral titer and the absence of clinical signs observed with the isolated CP biotype are related to a recurrent subclinical infection. Of note is that the BVDV isolated from the deer serum was capable of replicating in vitro in bovine cells (MDBK), which suggests that this isolate could infect both species. Further research is required to evaluate its transmission capacity and virulence in both species. This study is the first report of the detection and isolation of BVDV (Pestivirus A, subgenotype 1b, biotype CP) in water buffalo and fallow deer in Mexico. No other pestivirus species were detected. Further research is needed to better characterize the circulating pestiviruses in wild animal populations in Mexico since they can represent a source of infection for domestic species and vice versa. The present results contribute to understanding the genetic and epidemiological diversity of pestiviruses in the evaluated populations.

Acknowledgements

The research reported here was financed by the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica IN217919 “Identificación y caracterización genética de las cepas del virus de la diarrea viral bovina circulantes en poblaciones ganaderas de México”, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México (FMVZ-UNAM).

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Conflict of interest

The authors declare no conflict of interest.

Ethical approval

The sample collection protocol was approved by the Comité Interno para el Cuidado y Uso de los Animales, Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México (FMVZ-UNAM). Literature cited: 1.

Walker PJ, Siddell SG, Lefkowitz EJ, Mushegian AR, Adriaenssens EM, AlfenasZerbini P, et al. Changes to virus taxonomy and to the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2021). Arch Virol 2021;166(9):2633–2648.

Yeşilbağ K, Alpay G, Becher P. Variability and global distribution of subgenotypes of bovine viral diarrhea virus. Viruses 2017;9(6):E128.

Tautz N, Tews BA, Meyers G. The molecular biology of pestiviruses. Adv Virus Res 2015;(93):47–160.

Lértora WJ. Diarrea viral bovina: actualización. Rev Vet 2003;14(1):42–51.

Rondón I. Diarrea vial bovina: patogénesis e inmunopatología. Rev MVZ Córdoba 2006;11(1):694–704.

Strong R, La Rocca SA, Ibata G, Sandvik T. Antigenic and genetic characterisation of border disease viruses isolated from UK cattle. Vet Microbiol 2010;141(3–4):208–215.

Brock KV. The many faces of bovine viral diarrhea virus. Vet Clin North Am Food Anim Pract 2004;20(1):1–3.

Mattson DE, Baker RJ, Catania JE, Imbur SR, Wellejus KM, Bell RB. Persistent infection with bovine viral diarrhea virus in an alpaca. J Am Vet Med Assoc 2006;228(11):1762–1765.

620

Rev Mex Cienc Pecu 2022;13(3):612-624

Vilcek S, Nettleton PF. Pestiviruses in wild animals. Vet Microbiol 2006;116(1-3):1– 12.

10. Nelson DD, Dark MJ, Bradway DS, Ridpath JF, Call N, Haruna J, et al. Evidence for persistent Bovine viral diarrhea virus infection in a captive mountain goat (Oreamnos americanus). J Vet Diagn Invest 2008;20(6):752–759. 11. Duncan C, Ridpath J, Palmer MV, Driskell E, Spraker T. Histopathologic and immunohistochemical findings in two white-tailed deer fawns persistently infected with Bovine viral diarrhea virus. J Vet Diagn Invest 2008;20(3):289–296. 12. Passler T, Ditchkoff SS, Walz PH. Bovine viral diarrhea virus (BVDV) in white-tailed deer (Odocoileus virginianus). Front Microbiol 2016;(7):945. 13. Wolff PL, Schroeder C, McAdoo C, Cox M, Nelson DD, Evermann JF, et al. Evidence of bovine viral diarrhea virus infection in three species of sympatric wild ungulates in Nevada: life history strategies may maintain endemic infections in wild populations. Front Microbiol 2016;(7):292. 14. Salgado R, Hidalgo-Hermoso E, Pizarro-Lucero J. Detection of persistent pestivirus infection in pudú (Pudu puda) in a captive population of artiodactyls in Chile. BMC Vet Res 2018;14(1):37. 15. Houe H. Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections. Vet Microbiol 1999;64(2–3):89–107. 16. Houe H. Economic impact of BVDV infection in dairies. Biologicals 2003;31(2):137– 143. 17. Bennett RM, Christiansen K, Clifton-Hadley RS. Estimating the costs associated with endemic diseases of dairy cattle. J Dairy Res 1999;66(3):455–459. 18. Heuer C, Healy A, Zerbini C. Economic effects of exposure to bovine viral diarrhea virus on dairy herds in New Zealand. J Dairy Sci 2007;90(12):5428–5438. 19. Houe H, Pedersen KM, Meyling A. A computarized spread sheet model for calculating total annual national losses due to bovine viral diarrhoea virus infection in dairy herds and sensitivity analysis of selected parameters. In: Proc Second Symp Pestiviruses. Annecy, France. 1993:179–184.

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Rev Mex Cienc Pecu 2022;13(3):612-624

20. Gómez-Romero N, Basurto-Alcántara FJ, Verdugo-Rodríguez A, Bauermann FV, Ridpath JF. Genetic diversity of bovine viral diarrhea virus in cattle from Mexico. J Vet Diagn Invest 2017;29(3):362–365. 21. Kirkland PD, Frost MJ, King KR, Finlaison DS, Hornitzky CL, Gu X, et al. Genetic and antigenic characterization of Bungowannah virus, a novel pestivirus. Vet Microbiol 2015;178(3–4):252–259. 22. Mahony TJ, McCarthy FM, Gravel JL, Corney B, Young PL, Vilcek S. Genetic analysis of bovine viral diarrhoea viruses from Australia. Vet Microbiol 2005;106(1–2):1–6. 23. Vilcek S, Herring AJ, Herring JA, Nettleton PF, Lowings JP, Paton DJ. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch Virol 1994;136(3–4):309–323. 24. Ridpath JF, Lovell G, Neill JD, Hairgrove TB, Velayudhan B, Mock R. Change in predominance of Bovine viral diarrhea virus subgenotypes among samples submitted to a diagnostic laboratory over a 20-year time span. J Vet Diagn Invest 2011;23(2):185– 193. 25. Armstrong JA, Schulz JR. Agarose gel electrophoresis. Curr Protoc Essen Lab Tech 2015;10(1):7.2.1-7.2.22. 26. Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16(2):111–120. 27. Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985;39(4):783–791. 28. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol 2018;35(6):1547–1549. 29. Warner DR, Sakai D, Sandell LL. Mammalian cell culture. Curr Protoc Essen Lab Tech 2015;10(1):4.3.1-4.3.33. 30. Reed LJ, Muench H. A simple method of estimating fifty per cent endpoints. Am J Epidemiol 1938;27(3):493–497. 31. Deregt D, Tessaro SV, Baxi MK, Berezowski J, Ellis JA, Wu JTY, et al. Isolation of bovine viral diarrhoea viruses from bison. Vet Rec 2005;157(15):448–450.

622

Rev Mex Cienc Pecu 2022;13(3):612-624

32. Gong X, Liu L, Zheng F, Chen Q, Li Z, Cao X, et al. Molecular investigation of bovine viral diarrhea virus infection in yaks (Bos gruniens) from Qinghai, China. Virol J 2014;(11):29. 33. Becher P, Orlich M, Kosmidou A, König M, Baroth M, Thiel HJ. Genetic diversity of pestiviruses: identification of novel groups and implications for classification. Virology 1999;262(1):64–71. 34. Passler T, Walz PH, Ditchkoff SS, Brock KV, Deyoung RW, Foley AM, et al. Cohabitation of pregnant white-tailed deer and cattle persistently infected with Bovine viral diarrhea virus results in persistently infected fawns. Vet Microbiol 2009;134(3– 4):362–367. 35. Goyal SM, Bouljihad M, Haugerud S, Ridpath JF. Isolation of bovine viral diarrhea virus from an alpaca. J Vet Diagn Invest 2002;14(6):523–525. 36. Cantu A, Ortega-S JA, Mosqueda J, Garcia-Vazquez Z, Henke SE, George JE. Prevalence of infectious agents in free-ranging white-tailed deer in northeastern Mexico. J Wildl Dis 2008;44(4):1002–1007. 37. Fulton RW, Ridpath JF, Saliki JT, Briggs RE, Confer AW, Burge LJ, et al. Bovine viral diarrhea virus (BVDV) 1b: predominant BVDV subtype in calves with respiratory disease. Can J Vet Res 2002;66(3):181–190. 38. Odeón AC, Risatti G, Kaiser GG, Leunda MR, Odriozola E, Campero CM, et al. Bovine viral diarrhea virus genomic associations in mucosal disease, enteritis and generalized dermatitis outbreaks in Argentina. Vet Microbiol 2003;96(2):133–144. 39. Brownlie J. The pathogenesis of bovine virus diarrhoea virus infections. Rev Sci Tech 1990;9(1):43-59. 40. Falkenberg SM, Dassanayake RP, Walz P, Casas E, Neill JD, Ridpath JF. Frequency of bovine viral diarrhea virus detected in subpopulations of peripheral blood mononuclear cells in persistently infected animals and health outcome. Vet Immunol Immunop 2019;(207):46–52. 41. Rondena M, Lorenzi V, Binanti D, Gelmetti D, Pravettoni D, Finazzi M, et al. Immunedepletion related to bovine viral diarrhoea virus in a heifer with naturally occurring mucosal disease. J Comp Pathol 2009;141(4):306.

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Rev Mex Cienc Pecu 2022;13(3):612-624

42. Givens MD, Riddell KP, Edmondson MA, Walz PH, Gard JA, Zhang Y, et al. Epidemiology of prolonged testicular infections with bovine viral diarrhea virus. Vet Microbiol 2009;139(1–2):42–51.

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https://doi.org/10.22319/rmcp.v13i3.5997 Article

Evaluation of real-time polymerase chain reaction coupled to immunomagnetic separation (rtPCR-IMS) as an alternative method for the routine detection of Salmonella spp. in beef in Mexico

Gloria Marisol Castañeda-Ruelas a José Roberto Guzmán-Uriarte b José Benigno Valdez-Torres b Josefina León-Félix b*

Universidad Autónoma de Sinaloa. Facultad de Ciencias Químico-Biológicas. Culiacán, Sinaloa, México. b

Centro de Investigación en Alimentación y Desarrollo, AC., Biología Molecular y Genómica Funcional. Carretera a El Dorado Km 5.5, Col. Campo El Diez, 80129, Culiacán, Sinaloa, México.

*Corresponding author: ljosefina@ciad.mx

Abstract: Salmonella is a pathogenic bacterium considered a threat to the food industry, its timely detection being relevant. The objective of the study was to evaluate the real-time polymerase chain reaction coupled to immunomagnetic separation (rtPCR-IMS) as an alternative method to the Official Mexican Standard (NOM-114-SSA1-1994) for the detection of Salmonella in beef. The parameters evaluated were limit of detection, sensitivity, specificity, selectivity (inclusivity and exclusivity) and degree of agreement between both methods for the detection of Salmonella in presumptive and artificially contaminated beef samples. The incidence of Salmonella in presumptive beef samples (n= 60) ranged from 20.0 to 21.6 % by both methods. In the inoculated samples (n= 60), the detection rate of Salmonella by rtPCR-IMS (93.3 %) and NOM-114-SSA1-1994 (98.3 %) showed a match of 56 occasions with a negative deviation. The comparison of rtPCR-IMS and NOM-114-SSA1-1994 in beef 625

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reported an accuracy of 98.3 %, sensitivity of 98.2 %, specificity of 100 % and selectivity of 100 %. The limit of detection for both methods was 1-5 CFU·25 g-1 of beef. The statistical analysis indicates that the rtPCR-IMS is equivalent to the reference method for the detection of Salmonella in beef. These results warn of the high incidence of Salmonella in beef and propose rtPCR-IMS as an ideal and fast method for the control of Salmonella in the meat industry. Key words: Beef, Polymerase chain reaction (PCR), Salmonella, Immunomagnetic separation (IMS).

Received: 28/05/2021 Accepted: 03/02/2022

Introduction Salmonella is a group of Gram-negative bacteria that comprises >2,600 serotypes classified into two species, S. enterica (includes six subspecies) and S. bongori. Among these, Salmonella enterica subsp. enterica (>1,500 serotypes) is the main group responsible for diseases in man called “salmonellosis”(1). The main clinical manifestations are enteric fever (typhoid and paratyphoid) and gastroenteritis caused by typhoidal and nontyphoidal serotypes of Salmonella, respectively(2). The annual global estimate of salmonellosis is >25 million cases of enteric fever and 153 million cases of gastroenteritis with ~300 thousand deaths, which are mostly associated with the consumption of contaminated foods(3). Salmonellosis is currently one of the four major foodborne diseases (FBDs) worldwide(4). Salmonella is widely distributed in nature and can survive in a wide variety of foods (animal origin and vegetables), which have been identified as vehicles of transmission of the bacterium(2,4,5). The zoonotic nature of nontyphoidal serotypes of Salmonella points to animals as the main risk factor for exposing the bacterium to the environment and transferring the pathogen to humans during the production, handling or consumption of foods(6). Salmonella surveillance should be based on reliable detection methods that favor food safety(7). Detecting Salmonella in foods can be complex because the bacteria are often found in low concentrations in foods. Another aspect that hinders the detection of the microorganism in a food is the process of production, the background microorganisms and the type of food matrix(8). This can warn of a health risk and justify the need for timely methods for the detection of relevant pathogens such as Salmonella(7). 626

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The culture method is considered the gold standard for the isolation and detection of pathogenic microbes in foods(9). The culture method for the detection of Salmonella involves a stage of nonselective pre-enrichment, followed by selective enrichment and seeding on selective agars, and subsequent biochemical and serological characterization of presumptive colonies, allowing a negative or positive result to be obtained in 4 and 6-7 d, respectively(9). The consumption of time, labor and reagents involved in this method are an inconvenience for the rapid detection of Salmonella in the food industry(7). Currently, rapid molecular methods have been developed, such as the polymerase chain reaction (PCR) method and its real-time variant (rtPCR), which allow the detection of Salmonella in a short time (24-72 h) in various foods(10-12). Immunological, biochemical tests and biosensors have also been proposed as rapid methods(8). Molecular methods are useful as detection tools, reducing labor and response time compared to culture methods(12). However, the accuracy of molecular methods can be limited by the presence of inherent substances of the food (bile salts, bilirubin, hemoglobin, urea, polysaccharide, feces) and antigens and DNA, which can interfere with the results(13). Immunomagnetic separation (IMS) has been used for the isolation of Salmonella in different food matrices(8,14-16). IMS improves the sensitivity and specificity of detection of Salmonella in foods due to the anti-Salmonella polystyrene beads that capture the bacterium, and this can be identified by culture, immunological or molecular methods(17). Recently, methods that combine IMS and rtPCR technologies have been developed, which dispense with DNA extraction and concentrate the microorganism by IMS after a primary enrichment of <24 h(10,11,18). In Mexico, Salmonella represents an issue concerning public health and the food industry. The General Directorate of Epidemiology of Mexico annually reports 28,815 cases of typhoid fevers and 78,681 cases of other salmonellosis nationwide(19). But the identification of these cases as FBDs is not clarified. Despite the fact that, in Mexico, Salmonella has been identified as a contaminant of chicken meat(20,21), pork(21), beef (21-23), vegetables(24) and eggs(25). The timely detection of Salmonella in beef is of special interest given its high frequency (28.9 to 32.4 %) in this food(21-23) and the relevance of production (1,960 t) and annual per capita consumption (14.8 kg) of beef in Mexico(26). In addition, livestock and birds have been identified as reservoirs of Salmonella(27). In Mexico, the culture method is the gold standard for the detection of Salmonella in foods, whose process can be laborious and require a lot of time (4 to 7 days) to obtain final results(28). Therefore, the objective of this study was to perform an internal validation of the commercial method of rtPCR-IMS for the detection of Salmonella in beef, and to compare its efficiency with the reference culture method used in Mexico. 627

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Material and methods Description of the study The internal validation study of the real-time polymerase chain reaction method coupled to immunomagnetic separation (rtPCR-IMS) for the detection of Salmonella in beef was evaluated with respect to the reference culture method in Mexico “NOM-114-SSA11994”(28), as specified in the manual for the validation of alternative microbiological methods proposed by ISO16140:2003(29). The validation of the rtPCR-IMS method was performed on presumptive and artificially contaminated beef samples. The parameters evaluated were limit of detection, sensitivity, specificity, selectivity, and the degree of agreement between the methods.

Bacterial strains The Salmonella strain ATCC 35664 was used as a reference control for the validation assay. One pure colony was transferred to 30 mL of trypticasein soy broth (TSB; Becton Dickinson) and incubated for 4 h in a water bath at 35± 2 °C with constant stirring. An aliquot of the culture was used to inoculate 50 mL of TSB to obtain an absorbance (OD) of 0.1 at 600 nm. This subculture was incubated under the same conditions until obtaining an OD 1.0 (λ= 600 nm). Serial dilutions and plate count in Hektoen enteric agar (Becton Dickinson) were performed to standardize the concentrations of 1-5, 6-10, 1-15 and 16-30 CFU·100 μL-1. A list of 60 strains classified as Salmonella (n= 30) and non-Salmonella (n= 30) were used for the selectivity parameter (Table 1). The selection of non-Salmonella strains was based on the biochemical characteristics they share with Salmonella or because they are considered contaminants of the meat. Most of the non-Salmonella strains corresponded to collection cultures and were obtained commercially. Salmonella strains were obtained from the laboratory, whose identification was previously made with molecular tests. All strains were grown in TSB at 37 ºC for 24 h.

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Inclusivity test Salmonella strains S. Agona S. Albany S. Anatum S. B monofásica S. C1 monofasica S. Cayar S. Cholerasuis S. Enteritidis S. F S. Gaminara S. Give S. Haviana S. Infantis S. Luciana S. Meliagridis S. Minnesota S. Montevideo S. Muenchen S. Newport S. Oranienburg S. Saintpaul S. San Diego S. Senftenberg S. Sohanina Salmonella sp S. Typhimurium S. Thompson S. Weltevreden

Table 1: Microorganisms used for the selectivity test Exclusivity test No Reference Non-Salmonella strains No Reference 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1 1 1 1

CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C CIAD A.C

Bacillus subtilis Candida Albicans Citrobacter freundii Citrobacter freundii Enterobacter aerogenes Enterobacter aerogenes Enterobacter cloacae Enterococcus faecalis Escherichia coli Escherichia coli Eschrichia coli O157:H7 Klebsiella pneumoniae Listeria innocua Listeria ivanovvi Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Listeria monocytogenes Proteus mirabilis Proteus mirabilis Proteus vulgaris Pseudomonas aeroginosa Pseudomonas aeruginosa Rhodococcus equi Rhodococcus equi Shigella flexneri Gpo. B Shigella flexnieri Shigella sonnei Staphylococcus aereus Staphylococcus epidermis

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Ambiental ATCC 10231 LEM 04001 LEM-04001 LEM-04003 ATCC 13048 LEM-04013 LIBM-01003 LEM-01005 ATCC 25922 CENAPA700728 ATCC 13883 ATCC 33090 ATCC 19119 Ambiental ATCC 7694 ATCC 7644 Ambiental ATCC 12453 LEM-03011 LEM-06070 ATCC 27853 LEM-01002 LEM-01019 ATCC 6939 ATCC 12022 LEM-04004 ATCC 9290 ATCC 25923 ATCC 12228

The acronyms stands for: CIAD A.C. (Centro de Investigación en Alimentación y Desarrollo A.C.), CENAPA (Centro Nacional de Servicios de Diagnóstico en Salud Animal), ATCC (American Type Culture Collection), LEM (internal code), LIBM (internal code).

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Collection of beef samples For the study, 60 samples of raw beef consisting of ground beef (n= 20), inside round (n= 20) and peeled knuckle (n= 20) were collected from three markets located in the city of Culiacán, Sinaloa, Mexico. The number of beef samples was assigned according to the minimum recommendation suggested by ISO16140:2003(29). Samples of 500 g of beef were taken, which were placed in sterile plastic bags and transported in refrigeration to the laboratory for their further analysis. The study contemplated performing the analysis on presumptive samples to simulate the actual effects of contamination occurring in nature, and on artificially contaminated beef samples.

Analysis of presumptive beef samples Fifty grams of beef were homogenized with 50 ml of sterile distilled water in a grinder at 230 rpm for 2 min. Subsequently, the sample was divided equally into 25 mL portions for the method of rtPCR-IMS and NOM-114-SSA1-1994.

Analysis of artificially contaminated beef samples Samples of beef classified as negative for Salmonella by both methods were used. A 100 g portion of beef was homogenized with 100 mL of sterile distilled water for 2 min at 230 rpm and separated equally into two 50 mL portions; one portion was used as a negative control and the remaining portion for contamination of the beef with Salmonella. The contaminated portion was homogenized for 2 min at 230 rpm, and subsequently the homogenate was divided into two portions of 25 mL for their analysis by both methods. In parallel, a negative control under the same conditions was included. The selection of the inoculum corresponded to the limit of detection that allows a fractional recovery of the bacterium by any of the methods.

Limit of detection To establish the relative detection limit, five levels of inoculum of Salmonella were prepared: 0, 1-5, 6-10, 1-15 and 16-30 CFU·25 mL-1. From the beef samples classified as negative for Salmonella, 250 g of sample were taken and homogenized with 250 mL of sterile distilled water for 2 min at 230 rpm in an automatic homogenizer (Stomacher 400 Circulator). The homogenate was divided into 10 portions of 25 mL for their inoculation with Salmonella: portion 1-2 (0 CFU·25 mL-1), portion 3-4 (1-5 CFU·25 mL-1), portion 5-6 (6-10 CFU·25 mL-1), portion 7-8 (1-15 CFU·25 mL-1) and portion 9-10 (16-30 CFU·25 mL-1). The levels of inoculum added were corroborated by the method of plate count in Hektoen enteric agar, and the types of portions were evenly distributed for their analysis for both methods. The 630

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limit of detection will correspond to the smallest concentration of the inoculum that can be detected in the sample 50 % of the time by the methods(29).

Selectivity test The selectivity test of the methods was performed in vitro without the use of beef samples and the selection of the bacterial inoculum was performed according to the specifications of ISO16140:2003(29). For each bacterium (Table 1), an inoculum was standardized at an absorbance of 1.0 (λ= 600 nm) in both assays. For the exclusivity assay, a concentration of 104 CFU 100 μL-1 was adjusted in 225 mL of peptone water. Whereas, in the inclusivity assay, the concentration of 100 CFU·100 μL-1 was adjusted in 225 mL of the enrichment media used by the rtPCR-IMS (peptone water) and NOM-114- SSA1-1994 (lactose broth) methods, and the subsequent addition of 100 μL from a pool of the strains used in the exclusivity test. All cultures were evaluated by the methods of rtPCR-IMS and NOM-114SSA1-1994.

Reference culture method (NOM-114-SSA1-1994) Beef samples (25 mL) were enriched with 225 mL of lactose broth (Becton Dickinson) and incubated at 35 ± 2 °C for 24 ± 2 h. Subsequently, aliquots of 1 mL of the culture were transferred to 10 mL of Selenite Cystine broth (Becton Dickinson) and 10 mL of Rappaport Vassiliadis broth (Becton Dickinson), for their incubation at 35 ± 2 °C for 24 h. Once incubation was complete, 10 μL aliquots of the previous cultures were inoculated into Hektoen enteric agar (Becton Dickinson), Xylose Lysine Deoxycholate Agar (Becton Dickinson) and Salmonella-Shigella agar (Becton Dickinson) and incubated at 35 ± 2 °C for 24 h. From the agars, presumptive colonies (n= 3) of Salmonella were selected for their identification by primary biochemical tests (triple sugar iron agar, lysine and iron agar and urea broth), API 20E (Biomeriux NC) and serological tests based on the detection of polyvalent O antigen (InDRE)(28). In parallel, a negative control (medium without bacteria) and a positive control (Salmonella ATCC 35664) were included.

rtPCR-IMS method The detection of Salmonella was carried out according to the conditions of the supplier (www.biocontrolsys.com). All reagents necessary for the preparation of the rtPCR reaction for Salmonella (GDS Salmonella Tq 71008) are commercially available by Assurance GDS®, BioControl System Inc. A 25 mL portion of sample was enriched with 225 mL of buffered peptone water (Becton Dickinson) and incubated at 35 ± 2 °C for 24 h. Subsequently, 1 mL of the previous enrichment was transferred to the concentration plate containing 20 μl of concentration reagent (magnetic beads coated with anti-Salmonella antibodies) and mixed 631

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for 10 min in an automatic homogenizer (Vortex Mixed Biocontrol Bellevue System). The magnetic beads were removed with a magnetic pipette (Pick Pen™ Biocontrol System Bellevue), washed with 1 ml of the buffer for 7 sec and transferred to a plate containing 35 μL of the resuspension reagent. With a multichannel pipette, 20 μL of the resuspension buffer was transferred with the immunomagnetic beads and deposited in the PCR tubes, which previously contained the probe, oligonucleotides and Taq DNA polymerase (5-Prime). Finally, the tubes were placed in the rtPCR machine. In parallel, a negative control (medium without bacteria) and a positive control (Salmonella ATCC 35664) were included. All samples analyzed by the rtPCR-IMS assay were re-evaluated from the original enrichment using the reference method to verify the detection of Salmonella.

Statistical analysis The validation parameters of the rtPCR-IMS method were based on the comparison of positive agreements (PAs), negative agreements (NAs), positive deviations (PDs) and negative deviations (NDs) of the results obtained in the detection of Salmonella in the beef samples compared to the NOM-114-SSA1-1994 method. The parameters were calculated with the following formulas: Accuracy =

PAs+NAs PAs+NAs+PDs+NDs NAs

Specificity =

Sensitivity =

NAs+PDs PAs PAs+NDs

x 100%

(1)

x 100%

(2)

x 100%

(3)

False positives = 100 % − % sensitivity False negatives = 100 % − % specificity Discordant values (Y) = PDs + NDs

(4) (5) (6)

The degree of agreement between the methods (rtPCR-IMS and NOM-114-SSA1-1994) was determined by the kappa index and the McNemar test (χ²) with significance of 5 %.

Results Limit of detection Table 2 shows the capacity of the rtPCR-IMS and NOM-114-SSA1-1994 methods for the detection of different levels of Salmonella contamination in beef. The limit of detection for the rtPCR-IMS method was 1-5 CFU·25 g-1. No PCR products were obtained from the noninoculated samples.

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Sensitivity, specificity and accuracy Table 3 summarizes the agreements (PAs and NAs), deviations (PDs and NDs) and relative validation parameters of the rtPCR-IMS assay for the detection of Salmonella in the beef samples. The detection rate of Salmonella in presumptive samples (n= 60) by the rtPCR-IMS (21.6 %) and NOM-114-SSA1-1994 (20.0 %) analyses was not sufficient to calculate the validation parameters. In the 60 artificially contaminated beef samples (1-5 CFU·25 mL-1), the rtPCR-IMS and NOM-114-SSA1-1994 assays detected Salmonella in 56 (93.3 %) and 59 (98.3 %) times, respectively. The methods coincided in the detection of Salmonella on 59 occasions: 56 PAs and 3 NAs. Only one ND (detected by NOM-114 but not by rtPCR-IMS) was obtained. The rtPCR-IMS had a sensitivity of 98.2 % (56/57), specificity of 100 % (3/3) and accuracy of 98.3 % (59/60). The concordance indices (k= 0.85 and χ²= 1.0) and the discordant value (Y= 1) indicated that the rtPCR-IMS assay and the reference method (NOM-114-SSA1-1994) coincide in the statistical criteria (Table 3). All non-inoculated beef samples were negative for Salmonella by both methods.

Selectivity The rtPCR-IMS and NOM-114-SSA1-1994 methods had 100 % exclusivity and 100 % inclusivity. None of the methods reported cross-reactions. Figure 1 shows the amplifications of the rtPCR-IMS method corresponding to the inclusivity and exclusivity tests.

Discussion Salmonella represents a threat to public health and the food industry worldwide(5), and in Mexico it is no exception(19,20-25). The results of this work show the high persistence of Salmonella in presumptive beef samples (20 to 21.6 %), as previously shown by some studies in Mexico(21-23). Conventional microbiological methods serve as the basis for routine analysis in many food safety and public health laboratories due to the ease of use, reliability of the results, high sensitivity and specificity(8). However, the analysis time (5 to 7 d) of the culture methods is observed as a limitation. The incorporation of rapid molecular methods for the detection of Salmonella in foods allows early intervention and makes possible the preventive protection of the consumer(10,11,18). The limit of detection of the rtPCR-IMS and NOM-114-SSA1-1994 methods was 1-5 CFU·25 g-1 in beef, which corresponds to the lowest concentration evaluated. Of the total samples inoculated with 1-5 CFU·25 g-1, the alternative method yielded three negative repetitions, of which only two were confirmed as truly negative by the reference method 633

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(Table 2). The non-detection by the rtPCR-IMS method can be explained by the small amount (1-5 CFU·25 g-1) of Salmonella in the non-selective enrichment, probabilistic inoculation considerations or the effect of enrichment(30). Widjojoatmodjo et al(31) highlight the importance of the pre-enrichment prior to PCR detection to increase its sensitivity, given that most PCR methods require high concentrations of microorganisms for adequate detection. Similar to these results, Notzon et al(30) and O’Regan et al(32) reported a limit of detection of the rtPCR-IMS method in beef of 10-100 CFU·25 g-1, and in chicken meat of 1-10 CFU·25 g-1, respectively. These methods used an enrichment of 6 h(30) or 24 h(32) prior to detection by PCR. On the other hand, it is mentioned(10) that IMS is an alternative to avoid secondary enrichment, allowing the detection of 1-10 cells in an incubation period of 12-24 h. The limit of detection (1-5 CFU·25 g-1) observed with rtPCR-IMS would allow alignment with the national regulatory requirements (NOM-213-SSA1-2002) that require zero tolerance of Salmonella in 25 g of raw beef(33). The data obtained in the presumptive samples did not allow the determination of the validation parameters, because the calculations are made on a series of negative results obtained by the reference method, which cannot exceed twice the number of positive results as stipulated in the validation manual(29). So, this study explains the validation of the rtPCRIMS method for Salmonella analysis based on artificially contaminated beef samples. The degree of sensitivity, specificity, accuracy and agreement of the commercial method of rtPCR-IMS with the reference culture method validates its use for the analysis of Salmonella in beef, generating ideal results in a time of 24 h (Table 2). In addition, the rate of false positives (0 %) and false negatives (1.8 %) of the method are low. It is important to note that molecular methods do not replace culture techniques, since positive results must be confirmed by the reference method(34). Some previous studies have exposed the concordance of rtPCR-IMS protocols with the reference culture methods for the detection of Salmonella in beef(30) and chicken(32,35), highlighting their high degree of sensitivity (94-100 %), specificity (80-94 %) and accuracy (89-100 %). These characteristics determined in the commercial method of rtPCR-IMS (Table 3) can be attributed to the fact that the immunomagnetic beads contain antigens that allow the microbe of interest to be concentrated from non-selective enrichments, reducing the analysis time(35). In addition, the oligonucleotides used are able to detect different types of Salmonella strains (Figure 1).

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Figure 1: Amplifications of the rtPCR-IMS method for the inclusivity and exclusivity test

Figures a and b show the amplification of the 30 strains of Salmonella (25 different serotypes) and the 30 strains of non-Salmonella, respectively. The position of the lines respect to the threshold indicates a positive (upper) or negative (lower) result. Figures c and d show the amplification of the internal controls of the reaction (ICA) of the strains included in the inclusivity (a) and exclusivity (b) assay. Those of ICA lines exceeded the threshold so it is considered a valid reaction.

The negative deviation (ND) observed between the methods (Table 2) can be explained by the fact that the culture method contains several enrichment stages that favor the recovery of damaged cells and the growth of the microorganism of interest compared to the rapid methods(12). Also, the presence of Proteus, E. coli, Klebsiella aerogenes and Enterobacter in mucoid state in the enrichment broth can bind to the antibodies of the pearls, causing crossreactions and preventing the detection of Salmonella(35). It has been widely described that the type of matrix and its chemical components can affect the results of molecular methods(12-13). As for the three negative agreements between the methods, it can be attributed to the extremely low amount of the microorganism after enrichment or that there were no cells in the initial inoculum. The McNemar value (χ²= 1.0, P= 0.317) obtained in this study meets the non-significance parameter (χ²< 3.84)(29), and demonstrates that there is no difference between the rtPCR-IMS methods and the NOM-114-SSA1-1994 method for the detection of Salmonella in beef. In addition, the Kappa index reveals a high concordance (0.85 or 85 %) between the methods. Notzon et al(30) inferred the comparability of the alternative method of rtPCR-IMS with a

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concordance of 85 % (k= 0.85) and 87 % (k= 0.87) for the detection of Salmonella in artificially and naturally contaminated beef, respectively. A selective method is one that allows detecting the analyte being examined, and that can guarantee that the detected signal can only be a product of that specific analyte(29). In this sense, the rtPCR-IMS method was able to discriminate against Salmonella, since it detected the 30 strains of Salmonella corresponding to 25 different serotypes even in the presence of other microorganisms, and not generate interference with the strains other than Salmonella. Mercanoglu & Griffiths(36) have reported that the combination of rtPCR and IMS for the detection of Salmonella have a selectivity of 100 %, attributing this property to the effect of the immunomagnetic beads and the selection of the oligonucleotides used.

Conclusions and implications The results propose rtPCR-IMS as an efficient method for the rapid detection of Salmonella spp. in beef since it did not present differences with the reference method (NOM-114-SSA11994), providing the advantage of detecting the microorganism in a short time (24 h) and in a minimum concentration (1 CFU·25 g-1) and without causing cross-reactions with other microorganisms found as natural microbiota in beef. The incorporation of this type of methods in the food industry and microbiological laboratories will allow a rapid response to ensure food safety and prevent the risk of diseases. Additionally, health authorities are alerted to the high incidence of Salmonella in raw beef in order to include controls along the food chain. Acknowledgements Thanks to QFB. Jesús Héctor Carrillo Yáñez, person in charge of the Laboratory of Molecular Biology and Functional Genomics, for his technical support, and to Fund S00072006-1. SAGARPA-CONACYT. Project No. 48134 for financing the development of the research. Conflict of interest The authors declare that they have no conflicts of interest, financial or otherwise. Literature cited: 1. Ryan MP, O’Dwyer J, Adley CC. Evaluation of the complex nomenclature of the clinically and veterinary significant pathogen Salmonella. BioMed Res Int 2017;1-6.

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2. Eng SK, Pusparajaha P, Mutalibc NSA, Sera HL, Chand KG, Lee L. Salmonella: A review on pathogenesis, epidemiology and antibiotic resistance. Front Life Sci 2015;8(3):284– 293. 3. Kirk MD, Pires SM, Black RE, Caipo M, Crump JA, Devleesschauwer B, et al. World Health Organization estimates of the global and regional disease burden of 22 foodborne bacterial, protozoal, and viral diseases, 2010: A data synthesis. PLoS Med 2015;12(12): e1001921. 4. OMS. Organización Mundial de la Salud. Salmonella (non-typhoidal). Ginebra, 2020. https://www.who.int/es/news-room/fact-sheets/detail/salmonella-(non-typhoidal). 5. Jajere SM. A review of Salmonella enterica with particular focus on the pathogenicity and virulence factors, host specificity and antimicrobial resistance including multidrug resistance. Vet World 2019;12(4):504-521. 6. Andino A, Hanning I. Salmonella enterica: survival, colonization, and virulence differences among serovars. Sci World J 2015;2015:1-16. 7. Wang J, Li Y, Chen J, Hua D, Li Y, Deng H, Li Y, et al. Rapid detection of food-borne Salmonella contamination using IMBs-qPCR method based on pagC gene. Braz J Microbiol 2018;49:320–328. 8. Lee KM, Runyon M, Herrman TJ, Phillips R, Hsieh J. Review of Salmonella detection and identification methods: Aspects of rapid emergency response and food safety. Food Control 2015;47:264e276. 9. Ahmed OB, Asghar AH, Abd El-Rahim IH, Hegazy A. Detection of Salmonella in food samples by culture and polymerase chain reaction methods. J Bacteriol Parasitol 2014;5(3):1000187. 10. Mercanoglu TB & Aytac SA. Application of magnetic immuno-polymerase chain reaction assay for detection of Salmonella spp. in chicken meats. Euro Food Res Technol 2009;229(4):623-628. 11. Anderson A, Pietsch K, Zucker R, Mayr A, Müller-Hohe E, Messelhäusser U, Sing A et al. Validation of a duplex real-time PCR for the detection of Salmonella spp. in different food products. Food Anal Methods 2011;4:259–267. 12. Thung TY, Lee E, Wai GY, Pui CF, Kuan CH, Premarathne JM, Nurzafirah M et al. A review of culture-dependent and molecular methods for detection of Salmonella in food safety. Food Res 2019;3(6):1-6.

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13. Lim DV, Simpson JM, Kearns EA, Kramer MF. Current and developing technologies for monitoring agents of bioterrorism and biowarfare. Clin Microbiol Rev 2005;18(4)583607. 14. Skjerve E, Olsvik O. Immunomagnetic separation of Salmonella from foods. Int J Food Microbiol 1991;14(1):11-17. 15. Zheng Q, Mikš-Krajnik M, Yang Y, Xu W, Yuk HG. Real-time PCR method combined with immunomagnetic separation for detecting healthy and heat-injured Salmonella Typhimurium on raw duck wings. Int J Food Microbiol 214;186:6-13. 16. Zheng Q, Mikš-Krajnik M, Yang Y, Lee SC, Yuk HG. Evaluation of real-time PCR coupled with immunomagnetic separation or centrifugation for the detection of healthy and sanitizer-injured Salmonella spp. on mung bean sprouts. Int J Food Microbiol 2016;2(222):48-55. 17. Yang, X, Li H, Wu Q, Zhang J, Chen L. Comparison of direct culture, immunomagnetic separation/culture, and multiplex PCR methods for detection of Salmonella in food. Food Sci Technol Res 2015;21(5):671-675. 18. Jeníková G, Pazlarová J, Demnerová K. Detection of Salmonella in food samples by the combination of immunomagnetic separation and PCR assay. Int Microbiol 2000;3:225229. 19. DGE. Dirección General de Epidemiología. Distribución de casos nuevos de enfermedad por fuente de notificación en los Estados Unidos Mexicanos 2019. México, 2021. https://epidemiologia.salud.gob.mx/anuario/2019/morbilidad/nacional/distribucion_cas os_nuevos_enfermedad_fuente_notificacion.pdf . 20. Rodríguez R, Gómez F, Vázquez H, Corona JL, Mendoza MY. Presencia de Campylobacter y Salmonella en pollo a la venta en Gómez Palacio Durango, México. Rev Electrón Vet 2016;17(6):1-7 21. Villalpando-Guzmán S, Ramón C, Natividad-Bonifacio I, Curiel-Quesada E, QuiñonesRamírez EI, Vázquez-Salinas C. Frecuencia, susceptibilidad antimicrobiana y patrón de adherencia de Salmonella enterica aislada de carne de pollo, res y cerdo de la Ciudad de México. Rev Chil Infectol 2017;34(5):458-466. 22. Bello-Pérez LA, Ortiz-Dillanes DM, Pérez-Memije E, Castro-Domínguez V. Salmonella en carnes crudas: un estudio en localidades del estado de Guerrero. Salud Púb Méx 1989;32(1):74-79.

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23. Rubio M, Martínez JF, Hernández R, Bonilla C, Méndez RD, Núñez JF, Echeverry M. Detection of Listeria monocytogenes, Salmonella and Yersinia enterocolitica in beef at points of sale in Mexico. Rev Mex Cienc Pecu 2012;4(1):107-115. 24. Quiroz-Santiago C, Rodas-Suárez O, Vázquez C, Fernández FJ, Quiñonez-Ramírez EI, Vázquez-Salinas C. Prevalence of Salmonella in vegetables from México. J Food Protec 2009;72(6):1279–1282. 25. Mancera A, Vázquez J, Ontiveros ML, Durán S, López D, Tenorio V. Identificación de Salmonella enteritidis en huevo para consumo en la ciudad de México. Téc Pecu Méx 2005;43(2):229-237. 26. COMECARNE. Consejo Mexicano de Carne. Compendio estadístico 2018. México. 2018. https://www.inforural.com.mx/wp-content/uploads/2019/05/CompendioEstad%C3%ADstico-2018-VF.pdf . 27. Jiménez M, Martínez-Urtaza J, Chaidez C. Geographical and temporal dissemination of Salmonellae isolated from domestic animal hosts in the Culiacan Valley, Mexico. Microbial Ecol 2011;61:811-820. 28. DOF. Diario Oficial de la Federación. Norma Oficial Mexicana NOM-114-SSA1-1994. Método para la determinación de Salmonella en alimentos. México, 1994. 29. ISO. International Standardization Organization. Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods (ISO 16140:2003). International Organization for Standardization. Geneva, 2003. 30. Notzon A, Helmuth R, Bauer J. Evaluation of an immunomagnetic separation–real-time PCR assay for the rapid detection of Salmonella in meat. J Food Protec 2006;69(12):2896–2901. 31. Widjojoatmodjo MN, Fluit AC, Torensma R, Keller BHI, Verhoef J. Evaluation of the Magnetic Immuno PCR Assay for rapid detection of Salmonella. Eur J Clinic Microbiol Infec Dis 1991;10(11):935-938. 32. O'Regan E, McCabe E, Burgess C, McGuinness S, Barry T, Duffy G, Whyte P, Fanning S. Development of a real-time multiplex PCR assay for the detection of multiple Salmonella serotypes in chicken samples. BMC Microbiol 2008;21(8):156. 33. DOF. Diario Oficial de la Federación. Norma Oficial Mexicana NOM-213-SSA1-2002. Productos cárnicos procesados. Especificaciones sanitarias. Métodos de prueba. México. 2002. 34. Bindun SC, Kim HT, Benjakul S. Rapid pathogen detection tools in seafood safety. Curr Opin Food Sci 2018;20:92-99. 639

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35. Dos Santos RC, Conceiçao D, Nunes-Moreira A, Ramos RJ, Goularte FL, Carvalhal JB, Guimaraes-Aleixo JA. Detection of Salmonella sp in chicken cuts using immunomagnetic separation. Braz J Microbiol 2008;39(1):173-177. 36. Mercanoglu TB & Griffiths MW. Combination of immunomagnetic separation with realtime PCR for rapid detection of Salmonella in milk, ground beef, and alfalfa sprouts. J Food Protec 2005;68(3):557-561.

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Table 2: Limit of detection of Salmonella in beef samples by rtPCR-IMS and NOM-114-SSA1-1994 methods rtPCR-IMS NOM-114-SSA1-1994 No. of replication 1-5 CFU 6-10 CFU 11-15 CFU 16-30 CFU 0 CFU 1-5 CFU 6-10 CFU 11-15 CFU 16-30 CFU 0 CFU 1 +/+/+ +/+/+ +/+/+ +/+/+ -/-/+/+/+ +/+/+ +/+/+ +/+/+ -/-/2

+/+/+

-/-/-

+/+/+

-/-/-

+/+/+

-/-/-

+/+/+

-/-/-

+/+/-*

+/+/+

-/-/-

+/+/+

-/-/-

+/+/+

-/-/-

+/+/ -

+/+/+

-/-/-

+/ -/ -

+/+/+

-/-/-

+/+/+

-/-/-

+ = Salmonella sp. was detected in the sample; – = Salmonella sp. was not detected in the sample. *The assay was negative, but confirmation by the reference method was positive from the original enrichment.

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Table 3: Comparison of rtPCR-IMS and NOM-114-SSA1-1994 methods for the detection of Salmonella in beef Sample Presumptive

PD 7

NA 41

ND 7

No 14

(%) NDe

False Specificity negative (%) (%) NDe NDe

98.2

1.8

Results* PA 5

Contaminated** 56

Sensitivity

100

False Accuracy positive (%) (%) NDe NDe 0

98.3

χ²

Kappa

NDe NDe 1.0 0.85 (P=0.317)

*PA (positive agreement): Detection of the pathogen by both methods. NA (negative agreement): No detection of the pathogen by both methods. PD (positive deviation): Detection of the pathogen by the alternative method, but not by the reference method. ND (negative deviation): Detection of the pathogen by the reference method, but not by the alternative method. NDe (not determined). **The results correspond to beef samples inoculated with a concentration of 1-5 CFU·25 ml-1 of Salmonella ATCC 35664.

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https://doi.org/10.22319/rmcp.v13i3.5776 Article

Prevalence of Mycobacterium avium subsp. paratuberculosis and associated risk factors in dairies under mechanical milking parlorsystems in Antioquia, Colombia

Nathalia M. Correa-Valencia a Nicolás F. Ramírez-Vásquez a Jorge A. Fernández-Silva a*

Universidad de Antioquia. Facultad de Ciencias Agrarias. Centauro, Escuela de Medicina Veterinaria, Carrera 75 # 65 - 87, Ciudadela Robledo, Medellín, Colombia.

*Corresponding author: jorge.fernandez@udea.edu.co

Abstract: The present study aimed to determine Mycobacterium avium subsp. paratuberculosis (MAP) prevalence according to environmental samples and to explore the herd-level risk factors associated to MAP infection in dairy herds under mechanical milking parlor and pasture grazing-based systems. The study herds (n= 94) were located in 60 districts from five municipalities in the Northern region of the province of Antioquia, Colombia. Herds were visited once in 2016 to collect two composite environmental samples and to complete a risk assessment questionnaire. MAP identification was carried out using a quantitative real-time PCR method based on the IS900 sequence. A herd was considered as MAP-positive if one or both of the environmental samples were found positive by the molecular technique. The information on risk factors was analyzed using a multivariable logistic regression model. The apparent herd-level prevalence found was 14.9 % (14/94; 95 % CI: 7.7-22.1), ranging from 0 to 33.3 % at municipality-level. Herds where other than Pure-Holstein breeds were predominant (i.e. Jersey, Jersey crossbreeds) were more likely to be MAP-qPCR positively infected than those on which where pure-Holstein cattle was predominant (OR=3.7; 95 % CI: 1.1-15.2). The present study reports MAP prevalence in dairy herds in the province of Antioquia (Colombia), and the association between MAP environmental positivity with the predominant breed in the herd. Key words: Environmental sampling, Holstein, Jersey, Johne’s disease.

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Received: 19/08/2020 Accepted: 20/04/2021

Introduction Mycobacterium avium subsp. paratuberculosis (MAP) is the causal agent of Johne´s disease (JD)(1), a slow-developing chronic granulomatous enterocolitis(2). MAP is resistant to both environmental and chemical changes and can survive in the environment for up to a year(3,4). The disease has a worldwide distribution and related production losses are also described(5,6). Each of the currently available MAP-diagnostic tests presents advantages and disadvantages, depending on the matrix and the different stages of the infection and subsequent illness(7,8). Molecular detection of MAP using polymerase chain reaction (PCR) on environmental samples has been proposed for herd screening(8,9), with equivalent results to those obtained by culture(10). The sensitivity (Se) of the PCR (in every format) can vary due to the irregular fecal shedding of the organisms, whereas its specificity (Sp) is close to 100 % in all stages of the disease(11,12). Quantitative real-timePCR (qPCR) method has been found to be sensitive (≈60 %) and specific (≈97 %)(13,14), also allowing MAP detection and quantification on complex matrixes (e.g. milk, fecal samples)(15-17). The analysis of environmental samples using qPCR is considered nowadays as a cost-saving and easy-to-use approach to diagnose JD at herd-level and to classify the herd as infected or not, since it does not require sample collection from individual animals, reducing the inherent stress of the sampling process(16,18). The JD herd-level prevalence worldwide seems to be >18 %, with reports >50 %(19-21). In Colombia, an apparent herd-level seroprevalence appeared to be >50 %, according to ELISA-based studies(22,23). Nevertheless, other studies have reported lower prevalences, such as 3.6(24) and 4.1 %(25). It seems that the range of possible results about prevalence estimation in the country is wide, and depends largely on the diagnostic test used and on the population of study. In Colombia, data on animal or herd-level risk factors on JD in dairy herds are still limited and dairy production systems are diverse. Hence herd-level management practices and risk factors, which differ between herds and dairy production systems(26,27), demands the local definition of risk factors for the disease considering the local diversity of dairy production systems. This diversity is often overseen and reports on prevalence and risk factors commonly ignores this fact, delivering data which cannot be compared with other studies and results, even in the same region or country. In a previous recent cross-sectional study, 292 dairy herds with on-paddock milking facilities —mobile units, located in 61 different districts from six municipalities in Northern Antioquia (Colombia) were sampled with one composite environmental sample

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containing material from at least six different sites (subsamples) of the concentration of adult cattle and/or high traffic areas in grazing paddocks. In this study, herds with a history of mixed farming of cattle with other ruminants had higher odds of being MAP infected than herds without this feature(25). Other very common dairy production system in Northern Antioquia and in whole Colombia, based its milk production on mechanical milking parlor and pasture grazingbased systems. This system is hypothesized to have different prevalence levels as well as different herd-level risk factors in comparison to dairy herds with on-paddock milking facilities —mobile units, which was previously studied(25). Therefore, this cross-sectional study aimed to determine MAP herd-level prevalence according to IS900-qPCR results on environmental samples, and to explore herd-level risk factors associated to MAP infection in dairy herds under mechanical milking parlor-systems of the Northern region of the Province of Antioquia (Colombia).

Material and methods Study design and herd selection

A cross-sectional probabilistic study design was carried out in the Northern dairy region of the Province of Antioquia (Colombia) in 2016. Selected herds were distributed into five municipalities (San Pedro de los Milagros, Entrerríos, Santa Rosa de Osos, Donmatías, and Belmira), known for their considerable volumes of dairy production. The study area is found between 1,090 and 2,979 m asl, and the environmental temperature ranges from 12 to 16 °C. According to the Caldas-Lang climate classification, Santa Rosa de Osos, San Pedro de Los Milagros, Entrerríos, and Donmatías municipalities are classified as cold-humid, and Belmira municipality as cold-very humid(26). The herd was considered as the unit of analysis. The study was performed under a stratified random sampling design, with restitution and without replacement. A municipality- and district-level proportional distribution was considered in the study design, according to the adult cattle population in each level (cows and bulls >2 yr of age)(27). The selection of districts to be sampled into each of the five municipalities was defined according to their specific weight into the corresponding municipality, considering the first districts with the largest adult bovine population, until the quantity of their census reached the 70 % of the population into each municipality. Sample size was defined according to a formula for prevalence estimation from a finite population(28), including a priori JD-prevalence proportion estimation of 0.118 (11.8 %) from a previous report in the study region(25) —achieved under similar methodological

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conditions and population, a 95% confidence level, and a maximum acceptable error rate of 7 %. The upper limit of such report was considered. The sampling frame referred to 7,794 herds registered on the foot-and-mouth disease vaccination records of the five municipalities of study(27). From the listed herds, 94 herds in 60 districts were randomly selected, according to the sampling strategy and inclusion criteria (i.e. having adult cattle, mechanical milking in parlor facilities and pasture grazing-based systems, geographic accessibility, no previous history or report of JD or MAP detection by any method, disposition of the owner to participate).

Collection of the sample and questionnaire

Environmental sampling was carried out as reported previously by the literature(18,29), with some modifications due to particularities in the productive systems and facilities in the study region (e.g. maternity, quarantine and/or nursing area not always defined) and to budget restraints. Each study herd was visited once to collect two composite environmental samples and to fulfill the questionnaire. The first composite sample contained subsamples from at least six different points of concentration of adult cattle/high traffic areas (e.g. paddock, areas nearby waterers and feeders, alleyways, gutters, milking parlor holding areas). Each subsample was collected considering those not being previously exposed to direct sunlight. The second composite sample contained manure from the milking parlor collected from the manure storage lagoon, after mixing its content for at least 5 min before sampling. The subsamples from the lagoon were obtained from six different places of the perimeter by submerging the sampling container up to 10 cm beneath the surface. Each environmental sample was collected using a clean disposable plastic glove. Subsamples of each of the two collection places were pooled and mixed by hand at the farm. Then, approximately 20 g of each of the two pool samples (separately) was placed into a container. Definitive samples were preserved in refrigeration at 4 °C during transport to the research laboratory, where they were homogenized by hand for 5 min and then stored at -20 °C until DNA extraction. The same one-page questionnaire used and reported previously(25) was applied herein (available upon request).

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Laboratory analysis

Laboratory analysis was carried out as previously described(25). Briefly, a commercial DNA preparation kit (ZR Fecal DNA Kit™, Zymo Research, CA, USA) was used for the DNA isolation, and the protocol included a bead-beating prior step (Disruptor Genie® 120V, Thomas Scientific, Swedesboro, NJ, USA). A NanoDrop 2000® spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used to measure the purity and yield of nucleic acids at two wavelengths (A260 and A280 nm). DNA integrity was confirmed using an only-agarose gel on a representative sub-sample of each extraction batch (10 %). DNA extraction efficiency was confirmed by PCR using bacterial constitutive genes to the same sub-samples mentioned above. The extracted DNA was preserved at -20 °C until IS900-qPCR analysis (Bactotype MAP PCR Kit®, Qiagen, Leipzig, Germany). The analyzed sample was considered as positive when a FAM/MAX channels signal was produced or strongly positive if a FAM-only signal was emitted, with a Ct≤40 and a sigmoid-pattern curve, according to MIQE guidelines(30).

Statistical analysis

Statistical analysis was carried out as previously described(25). The independent variable was the IS900-qPCR MAP-infection herd-status (positive/negative). All the information was analyzed using Stata 15.0 (StataCorp, 2017, College Station, Texas, USA) for the descriptive and regression modeling. Descriptive statistics were computed for all the variables of interest. A complex survey analysis was considered, according to a districtlevel cluster-effect and to the stratified design of the study. Univariable analysis was performed to assess unconditional associations between the outcome (MAP-herd status) and each independent predictor using simple logistic regression. Associations with a P≤0.20 were considered for inclusion in the multivariable logistic regression model. Evaluation of potential confounders was then performed by assessing the change in the β-coefficient of the variables of the adjusted model compared to the non-adjusted model. The variables to be explored as confounders (i.e. herd size, predominant breed) were considered according to literature. Biologically plausible interactions were studied between significant variables from the multivariable models, as well as the 2-way interactions between significant predictors with a significant unconditional association with the dependent variable. Confounders were only retained if a change greater than 15% was observed, regardless of the significance of the coefficient of the confounding variable in the model. Independent variables included in the final model were selected according to a backward-stepwise procedure (entry P=0.20; removal P=0.25). The final model is presented considering Odds Ratios (OR) with 95 % CIs. The model fit was assessed using the Hosmer-Lemeshow goodness-of-fit test(28).

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Results Two environmental samples were collected from each of 94 dairy herds under mechanical milking parlor and pasture grazing-based systems, located in 60 different districts in five municipalities of the Province of Antioquia (Colombia). None of the herds were housed or semi-housed. The 2.1 % (2/94) of the primarily eligible herds did not approve to be visited when they were first contacted by phone and, a 6 % of the phone numbers were out of service/not registered. The non-participating herds were considered as big dairy herds (>30 milking cows) for the Colombian context, and mainly located in the municipalities of Donmatías and Entrerríos, according to the Province´s census records. The apparent herd-level prevalence found was 14.9 % (14/94; 95 %CI: 7.7-22.1), ranging from 0 to 33.3 % at municipality-level (Table 1). Tables 2 and 3, show the herd-level characteristics and management practices considered as predictors for the MAP-risk factor approach. Table 1: Municipality-level prevalence of Mycobacterium avium subsp. paratuberculosis in the Province of Antioquia, Colombia (2016)

Belmira

Sample weight (%) 3.2

Herds of study 3

No. of positive herds (%) 0 (-)

Santa Rosa de Osos Entrerríos San Pedro de Los Milagros Donmatías Total

16.0 26.6 41.5 12.7 100

15 25 39 12 94

0 (-) 6 (24.0) 4 (10.3) 4 (33.3) 14 (14.9)

Municipality

Table 2: Herd-level characteristics in dairies from the Northern region in the Province of Antioquia, Colombia (2016)

17 77

DIS (%) 18.1 81.9

OR (95%CI) 1.4 (0.3-6.9)

71 9

81 13

86.2 13.8

3.2 (0.8-12.2)

0.096*

73 7

85 9

90.4 9.6

0.6 (0.1-3.1)

0.871

Predictor

CAT

PH (n)

NH (n)

Herd size

≤ 30 >30

2 12

15 65

Predominant breed

Pure Holstein Othera Yes No

10 4 12 2

Availability of veterinary assistance

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P-value 0.514

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Cattle purchasing practices (use of replacement calves/heifers in the last 10 yr) Foreign animals grazing in own pastures Own animals grazing in nonproper pastures Co-farming in the last 2 yr of the cattle with other MAP-susceptible ruminants (e.g. goats, sheep, buffaloes) Ruminants species co-farming with the cattle in the last 2 yr

Yes No

4 10

37 43

41 53

43.6 56.4

0.5 (0.1-1.6)

0.226

Yes No

1 13

1 79

2 92

2.1 97.9

6.1 (0.4-103.3)

0.262

Yes No

0 14

4 76

4 90

4.3 95.7

Yes No

3 11

17 63

20 74

21.3 87.7

1.0 (0.3-4.0)

0.938

Goats Sheep Sheep and goats Not applicable

2 0 1 11

8 8 1 63

10 8 2 74

10.6 8.5 2.2 78.7

5.7 (1.5-20.9) 0 1.4 (0.2-11.6)

0.099

Good farming practices-status (GFP; according to the ICA) Bovine tuberculosis status (tuberculosis-free according to the ICA) Producer´s knowledge about the disease

Yes No

9 5

33 47

42 52

44.7 55.3

2.6 (0.8-8.4)

0.111*

Yes No

11 3

57 23

68 26

72.3 27.7

1.5 (0.4-5.8)

0.572

4 Someb 10 Never heard about it before

0 70

14 80

14.9 85.1

0.4 (0.1-1.4)

JD-compatible symptoms´ history

Yesc Never

17 63

20 74

21.3 87.7

1.0 (0.3-4.0)

0.938

3 11

IN 0.773

CAT= categories; PH= positive herds; NH= negative herds; DIS= distribution; IN= inestimable. ICA= Instituto Colombiano Agropecuario. OR= Odds Ratio. CI= Confidence interval. a Includes: Pure Jersey and Jersey- crossbreeds. b Includes: Recognizes the name only, some basics, and fairly knowledgeable. c Includes: At present and/or in the last 2 yr. * Variables used for the multivariable analysis (P≤0.20).

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Table 3: Herd-level management practices in dairies from the Northern region in the Province of Antioquia, Colombia (2016) Predictor

CAT

PH (n)

Manure spreading as fertilizer in the pastures Typical time of separation of the newborn calf from their dam after birth (in days) Calves ≤ 6 mo old in direct contact with adult cattle

Yes No

14 0

NH (n) 77 3

≤1 ≥2

4 10

Yes No

From multiple cows From its own dam Unsalable milk Other sourcesa

Source of colostrum fed to calves Source of milk fed to unweaned calves

OR (95% CI) 0

Pvalue IN

22.3 77.4

0.68 (0.2-2.4)

0.544

12 82

12.8 87.2

1.2 (0.2-6.0)

0.854

0 80

0 94

100.0

30 50

35 59

37.2 67.8

1.08 (0.3-3.5)

0.899

DIS (%)

91 3

96.8 3.2

17 63

21 73

2 12

10 70

0 14

5 9

CAT= categories; PH= positive herds; NH= negative herds; OR= Odds Ratio. CI= confidence interval; IN= inestimable. a Includes: Milk without antibiotic (salable milk) and milk replacer. * Variables used for the multivariable analysis (P≤0.20).

The variables with an average of “0” between herds with MAP-positive/negative status were excluded from logistic regressions (i.e. own animals grazing in non-proper pastures, producer´s knowledge about the disease, manure spreading as fertilizer in the pastures, source of colostrum fed to calves). The variables herd size and cattle purchasing practices were included in the analysis as potential confounders, but the relative change in the coefficients was <15%, so they were not furtherly explored. The final multivariable logistic regression model for MAP-positive status in the dairies of study showed that herds where other than pure-Holstein breeds were predominant (i.e. Jersey, Jersey crossbreeds) were more likely to be MAP-qPCR positively infected using environmental sampling than those on which where pure-Holstein cattle was predominant (OR= 3.7; 95 % CI: 1.1-15.2). 650

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Discussion The present study was carried out to determine MAP herd-level prevalence according to environmental samples in 94 herds in five different municipalities. In addition, the study aimed to explore the herd-level risk factors associated with MAP infection in dairies under mechanical milking parlor and pasture grazing-based systems in the Northern Antioquia, Colombia. All herds found positive to MAP-qPCR were considered as infected, based on the fact that a MAP-elimination source leads to environmental fecal contamination, and therefore to the risk of ingestion by susceptible cattle(31). The apparent MAP herd-level prevalence of 14.9 % estimated from the present study (033.3 % at municipality-level), appears to be lower than those reported for cattle in North American, European, and Latin American and Caribbean cattle regardless of the dairy or beef production system(19-21). At a national scale, results from a recent study carried out in the same region in dairy herds with in-paddock milking facilities found an apparent prevalence of 4.1 %(25) based on molecular detection of MAP in environmental samples using a quantitative real-time PCR method based on the IS900 MAP-sequence. Differences in prevalence estimations of these two dissimilar dairy production systems (even being both under rotational grazing systems in most cases) and milking procedures could be due, hypothetically, to a higher metabolic load and consequent stress for individuals, which must walk at least twice a day to and from the milking parlor compared to those cows in dairy herds with in-paddock milking facilities, in which cows remain on pastures grazing most of the day and it is the milker who approaches them for milking. Cows that have to walk several times per day could have a compromised immunity that could favor the success of intestinal colonization by MAP and the formation of granulomatous lesions, and the consequent elimination of the agent to the environment(2,32), as well as the infection by other pathogens. In addition, the higher apparent prevalence could be due to a higher probability of detection of positive herds in the environment when two samples of each are collected, as followed in this case(16,33). These proposed arguments need further research approaches. A precise place known as appropriate to define a herd-level MAP-positive finding is the manure storage lagoon(34,35). Its considerations in the study region seem not to be a representative characteristic of the local/regional dairy systems in Colombia, taking into account that only 1 out of 4 dairies in the Province counts on this facility(27). Nevertheless, such dairies were a representative source of the positive findings, since 8 of the 14 herds found as MAP-qPCR positive were detected using the samples from the lagoons, whereas five of the positives were from the adult cattle concentration and/or high traffic area, and one from both sampling places. This may be an additional explanation when comparing the prevalence result of this work with that previously reported(25), as mentioned.

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It was found that herds where other than pure-Holstein breeds were predominant (namely, Jersey and Jersey crossbreeds) were more likely to be MAP-qPCR positively infected compared to those on which pure Holstein was the predominant one. An apparent higher susceptibility to JD for other than pure Holstein-breed cows have been reported in previous studies(36-38). Channel Island breeds (i.e. Jersey, Guernsey) have been suggested to be more susceptible to JD, based on evidence that the clinical disease has more frequently been reported in these breeds than any other breeds(37). However, the reason for this remains unknown. It has been hypothesized that this susceptibility may be related to increased exposure rather than increased susceptibility or may be confounded by some factors that play important role in the development of clinical disease such as lower culling rate in Channel Island breeds, just to give an example, but that information is not available from this study. Susceptibility to infection is suspected, then, to have a genetic component, and moderated values for heritability of infection and susceptibility have been reported and some approaches have been made so far on the topic. In this respect, the use of genetic selection as a control tool for JD is a relatively new approach. The phenotype (infection status) shown by some animals is a combination of genetically determined factors (susceptibility/resistance/tolerance genes) and environmental factors (exposure to MAP)(12). Susceptibility is evidenced by infection and progression to the clinical stage; resistance is characterized by the absence of infection or successfully fighting an infection and eliminating the pathogen from the body; and, tolerance is characterized by infection and a subclinical status. It is expected that the genetic variations of the host contribute to modify the response of the animal to the exposure to the agent and reaching one of the three triggering challenge scenarios(39). In the case of JD, the research objectives so far have been focused mainly on the assessment of susceptibility. This is given mainly for reasons of practicality, since resistance or tolerance to infection should be evaluated through challenge studies, in which the animals are exposed to equal doses of the pathogen and subsequently their response would be evaluated over a long period of time, being an expensive approach (due to the type of species), in the long term (due to the pathogenesis of the disease), and to reach acceptable conclusions the group of animals must be large(40). Nevertheless, stablishing a more MAP-resistant population through breeding programs should not be considered a complete solution to control the disease, but rather as a tool to prevent or reduce the incidence of infection(41). Milk and colostrum can carry MAP, because of fecal contamination of teats or by being excreted from the udder(7). From a local point of view, other authors have reported that the odds of being a seropositive herd were lower in those feeding calves with pooled colostrum from several cows compared to those to herds feeding calves with colostrum from their own dams(42). This previous study was carried out on 14 dairies, located in the municipalities of Belmira and San Pedro de los Milagros (Province of Antioquia), two of the five municipalities included in this study. Their results are in contrast to previous knowledge of the risk of being seropositive represented by the use of colostrum from 652

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multiple cows vs own dam´s(7). Results in this work reported that all the colostrum given to the calves is from their own dams (100 % of the herds). Other studies have related feeding antibiotic-contaminated or other discard-milk to young animals to be a significant risk factor for MAP spread(43). The results indicated that milk replacers and salable milk (without antibiotics) were the main sources used to feed unweaned calves. Nevertheless, according to the experience, to use discarded milk to feed the calves is still a common practice in the systems of study in Colombia, increasing the odds of within-herd transmission of, not only MAP, but other infectious agents.

Conclusions and implications The apparent prevalence found in the herds with in-paddock milking facilities of the present study was 14.9 %, varying from 0 to 33.3 % between the municipalities of study. In addition, it was found that dairies -where other than pure-Holstein breeds were predominant- were more likely to be MAP-qPCR positively infected using environmental sampling than those on which pure-Holstein was the predominant one (OR= 3.7; 95 %CI: 1.1-15.2), which does not mean that Holstein cattle are resistant to MAP infection. Nevertheless, this feature should be taken into account for JD´s control, particularly in dairies in Colombia under the same dairy production system than the ones considered herein.

Acknowledgments

Authors thank the Convocatoria Programática 2014-2015: Área de Ciencias de la Salud, Universidad de Antioquia (grant #8714-2015-2042), Medellín (Colombia). To Dr. Christine Gaunitz (Qiagen, Leipzig, Germany) for her accompaniment in the interpretation of the PCR results. To Gentech Biosciences (Colombia) for all technical and laboratory support.

Conflict of interest

The authors state that they have no conflicts of interest to declare.

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Literature cited: 1.

Sweeney RW. Transmission of paratuberculosis. Vet Clin North Am Food Anim Pract 1996;12(2):305-312.

Clarke CJ. The pathology and pathogenesis of paratuberculosis in ruminants and other species. J Comp Pathol 1997;116:217-261.

Whittington RJ, Marsh IB, Reddacliff LA. Survival of Mycobacterium avium subsp. paratuberculosis in dam water and sediment. Appl Environ Microbiol 2005;71(9):5304-5308.

Elliott GN, Hough RL, Avery LM, Maltin C, Campbell CD. Environmental risk factors in the incidence of Johne’s disease. Crit Rev Microbiol 2014;7828:1-20.

Kudahl AB, Østergaard S, Sørensen JT, Nielsen SS. A stochastic model simulating paratuberculosis in a dairy herd. Prev Vet Med 2007;78(2):97-117.

McAloon CG, Whyte P, More SJ, Green MJ, O’Grady L, Garcia A, Doherty ML. The effect of paratuberculosis on milk yield—A systematic review and metaanalysis. J Dairy Sci 2016;99:1449-1460.

Nielsen SS, Bjerre H, Toft N. Colostrum and milk as risk factors for infection with Mycobacterium avium subspecies paratuberculosis in dairy cattle. J Dairy Sci 2008;91:4610-4615.

Stevenson K. Diagnosis of Johne’s disease: current limitations and prospects. Cattle Pract 2010;18:104-109.

Collins MT, Gardner IA, Garry FB, Roussel AJ, Wells SJ. Consensus recommendations on diagnostic testing for the detection of paratuberculosis in cattle in the United States. J Am Vet Med Assoc 2006;229:1912-1919.

10. Douarre PE, Cashman W, Buckley J, Coffey A, O’Mahony JM. Isolation and detection of Mycobacterium avium subsp. paratuberculosis (MAP) from cattle in Ireland using both traditional culture and molecular based methods. Gut Pathog 2010;2:1-7. 11. Eamens GJ, Whittington RJ, Marsh IB, Turner MJ, Saunders V, Kemsley PD, Rayward D. Comparative sensitivity of various faecal culture methods and ELISA in dairy cattle herds with endemic Johne’s disease. Vet Microbiol 2000;77:357-367. 12. Sweeney RW, Collins MT, Koets AP, Mcguirk SM, Roussel AJ. Paratuberculosis (Johne’s disease) in cattle and other susceptible species. J Vet Intern Med 2012;26:1239-1250. 13. Aly S, Mangold B. Correlation between Herrold egg yolk medium culture and realtime quantitative polymerase chain reaction results for Mycobacterium avium subspecies. J Vet 2010;683:677-683. 654

Rev Mex Cienc Pecu 2022;13(3):643-657

14. Logar K, Kopinč R, Bandelj P, Starič J, Lapanje A, Ocepek M. Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: Comparison with faecal culture, milk real-time PCR and milk ELISA. BMC Vet Res 2012;8:1-10. 15. Alinovi CA, Ward MP, Lin TL, Moore GE, Wu CC. Real-time PCR, compared to liquid and solid culture media and ELISA, for the detection of Mycobacterium avium ssp. paratuberculosis. Vet Microbiol 2009;136:177-179. 16. Donat K, Kube J, Dressel J, Einax E, Pfeffer M, Failing K. Detection of Mycobacterium avium subspecies paratuberculosis in environmental samples by faecal culture and real-time PCR in relation to apparent within-herd prevalence as determined by individual faecal culture. Epidemiol Infect 2015;143:975-985. 17. Soumya MP, Pillai RM, Antony PX, Mukhopadhyay HK, Rao VN. Comparison of faecal culture and IS900 PCR assay for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faecal samples. Vet Res Commun 2009;33:781-791. 18. Wolf R, Barkema HW, De Buck J, OrseL K. Sampling location, herd size, and season influence Mycobacterium avium ssp. paratuberculosis environmental culture results. J Dairy Sci 2015;98:275-287. 19. Wells SJ, Wagner BA. Herd-level risk factors for infection with Mycobacterium paratuberculosis in US dairies and association between familiarity of the herd manager with the disease or prior diagnosis of the disease in that herd and use of preventive measures. J Am Vet Med Assoc 2000;216:1450-1457. 20. Nielsen SS, Toft N. A review of prevalences of paratuberculosis in farmed animals in Europe. Prev Vet Med 2009;88:1-14. 21. Fernández-Silva JA, Correa-Valencia NM, Ramírez N. Systematic review of the prevalence of paratuberculosis in cattle, sheep, and goats in Latin America and the Caribbean. Trop Anim Health Prod 2014;46:1321-1340. 22. Fernández-Silva JA, Abdulmawjood A, Akineden Ö, Bülte M. Serological and molecular detection of Mycobacterium avium subsp. paratuberculosis in cattle of dairy herds in Colombia. Trop Anim Health Prod 2011;43:1501-1507. 23. Benavides B, Arteaga AV, Montezuma CA. Estudio epidemiológico de paratuberculosis bovina en hatos lecheros del sur de Nariño, Colombia. Rev Med Vet 2016;31:57-66. 24. Correa-Valencia NM, Ramírez NF, Olivera M, Fernández-Silva JA. Milk yield and lactation stage are associated with positive results to ELISA for Mycobacterium avium subsp. paratuberculosis in dairy cows from Northern Antioquia, Colombia: A preliminary study. Trop Anim Health Prod 2016;48:1191-1200.

655

Rev Mex Cienc Pecu 2022;13(3):643-657

25. Correa-Valencia NM, Ramírez NF, Arango-Sabogal JC, Fecteau G, Fernández-Silva JA. Prevalence of Mycobacterium avium subsp. paratuberculosis infection in dairy herds in Northern Antioquia (Colombia) and associated risk factors using environmental sampling. Prev Vet Med 2019;170:104739. 26. Gobernación de Antioquia, Fichas municipales de Antioquia 2015-2016. Colombia. http://www.antioquia.gov.co/planeacion/fichas_municipales_web/index.html. 2016. 27. Fedegan (Federación Nacional de Ganaderos). Registro de vacunación, primer ciclo 2015 (official restricted access material). 28. Dohoo I, Martin W, Stryhn H. Veterinary epidemiologic research. VER Inc., 8 Berkeley Way, Charlottetown, Prince Edward Island, Canada. 2014. 29. Corbett CS, Naqvi SA, Bauman CA, De Buck J, Orsel K, Uehlinger F, Kelton DF, Barkema HW. Prevalence of Mycobacterium avium ssp. paratuberculosis infections in Canadian dairy herds. J Dairy Sci 2018;101(12):11218-11228. 30. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009;55:611-622. 31. Elliott GN, Hough RL, Avery LM, Maltin C, Campbell CD. Environmental risk factors in the incidence of Johnes disease. Crit Rev Microbiol 2015;41:488-507. 32. Nielsen SS, Enevoldsen C, Gröhn YT. The Mycobacterium avium subsp. paratuberculosis ELISA response by parity and stage of lactation. Prev Vet Med 2001;54:1-10. 33. Wolf R, Barkema HW, De Buck J, Slomp M, Flaig J, Haupstein D, Pickel C, Orsel K. High herd-level prevalence of Mycobacterium avium subspecies paratuberculosis in Western Canadian dairy farms, based on environmental sampling. J Dairy Sci 2014;97:6250-6259. 34. Pillars RB, Grooms DL, Kaneene JB. Longitudinal study of the distribution of Mycobacterium avium subsp. paratuberculosis in the environment of dairy herds in the Michigan Johne’s disease control demonstration herd project. Can Vet J 2009;50(10):1039-1046. 35. Pillars RB, Grooms DL, Woltanski JA, Blair E. Prevalence of Michigan dairy herds infected with Mycobacterium avium subspecies paratuberculosis as determined by environmental sampling. Prev Vet Med 2009;89:191-196. 36. NcNab WB, Meek AH, Duncan JR, Martin SW, Van Dreumel AA. An epidemiological study of paratuberculosis in dairy cattle in Ontario: study design and prevalence estimates. Can J Vet Res 1991;55(3):246-251.

656

Rev Mex Cienc Pecu 2022;13(3):643-657

37. Çetinkaya B, Erdogan H, Morgan K. Relationships between the presence of Johne’s disease and farm and management factors in dairy cattle in England. Prev Vet Med 1997;32:253-266. 38. Jakobsen MB, Alban L, Nielsen SS. A cross-sectional study of paratuberculosis in 1155 Danish dairy cows. Prev Vet Med 2000;46:15-27. 39. Gonda MG, Chang YM, Shook GE, Collins MT, Kirkpatrick BW. Genetic variation of Mycobacterium avium ssp. paratuberculosis infection in US Holsteins. J Dairy Sci 2006;89:1804-1812. 40. Kirkpatrick MS, Shook GE. Genetic susceptibility to paratuberculosis. Vet Clin Food Anim 2011;27:559-571. 41. Kirkpatrick BW. Genetics of host susceptibility to paratuberculosis. In: Behr MA, Collins DM, editor. Paratuberculosis: Organism, Disease, Control. CAB International, Oxfordshire, England, 2010:50–55. 42. Fernández-Silva JA, Ramírez N, Correa-Valencia NM. Factors associated with Mycobacterium avium subsp. paratuberculosis in dairy cows from Northern Antioquia, Colombia. Rev Colomb Cienc Pecu 2016;30:48-59. 43. Ridge SE, Baker IM, Hannah M. Effect of compliance with recommended calfrearing practices on control of bovine Johne’s disease RIDGE2005. Aust Vet J 2005;83:85-90.

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https://doi.org/10.22319/rmcp.v13i3.6015 Article

Effects of nutrition in the final third of gestation of beef cows on progeny development

John Lenon Klein a* Sander Martinho Adams a Amanda Farias de Moura b Daniele Borchate a Dari Celestino Alves Filho a Dieison Pansiera Antunes a Fabiana Moro Maidana c Gilmar dos Santos Cardoso a Ivan Luiz Brondani a Ricardo Gonçalves Gindri a

Federal University of Santa Maria (UFSM). Department of Animal Science, Santa Maria, RS, Brazil. b

State Technical School of Professional and Technological Education of Diamantino, MT, Brazil. c

Federal University of Rio Grande do Sul (UFRGS). Department of Animal Science, Porto Alegre, RS, Brazil.

Corresponding author: johnlenonklein@yahoo.com.br

Abstract: The restriction of nutrient intake by beef cows during pregnancy may influence the progeny postpartum growth potential, therefore, the objective of the present study was to evaluate the effects of nutritional restriction and adequate nutrition or overfeeding during the final third of gestation of the crossbred cows (Charolais x Nellore) kept in the Pampa 658

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biome, on the productive performance of the progeny up to 15 mo of age. Eighty-three (83) cows were divided into: control cows on natural pasture under nutritional restriction (RES); Supplementation to meet 100 % of requirements (REQ); Supplementation above requirements (HIGH). REQ and HIGH calves had higher body weight at birth compared to calves from RES cows (39.28, 39.13 vs 34.58 kg), without influences on postnatal performance. Females from REQ and HIGH cows presented better postnatal performance and consequently higher weight at 12 mo of age in compared to offspring RES cows (300.71 and 311.79 vs 259.47 kg). These female calves reached 60 % of early adult weight (358 and 345 vs 405 d) and had a higher percentage of breeding at twelve months of age (73.98 and 84.08 vs 34.08 %) than females from RES cows. Supplementing cows to meet 100% requirements, as well as overfeeding during the final third of gestation, improves offspring performance at twelve months of age, with males and females responding differently to maternal nutritional insults during this period. Key words: Calves, Fetal growth, Nutritional restriction, Myogenesis.

Received; 08/07/2021 Accepted: 18/01/2022

Introduction In the calf production system, beef cows are kept in lower quality forage systems and go through periods of lower nutrient supply during pregnancy. Food shortages are common in many regions of the world, resulting in cows under food restriction during pregnancy(1). This low nutrient intake is associated with future progeny development(2). In these situations of low nutrient supply, supplementation during pregnancy is an alternative to avoid negative effects of nutritional deficiency during the fetal period on progeny productivity(3). According to Wilson et al(4), dietary nutrient intake affects fetal development as well as future progeny performance. Working with crossbred cows (Angus x Hereford), Bohnert et al(5), who obtained heavier male calves at birth (40.8 vs 39.3 kg) and at weaning (191.0 vs 183.0 kg), when they were born from supplemented cows with distillery grains during final pregnancy. Providing two levels of total digestible nutrients in the final third of pregnancy (100 and 12 5% of requirements) for crossbred Angus x Simmental cows, Wilson et al(4) also observed an increase in male calves weight at birth (41 vs 44 kg) when the cow's energy intake increased. Better progeny development is the result of better skeletal muscle formation during pregnancy, as female nutrition during this period results in greater synthesis and growth of muscle fibers, as well as increased formation of

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intramuscular adipocytes, thus favoring the production of quality progeny meat(2). Some researchers(3) added that a decrease in the number of muscle fibers formed during pregnancy due to fetal programming reduces muscle mass and negatively influences performance of the animals. In addition to improving offspring meat production, cow nutrition during pregnancy also affects productive potential of females. Funston et al(6) observed a higher weight at weaning (232 vs 225 kg) and lower age at puberty (352 vs 366 d) for heifers from crossbred Red Angus x Simmental cows supplemented on winter native pasture and/or corn crop residues during late gestation. Studying the effects of maternal nutrition in the second and third trimester of pregnancy, it was(7) demonstrated that females from crossbred cows (¼ Angus, ¼ Hereford, ¼ Pinzgauer and ¼ RedPoll) submitted to 125 % of the nutritional requirements in the last trimester of pregnancy had a higher calving rate and conceived first during the breeding season than calves of cows receiving 100 and 75 % of the requirements. Based on the hypothesis that the greater intake of nutrients by the pregnant cow improve the productivity of the progeny after birth, the objective of the present study was to evaluate the effects of nutritional restriction and adequate nutrition or overfeeding during the final third of gestation of the crossbred cows (Charolais x Nellore) kept in the Pampa biome, on the productive performance of the progeny up to 12 mo of age.

Material and methods The protocols used in the experiment were approved by the Animal Use Ethics Committee (CEUA) of the Federal University of Santa Maria, under protocol No. 7920140617 approved on 12/07/2017.

Animals and study factors

The study was carried out at the Department of Animal Science of the Federal University of Santa Maria, Santa Maria - RS, Brazil. The experimental area is located at an average altitude of 95 m, with 29° 43' south latitude and 53° 42' west longitude. The climate of the region is "Cfa" (humid subtropical), according to Köppen's classification, with average annual rainfall between 1600 to 1900 mm, temperature of 18.8 °C, with a minimum average of 9.3 °C and average maximum of 24.7 ºC(8). A total of 83 beef cows were used, along with their progeny, from the rotary crossbreed between Charolais (CH) and Nelore (NE), which were previously distributed according to age (4 to 12 yr) and percentage of Nelore blood. After diagnosis of pregnancy to

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determine the time of pregnancy, they were divided into three treatments according to nutritional level in the last third of pregnancy: 28 control cows on natural pasture under nutritional restriction (RES); 28 cows on natural pasture supplemented to meet 100 % energy and protein requirements (REQ); 27 cows on natural pasture with supplementation above energy and protein requeriments (HIGH). Diets were calculated by nutritional requirements in the last third of gestation of 475 kg body weight cows, consuming 2.1 % of the live weight of forage dry matter, as recommended by the National Research Council – NRC(9). The concentrate supplement (Table 1) was considered an additive to forage intake, providing 0.28 and 0.98 % of the body weight of supplementation for the treatments REQ and HIGH, respectively. This nutritional plan allowed daily weight gains of -0.103; 0.025 and 0.207 kg d-1 during the last third of pregnancy for the RES, REQ and HIGH treatments, respectively, resulting in better body condition of these cows at birth (2.81, 2.92 and 2.99 points, in the same order), following the scale of 1 to 5 points, where 1 is classified as very thin and 5 is very fat. Cows that calved males showed lower daily weight gain during the last third of gestation (-0.02 vs 0.11 kg d-1) and calved with lower body condition than those that carried females (2.89 vs 2.93 points). The animals were kept in four natural pasture paddocks, with areas of 20, 21, 41, and 47 ha, and a mineral salt supplement (ProduBeef 60P® - with minimum values of 170, 60 and 130 g kg-1 of calcium, phosphorus and sodium, respectively) was available in each paddock, with free access. The native pasture was composed mostly of the summer species, which is of African origin, called capim-annoni (Eragrostis plana Ness), and by other warm season grass species, Paspalum notatum, Axonupus affinis, and Desmodium incanum. The cows for each treatment were managed in groups. The paddocks were rotated between treatments every 28 d to reduce the experimental error. The mass and forage supply in the paddocks were 4144.72 kg dry matter ha-1 and 11.22 kg dry matter per 100 kg of live animal weight. The average stocking rate was equivalent to 275.21 kg ha-1 body weight. Supplementation was provided daily at 1100 h, beginning on August 15, 2017, and lasted until the date of the cows calving. The supplementation period was 95 and 92 d for REQ and HIGH cows, respectively.

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Table 1: Composition of the concentrated fraction and nutrient consumption by the cows in the last third of pregnancy Treatments1 Diet fraction RES REQ HIGH Ingredients of the concentrated fraction (% dry matter) Milled corn 81.78 Soybean meal 17.22 Urea 1.00 Bromatological composition of the concentrated fraction (% dry matter) Crude protein 18.00 Total digestible nutrients 85.00 Dry matter and nutrient intake for pregnant cows weighing 475 kg Native forage2 , kg d-1 9.98 9.98 Concentrated supplement, kg d-1 1.32 -1 Total digestible nutrients, kg d 4.69 5.81 -1 Crude protein, kg d 0.45 0.70 Total digestible nutrients3 % dry matter 88.50 109.60 3 Crude protein , % dry matter 60.00 93.40

91.74 7.26 1.00 15.00 85.00 9.98 4.69 8.60 1.15 162.30 153.30

Composition of native forage: Crude protein 4.5%; Total digestible nutrients 47.0%. RES= cows on natural pasture under nutritional restriction; REQ= cows supplemented to meet 100% requirements; HIGH= cows supplemented above requirements. 2 Forage consumption estimated at 2.1% of body weight(9). 3 Consumption in relation to daily requirements of total digestible nutrients (5.30 kg) and crude protein (0.75 kg) described in NRC(9). 1

After calving, the same management conditions were maintained for all treatments. Calving took place between October 25 and December 15, 2017. At the day of birth, the set of mothers and calves were taken to the management center for weighing and care of the offspring, and then kept on Tifton-85 (Cynodon ssp) pasture for 3 wk. After this period, the animals were relocated in natural pastures until the time of conventional offspring weaning, where they were presented on average 165 ± 17 days of age.

Growth of calves

After weaning, the progeny remained for 30 d in tifton-85 pasture receiving 1 % of live weight of concentrated supplementation. The calf supplement consisted of 57.2 % ground corn, 38.14 % soybean meal and 4.66 % mineral salt (ProduBeef 60P®). The combination of these ingredients showed 22.20 % crude protein and 83.54 % total digestible nutrients. Subsequently, they were kept in Black Oat + Ryegrass (Avena strigosa + Lolium multiflorum) intercropping pasture, up to 12 mo of age. The pasture showed 17.84, 59.97 and 57.36 % of crude protein, total digestible nutrients and neutral detergent fiber, respectively, samples obtained by cutting the forage. All chemical analyzes of the concentrate and forage were performed at the Laboratory of Bromatological analyzes at the

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Federal University of Santa Maria. The offspring remained in pasture between June 5, 2018 and November 6, 2018, totaling 155 d. Forage mass and supply in the period were 1087.56 kg ha-1 of dry matter and 7.61 kg of dry matter per 100 kg of live animal weight. The average stocking rate in this period was 894.83 kg ha-1 body weight. Because the calves were kept in a single group, it was not possible to quantify the animals' forage intake, however, the dry matter intake estimate for this growing category is close to 2.40 % of body weight(9).

Progeny performance measures Males and female’s performance was evaluated separately by weighing at birth, at weaning and at 265 and 335 d of age (± 17 d). The average daily gain of the animals was calculated by dividing the total weight gain by the number of days between the weights, and the offspring weights were later adjusted to 205, 270 and 365 d of age, according to the following equations: PAJUST205 = (ADG birth at Weaning) x 205 + Weight at birth; PAJUST270 = (ADG Weaning at 270 d) x 65 + PAJUST205; PAJUST365 = (ADG Weaning at 365 d) x 160 + PAJUST205, where ADG = average daily gain. To complement the performance evaluations, body measurements of the males were taken, together with the weightings. The body compacity of the calves was calculated following the methodology described by Cattelam et al(10), through the quotient of weight by body length, being subsequently adjusted for the 205 and 365 d. Female reproductive aptitude was evaluated based on the target reproductive weight of 285 kg, which represents about 60 % of the adult weight (AW), as recommended by the NRC(9).

Experimental design and statistical analysis

The experimental design used for both males (n= 42) and females (n= 41) was completely randomized with three treatments and varied number of repetitions. The normality of the residues was analyzed by the Shapiro-Wilk test. Transformations and outliers were eliminated when necessary. Subsequently, the data were subjected to analysis of variance by the F test through the PROC GLM procedure, and when significance was found, the means were compared by Tukey test at a 5% significance level (P<0.05). Statistical analyzes were performed using the SAS® Studio University Edition statistical (11) package using the following mathematical model:

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Yijk = µ + Ni + Ij+ Zk+εijk Where, γijk: dependent variables; μ: mean of all observations; Ni: effect of the i-th prenatal nutritional level; Ij: effect of the covariate cow age; Zk: effect of co-variable percentage Nellore breed on cows; εijk: effect of residual random error (error b).

Results Male performance

The performance of the offspring of cows subjected to different nutritional levels in the last third of gestation is shown in Table 2. REQ and HIGH cows produced heavier male calves at birth in relation to the RES cows (39.28 and 39.13 vs 34.58 kg, respectively). Performance of male calves during the lactation phase was not influenced by the cow's nutritional level in the last third of pregnancy (P= 0.1375), with weight adjusted at 205 days of age respectively of 199.75, 220.76 and 215.93 kg body weight for the treatments RES, REQ and HIGH. Weight at 205 d of age is a result of the similarity (P=0.2471) of male daily weight gain during lactation, with an average value of 0.85 kg d-1. Similar behavior was observed for post-weaning performance of males, which was not influenced by maternal nutrition during the last trimester of pregnancy (Table 2). Males had an average weight of 229.18 and 302.12 kg of body weight respectively at 270 d (P = 0.1305) at 365 d of age (P= 0.3603). Male body weight at 365 d of age is a result of the similarity in performance of these animals during the post-weaning period (P=0.8582). The average daily weight gain of males in this period was equivalent to 0.56 kg d-1 body weight, performance that can be considered lower than expected for animals in the initial phase of growth in cultivated pasture. Performance during the postnatal period up to twelve months of age was not influenced by the cow's nutritional level in the last third of gestation (P=0.5829), where male calves had a daily gain equivalent to 0.70 kg d-1 of body weight from birth to twelve months of age. Likewise, there was no effect of the nutritional level of the cow during pregnancy (P>0.05) on the body length of the calves. However, body compacity at birth was higher in REQ and HIGH calves compared to RES (0.59 and 0.59 vs 0.54 kg cm-1, respectively), showing greater potential for muscle production per centimeter of carcass.

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Table 2: Effects of the nutritional level of beef cows during final third of gestation on the postpartum performance of male calves Treatments1 (n) PPerformance RES (14) REQ (16) HIGH (12) value Pre-weaning performance at seven months age Birth weight, kg 34.58b ± 1.32 39.28a ± 1.24 2 PAJUST205 kg 199.75 ± 7.71 220.76 ± 7.25 -1 Birth-Weaning, kg d 0.80 ± 0.03 0.88 ± 0.03 Body length, cm 63.58 ± 1.19 64.56 ± 1.15 Body compacity, kg cm-1 0.54b ± 0.13 0.59a ± 0.13 Post-weaning performance at nine months of age PAJUST2702 kg 215.97 ± 8.28 238.67 ± 7.50 Weaning-265 d, kg d-1 0.28 ± 0.04 0.28 ± 0.03 Body length, cm 108.18 ± 1.69 111.50 ± 1.62 Body compacity, kg cm-1 1.77 ± 0.05 1.93 ± 0.05 Post-weaning performance at twelve months of age PAJUST3652 , kg 289.15 ± 10.96 308.52 ± 10.30 Weaning-335 d, kg d-1 0.56 ± 0.03 0.56 ± 0.03 -1 Birth-335 d kg d 0.68 ± 0.02 0.71 ± 0.02 Body length, cm 125.78 ± 1.97 128.12 ± 1.89 Body compacity, kg cm-1 2.25 ± 0.08 2.38 ± 0.07

39.13a ± 1.43 215.93 ± 8.35 0.86 ± 0.03 64.51 ± 1.37 0.59a ± 0.15

0.0248 0.1375 0.2471 0.8130 0.0212

232.91 ± 8.66 0.26 ± 0.04 110.94 ± 1.87 1.94 ± 0.05

0.1305 0.9498 0.3447 0.0813

308.72 ± 11.87 0.58 ± 0.04 0.72 ± 0.03 125.12 ± 2.18 2.45 ± 0.08

0.3603 0.8582 0.5829 0.5388 0.2438

Values are predicted means ± standard errors mean (SEM). abc Distinct letters on the same line differ by Tukey test (P<0.05). 1 RES = cows on natural pasture under nutritional restriction; REQ = cows supplemented to meet 100% requirements; HIGH = cows supplemented above requirements. 2 Age adjusted weight of calves.

Female performance

Unlike male performance, female birth weight was not influenced (P= 0.3730) by cow's nutritional level in the last third of gestation (Table 3), with average value equivalent to 34.08 kg body weight. However, the female calves initial development during the lactation phase resulted in higher body weight adjusted at 205 d of age for the progeny of REQ and HIGH cows compared to those born from RES (211.58 and 210.95 vs 187.76 kg, respectively). This result is related to the higher average daily gain of these females during lactation (P=0.0336) in relation to the female calves from RES cows (0.86 and 0.86 vs 0.75 kg d-1, respectively). In addition to better performance during the lactation phase, female calves of REQ and HIGH cows presented higher post-weaning performance than those born to RES cows, resulting in higher body weights at both 270 (P=0.0105) and 365 d of age (P=0.0021). The female calves presented body weights of 200.93, 224.89 and 233.15 kg at 9 mo and 259.47, 300.71 and 311.79 kg at 12 mo of age respectively for the RES, REQ and HIGH treatments. 665

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The superiority in body weight of females can be explained by the daily body weight gains in the post-weaning period offspring (Table 3). In this period, offspring of HIGH cows showed higher gains than those born from RES cows at both 265 (0.34 vs 0.17 kg d-1) and 335 d of age (0.45 vs 0.63 kg d-1). Female calves from REQ cows showed similar performance to the other groups studied during the growth phase, with daily gains of 0.21 and 0.55 kg of body weight at 270 and 365 d of age, respectively. The performance from birth to 12 mo of females was higher in female calves from REQ and HIGH cows (P=0.0019) in relation to those born from RES (0.70 and 0.74 vs 0.60 kg d-1, respectively). Cow nutrition during the last third of gestation improved female calves' reproductive aptitude at twelve months of age (Table 3). Female calves from REQ and HIGH cows presented higher percentage of adult weight at twelve months of age (P=0.0021) when compared to those born from RES (63.19 and 65.64 vs 54.65 %, respectively), a result that is related to the higher postnatal performance of these females and consequently higher body weight at that age. Table 3: Effects of the nutritional level of beef cows during final third of gestation on the postpartum performance of female calves Treatments1 (n) PPerformance RES (14) REQ (12) HIGH (15) value Post weaning performance Birth weight, kg PAJUST2052 , kg Birth-weaning, kg d-1 Post weaning performance PAJUST2702, kg Weaning-265d, kg d-1 PAJUST3652 , kg Weaning-335d, kg d-1 Birth-335d, kg d-1 Adult weight-335 d, %3 1

32.79 ± 1.25 187.76b ± 6.46 0.75b ± 0.02

35.45 ± 1.38 211.58a ± 7.28 0.86a ± 0.03

34.01 ± 1.24 210.95a ± 7.08 0.86a ± 0.03

0.3730 0.0266 0.0336

200.93b ± 7.08 0.17b ± 0.03 259.47b ± 9.60 0.45b ± 0.03 0.60b ± 0.02 54.65b ± 2.02

224.89a ± 7.71 0.21ab ± 0.03 300.71a ± 10.83 0.55ab ± 0.03 0.70a ± 0.02 63.19a ± 2.28

233.15a ± 7.48 0.34a ± 0.03 311.79a ± 10.51 0.63a ± 0.03 0.74a ± 0.02 65.64a ± 2.21

0.0105 0.0103 0.0021 0.0043 0.0019 0.0021

Values are predicted means ± standard errors mean (SEM). RES = cows on natural pasture under nutritional restriction; REQ = cows supplemented to meet 100% requirements; HIGH = cows supplemented above requirements. 2 Age adjusted weight of calves. 3 Females to reach 60% of adult weight (285 kg body weight). abc Distinct on the same line differ by Tukey test (P<0.05).

Discussion Among the factors that may alter the uterine environment during pregnancy, it can be highlight the effects of maternal nutrition, where both malnutrition and overnutrition can modify calf metabolism and physiology after birth(12), with reflections on the potential for

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progeny production. In our study, the effects of different nutritional levels of beef cows during the last third of gestation were different between male and female progeny, resulting in a separate discussion for the categories.

Male performance

Meeting the requirements or supplementing cows above the maintenance requirements for protein and energy in the final third of gestation provided higher nutrient intake by pregnant cows, an aspect that may have improved the nutritional supply for the fetus and consequently the formation of muscle tissue. Improvement in maternal nutrition resulted in greater birth weight of REQ and HIGH males. Du et al(13) state that at this stage of gestation there is the completion of muscle hyperplasia and hypertrophy of preformed fibers, as well as the beginning of adipocyte formation in the fetal skeletal muscle. According to Funston et al(6), the development of fetal skeletal muscle has low nutritional priority during pregnancy, a factor that impairs the formation of muscle tissue in situations of low nutrient intake. Corroborating our study, the improvement in body weight at birth of male calves born to cows with higher nutritional status in pregnancy has been reported by several authors who have studied the effects of maternal nutrition on male progeny performance(5,14,15), which justify this result to the effects of fetal programming. The greater body compactness of REQ and HIGH calves compared to RES (Table 3) may be related to changes in muscle fiber hyperplasia and/or hypertrophy that occur in the final third of pregnancy and were favored by better nutrition of the pregnant cow, as explained earlier(12). Maresca et al(16) calculated the body mass index through the quotient between calf weight at birth by the square root of body length, and obtained a higher muscle index in calves born from Aberdeen Angus cows fed with a higher level of protein in the diet during late gestation. Body compacity represents greater deposition of muscle per unit of body measurement, an aspect that can increase the edible portion of the carcass. The effects of maternal nutrition during pregnancy were not evidenced after male calves' birth, with similarity (P>0.05) in the adjusted weights for 205, 270 and 365 d of males. This result is related to the daily weight gain of male calves after birth. However, offspring of REQ and HIGH cows were about 10.51 and 8.10 % higher at 105 d of age than males born from RES cows. Higher weaning weights of calves born to cows supplemented with distillery grains in the final third of gestation were obtained by Larson et al(17) and Bohnert et al(5), who worked with Red Angus x Simmental and Angus x Hereford cows, respectively. Marques et al(18) observed greater weight at weaning of male calves born to Angus x Hereford cows who gained body condition score in the second or third trimester of pregnancy, compared to those fed to gain body condition early in pregnancy or who were supplemented to 667

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maintain adequate and inadequate body condition score throughout pregnancy, demonstrating that nutritional stimulation during the stages of myogenesis and adipogenesis results in a male progeny with greater productive potential. The post-weaning performance of males was similar between the nutritional levels of cows during the last third of pregnancy (Table 2). A meta-analytical study(19) demonstrated that the weight gain of the cow in pregnancy improves the performance of the progeny, and complements that these effects of the best maternal nutrition in the pregnancy are most evident in our initial two months of life. In general, the literature has shown that fetal programming results are more clearly expressed in the first months of progeny life, especially when related to male performance(5,14,17). These authors found no differences in weight after weaning, however, comment that the superior body weight observed in the offspring of cows with higher nutritional level during pregnancy, persists or even increases during the growth phases of these animals. Even without obtaining the forage intake of the calves, the stress caused by weaning and subsequent adaptation to the Black Oat + Ryegrass pasture may have limited the productive potential of the progeny REQ and HIGH during the initial growth period up to 270 d of age, where calves from RES cows had their performance favored in this period (Table 2). Webb et al(20) state that nutritional restriction during gestation ends up producing an “economic” phenotype, which according to Greenwood et al(21), have a greater capacity for metabolic adaptation to less favorable environments during the postnatal life, may show compensatory gains in challenging environments after birth(22). These structural and functional changes in the organs serve to allow a rapid adaptation of the developing fetus to the pressure of uterine environmental selection(23), preparing the organism to survive in similar environments in adult life.

Female performance

The effects of fetal programming were also observed in the female calves, however, with distinct effects to those observed in males. Unlike that observed in male progeny, female birth weight was similar between the nutritional levels of cows in the last third of gestation (Table 3). Similar results were observed(6,17)),who studied the effects of fetal programming on the performance of contemporary males and females Red Angus x Simmental. These authors reported different behaviors for the development of males and females, where the males showed differences in birth weight, but similar performances in the later stages of growing. When evaluating the females, Funston et al(6) observed similar weights at birth, however, in the postnatal performance, the daughters of cows that received protein supplementation during late gestation presented greater weight at weaning, and this weight difference was maintained until the beginning of reproduction. 668

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In general, females have lower birth weight compared to males, which may represent lower nutritional demand for cow during pregnancy and consequently lower effects of fetal programming on female calves birth weight, as observed in the present study, where offspring of REQ and HIGH cows were only 5.91 % heavier at birth compared to those born from RES cows. Females were on average 10.5 0 % lighter at birth than males, which may justify the hypothesis that females have lower nutritional requirements in the fetal period. This theory is even more evident when is observed that cows that calved females had higher average daily gain during the supplementation period (0.11 vs -0.02 kg d-1) and calved with a better body condition score (2.93 vs 2.89 points) compared to those cows which calved male, that is, the excess nutrients were stored as body reserves due to the lower nutritional demands for the fetus. The effects of fetal programming were more clearly expressed on the postnatal performance of females (Table 3), with calves REQ and HIGH on average being 23.50 kg heavier at 7 mo of age when compared to offspring RES. Higher weaning weights of females born to cows supplemented in the last trimester of pregnancy were also observed(24), with crossbred Red Angus cows, and by Funston et al(6), being this group of females respectively 7.5 and 8.0 kg heavier in relation to the female calves of cows with lower nutritional level in the same period. Cushman et al(7) and Shoup et al(25), when studying crossbred cows (¼ Angus, ¼ Hereford, ¼ Pinzgauer and ¼ RedPoll) and Angus x Simmental, respectively, did not observe differences in performance during lactation until weaning of female calves of cows that received or not supplementation during pregnancy, justifying this result by the similarity in weight gains in the lactation phase. Growth superiority of female of REQ and HIGH cows persisted and increased after the lactation period, and at twelve months of age, these females had respectively 15.89 and 20.16 % more body weight compared to those born from RES. According to Greenwood et al(21), feeding restriction during fetal development may limit progeny growth capacity during the postnatal period, with slower offspring growth lasting until 30 mo of age. Delayed development of females during adulthood can result in losses and decline in their productive potential. As a consequence of the higher body development of the REQ and HIGH females during the growing, they had a higher percentage of adult weight at twelve months of age (Table 3). Shoup et al(25) did not observe differences in the percentage of adult weight presented by offspring of non-supplemented cows or who received low or high amounts of gestational concentrate, with an average of 51 % of adult weight. Higher percentage of adult mating weight may reflect better reproductive rates, since the female needs to gain less weight during pregnancy until the first delivery. In addition to improving the body structure of females, some authors(7,24) observed higher calving rate in the first 21 d of the birth season in female calves of cows with higher nutrient intake in the last trimester of gestation, indicating that these females conceive at the beginning of breeding, which may result in greater productive longevity of female calves of cows with higher nutritional level during pregnancy(6).

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In this sense, it is evident the influence exerted by the best maternal nutrition during pregnancy on the productive potential of the progeny. However, the provision of nutritional requirements or the overfeeding of cows in the final third of gestation did not change the performance of the offspring evaluated until twelve months of age. It is also noteworthy that the effects of fetal programming on the postnatal performance of males and females may be different during the growth phases.

Conclusions and implications In summary, the results of this study demonstrate that both supplementation above nutritional requirements, as well as meeting 100 % of the requirements for beef cows during the final third of gestation improves the birth weight of males, but mainly the postnatal performance of females up to 12 mo of age, resulting in higher adult weight at 365 d of age for these heifers. Therefore, the male and female progeny respond differently to intrauterine changes caused by maternal nutrition during the last third of pregnancy, and further investigation is needed. Furthermore, overfeeding cows in the final third of gestation does not improve the performance of offspring until 12 mo of age for the parameters evaluated.

Acknowledgments

To the support team of professors and undergraduate and postgraduate students of the Agronomy, Veterinary Medicine and Animal Science courses for their assistance in the operational activities that enabled the execution of this work.

Conflict of interest declaration

The authors declare they have no conflicts of interest with the work presented in this review article.

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Literature cited: 1. Gutiérrez V, Espasandín AC, Machado P, Bielli A, Genovese P, Carriquiry M. Effects of calf early nutrition on muscle fiber characteristics and gene expression. Livest Sci 2014;(167):4018-416. 2. Du M, Huang Y, Das AK, Duarte MS, Dodson MV, Zhu MJ. Manipulating mesenchymal progenitor cell differentiation to optimize performance and carcass value of beef cattle. J Anim Sci 2013;(91):1419-1427. 3. Du M, Wang B, Fu X, Yang Q, Zhu MJ. Fetal programming in meat production. Meat Sci 2015;(109):40-47. 4. Wilson TB, Faulkner DB, Shike DW. Influence of prepartum dietary on beef cow performance and calf growth and carcass characteristics. Livest Sci 2016;(184):2127. 5. Bohnert DW, Stalker LA, Nyman A, Falck SJ, Cooke RF. Late gestation suplementation of beff cows differing in body condition score: Effects on cow and calf performance. J Anim Sci 2013;(91):5485-5491. 6. Funston RN, Martin JL, Adams DC, Larson DM. Winter grazing system and supplementation of beef cows during late gestation influence heifer progeny. J Anim Sci 2010;(88):4094-4101. 7. Cushman RA, McNeel AK, Freetly HC. The impact of cow nutrient status during the second and third trimesters on age at puberty, antral follicle count, and fertility of daughters. Livest Sci 2014;(162):252-258. 8. Alvares, CA, Stape, JL, Sentelhas, PC, Gonçalves, JLM, Sparovek, G. KÖPPEN’S climate classification map for Brazil. Meteorologi Schezeit Schrift 2014;(22):211728. 9. NRC. National Research Council. Nutrient requirements of beef cattle. Oklahoma State University: Division of Agricultural Sciences and Natural Resources, Oklahoma, USA: National Academy Press; 1998. 10. Cattelam J, Argenta FM, Alves FDC, Brondani IL, Pacheco PS, Pacheco RF, et al. Characteristics of the carcass and quality of meat of male and female calves with different high-grain diets in confinement. Semina: Ciências Agrárias 2018;(39): 667681. 11. SAS. Statistical Analysis System. User’s guide version 3.5 SAS® Studio University Edition. Cary, North Carolina; USA: SAS Inst. Inc. 2016. 12. Tsuneda PP, Hatamoto-Zervoudadakls LK, Duarte Júnior MF, Silva LES, Delbem RA, Motheo TF. Efeitos da nutrição materna sobre o desenvolvimento e performance reprodutiva da prole de ruminantes. Investigação 2017;(16):56-61.

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13. Du M, Tong J, Zhao J, Underwood KR, Zhu MJ, Ford SP, Nathanielsz PW. Fetal programming of skeletal muscle development in ruminant animals. J Anim Sci 2010;(88):51-60. 14. Underwood KR, Tong JF, Price PL, Roberts AJ, Grings EE, Hess BW et al. Nutrition during mid to late gestation affects growth, adipose tissue deposition, and tenderness in cross-bred beef steers. Meat Sci 2010;(86):588-593. 15. LeMaster CT, Taylor RK, Ricks RE, Long NM. The effects of late gestation maternal nutrient restriction whit or without protein supplementation on endocrine regulation of newborn and postnatal beef calves. Theriogenology 2017;(87):64-71. 16. Maresca S, Lopez Valiente S, Rodriguez AM, Long NM, Pavan E, Quintans G. Effect of protein restriction of bovine dams during late gestation on offspring postnatal growth, glucose-insulin metabolism and IGF-1 concentration. Livest Sci 2018;(212):120-126. 17. Larson DM, Martin JL, Adams DC, Funston RN. Winter grazing system and supplementation during late gestation influence performance of beef cows and steer progeny. J Anim Sci 2009;(87):1147-1155. 18. Marques RS, Cooke RF, Rodrigues MC, Moriel P, Bohnert DW. Impacts of cow body condition score during gestation on weaning performance of the offspring. Livest Sci 2016;(191):174-178. 19. Klein JL, Machado DS, Adams SM, Pötter L, Alves-Filho DC, Brondani IL. Beef cow weight variations during gestation and offspring performance: a meta-analysis. Semina: Ciências Agrárias 2010;(42):3961-3976. 20. Webb MJ, Block JJ, Funston RN, Underwood KR, Legako JF, Harty AA et al. Influence of maternal protein restriction in primiparous heifers during mid and/or late-gestation on meat quality and fatty acid profile of progeny. Meat Sci 2019;(152):31-37. 21. Greenwood PL, Thompsom AN, Ford SP. Posnatal consequences of the maternal environment and growth during prenatal life for productivity of ruminants. In: greenwood PL et al. editors. Managing the prenatal environment to enhance livestock productivity. Springer Dordrecht Heldeliberg London New York, 2010:336. 22. Ramírez M, Testa LM, Valiente SL, La Torre E, Long NM, Rodriguez AM et al. Maternal energy status during late gestation: Effects on growth performance, carcass characteristics and meat quality of steers progeny. Meat Sci 2020;(164):1-7. 23. Reynolds LP, Borowicz PP, Caton JS, Crouse MS, Dahlen CR, Ward AK. Developmental programming of fetal growth and development. Vet Clin Food Anim 2019;(35):229-247.

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24. Martin JL, Vonnahme KA, Adams DC, Lardy GP, Funston RN. Effects of dam nutrition on growth and reproductive performance of heifer calves. J Anim Sci 2007;(85):841-847. 25. Shoup LM, Ireland FA, Shike DW. Effects of dam prepartum supplement level on performance and reproduction of heifer progeny. Italian J Anim Sci 2017;(16):7581.

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https://doi.org/10.22319/rmcp.v13i3.6039 Article

Establishment of tropical forage grasses in the Cerrado biome

Antonio Leandro Chaves Gurgel a* Gelson dos Santos Difante a Carolina Marques Costa a João Virgínio Emerenciano Neto b Gustavo Henrique Tonhão a Luís Carlos Vinhas Ítavo a Alexandre Menezes Dias a Iuri Mesquita Moraes Vilela a Vivian Garcia de Oliveira a Pâmella Cristina da Silva Lima a Andrey William Alce Miyake a

Universidade Federal de Mato Grosso do Sul, Faculdade de Medicina Veterinária e Zootecnia. Avenida Senador Filinto Müler, 2443 - Pioneiros, 79074-460, Campo Grande, Mato Grosso do Sul, Brasil. b

Universidade Federal do Rio Grande do Norte, Unidade Acadêmica Especializada em Ciências Agrárias. Macaíba, Rio Grande do Norte, Brasil.

* Corresponding author: antonioleandro09@gmail.com

Abstract: This study was carried out to evaluate the time for the establishment of tropical forage grasses in the “Cerrado” biome, based on morphogenetic and structural traits. Three Brachiaria brizantha (Syn. Urochloa brizantha) cultivars (Paiaguás, Ipyporã and Marandu) and two Panicum maximum (Syn. Megathyrsus maximus) cultivars (Quênia and Tamani) were distributed in a randomized-block design with four replicates. 674

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Morphogenetic and structural traits of the pasture were assessed from d 35 to d 65 after sowing, at seven-day interval. Canopy height rose linearly with the establishment period, in all cultivars. In the Megathyrsus cultivars, tiller density decreased as the experimental period progressed, whereas the number of tillers in the Urochloa cultivars increased. The cultivars Ipyporã and Marandu had the highest leaf appearance rates. The lowest leaf elongation rates occurred in the cultivars Paiaguás, Ipyporã and Tamani, and the highest elongation rates in cv. Quênia. As a result, cv. Quênia showed the highest values of final leaf length (64.9 cm) and leaf blade mass (3,352.9 kg DM ha-1). The higher senescence rate of cv. Tamani (2.1 cm tiller-1 d-1) resulted in the highest percentage of dead material (1,815.5 kg ha-1) being found in the herbage mass of this cultivar. Cultivars Paiaguás, Marandu and Tamani were established at 44 d, whereas cv. Quênia and Ipyporã were established at 51 and 58 d after sowing, respectively, in the Brazilian Cerrado. Key words: Morphogenesis, Megathyrsus maximus, Pasture, Urochloa brizantha.

Received: 10/08/2021 Accepted: 23/02/2022

Introduction Forage plants are the main source of feed for ruminants in Brazil and contribute significantly to food production. In the Cerrado Biome, a relevant region for Brazilian livestock, Urochloa brizantha and Megathyrsus maximus genera are the most used due to their high yield potential and adaptability to the tropical climate(1,2). However, most cultivated areas are degraded or in the process of degradation, which has constituted a major obstacle to the expansion and intensification of animal production in grazing systems(3). Proper establishment of forage species is fundamental for perenniality and productivity of a pasture(4). Establishment phase is a critical moment in the formation of pastures. Oftentimes, it represents the beginning of the degradation process or the implementation of a perennial and productive pasture, depending on the interaction between soil, plant and climate. Efficient soil correction and fertilization practices; the choosing the appropriate period for sowing the pasture; and the right moment for the first grazing are essential to ensure plant germination and growth(5). Understanding the evolution of the pasture structure throughout the establishment period allows for greater assertiveness about the time for the first grazing to be performed. In addition, understanding the morphological and structural changes of forage cultivars

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when subjected to different soil-climatic conditions allows to identify their mechanisms of adaptation to the environment and helps in choosing those cultivars with more vigorous establishment(6). In view of the above-described scenario, this study was undertaken to evaluate the time for the establishment of tropical forage grasses in the Cerrado Biome based on morphogenetic and structural traits.

Material and methods The experiment was carried out from December 15, 2020, to February 19, 2021, on the Fazenda Escola farm, of the Federal University of Mato Grosso do Sul, located in the municipality of Terenos - MS, Brazil (20°26'31" S, 54°51'36" W, 437 m asl). The climate of the region is classified as tropical rainy savanna (Aw subtype), characterized by the seasonal distribution of rainfall (Köppen). Temperature data were obtained from the INMET database and the precipitation data (Figure 1) were recorded from a rain gauge installed at the experiment site. The accumulated precipitation during the experimental period was 453 mm. Figure 1: Rainfall and minimum (Tmin) and maximum (Tmax) temperatures during the experimental period

The soil in the experimental area is classified as a Red Oxisol with a very clayey texture(7). Before sowing, a soil sample was taken from the 0-20 cm layer for chemical analysis (Table 1). Based on these results, fertilization was carried out at sowing with 70.0 kg ha-1 of P2O5 and 35 kg ha-1 of K2O.

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Table 1: Chemical characteristics of the soil in the experimental area, in the 0-20-cm depth layer 2+ 2+ + 3+ pH* Ca Mg K Al H+Al SB CEC BS OM P mg -3 5.9 -------------------------------cmolc dm ------------------------------- -----%----- dm-3 5.9 4.0 0.11 5.7 10.0 15.7 63.7 3.4 8.3 *pH in water 1:2.5; SB: sum of bases (Ca + Mg + K); CEC: cation-exchange capacity at pH 7.0 [SB+(H+Al)]; BS: base saturation [(SB/CEC) * 100]; OM: organic matter.

The experimental design was a randomized blocks with five treatments and four replications. The treatments consisted of three Urochloa brizantha cultivars (Paiaguás, Ipyporã and Marandu) and two Megathyrsus maximus cultivars (Quênia and Tamani). The experimental area (3.14 ha) was divided in four blocks of 7,850 m2. Each block was composed of five plots measuring 1,570 m². The soil was mechanically prepared with deep plowing and level-disc harrowing. Sowing was carried out by broadcasting on December 15, 2020. The sowing rate was calculated as described by Dias-Filho(4), considering a seed value for cultivation of 60 % for the Urochloa brizantha cultivars and 40 % for the Megathyrsus maximus cultivars. A road roller was used to increase the soilseed contact. The evaluation spanned five weeks, from d 30 to d 65 after sowing. Morphogenetic and structural traits of the pastures were assessed at seven-day intervals. Canopy height (cm) was measured in 15 representative points per experimental plot, using a millimetric ruler. The canopy height at each point corresponded to the average height of the curvature of the leaves around the ruler. Tiller density (TD, tillers m-2) was evaluated at three points per experimental plot, by counting all the tillers within a 0.50 × 0.50 m square frame (0.25 m2). The sampling points were fix throughout the experimental period, marked with wooden stakes. Morphogenetic and structural traits of the forage canopy were evaluated using the tillertagging technique. Three tillers were marked per experimental plot using colored threads and measured weekly with a ruler graduated in centimeters. The heights of pseudostem and extended tiller and the length of each leaf were measured every seven days to estimate the following variables: leaf appearance rate (LAR, leaves tiller-1 d-1); phyllochron (days leaf-1 tiller-1); leaf elongation rate (LER, cm tiller-1 d-1); stem elongation rate (SER, cm tiller-1 d-1); final leaf length (FLL, cm tiller-1); leaf senescence rate (LSR, cm tiller-1 d-1); number of live leaves (NLL, leaves tiller-1); and leaf lifespan (LLS, d), as proposed by Lemaire and Chapman(8). The cut to determine the forage mass and morphological components occurred when the canopy intercepted, 95.0 ± 3.5 % of the incident light, 65 d after sowing. The light intercepted by the canopy was estimated using a canopy analyzer (PAR Ceptometer - 80 AccuPAR Linear PAR / LAI; DECAGON Devices), at 15 random points per

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experimental unit. At each point, a reading was taken at the top of the forage canopy and another at 10 cm of the ground. Thus, at 65 d after sowing, the herbage dry mass (DM, kg DM ha-1 by cutting the herbage contained within three 1-m² squares per experimental plot. The samples were weighed and dried in a forced-air oven at 55 ºC until constant weight and then weighed again to determine the herbage dry mass. To evaluate the morphological components of the herbage, three sub-samples were extracted from the samples collected to determine HM. These were separated into leaf (leaf blade), stem (stem + sheath), dead material and undesirable plants. Leaf: stem ratio was calculated as the ratio between leaf blade dry mass (LBM, kg ha-1) and stem mass (SM, kg ha-1). Canopy height, TD and NLL data were subjected to analysis of variance, considering a randomized-block design with repeated measures over time. The effect of cultivars was allocated to the plot, and days after sowing (30, 37, 44, 51, 58 and 65 d) to the subplot (repeated measurements over time). The following model was used: Yijk = μ + Ci + Bj + αij + Dk + CDik + βijk, where: Yijk= value observed in cultivar i, block j and day k; μ= overall-mean effect; Ci= effect of cultivar i; Bj= effect of block j; αij : effect of random error attributed to the plot; Dk= effect of day after sowing k; CDik= interaction effect between cultivar and day; βijk= random error attributed to the subplot. When significant by the F-test, the cultivars were compared by Tukey’s test at a significance level of 5%, whereas the effect of days after sowing was analyzed using regression equations. The remaining variables were subjected to analysis of variance according to the following model: Yij = μ + Ci + Bj + αij, where: Yij= value observed in cultivar i and block j; μ= overall-mean effect; Ci= effect of cultivar i; Bj= effect of block j; and αij= random-error effect. When significant by the F-test, the effects of the cultivars were analyzed by Tukey’s test at 5% significance. All statistical analyzes were performed using the MIXED procedure, in SAS ver. 9.1. 678

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Results The interaction cultivar by day after sowing was significative (P<0.05) for canopy height, TD and NLL. Canopy height increased linearly for all cultivars, with estimated daily increments of 1.52, 0.95, 1.21, 2.53 and 1.01 cm for Paiaguás, Ipyporã, Marandu, Quênia and Tamani, respectively (Table 2). On d 30 after sowing, cultivar Quênia had a greater height than cultivar Ipyporã, but there was no difference among the other cultivars. On d 37, the cultivar Quênia presented a higher canopy than the cultivars Ipyporã and Paiaguás. In the other periods, the cultivar Quênia had the largest canopy, followed by cvs. Marandu, Paiaguás and Tamani. Tiller density increased linearly for Urochloa brizantha cultivars and decreased linearly for Megathyrsus maximus cultivars during the establishment period. The estimated increase in number of tillers in Paiaguás, Ipyporã and Marandu was 10.2, 10.8 and 6.6 tillers m-2 d-1, respectively. In Quênia and Tamani, the tiller population decreased by 5.09 and 29.67 tillers m-2 d-1, respectively. The TD at 30, 37, 44 and 51 d after sowing was higher in the Tamani cultivar. On the other hand, on day 58 after sowing, Tamani presented higher TD than Ipyporã, Marandu and Quênia, with no differences between the last two and Paiaguás. Finally, on d 65, the TD of the Tamani above that observed in Marandu and Quênia (Table 3). Quênia maintained a constant NLL since the beginning of the evaluation period. In Marandu, there was an estimated daily increase of 0.05 leaves tiller-1. In the other cultivars, the NLL fitted a second degree-linear regression. Paiaguás and Ipyporã reached the maximum NLL at 42.5 and 47.0 d after sowing, respectively. Tamani reached an estimated minimum NLL at 58.3 d. 30 d after sowing, no differences were observed in the NLL between cultivars. At 37 d, a difference was found between the Paiaguás and Tamani. In the other periods (30, 44, 51, 58 and 65) the Urochloa brizantha cultivars presented a higher NLL (Table 4). Leaf lifespan did not differ between the cultivars (Table 5). Ipyporã and Marandu showed higher LAR than Tamani, whereas Paiaguás and Quênia showed intermediate values. The phyllochron differed between Tamani and Ipyporã, which presented the highest and lowest values, respectively, while the cultivars Paiaguás, Marandu and Quênia presented intermediate values. The lowest LER were observed in Paiaguás, Ipyporã and Tamani, and the highest in Quênia, which in turn was above Marandu. As a result, Quênia showed the highest FLL. Paiaguás and Quênia had the highest SER. Lastly, LSR was highest in Quênia and lowest in Marandu, while Paiaguás, Ipyporã and Tamani showed intermediate values (Table 5). Herbage mass did not differ between cultivars (Table 6). Quênia produced the largest LBM, whereas Paiaguás and Ipyporã showed the lowest, the others showed intermediate

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values. Stem mass was larger in Marandu and Quênia than in Ipyporã and Tamani. The SM of Paiaguás was similar to the others. Cultivar Tamani showed the largest dead material mass (DMM). The only difference for undesirable-plant mass (UPM) was found between Paiaguás and Tamani, whereas the Tamani pastures did not have UPM. The highest leaf:stem ratio occurred in Tamani, Ipyporã and Quênia.

Discussion The linear increase in canopy height is consistent with the establishment period (Table 2), since when the forage is in the initial phase of vegetative growth, an increase in the average height of the sward over the days is observed, regardless of the cultivar(5,6). The recommended height to interrupt growth is around 30 cm for Brachiaria cultivars(9,10,11), 70 cm for Quênia(12) and 35 cm for Tamani(13). Paiaguás, Marandu and Tamani reached the height recommended for growth interruption by d 44 after sowing, whereas Quênia and Ipyporã reached this value at 51 and 58 d, respectively. The high tiller population observed at all assessment periods in Quênia and Tamani may partly explain the reduction in DT (Table 3). Higher tiller populations during pasture vegetative growth promote greater intraspecific competition for light, reducing the amount and quality of light reaching the base of the canopy(14), which results in tiller mortality(15). Therefore, the time when the cultivars reached the recommended height to stop their growth would be adequate to carry out the first grazing The number of live leaves differed between cultivars (Table 4). In Paiaguás and Ipyporã, there was an increase in NLL until it reached its maximum value, at 42.5 and 47.0 d after sowing, respectively. From that point, for each new leaf that appeared, another one started to die. However, the linear increase in NLL in cv. Marandu indicates that this cultivar did not reach the maximum value during its establishment. The always-higher NLL in Urochloa brizantha cultivars in comparison to Megathyrsus maximus cultivars can be attributed to their genetics, since the number of leaves formed in Urochloa brizantha cultivars is greater than in plants of Megathyrsus maximus(5,6,16). Among the Megathyrsus maximus cultivars, Quênia maintained a constant NLL since the beginning of the evaluation period (30 days after sowing). On the other hand, in cv. Tamani, NLL decreased up to a minimum value that occurred on day 58 after sowing. Subsequently, there was an increase in the number of leaves per tiller. This behavior was due to the high TD of cv. Tamani, which promoted a compensatory effect, since, canopies with a high TD have shorter tillers, but these have low growth rates and vice-versa(17,18). This compensatory mechanism is evidenced by the morphogenetic variables evaluated during the establishment period, since Tamani was the one with fewest leaves, which in turn increased the phyllochron (Table 3).

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For tropical pastures in a vegetative stage, morphogenesis can be described by LAR, LER, LLS(18) and SER(19). These traits are genetically determined and, as such, vary according to the evaluated genotype. Furthermore, variations in morphogenesis determine structural traits of the pasture(15,19). Therefore, for a consistent interpretation of these variables, the interactions between them must be taken into account. The variations in LER reflected the morphological differences between the cultivars, especially in FLL, a genetically determined structural trait of the forage canopy(18). As described by Lemaire and Chapman(8), LER tends to follow the behavior of FLL. This association could be observed in cv. Quênia, which showed the highest values for both LER and FLL (Table 5). Marandu, Paiaguáis and Quênia showed greater stem elongation of all because these cultivars are taller (Table 2), which evidences the association between SER and the height of each cultivar(20). The higher LSR observed in cv. Quênia can be attributed to its higher rates of leaf appearance and elongation. With new tissues growth, an increase of the rate of senescence of older tissues are expected, due to the plant renewal process(18). Cultivar Marandu had the lowest LSR, which can be explained because this cultivar did not reach the maximum NLL (Table 4), and senescence in forage plants is enhanced after the maximum NLL has emerged(19). The similar HM between the cultivars may reflect the negative correlation between height and TD(21), which induces a compensation in HM. Cultivar Quênia had the highest values of rate of leaf appearance, elongation, and FLL. This combination of factors was responsible for the highest LBM occurring in this cultivar. Stem mass followed the trend observed for SER. Luna et al(16) reported a similar result, in which the stem accumulation rate behaved similarly to SER, regardless of the species evaluated. The greater DMM contribution to the HM in cv. Tamami was a result of the high LSR and tiller mortality found in this cultivar (Tables 3 and 4). Due to its high TD, it is possible that Tamani would have quickly reached the critical leaf area index(22). Subsequent increases in leaf area index lead to reduced leaf accumulation and increased leaf and tiller mortality(23). On the other hand, this increased competition for light decreased the number of undesirable plants in the Tamani pastures. These findings suggest that all cultivars exhibited vigorous establishment, given their high rates of tissue renewal, which promoted changes in the structural traits of the pasture(24). As a consequence, herbage mass, morphological composition and tiller population were modified.

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Conclusions and implications It was found that, depending on the morphogenetic and structural characteristics, the establishment time in the Cerrado Biome is 44 days after sowing for cvs. Paiaguás, Marandu and Tamani; and 51 and 58 days after sowing for cvs. Quênia and Ipyporã, respectively. Morphogenic traits are genetically determined and, as such, vary between genotypes; in addition, variations in morphogenesis determine structural characteristics of the pasture. Evaluating the morphogenetic and structural variables simultaneously allows observing the dynamics of tissue emergence and death both within a tiller and for the entire tiller population. Thus, allowing greater precision at the ideal time to stop the growth of grasses.

Acknowledgments

The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Brasil (CAPES) - Finance Code 001. Thanks also to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Fundação de Apoio ao Desenvolvimento da Educação, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT) and the Universidade Federal de Mato Grosso do Sul for their support. Literature cited: 1.

Euclides VPB, Montagner DB, Macedo MCM, Araújo AR, Difante GS, Barbosa RA. Grazing intensity affects forage accumulation and persistence of Marandu palisadegrass in the Brazilian savannah. Grass Forage Sci 2019;75(1):1-13.

Véras ELL, Difante GS, Gurgel ALC, Costa ABG, Rodrigues JG, Costa CM, et al. Tillering and structural characteristics of Panicum cultivars in the Brazilian semiarid region. Sustainability 2020;12(9):3849.

Pereira OJR, Ferreira LG, Pinto F, Baumgarten L. Assessing pasture degradation in the Brazilian Cerrado based on the analysis of MODIS NDVI time-series. Remote Sens 2018;10(11):1761.

Dias-Filho MB. Formação e manejo de pastagens. Embrapa Amazônia OrientalComunicado Técnico 2012;235:1-9.

Rodrigues JG, Difante GS, Gurgel ALC, Veras ELL, Costa ABG, Pereira MG, et al. Establishment of Brachiaria cultivars in the soil-climatic conditions of the Brazilian semi-arid region. Acta Sci Anim Sci 2021;43: e51802.

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Costa ABG, Difante GS, Gurgel ALC, Veras ELL, Rodrigues JG, Pereira MG, et al. Morphogenic and structural characteristics of Panicum cultivars during the establishment period in the Brazilian Northeast. Acta Sci Anim Sci 2021;43:e50984.

EMBRAPA. Empresa Brasileira de Pesquisa Agropecuária. Sistema Brasileiro de Classificação de Solos. 3th ed. Rio de Janeiro, Brasil: Centro Nacional de Pesquisa de solo; 2013.

Lemaire G, Chapman D. Tissue flows in grazed plant communities. In: Hodgson J, Illius AW, editores. The ecology and management of grazing systems. 1rst ed. Wallingford, UK: CABI Publishing; 1996:3-29.

Giacomini AA, Silva SC, Sarmento DOL, Zeferino CV, Souza Júnior SJ, Trindade JK, et al. Growth of Marandu palisadegrass subjected to strategies of intermittent stocking. Sci Agric 2009;66(6):733-741.

10. Euclides VPB, Montagner DB, Barbosa RA, Valle CB, Nantes NN. Animal performance and sward characteristics of two cultivars of Brachiaria brizantha (BRS Paiaguás and BRS Piatã). R Bras Zootec 2016;45(3):85-92. 11. Echeverria JR, Euclides VPB, Sbrissia AF, Montagner DB, Barbosa RA, Nantes NN. Forage accumulation and nutritive value of the Urochloa interspecific hybrid 'BRS RB331 Ipyporã' under intermittent grazing. Pesqui Agropecu Bras 2016;51(7): 880889. doi:10.1590/S0100-204X2016000700011. 12. Andrade CM, Farinatti LH, Nascimento HL, Abreu ADQ, Jank L, Assis GM. Animal production from new Panicum maximum genotypes in the Amazon biome, Brazil. Trop Grassl Forr Trop 2013;1(1):36-38. 13. Tesk CR, Cavalli J, Pina DS, Pereira DH, Pedreira CG, Jank L, et al. Herbage responses of Tamani and Quênia guinea grasses to grazing intensity. Agron J 2020; 112(3):2081-2091. 14. Montagner DB, Nascimento Júnior D, Vilela HH, Sousa BML, Euclides VPB, Silva SC, et al. Tillering dynamics in pastures of guinea grass subjected to grazing severities under intermittent stocking. R Bras Zootec 2012;41(3):544-549. 15. Sbrissia AF, Schmitt D, Duchini PG, da Silva SC. Unravelling the relationship between a seasonal environment and the dynamics of forage growth in grazed swards. J Agron Crop Sci 2020;206(5):630-639. 16. Luna AA, Difante GS, Montagner DB, Emerenciano Neto JV, Araújo IMM, Oliveira LEC. Características morfogênicas e acúmulo de forragem de gramíneas forrageiras sob corte. Biosc J 2014;30(6):1803-1810.

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17. Sbrissia AF, Duchini PG, Zanini GD, Santos GT, Padilha DA, Schmitt D. Defoliation strategies in pastures submitted to intermittent stocking method: Underlying mechanisms buffering forage accumulation over a range of grazing heights. Crop Sci 2018;58(2):945-954. 18. Gastal F, Lemaire G. Defoliation, shoot plasticity, sward structure and herbage utilization in pasture: Review of the underlying ecophysiological processes. Agriculture 2015;5(1):1146-1171. 19. Difante GS, Nascimento Júnior D, Silva SC, Euclides VPB, Montagner DB, Silveira MCT, et al. Características morfogênicas e estruturais do capim-marandu submetido a combinações de alturas e intervalos de corte. Rev Bras Zootec 2011;40(5):955963. 20. Sousa BML, Nascimento Júnior D, Silva SC, Monteiro HCF, Rodrigues CS, Fonseca DM, et al. Morphogenetic and structural characteristics of Andropogon grass submitted to different cutting heights. Rev Bras Zootec 2010;39(10):2141-2147. 21. Véras ELL, Difante GS, Gurgel ALC, Costa CM, Emerenciano Neto JV, Rodrigues JG, et al. Tillering capacity of Brachiaria cultivars in the Brazilian Semi-Arid Region during the dry season. Trop Anim Sci J 2020;43(2):133-140. 22. Euclides VPB, Valle CB, Macedo MCM, Almeida RG, Montagner DB, Barbosa RA. Brazilian scientific progress in pasture research during the first decade of XXI century. Rev Bras Zootec 2010;39(Suppl Spec):151-168. 23. Zanine A, Farias L, Ferreira D, Farias L, Ribeiro M, Souza A, et al. Effect of season and nitrogen fertilization on the agronomic traits and efficiency of Piatã grass in Brazilian savanna. Agriculture 2020;10(8):337. 24. Sousa CCC, Montagner DB, Araújo AR, Euclides VPB, Difante GS, Gurgel ALC, et al. The soil-plant interface in Megathyrsus maximus cv. Mombasa subjected to different doses of nitrogen in rotational grazing. Rev Mex Cienc Pecu 2021;12(4): 1098-1116.

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Table 2: Canopy height (cm) of tropical forage grasses in different evaluation periods during establishment in the Brazilian Cerrado biome Days after sowing P-value Cultivar Regression equation R2 (%) 30 37 44 51 58 65 L Q Paiaguás 12.1ab 16.0b 27.9b 40.1cb 47.2cb 60.3b 0.001 0.522 Y = -33.30 + 1.42x 98.5 b b b c c c Ipyporã 8.5 12.8 20.5 26.8 30.6 43.0 0.001 0.554 Y = -21.23 + 0.95x 97.6 ab ab b b b cb Marandu 17.6 24.8 33.4 44.2 51.5 58.6 0.001 0.896 Y = -19.09 + 1.21x 99.6 a a a Quênia 28.5 37.0 57.3ª 69.5ª 87.1ª 119.7 0.001 0.101 Y = -53.45 + 2.53x 96.5 ab ab b cb cb cb Tamani 19.5 24.1 35.8 40.1 42.5 57.1 0.001 0.765 Y = -11.56 + 1.01x 95.5 abc

L= linear; Q= quadratic. Y is the dependent variable and X is the independent variable (days after sowing). Lowercase letters in the same column differ from each other by Tukey’s test (P<0.05); Standard error of the mean= 4.38;

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Table 3: Tiller density (tillers m-2) of tropical forage grasses in different periods of evaluation during establishment in the Brazilian Cerrado biome Days after sowing P-value Cultivar Regression equation R2 (%) 30 37 44 51 58 65 L Q Paiaguás 210.0c 245.7c 318.0b 412.3b 487.0ab 546.0ab 0.001 0.821 Y = -114.50 + 10.20x 98.8 Ipyporã

166.0c

229.0c

314.0b

396.7b

445.7b

546.7ab

0.001

0.934

Y = -161.46 + 10.76x

99.5

Marandu

221.0c

257.7c

354.3b

422.7b

445.7b

417.7b

0.002

0.270

Y = 39.93 + 6.59x

84.5

Quênia

602.0b

583.6b

531.7b

435.0b

456.5b

448.2b

0.016

0.538

Y = 751.27 - 5.09x

83.3

Tamani

1534.4a

1605.0a

1617.3a

864.4a

737.2a

742.0a

0.001

0.136

Y = 2594.20 - 29.67x

77.3

abc

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Table 4: Number of live leaves per tiller in tropical forage grasses in different periods of evaluation during establishment in the Brazilian Cerrado biome Days after sowing P-value Cultivar Regression equation R2 (%) 30 37 44 51 58 65 L Q Paiaguás 5.3a 6.3a 7.5a 6.0a 5.1ab 5.7ab 0.227 0.002 Y = -1.26 + 0.34x - 0.004x2 39.0 Ipyporã

4.0a

5.6ab

6.6ab

6.1a

5.4ab

5.9ab

0.105

0.001

Y = -5.61 + 0.47x - 0.005x2

71.8

Marandu

4.9a

5.2ab

6.0ab

6.4a

6.6a

6.3a

0.001

0.117

Y = 3.61 + 0.05x

81.7

Quênia

4.5a

5.2ab

5.2b

4.3b

4.5b

0.367

0.326

Y = 4.7

Tamani

4.9a

4.5b

3.8c

2.8b

2.8c

3.5c

0.081

0.008

Y = 12.87 - 0.35x + 0.003x2

86.5

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Table 5: Structural and morphogenetic traits of tropical forage grasses during the establishment period in the Brazilian Cerrado biome Cultivar Variable SEM P-value Paiaguás Ipyporã Marandu Quênia Tamani LAR, leaves tiller-1 d-1 0.16ab 0.18a 0.17a 0.15ab 0.13b 0.01 0.0181 Phyllochron, days leaf-1 tiller-1

6.4ab

5.7b

5.8ab

6.9ab

8.1a

0.50

0.0355

LER, cm tiller-1 d-1

5.4c

5.3c

7.8b

10.8a

5.4c

0.48

0.0001

SER, cm tiller-1 d-1

1.25a

0.52b

1.1a

1.0a

0.19b

0.08

0.0001

LSR, cm tiller-1 d-1

1.7ab

1.5ab

1.2b

2.5a

2.1ab

0.26

0.0312

FLL, cm

30.2b

29.1b

43.1b

64.9a

36.0b

3.50

0.0001

LLS, days

37.9

32.0

34.4

32.7

28.2

2.40

0.2026

LAR= leaf appearance rate; LER= leaf elongation rate; SER= stem elongation rate; LSR= leaf senescence rate; FLL= final leaf length; LLS= leaf lifespan; SEM= standard error of the mean. abc Lowercase letters in the same row differ from each other by Tukey’s test (P<0.05).

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Table 6: Structural traits of tropical forage grasses during the establishment period in the Brazilian Cerrado biome Cultivar Variable SEM Paiaguás Ipyporã Marandu Quênia Tamani HM, kg DM ha-1 5405.0 4736.5 5766.5 6884.8 6007.5 529.5

P-value

LBM, kg DM ha-1

1654.6b

1962.7b

2533.1ab

3352.8a

2571.7ab

258.1

0.0131

SM, kg DM ha-1

1879.9ab

1427.1b

2345.7a

2429.6a

1619.3b

209.2

0.0353

DMM, kg DM ha-1

546.7b

449.2b

484.3b

839.4b

1816.5a

96.1

0.0001

UPM, kg DM ha-1

1444.9a

937.8ab

403.4ab

263.0ab

0.0b

279

0.0378

Leaf:stem ratio

0.9b

1.5a

1.1b

1.4a

1.59a

0.1

0.0436

0.1554

HM= herbage mass; LBM= leaf blade mass; SM= stem mass; DMM= dead material mass; UPM= undesirable-plant mass; SEM= standard error of the mean. ab Lowercase letters in the same row differ from each other by Tukey’s test (P<0.05).

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https://doi.org/10.22319/rmcp.v13i3.5786 Article

Effectiveness of zilpaterol hydrochloride in lamb finishing: Patent vs. Generic

Arnulfo Vicente Pérez a,b Leonel Avendaño-Reyes a Juan E. Guerra-Liera b Rubén Barajas Cruz b Ricardo Vicente-Pérez c M. Ángeles López-Baca a Miguel A. Gastelum Delgado b Alfonso J. Chay-Canul d Ulises Macías-Cruz a*

Universidad Autónoma de Baja California. Instituto de Ciencias Agrícolas, 21705, Valle de Mexicali, BC., México. b

Universidad Autónoma de Sinaloa. Facultad de Medicina Veterinaria y Zootecnia, Culiacán, Sinaloa, México. c

Universidad de Guadalajara. CUCSUR-Departamento de Producción Agrícola, Autlán de Navarro, Jalisco, México. d

Universidad Juárez Autónoma de Tabasco. División Académica de Ciencias Agropecuarias, Villahermosa, Tabasco, México.

*Corresponding author: ulisesmacias1988@hotmail.com, umacias@uabc.edu.mx

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Abstract: The objective of this study was to compare the effect of the patent vs. generic sources of zilpaterol hydrochloride (ZH) on the productive performance, carcass characteristics, primary cut yields, and meat quality of lambs finished in feedlot. Thirty (30) Dorper×Pelibuey male lambs were distributed into 10 blocks, each with three lambs of similar initial live weight which were randomly assigned to the following treatments: 1) without ZH (control), 2) with patent ZH (PZH), and 3) with generic ZH (GZH). Treatment means were compared through two orthogonal contrasts: control vs. ZH (PZH+GZH) and PZH vs. GZH. ZH did not affect (P≥0.15) the productive performance, carcass weight, backfat thickness, or fat percentages (kidney-pelvic-heart, mesenteric or omental), but increased (P≤0.05) Longissimus dorsi muscle area and yields of carcass, shoulder, leg, and plain loin. As for the meat quality, ZH did not affect (P≥0.24) pH and shear force, but reduced (P<0.05) redness, yellowness, and chroma color values at 24 h post mortem, as well as the redness value (P<0.01) at 14 days of aging. With exception of carcass yield which tended (P=0.07) to increase with PZH, all measured variables were similar (P0.14) between PZH and GZH. It has been concluded that both types of ZH at a dose of 0.10 mg per kg of live weight promote muscular hypertrophy in finishing lambs; however, this dosage is not sufficient to result in a better productive performance or carcass weight. Key words: Adrenergic agonists, Meat quality, Carcass characteristics, Hair sheep.

Received: 29/08/2020 Accepted: 13/07/2021

Introduction The supply of sheep meat in Mexico is lower than the demand for it (~30 %). So, the sheep meat industry is constantly searching for low cost strategies that help increase feed efficiency and weight gain(1). In the last two decades, several studies have shown that zilpaterol hydrochloride (ZH) effectively promotes growth in finishing lambs in feedlot, as it improves feed efficiency, growth rate, carcass weight and yield, and Longissimus dorsi muscle area, as well as decreases internal and external fat deposition in the body(2–5). Despite this, the dietary addition of ZH also decreases the lamb meat quality, specifically maintaining a high ultimate pH, which causes discoloration and increases meat toughness(6,7).

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Although, the results on the use of ZH in intensive fattening of lambs have mostly been positive, this product has increased economic cost and, in consequence, farmers have exhibited some resistance to adopting the use of ZH in sheep feeding. Once the patent of ZH had expired, various pharmaceutical companies began to produce generic ZH, and today they sell it 23 % cheaper than patent ZH. Recent studies carried out in sheep(8) and bulls(9) proved that, patent and GrofactorTM generic ZH are similarly effective as growth promotors at the dosage recommended on the label (0.15 mg per kg of live weight [LW]). For their part, Avendaño-Reyes et al(10) evidenced that the optimal dose of this generic ZH for hair sheep finished in feedlot is lower (0.10 mg per kg of live weight [LW]) than that recommended on the label of all ZH brands (i.e. 0.15 mg per kg of LW). Based on the above, the generic ZH said could be used to reduce costs derived from the use of this technology, as it has a lower market price and its required dose (33.3 %) is lower than that indicated for patent ZH(11). Despite the history of generic ZH effectiveness in the finishing lambs, it is necessary to verify whether the effectiveness of generic ZH is comparable to that of patent ZH at a dose of 0.10 mg per kg of LW. It is noteworthy that the molecule of ZH in this generic adrenergic agonist is bioequivalent to that of patent ZH, but differs in the manner in which it is attached to the vehicle, which may reduce its bioavailability and its mode of action(10). Therefore, the objective of the present study was to compare the effect of the source of ZH (patent vs. generic) at a daily dose of 0.10 mg per kg of LW on productive performance, carcass characteristics, primary cut yields, and meat quality in hair male lambs finished in feedlot.

Material and methods Location of the experiment

All management and care procedures of animals were carried out according to the Mexican Official Norms NOM-051-ZOO-1995 (Humane care of animals during mobilization) and NOM-033-ZOO-1995 (Slaughter of domestic and wild animals). The study was performed in spring at the sheep experimental unit of the Instituto de Ciencias Agrícolas, Universidad Autónoma de Baja California, located in the Valley of Mexicali, Baja California, México (32.8o N, 114.6o W).

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Animals and their experimental management

Thirty entire male lambs from the Dorper × Pelibuey cross (initial LW= 36.9 ± 6.9 kg and age= 5 mo) were utilized, having been adapted to individual pens and basal diet (Table 1) for 20 days previous to the onset of the experiment. The animals received two injections at the beginning of the adaptation period, an intramuscular one administering 1.0 ml of vitamins (Vigantol ADE Fuerte; Bayer, Mexico City) and a subcutaneous one with 0.5 ml of ivermectine (Sanfer, Mexico City). The individual pens were provided with food and water troughs and shade. The basal diet was formulated for a daily weight gain of 300 g in finishing lambs (2.8 Mcal of metabolizable energy per kg of dry matter [DM] and 16 % crude protein)(12). Feed samples were collected on a weekly basis, and at the end of the experimental period they were brought together in order to obtain two subsamples, which were analyzed to determine its chemical composition(13,14,15). In general, the diet was offered twice a day (at 0700 and 1800 h) during both the adaptation and the experimental period, guaranteeing a daily rejection rate of at least 10 %, while water was available ad libitum. Table 1: Ingredients and chemical composition of the experimental diet Ingredients (%)* Chemical makeup (% DM) Alfalfa hay 17.5 Dry matter Wheat straw 11.0 Organic matter Ground wheat grains 60.0 Crude protein Soybean meal 7.0 Ethereal extract Soybean oil 2.0 Neutral detergent fiber Limestone 1.0 Acid detergent fiber Dicalcium phosphate 1.0 Ashes Common salt 0.5 Calcium Phosphorus Metabolizable energy, Mcal/kg

94.2 92.9 15.1 4.2 17.9 10.1 7.1 0.87 0.54 2.9

* The amount of each ingredient was calculated on a wet basis. DM= dry matter.

Experimental design

A 32-d productive performance test was carried out after the adaptation period. On the first day of the test, all fasting male lambs were weighed individually (after 12 h without feed or water) and then grouped into 10 blocks, each containing three lambs of similar initial LW

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(blocking factor). Thus, the male lambs in each block were randomly assigned to the treatments, which consisted in offering them a basal diet that included: 1) 0 mg of ZH / kg of LW (control); 2) 0.10 mg of patent ZH / kg of LW (ZilmaxTM, Intervet, Mexico City, Mexico; PZH), and 3) 0.10 mg of generic ZH per kg of LW (GrofactorTM, Virbac Mexico, Guadalajara, Mexico; GZH). All male lambs were individually weighed every 10 d in order to adjust the amount of ZH in PZH and GZH treatments. The daily dose of the product was mixed with 30 g of ground wheat grains and offered in the morning, before the basal diet. The control group was fed 30 g ground wheat grains at the same time as in the PZH and GZH groups. Through the feedlot test, treatments were offered during the first 30 d, and the last two days were utilized as withdrawal period. The fasting male lambs were then transported to the meat workshop (located at a distance of 200 m from the pens), where they were slaughtered by the disgorging method.

Productive performance

The variables evaluated for productive performance were initial and final LW (kg), average daily gain (DWG= TWG/32; kg/d/animal), total weight gain (TWG= final LW – initial LW; kg/period), daily dry matter intake (DMI= kilograms of fresh food ingested × [% DM/100], kg/animal), and feed efficiency (DWG/DMI). Lambs were fasting when the individual LW on day 1 (initial) and 33 (final) of the feedlot test was recorded, and these data were then used to calculate DWG and TWG.

Body offal and carcass characteristics

The evaluation of offal and carcass characteristics was carried out as described by AvendañoReyes et al(10). After slaughter, the bodies were eviscerated, and the weights of each organ, viscera and offal (skin, head, feet, testicles, blood, heart, liver, kidneys, lungs, spleen, full and empty gastrointestinal tract, rumen, intestines, and omental and mesenteric fat, as well as the fat surrounding kidneys, pelvic cavity and heart [KPH]) were recorded. The gastrointestinal content was estimated by difference between the weights of the empty and full gastrointestinal tract, while empty LW was calculated by subtracting the gastrointestinal content weight from the final LW. Thus, the weights of all organs, viscera and offal were expressed as percentages of the empty LW. The hot carcass was weighed (HCW) and then cooled at 4 ºC during 24 h in order to record cold carcass weight (CCW), conformation, carcass length, thoracic depth, leg length and

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perimeter, Longissimus dorsi muscle (LM) area, and fat thickness. The carcass conformation was assessed on an 8-point scale where 1 is bad and 8 is excellent(16). A flexible measuring tape was utilized to take the carcass morphometric measurements(17). Both LMA and fat thickness were measured between the 12th and 13th rib, performing a perpendicular cut to the loin at that height. The LM area was measured using a dot square grid (64 mm2), while the fat thickness was determined with a caliper. Finally, the carcass yield was estimated by expressing the HCW as a percentage of the empty LW.

Primary cut yields

Carcasses were cut along the middle line and the right half carcass was then divided into the following primary cuts(18): forequarter, hindquarter, neck, shoulder, ribs, loin, plain loin, leg, and breast and flank. The right half-carcass and each primary cut were weighed; the yield of each cut was then calculated by expressing its weight as a percentage of the half-carcass weight.

Meat quality

The meat quality was assessed in the LM. A piercing electrode (HACH model PHW57-SS, Colorado, USA) attached to a pH meter (HACH model H160G, HACH, Colorado, USA) was inserted into the carcass loin, between the 12th and the 13th rib, in order to record pH at 45 min and 24 h post mortem. Subsequently, the LM was dissected from the loin primary cut, and color parameters (a* [redness], b* [yellowness], L* [luminosity], C* [color saturation index], and h* [hue angle]) were measured at 24 h post mortem, using a portable colorimeter (X-rite model SP60, Michigan, USA). Finally, the LM was vacuum-packed and refrigerated at 4 ºC during 14 d, after which pH and color parameters were measured again, likewise shear force in matured meat was also measured. So, after the aging period, the LM was unpacked and exposed to blooming during 30 min before quality measurements. The pH was measured in a homogenized mixture of 5 g of meat and 25 ml of distilled water, using a liquid potentiometer (Hanna Instruments Digital model HI-2210, Woonsocker, Rhode Island). Color parameters were measured using the methodology described above in the evaluation at 24 h post mortem. Color measurements were carried out in triplicate, and, finally, the average for each parameter was estimated and recorded. For shear force, two (2.5 cm thick) LM steaks were cooked on an electric grill until they attained an internal temperature of 71 °C; the steaks were then cooled to ambient temperature (~25 ºC), and five prisms with a 1.27 cm diameter were obtained, in which the shear force was measured using a Warner-Bratzler

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shear machine (Salter 235, Manhattan, KS, USA). The shear force per sample was recorded, averaging the three most homogenous values (variation coefficient <5 %).

Statistical analysis

All data were analyzed using the ANOVA procedure of the SAS statistic software(19), applying the statistical model of a randomized complete block design. The model included to initial LW as blocking factor and the type of ZH as treatment. Means were compared through two orthogonal contrasts, establishing P≤0.05 as differences, and 0.05>P≤1.0 as trend. The first contrast compared the control group against the use of any source of ZH (C1: control vs PZH+GZH), and the second contrast compared between the sources of ZH (C2: PZH vs GZH).

Results Control versus zilpaterol hydrochloride Regardless of the source of ZH, the dietary addition of this β2-adrenergic agonist (β2-AA) did not affect (P≥0.15) the productive performance of animals (Table 2). In carcass, ZH increased carcass yield (P≤0.01) and LM area (P≤0.05); likewise, it exhibited a tendency (P=0.09) to improve conformation, but it did not affect (P=0.09) HCW, CCW, carcass morphometric measurements, or any variable associated with internal or external fat deposition (Table 3). As for the primary cut yields, ZH increased (P≤0.03) yields of forequarter, leg and plain loin, but reduced (P≤0.01) yields of hindquarter and shoulder, without affecting (P≥0.27) any of the other primary cuts (Table 4). In body offal (weights expressed as a percentage of the empty LW), the dietary addition of ZH reduced (P≤0.05) the weights of skin, liver, kidneys, spleen, and rumen, and exhibited a tendency (P=0.06) to decrease the feet weight (Table 5). The weight of the rest of body offal did not vary (P≥0.12) with the ZH inclusion.

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Table 2: Productive performance of male lambs fed patent (PZH) or generic (GZH) zilpaterol hydrochloride Treataments Contrasts* Variables (kg) Control GZH PZH SE C1 C2 Initial weight 36.9 36.8 36.9 2.19 0.98 0.96 Final weight 46.1 46.6 46.8 2.58 0.85 0.96 Total weight gain 9.18 9.80 9.85 0.69 0.45 0.96 Daily weight gain 0.30 0.31 0.31 0.02 0.32 0.96 Daily DM intake 1.59 1.51 1.56 0.06 0.26 0.62 Feed efficiency 0.18 0.20 0.20 0.01 0.15 0.28 * C1= control s. PZH+GZH; C2= PZH vs GZH. SE= standard error; DM= dry matter.

Table 3: Carcass characteristics of male lambs fed patent (PZH) or generic (GZH) zilpaterol hydrochloride Treatament Contrasts* Variables Control GZG PZH SE C1 C2 Hot carcass weight, kg 22.00 22.59 23.52 1.32 0.51 0.62 Cold carcass weight, kg 21.82 22.42 23.36 1.31 0.51 0.61 Carcass yield, % 53.67 54.63 55.87 0.46 0.01 0.07 2 LM area, cm 13.42 15.69 16.88 1.19 0.05 0.48 Conformation, points 6.17 6.40 6.80 0.20 0.09 0.17 Carcass length, cm 55.15 54.82 54.58 0.92 0.69 0.85 Leg length, cm 36.20 34.90 36.15 0.58 0.35 0.14 Leg perimeter, cm 47.23 49.91 47.97 2.04 0.50 0.50 Thoracic depth, cm 15.85 15.38 15.48 0.33 0.30 0.83 Fat thickness, mm 1.15 1.15 1.10 0.16 0.90 0.83 Omental fat, % 2.08 2.32 2.27 0.23 0.46 0.90 Mesenteric fat, % 1.87 1.85 1.66 0.16 0.36 0.21 KPH fat**, % 1.49 1.74 1.62 0.14 0.28 0.55 * C1= control vs PZH+GZH; C2= PZH vs GZH. SE= standard error; LM= Longissimus dorsi muscle; KPH fat= Total fat located around kidneys, pelvic cavity and heart.

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Table 4: Primary cut yields of male lambs fed patent (PZH) or generic (GZH) zilpaterol hydrochloride Treatment Contrasts** Variables (%)* Control GZH PZH SE C1 C2 Forequarter 54.59 53.04 52.50 0.39 <0.01 0.34 Neck 4.06 4.07 3.93 0.25 0.83 0.68 Ribs 9.72 9.83 9.71 0.27 0.88 0.73 Loin 9.70 10.2 9.95 0.25 0.27 0.54 Shoulder 31.0 28.9 29.9 0.67 0.01 0.96 Hindquarter 45.4 47.0 47.4 0.39 <0.01 0.34 Leg 31.3 32.5 32.6 0.47 0.03 0.90 Plain loin 8.80 9.35 9.47 0.22 0.03 0.70 breast and flank 5.25 5.00 5.34 0.38 0.85 0.54 * Yields were calculated by expressing the weight of each cut as a percentage of the half-carcass weight. ** C1= control vs PZH+GZH; C2= PZH vs GZH. SE= standard error.

Table 5: Percentage in offal of male lambs fed patent (PZH) or generic (GZH) zilpaterol hydrochloride Treatment Contrasts** Variables (%)* Control GZH PZH SE C1 C2 Blood 4.55 4.38 4.29 0.13 0.18 0.61 Feet 2.44 2.25 2.33 0.06 0.06 0.35 Head 5.61 5.68 5.71 0.16 0.69 0.88 Skin 9.69 8.99 8.68 0.24 <0.01 0.37 Heart 0.48 0.45 0.45 0.01 0.19 0.79 Liver 2.33 2.17 2.06 0.06 0.01 0.24 Kidneys 0.30 0.27 0.28 0.01 0.03 0.64 Lungs 1.81 1.95 1.85 0.10 0.52 0.51 Spleen 0.22 0.17 0.18 0.01 0.01 0.66 Rumen 3.41 3.22 3.13 0.09 0.05 0.52 Intestine 3.22 2.84 2.90 0.17 0.12 0.81 Testicles 1.53 1.51 1.57 0.07 0.93 0.54 * Weights of each organ or viscera were expressed as percentages of the empty live weight. ** C1= control vs PZH+GZH; C2= PZH vs GZH; SE= standard error.

In the case of meat quality, ZH did not affect (P≥0.23) the post-mortem pH at 45 min, 24 h, or 14 d, but decreased (P≤0.01) a* and C* values, and tended (P=0.07) to reduce b* values at 24 h (Table 6). After 14 d post mortem, dietary inclusion of ZH reduced (P<0.01) a* values and increased (P=0.05) h* values, while it had no effects (P=0.95) on shear force. Values of L* were not affected (P≥0.65) by ZH in any of the evaluated time periods.

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Table 6: Meat quality of male lambs fed patent (PZH) or generic (GZH) zilpaterol hydrochloride Treatment Contrasts** Variables Control GZH PZH SE C1 C2 pH45 min 6.54 6.59 6.57 0.05 0.60 0.82 Evaluation at 24 h post mortem pH 5.83 5.93 5.92 0.06 0.24 0.88 L* 39.1 37.7 38.9 1.30 0.65 0.51 a* 10.3 8.17 9.01 0.44 <0.01 0.19 b* 9.51 8.46 8.82 0.38 0.07 0.51 C* 13.7 12.0 12.6 0.43 0.01 0.34 h* 43.8 44.3 44.4 1.33 0.76 0.92 Evaluation after 14 d post mortem pH 5.83 5.95 5.98 0.08 0.23 0.81 2 Shear force, kg/cm 2.04 1.87 2.25 0.24 0.95 0.28 L* 41.2 42.5 41.1 1.20 0.70 0.39 a* 9.15 7.76 7.98 0.36 <0.01 0.66 b* 10.26 9.74 9.19 0.50 0.21 0.45 C* 13.8 12.5 13.2 0.56 0.18 0.39 h* 45.9 51.3 49.5 1.66 0.05 0.45 ** C1= control vs PZH+GZH; C2= PZH vs GZH; SE= standard error. L*= Luminosity, a*= redness, b*= yellowness, C*= chroma, and h*= hue angle.

Patent versus generic zilpaterol hydrochloride

Productive performance (Table 2), carcass characteristics (except for carcass yield; Table 3), primary cut yields (Table 4), weight of the offal expressed as a percentage of the empty LW (Table 5), and meat quality (Table 6) exhibited no variation (P≥0.23) between PZH and GZH. The dietary addition of PZH showed a tend (P = 0.07) to increase carcass yield compared to GZH.

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Discussion Control versus zilpaterol hydrochloride

The addition of ZH to the finishing diet did not improve productive performance or carcass weight in hair male lambs. These results were not expected, as most studies including this β2-AA report a greater DWG, feed efficiency, HCW, and CCW, with little consistent effects on DMI(4,5,8,18,20-22). One might think that having administered a dose below the one recommended by the company (0.10 vs 0.15 mg/kg of LW) was the cause of this lack of effects, as most of those studies that report improvement added the product according to the indications on the label. Nevertheless, a previous study evidenced that 0.10 mg/kg of LW is an optimal dose for finishing male lambs in feedlot when this type of generic ZH is used(10). Notably, those studies in which ZH improved productive performance and carcass weight, the control group exhibited lower DWG (168-280 g/d) than that determined in the present study. So, results in terms of productive performance and carcass weight may be due to the fact that the lambs expressed their maximum genetic potential for growth without the need to use ZH and, in consequence, this promoter had a limited margin to exert its positive effects on DWG and carcass weight. The literature indicates that hair lambs have a DWG of approximately 250 g/d (23), i.e., 50 g less than that observed in the control group of this study which are also hair sheep. According to this finding, Mersmann(24) mentions that the effectiveness of β2-AA for improving body mass gain may be limited by the animal genetic potential. While ZH did not affect LW gain or carcass weight in male lambs; this product improved carcass yield, LM area and yields of certain primary cuts (i.e., leg and plain loin). This suggests that the β2-AA promoted muscular hypertrophy and, therefore, increased muscular mass deposition, although to a limited extent. The increase in carcass yield may be a consequence of the greater muscle mass deposition in body regions as plain loin and legs. These findings agree with results of other studies(5,10); still in these, the effect was more pronounced which further supports the idea that external factors were more important than ZH itself in inducing the maximum expression of genetic growth potential by the sheep, limiting the mechanism of action of the β2-AA(24). The dietary inclusion of ZH did not affect the body internal or external fat deposition, but reduced the weights of some offal (skin, liver, kidneys, spleen, rumen, and feet). This suggests that the muscular hypertrophy observed in loin and legs of the male lambs was caused by an β2-AA-promoted redistribution of nutrients from offal tissue toward the formation of skeletal muscle as previously reported with the addition of generic ZH(8,10,22).

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On the other hand, feeding male lamb with ZH has been mainly associated with an increase in meat pH, which causes a reduction in color values and an increase in meat toughness(6,7,22). However, there is evidence that ZH decreases meat color even with a normal pH(25). In the present study, ZH did not affect post mortem pH (45 min, 24 h, or 14 d); this could partially explain because shear force and luminosity of the meat did not change with the daily inclusion of this β2-AA. Given results for pH, the meat from ZH-treatment unexpectedly discolored without losing luminosity at 24 h post mortem; this effect persisted to a lesser extent after 14 d of maturation. Although the pH at 24 h and 14 d post mortem exhibited no significant change, the pH values in ZH meat (>5.9) remained above the normal range (≤5.8)(26). This may have caused an increase in the mitochondrial respiration of oxidative fibers and a greater competition for oxygen with oxymyoglobin, increasing desoxymyoglobin and metmyoglobin concentrations—pigments which favor meat discoloration(27).

Patent versus generic zilpaterol hydrochloride

Male lambs fed PZH and GZH exhibited no differences in their productive performance, carcass characteristics, primary cut yields, or meat quality, suggesting that both molecules are similarly effective as growth promoters at a daily dose of 0.10 mg per kg of LW in this type of animals. Although, this twofold finding must be taken with caution, since, as stated above, the dietary addition of patent and generic ZH in the sheep feeding promoted muscular hypertrophy, though not at a sufficient level to result in higher DWG, feed efficiency, or carcass weight. Nevertheless, the fact that generic ZH at the dose used promotes a degree of muscular hypertrophy comparable to that observed with patent ZH in finishing male lambs is rescued. This may be due to the fact that both products are similar in bioequivalence, chirality, and isomeric determination(9). Despite this, they differ as they are attached to the vehicle (ground cob); while the patent ZH molecule is attached around the granules of the vehicle, the generic ZH molecule remains unattached to this(10). This could reduce the bioavailability and, consequently, the effectiveness of the product. Results of this study suggest that the manner in which the molecule is attached in the vehicle, it is not a factor that decreases or enhances the functioning of ZH on the growth of hair male lambs finished in feedlot. As in the present study, other authors(8) found no difference in productive performance, carcass characteristics, wholesale cut yields, or noncarcass component weights between lambs fed patent and generic ZH. On the other hand, in bulls, the source of ZH did not affect growth, carcass characteristics or meat quality(9).

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It should be noted that neither of the sources of ZH promoted lipolysis in order to exert its hypertrophic effects on skeletal muscle(25). This had already been documented using generic ZH(8,10,22); however, patent ZH had regularly caused a lipolytic effect in fattening sheep(4,20,28). This suggests that the mechanism of lipolytic action associated to patent ZH depends on the dose, as doses of 0.15 mg per kg of LW were used in previous studies, while the present study used 33.3 % less. Nevertheless, further research is required to confirm this finding.

Conclusions and implications The daily addition of 0.10 mg / kg of patent or generic ZH in the finishing diet of hair male lambs substantially promotes muscular hypertrophy in loin and legs, resulting in greater carcass yield, but not in better growth rate, feed efficiency, carcass weight, or meat color. Finally, further studies using different doses of AA-β2 and sheep genotypes are recommended in order to determine whether generic ZH is equally effective as patent ZH for growth promotion.

Acknowledgments

The authors wish to express their gratitude to the Academic Group of Animal Physiology and Genetic for their support for this study, having financed and conducted it in its entirety in their facilities. They are grateful as well to the graduate students (Erick Gómez Aranda, Ricardo Vicente Pérez, Yolanda Osorio Marín, and Arnulfo Vicente Pérez) and laboratory technicians who collaborated in carrying out field phase. The first author also thanks Conacyt for the scholarship it granted him to carry out his doctoral studies. Literature cited: 1. Hernández-Marín JA, Valencia-Posadas M, Ruíz-Nieto JE, Mireles-Arriaga AI, CortezRomero C, Gallegos-Sánchez J. Contribución de la ovinocultura al sector pecuario en México. Agroproductividad 2017;10(3):87-93. 2. Macías-Cruz U, Álvarez-Valenzuela FD, Torrentera-Olivera NG, Velázquez-Morales JV, Correa-Calderón A, Robinson PH, et al. Effect of zilpaterol hydrochloride on feedlot performance and carcass characteristics of ewe lambs during heat-stress conditions. Anim Prod Sci 2010;50(10):983.

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3. Ríos Rincón FG, Barreras-Serrano A, Estrada-Angulo A, Obregón JF, Plascencia-Jorquera A, Portillo-Loera JJ, et al. Effect of level of dietary zilpaterol hydrochloride (β 2agonist) on performance, carcass characteristics and visceral organ mass in hairy lambs fed all-concentrate diets. J Appl Anim Res 2010;38(1):33-38. 4. Lopez-Carlos MA, Ramirez RG, Aguilera-Soto JI, Rodríguez H, Carrillo-Muro O, Méndez-Llorente F. Effect of two beta adrenergic agonists and feeding duration on feedlot performance and carcass characteristics of finishing lambs. Livest Sci 2011;138(1-3):251-258. 5. Rojo-Rubio R, Avendaño-Reyes L, Albarrán B, Vázquez JF, Soto-Navarro SA, Guerra JE, et al. Zilpaterol hydrochloride improves growth performance and carcass traits without affecting wholesale cut yields of hair sheep finished in feedlot. J Appl Anim Res 2018;46(1):375-379. 6. Dávila-Ramírez JL, Avendaño-Reyes L, Macías-Cruz U, Torrentera-Olivera NG, Zamorano-Garcia L, Peña-Ramos A, et al. Effects of zilpaterol hydrochloride and soybean oil supplementation on physicochemical and sensory characteristics of meat from hair lambs. Small Ruminant Res 2013;114(2-3):253-257. 7. Dávila-Ramírez JL, Avendaño-Reyes L, Macías-Cruz U, Peña-Ramos EA, Islava-Lagarda TY, Zamorano-García L, et al. Fatty acid composition and physicochemical and sensory characteristics of meat from ewe lambs supplemented with zilpaterol hydrochloride and soybean oil. Anim Prod Sci 2017;57(4):767. 8. Rivera-Villegas A, Estrada-Angulo A, Castro-Pérez BI, Urías-Estrada JD, Ríos-Rincón FG, Rodríguez-Cordero D, et al. Comparative evaluation of supplemental zilpaterol hydrochloride sources on growth performance, dietary energetics and carcass characteristics of finishing lambs. Asian-Australas J Anim Sci 2019;32(2):209-216. 9. Avendaño-Reyes L, Meraz-Murillo FJ, Pérez-Linares C, Figueroa-Saavedra F, Correa A, Álvarez-Valenzuela FD, et al. Evaluation of the efficacy of Grofactor, a beta-adrenergic agonist based on zilpaterol hydrochloride, using feedlot finishing bulls. J Anim Sci 2016;94(7):2954-2961. 10. Avendaño-Reyes L, Torrentera-Olivera NG, Correa-Calderón A, López-Rincón G, SotoNavarro SA, Rojo-Rubio R, et al. Daily optimal level of a generic beta-agonist based on zilpaterol hydrochloride for feedlot hair lambs. Small Ruminant Res 2018;165:48-53. 11. Ortiz Rodea A, Amezcua Barbosa M, Partida De La Peña JA, González Ronquillo M. Effect of zilpaterol hydrochloride on animal performance and carcass characteristics in sheep: A meta-analysis. J Appl Anim Res 2016;44(1):104-112.

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12. NRC. Nutrient requirements of small ruminants: Sheep, goat, cervids, and new world camelids. Washington, D.C., USA: National Academic Press; 2007. 13. AOAC. Official methods of analysis. 15th ed. Washington, DC: Association of Official Analytical Chemists; 1990. 14. Van Soest PJ, Robertson JB, Lewis BA. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. J Dairy Sci 1991;74(10):3583-3597. 15. NRC. Nutrient requirements of sheep. 6th ed. Washington, D.C., USA: National Academy Press; 1985. 16. Smith GC, Griffin DB, Kenneth JH. Meat evaluation handbook. First ed. American Meat Science Association, ed. IL: American Meat Science Association; 2001. 17. Párez-Casanova PMP. Morphometric dimensions allow differentiation of lamb carcasses for some breeds. Egyptian J Sheep Goat Sci 2013;8(1):167-170. 18. Avendaño-Reyes L, Macías-Cruz U, Álvarez-Valenzuela FD, Águila-Tepato E, Torrentera-Olivera NG, Soto-Navarro SA. Effects of zilpaterol hydrochloride on growth performance, carcass characteristics, and wholesale cut yield of hair-breed ewe lambs consuming feedlot diets under moderate environmental conditions. J Anim Sci 2011;89(12):4188-4194. 19. SAS institute. SAS/STAT: User’s Guide Statistics Released. 2004. 20. Estrada-Angulo A, Barreras-Serrano A, Contreras G, Obregón JF, Robles-Estrada JC, Plascencia A, et al. Influence of level of zilpaterol chlorhydrate supplementation on growth performance and carcass characteristics of feedlot lambs. Small Ruminant Res 2008;80(1-3):107-110. 21. Macías-Cruz U, Avendaño-Reyes L, Álvarez-Valenzuela FD, Torrentera-Olivera NG, Meza-Herrera C, Mellado Bosque M, et al. Crecimiento y características de canal en corderas tratadas con clorhidrato de zilpaterol durante primavera y verano. Rev Mex Cien Pecu 2013;4(1):1-12. 22. López-Baca MÁ, Contreras M, González-Ríos H, Macías-Cruz U, Torrentera N, Valenzuela-Melendres M, et al. Growth, carcass characteristics, cut yields and meat quality of lambs finished with zilpaterol hydrochloride and steroid implant. Meat Sci 2019;158:107890. 23. Muñoz-Osorio GA, Aguilar-Caballero AJ, Sarmiento-Franco LA, Wurzinger M, CámaraSarmiento R. Technologies and strategies for improve hair lamb fattening systems in a tropical region: A review. Rev Ecosist Rec Agropec 2016;3(8):267-277. 704

Rev Mex Cienc Pecu 2022;13(3):690-705

24. Mersmann HJ. Overview of the effects of beta-adrenergic receptor agonists on animal growth including mechanisms of action. J Anim Sci 1998;76(1):160-172. 25. Partida PJM, Casaya RTA, Rubio LMS, Méndez MRD. Efecto del clorhidrato de zilpaterol sobre las caracterísiticas de la canal en cruzas terminales de corderos Kathadin. Vet Mex 2015;2(2):1-13. 26. Ponnampalam EN, Hopkins DL, Bruce H, Li D, Baldi G, Bekhit AE. Causes and contributing factors to “dark cutting” meat: Current trends and future directions: A review. Compr Rev Food Sci Food Saf 2017;16(3):400-430. 27. Suman SP, Joseph P. Myoglobin chemistry and meat color. Annu Rev Food Sci Technol 2013;4(1):79-99. 28. Dávila-Ramírez JL, Macías-Cruz U, Torrentera-Olivera NG, González-Ríos H, SotoNavarro SA, Rojo-Rubio, et al. Effects of zilpaterol hydrochloride and soybean oil supplementation on feedlot performance and carcass characteristics of hair-breed ram lambs under heat stress conditions. J Anim Sci 2014;92(3):1184-1192.

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https://doi.org/10.22319/rmcp.v13i3.6022 Article

Forage availability in Xaraés grass pastures subjected to nitrogen sources of the slow and fast release

Luís Henrique Almeida Matos a Carlindo Santos Rodrigues a,b* Douglas dos Santos Pina a Vagner Maximino Leite a Paula Aguiar Silva a Taiala Cristina de Jesus Pereira a Gleidson Giordano Pinto Carvalho a

School of Veterinary Medicine and Animal Science, Federal University of Bahia, Adhemar de Barros Avenue, 500, Ondina, Zipcode 40170-110, Salvador, Bahia, Brazil. b

Federal Institute of Education, Sciences and Technology Baiano, Bahia, Brazil.

Corresponding author: carlindo.rodrigues@ifbaiano.edu.br

Abstract: The N-(n-butyl) thiophosphoric triamide (NBPT), a urease inhibitor, has been reported as one of the most promising compounds to reduce losses by volatilization, and to maximize the use of urea nitrogen (N) in agricultural systems. A field study was carried out to examine urease inhibitors' potential about volumetric density and forage mass grass (Brachiaria brizantha cv. Xaraés) to N application. The experiment was carried out from September 2017 to September 2018. The experimental design used was complete randomized blocks in the 3×2×4 factorial array, considering: three periods of the year (wet season, dry season, and the transition), two sources of urea (conventional urea and NBPT–treated urea), and four N rates (0, 80, 160 and 240 kg N ha-1 yr-1), replicated three times. Nitrogen sources promoted a

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positive effect (P<0.0001) on bulk density, forage mass, and in the grazing stratum during the wet season and the transition season, with increasing N rates in pastures. The leaf: stem ratio decreased linearly (P<0.0045) as increased N rates, and the higher ratio during the wet season and lower in the dry season of the year. For the rates of 80 kg N ha-1 yr-1, there was a significant difference (P=0.0042) between sources, with greater (P=0.0006) forage mass of 0–30 cm, post-grazing forage mass (P=0.0042) and forage volumetric density (P=0.0006), when utilized the conventional urea. The application of N, regardless of the source, provides an increase in forage mass and volumetric density in Xaraés grass pastures up to a dose of 240 kg N ha-1 yr-1, in the transition season and wet season. Key words: Brachiaria brizantha, Nutrient use efficiency, Pastures, Ammonia volatilization.

Received: 14/07/2021 Accepted: 31/01/2022

Introduction Nitrogen fertilization has been used as an important strategy to increase forage supply in quantity and quality. Nitrogen (N) is an important constituent of proteins and the main nutrient for maintaining productivity. When applied, it is assimilated by the plants, promoting the increase of cellular constituents(1) and, consequently, increasing the regrowth vigor and the total production of green dry matter of the plant under favorable climatic conditions. Urea [CO(NH2)2] is the fertilizer that has had more problems with the topdress soil due to N losses through volatilization of NH3(2,3). Changes in the amount of N available in the system and in the nitrate:ammonium ratio in the soil solution affect N recovery and use efficiency, dry matter yield, and chemical composition of pastures(4). Nevertheless, this source is considered one of the most important due such as high N concentration (46 % N) and lower production costs compared to other N sources(5-9). Many researchers have worked in order to mitigate NH3 losses from urea treating it with a urease inhibitor, of which N-(n-butyl) thiophosphoric triamide (NBPT) is the most studied and utilized compound (10-17). Even though most studies have proved the potential of urea treated with NBPT-based products to reduce NH3 losses(18-21), the benefits of urea treated

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with NBPT compared with untreated urea are less consistent to increase forage production, with no yield difference under some conditions(23,24,25). Such inconsistencies in certain studies probably are associated with the weather and soil conditions at the time of fertilizer application. The increasing N rates(26,27), the application of urea over to soils with high moisture and temperature, usually cause enhanced NH3 loss(20,26) and hence, makes the use of urease inhibitors more attractive as a tool to increase N use efficiency. Conversely, low temperature or dry conditions may limit urea hydrolysis and, thus NH3 losses(20,28). In view of the above, there are uncertainties regarding the advantages of using NBPT-treated urea under such conditions for increasing pasture yield. This study aimed to evaluate the effect of the potential of urease inhibitors regarding forage volumetric density and in the forage mass grass (Brachiaria brizantha cv. Xaraés) to N application.

Material and methods Location of the experiment and climatic conditions

The experiment was carried out from September 2017 to September 2018, on the Talitha farm located in the district of Monte Gordo, Camaçari city, state of Bahia, Brazil, located at 12°41'51" latitude, 38°19'27" longitude, and 36 m altitude. The average annual temperature is around 23.3°C and an average rainfall of 1,466.5 mm. The soil in the experimental area with free sand soil showed the following chemical and physical characteristics: organic matter (OM)= 21.0 g dm-3; pH (H2O)= 5.3; P= 4.0 mg dm-3; K= 0.2 mmolc dm-3; Ca= 13.0 mmolc dm-3; Mg= 7.0 mmolc dm-3; Na= 0.0 mmolc dm-3; Al= 0.0 mmolc dm-3; H + Al = 18.0 mmolc dm-3; SB= 20.0 mmolc dm-3; CTC= 38.0 mmolc dm-3; V= 53%; sand= 894 g dm-3; slime 18 g dm-3; clay= 88 g dm-3.

Treatments and experimental design

The experimental design used was complete randomized blocks in the 3 × 2 × 4 factorial array, considering: three periods of the year (wet season, dry season, and the transition), two sources of urea (conventional urea and NBPT-treated urea), and four rates N (0, 80, 160 and 240 kg N ha-1 yr-1), with three replications. The experimental period lasted 380 d, being

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monitored temperature, rainfall index (Figure 1), and water balance (Figure 2) using a water storage capacity of 50 mm(29).

40 35 30 25 20 15 10 5 0

350 300 250 200 150 100 50 0

Rainfall (mm)

Temperature co

Figure 1 : Precipitation index and monthly average temperature (2017 to 2018)

Rainfall Max Min Mean

The fertilizing and planting operations were implemented on June 26, 2016. For planting 15 kg ha-1 was used of Brachiaria brizantha cv. Xaraés seeds, 70 kg P2O5 ha-1 (single superphosphate), 60 kg KCL ha-1 (potassium chloride), and 50 kg of N ha-1 in coverage, throughout the experimental area. The total experimental area including corridors, management area, and spacing between plots was 0.66 ha, divided into three blocks, each plot measuring 10 m × 10 m, totaling 100 m2. All plots received an application of 30 kg P2O5 ha-1, 200 kg KCL ha-1. The application of N was done in coverage, except in the 0 rates of N. Superphosphate was applied in a single dose in the first cycle, but potassium chloride and N were divided into four applications of equal amounts (beginning and end of the rainy period). Figure 2: Extract from the average water balance (2017 to 2019)

The monitoring of the canopy height started after post-grazing, performed three times a week until reaching pre-grazing height (30 cm). Twelve readings were performed for each experimental unit using a graduated stick and a radiographic film sheet, according to 709

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Pequeno(30). The pasture defoliation was carried out by adding or removing regulating animals (“mob grazing”)(31) simulating a rotational grazing scenario.

Traits measured

The forage cuts were performed at a height above 15 cm, which resulted in the forage mass in the grazing stratum, and at ground level (0 – 15 cm in height) in the pre-grazing, the result of the sum of these cuts corresponded to the mass of forage of 0–30 cm. As for the postgrazing forage mass sampling, the cuts were performed at ground level. Forage samples corresponding to each of the canopy strata were weighed and then placed in an air forced circulation oven at 65 °C until constant weight. Subsequently, the samples were used for the analysis of dry matter (DM) content using the Next Infrared Reflectance Spectroscopy System (NIRS) according to procedures Marten et al (32). The reflectance data of the samples, in the wavelength range of 700–2,500 nm, were stored by a spectrometer (model Unity Scientific SpectraStar ™ 2500 XL). With the result of dry matter yield (DMY), the forage volumetric density was determined calculated by dividing the forage mass in the pre-and post-grazing by the corresponding pasture height, with values expressed in kg ha-1 cm-1 of DM, according to the methodology of Stobbs(33). Thus, the forage volumetric density (kg DMY ha-1 cm-1) was obtained, corresponding to the stratum from 0 to 30 cm in height, and the volumetric density of the grazing stratum (kg DM ha-1 cm-1) from 15 to 30 cm in height. After removing the animals, two groups of ten tillers were identified at random in different areas of the experimental unit (paddock)(34), and at the end of the grazing cycle, they were cut close to the soil surface. Then, the morphological separation in leaf, stem, and dead material was carried out with respective weighing and drying at 65 oC for 72 h for further analysis. Subsequently, the leaf:stem ratio was determined by dividing leaf grams and stem grams.

Statistical analysis

The variables were subjected to analysis of variance using the PROC MIXED of SAS (Statistical Analysis System - version 9.2 for Windows®) as described by model below: Yijkl=µ + Bi +Sj + Dk+ (SxD)jk + eijk +Pl + (SxP)jl + (DxP) kl + (SxDxP)jkl + Ɛijkl

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Where: Yijkl = observed value; μ= overall mean; Bi= random effect of the blocks; Sj = fixed effect of the N source; Dk= fixed effect of N rate; (SxD)jk= interaction effect of source x rate; eijk = random error associated with source and rate of N; Pl = fixed effect of the period of the year; (SxP)jl = interaction effect of source x period; (DxP) jL= interaction effect of rate x period; (SxDxPx) jkl = interaction effect of source x rate x period; Ɛijkl = random error associated with period effect. The results for the of quantitative factors (rate) were evaluated by regression analysis, and for qualitative factors (source and period) the Tukey test, both considering 5% probability to type I error.

Results Forage mass (0–30 cm) varied with sources of urea (P=0.0145), periods of the year (P=0.0230), N rates (P<0.0001), and interaction (P=0.0020). In the interaction, there was a positive linear effect (P≤0.05) for the conventional urea and NBPT-treated urea, in the forage mass of 0–30 cm, as there was an increase in rates in pastures, however with similar mass values between sources. However, for the rate of 80 kg of N ha-1 yr-1 there was a significant difference (P=0.0006) between the sources (conventional urea and NBPT-treated), with the means, respectively, of 4,093.28 and 3,450.44 kg DM ha-1 (Table 1). A positive linear effect was observed during the wet season and the in transition, resulting in more N pasture growth as N rate increased. However, in the dry season, the N rate had no effect on pasture growth (Table 1). The pastures that did not receive fertilization and those that received rates of 80 kg of N ha-1 yr-1, the forage mass (0–30 cm) did not show significant differences (P>0.05) with the periods of the year. The rates of 160 (P=0.0372) and 240 kg of N ha-1 yr-1 the forage mass was higher (P≤0.05) during the wet season and the transition when compared to the dry season. For forage mass in the grazing stratum (15–30 cm), significant effects (P≤0.05) were observed too for the periods of the year, N rates, and interaction. In the interaction, there

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was a positive linear effect during the wet season (P<0.0001) and the transition (P<0.0001) season, plants that had more water were able to respond to the N added. However, for the dry season there was no rates effect (P>0.05) when adjusted to the linear and quadratic functions (Table 1). When comparing periods of the year with N fertilization rates, the forage mass in the grazing stratum did not differ (P>0.05) for pastures that did not receive fertilization and those that received rates of 160 kg of N ha-1 yr-1. However, for the rates of 80 and 240 kg of N ha-1 yr-1, the periods during the wet season and the transition, presented higher mass production in the potentially grazing stratum (Table 1). Table 1: Forage available by stratum of the Xaraés grass in response to the N rates during periods of the year (wet season, dry season, and the transition) Rates (kg N ha-1 yr-1) Effect Period of year 0 80 160 240 L Q -1 Forage mass (0 – 30 cm) (kg DM ha ) Wet 3136.2a 3765.5a 3876.1a 4023.9a <0.00011 0.2534 Dry 3477.9a 3730.9a 3339.2b 3350.3b 0.2393 0.4207 a a b a 2 Transition 3360.9 3819.2 3527.9 4330.5 <0.0001 0.1022 P-value 0.2456 0.9174 0.0372 <0.0001 -1 Forage mass in the grazing stratum (kg DM ha ) Wet 1005.8 a 1152.6 a 1164.1 a 1259.3 a <0.00013 0.4761 Dry 1021.0a 904.5 b 1048.2 a 990.9 b 0.8301 0.5496 a a a a 4 Transition 947.1 1121.9 1105.7 1338.1 <0.0001 0.5228 P-value 0.4946 0.0001 0.1355 <0.0001 -1 Post-grazing forage mass (kg DM ha ) Wet 1879.0b 2346.5a 2250.5a 2481.8a 0.00045 0.4476 Dry 2394.6a 2226.7a 2344.4a 2245.1ab 0.4604 0.7318 b a a b Transition 2045.3 2083.1 2164.8 2050.4 0.8395 0.2411 P-value 0.0022 0.1836 0.4484 0.0136 ab

L= linear; Q= quadratic; N= nitrogen; DM= Dry matter. Mean values followed by different letters, in the same column, are different at 5 % probability by Tukey’s test. Regression equations: 1Ŷ = 3.4671x + 3284.4 R² = 0.84; 2Ŷ = 3.2719x + 3367 R² = 0.63; 3Ŷ = 0.9649x + 1029.7 R² = 0.91;4Ŷ = 1.4463x + 954.66 R² = 0.87; 5Ŷ = 2.1404x + 1982.6 R² = 0.73

In post-grazing, the forage mass of the cultivar Xaraés a significant effect (P≤0.05) was observed depending on the period of the year, period × N rates interaction, and source × N rates interaction. In the interaction, a positive linear effect was observed during the wet season as N rates increased (Table 1). The pastures, where fertilizer was not applied, had

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higher (P=0.0022) forage mass during the dry season. However, no differences (P>0.05) were observed for the rates of 80 and 160 kg of N ha-1 yr-1 over the period of the year. Pastures that received rates of 240 kg of N ha-1 yr-1 had higher forage mass during the wet season, but the dry season was similar (P>0.05) to the transition season. In the interaction, the fast-release N source (conventional urea) was not influenced (P>0.05) by the rates when adjusted to the functions linear and quadratic for forage mass after grazing. However, the interaction showed a positive linear effect (P= 0.0358), as the N rates increased, when using the NBPT-treated urea. For the rate of 80 kg N ha-1 yr-1, there was a significant difference (P=0.0042) between sources, with higher post-grazing forage mass using conventional urea and lower the NBPT-treated urea, with means, respectively of 2,391.94 and 2,045.60 kg of DM ha-1. Table 2: Forage mass and volumetric density in Xaraés grass pastures in response to N application under pre and post grazing conditions Rates (kg N ha-1 yr-1) Effect Source 0 80 160 240 L Q -1 Forage mass of 0–30 cm (kg DM ha ) Urea 3325.0 4093.3 3702.4 3891.6 0.00171 0.1816 2 NBPT 3325.0 3450.4 3459.7 3911.6 0.0172 0.3521 P-value 1.0000 0.0006 0.1690 0.9052 -1 Post-grazing forage (kg DM ha ) Urea 2106.3 2391.9 2321.0 2188.8 0.7046 0.2139 NBPT 2106.3 2045.6 2185.5 2329.4 0.03583 0.0534 P-value 1.0000 0.0042 0.2439 0.2268 -1 Forage volumetric density (kg DM ha cm-1) Urea NBPT P-value

110.8 110.8 1.0000

136.4 115.0 0.0006

123.4 115.3 0.1689

129.7 130.4 0.9049

0.00174 0.01735

0.1816 0.3520

L= linear; Q= quadratic; N= nitrogen; DM= Dry matter; NBPT= N-(n-butyl) thiophosphoric triamide. Regression equations: 1Ŷ = 1.6363x + 3556.7 R² = 0.27; 2Ŷ = 2.2112x + 3271.3 R² = 0.79; 3Ŷ = 1.0115x + 2045.3 R² = 0.73; 4Ŷ = 0.0546x + 118.55 R² = 0.27; 5Ŷ = 0.0737x + 109.04 R² = 0.79

For volumetric density (0–30 cm), under pre-grazing conditions, there were significant differences (P≤0.05) for source, period of the year (P=0.0231), N rates (P<0.0001), source × N rates interaction (P=0.0305), and period × N rates interaction (P<0.0020). In the interaction, a positive linear effect (P≤0.05) was observed during the wet season and the transition; plants that had more water were able to respond to the N added. In the dry season, they were not influenced (P>0.05) by the N rates when they adjusted to a linear and quadratic (Table 3).

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Table 3: Forage volumetric density in Xaraés grass pastures in response to N doses during periods of the year (wet season, dry season, and the transition) Rates (kg N ha year) Effect Period of year 0 80 160 240 L Q -1 -1 Forage volumetric density (kg DM ha cm ) Wet 104.5 a 125.5 a 129.2 a 134.1 a <0.00012 0.2535 Dry 115.9 a 124.4 a 111.3 b 111.7 b 0.2392 0.4206 a a ab a 1 Transition 112.0 127.3 117.6 144.4 <0.0001 0.1022 P-value 0.2456 0.9174 0.0372 <0.0001 -1 Volumetric density in the grazing stratum (kg DM ha cm-1) Wet 67.1 a 76.8 a 77.6 a 84.0 a <0.00014 0.4760 Dry 68.1 a 60.3b 69.9 a 66.1b 0.8304 0.5493 Transition 63.1 a 74.8 a 73.7 a 89.2 a <0.00013 0.5231 P-value 0.4946 0.0001 0.1357 <0.0001 ab

L= linear; Q= quadratic; N= nitrogen; DM= Dry matter. Mean values followed by different letters, in the same column, are significantly different at 5 % probability by Tukey’s test. Regression equations: 1Ŷ= 0.1156x + 109.48 R² = 0.84; 2Ŷ= 0.1091x + 112.24 R² = 0,63; 3Ŷ= 0.0643x + 68.649 R² = 0.91 4Ŷ= 0.0964x + 63.648 R² = 0.87.

When evaluating the volumetric density in the grazing stratum, there was a significant effect (P<0.0025) in the period of year × N rates interaction. The interaction showed a positive linear effect was during the wet season and the transition, as there was an increase in N rates in the pastures. However, during the dry season, they were not influenced (P>0.05) by the rates when the adjusted function was linear and quadratic (Table 1). The leaf:stem ratio was influenced (P≤0.05) by the periods of the year and N rates. Higher leaf:stem ratio was observed during the wet season and lower in the dry season (Figure 3a). The N sources did not influence the leaf:stem ratio (P>0.05). However, the increase in N rates in pastures reflected a linear reduction (P≤0.05) in the leaf:stem ratio (Figure 3b).

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Figure 3: Leaf: stem ratio in Xaraés grass in response to N rates during periods of the year [wet season (W), dry season (D), and the transition (T)] 2.25 (a)

Leaf: stem Ratio

Leaf: Steam ratio

2.5

1.56 (b) 1.27 (c)

1.5 1 0.5

0 W

a abc

Ŷ = -0.0012x + 1.8334 R² = 0.92

1.90 1.85 1.80 1.75 1.70 1.65 1.60 1.55 1.50

D Period of the year

160

240

Nitrogen rates ( Kg N Ha-1 Year-1)

b Means followed by the same letter in the column do not differ from each other by the Tukey test, at the 5% probability level.

Discussion Nitrogen is an important constituent of proteins and the main nutrient for maintaining productivity(35). When applied, it is assimilated by plants, promoting the increase of cellular constituents(1). Furthermore, it strongly influences the appearance and elongation of leaves(36,37).Thus, N fertilization acts directly on the growth rate, which in turn affects the increase and availability of forage mass in the pasture. The forage mass, characterized by the height of the canopy in the pre-grazing, was influenced by the rates of N, which promoted a positive linear effect in the wet season and the transition (Table 1), however in different magnitudes, according to the slope coefficient of the straight line. In the transition period for each kilogram of N applied, increases of 3.2719 kg DM ha-1, and in the wet season values of 3.4671 kg DM ha-1 of available forage mass can be expected. These variations in the magnitude of responses to N fertilization can also be related to weather conditions throughout the year (Figure 1), with temperature and humidity in the favorable range for the development of Xaraés grass. According to Minson(38), the availability of forage mass must be greater than 2,000 kg of DM ha-1, as lower values, promote longer grazing time and reduced pasture consumption by the animals. It is noteworthy that pastures that did not receive N fertilization presented values higher than the above, with averages of 3,360.9; 3,477.9, and 3,136.2 kg MS ha-1 in the transition season, dry, and the wet season, respectively (Table 1). This situation can be

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attributed to defoliation management, with a height goal established respecting the ecophysiological limits of the forage plant. The grazing strategy was defined according to the recommendations of Pedreira et al(39) and Sousa et al(40) for the cultivar Xaraés, under intermittent stocking, with the entry of animals to pasture occurring with a pre-grazing height of 30 cm, corresponding to 95 % of light interception (IL), and exit when lowered to 15 cm. Thus, the range of 15–30 cm in height was considered to determine the forage mass available in the grazing stratum (Table 1), which responded to N rates with a pattern similar to the forage mass of 0–30 cm. However, the forage mass in the grazing stratum, in theory, is what will be consumed by the animal during the time of occupation of the paddock. Thus, this stratum will directly influence the animal response, since, in practice, the availability of forage mass is associated with individual consumption by the animals and, consequently, greater performance(41). However, the responses of plants and animals under grazing are conditioned by the structure of the forage canopy(42), which has been characterized by variables such as canopy light interception, sward height, forage mass, and volumetric density. Considering that the pre-grazing height of the pastures was the same for all treatments, fixed at 30 cm. Thus, the variations obtained in the structure of pastures throughout the experimental period were results isolated and/or from the interaction of the sources of variations, of the periods of the year (wet season, dry season, and the transition), of the rates (0, 80, 160, and 240 kg of N ha-1 yr-1) and sources N (conventional urea and NBPT-treated). The forage volumetric density (Tables 2 and 3) and the leaf:stem ratio (Figure 3), evaluated in this study add to the results obtained for forage mass, since they are the relevant components in the structure of the pasture that influence behavior ingestive of the grazing animals(43). A linear increasing effect was observed for the forage volumetric density and the volumetric density of the grazing stratum during the transition season, and the wet season (Table 3). The forage volumetric density, for each kilogram of N, applied, corresponded to the averages of 0.1091 and 0.1156 kg DM ha-1cm-1 for the wet season and the transition, respectively. However, for the volumetric density of the grazing stratum, these increases were 0.0964 and 0.0643 kg DM ha-1cm-1 respectively. Increases in forage volumetric density favor the apprehension by the animals during grazing(44), preferably with a greater proportion of leaf blades. The main plant structures that make up the forage volumetric density in the pasture are the leaf blade, stalk, and the ratio (leaf blade/stem), which constitute a relevant tool for the management of forage plants. In the critical limit condition, since a leaf/stem ratio less than 1 means greater production of stems, and these increase biomass production, implying a reduction in the quality of forage produced(45).

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The leaf:stem ratio had a negative linear effect, with reductions of 0.0012 points for each kg of N applied to pastures (Figure 3b). However, despite the decreasing slope coefficient, the lowest value of this relationship was above the recommended limit, with a ratio of 1.55 points for rates of 240 kg of N ha-1 yr-1(Figure 3b). This decrease can be explained by greater plant growth, but particularly the higher growth of the stems, with higher rates associated with temperature and rainfall conditions during the wet season, and the transition. Increases in the levels of N available to the plant cause an increase in tiller density(46), followed by an increase in the plant growth rate, which can promote early competition for light in the canopy, favoring elongation of the pseudostem(47-50), thus resulting in a reduction in the leaf:stem ratio. Nonetheless, the adopted pasture management strategy avoided excessive culm accumulation, and although present, they were of younger culms that were easily harvested by the animal, that is, consisting basically of pseudostem(39), formed by leaf sheath invaginations. The blade:stem ratio was influenced by the periods of the year (Figure 3a), with the highest ratio in the wet season (2.25) and the lowest (1.27) in the dry season. Regardless of the season, the blade:stem ratio found in this study are above the pre-established critical limit, configuring a smaller proportion of stalk and demonstrating that the grazing strategy was efficient in controlling stalk elongation, ensuring a better quality of available forage. However, it is important to note that during the dry season, the forage mass of 0–30 cm did not differ from the wet season and the transition in pastures without N fertilization and with 80 kg of N ha-1 yr-1 (Table 1). For forage mass in the grazing stratum, this condition too was observed in pastures fertilized with 80 and 160 kg of N ha-1 yr-1. Considering the leaf:stem ratio in the period of the year (Figure 3b), this situation demonstrates that the predominance of the stalk proportion, which can affect forage quality, impacting in intake, and animal performance. The available forage mass is the result of forage accumulation during the regrowth period, which in turn is influenced by the post-grazing residue. There was a positive linear effect in the wet season (P<0.0004) for post-grazing forage mass (Table 1), for every kilogram of N applied, approximately 2,1404 kg DM ha-1. This condition provides vigorous regrowth after grazing since greater green leaf remnant can be translated as greater photosynthetic apparatus for the plant to initiate regrowth. Regarding fast release sources (conventional urea) and slow release sources (urea treated with NBPT) of N, it was expected that the use of protected urea would promote higher production of forage mass in all treatments with NBPT; due to the slow release of the N, since it would have lower losses of NH3 by volatilization with greater use of N by the plant. However, this did not occur, only at the rate of 80 kg of N ha-1 yr-1, differences were verified for forage mass in the pre-and post-grazing, and consequently, in volumetric density, with higher values in pastures fertilized with conventional urea (Table 2). 717

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These results can be attributed to the synergistic effect of some factors: such as crop residues(51,52) which in pastures would be the post grazing residue, concentration of NBPTtreated urea(6), and climatic conditions with high temperatures and soil moisture during the period of fertilizer application(51,52,53). In the present study, efficient grazing management promoted adequate grazing residue (Table 1) and consequently increased soil vegetation cover, which possibly affected the efficiency of NBPT-treated urea, when applied superficially in systems with leftovers. crop or post-grazing residue, which provides greater ground cover, the efficiency of NBPT-treated urea may be low due to the high urease activity(51). The source NBPT-treated urea used is a commercial product, which in Brazil is marketed at a concentration of 530 mg kg-1(52). In a work carried out by the same author, using NBPTtreated urea in sugarcane cultivation with green straw cover, the amount of straw in the soil affected the efficiency of NBPT-treated urea. In this case, the recommendation was to double the 530 mg kg-1 concentration of NBPT in urea, as a way to increase its efficiency. Possibly, associated with the post grazing residue at the time of fertilization, the concentration of NBPT-treated urea has contributed to the results found, being indicative of evaluations of the use of NBPT in pastures with higher concentrations. In addition, fertilizer applications were preceded by rain (Figures 1 and 2) and with favorable temperature, both in the wet season and the transition, a situation that favors the acceleration of NBPT degradation and increased NH3 volatilization(54,55). The correct grazing management ensured adequate post-grazing residue, and the increase in N rates reflected in greater forage production, affecting the quantity and the leaf-stem ratio, that was an indirect way of inferring the quality of the forage, and efficiency in the use of the forage produced. However, it may have compromised the efficiency of slow-release urea (NBPT) in promoting an increase in forage mass productivity.

Conclusions and implications Slow release nitrogen source in Xaráes grass pasture managed with pre- and post-grazing heights of 30 and 15 cm, has no effect on forage availability. Therefore, the application of N, regardless of the sources of slow or fast release, provides an increase in forage mass and volumetric density in pasture up to the rate of 240 kg N ha-1 year-1, during the wet season and the transition. It is necessary to carry out research, evaluating the increase in the concentration of NBPT-treated urea in tropical grass pastures with different post-grazing height.

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Acknowledgments

The authors gratefully acknowledge CNPq and CAPES for the scholarships awarded. Literature cited: 1.

Van Soest PJ. Nutritional ecology of the ruminant. 2. ed. Ithaca: Cornell Universtity 1994.

Gao WL, Yang H, Kou L, Li SG. Efeitos da deposição de nitrogênio e adubação nas transformações de N em solos florestais: uma revisão. J Solos e Sed 2015;15(4):863879.

Cameron KC, Di HJ, Moir JL. Perdas de nitrogênio do sistema solo/planta: uma revisão. Ann Appl Bio 2013;162 (2):145-173.

Primavesi AC, Primavesi O, Corrêa LA, Silva AG, Cantarella H. Nitrate leaching in heavily nitrogen fertilized coastcross pasture. R Bras Zootec 2006;35:683-690.

Bortoletto-Santos R, Guimarães GGF, Roncato Junior V, Cruz DF, Polito WL, Ribeiro C. Biodegradable oil-based polymeric coatings on urea fertilizer: N release kinetic transformations of urea in soil. Sci Agric 2020;77(e20180033). https://doi.org/10.1590/1678-992x-2018-0033.

Cantarella H, Otto R, Soares JR, Silva AGB. Agronomic efficiency of NBPT as a urease inhibitor: A review. J Adv Res 2018;13:19-27. https://doi.org/10.1016/j.jare.2018.05.008.

Guimarães GG, Mulvaney RL, Cantarutti RB, Teixeira BC, Vergütz L. Value of copper, zinc, and oxidized charcoal for increasing forage efficiency of urea N uptake. Agric Ecosyst Environ 2016; 224:157-165.

Ibrahim KRM, Babadi FE, Yunus R. Comparative performance of different urea coating materials for slow release. Particuology 2014;17:165-172. https://doi.org/10.1016/j.partic.2014.03.009.

Ni B, Liu M, Lü S. Multifunctional slow-release urea fertilizer from ethylcellulose and superabsorbent coated formulations. Chem Eng J 2009;155(3):892-898. https://doi.org/10.1016/j.cej.2009.08.025.

10. Lasisi AA, Akinremi OO, Zhang Q, Kumaragamage D. Efficiency of fall versus spring applied urea‐based fertilizers treated with urease and nitrification inhibitors I. Ammonia volatilization and mitigation by NBPT. Soil Sci Soc Am J 2020. https://doi.org/10.1002/saj2.20062. 719

Rev Mex Cienc Pecu 2022;13(3):706-724

11. Silva AGB, Sequeira CH, Sermarini RA, Otto R. Urease inhibitor NBPT on ammonia volatilization and crop productivity: a meta-analysis. Agron J 2017;109(1):1. https://doi.org/10.2134/agronj2016.04.0200. 12. Singh J, Kunhikrishnan A, Bolan NS, Saggar S. Impact of urease inhibitor on ammonia and nitrous oxide emissions from temperate pasture soil cores receiving urea fertilizer and cattle urine. Sci Total Environ 2013;65:56–63. 13. Halvorson AD, Snyder CS, Blaylock AD, Del Grosso SJ. Enhanced-efficiency nitrogen fertilizers: Potential role in nitrous oxide emission mitigation. Agron J 2014;106(2): 715–722. https://doi.org/10.2134/agronj2013.0081. 14. Trenkel ME. Slow-and controlled-release and stabilized fertilizers: An option for enhancing nutrient use efficiency in agriculture. International Fertilizer Industry Association (IFA), Paris. 2010. 15. Watson CJ, Laughlin RJ, McGeough KL. Modification of nitrogen fertilizers using inhibitors: Opportunities and potentials for improving nitrogen use efficiency. Int Fert Soc Proc. Colchester, UK. 2009; 658. 16. Gioacchini P, Nastri A, Marzadori C, Giovannini C, Antisari LV, Gessa C. Influence of urease and nitrification inhibitors on N losses from soils fertilized with urea. Biol Fertil Soils 2002;36:129–135. https://doi.org/10.1007/s00374-002-0521-1. 17. Carmona G, Christianson CB, Byrnes BH. Temperature and low concentration effects of the urease inhibitor N-(n-butyl) thiophosphoric triamide (n-BTPT) on ammonia volatilization from urea. Soil Biol Biochem 1990;22(7):933–937. https://doi.org/10.1016/0038-0717(90)90132-J. 18. Chagas PHM, Gouveia GCC, Costa GGS, Barbosa WFS, Alves AC. Volatilização de amônia em pastagem adubada com fontes nitrogenadas. J Neotrop Agric 2017;4(2):7680. 19. Soares JR, Cantarella H, Menegale MLC. Ammonia volatilization losses from surfaceapplied urea with urease and nitrification inhibitors. Soil Biology Biochem 2012;52:82– 89. https://doi.org/10.1016/j.soilbio.2012.04.019 20. Cantarella H, Trivelin PCO, Contin TLM, Dias FLF, Rossetto R, Marcelino R, Coimbra RB, Quaggio JA. Ammonia volatilization from urease inhibitor-treated urea applied to sugarcane trash blankets. Sci Agric 2008;65(4):397-401.

720

Rev Mex Cienc Pecu 2022;13(3):706-724

21. Watson CJ, Miller H, Poland P, Kilpatrick DJ, Allen MDB, Garrett MK, Christianson C. Soil properties and the ability of the urease inhibitor N- (n-butyl) thiophosphoric triamide (n BTPT) to reduce ammonia volatilization from surface-applied urea. Soil Biol Biochem 1994;26(9):1165–1171. https://doi.org/10.1016/0038-0717(94)90139-2. 22. Silveira ML, Vendramini JMB, Sellers B, Monteiro FA, Artur AG, Dupas E. Bahiagrass response and N loss from selected N fertilized sources. Grass Forage Sci 2015;70(1):154-160. 23. Zavaschi E, Faria LDA, Vitti GC, Nascimento CADC, Moura TAD, Vale DWD, et al. Ammonia volatilization and yield components after application of polymer-coated urea to maize. R Bras Ciênc Solo 2014;38(4):1200-1206. https://doi.org/10.1590/S010006832014000400016. 24. Espindula MC, Rocha VS, Souza MA, Capanharo M, Paula GS. Rates of urea with or without urease inhibitor for topdressing wheat. Chil J Agric Res 2013;73(2):160–167. https://doi.org/10.4067/S0718-58392013000200012. 25. Massey CG, Norman RJ, Jr EEG, DeLong RE, Golden BR. Bermuda grass forage yield and ammonia volatilization as affected by nitrogen fertilization. Soil fertility and plant nutrition. Soil Sci Soc Am J 2011;75:638–648. 26. Pan B, Lam SK, Mosier A, Luo Y, Chen D. Ammonia volatilization from synthetic fertilizers and its mitigation strategies: a global synthesis. Agric Ecosyst Environ 2016; 232:283-289. https://doi.org/10.1016/j.agee.2016.08.019. 27. Turner DA, Edis RB, Chen D, Freney JR, Denmead OT, Christie R. Determination and mitigation of ammonia loss from urea applied to winter wheat with N- (n-butyl) thiophosphorictriamide. Agric Ecosyst Environ 2010;37(3–4):261-266. 28. Schraml M, Gutser R, Maier H, Schmidhalter U. Ammonia loss from urea in grassland and its mitigation by the new urease inhibitor 2-NPT. J Agric Sci 2016;154(8):14531462. https://doi.org/10.1017/S0021859616000022. 29. Thornthwaite CW, Mather RJ. The water balance. New Gersey: Laboratory of climatology 1955;104. 30. Pequeno DNL. Intensidade como condicionante da estrutura do dossel e da assimilação de carbono de pastos de capim Xaraés [Brachiaria brizantha (A. Rich) Stapf. cv. Xaraés sob lotação continua .75f. Escola Superior de Agricultura “Luiz de Queiroz” – Esalq, 2010.

721

Rev Mex Cienc Pecu 2022;13(3):706-724

31. Mislevy P, Mott GO, Martin FG. Screening perennial forages by mob grazing technique. In: Smith JA, Hays VW, eds. Proc. Int. Grassl. Congr. 14th, Lexington, KY. 15–24 June 1981. Boulder, CO: Westview Press; 1983:516-519. 32. Marten GC, Shenk JS and Barton II FE. Near-infrared reflectance spectroscopy (NIRS), analysis of forage quality. Washington: USDA, ARS (Agriculture Handbook, 643), 1985. 33. Stobbs, THA. The effect of plant structure on the intake of tropical pasture. I. Variation in the bite size of grazing cattle. Aust J Agric Res 1973;24(6):809-819. 34. Grant SA, Marriot CA. Detailed studies of grazed sward-techniques and conclusions. J Agric Sci 1994;122(1):1-6. 35. Galindo FS, Buzetti S, Teixeira Filho MCM, Dupas E, Ludkiewicz MGZ. Application of different nitrogen doses to increase nitrogen efficiency in Mombasa guinegrass (Panicum maximum cv. Mombasa) at dry and rainy seasons. Aust J Crop Sci 2017;11 (12):1657-1664. 36. Pereira LET, Paiva AJ, Guarda VD, Pereira PM, Caminha FO, Silva SC. Eficiência de aproveitamento da forragem do capim-marandu em estoque contínuo submetido à fertilização com nitrogênio. Sci Agric 2015;72(2):114-123. https://doi.org/10.1590/0103-9016-2014-0013. 37. Martuscello J, Rios J, Ferreira M, Assis J, Braz T, Cunha D. Produção e morfogênese de capim BRS Tamani sob diferentes doses de nitrogênio e intensidades de desfolhação. Boletim de Indústria Animal 2019;76:1-10. https://doi.org/10.17523/bia.2019.v76.e144.1 38. Minson DJ . Forage in ruminant nutrition. San Diego: Academic Press, 1990. 39. Pedreira BC, Pedreira CGS, Silva SC. Herbage accumulation during regrowth of Xaraés palisadegrass submitted to rotational stocking strategies. R Bras Zootec 2009;38 (4):618-625. 40. Sousa BMDL, Nascimento Júnior DD, Rodrigues CS, Monteiro HCDF, Silva SCD, Fonseca DMD, Sbrissia AF. Características morfogênicas e estruturais do capim-xaraés submetido a alturas de corte. R Bras Zootec 2011;40(1):53-59. 41. Hodgson J. Grazing management. Science into practice. Longman Group UK, 1990. 42. Carvalho PDF, Ribeiro Filho HMN, Poli CHEC, Moraes AD, Delegarde R. Importância da estrutura da pastagem na ingestão e seleção de dietas pelo animal em pastejo. Reunião Anual da Sociedade Brasileira de Zootecnia 2001;38:871.

722

Rev Mex Cienc Pecu 2022;13(3):706-724

43. Stobbs THA. The effect of plant structure on the intake of tropical pasture. I. Variation in the bite size of grazing cattle. Aust J Agric Res 1973;24(6):809-819. 44. Palhano AL, Carvalho PCDF, Dittrich JR, Moraes AD, Barreto MZ, Santos MCFD. Estrutura da pastagem e padrões de desfolhação em capim-mombaça em diferentes alturas do dossel forrageiro. R Bras Zoote 2005;34(6):1860-1870. 45. Brâncio PA, Euclides VPB, Nascimento Júnior DD, Fonseca DMD, Almeida RGD, Macedo MCM, Barbosa RA. Avaliação de três cultivares de Panicum maximum Jacq. sob pastejo: disponibilidade de forragem, altura do resíduo pós-pastejo e participação de folhas, colmos e material morto. R Bras Zootec 2003;32(1):55-63. 46. Santos MER, Souza BDL, Rocha GDO, Freitas CAS, Silveira MCT, Sousa DOC. Estrutura do dossel e características de perfilhos em pastos de capim-piatã manejados com doses de nitrogênio e períodos de diferimento variáveis. Cienc Anim Bras 2017; 18:1-13. 47. Gastal F, Nelson CJ. Nitrogen use within the growing leaf blade of tall fescue. Plant Physiology 1994;105(1):191-197. 48. Cruz, P, Boval, M. Effect of nitrogen on some morphogenetic traits of temperate and tropical perennial forage grasses. In: Lemaire G, Hodgson J, Moraes A, editors. Grassland ecophysiology and grazing ecology. Centre for Agriculture and Biosciences International; London, UK. 2000:151-168. 49. Sbrissia AF, Silva SC. O ecossistema de pastagens e a produção animal. Anais da Reunião Anual da Sociedade Brasileira de Zootecnia. Sociedade Brasileira de Zootecnia: Brasília, DF, Brazil. 2001. 50. Mesquita P, Silva SC, Paiva AJ, Caminha FO, Pereira LET, Guarda VD, Nascimento Júnior D. Structural characteristics of marandu palisadegrass swards subjected to continuous stocking and contrasting rhythms of growth. Sci Agric 2010;67(1):23-30. https://doi.org/10.1590/S0103-90162010000100004. 51. Tasca FA, Ernani PR, Rogeri DA, Gatiboni LC, Cassol PC. Volatilização de amônia do solo após a aplicação de ureia convencional ou com inibidor de uréase. Rev Bras Ciência do Solo 2011;35(2):493-502. https://doi.org/10.1590/S0100-06832011000200018. 52. Mira AB, Cantarella H, Souza-Netto GJM, Moreira LA, Kamogawa MY, Otto R. Optimizing urease inhibitor usage to reduce ammonia emission following urea application over crop residues. Agric, Ecosyst Environmen 2017;248:105–112. https://doi.org/10.1016/j.agee.2017.07.032.

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Rev Mex Cienc Pecu 2022;13(3):706-724

53. Bouwmeester RJB, Vlek PLG, Stumpe JM. Effect of environmental factors on ammonia volatilization from a urea-fertilized soil. Soil Sci Soc Am J 1985;49(2):376. https://doi.org/10.2136/sssaj1985.03615995004900020021x. 54. Engel R, Williams E, Wallander R, Hilmer J. Apparent persistence of N- (n-butyl) thiophosphoric triamide is greater in alkaline soils. Soil Sci Soc Am J 2013;77(4): 1424. https://doi.org/10.2136/sssaj2012.0380. 55. Suter HC, Pengthamkeerati P, Walker C, Chen D. Influence of temperature and soil type on inhibition of urea hydrolysis by N- (n-butyl) thiophosphoric triamide in wheat and pasture soils in south-eastern Australia. Soil Res 2011;49(4):315. https://doi.org/10.1071/sr10243.

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https://doi.org/10.22319/rmcp.v13i3.5564 Review

Diagnosis, prevention and control of diseases caused by Chlamydia in small ruminants. Review

Fernando de Jesús Aldama a Roberto Montes de Oca Jiménez a* Jorge Antonio Varela Guerrero a

Universidad Autónoma del Estado de México. Facultad de Medicina Veterinaria y Zootecnia. Toluca, Estado de México, México.

* Corresponding author: romojimenez@yahoo.com

Abstract: The species that make up the genus Chlamydia affect a wide range of animal hosts, causing various pathologies. Chlamydia abortus (C. abortus), Chlamydia psittaci (C. psittaci) and Chlamydia pecorum (C. pecorum) are the most clinically relevant in small ruminants worldwide, since they have been related to reproductive, ocular and digestive tract problems respectively; two of these (C. abortus and C. psittaci) represent a potential zoonotic risk to humans. The diagnosis of infections by organisms of this genus is complicated; since, in most cases, there are no clinical signs that indicate the presence of the agent in affected animals. Currently, in European countries, the prevention and control mainly of C. abortus is carried out through the administration of commercial attenuated immunogens; however, their use has not shown satisfactory results in the protection of susceptible animals. Therefore, the implementation of new immunization options based on the utilization of recombinant proteins is the line of research that is currently taking the most prominence. Additionally, the use of proteins with immunogenic potential could be important tools for the diagnosis, prevention and control of these pathogens. Due to this, the present review focused on recapitulating the most current studies focused on the experimental use of different immunogenic proteins of Chlamydia spp. used worldwide, highlighting their innovation and results obtained in experimental models.

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Key words: Chlamydia, Diagnosis, PCR, Recombinant Proteins, Vaccines.

Received: 28/11/2019 Accepted: 02/12/2020

Introduction The bacteria of the genus Chlamydia are Gram-negative, obligate intracellular organisms that are characterized by sharing a unique biphasic development cycle, they have two morphological structures called: elementary body (EB), which is the infective form, and the reticulate body (RB), form of the metabolically active bacterium(1). These bacteria cause a wide range of diseases in different animal host and man(1,2). Within the genus, a total of twelve species have been reported(3): C. trachomatis, C. muridarum, C. suis, C. psittaci, C. abortus, C. caviae, C. felis, C. pneumoniae, C. pecorum, C. avium, C. gallinacea, C. poikilothermis and four species candidates: C. ibidis, C. serpentis, C. corallus and C. sanzinia(4-6). Some of these species tend to affect production animals; in addition, they represent a potential zoonotic risk to humans(7). The main species of the genus identified in these animal species are: C. abortus, C. psittaci and C. pecorum; in addition, the pathologies associated with them have been widely documented(1,2,8). The pathologies related to these organisms are diverse, among which the following stand out: abortions, keratoconjunctivitis and problems in the digestive tract(2,9); however, C. abortus is the most important in livestock production, generating greater losses in flocks than due to the presence of C. psittaci and C. pecorum(1,2). Due to the importance for public and animal health that these organisms represent, this review focused on compiling the most recent studies on the development of diagnostic tests, treatment and control of infections caused by these bacterial species, where studies focused on the production of recombinant proteins stand out, which have been the subject of study in recent years.

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Species of the genus Chlamydia affecting small ruminants Chlamydia abortus

Causative agent of Ovine Enzootic Abortion (OEA), a disease that is widely distributed worldwide, which causes economic losses in countries that are engaged in livestock activity. The disease causes abortion in pregnant ewes in the last third of gestation or, in some cases, the birth of weak lambs that do not exceed 48 h of life(1,2). It is currently considered the most important pathology of chlamydial origin; since it represents a potential occupational zoonotic risk and for pregnant women who are in contact with infected animals(2). In this order, different reports have been made in which, in addition to causing abortions, it causes other conditions, such as: febrile illness, development of disseminated intravascular coagulation, acute renal failure and pulmonary edema(10), septicemia and important lesions in the liver, kidney and heart; it is worth mentioning that these pathologies occurred after the abortion(11). It is also described as a causative agent of pelvic inflammatory disease(12). In Mexico, the prevalence of antibodies against C. abortus was reported in exposed risk groups (workers and veterinarians) who were in contact with flocks with a history of abortion(13). Finally, it has also been reported as a causative agent of pneumonia problems(14), thus demonstrating the zoonotic potential of this bacterial species.

Chlamydia psittaci

Bacterium of avian origin that causes psittacosis in birds and with proven potential zoonotic risk. In the last five years, different studies have been carried out, evidencing the risk that this bacterial species represents for humans, mainly due to contact with infected birds(2). In the first instance causing psittacosis, ornithosis(2) or atypical pneumonia(15). It has also been reported as a causative agent of genital infections in women(16). It has been identified in patients with respiratory diseases; for example, pneumonia in farmers who worked with infected animals(17). Similarly, it has been linked to pneumonia, pertussis and conjunctivitis(18). Not least, associating the possible risk factors involved in the contagion of psittacosis in people who handle birds(19,20). Recently, it was reported as a causative agent in an outbreak related to severe respiratory disease among workers of poultry slaughter plants in the USA(21). There are currently no reports of contagion of C. psittaci from mammals to humans.

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Chlamydia pecorum

This bacterium lodges naturally in the digestive tract and causes enteritis; in most cases, it occurs subclinically, thus avoiding the timely detection of the disease(22). C. pecorum has also been associated with other diseases, among them: arthritis, keratoconjunctivitis, encephalomyelitis, infertility, pneumonia and mastitis, and causing economic losses in production units(22). Despite the wide variety of pathologies with which this bacterial agent has been related, the zoonotic potential of this species of Chlamydia is still unknown(23).

Transmission It has been widely documented that the way in which these organisms are mostly transmitted is through the oronasal route(1). C. abortus is excreted by infected ewes through vaginal fluids or placental remains, which contaminate water or food, the entry to susceptible animals is through the intake of these(2). C. psittaci is usually contained in the feces of birds and small ruminants, therefore, the main route of transmission of this pathogen is through the inhalation of aerosols contaminated by feces, in feeders or outdoor areas(7). Transmission of C. pecorum is thought to take place by the oral-fecal route or by ingestion or inhalation of bacteria contained in secretions of infected animals. Some studies have suggested transmission as a result of factors such as mutual grooming, inhalation and overcrowding(22).

Diagnosis Due to the variety of clinical pictures, animal hosts and since these agents are often diagnosed in combination with other infectious agents, a definitive diagnosis usually requires laboratory tests in most cases(24). The diagnosis of diseases of chlamydial origin is complicated and requires complex methodologies that require highly trained personnel to be able to carry out an ideal diagnosis(24). To make the diagnosis of Chlamydia species, the samples by choice are swabs (vaginal, conjunctival and/or rectal) preserved in a special transport medium for Chlamydia spp., sucrose-phosphate-glutamate (SPG)(24,25); on the other hand, the diagnosis can be made by indirect (ELISA) or direct methods (cell culture and PCR)(24). 728

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Enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) is an indirect diagnostic test that detects antibodies in serum of affected individuals against specific antibodies of bacteria of this genus(2,24), there are currently commercially available ELISA tests, among the disadvantages of this type of tests are cross-reactions between species (C. abortus and C. pecorum), which makes specific diagnosis difficult(2). The assays that evaluate different antigens for the detection of species of Chlamydia spp. have been diverse. First, evaluating fragments of the MOMP through an indirect ELISA test (rOMP91B iELISA), which showed a sensitivity and specificity of 84.2 and 98.5 % respectively; the study showed that the indirect ELISA test was better at differentiating animals infected with C. abortus and C. pecorum(26). Later, another study evaluated different recombinant antigens, all of these were identified from the Polymorphic Outer Membrane Protein or POMP90. Of the 11 fractions identified, OMP903 and OMP90-4 were the most effective, showing a sensitivity of 95.7 and 94.3 % respectively and a specificity of 100 % for both. The findings of the study revealed that the ELISA test with the fragment rOMP90-4 was more sensitive than that of rOMP90-3, since it identified more positive samples for OEA and, in addition, both were superior to the complement fixation test (CFT)(27). Additionally, and in a complementary way to these studies, a study was carried out in which four experimental ELISA tests based on complete EB of C. abortus (EB), a preparation from the outer membrane of complete bacteria (SolPr) and two recombinant fragments of POMP90 (rOMP90-3 and rOMP90-4), were evaluated against three commercial tests, the CHEKIT1 Chlamydophila abortus test, Pourquier1 ELISA Chlamydophila abortus and ImmunoComb Ovine Chlamydophila Antibody. The results during the test showed that the commercial ELISA test InmunoComb obtained the highest sensitivity (98.4 %) compared to the others; however, the specificity determined (65.4 %) was lower than all tests evaluated. The results at the end of the study determined that, of the eight ELISA tests evaluated, the test that offers the best results in terms of sensitivity and specificity was the ELISA test based on the recombinant fragment rOMP903, with values of 96.8 % and 100 % respectively, this study showed that this experimental ELISA test can be an adequate alternative for the serological diagnosis of OEA(28). A study carried out in 2018 adds to these results, which compared three commercial tests (IDvet, MVD-Enfer and LSI) for the detection of antibodies against C. abortus in ewes, vaccinated animals were evaluated for different periods of time to measure the production of antibodies between animals that aborted and animals that reached term, the results revealed that the most sensitive test was the LSI (94.74 %) followed by MVD-Enfer (78.95 %) and finally IDvet (73.68 %), all three kits detected high levels of antibodies in ewes that aborted compared to those that had lambs without complications. The most sensitive test in this study is based on the identification of chlamydial lipopolysaccharide (LPS), which shows cross-

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reaction with all species of the genus Chlamydia, it is determined suitable for identifying ewes infected with any species of Chlamydia, but it is not considered specific for C. abortus(29). Finally, a study revealed the disadvantages of this type of commercial tests in terms of cross-reactions between C. abortus and C. pecorum, carrying out the evaluation in different flocks, serological tests revealed a low seropositivity of C. abortus using an ELISA test based on peptides (1.2 %) in Australian ewes and a moderate seropositivity in a Swiss flock with a clinical history of abortion associated with C. abortus (26.9 %). Using CFT and ELISA tests, seropositivity was significantly higher, suggesting cross-reactivity between these two species. Additionally, using a real-time PCR test to detect DNA of C. pecorum in Australian animals seropositive for Chlamydia spp., it was concluded that the seropositivity of Chlamydia may be related to cross-reactivity with endemic infections of C. pecorum(30). Due to the disadvantages shown by this type of tests, it is advisable to complement them with a more specific one such as PCR tests(24).

Cell culture

Due to their obligate intracellular nature, these bacteria require live media for their isolation; due to this, cell culture (McCoy cells by choice) is currently used for this purpose(24), until a few years ago, this technique was considered the gold standard for the diagnosis of Chlamydia spp.(1); however, the development of new methodologies, such as nucleic acid amplification (PCR and sequencing), employed to improve diagnosis are currently considered the gold standard for diagnosing Chlamydia spp. infections(8). Other tests such as polymerase chain reaction, which is a much more specific technique for the detection and typing of species of Chlamydia spp., is used much more frequently because it has a greater sensitivity and specificity compared to cell culture(2).

Polymerase chain reaction test

This test detects specific DNA of any organism; therefore, it is a much more sensitive and specific test than cell culture when identifying the genus and species involved in the affected individuals(24). In the last 15 yr, the use of this technique has become very relevant, and its different variants reveal better results compared to those mentioned above, such as: i) processing a greater number of samples, ii) less time to obtain results, iii) the use of different types of samples for diagnosis, iv) organisms do not have to be one hundred percent viable and v) greater sensitivity and specificity. Since its use in veterinary diagnostic laboratories was implemented, the genes used to identify these bacterial agents have been different: major

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outer membrane protein (MOMP), polymorphic membrane proteins (Pmps), 16S and 23S(24). PCR methodologies for the specific detection of Chlamydia species are varied, to mention a few: The PCR test “Touchdown enzyme time release” to amplify different DNA sequences in the variable regions of the spacer rRNA genes 16S and 16S-23S specific for the identification of species of this bacterial genus; for example, Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia psittaci(31). Another variant of this test, the PCRRFLP test that identifies in the first instance the presence of the omp2 gene specific to the family Chlamydiaceae and later, by digestion with the restriction enzyme AluI, has the ability to identify a total of nine species of the genus Chlamydia, among these, C. abortus, C. pecorum(32) and C. psittaci(33). Additionally, a variant focused on the 16S gene specific to the family Chlamydiaceae but using a real-time PCR test demonstrated high specificity when evaluating samples of different Chlamydia species against other bacterial genera(34); additionally, this variant can also be used for the specific identification of Chlamydia species using specific primers for each one(35). Later, a multiplex PCR test was developed for the identification of C. abortus, C. pecorum and Coxiella burnetii, involved as causative agents of abortion, this test, unlike the aforementioned ones, helps to simultaneously identify the three species, this test proved to be highly specific and rapid for the detection of these bacterial agents(36).

Treatment Antibiotics

For the treatment of pathologies of chlamydial origin, antibiotics are the drugs of choice, since bacterins, in the case of OEA, are only available in some European countries and the costs required for their implementation are very high(2,7). The administration of tetracycline, penicillin and chloramphenicol for the treatment of infections caused by these bacterial agents has been shown to inhibit the growth of these organisms(24), it is important to emphasize that the use of antibiotics should be in a controlled manner to minimize the development of resistance by the pathogen. Although antibiotics serve to reduce losses due to these pathogens, these types of treatments do not eliminate bacteria; since affected animals continue to shed the organisms; therefore, their prophylactic use is not recommended(37). In European countries, in addition to the use of antibiotics, the administration of bacterins is also implemented in the case of OEA to prevent and control the disease in flocks with susceptible animals, this is because it is the only disease of chlamydial origin for which there are commercially available bacterins(2).

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Immunogens

In the past century, immunogens intended for the treatment and prevention of diseases worldwide have been one of the greatest achievements of public health; in this area, between 2 and 3 million lives are saved due to the implementation of these health measures(38). In the veterinary field, technological advances in the development of immunogens for disease control have had an important enhancement in the last 25 yr, from the use of complete bacteria, whether alive or dead, to the use of DNA immunogens, which offer safer measures for both the animal and the veterinarian who administers them(39). In the last 70 yr, studies for the development of immunogens to combat diseases of chlamydial origin in animal species, mainly livestock (sheep, goats, cattle and pigs), focus on preventing economic losses in production units; however, the main objective is to preserve human health due to the zoonoses that some of these represent. Vaccine trials against Chlamydia spp. have increased in the last 10 yr, in these studies, the protein most used in the challenges against Chlamydia spp. has been the MOMP(40). Additionally, studies focused on the search for specific antigens of this surface protein have been carried out for a differentiation between species(41). Later, different types of antigens have been used in the development of immunogens against chlamydial agents, the first tests traditionally used the EB, which were inactivated by treatments with ultraviolet light or live (attenuated) fixed with formalin. Later, in the mid-1990s, approaches in the use of other antigens for the development of subunit vaccines, such as recombinant proteins, synthetic peptides, expression vectors and DNA, began to be used for the challenge in murine models mainly(40). In the case of C. pecorum, there are only two trials of vaccines against this agent, which have been challenged in murine models, although the results revealed an immune response in animals, these should be treated with caution because clinical cases of abortions are rarely related to C. pecorum(42). Finally, although there are commercially available vaccines for the control and prevention of C. abortus, it is well documented that in the case of the live attenuated 1B C. abortus vaccine, it has the potential to reactivate and cause the disease in immunized animals(43-45). Of the total vaccine challenges against Chlamydia spp., only 5 % have been focused on C. abortus, evaluated in mice, cows, ewes and guinea pigs(40), the studies evaluate some proteins, mainly Pmps, looking for different variations or mutations that can serve as key points for the prevention of OEA(46).

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Subunit vaccines, new technologies for the development of serodiagnostic tests and vaccine prototypes To date, different studies that have identified these possible candidates, both for the diagnosis and for the development of vaccines(2), have been developed. Immunoreactive proteins, expressed in infected animals, have been proposed as new candidates as marker antigens for the diagnosis and use of different virulence genes that can be used for the development of prototypes of subunit vaccines for the prevention and control of diseases caused by species of Chlamydia spp.(47). In the case of C. abortus, several studies have been carried out, evaluating different proteins. A study evaluated the humoral immune response triggered by some surface proteins (MOMP, MIP, Pmp13G) and associated with virulence (CPAF, TARP, SINC), this study showed that ewes that aborted showed a strong antibody response to surface antigens. Additionally, they identified that the most specific antigen for the serodiagnosis of human infections by C. abortus was Pmp13G; this protein did not show cross-reactivity with other species of Chlamydia spp. that affect humans(47). Regarding the proteins currently used for the development of vaccine prototypes, studies have focused on three proteins that play a major role in the development cycle of the bacterium(40). Other proteins of this bacterial genus used as antigens in vaccine trials are the membrane surface proteins of Chlamydia spp., which have been shown to have highly conserved regions(48). Heat shock proteins (HSPs) and chlamydia protease-like activity factor (CPAF) proteins have also been considered as suitable candidates for the development of immunogens for the control and prevention of diseases of chlamydial origin, these have been shown to cause a strong inflammatory response in the host(49). Another study evaluates three different types of vaccines: DNA vaccine, phage vaccine (OmpA) and a commercial live attenuated, freeze-dried vaccine based on the strain 1B of Chlamydia abortus. Although the phage vaccine offers good results, it does not exceed that offered by the commercial vaccine; however, the study concludes that this novel vaccine administration system offers advantages that far outweigh commercial vaccines, such as: handling, more efficient safety and relatively cheaper production(50). Other tests evaluated a combination with the polysaccharide Lycium barbarum (LBP3a), the results demonstrated good protection in mice challenged with C. abortus using a polysaccharide LBP3a combined with a DNA vaccine encoding the MOMP of C. abortus(51). Later, other proteins have been proposed, such as those belonging to the family of Pmps, these have been shown to have the immunogenic potential for the development of immunogens against C. abortus(2). One study evaluated Pmp18D in two different formulations, FL (tyrosine kinase 3 ligand type Fms; Flt3L) and Vibrio cholerae ghosts (VCGs), to induce innate and cross-protective immunity against genital C. abortus infection.

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The evaluation was carried out with the regulation of the expression of the protein, by the activation and differentiation of different cell types. The results showed that the formulation that offers the best results is Pmp18D+VCG(52), as well as another variant, using an Nterminal fragment of this protein, called Pmp18D.1, the study evaluated its ability to induce an innate immune response in dendritic cells and activate the signaling pathways involved in the secretion of IL-1β(53). Other proteins used as combined antigens (MIP and CPAF) demonstrated 50 % efficacy against a commercial live attenuated vaccine; additionally, although there is release of the pathogen by immunized animals, these are released in smaller quantities compared to the negative control. Nevertheless, it was possible to observe in this study that, when these two proteins are administered individually, it has no effect against the experimental infection(54). Several studies have been carried out against C. psittaci, using different types of proteins. Initially focused on the evaluation of the persistence of a DNA vaccine (pcDNA1-MOMP) and the expression of the recombinant protein (rMOMP) from the MOMP of an avian strain of C. psittaci, which causes respiratory problems in turkeys, the results showed that the persistence of the vaccine was 10 weeks and the expression of the protein was proportional to the persistence time(55). Subsequently, using a recombinant adenovirus vaccine using the same protein, it was evaluated in birds against avian chlamydia, the results of this study showed that this vaccine was safe, and that the protection rate reached up to 90 % in the challenged animals. Although the period of protection of the vaccine was six months, it is emphasized that the growth periods of the birds used for meat are approximately similar. However, birds intended for laying should be vaccinated twice because they have longer life spans(56). On the other hand, Pmps have also been used as candidates for the development of vaccines against C. psittaci; in this sense, a study developed and examined a recombinant vaccine administered due to the herpesvirus of turkeys, using the 5’ end of the PmpD gene, which encodes the N-terminal fraction of this (pmpD-N). The evaluation of the recombinant virus (rHVT-pmpD-N) in the challenged birds revealed increased levels of specific antibodies against PmpD and a proliferation of specific lymphocytes against it. After the challenge with the strain C. psittaci CB7, a significant decrease in respiratory distress, lesions and bacterial load was found in the challenged group(57). One study evaluated the efficacy of vaccination with plasmid proteins to prevent lung infection by C. psittaci in mice, in which a recombinant protein of CPSIT_p8 is used, which belongs to an important virulence factor in the form of a highly conserved “cryptic” plasmid of 7.5 kb. A recombinant of this protein was produced and challenged in a murine model. The results in this study showed that immunization significantly decreased the bacterial load in the lungs of the challenged mice, a lower level of IFN-γ was also observed. Its results conclude that the recombinant protein evaluated in this study induces significant protective immunity against C. psittaci and that it could be

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considered as a candidate for the development of a new vaccine for the prevention of infections caused by this bacterium(58). However, other proteins involved in the virulence of this bacterium, such as chlamydial inclusion membrane proteins (Incs), have also been used as candidates in the development of vaccine prototypes. One study employed a recombinant of the transmembrane head protein CPSIT_0846 and challenged mice with respiratory tract infection caused by C. psittaci, the study revealed a strong cytokine profile with high levels of IFN-γ; similarly, a strong humoral immune response was detected in the challenged mice, with high titers of specific IgG antibodies. The strong immune response correlated with a significantly reduced bacterial concentration and a decrease in the inflammatory pathology in the lungs of the mice after the challenge. The results of this study suggest that the protein CPSIT_0846 may be a possible candidate antigen for the development of a vaccine to induce protection against this type of infections(59). For the detection of antibodies against C. psittaci, an ELISA test based on the N-terminal fragment of the PmpD (PmpD-N) was developed, the tests were performed to determine its sensitivity and specificity in experimentally infected and uninfected birds. The results of this study revealed that the ELISA-PmpD-N had a sensitivity and specificity of 97.9 %, 100 % respectively; in addition, there was no cross-reaction with positive serums for other avian pathogens. The results concluded that this protein fraction (PmpD-N) can be used as an antigen for the diagnosis of C. psittaci infections in birds(60). It should be noted that all studies have been carried out in C. psittaci of avian origin; however, since C. psittaci is genetically related to C. abortus(61), with this evidence, the idea of focusing studies on the variant that affects small ruminants can be contemplated. In C. pecorum, the most recent study focused on the development of vaccine prototypes using surface proteins has been carried out in experimentally infected ewes and evaluating two recombinant proteins: rMOMP and rPmpG, this study identified B-cell epitopes in asymptomatic animals, with arthritis related to this agent and animals immunized with a recombinant vaccine of these proteins. The results of this study conclude that these tests can help improve diagnostic tests for this agent in sheep flocks(62). Later, a study evaluates a direct ELISA test using two recombinant protein antigens of this bacterial species (rPmpG and rMOMP-G) and using the Pepscan method, a mapping and characterization of B-cell epitopes in these proteins was carried out in lambs with asymptomatic C. pecorum infections, with polyarthritis associated with C. pecorum and vaccinated with recombinant proteins. The results revealed that there is an immune response of antibodies against PmpG in the natural infection. Antibodies against MOMP-G increased in animals with polyarthritis. Finally, an epitope response was identified in immunized lambs and in naturally infected lambs(63). 735

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Conclusions Studies focused on the identification of immunoreactive proteins for the development of ELISA tests and vaccine prototypes against diseases caused by species of the genus Chlamydia that affect small ruminants have become very relevant in recent years, due to the importance in public health, animal welfare and economic importance that they represent. Immunoassays with specific proteins of each species, such as Pmps, can be a key point to avoid cross-reactions between species, which would reduce erroneous results in veterinary diagnostic laboratories. C. abortus is the species of the genus that has been the most importance in the last decade; since the available commercial vaccines have not given satisfactory results for the prevention and control of OEA; in addition to the biological risk that it represents. The use of subunit vaccines as an option for prototype development has good levels of safety compared to commercial vaccines; since they do not represent a risk to the personnel who handles them and offer results equal to or superior to those offered by them.

Conflicts of interest

The authors declare that they have no conflict of interest. Literature cited: 1. Longbottom D, Coulter LJ. Animal chlamydioses and zoonotic implications. J Comp Pathol 2003;128:217–244. 2. Rodolakis A, Laroucau K. Chlamydiaceae and chlamydial infections in sheep or goats. Vet Microbiol 2015;181:107-118. 3. Sachse K, Bavoil PM, Kaltenboeck B, Stephens R, Kuo CC, Rosselló-Móra R. et al. Emendation of the family Chlamydiaceae: Proposal of a single genus, Chlamydia, to include all currently recognized species. Syst Appl Microbiol 2015;38:99–103. 4. Vorimore F, Hsia R-ching, Huot-Creasy H, Bastian S, Deruyter L, Passet A. et al. Isolation of a new Chlamydia species from the Feral Sacred Ibis (Threskiornis aethiopicus): Chlamydia ibidis. PLoS One 2013;8(9):e74823.

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5. Taylor-Brown A, Bachmann NL, Borel N, Polkinghorne A. Culture-independent genomic characterisation of Candidatus Chlamydia sanzinia, a novel uncultivated bacterium infecting snakes. BMC Genomics 2016;17(1):710. 6. Staub E, Marti H, Biondi R, Levi A, Donati M, Leonard CA, et al. Novel Chlamydia species isolated from snakes are temperature-sensitive and exhibit decreased susceptibility to azithromycin. Sci Rep 2018;(1):5660 7. Bommana S, Polkinghorne A. Mini review: Antimicrobial control of chlamydial infections in animals: Current practices and issues. Front Microbiol 2019;10:1-9. 8. Borel N, Polkinghorne A, Pospischil A. A review on chlamydial diseases in animals: still a challenge for pathologists? Vet Pathol 2018;55:374-390. 9. Jelocnik M, Laurence M, Murdoch FR, Polkinghorne A. Detection of Chlamydiaceae in ocular swabs from Australian pre-export feedlot sheep. Aust Vet J 2019;97(10):401403. 10. Johnson F, Matheson BA, Williams H, Laing AG, Jandial V, Davidson-Lamb R, et al. Abortion due to infection with Chlamydia psittaci in a sheep farmer’s wife. Br Med J 1985;290:592-594. 11. Pospischil A, Thoma R, Hilbe M, Grest P, Gebbers FO. Abortion in woman caused by caprine Chlamydophila abortus (Chlamydia psittaci serovar 1). Swiss Med Wkly 2002;132:64-66. 12. Walder G, Meusburger H, Hotzel H, Oehme A, Neunteufel W, Dierich MP, et al. Chlamydophila abortus Pelvic Inflammatory Disease. Emerg Infect Dis 2003;9(12):1642-1644. 13. Barbosa Mireles MA, Salazar García F, Fernández Rosas P, Montes de Oca-Jiménez R. Detection of serologic antibodies against Chlamydophila Abortus in two groups of people exposed to risk in ovine farms in Xalatlaco, Mexico. Trop Subtrop Agroecosystem 2013;16:423-429. 14. Ortega N, Caro MR, Gallego MC, Murcia-Belmonte A, Álvarez D, del Río L, et al. Isolation of Chlamydia abortus from a laboratory worker diagnosed with atypical pneumonia. Irish Vet J 2016;69:78. 15. Fossádal ME, Grand M, Gaini S. Chlamydophila psittaci pneumonia associated to exposure to fulmar birds (Fulmaris glacialis) in the Faroe Islands. Infect Dis (Auckl) 2018;50:817-821.

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Rev Mex Cienc Pecu 2022;13(3):725-742

16. Osman KM, Ali HA, Eljakee JA, Gaafar MM, Galal HM. Antimicrobial susceptibility and molecular typing of multiple chlamydiaceae species isolated from genital infection of women in Egypt. Microb Drug Resist 2012;18:440-445. 17. Lagae S, Kalmar I, Laroucau K, Vorimore F, Vanrompay D. Emerging Chlamydia psittaci infections in chickens and examination of transmission to humans. J Med 2014;63:399-407. 18. Cadario ME, Frutos MC, Arias MB, Origlia JA, Zelaya V, Madariaga MJ, et al. Epidemiological and molecular characteristics of Chlamydia psittaci from 8 human cases of psittacosis and 4 related birds in Argentina. Rev Argent Microbiol 2017;49:323327. 19. Čechová L, Halánová M, Babinská I, Danišová O, Bartkovský M, Marcinčák S, et al. Chlamydiosis in farmed chickens in slovakiaand zoonotic risk for humans. Ann Agric Environ Med 2018;25:320-325. 20. Tolba HMN, Abou Elez RMM, Elsohaby I. Risk factors associated with Chlamydia psittaci infections in psittacine birds and bird handlers. J Appl Microbiol 2019;126:402410. 21. Shaw KA, Szablewski CM, Kellner S, Kornegay L, Bair P, Brennan S, et al. Psittacosis outbreak among workers at chicken slaughter plants, Virginia and Georgia, USA, 2018. Emerg Infect Dis 2019;25(11):2143–2145. 22. Walker E, Lee EJ, Timms P, Polkinghorne A. Chlamydia pecorum infections in sheep and cattle: A common and under-recognised infectious disease with significant impact on animal health. Vet J 2015;206:252–260. 23. Rodolakis A, Mohamad KY. Zoonotic potential of Chlamydophila. Vet Microbiol 2010;140:382. 24. Sachse K, Vretou E, Livingstone M, Borel N, Pospischil A, Longbottom D. Recent developments in the laboratory diagnosis of chlamydial infections. Vet Microbiol 2009;135:2-21. 25. Mora Diaz JC, Díaz Aparicio E, Herrera López E, Suarez Güemes F, Escalante Ochoa C, Jaimes Villareal S, et al. Aislamiento de Chlamydia abortus en rebaños caprinos lecheros y su relación con casos de aborto en Guanajuato, México. Vet Mex 2015;2:11. 26. Longbottom D, Psarrou E, Livingstone M, Vretou E. Diagnosis of ovine enzootic abortion using an indirect ELISA (rOMP91B iELISA) based on a recombinant protein fragment of the polymorphic outer membrane protein POMP91B of Chlamydophila abortus. FEMS Microbiol Lett 2001;195:157-161.

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27. Longbottom D, Fairley S, Chapman S, Psarrou E, Vretou E, Livingstone M. Serological diagnosis of ovine enzootic abortion by enzyme-linked immunosorbent assay with a recombinant protein fragment of the polymorphic outer membrane protein POMP90 of Chlamydophila abortus. J Clin Microbiol 2002;40:4235-4243. 28. Wilson K, Livingstone M, Longbottom D. Comparative evaluation of eight serological assays for diagnosing Chlamydophila abortus infection in sheep. Vet Microbiol 2009;135:38-45. 29. O’Neill LM, O’Driscoll, Markey B. Comparison of three commercial serological tests for the detection of Chlamydia abortus infection in ewes. Irish Vet J 2018;71:1-9. 30. Bommana S, Jelocnik M, Borel N, Marsh I, Carver S, Polkinghorne A. The limitations of commercial serological assays for detection of chlamydial infections in Australian livestock. J Med 2019;68:627-632. 31. Madico G, Quinn TC, Boman J, Gaydos CA. Touchdown enzyme time release-PCR for detection and identification of Chlamydia trachomatis, C. pneumoniae and C. psittaci Using the 16S and 16S-23S spacer rRNA genes. J Clin Microbiol 2000;38:1085-1093. 32. Marsilio F, Di Martino B, Di Francesco CE, Meridiani I. Diagnosis of ovine chlamydial abortions by PCR-RFLP performed on vaginal swabs. Vet Res Commun 2005;29:99106. 33. Hartley JC, Kaye S, Stevenson S, Bennett J. PCR Detection and molecular identification of Chlamydiaceae species. J Clin Microbiol 2001;39:3072-3079. 34. Condon K, Oakey J. Detection of Chlamydiaceae DNA in veterinary specimens using a family-specific PCR. Lett Appl Microbiol 2007;45:121-127. 35. Nordentoft S, Kabell S, Pedersen K. Real-time detection and identification of Chlamydophila species in veterinary specimens by using SYBR green-based PCR assays. Appl Environ Microbiol 2011;77:6323-6330. 36. Berri M, Rekiki A, Boumedine K, Rodolakis A. Simultaneous differential detection of Chlamydophila abortus, Chlamydophila pecorum and Coxiella burnetii from aborted ruminant’s clinical samples using multiplex PCR. BMC Microbiol 2009;9:130. 37. Essig A, Longbottom D. Chlamydia abortus: new aspects of infectious abortion in sheep and potential risk for pregnant women. Curr Clin Microbiol Reports 2015;2:22-34. 38. Delany I, Rappuoli R, De Gregorio E. Vaccines for the 21st century. EMBO Mol Med 2014;6(6):708-720.

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39. Francis MJ. Recent advances in vaccine technologies. Vet Clin North Am - Small Anim Pract 2018;48:231-241. 40. Phillips S, Quigley BL, Timms P. Seventy years of Chlamydia vaccine research Limitations of the past and directions for the future. Front Microbiol 2019;10:1-18. 41. Hoelzle LE, Hoelzle K, Wittenbrink MM. Recombinant major outer membrane protein (MOMP) of Chlamydophila abortus, Chlamydophila pecorum, and Chlamydia suis as antigens to distinguish chlamydial species-specific antibodies in animal sera. Vet Microbiol 2004;103:85-90. 42. Rekiki A, Bouakane A, Rodolakis A. Combined vaccination of live 1B Chlamydophila abortus and killed phase I Coxiella burnetii vaccine does not destroy protection against chlamydiosis in a mouse model. Can J Vet Res 2004;68(3):226–228. 43. García-Seco T, Pérez-Sancho M, Salinas J, Navarro A, Díez-Guerrier A, García N, et al. Effect of preventive Chlamydia abortus vaccination in offspring development in sheep challenged experimentally. Front Vet Sci 2016;3:67. 44. Laroucau K, Aaziz R, Vorimore F, Menard MF, Longbottom D, Denis G. Abortion storm induced by the live C. abortus vaccine 1B strain in a vaccinated sheep flock, mimicking a natural wild-type infection. Vet Microbiol 2018;225:31-33. 45. Longbottom D, Sait M, Livingstone M, Laroucau K, Sachse K, Harris SR, et al. Genomic evidence that the live Chlamydia abortus vaccine strain 1B is not attenuated and has the potential to cause disease. Vaccine 2018;36:3593-3598. 46. Burall LS, Rodolakis A, Rekiki A, Myers GSA, Bavoil PM. Genomic analysis of an attenuated Chlamydia abortus live vaccine strain reveals defects in central metabolism and surface proteins. Infect Immun 2009;77(9):4161–4167. 47. Forsbach-Birk V, Foddis C, Simnacher U, Wilkat M, Longbottom D, Walder G, et al. Profiling antibody responses to infections by Chlamydia abortus enables identification of potential virulence factors and candidates for serodiagnosis. J Clin 2013;8:1-15. 48. Hagemann JB, Simnacher U, Longbottom D, Livingstone M, Maile J, Soutschek E, et al. Analysis of humoral immune responses to surface and virulence-associated Chlamydia abortus proteins in ovine and human abortions by use of a newly developed line immunoassay. J Clin Microbiol 2016;54:1883-1890. 49. Vasilevsky S, Stojanov M, Greub G, Baud D. Chlamydial polymorphic membrane proteins: Regulation, function and potential vaccine candidates. Virulence 2016;7(1):11–22.

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50. Li W, Guentzel MN, Seshu J, Zhong G, Murthy AK, Arulanandam BP. Induction of cross-serovar protection against genital chlamydial infection by a targeted multisubunit vaccination approach. Clin Vaccine Immunol 2007;14(12):1537-1544. 51. Ling Y, Liu W, Clark JR, March JB, Yang J, He C. Protection of mice against Chlamydophila abortus infection with a bacteriophage-mediated DNA vaccine expressing the major outer membrane protein. Vet Immunol Immunopathol 2011;144:389–395. 52. Ling Y, Li S, Yang J, Yuan J, He C. Co-administration of the polysaccharide of Lycium barbarum with DNA vaccine of Chlamydophila abortus augments protection. Immunol Invest 2011;40:1–13. 53. Pan Q, Pais R, Ohandjo A, He C, He Q, Omosun Y, et al. Comparative evaluation of the protective efficacy of two formulations of a recombinant Chlamydia abortus subunit candidate vaccine in a mouse model. Vaccine 2015;33:1865–1872. 54. Pan Q, Zhang Q, Chu J, Pais R, Liu S, He C, et al. Chlamydia abortus Pmp18.1 induces IL-1β secretion by TLR4 activation through the MyD88, NF-κB, and caspase-1 signaling pathways. Front Cell Infect Microbiol Frontiers 2017;7:514. 55. O’Neill LM, Keane OM, Ross PJ, Nally JE, Seshu J, Markey B. Evaluation of protective and immune responses following vaccination with recombinant MIP and CPAF from Chlamydia abortus as novel vaccines for enzootic abortion of ewes. Vaccine 2019;37:5428–5438. 56. Loots K, Vleugels B, Ons E, Vanrompay D, Goddeeris BM. Evaluation of the persistence and gene expression of an anti-Chlamydophila psittaci DNA vaccine in turkey muscle. BMC Vet Res 2006;2:18. 57. Qiu C, Zhou J, Cao XA, Lin G, Zheng F, Gong X. Immunization trials with an avian chlamydial MOMP gene recombinant adenovirus. Bioeng Bugs 2010;1:267-273. 58. Liu S, Sun W, Chu J, Huang X, Wu Z, Yan M, et al. Construction of recombinant HVT expressing PmpD, and immunological evaluation against Chlamydia psittaci and Marek’s disease virus. PLoS One 2015;10(4):e0124992. 59. Liang M, Wen Y, Ran O, Chen L, Wang C, Li L, et al. Protective immunity induced by recombinant protein CPSIT_p8 of Chlamydia psittaci. Appl Microbiol Biotechnol 2016;100:6385-6393. 60. Ran O, Liang M, Yu J, Yu M, Song Y, Yimou W. Recombinant protein CPSIT 0846 induces protective immunity against Chlamydia psittaci infection in BALB/c mice. Pathog Dis 2017;75:18.

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61. Liu SS, Chu J, Zhang Q, Sun W, Zhang TY, He C. Development of a novel PmpD-N ELISA for Chlamydia psittaci infection. Biomed Environ Sci 2016;29:315-322. 62. Pannekoek Y, Dickx V, Beeckman DSAB, Jolley KA, Keijzers WC, et al. Multi locus sequence typing of Chlamydia reveals an association between Chlamydia psittaci genotypes and host species. PLoS One 2010;5(12):e14179. 63. Desclozeaux M, Jelocnik M, Whitting K, Saifzadeh S, Bommana S, Potter A, et al. Safety and immunogenicity of a prototype anti-Chlamydia pecorum recombinant protein vaccine in lambs and pregnant ewes. Vaccine 2017;35(27):3461–3465.

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https://doi.org/10.22319/rmcp.v13i3.6103 Review

Ingestion behavior and forage intake by grazing cows in temperate climate. Review

Juan Daniel Jiménez Rosales a Ricardo Daniel Améndola Massiotti a*

Universidad Autónoma Chapingo. Departamento de Zootecnia, Posgrado en Producción Animal. km. 38.5 Carretera México-Texcoco, 56230, Chapingo, Estado de México. México.

Corresponding author: r_amendola@yahoo.com

Abstract: The objective was to review, based on predominantly recent publications, the knowledge on the components of the ingestion behavior (IB) of cows that graze in a temperate climate, and their relationship with the characteristics of the pastures that regulate the daily forage intake (FI). The components of IB that regulate FI are bite mass (BM, g DM bite-1), bite rate (BR, bites min-1), intake rate (IR, g DM min-1) and grazing time (GT, min d-1). The mass, height and density of pasture forage affect BM and consequently, FI. Pasture height is related to IB components and is useful for assessing FI. Based on studies in temperate pastures in a vegetative state, it is highlighted that the FI of cows increases with increases in pasture height, because they harvest bites of greater BM, which allows them to obtain high IRs. But there is evidence that IR may decrease in pastures that are too tall; to process larger bites, cows reduce their BR and execute more compound and chewing jaw movements. In contrast, in short pastures, cows increase their BR and GT, to remedy the reduction in IR due to harvesting lighter weight bites, although this does not fully compensate for the decrease in IR. Therefore, to maintain high IRs, cows should not be forced to consume forage at high grazing intensities. Key words: Intake rate, Grazing time, Bite rate, Bite mass, Pasture height.

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Received: 28/11/2021 Accepted: 22/02/2022

Introduction Dry matter (DM) intake and diet digestibility determine the supply of nutrients to housed or grazing cows, and consequently affect their production. Therefore, understanding the factors that affect the short-term forage intake by grazing cows is important for grazing management, even more so when the intake and, therefore, the production of grazing cows is lower than in housed cows. Forage intake by grazing cattle can be evaluated from the ingestion behavior they exhibit during foraging, since they adapt this behavior according to characteristics of the pasture(1) and several factors such as the chemical composition of the forage (neutral detergent fiber, soluble carbohydrates and crude protein), the stimuli of the products of rumen fermentation (N-NH3 and volatile fatty acids), the hormones of hunger (ghrelin), satiety (leptin and melatonin), rumen filling(2), post-ingestive consequences caused by the content of secondary metabolites in plants(3), supplementation, and physiological and nutritional status(4). However, in grazing, the bite mass along with pasture characteristics such as: mass, height and disappearance of forage are factors that regulate intake and, in one study, they explained 78 % of the variations in the weight gain of cattle(5). Corroborating this result, in a recently published meta-analysis(6), which included 103 publications with 278 experiments, it was confirmed that bite mass (BM) is a fundamental component of ingestion behavior (IB) in grazing; since it is sensitive to the main characteristics of the pasture canopy and is a determining factor for the intake rate (IR) and forage intake (FI). Due to the importance of forage intake in grazing animal production and due to the effects of the characteristics of the induced grassland (pasture) on ingestion behavior, the purpose of this review is to characterize the components of the ingestion behavior of grazing cows in temperate climate, based on mainly recent research results, made mostly in homogeneous temperate climate pastures (monophyte, with little spatial variation in the vertical and horizontal axes). These studies have made it possible to advance in the understanding of the functional relationships between pasture characteristics and the dimensions of the bite and IR(7). The predominance of the study of the ingestive behavior of cattle in homogeneous pastures is due to the greater difficulty in the methodology to evaluate this behavior in more heterogeneous vegetation, with plants that differ in their morphology and structure(8).

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At the beginning of this document, the components of ingestion behavior and the space-time scales of the grazing environment are defined and described; then, concepts about bite dimensions are presented, to analyze their relationship with forage density and impact on forage intake. Later, the importance of pasture height and its reduction, as measures of forage abundance, on ingestion behavior and intake is addressed. At the end, the pattern of cow grazing activity and results of ingestion behavior and intake are described.

Ingestion behavior and grazing scales Knowledge of the IB of cows is essential to understand and manage their FI. In the analysis of the IB of cows to evaluate FI (g DM d-1), components of the behavior of the animals and attributes of the pasture are included (Figure 1). The components are BM (g DM bite-1), bite rate (BR, bites min-1), IR (g DM min-1) and grazing time (GT, min d-1). Cows collect forage at different hierarchical scales of space and time of the grazing environment, making decisions, which together are known as ingestion behavior, equivalent to foraging dynamics (Figure 2)(9). Figure 1: Components of the ingestion behavior of grazing cows(1,10,11).

The smallest scale of foraging dynamics is the bite, and its obtaining is defined as the placement of forage in the mouth and its detachment from the rest of the plant by movements of the mouth and head(8,12). With movements of the lips, tongue and jaws, the bite is

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apprehended and accommodated inside the mouth and with movements of the head, the tension to achieve the rupture of the forage is obtained. During this operation, cattle use their tongue to introduce forage into their mouths and to expand the bite area(13). Figure 2: Spatial and temporal scales of grazing behavior of large herbivores(9,11,12)

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The next spatial scale is the feeding station, an area in which the animal selects and takes bites with neck movements without moving its front legs(14). The level immediately above is the patch, which consists of a set of feeding stations(13). Foraging at these scales can last from 1 sec to 30 min, on areas that range from a few cm2 to 1 ha(9). The next scale is the grazing site, which is made up of different patches where the cow consumes forage during a grazing session(13). The time of permanence at this scale varies between 1 and 4 h, on areas that range from 1 to 4 ha(9). Finally, the upper scale of grazing behavior is the grazing field, which involves more time (weeks) and space (km2). This scale can be reached by herds of cattle that, during grazing, move within large grasslands(15). The time of permanence of large herbivores (including cattle) at the different scales is generally a function of the quantity and quality of the forage found there(9); in addition to other factors that include the topography of the area(16), the location of the water(17), the season of the year(18), as well as components of the social behavior of cattle(19). The genotype of the animal is a factor in which contradictory results have been found; in a study, the grazing of Beefmaster × Simford cows was compared with that of smaller Baladi cows, the Baladi cows exhibited greater grazing time and distance walked(18). In another study, early-maturing (Angus and Hereford) and late-maturing (Limousin, Charolais) young bulls were compared, and no differences were found in ingestive behavior and intake between genotypes(20). Nevertheless, forage intake in F1 Hereford × Angus cows during gestation was lower than in Hereford and Angus cows(21). In the research on FI by dairy cows in pastures, short-term studies on bite scales(22,23), feeding station(24,25), patch(26,27) and grazing site(16) have predominated. One reason that research has focused more on the short term is because few researchers have access to automatic recording of IB and personnel trained to make long-term observations(7). However, precision livestock farming, which includes the use of technological tools to assess cattle behavior(28), can contribute to making management decisions in real-time and better understanding the plantanimal relationship in grazing systems. In this regard, there are several sensors and devices that have been used for the monitoring of jaw movements and the ingestion behavior of grazing cattle(12). The FI of grazing animals can be described arithmetically as the product of two components, IR and GT(1) and, in turn, IR as the product of BR and BM(29). For its part, BR is determined by jaw movements (Figure 1) that can be differentiated with equipment that registers either mechanical signals(30) or acoustic signals(31). Jaw movements are for chewing, bite and compound (chewing-bite)(23), the latter have been identified exclusively with acoustic analysis(32). A recently published meta-analysis(6) that involved 103 publications with 278 experiments confirmed that BM is a fundamental component of IB in grazing, as it is sensitive to the main characteristics of the pasture canopy and is a determining factor for IR and FI. 747

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To understand the foraging strategy of grazing animals and to be able to optimize the FI between the scales, more than two decades ago, it was postulated in a model that the movement between the smaller scales is governed by the IR of the hierarchical scales immediately below (Figure 2)(33). For instance, when a cow harvests bites within a feeding station, grazing may continue until the IR of the last bites falls below a threshold. If that situation occurs, the cow will move to the next upper hierarchical scale, in which the harvest of bites will be carried out at the patch level, and there in turn it will remain until the IR reaches the lower threshold. In this regard, it has been highlighted that the time of permanence of animals harvesting bites at each feeding station reflects the condition of the forage canopy; when the structural quality of the forage is better in leaf/stem ratio, mass, height and density of forage, the time of permanence will be greater(13). In a study, it was documented that cattle and sheep remained less between feeding stations and moved more quickly between them in native pastures of southern Brazil of low height (4 and 8 cm) than of greater height (12 and 16 cm)(14). Likewise, in another study, it was obtained that the time of permanence of steers in the feeding station of ryegrass (Lolium multiflorum Lam.) with black oats (Avena strigosa Schreb) increased with increases in pasture height (10, 20, 30 and 40 cm)(34). In relation to the FI at the feeding station level, in a study, it was obtained that steers grazing wheat (Triticum aestivum L.) of greater height (23.6 cm) had 1.9 times higher IR of forage and harvested more bites in the area grazed per feeding station than in pastures of lower height (20.4 and 19.5 cm)(35). This is evidence that the foraging strategy used by cattle to achieve a high forage intake during grazing is to increase their IR in patches with high amounts of forage (greater height) and move more quickly between feeding stations when they find patches of lower forage supply (lower height). The distance between potential patches for forage intake is also important in IR components. In this regard, it was obtained that the number of bites, the times of permanence, the speed of movement and the proportion of total forage consumed by cows in patches of alfalfa (Medicago sativa L.) and fescue (Festuca arundinacea L.) increased and the IR decreased with the increase in the distance between patches (1, 4 and 8 m)(27); the cows made a more uniform use of the species as the distance between patches increased.

Bite dimensions

The FI of a grazing animal is related to the capacity of its harvesting apparatus(11) and to the dynamics of the functional response, which is the relationship between IR and some variables

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that describe the abundance of forage in the grazing area, for example, biomass, height and density of the forage(36). Therefore, the FI per bite can be evaluated by bite dimensions and forage bulk density (FBD). BM can be quantified with the use of arithmetic, as the product of bite volume (BV) and FBD(1). BV is not the volume of the oral cavity, but the volume occupied by the forage in the pasture that is harvested with the bite, which has been considered a cylinder of determined depth and area (Figure 1). As a result of a meta-analysis, it was published that there is a curvilinear relationship between BV (y) and pasture height (x), y=9.63*(1-exp (0.00125x)), n=90, RMSE=0.30, due to the responses between the depth and the area of the bite due to the effect of the increase in pasture height(10). Bite depth can be defined as the difference between the initial mean height of the tillers and the mean height of the tillers measured after grazing(8) and, in grass pastures, it can be known from the measurement of the length of the extended tillers before and after grazing(14). In addition, results obtained in different species of forages and ruminants show that there is a linear relationship between the depth of the bite (y) and the length of the extended tillers (x), y= 1.1 + 0.52x, R2 =0.84, n =203, which highlights that the depth of the bite corresponds to 52 % of the length of the tillers(1). The slope value of the previous model approximates the slope obtained with another model based on data from experiments conducted with cattle, which indicated that the depth of the bite (y) increases linearly with the height of the pasture (x), y=1.41+0.44x, RMSE= 1.4, n= 149(10). Based on the above, there is evidence(1,10) to support that the depth of the bite in cattle is close to 50 % of the height of the pasture. However, in a previous experiment on bite depth(26), it was concluded that caution should be exercised with this concept of proportionality, despite the fact that, in the same experiment, bite depth values of 40 to 55 % of the height of an English ryegrass (Lolium perenne L.) pasture were documented in cows. It was also reported that the depth of the bite increased progressively during the first 10 to 20 bites, which indicates that cattle can be cautious when evaluating the patches during grazing(26). The area of the bite in studies with manually built plots is calculated as the quotient of the total grazed area between the number of bites made(37). The area of the bite in cattle increases with the height of the pasture, with a curvilinear relationship and a theoretical maximum of 153.6 cm2(10). FBD refers to the relationship between the mass of the forage and the volume occupied by that mass in the pasture. In studies on bite dimensions, FBD is estimated by the quotient of the forage mass and the volume of the canopy stratum(38). The decrease in FBD has a positive effect on the bite area of cattle; at lower FBD values, the bite area was close to 130 cm2, while for high FBD values, the bite area had a minimum value of 33 cm2(10). The height and density of temperate pastures do not vary independently but are usually inversely related. In very short pastures (generally denser), larger areas of bite are not possible, since the short components (essentially leaves) that are at the edges of the area of 749

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the bite that the animal tries to take escape capture by the tongue and grasp by the teeth and the dental arcade(12). On the contrary, in the temperate climate pastures of greater height in a vegetative state, the depth and the area of the bite are greater because the apprehension of the components of the canopy is facilitated(13). In these cases, the cattle execute compound jaws movements; the animal takes a new bite when it is still chewing the bite it previously took(23).

The height of the pasture in the intake

So far, the effects of pasture height and FBD on bite dimensions have been highlighted. But it is important to discuss the impact that the height of the pasture has on the FI in grazing and to explain how the latter is regulated by IB components and by BM(6,10). Nevertheless, in temperate and tropical pastures, in a vegetative state, FI will be positively affected by the height of the pasture, as long as canopies with high density of green leaves are maintained(29,39,40). The increase in FI by grazing dairy cows is a classic response to the increase in pasture height, which is explained by the changes that occur in the components of IB during grazing, also by the effect of changes in pasture height (Figure 3). This was evidenced from a classic work conducted with ewes in temperate pastures(40) and in recent years, results obtained with beef cattle in tropical pastures(29) confirmed the same. The classic response of IR and BM due to the effect of pasture height is that these variables increase with the increase in height(29,40,41,42). However, recent studies in which different pasture heights were evaluated in Cynodon sp. (10, 15, 20, 25, 30 and 35 cm) and Avena strigosa Schreb (15, 20, 25, 30, 35, 40, 45 and 50 cm) highlighted that, although the same pattern was found, IR and BM decreased in the tallest pastures that were evaluated(24); the results showed a functional response in the form of a dome, where the IR of forage was higher at intermediate pasture heights in both species (39.2 g DM min-1 with 19 cm in Cynodon and 54 g DM min-1 with 29.3 cm in oats)(43). The authors of the work highlighted that this response was the result of changes in BM in tall pastures (30 and 35 cm in Cynodon, 45 and 50 cm in oats) and attributed the decrease in BM to a reduction in its volume, due to a smaller bite area driven by the selective behavior of the animals, since it was not related to pasture restrictions that limited the formation of the bite(43). With the increase in pasture height, cattle also increase the total jaw movements per gram of DM consumed(38), because animals must execute a greater number of jaw movements per each bite because these are of greater mass and, therefore, they require a greater number of chews so that the forage can be swallowed(6,41). In summary, BM and FI in temperate pastures, in a vegetative state, can be higher in pastures with intermediate(43) and tall

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heights(29), and to process larger bites, cattle increase the number of compound jaw movements(23). Another important change in the IB of grazing cows in response to the increase in pasture height is the reduction in BR and GT (Figure 3); the first is a consequence of the greater amount of forage apprehended per bite, which implies an increase in the time per bite(24) due to the increase in the number of chewing jaw movements(34). In other words, as cattle apprehend more forage per bite, they spend more time chewing, which postpones taking the next bite(6,34).

The reduction of the height of the pasture in consumption

So far, it has been mentioned how FI is explained by the IB of the animals and by structural characteristics of the pasture canopy (amount of forage, FBD and pasture height) that regulate BM, based on studies where these variables were evaluated in plants of different heights in a vegetative state. However, it is important to discuss the effect of the reduction in pasture height on FI, which occurs with the rapid depletion of the forage resource that occurs during grazing throughout the day. The harvest of bites during grazing is carried out through horizons of the forage canopy, at a more or less constant depth of 50 % of the height of the pasture(11). Cattle begin grazing at horizon 1 (H1), consume 50 % of the height of the forage and then continue to horizon 2 (H2), where they will harvest approximately the same proportion of height corresponding to the horizon, until they reach horizon 3 (H3) and end at a grazing limit height (Figure 1). In a study conducted with cattle, it was obtained that the depth, area and BM in wheat, sorghum (Sorghum sacharatum L.) and alfalfa (Medicago sativa L.) varied across grazing horizons(22). From H1 to H3, bite depths decreased by 76, 78 and 70 %, bite areas decreased by 44, 56 and 56 % and consequently BMs were lower by 61, 71 and 87 % for wheat, sorghum and alfalfa, respectively. In the vertical structure of the pasture is part of the cause of the variation in the dimensions of the bite because the increase of the pseudostem, stem and dead material from the surface of the pasture to its base become barriers to the formation of the bite(11,38), and consequently they modify the BM and in turn the FI. It has also been documented that the pseudostem and the heights of regrowth and dead material within the forage canopy are partial regulators of the bite depth(26). Therefore, the decrease in the bite area from H1 to H3 is due to the decrease in leaves at the base of the forage canopy and due to the difficulty of apprehension of the

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forage that escapes the sweep of the tongue(22). In addition, the increase in FBD in the lower strata of the pastures(38,39) negatively affects the bite area(10). Based on the above, there is evidence to highlight that the decrease in FI that occurs with the reduction of the height of the pasture, when animals are forced to harvest to the lowest horizon of the pasture, is due to the harvest of small bites. Strip grazing is associated with situations of rapid depletion of the forage resource and when the levels of reduction of pasture height in this situation are not controlled, FI can be negatively affected(44). Nevertheless, in situations of different management, in which the pastures do not exhibit changes in their condition during the day, IR is constant, and the intake behavior is similar throughout the day(29). Rotatinuous grazing (low intensity and high grazing frequencies) is a management strategy to maintain high forage IRs since animals harvest most bites at H1(1,45). Therefore, it is important to note that the control of grazing intensity during pasture management is a useful management measure to have an idea of the level of FI. For example, in order to maximize the FI in dairy cows grazing Lolium arundinaceum, it has been highlighted that grazing management should be carried out at low grazing intensities; residual forage heights of 12 and 15 cm in autumn-winter and spring, respectively(25). While in cattle under grazing of black oats cv. Iapar 61, Cynodon sp. cv. Tifton 85(24) and sorghum (Sorghum bicolor L.)(46), it was found that high IRs remained up to a reduction level of 40 % of the height of the pasture. Shorter residual forage not only translates into lower intake, but also into lower selectivity, which contributes to a lower intake of digestible organic matter, and therefore lower milk production(25). Grazing cattle and sheep are able to increase their BR and GT in situations of low BM (Figure 3) as a behavioral strategy to partially compensate for the reduction in IR(6,7). However, the decrease in the amount of forage ingested per bite is not fully compensated and, as a result, FI is lower(7). Due to differences in animal behavior, the response occurs in continuous and rotational grazing but not in strip grazing; in these cases, height becomes a very limiting factor, causing the animals to choose not to graze for a longer time due to the low compensation associated with very small bite masses(20,47).

Grazing time and forage intake

During the day, the cows carry out grazing, rumination and other activities. Some results of the time that cows spend on each activity in temperate climate in grazing of English ryegrass are shown in Table 1. Cows in temperate pastures exhibit different grazing sessions during the day, between three and four sessions, two of greater intensity, at noon and before dusk(2).

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In a study on grazing of English ryegrass with morning forage allocation, cows spent between 70 and 80 % of their time on grazing after morning and evening milkings(48). In this regard, in another study(49), it was found that, as the first 4 h after morning milking (after 0800 h) and evening milking (after 1500 h) elapsed, the percentage of cows in grazing decreased from 94 to 35 % (morning milking) and from 87 to 9 % (afternoon milking). In relation to rumination and inactivity times for dairy cows, they spend more than 70 % of their time on these activities at night(48,49). Although the greatest rumination time occurs during the night, there are also periods of rumination in the day. Rumination at night is associated with the natural behavior exhibited by ruminants; at dusk they consume forage as quickly as possible and reserve the rumination for the night when they hide with relative safety and the risks of being preyed on decrease(2). Because the total daily grazing time is the cumulative result of all grazing sessions or events, for FI estimation purposes, the GT must be active grazing, which is determined by the number and length of grazing sessions (GSs) during the day(13). GS by definition refers to a long sequence of grazing, which is characterized by a minimum of 20 min of active grazing, whose interruption occurs by the performance of any other activity, also for a minimum period of 20 min(50). From studies conducted on rotational grazing of pastures dominated by English ryegrass, it has been found that dairy cows can perform around 5.6 to 10.0 GSs throughout the day (Table 1) and the length of the GS is approximately 90 min. The number of GSs and their duration have been associated with the quality and quantity of forage; if the mass of available forage is high, the number of GSs is higher and their duration is shorter(25). In these situations, cattle become more selective and can therefore harvest higher quality forage in less time(50). FI in dairy cows grazing pastures dominated by English ryegrass is approximately 3 % of their live weight (LW), based on the integration of results on reported IB components (48,51) (Table 1). Nevertheless, the variation around this mean can be high due to pasture, grazing management and animal factors. In a review of temperate pasture grazing, FI values of 1.6 to 3 % of LW were reported in dairy cows.

Intake rate of different categories

Based on the results in Table 1, the BR is similar between heifers and adult cows (59 bites min-1 on average), while the average BM is higher in adult animals (0.41 g) than in young animals (0.22 g). This is due to the greater length in the arcade of the incisors of the cows

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because it determines the area and volume of the bite and consequently the BM(10). Therefore, the average IR reported in heifers is lower (12.8 g DM min-1) than in cows (24 g DM min-1).

Conclusions Although cows on low-height (short) pastures have the ability to increase BR and GT, as a behavioral strategy to remedy the reduction in IR due to harvesting lower weight bites, the animals do not compensate for the decrease in the amount of forage ingested per bite and, as a result, FI may be low. Therefore, when cows are kept in very short pastures or in situations of overgrazing, it is certain that IRs will be low and consequently the daily forage intake will be lower. Due to the relationship of pasture height and IB components and FI, in practice the monitoring of pasture height can be a useful tool for assessing FI and maintaining high IRs in cattle. However, in Mexico it is necessary to develop studies focused on evaluating the IR of grazing cattle at different pasture heights, which allow generating practical implications for grazing management.

Acknowledgements and conflict of interest

The authors thank the support of CONACYT for the doctoral studies of the first author and declare that they do not have any conflict of interest. Literature cited: 1. Carvalho PCF. Harry Stobbs Memorial Lecture: Can grazing behavior support innovations in grassland management? Trop Grassl 2013;1(2):137-155. https://doi.org/10.17138/tgft(1)137-155. 2. Gregorini P. Diurnal grazing pattern: its physiological basis and strategic management. Anim Prod Sci 2012;52:416-430. http://dx.doi.org/10.1071/AN11250. 3. Villalba JJ, Provenza FD, Catanese F, Distel RA. Understanding and manipulating diet choice in grazing animals. Anim Prod Sci 2015;55:261-271. http://dx.doi.org/10.1071/AN14449. 4. Sheahan AJ, Kolver ES, Roche JR. Genetic strain and diet effects on grazing behavior, pasture intake, and milk production. J Dairy Sci 2011;94(7):3583-3591. http://dx.doi.org/10.3168/jds.2010-4089.

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5. Carvalho PCF, Bremm C, Mezzalira JC, Fonseca L, Trindade JK, Bonnet OJF, et al. Can animal performance be predicted from short-term grazing processes? Anim Prod Sci 2015;55:319-327. https://doi.org/10.1071/AN14546. 6. Boval M, Sauvant, D. Ingestive behaviour of grazing ruminants: Meta-analysis of the components linking bite mass to daily intake. Anim Feed Sci Technol 2021;278 115014. https://doi.org/10.1016/j.anifeedsci.2021.115014. 7. Chilibroste P, Gibb MJ, Soca P, Mattiauda DA. Behavioural adaptation of grazing dairy cows to changes in feeding management: do they follow a predictable pattern? Anim Prod Sci 2015;55:328-338. https://doi.org/10.1071/AN14484. 8. Yayota M, Doi K, Kawamura K, Ogura S. Monitoring foraging behavior in ruminants in a diverse pasture. J Integr Field Sci 2017;14:39-47. 9. Bailey DW, Provenza FD. Mechanisms determining large-herbivore distribution. In: Prins HHT, Langevelde F, editors. Resource ecology: Spatial and temporal dynamics of foraging. Dordrecht, The Netherlands: Springer; 2008:7-28. 10. Boval M, Sauvant D. Ingestive behaviour of grazing ruminants: meta-analysis of the components of bite mass. Anim Feed Sci Technol 2019;251:96-111. https://doi.org/10.1016/j.anifeedsci.2019.03.002. 11. Benvenutti MA, Cangiano CA. Características de las pasturas y su relación con el comportamiento ingestivo y consumo en pastoreo. En: Cangiano CA, Brizuela MA, editores. Producción Animal en Pastoreo. Buenos Aires, Argentina: Instituto Nacional de Tecnología Agropecuaria; 2011:259-290. 12. Andriamandroso AHL, Bindelle J, Mercatoris B, Lebeau F. A review on the use of sensors to monitor cattle jaw movements and behavior when grazing. Biotechnol Agron Soc Environ 2016;20:1-14. https://doi.org/10.25518/1780-4507.13058. 13. Carvalho PCF, Trindade JK, Bremm C, Mezzalira JC, Fonseca L. Comportamiento ingestivo de animais em pastejo. In: Reis RA, Bernardes TF, Siqueira GR, editores. Forragicultura: Ciência, Tecnologia y Gestão dos Recursos Forrageiros. Jaboticabal, SP, Brasil: Gráfica Multipress; 2013:525-545. 14. Gonçalves EN, Carvalho PCF, Devincenzi T, Lopes MLT, Freitas FK, Jacques AVA. 15. Relações planta-animal em ambiente pastoril heterogêneo: padrões de deslocamento e uso de estações alimentares. Rev Bras Zootec 2009;38(11):2121-2126. https://doi.org/10.1590/S1516-35982009001100008.

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Rev Mex Cienc Pecu 2022;13(3):743-762

16. Bailey DW, Stephenson MB, Pittarello M. Effect of terrain heterogeneity on feeding site selection and livestock movement patterns. Anim Prod Sci 2015;55:298-308. http://dx.doi.org/10.1071/AN14462. 17. Larson-Praplan S, George MR, Buckhouse JC, Laca EA. Spatial and temporal domains of scale of grazing cattle. Anim Prod Sci 2015;55:284-297. https://doi.org/10.1071/AN14641. 17. Brizuela MA, Cibils A. Implicancias de la carga animal, distribución de los animales y métodos de pastoreo en la utilización de praderas. En: Cangiano CA, Brizuela MA, editores. Producción Animal en Pastoreo. Buenos Aires, Argentina: Instituto Nacional de Tecnología Agropecuaria; 2011:349-376. 18. Dolev A, Henkin Z, Brosh A, Yehuda Y, Ungar ED, Shabtay A, et al. Foraging behavior of two cattle breeds, a whole-year study: II. Spatial distribution by breed and season. J Anim Sci 2014;92:758-766. https://doi.org/10.2527/jas2013-6996. 19. Hirata M, Matsubara A, Uchimura M. Effects of group composition on social foraging in cattle: inclusion of a leader cow in replacement of a follower facilitates expansion of grazing distribution patterns of beef cows. J Ethol 2022;40:71-78. https://doi.org/10.1007/s10164-021-00731-0. 20. Doyle PR, McGee M, Moloney AP, Kelly AK, O’Riordan EG. Effect of post-grazing sward height, sire genotype and indoor finishing diet on steer intake, growth and production in grass-based suckler weanling-to-beef systems. Animals 2021;11:2623. https://doi.org/10.3390/ani11092623. 21. Do Carmo M, Genro TCM, Cibils AF, Soca PM. Herbage mass and allowance and animal genotype affect daily herbage intake, productivity, and efficiency of beef cows grazing native subtropical grassland. J Anim Sci 2021;99(10):skab279. https://doi.org/10.1093/jas/skab279. 22. Cangiano CA, Galli JR, Pece MA, Dichio L, Rozsypalek SH. Effect of liveweight and pasture height on cattle bite dimensions during progressive defoliation. Aust J Agric Res 2002;53:541-549. https://doi.org/10.1071/AR99105. 23. Galli JR, Cangiano CA, Pece MA, Larripa MJ. Monitoring and assessment of ingestive chewing sounds for prediction of herbage intake rate in grazing cattle. Animal 2018;12:973-982. https://doi.org/10.1017/S1751731117002415. 24. Mezzalira JC, Carvalho PCF, Fonseca L, Bremm C, Cangiano C, Gonda HL, et al. Behavioural mechanisms of intake rate by heifers grazing swards of contrasting structures. Appl Anim Behav Sci 2014;153:1-9. https://doi.org/10.1016/j.applanim.2013.12.014.

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Rev Mex Cienc Pecu 2022;13(3):743-762

25. Menegazzi G, Giles PY, Oborsky M, Fast O, Mattiauda DA, Genro TCM, et al. Effect of post-grazing sward height on ingestive behavior, dry matter intake, and milk production of Holstein dairy cows. Front Anim Sci 2021;2:742685. https://doi.org/10.3389/fanim.2021.742685. 26. Griffiths WM, Hodgson J, Arnold GC. The influence of sward canopy structure on foraging decisions by grazing cattle II. Regulation of bite depth. Grass Forage Sci 2003;58:125-137. https://doi.org/10.1046/j.1365-2494.2003.00360.x. 27. Utsumi SA, Cangiano CA, Galli JR, McEachern MB, Demment MW, Laca EA. Resource heterogeneity and foraging behaviour of cattle across spatial scales. BMC Ecol 2009;9:1-10. https://doi.org/10.1186/1472-6785-9-9. 28. Pulina G, Dias FAH, Stefanon B, Sevi A, Calamari L, Lacetera N, et al. Sustainable ruminant production to help feed the planet. Ital J Anim Sci 2017;16(1):140-171. http://dx.doi.org/10.1080/1828051X.2016.1260500. 29. Da Silva SC, Gimenes FMA, Sarmento DOL, Sbrissia AF, Oliveira DE, HernándezGaray, et al. Grazing behaviour, herbage intake and animal performance of beef cattle heifers on Marandu palisade grass subjected to intensities of continuous stocking management. J Agric Sci 2013;151:727-739. https://doi.org/10.1017/S0021859612000858. 30. Rombach M, Münger A, Niederhauser J, Südekum K-H, Schori F. Evaluation and validation of an automatic jaw movement recorder (RumiWatch) for ingestive and rumination behaviors of dairy cows during grazing and supplementation. J Dairy Sci 2018;101:2463-2475. https://doi.org/10.3168/jds.2016-12305. 31. Vanrell, SR, Chelotti, JO, Bugnona, LA Rufiner, HL Milone, DH, Laca, EA, Galli, JR. Audio recordings dataset of grazing jaw movements in dairy cattle. Data Brief 2020, 30:105623. https://doi.org/10.1016/j.dib.2020.105623. 32. Galli JR, Milone DH, Cangiano CA, Martínez CE, Laca EA, Chelotti JO, et al. Discriminative power of acoustic features for jaw movement classification in cattle and sheep. Bioacoustics 2020;29(5):602-616. https://doi.org/10.1080/09524622.2019.1633959. 33. Laca EA. Modelling spatial aspects of plant–animal interactions. In: Lemaire G, et al editors. Grassland ecophysiology and grazing ecology. Wallingford, UK: CAB International; 2000:209-231.

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Rev Mex Cienc Pecu 2022;13(3):743-762

34. Baggio C, Carvalho PCF, Da Silva JLS, Anghinoni I, Lopes MLT, Thurow JM. Padrões de deslocamento e captura de forragem por novilhos em pastagem de azevém-anual e aveia-preta manejada sob diferentes alturas em sistema de integração lavoura-pecuária. Rev Bras Zootec 2009;38(2):215-222. https://doi.org/10.1590/S151635982009000200001. 35. Gregorini P, Gunter AS, Beck PA, Caldwell J, Bowman MT, Coblentz WK. Short-term foraging dynamics of cattle grazing swards with different canopy structures. J Anim Sci 2009; 87:3817-3824. https://doi.org/10.2527/jas.2009-2094. 36. Searle KR, Shipley LA. The comparative feeding behaviour of large browsing and grazing herbivores. In: Gordon IJ, Prins HHT editors. The ecology of browsing and grazing. Heidelberg, Germany: Springer 2008;117-148. 37. Benvenutti MA, Poppi DP, Crowther R, Spinks W, Moreno FC. The horizontal barrier effect of stems on the foraging behaviour of cattle grazing five tropical grasses. Livest Sci 2009;126:229–238. https://doi.org/10.1016/j.livsci.2009.07.006. 38. Fonseca L, Carvalho PCF, Mezzalira JC, Bremm C, Galli JR, Gregorini P. Effect of sward surface height and level of herbage depletion on bite features of cattle grazing Sorghum bicolor swards. J Anim Sci 2013;91:4357-4365. https://doi.org/10.2527/jas.2012-5602. 39. Brink GE, Soder KJ. Relationship between herbage intake and sward structure of grazed temperate grasses. Crop Sci 2011;51:2289-2298. https://doi.org/10.2135/cropsci201().10.0600. 40. Penning PD, Rook AJ, Orr RJ. Patterns of ingestive behaviour of sheep continuously stocked on monocultures of ryegrass or white clover. Appl Anim Behav Sci 1991;31:237-250. https://doi.org/10.1016/0168-1591(91)90008-L. 41. Gonçalves EN, Carvalho PCF, Kunrath TR, Carassai IJ, Bremm C, Fischer V. Relações planta-animal em ambiente pastoril heterogêneo: processo de ingestão de forragem. Rev Bras Zootec 2009;38(9):1655-1662. 42. Silva GP, Fialho CA, Carvalho LR, Fonseca L, Carvalho PCF, Bremm C, et al. Sward structure and short-term herbage intake in Arachis pintoi cv. Belmonte subjected to varying intensities of grazing. J Agric Sci 2017;156:92-99. https://doi.org/10.1017/S0021859617000855. 43. Mezzalira JC, Bonnet OJF, Carvalho PCF, Fonseca L, Bremm C, Mezzalira CC, et al. Mechanisms and implications of a type IV functional response for short-term intake rate of dry matter in large mammalian herbivores. J Anim Ecol 2017;86:1159-1168. https://doi.org/ 10.1111/1365-2656.12698.

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Rev Mex Cienc Pecu 2022;13(3):743-762

44. Piña LF, Balocchi OA, Keim JP, Pulido RG, Rosas F. Pre-grazing herbage mass affects grazing behavior, herbage disappearance, and the residual nutritive value of a pasture during the first grazing session. Animals 2020;10(2):212 doi:10.3390/ani10020212. 45. Savian JV, Schons RMT, Mezzalira JC, Neto AB, da Silva Neto GF, Benvenutti MA, et al. A comparison of two rotational stocking strategies on the foraging behaviour and herbage intake by grazing sheep. Animal 2020;14(12):2503-2510. https://doi.org/ 10.1017/S1751731120001251. 46. Fonseca L, Mezzalira JC, Bremm C, Filho RSA, Gonda HL, Carvalho PCF. Management targets for maximizing the short-term herbage intake rate of cattle grazing in Sorghum bicolor. Livest Sci 2012;145:205-211. https://doi.org/10.1016/j.livsci.2012.02.003. 47. Castro ME, Andriamandroso ALH, Beckers Y, Rond L, Montufar C, Da Silva NGF, et al. Analysis of the nutritional and productive behaviour of dairy cows under three rotation bands of pastures, Pichincha, Ecuador. J Agric Rural Dev Trop Subtrop 2021;122(2): 289-298. https://doi.org/10.17170/kobra-202112035148. 48. Henriquez-Hidalgo D, Hennessy D, Gilliland T, Egan M, Mee JF, Lewis E. Effect of rotationally grazing perennial ryegrass white clover or perennial ryegrass only swards on dairy cow feeding behaviour, rumen characteristics and sward depletion patterns. Livest Sci 2014;169:48-62. https://doi.org/10.1016/j.livsci.2014.09.002. 49. Sheahan AJ, Boston RC, Roche JR. Diurnal patterns of grazing behavior and humoral factors in supplemented dairy cows. J Dairy Sci 2013;96:3201-3210. https://doi.org/10.3168/jds.2012-6201 10.3168/jds.2012-6201. 50. Baggio C, Carvalho PCF, Da Silva JLS, Rocha LM, Bremm C, Santos DT, et al. Padrões de uso do tempo por novilhos em pastagem consorciada de azevém anual e aveia-preta. Rev Bras Zootec 2008;37(11):1912-1928. https://doi.org/10.1590/S151635982008001100002. 51. Rutter SM, Orr RJ, Penning PD, Yarrow NH, Champion RA. Ingestive behaviour of heifers grazing monocultures of ryegrass or white clover. Appl Anim Behav Sci 2002;76:1-9. https://doi.org/10.1016/S0168-1591(01)00205-2. 52. Ganche E, Delaby L, O’Donovan M, Boland TM, Kennedy E. Short-term response in milk production, dry matter intake, and grazing behavior of dairy cows to changes in postgrazing sward height. J Dairy Sci 2014;97:3028-3041. https://doi.org/10.3168/jds.2013-7475.

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53. Prendiville R, Lewis E, Pierce KM, Buckley F. Comparative grazing behavior of lactating Holstein-Friesian, Jersey, and Jersey × Holstein-Friesian dairy cows and its association with intake capacity and production efficiency. J Dairy Sci 2010;93:764-774. https://doi.org/10.3168/jds.2009-2659. 54. Kennedy E, McEvoy M, Murphy JP, O’Donovan M. Effect of restricted access time to pasture on dairy cow milk production, grazing behaviour, and dry matter intake. J Dairy Sci 2009;92:168-176. https://doi.org/10.3168/jds.2008-1091.

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Figure 3: Forage intake and components of ingestion behavior in response to pasture height(29,40)

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Table 1: Components of ingestion behaviour, grazing times, rumination and inactivity in dairy cows under grazing in temperate climate pastures Dairy cows Heifers English English English Trifolium (52) (53) (51) ryegrass ryegrass ryegrass repens(51) Forage intake, kg DM 100 kg LW-1 3.3 3.0 3.0 2.4 -1 Forage intake, kg DM d 15.2 15.5 6.9 5.5 -1 Grazing time, min d 629 646 536 436 -1 Intake rate, g DM min 24.2 23.9 12.8 12.7 -1 Bite rate, bites min 62 57 61 55 Bite mass, g 0.39 0.42 0.21 0.23 Times of grazing, rumination and other activities in dairy cows English Ryegrass (80%) English Ryegrass (80%) (48) (48) (53) ryegrass and clover ryegrass and clover(54) Grazing time, min d-1 622 591 646 549 -1 Grazing, sessions d 6.5 5.6 10.0 9.6 -1 Session duration, min period 99 120 79.5 63 -1 Rumination, min d 431 402 426 401 -1 Other activities, min d 388 447 368 490

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https://doi.org/10.22319/rmcp.v13i3.5985 Review

Flow cytometry, a universe of possibilities in the veterinary field. Review

Luvia Enid Sánchez-Torres a* Alejandra Espinosa-Bonilla b Fernando Diosdado-Vargas c

Instituto Politécnico Nacional. Escuela Nacional de Ciencias Biológicas, Departamento de Inmunología. Prolongación de Carpio y Plan de Ayala s/n, Colonia Santo Tomás, Alcaldía Miguel Hidalgo, 11340, Ciudad de México, México. b

Instituto Politécnico Nacional. Escuela Nacional de Ciencias Biológicas, Central de Instrumentación. México. c

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Ciudad de México, México.

* Corresponding author: luviasanchez@hotmail.com

Abstract: Flow cytometry is a technology that has helped to rapidly advance many diverse areas of science by allowing the simultaneous measurement of multiple characteristics of each of the individual particles or cells in a sample as they pass at high speed through an area illuminated by one or more lasers. The information obtained includes data on the size and internal complexity, as well as other parameters inherent to each of the particles present in the sample, which are captured by the equipment as light signals. The most common particles analyzed in flow cytometers are cells, so the expression of molecules on their surface and inside, viability, functionality, cell proliferation, DNA content, cytokine production and many others can be analyzed. These determinations can be carried out by using antibodies coupled to fluorochromes or by using molecules whose fluorescence depends on the characteristic to be evaluated. Some flow cytometers are also sorters, which means that the equipment can physically sort those cells that exhibit the characteristics of interest; in addition, it is feasible

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that once they have been purified, they can be used in subsequent experiments. This review focuses on the fundamentals of flow cytometry and its main applications, which offer a great window of opportunity in the veterinary field, both in research and in the clinic. Key words: Flow Cytometry, Immunophenotyping, Viability, Cell Death, DNA Analysis, Cytokines.

Received: 28/04/2021 Accepted: 17/11/2021

General information on flow cytometry Flow cytometry (FC) allows the simultaneous analysis of several individual characteristics of cells or particles in suspension as they pass through one or more laser light beams. Flow cytometers can read thousands of cells per second with the possibility of regulating the speed of analysis, so that multiparametric analysis and the speed at which it is performed are two of its main and most powerful advantages. The type of sample that can be used is very diverse and includes blood, purified cell populations, cell lines, cell suspensions from solid organs, nuclei extracted from paraffin blocks, cell organelles, liposomes, extracellular vesicles, and body fluids, among others. The cells that can be studied in a flow cytometer can come from different animal and plant species, and it is even possible to perform studies directly on microorganisms(1-5). Due to the possibility of studying also inert particles, it is feasible to analyze and quantify molecules in solution, which can be in samples of serum, plasma, urine, cerebrospinal fluid, colostrum, semen, culture supernatants, etc.(6,7). Given its great versatility, FC is currently one of the most widely used techniques in several areas. The relevance of its contributions and the transcendence of the results obtained has led to the integration of national and international associations that have made it possible to share information and establish unified protocols (Optimized Multicolor Immunofluorescence Panels, "OMIP") for various applications, mainly those related to the characterization of cell populations and clinical diagnostics, some of which are focused on the veterinary field(8-11), as well as specialized magazines on the subject such as "Cytometry"(12). The use of this technology in the veterinary field has been increasing slowly and gradually; multiple applications have been reported not only in research but also in the clinic, both for companion animals and wildlife animals. The aspects of veterinary medicine that have benefited most from FC are diagnosis, prognosis and artificial insemination(13-19). Unfortunately, in Mexico,

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its use in the veterinary clinic is scarce, as no laboratories have any of these devices. And although, in the area of veterinary research, several educational institutions do have equipment in which this type of analysis can be carried out, little use is made of this technology. For this reason, in the present document, it was intend to emphasize its versatility and make known the multiple areas of opportunity that exist in the veterinary field to benefit from all the applications of flow cytometry, especially in the clinic.

Fundamentals of flow cytometry

Properties that can be measured by a flow cytometer include the size, internal complexity and fluorescence intensity of the cells analyzed. All these parameters are determined on a relative basis and no absolute values are generated, unless reference standards and controls are used(20,21). The particles or cells to be analyzed must be in suspension, so that they are picked up by the equipment and directed to a physical space called by some authors as "interrogation point", which is the place where the laser beam or beams of the equipment hit the cells (Figure 1A). When the laser beam hits individual cells in the sample, it causes the light to be scattered in various directions, which provides information about their relative size and complexity (Figure 1B); if there are also fluorescent molecules present in the cell, the equipment captures the fluorescence emitted by these molecules, which can provide information about the expression of molecules and some cellular functions, among other characteristics, as detailed below (Figure 1C). The magnitude of each of the signals for size, complexity and fluorescence is recorded for each and every cell in the sample that passes through the laser beam, which allows this technology to perform an individual cell-by-cell analysis. Finally, the emitted signals are collected and transformed into values that can be analyzed by a computer and easily interpreted by the users (Figure 1C) (22).

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Figure 1: General operation of a flow cytometer

The sample is taken to the physical site, called the interrogation point, where the laser beam hits each of the cells. The alignment of the cells is achieved thanks to the pressure difference at which the solution circulates outside the sample (sheath fluid), generating the hydrodynamic focusing of the cells (A). When the laser beam hits each cell, it is deflected based on the cell’s size and internal complexity; these signals are detected in the forward scatter (FSC) and side scatter (SSC) detectors, respectively (B). Detectors of size and complexity, as well as detectors that pick up fluorescent signals, take the information to a computer in order to display it in easy-to-interpret graphs (C). Image partially created with BioRender.

Components of a flow cytometer

Flow cytometers are composed of three main systems: fluid system, optical system and electronic system(2,23). The fluid system takes the sample and directs the cells to the interrogation point. In order for the particles to be best illuminated, they must pass one by one through the center of the laser beam. This is achieved thanks to the hydrodynamic approach, which favors the alignment of the cells in the flow thanks to the pressure difference between the cell suspension and the liquid on the outside, called sheath fluid, as shown in Figure 1A(24,25). The optical system is composed of two systems, the excitation system (laser beams) and the signal collector(26). When the laser encounters a cell, it is scattered depending on the physical properties of the cell, in particular its size and internal complexity. The scattered light is captured by a front-end detector (forward scatter = FSC), and the value reported is proportional to the cell surface or size of the illuminated particle. On the other hand, the laterally scattered light is captured by another detector located at 90° to the laser (side scatter 766

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= SSC), in this case, the value generated is proportional to the internal complexity of the cell or particle(22-23). A representative scheme is shown in Figure 1B. Based on the FSC and SSC values of each cell in the sample, the electronic system of the cytometers constructs graphs that allow to place in different positions those cells in the sample that have sufficient differences between them in terms of their size or complexity. I.e., if is taken a peripheral blood sample, lyse its erythrocytes, and pass it through a cytometer, lymphocytes will appear in the graph in a different position than neutrophils, since the former are small and their internal complexity is low, while neutrophils are larger and are more complex inside because their nucleus is multilobed and has a large number of granules (Figure 1C)(1-2). It is worth mentioning that the location on the FSC and SSC graph does not give the identification of the cell populations; as will be seen later, the use of antibodies is required in order to precisely define the identity of the cells present and the proportion in which each one is found in the sample being analyzed. The electronic system is responsible for converting the optical signals into proportional electronic signals or voltage pulses. Light signals are generated as each cell passes through the laser beam. These light signals are transformed into electronic signals by photodetectors and, based on their intensity, are assigned a relative value on a scale. Signals with identical intensities accumulate at the same scale value, which increases the peak height and signals with higher intensities are plotted at higher values on the graph scale. Finally, all these values are presented in such a way that users can interpret the results obtained with the equipment, as shown below(22,25,27). In addition to the information that can be obtained about cells or particles with respect to their size and internal complexity, information about other characteristics can be analysed based on fluorescent signals(28). A fluorescent compound or fluorochrome is capable of absorbing light within a certain range of wavelengths and, consequently, of emitting at a wavelength longer than the absorption wavelength. The range of wavelengths in which a fluorescent compound can be excited is called the absorption spectrum, and the range of wavelengths of the photons emitted is called the emission spectrum(25). The fluorescence that can be detected in a cytometer can be intrinsic or extrinsic, i.e. it can come from molecules that are part of the cell (riboflavin, NADPH, tryptophan, tyrosine, etc.) and are therefore often referred to as autofluorescence, or it can come from some fluorescent reagent that was added to the sample(1). The most commonly used reagents in FC are antibodies, which bind to the molecule against which they are manufactured and which they are meant to detect. These antibodies, called primary antibodies, may be labeled with a fluorochrome (Figure 2A) or require a second antibody, or secondary antibody, that will recognize the primary antibody, and the secondary antibody may be the one that is conjugated to the fluorochrome (Figure 2B)(23). Some of the applications of cytometry may use as tools,

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instead of antibodies, molecules whose emission wavelength or fluorescence intensity depends on the cellular characteristic to be evaluated (Figure 2C). Figure 2: Flow cytometry staining with antibodies and fluorescent molecules

The expression of molecules in cells can be analyzed using cell-specific antibodies, which can be linked to a fluorochrome (green star) (A), or it may require a second antibody coupled to a fluorochrome to recognize the primary antibody (B). For some applications, molecules whose fluorescence indicates certain characteristic or function of the cell are used. The use of propidium iodide (PI), a fluorescent molecule added to the sample is exemplified in (C); living cells do not allow the fluorochrome to pass into their interior and therefore, they do not fluoresce, unlike dead cells, whose membrane is damaged and allows PI to pass through, staining the nucleic acids of the cell.

Current FC is considered multiparametric because it is possible to evaluate, in addition to size and complexity, several phenotypic and functional characteristics in the same cell simultaneously(22,29). The number of fluorochromes and, consequently, the number of characteristics that can be analyzed in the same sample depends on the model and configuration of the cytometer used, as well as on the characteristics of each fluorochrome to be utilized. The most commonly used laser in CF is the argon laser, which has a wavelength of 488 nm (blue). Most current flow cytometers have more than one laser beam, which increases their analytical potential and versatility(26). The fluorochromes used in a cytometer are those that can be excited by one of the equipment's lasers beams and captured by its fluorescence detectors. The wavelengths captured by each fluorescence detector are defined by the filters in the equipment. Some applications that facilitate the selection of fluorochromes based on

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the configuration of the available equipment include BD Bioscience Spectrum Viewer, Biolegend Spectra Analyzer, FluoroFinder, and ThermoFisher Fluorescence Spectra Viewer.

Types of graphs and their interpretation

The data provided by flow cytometers for each and every cell in a sample must be presented in a way that makes interpretation easy, fast and integrative. Histograms are used to analyze a single parameter captured by the equipment, which is displayed on the x-axis, while the yaxis represents the number of events —cells or particles that meet that characteristic. Histograms are actually frequency graphs (Figure 3). With this type of graph, it is possible to distinguish between events that have the characteristic evaluated on the x-axis and those that do not have it. The simplest interpretation of this type of graph is all or nothing, i.e., either it has or does not have the molecule to be identified; however, the usefulness of this type of graph goes beyond this, since it allows to know how much it expresses it and to compare expression levels of a molecule between cells of the same sample or between samples that are in different conditions. In order to establish the percentage of positive cells for the label, a sample to which the fluorescent label was not added must be given to the cytometer to serve as a reference point between negative and positive signal. Figure 3 shows an example in which the expression of the CD3 molecule is evaluated in the cells of a sample. Figure 3A shows the histogram of an unstained sample, which implies that, when reading a stained sample, all cells appearing on the right (in the values of the scale comprising M1, Figures 3B and 3C) will be positive for the mark being determined, whereby it is possible to obtain the percentage of these cells(1,23,30). Figure 3: Unidimensional analysis

Histograms display only one feature at a time. In order to find out if the cells express the molecule of interest (CD3), first, a sample that has not been stained is introduced (A) and it establishes the values on the scale at which the cells will be considered positive (M1). Histograms (B) and (C) are shown for two different stained samples with 25 % and 70 % of cells expressing the CD3 molecule, respectively.

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Dot plots, density plots, or contour plots are examples of graphs that allow two parameters to be correlated at the same time, one plotted on the x-axis, and the other, on the y-axis. The difference between these plots is the way in which the interaction is presented. The location of each cell on the graph is similar to what happens with coordinates on a Cartesian plane, where the position of each cell will depend on its individual values for the characteristic plotted on x and for the one plotted on y (Figure 4A). With this type of representation, is possible distinguish at least four possibilities: cells that exhibit neither of the two characteristics being evaluated and are located in the lower left quadrant; those that exhibit only one or the other and will, therefore, be displayed in the upper left or lower right quadrants, and those that exhibit both and are therefore located in the upper right quadrant (Figure 4B). When the results are analyzed, the flow cytometer will report the percentage of cells that have the characteristic(s) of interest(23,30). Figures 4C-E show the biparametric analysis of a thymus suspension stained with an anti-CD4-FITC antibody (feature 1) and an anti-CD8-PE antibody (feature 2). It can be observed that the sample comprises 4 % of cells expressing neither CD4 molecule nor CD8 molecule, 17 % expressing only CD4, 6 % expressing only CD8, and 73 % of cells expressing both CD4 and CD8. Visualization of the expression and coexpression of both molecules can be done in dot plots (Figure 4C), density plots (Figure 4D), or contour plots (Figure 4E). Figure 4: Two-dimensional analysis

Two characteristics are displayed simultaneously, and correlations can be established between them. Each cell (point) has a value on the x and y-axis, as on a Cartesian plane (A); General interpretation for twodimensional plots (B); Examples of two-dimensional plots: Dot plot (C), density plot (D) and contour plot (E) of the same sample.

Undoubtedly, one of the areas of greatest progress in recent years in FC has to do with the development of increasingly robust software, which utilizes data mining and machine

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learning to make complex multivariate analysis possible. This has frequently allowed finding FC results organized in heat maps or in principal component analysis, among many other strategies, thanks to which the analysis of many parameters can be integrated into a single graph and to compare the results between individuals or conditions. The files generated by flow cytometers use the ".fcs" (flow cytometry standard) file format, which facilitates the analysis of files generated on different platforms, regardless of the flow cytometer on which the samples were acquired(22,31,32).

“Cell sorting”

Some flow cytometers are capable of sorting, that is, of enriching a particular population, which is physically separated from the rest of the cells in the sample; at the end, the purified or enriched population or populations of interest are obtained aseptically in individual tubes or in the wells of culture plates, with the option of performing subsequent studies with the cells thus obtained, such as microscopy, cell culture and molecular biology analyses, among others(33,34).

Classical applications of flow cytometry As mentioned above, FC is an extremely versatile technology that evaluates several parameters simultaneously in each cell, allowing correlations to be established between them. Not only can FC analyze the presence of molecules on the surface or inside cells: it can also quantify them. It is possible to perform functional tests, biological evaluations of compounds in both eukaryotic and prokaryotic cells, purify cell populations even when they are present in a low proportion in the sample, or quantify soluble molecules and evaluate cell proliferation, cell viability, cell death, metabolic activity and intracellular signaling, just to mention some of them; Another very important aspect is that, by being able to evaluate many of these characteristics simultaneously, the sample volume required is reduced(35,36). The techniques described below have application in both human and veterinary medicine, and within the latter, both in domestic animals and wildlife. It is worth mentioning that, on some occasions, the limitation is the availability in the market of antibodies that recognize specific molecules of the different animal species, but given the importance of the results that can be obtained by FC, the availability of antibodies with different specificities has grown in recent years, facilitating the work in the veterinary area both in research and in the clinic, especially for diagnosis. In addition, interspecies reactivity of different antibodies has been reported, which allows their use even in those for which they were not originally produced(37).

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Immunophenotype

Immunophenotyping refers to the characterization of cell populations based on the molecules expressed by a cell, allowing their identification through the use of antibodies(28). In order to perform an immunophenotyping study, the antibodies used recognize particular molecules in the cells; based on their presence or absence, or on their level of expression, the identification or characterization is performed. It must keep in mind that there are molecules that are expressed in several cell types, while the expression of others is exclusive to certain cell populations or subpopulations, which implies that a correct selection of antibodies must be made. It should be taken into account that the molecules that identify each cell population may depend on the animal species being worked with(38-42). The detection of the different molecules of interest is performed using antibodies labeled with fluorochromes, so if a beam of light that excites the fluorochrome is shone on it, it will emit fluorescence at a certain wavelength that will be captured by a detector which will then identify the fluorochrome and, consequently, the molecule to which the antibody was bound. Identification or characterization of cell populations can be as detailed as needed. Some cytometers, as previously mentioned, only allow the simultaneous evaluation of 1-4 parameters based on fluorescence, i.e. only a maximum of four antibodies that recognize different molecules can be used, while in other equipment, the expression of more than 20 molecules can be studied in the same tube, which expands the possibilities and the detail of the characterization and quantification of cell populations(28). Figures 4C-E are examples of immunophenotyping of different samples using two antibodies: an anti-CD4 and an antiCD8. Immunophenotyping has proved to be of vital importance in the diagnosis and prognosis of several veterinary diseases and a very adequate complement to the conventional morphological study. It is feasible to detect modifications in the proportions of the different cell populations, as well as to characterize neoplastic cells based on their phenotypic markers(43), which allows an accurate diagnosis to be made. Currently, one of the main uses of immunophenotyping has been in the detection and characterization of hemato-oncological processes, both lympho- and myeloproliferative in small species(13,14,16,44). As an example, it can be mentioned the case of canine lymphomas, which are the most frequent type of hematological tumor; 30 to 40% are T-cell lymphomas, and the rest are B-cell lymphomas, which can be differentiated according to the phenotypic markers they present. The antibody panels utilized can lead to a finer characterization that allows, if necessary, to identify even different subtypes. Information on the antibodies, the phenotypic markers of each population and cell subpopulation, as well as the analysis strategy can be consulted in different papers published recently(45), in which it is mentioned that FC can also be used to follow up on the

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treatment and detect in these animals minimal residual disease, which may indicate a relapse. Thanks to FC, certain phenotypes with a worse prognosis have been identified(45). Interestingly, some antibodies obtained for humans show cross-reactivity with canine antigens, which allows their use. The rapidity with which the diagnosis can be obtained is an additional advantage. On the other hand, in dairy herds, the quantification of somatic cells in milk is a common strategy to detect the presence of clinical and subclinical infections in cows. FC allows a differential count of the cell populations and subpopulations present in milk, which has been suggested as an excellent alternative to identify inflammatory processes in the udder, even when somatic cell counts are low. Early detection of infectious processes prevents their progression and thus, produces changes in the quantity and quality of milk, reducing possible economic losses(40,46).

Cell viability

Determining the viability of the cells in a sample allows to assess the state in which they are, a factor that can constitute an internal quality control within the laboratory(47,48). The viability of the cells in a sample depends on many factors, including the collection procedure, transport (if necessary), sample processing during staining, storage, etc., so that if the sample exhibits low viability, it is necessary to determine which factor or factors are affecting this biological parameter. It is important to mention that the presence of dead cells in a sample favors the binding of antibodies independently of their specificity, so if dead cells are not removed from the analysis, they can lead to erroneous results and conclusions due to the presence of falsepositive events(48,49). On the other hand, it is possible that there is a decrease in viability (and therefore an increase in cell death) as a consequence of infection, treatment with chemotherapeutics, in vitro cell stimulation, etc., an event that should attract attention and require a detailed study of the causes and implications. In the particular case of chemotherapeutics, an increase in the percentage of dead cells may indicate a good response to treatment. Reagents to quantify the percentage of viability (or cell death) or to exclude dead cells from a FC assay fall into two main groups. In the first case, the rationale implies that since dead cells have a damaged cell membrane, the fluorochrome enters and stains the dead cell, whereas living cells do not pick up the fluorochrome because their membrane is intact, and the two can be distinguished as stained and unstained, respectively (Figure 2C). Examples of such molecules are propidium iodide (PI) and 7-amino, actinomycin D (7-AAD); once stained, the cells cannot be fixed. In the second case, once the reagent is added, it is washed

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and the cells are fixated, which means that the reading in the cytometer does not have to be carried out immediately, and the samples can be stored. In this case, the fluorescent molecules used are covalently bound to cell proteins. In living cells, since the membrane is intact, the only proteins that will react with the reagent will be those on the surface. In the case of dead cells, the reagent will also react with the proteins inside the cell and stain them more intensely than live cells, making it possible to distinguish between them(47,50). The graphs obtained in both cases and their interpretation are similar regardless of the type of reagent used. The determination of sperm viability before and after the cryopreservation process is an example of the routine use of this application of FC and constitutes an indispensable evaluation in artificial insemination in different animal species(51,52).

Cell death

The study of cell death and the possible mechanisms that can induce it has been a topic of great interest. Since the description of apoptosis and subsequently of all the other mechanisms described to date, the techniques to demonstrate, quantify and characterize apoptosis have increased year by year, given the greater knowledge of the signaling pathways that are activated and lead to cell death(49,53,54). Cell death can be studied generally from two perspectives. The first involves establishing the percentage of dead cells in a cell suspension, as well as to establish the effect of different stimuli without the mechanism of death being of interest. This type of determinations is very common when performing biological activity studies of new molecules with possible antibiotic or antineoplastic activity. For the above, stainings similar to those described for viability are performed, but what is reported is the percentage of dead cells in the sample analyzed and, as already mentioned, the studies can be performed not only on infected eukaryotic cells, but also on microorganisms directly, which is very helpful for antibiotic resistance studies and the evaluation of new antimicrobial molecules(55,56). When it is necessary to identify the mechanism of cell death, it is important to establish a strategy to separate the potential biological events involved in cell death, such as the expression of specific molecules of each death mechanism, the activation of enzymes, the alteration of cellular functions such as mitochondrial membrane potential and the production of reactive oxygen species, the rearrangement of phospholipids in the membrane, the fragmentation of DNA, and the production of reactive oxygen species, etc. These events are related to the different types of cell death described so far and the signaling pathways that are activated in each one(53,57). To date, more than ten different mechanisms of cell death are known, some of which are interconnected. One of the most frequent uses of cell death

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analysis in the veterinary field is to complete the study of semen quality in different animal species(52).

Functional tests

FC also allows the functional capacity of cells to be evaluated; the detection of alterations in the normal capacities of cells can be indicative of pathologies, and their identification can aid in the diagnosis. The following are some of the techniques most commonly used and reported in the literature. Phagocytosis. Phagocytosis is part of the innate immune response mechanisms that allows the containment of infections, that alterations in any of the steps of this process have repercussions on the health of the animals. Neutrophils are the most abundant phagocytic cells in the blood circulation of many mammals, although their proportion may vary; in most carnivores and horses they account for more than 50 % of the cells in blood, in pigs they are at 50 %, while in rodents and ruminants, they are at an average of 25 %. In reptiles, birds, rabbits and fish, phagocytic cells are called heterophils, and their percentage is variable among them(58). FC allows the study of phagocytosis from several points of view; the most common is by measuring the capacity to phagocytose particles or by evidencing the intracellular biochemical changes that occur in the cells after phagocytosis and that lead to the intracellular destruction of the microorganisms. In the first case, bioindicators such as bacteria or yeast can be used, or inert particles coupled to a fluorochrome, so that the percentage of cells that phagocytose and the level of phagocytosis can be determined(59). In addition, it is feasible to monitor the pH change once the biomarkers or inert particles have been phagocytosed. For this purpose, fluorescent probes sensitive to pH changes are used, which allows monitoring each stage of the phagocytosis process, from the formation of phagosomes to their fusion with lysosomes (phagolysosomes), given the differences in the pH between the two compartments (pH 6.7 and 4.7, respectively)(60,61). This type of analysis allows the detection of alterations in phagocytosis, which have been reported in veterinary diseases such as anaplasmosis, chlamydiosis, canine parvovirus, leukocyte adhesion deficiency, and chronic granulomatous disease, among others(58,62). Cell activation. Activation of cells is the result of their interaction with foreign agents or mitogens and may occur in vivo or in vitro; in some cases, activation is manifested by the expression de novo, or as increased basal expression of characteristic molecules, and may or may not be accompanied by cell proliferation and synthesis of soluble molecules such as

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cytokines and chemokines that are released into the microenvironment, among other events. The detection of activated cells in an organism implies that an immune response is being elicited either against a microorganism or against a vaccine agent, and, together with immunophenotyping, it can provide important information in cases of infectious diseases. Proliferation. Cell proliferation can be assessed in vitro and in vivo by adding nucleotide analogues such as bromodeoxyuridine (BrdU) so that, if there is proliferation and, therefore, DNA synthesis, this molecule is incorporated into the new nucleic acid chains. Antibodies directed against BrdU labeled with fluorochromes are used to determine whether the molecule was incorporated; thus, if the cells proliferated, they will give a fluorescent signal, and then it will be possible to quantify the percentage of positive cells(63). It is worth mentioning that BrdU has other uses, such as evidencing DNA fragmentation; therefore, the rationale for its use in each case and the interpretation of the results should not be confused. Another technique used is DNA analysis. In this case, molecules are used that bind to DNA in a stoichiometric manner and therefore fluoresce with an intensity that is proportional to the nucleic acid content(64); thus, cells that are in the G0 or G1 phase of the cell cycle will be located at a value on the scale that is half the value at which cells in early G2 or mitosis (M) phase, prior to cytokinesis, are located. Cells that are in the different stages of the S phase, i.e. from the beginning to the end of the duplication of genetic material, will be located between the position of cells in G0/G1 and G2/M (Figure 5A). When a cell population is proliferating, as occurs in cancer, the percentage of cells in S phase and in G2/M will increase with respect to the control sample in which there is no proliferation. In this case, permeabilization of the cells is also necessary so that the fluorochrome can enter the nucleus and bind to the DNA. Among the most commonly used molecules are PI, 7-aminoactinomycin D (7-AAD) and Hoechst 33342. The study of the cell cycle can be extended and completed with the analysis of molecules involved in each of the phases of the cycle by using the multiparametric potential of the FC. It is worth mentioning that this type of analysis can also identify the presence of cells with fragmented DNA, which appear in what is known as the "Sub-G0 peak" (Figure 5B), which correlates with some types of cell death. With this technique it is also possible to assess genome stability by comparing the ploidy of the G0/G1 peak between tumor cells and healthy cells(64,65). In addition to the above, and as mentioned below, this technique allows distinguishing between X and Y spermatozoa based on their DNA content. Perhaps the most widely used technique to assess proliferation is the carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assay. This technique utilizes the compound CFDA, which is non-fluorescent and lipophilic and can penetrate the cell membrane; once inside, the action of esterases in living cells converts it into CFSE, which fluoresces. This molecule binds to cellular proteins, leaving all the cells in the sample marked; if the cells proliferate, each of the daughter cells will have half the fluorochrome of the cell from which 776

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it came, whereby it will be possible to establish the percentage of proliferating cells and proliferation cycles based on the progressive dilution or reduction of the label with each cell division(66). Figure 5: DNA content analysis

Cells are fixed and stained with propidium iodide. DNA analysis allows quantification of the percentage of cells in different phases of the cell cycle: G0/G1, S and G2/M based on the relative DNA content. The intensity of the staining is directly proportional to the amount of DNA in the cells at each stage (A). This type of analysis also makes it possible to quantify the percentage of cells with fragmented DNA, a peak identified as Sub-G0 (B).

In addition to the above, the expression of Ki67, a molecule located in the nucleus that is associated with cell proliferation, can be detected using an antibody directed against it as proposed in OMIP-065(14), which is the first optimized FC staining panel specific for dogs. Ki67 detection has been associated with the most aggressive canine cancers and can therefore be taken as a prognostic biomarker(67). Cytokine production. Cytokines are soluble molecules that are synthesized in response to a stimulus, and their main function is cell-to-cell communication. There are two general strategies to detect cytokine production: one that detects them intracellularly, allowing simultaneous identification of the producing cells(68), and one in which they can be quantified in a solution once they have been released into the microenvironment in which they are found (plasma, serum or other body fluid, or in the culture medium if it is an in vitro system). In the latter strategy, it is not possible to know which cell produced them unless the cells have been previously purified. In the first case, a reagent that prevents the secretion of cytokines such as brefeldin or monensin must be added to the cells, which are then permeabilized, after which antibodies labeled with fluorochromes directed against the cytokines to be determined are added(69). In the second case, the detection is made in a solution —either in a body fluid or in the culture medium. This strategy utilizes particles that have two characteristics; they

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emit fluorescence and, attached to their surface, they have antibodies directed against the cytokine or molecule of interest, which will act as capture antibodies (Figure 6A). The particles, or beads, as they are also known, have a particular fluorescence intensity depending on the molecule recognized by the antibodies on their surface, so that several soluble molecules can be detected in the same sample(7). Soluble molecules are quantified based on the fluorescence intensity emitted by a reporter antibody added at the end and coupled to a fluorochrome different from that of the bead; Consequently, the fluorescence intensity of the reporter antibody is directly proportional to the concentration of the molecule under study (Figure 6B-D). Thanks to the use of standards of known concentration of each molecule to be quantified, the concentration present in the sample can be obtained. Based on the above, the analysis of the results is biparametric: the intensity of the bead identifies the molecule to be quantified, and the fluorescence intensity of the reporter antibody, its concentration in the sample. Figure 6: Pearl arrangements

The most common ones utilize fluorescent inert particles (red circle) to which capture antibodies for the molecule to be quantified are attached (A). If the soluble molecule to be quantified (green circle) is present in the sample, it will bind to the antibodies. In the last step, a "reporter" antibody is added, which is bound to a fluorochrome (orange star) that is different from that of the bead, and, consequently, its fluorescence intensity will be proportional to the amount of molecule to be quantified. In this application, there are standards of known concentration; therefore, it is a quantitative technique. In B, C and D, three possible results of a bead array are schematized. Sample that does not have the molecule to be quantified (A), sample that has the molecule, but in a small quantity (C), and sample that has a high concentration of the molecule (D). The concentration is directly proportional to the fluorescence intensity emitted by the reporter antibody. Beads with capture antibodies for different molecules are used in the same assay. Image created with BioRender.

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Commercial kits known as "bead arrays" or "bead-based multiplex assays" for quantifying molecules in a solution are available on the market. Unlike ELISA assays, in which a given volume of sample is used to quantify each molecule, here, multiple molecules can be analyzed simultaneously with the same volume(6,70). Given their great versatility and potential in several areas, equipment has been developed for the reading of this type of assays that can detect up to 500 soluble molecules with a maximum sample volume of 50 l, which can be chosen and requested from the manufacturer, based on the particular needs of the buyer. Although commercial supply is still limited in the veterinary field, Christopher-Hennings et al. have proposed this type of assay as an excellent alternative for the quantification of cytokines, chemokines, hormones, pathogens and antibodies in the veterinary field(71). As an example of this, we can mention the serological diagnosis of leishmaniasis in dogs, in which recombinant antigens of the parasite are attached to the beads, rather than to capture antibodies, and what is detected in the sample are antibodies, so that the reporter antibodies recognize the canine antibodies present in the sample(72). Sperm sexing. Based on DNA analysis, X chromosomes can be differentiated from Y chromosomes in a sperm sample of any animal species, since the former have a higher content of genetic material. It is estimated that, in general terms, this difference is approximately 3 to 4 %, and, given the high sensitivity of flow cytometers, the two types of chromosomes can be differentiated and even physically separated into different tubes thanks to the "sorter" function of some equipment, which attains a degree of purity of over 90 %. Separated spermatozoa have been used in artificial insemination with varied results depending on the animal species(73,74). It should not be forgotten that spermatozoa can be evaluated for several of the parameters mentioned above, such as the expression of molecules on their surface or intracellularly, the integrity of their membrane, the presence of molecules involved in some of the types of cell death, etc. Therefore, the studies that can be performed on spermatozoa go beyond DNA analysis, allowing studies on the quality and function of the spermatozoa(19).

Concluding remarks The main use of FC in the veterinary field is currently in research; the contributions that have been made through the use of this technology have had a great impact on the knowledge of cells, organs and systems under certain physiological and pathological conditions in different species. The use of flow cytometers in the clinical area has many advantages, given their high sensitivity, specificity and versatility in terms of the tests that can be performed with them. The cost of these devices and reagents, in addition to their scarce availability, has become an obstacle to their implementation in Mexico in veterinary medicine. Fortunately, both

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situations have been overcome thanks to the great demand for this kind of equipment in different areas, which has reduced its price and expanded the availability of reagents, making their use gradually more accessible and opening the possibility of their introduction into veterinary clinical laboratories in our country. In Mexico, there are several alternatives for training in FC, both theoretical and practical, which are provided in some universities in the country, as well as at the National Laboratory of Flow Cytometry (Laboratorio Nacional de Citometría de Flujo, LabNalCit) and in the Cytometry Chapter of the Mexican Society of Immunology, the latter being a pioneer in the teaching and dissemination of the use of flow cytometers and the correct interpretation of the results obtained with this technology.

Conflict of interests

The authors declare that they have no conflict of interest. Literature cited: 1. Shapiro HM. Practical flow cytometry. 4th ed. New Jersey, USA: Wiley; 2003. 2. Cossarizza A, Chang HD, Radbruch A, Acs A, Adam D, Adam-Klages S, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (Second ed). Eur J Immunol 2019;49(10):1457-973. 3. D’Hondt L, Höfte M, Van Bockstaele E, Leus L. Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status. Mol Plant Pathol 2011;12(8):815-28. 4. Zamora JLR, Aguilar HC. Flow virometry as a tool to study viruses. Methods 2018;134– 135:87-97. 5. Duquenoy A, Bellais S, Gasc C, Schwintner C, Dore J, Thomas V. Assessment of gramand viability-staining methods for quantifying bacterial community dynamics using flow cytometry. Front Microbiol 2020;11:1469. 6. Mandy FF, Nakamura T, Bergeron M, Sekiguchi K. Overview and application of suspension array technology. Clin Lab Med 2001;21(4):713-729. 7. Morgan E, Varro R, Sepulveda H, Ember JA, Apgar J, Wilson J, et al. Cytometric bead array: a multiplexed assay platform with applications in various areas of biology. Clin Immunol 2004;110(3):252-66.

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Rev Mex Cienc Pecu 2022;13(3):763-786

8. Pantelyushin S, Ranninger E, Bettschart-Wolfensberger R, vom Berg J. OMIP-065: Dog immunophenotyping and t-cell activity evaluation with a 14-color panel. Cytometry A 2020;97(10):1024-7. https://pubmed.ncbi.nlm.nih.gov/32583607/. 9. Donaldson MM, Kao SF, Foulds KE. OMIP-052: An 18-color panel for measuring Th1, Th2, Th17, and Tfh responses in rhesus macaques. Cytometry A 2019;95(3):261-263. https://europepmc.org/articles/PMC6414258. 10. Pedreira CE, Costa E, Lecrevise Q, Grigori G, Fluxa R, Verde J, et al. From big flow cytometry datasets to smart diagnostic strategies: The EuroFlow approach. J Immunol Methods 2019;475. 11. Kalina T. Reproducibility of flow cytometry through standardization: Opportunities and challenges. Cytometry A 2020;97(2):137-147. 12. International Clinical Cytometry Society. https://www.cytometry.org/web/journal.php 13. Marconato L, Comazzi S, Aresu L, Riondato F, Stefanello D, Ferrari R, et al. Prognostic significance of peripheral blood and bone marrow infiltration in newly-diagnosed canine nodal marginal zone lymphoma. Vet J 2019;246:78–84. https://www.sciencedirect.com/science/article/pii/S1090023319300115. 14. Meichner K, Stokol T, Tarigo J, Avery A, Burkhard MJ, Comazzi S, et al. Multicenter flow cytometry proficiency testing of canine blood and lymph node samples. Vet Clin Pathol 2020;49(2):249-257. 15. Dudley A, Byron JK, Burkhard MJ, Warry E, Guillaumin J. Comparison of platelet function and viscoelastic test results between healthy dogs and dogs with naturally occurring chronic kidney disease. Am J Vet Res 2017;78(5):589-600. 16. Thamm DH, Gustafson DL. Drug dose and drug choice: Optimizing medical therapy for veterinary cancer. Vet Comp Oncol 2020;18(2):143-151. 17. Seidel GEJ. Sexing mammalian sperm - Where do we go from here? J Reprod Dev 2012;58(5):505-509. 18. Petrunkina AM, Harrison RAP. Fluorescence technologies for evaluating male gamete (dys)function. Reprod Domest Anim 2013;48 (Suppl 1):11-24. 19. Ortega-Ferrusola C, Gil MC, Rodríguez-Martínez H, Anel L, Peña FJ, Martín-Muñoz P. Flow cytometry in Spermatology: A bright future ahead. Reprod Domest Anim 2017;52(6):921-931. 781

Rev Mex Cienc Pecu 2022;13(3):763-786

20. Picot J, Guerin CL, Le Van Kim C, Boulanger CM. Flow cytometry: retrospective, fundamentals and recent instrumentation. Cytotechnology 2012;64(2):109-130. 21. Mizrahi O, Ish Shalom E, Baniyash M, Klieger Y. Quantitative flow cytometry: concerns and recommendations in clinic and research. Cytometry B Clin Cytom 2018;94(2):2118. 22. McKinnon KM. Flow cytometry: An overview. Curr Protoc Immunol 2018;120:5.1.15.1.11. 23. Macey MG. Flow cytometry: Principles and applications. 1st ed. Marion G. Macey, editor. Flow Cytometry: Principles and Applications. London UK: Humana Press Inc.; 2007:1-290. 24. Kachel V, Fellner-Feldegg H, Menke E. Hydrodynamic properties of flow cytometry instruments. Flow Cytom Sorting 1990;27-44. 25. Büscher M. Flow cytometry instrumentation - An Overview. Curr Protoc Cytom 2019;87(1):e52. 26. Shapiro HM, Telford WG. Lasers for flow cytometry: Current and future trends. Curr Protoc Cytom 2018;83:1.9.1-1.9.21. 27.

Snow C. Flow cytometer electronics. http://doi.wiley.com/10.1002/cyto.a.10120.

Cytometry

2004;57A(2):63–9.

28. Delmonte OM, Fleisher TA. Flow cytometry: Surface markers and beyond. J Allergy Clin Immunol 2019;143(2):528-537. 29. Maciorowski Z, Chattopadhyay PK, Jain P. Basic multicolor flow cytometry. Curr Protoc Immunol 2017;117:5.4.1-5.4.38. 30. Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A. Flow cytometry: basic principles and applications. Crit Rev Biotechnol 2017;37(2):163-176. 31. Wang S, Brinkman RR. Data-Driven flow cytometry analysis. Methods Mol Biol 2019;1989:245-265. 32. Montante S, Brinkman RR. Flow cytometry data analysis: Recent tools and algorithms Int J Lab Hematol 2019;41 (Suppl 1):56-62.

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33. Göttlinger C, Mechtold B, Meyer KL, Radbruch A. Setup of a flow sorter. In: Flow cytometry and cell sorting. Springer Berlin Heidelberg; 1992:153-158. 34. Jayasinghe SN. Reimagining flow cytometric cell sorting. Advanced biosystems. WileyVCH Verlag; 2020(4).doi.org/10.1002/adbi.202000019. 35. Claassen M. Shooting movies of signaling network dynamics with multiparametric cytometry. Curr Top Microbiol Immunol 2014;377:177-189. 36. Goetz C, Peng LJ, Aggeler B, Bonnevier J. Phenotyping CD4+ hTh2 cells by flow cytometry: Simultaneous detection of transcription factors, secreted cytokines, and surface markers. Methods Mol Biol 2017;1554:175-184. 37. Conrad ML, Davis WC, Koop BF. TCR and CD3 antibody cross-reactivity in 44 species. Cytometry A 2007;71(11):925-933. 38. Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay. Clinics in Laboratory Medicine. Elsevier; 2007;(27):469-85. 39. Herold NC, Mitra P. Immunophenotyping. In Treasure Island (FL); 2021. 40. Grandoni F, Scatà MC, Martucciello A, Carlo E De, Matteis G De, Hussen J. Comprehensive phenotyping of peripheral blood monocytes in healthy bovine. Cytometry A 2021. https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.24492. 41. Agulla B, García-Sancho M, Sainz Á, et al. Isolation and immunophenotyping by flow cytometry of canine peripheral blood and intraepithelial and lamina propria duodenal T lymphocytes. Vet Immunol Immunopathol 2021;239. https://pubmed.ncbi.nlm.nih.gov/34352607/. 42. Rütgen BC, Baszler E, Weingand N, Wolfesberger B, Baumgartner D, Hammer SE, et al. Composition of lymphocyte subpopulations in normal and mildly reactive peripheral lymph nodes in cats. J Feline Med Surg 2021;65-76. https://doi.org/101177/1098612X211005310. 43. Burkhard MJ, Bienzle D. Making sense of lymphoma diagnostics in small animal patients. Clin Lab Med 2015;35(3):591–607. http://www.labmed.theclinics.com/article/S0272271215000505/fulltext. 44. Giantin M, Vascellari M, Lopparelli RM, Ariani P, Vercelli A, Morello EM, et al. Expression of the aryl hydrocarbon receptor pathway and cyclooxygenase-2 in dog tumors. Res Vet Sci 2013;94(1):90-99. 783

Rev Mex Cienc Pecu 2022;13(3):763-786

45. Comazzi S, Riondato F. Flow cytometry in the diagnosis of canine T-cell lymphoma. Front Vet Sci 2021;8. https://pubmed.ncbi.nlm.nih.gov/33969027/. 46. Pilla R, Bonura C, Malvisi M, Snel GG, Piccinini R. Methicillin-resistant Staphylococcus pseudintermedius as causative agent of dairy cow mastitis. Vet Rec 2013;173(1):19. https://pubmed.ncbi.nlm.nih.gov/23723102/. 47. Coder DM. Assessment of cell viability. Curr Protoc Cytom 2001;Chapter 9:Unit 9.2. https://doi: 10.1002/0471142956.cy0902s15. 48. Johnson S, Nguyen V, Coder D. Assessment of cell viability. Curr Protoc Cytom 2013;64(1):9.2.1-9.2.26. https://onlinelibrary.wiley.com/doi/10.1002/0471142956.cy0902s64. 49. Galluzzi L, Aaronson SA, Abrams J, Alnemri ES, Andrews DW, Baehrecke EH, et al. Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes. Cell Death Differ 2009;16(8):1093-1107. 50. Perfetto SP, Chattopadhyay PK, Lamoreaux L, Nguyen R, Ambrozak D, Koup RA, et al. Amine reactive dyes: an effective tool to discriminate live and dead cells in polychromatic flow cytometry. J Immunol Methods 2006;313(1–2):199-208. 51. Jäkel H, Henning H, Luther AM, Rohn K, Waberski D. Assessment of chilling injury in hypothermic stored boar spermatozoa by multicolor flow cytometry. Cytometry A 2021;99(10):1033–1041. https://onlinelibrary.wiley.com/doi/full/10.1002/cyto.a.24301. 52. Caamaño JN, Tamargo C, Parrilla I, Martínez-Pastor F, Padilla L, Salman A, et al. Postthaw sperm quality and functionality in the autochthonous pig breed Gochu asturcelta. Anim 2021;11(7):1885. https://www.mdpi.com/2076-2615/11/7/1885/htm. 53. Wlodkowic D, Telford W, Skommer J, Darzynkiewicz Z. Chapter 4 - Apoptosis and Beyond: Cytometry in studies of programmed cell death. Methods Cell Biol 2011;103: 55–98. https://www.sciencedirect.com/science/article/pii/B9780123854933000048. 54. Programmed Necrosis. Methods and protocols. Anticancer Res 2018;38(11):6585–6. https://ar.iiarjournals.org/content/38/11/6585.6. 55. Ambriz-Aviña V, Contreras-Garduño JA, Pedraza-Reyes M. Applications of flow cytometry to characterize bacterial physiological responses. Biomed Res Int 2014. https://pubmed.ncbi.nlm.nih.gov/25276788/.

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Rev Mex Cienc Pecu 2022;13(3):763-786

56. Andrade OS, Chacón VKF, Correa BJ, Rodríguez VLM, Moorillón GVN, Sánchez TLE. Rational design of new leishmanicidal agents: In silico and in vitro evaluation. The Battle Against Microbial Pathogens: Basic Science, Technological Advances and Educational Programs. Méndez-Vilas A. editor. Formatex Research Center. ISSBN: (13):2015 ;978-84-942134-6-5. 57. Galluzzi L, Vitale I, Aaronson SA, Abrams JM, Adam D, Agostinis P, et al. Molecular mechanisms of cell death: Recommendations of the nomenclature committee on cell death. Cell Death and Differentiation 2018;25:486–541. https://doi.org/10.1038/s41418-017-0012-4. 58. Fingerhut L, Dolz G, de Buhr N. What is the evolutionary fingerprint in neutrophil granulocytes?. Int J Mol Sci 2020;21(12):4523. doi: 10.3390/ijms21124523. 59. Lehmann AK, Sornes S, Halstensen A. Phagocytosis: measurement by flow cytometry. J Immunol Methods 2000;243(1–2):229-242. 60. Neaga A, Lefor J, Lich KE, Liparoto SF, Xiao YQ. Development and validation of a flow cytometric method to evaluate phagocytosis of pHrodoTM BioParticles® by granulocytes in multiple species. J Immunol Methods 2013;390(1):9-17. https://www.sciencedirect.com/science/article/pii/S0022175911001517. 61. Simons ER. Measurement of phagocytosis and of the phagosomal environment in polymorphonuclear phagocytes by flow cytometry. Curr Protoc Cytom 2010;51(1):9.31.1-9.31.10. https://onlinelibrary.wiley.com/doi/10.1002/0471142956.cy0931s51. 62. du Preez K, Rautenbach Y, Hooijberg EH, Goddard A. Oxidative burst and phagocytic activity of phagocytes in canine parvoviral enteritis. J Vet Diagn Invest 2021;33(5):884893. https://doi: 10.1177/10406387211025513. 63. Rothaeusler K, Baumgarth N. Assessment of cell proliferation by 5-bromodeoxyuridine (BrdU) labeling for multicolor flow cytometry. Curr Protoc Cytom 2007;Chapter 7:Unit7.31. 64. Darzynkiewicz Z, Traganos F, Zhao H, Halicka HD, Li J. Cytometry of DNA replication and RNA synthesis: Historical perspective and recent advances based on “click chemistry.” Cytometry A 2011;79A(5):328-37. http://doi.wiley.com/10.1002/cyto.a.21048. 65. Jacobberger JW, Sramkoski RM, Stefan T, Woost PG. Multiparameter cell cycle analysis. In: Methods in molecular biology. Humana Press Inc.; 2018:203-247. 785

Rev Mex Cienc Pecu 2022;13(3):763-786

66. Lyons AB, Blake SJ, Doherty KV. Flow cytometric analysis of cell division by dilution of CFSE and related dyes. Curr Protoc Cytom 2013;64:9.11.1-9.11.12. 67. Rout ED, Avery PR. Lymphoid Neoplasia: Correlations between morphology and flow cytometry. Vet Clin North Am Small Anim Pract 2017;47(1):53-70. 68. Yin Y, Mitson-Salazar A, Prussin C. Detection of intracellular cytokines by flow cytometry. Curr Protoc Immunol 2015;110:6.24.1-6.24.18. 69. Lamoreaux L, Roederer M, Koup R. Intracellular cytokine optimization and standard operating procedure. Nat Protoc 2006;1(3):1507-1516. 70. Varro R, Chen R, Sepulveda H, Apgar J. Bead-based multianalyte flow immunoassays: the cytometric bead array system. Methods Mol Biol 2007;378:125-52. 71. Christopher-Hennings J, Araujo KP, Souza CJ, et al. Opportunities for bead-based multiplex assays in veterinary diagnostic laboratories. J Vet Diagn Invest 2013;25(6):671-91. Available from: https://pubmed.ncbi.nlm.nih.gov/24153036/. 72. Ker HG, Coura-Vital W, Valadares DG, Aguiar-Soares RDO, de Brito RCF, Veras PST, et al. Multiplex flow cytometry serology to diagnosis of canine visceral leishmaniasis. Appl Microbiol Biotechnol 2019;103(19):8179-90. https://link.springer.com/article/10.1007/s00253-019-10068-x. 73. Yadav SK, Gangwar DK, Singh J, Tikadar CK, Khanna VV, Saini S, et al. An immunological approach of sperm sexing and different methods for identification of Xand Y-chromosome bearing sperm. Vet World 2017;10(5):498-504. 74. Squires E. Current reproductive technologies impacting equine embryo production. J Equine Vet Sci 2020;89:102981. https://www.sciencedirect.com/science/article/pii/S0737080620300721.

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Presence of pyrrolizidine alkaloids in honey and the effects of their consumption on humans and honeybees. Review

Laura Yavarik Alvarado-Avila a Yolanda Beatriz Moguel-Ordóñez b Claudia García-Figueroa a Francisco Javier Ramírez-Ramírez a Miguel Enrique Arechavaleta-Velasco a*

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Centro Nacional de Investigación Disciplinaria en Fisiología y Mejoramiento Animal. Km. 1, Carretera a Colón, 76280, Colón, Querétaro. México. b

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias. Centro de Investigación Regional Sureste. Campo Experimental Mocochá. México.

*Corresponding author: arechavaleta.miguel@inifap.gob.mx

Abstract: Honey produced by honeybees (Apis mellifera L.) is a natural food whose composition depends on the floral origin, the geographical region and the climate where it is produced. Honeybees produce honey from the nectar of flowers, so honey may have secondary metabolites such as pyrrolizidine alkaloids, which are produced by some plants as defense mechanisms against insects and herbivorous animals. Pyrrolizidine alkaloids are toxic to humans and honeybees since they are mutagenic, carcinogenic and hepatotoxic to humans, and in honeybees they produce deterrent effects on feeding, reduce trophallaxis among worker honeybees and can cause the death of honeybees in a colony. The objective of this paper was to review the origin and chemical characteristics of pyrrolizidine alkaloids, the

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presence of these compounds in honey, their toxicity to both humans and honeybees, and the food regulation that establishes the limits of daily consumption of pyrrolizidine alkaloids. Key words: Honey, Pyrrolizidine alkaloids, Consumption, Honeybees, Humans.

Received: 10/06/2021 Accepted: 03/02/2022

Introduction Honey produced by honeybees (Apis mellifera L.) is a natural food composed mainly of carbohydrates, water, amino acids, organic acids, vitamins and minerals(1). The composition, color, aroma and taste of honey depend mainly on its floral origin, as well as on the geographical region and climate where it is produced(2,3). Honeybees produce honey from the nectar of flowers, so sometimes honey contains compounds generated by plants as defense mechanisms in response to biotic and abiotic stressors. Some of these compounds are pyrrolizidine alkaloids, which are secondary metabolites that plants use to protect themselves from herbivorous animals, insects and some microorganisms(4,5). Pyrrolizidine alkaloids may be present in the nectar of flowers. When honeybees forage nectar containing these alkaloids, they produce honey with these compounds and that is when honey becomes a source of pyrrolizidine alkaloids for both honeybees and humans who consume it(6,7,8,9). Honey is a product of commercial importance in Mexico, in 2019 the value of production was approximately 2.489 billion pesos, in that year, 61,986 t were produced and approximately 42,150 t were exported. These production and export volumes place Mexico in tenth place as a producer and in fourth place as an exporter of honey worldwide(10,11). The objective of this paper is to review the presence of pyrrolizidine alkaloids in honey, the origin and characteristics of these compounds, the effects of consuming this type of alkaloids for both humans and honeybees, and the food regulation that establishes the limits of daily consumption of pyrrolizidine alkaloids for humans.

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Origin of pyrrolizidine alkaloids Plants face biotic and abiotic stressors that affect their development, such as damage caused by herbivorous animals, insects and microorganisms(4,5). In response to these stressors, plants produce secondary metabolites such as alkaloids, which have a deterrent and toxic effect as they are not palatable, and some affect the central nervous system. The production of secondary metabolites is one of the defense systems most used by plants against herbivorous animals and insects(12). Currently, about 20,000 secondary metabolites are known, which are divided into two groups: nitrogenous and non-nitrogenous. Alkaloids belong to the group of nitrogenous metabolites and are heterocyclic compounds synthesized through amino acids such as tyrosine and arginine. Alkaloids are divided into five groups: pyrrolizidine alkaloids, isoquinoline alkaloids, quinolizidine alkaloids, tropane alkaloids and indole alkaloids(5,13). Pyrrolizidine alkaloids are produced by more than 560 species of plants, distributed mainly in the families: Asteraceae, Boraginaceae, Apocynaceae and in the genus Crotalaria of the family Fabaceae, although there are also some plants of the families Celastraceae, Convolvulaceae and Ranunculaceae and of the genera Liparis, Malaxis and Cysis of the family Orchidaceae that also produce pyrrolizidine alkaloids(14,15).

Chemical structure of pyrrolizidine alkaloids

The chemical structure of pyrrolizidine alkaloids consists of an acid fragment called necic acid (Figure 1a) and a bicyclic necine base (Figure 1b) with a hydroxymethyl substituent at position 1 and a hydroxyl group at position 7. This necine base may have a saturation (Figure 2a) or an unsaturation (Figure 2b) between positions 1 and 2(16,17). Figure 1: Basic structure of pyrrolizidine alkaloids: a) necic acid; b) bicyclic necine(17)

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Pyrrolizidine alkaloids can be found in two forms, one is in the form of a free base or tertiary alkaloid (Figure 2a and 2b) and the other is in the form of N-oxides (Figure 2c). The free base or tertiary alkaloid is the conjugate form of an amine, which, in the case of alkaloids, is a tertiary amine, which gives the property of being tertiary alkaloids, when this free base has an unsaturation between positions 1 and 2, the toxic version of the alkaloid appears, the presence of this unsaturation is a fundamental characteristic for its toxicity(17,18). Figure 2: Different forms of appearance of pyrrolizidine alkaloids: a) bicyclic necine with a saturation between positions 1, 2; b) bicyclic necine with an unsaturation between positions 1, 2; c) basic structure of pyrrolizidine alkaloids in their N-oxide form(17)

The N-oxide form is a product of the oxidation of pyrroles, and in this form the pyrrolizidine alkaloids are not toxic, but these can be reduced and transformed into tertiary alkaloids, in the same way, tertiary alkaloids can be oxidized to their corresponding N-oxide; so pyrrolizidine alkaloids have the ability to change from one form to another(19,20).

Classification of pyrrolizidine alkaloids

There are two main systems for classifying pyrrolizidine alkaloids, one is based on the combination of the necine base with necic acid and their binding patterns, while the other is based on the chemical structure of the necine base(14,21). In the first classification system, there are five types of pyrrolizidine alkaloids. Those of the senecionine type synthesized by the plants of the genera Senecio and Eupatorium of the family Asteraceae. Those of the lycopsamine type synthesized mainly by plants of the families Boraginaceae and Apocynaceae. The triangularine-type alkaloids, which are produced by several species of the genera Senecio and Boraginaceae. Those of monocrotaline type, which are produced mainly by plant species of the genus Crotalaria and some species of the family Boraginaceae. The phalaenopsine-type alkaloids, found in plants

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of the family Orchidaceae. Those of the loline type produced by the plant Lolium cuneatum and plants of the genus Adenocarpus of the family Fabaceae. Alkaloids of the miscellaneous type, mainly found in the genus Crotalaria of the family Fabaceae. Most of the known pyrrolizidine alkaloids are of the senecionine and lycopsamine types(14). In the second classification system, which is derived from the different necine bases, there are four types of pyrrolizidine alkaloids: those of the retronecine type, those of the heliotridine type, those of the otonecine type and those of the platynecine type. The base of the alkaloids of the retronecine, heliotridine and otonecine types has an unsaturation between positions 1 and 2, so they are considered of greater toxicity than those of the platynecine type(15,21).

Toxicity of pyrrolizidine alkaloids

The level of toxicity of pyrrolizidine alkaloids depends mainly on their chemical structure, the pathways involved in the metabolism of the alkaloids, the rate of detoxification and the variations of each individual(22). For pyrrolizidine alkaloids to be toxic, they must have a double bond between positions 1 and 2 and an ester group at position C-7, at position C-9 or at both positions of the necine base(23). Pyrrolizidine alkaloids are considered pretoxins, for them to cause damage, it is necessary that they be activated via hepatic metabolism, through the normal mechanisms of oxidative detoxification. In this way, the alkaloids that are consumed in their tertiary form are transformed into pyrrolic metabolites with the participation of the cytochrome P-450 monooxygenase enzyme(20,24,25). On the other hand, when the non-toxic form of pyrrolizidine alkaloids is consumed, they can be reduced to their corresponding tertiary alkaloid (toxic form), absorbed and activated via the liver(22,26,27). The toxic effect of alkaloids is because they interrupt the transmission of the neuronal signal by affecting neuronal receptors, ion channels and enzymes responsible for the degradation of neurotransmitters and second messengers. In addition, they have the ability to intercalate in DNA, as well as to stop protein synthesis, induce apoptosis and inhibit the activity of enzymes involved in carbohydrate metabolism(28).

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Studies conducted on Drosophila melanogaster indicate that pyrrolizidine alkaloids are mutagenic and genotoxic (Table 1)(29,30,31). Similarly, a study developed with rats and mice that were fed with the alkaloid riddelliine showed a significant increase in the presence of different types of cancer such as hemangiosarcoma, and tumors such as hepatocellular adenoma, alveolar adenoma and bronchiolar adenoma(32). Table 1: Pyrrolizidine alkaloid concentrations at which mutagenic or genotoxic effects occur in Drosophila melanogaster(29,30,31) Type Pyrrolizidine alkaloid Concentration in moles Effect -6 -4 Jacoline 25x10 -5x10 Genotoxic -2 Platiphylline 2x10 Mutagenic -6 -4 Retrorsine 25x10 -5x10 Genotoxic -6 -5 Seneciphylline 5x10 - 5x10 Genotoxic -5 Seneciphylline 1x10 Mutagenic -4 Senecionine Seneciphylline 1x10 Mutagenic -3 Seneciphylline 1x10 Mutagenic -6 -4 Senecionine 5x10 - 1x10 Genotoxic -2 Senecionine 2x10 Mutagenic -3 -2 Senkirkine 5x10 a 5x10 Genotoxic -5 Senkirkine 1x10 Mutagenic -5 -4 7-acetyl intermidine 1x10 - 1x10 Genotoxic -5 -4 7-acetyl lycopsamine 1x10 - 1x10 Genotoxic -2 Echimidine 2x10 Mutagenic -2 Echinatine 2x10 Mutagenic Lycopsamine -3 -2 Indicine 1x10 - 1x10 Genotoxic -4 -3 Intermedine 5x10 -1x10 Genotoxic -3 Lycopsamine 1x10 Genotoxic -4 -3 Indicine N-oxide 25x10 -25x10 Genotoxic -4 Monocrotaline 25x10 -0.10 Genotoxic Monocrotaline -2 Monocrotaline 2x10 Mutagenic -4 Retronecine Symlandine 1x10 Genotoxic -6 -4 Heliotrine 25x10 -5x10 Genotoxic -2 Heliotrine Heliotrine 2x10 Mutagenic -2 Lasiocarpine 2x10 Mutagenic

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Effects of pyrrolizidine alkaloid intake on human health

The presence of these alkaloids in foods is a hazard to human health, cases of exposure to pyrrolizidine alkaloids in humans occur due to the consumption of foods containing these alkaloids, or due to the consumption of medicines and food supplements made from medicinal plants. There are two types of poisoning from the intake of pyrrolizidine alkaloids. Chronic poisoning is the most frequent(33) and mainly causes delayed and progressive liver damage, hepatic and pulmonary vein occlusions and damage to blood vessels. In addition, to a lesser extent, it can cause damage to the kidney, the gastrointestinal tract, bone marrow, pancreas, megalocytosis and inhibition of mitosis(24,34,35,36). Acute poisoning occurs less frequently and causes effects such as hepatomegaly and ascites(36), it is estimated that it causes mortality in 20 % of people who suffer from acute poisoning, while 50 % recover and the remaining 30 % can develop chronic hepatic veno-occlusive disease years after poisoning(37). In addition, it is known that the alkylating agents that are formed by the biotransformation of pyrrolizidine alkaloids in the body have a synergistic effect with aflatoxins and with the virus that causes hepatitis, which causes different types of liver cancer(36,38).

Effects of pyrrolizidine alkaloid intake on honeybees

There are insects that have mechanisms to avoid the adverse effects of consuming pyrrolizidine alkaloids. Honeybees do not have these mechanisms, since they can’t perform the metabolic conversion from the toxic form of the alkaloid to the non-toxic form and honeybees do not have a specific system to keep the alkaloids in their non-toxic form, so they transform up to 69 % of the pyrrolizidine alkaloids they ingest in non-toxic form to their toxic form in the intestine due to a reduction of the N-oxide and absorb them passively into the hemolymph since these are fat-soluble(18,39). Once the alkaloids are in the hemolymph, the free base functions as a substrate for the cytochrome P450 oxidase enzyme, which is part of xenobiotic metabolism, and this free base is transformed into pyrroles. The products resulting from this bioactivation are highly reactive and mutagenic alkylating agents(24,39).

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A study conducted in Germany indicates that concentrations of 2 % of unsaturated pyrrolizidine alkaloids in a sucrose solution produce harmful effects on honeybees and can cause the death of up to 70 % of honeybees in a period of 48 h under laboratory conditions. This study, showed that the presence of pyrrolizidine alkaloids in the nectar affects trophallaxis, since honeybees that collected sugar syrup with a concentration of 2 % of pyrrolizidine alkaloids could only transfer 4 % of the volume of the syrup they stored in the honey stomach to other honeybees, while honeybees that were fed sugar syrup with pyrrolizidine alkaloid concentrations of 0.2 % were able to transfer more than 15 % of the volume of syrup stored in the honey stomach to other honeybees(18).

Regulation and consumption limits of pyrrolizidine alkaloids

The World Health Organization recommends reducing food contamination by pyrrolizidine alkaloids to the lowest possible level, as well as monitoring honeys that are produced in regions where it is known that there is a risk of contamination with pyrrolizidine alkaloids(36). However, to date there is no regulation for the presence of these alkaloids in honey since the limits to establish the criteria for acceptance or rejection in the commercialization of this food have not been determined. Some countries have established limits on the consumption of pyrrolizidine alkaloids based mainly on the use of drugs and food supplements made from medicinal plants. Germany established that products that contain pyrrolizidine alkaloids and that are taken orally must have the warning label “not to be used during pregnancy and lactation” and through the Federal Institute for Risk Assessment (BfR, for its acronym in German) recommends a limit of intake of pyrrolizidine alkaloids of 0.007μg/kg of body weight/day, as this dose is unlikely to cause harmful effects such as cancer. In addition, for treatments using drugs made from medicinal plants whose duration is less than six weeks, the daily intake limit of pyrrolizidine alkaloids is 1μg/day; if treatment must be followed for more than six weeks, the daily intake limit of these is reduced to 0.1μg(40,41). The United Kingdom Committee on Toxicity recommends a pyrrolizidine alkaloid intake limit of 0.007μg/kg of body weight/d(42). In the Netherlands, the National Institute for Public Health and the Environment (RIVM, for its acronym in Dutch) established a maximum consumption limit for pyrrolizidine alkaloids of 0.1μg/kg of body weight per day in order to reduce the risk of cancer due to the intake of high concentrations of these alkaloids(43,44).

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The United States Food and Drug Administration (FDA) withdrew from the market edible products containing pyrrolizidine alkaloids or originating from the comfrey plant (Symphytum spp.) as they were considered to cause severe health damage. However, a specific intake limit has not been established due to lack of information(45). In Japan, the Ministry of Health, Labour and Welfare banned the commercialization of comfrey (Symphytum spp.) and all foods containing it(46). In Australia and New Zealand, only chronic poisoning by pyrrolizidine alkaloids is considered a risk to human health, so a tolerable daily intake of pyrrolizidine alkaloids of 1μg/kg of body weight was established(47). The Joint Code of Food Standards in Australia and New Zealand prohibits the intentional addition of some pyrrolizidine alkaloid-producing plants to foods, such as Crotolaria spp., Echium plantagineum, Echium vulgare and Heliotropium spp., among others(48). Likewise, in Australia and New Zealand there are specific measures to reduce the concentration of pyrrolizidine alkaloids in honey, which consist of mixing honeys that are known to come from plants that produce these alkaloids with honeys that come from plants that do not produce them(49).

Pyrrolizidine alkaloids in honey

The presence of pyrrolizidine alkaloids in honey has been reported in several studies conducted in different countries and they report different levels of concentration and plant origins of the alkaloids. In a study conducted in the United States, it was found that honey obtained from the plant Senecio jacobea in the state of Oregon had a concentration of up to 3.9 μg/g of this type of alkaloids(7). In New Zealand, honeys were found with concentrations of pyrrolizidine alkaloids ranging from 0.017 to 2.85 μg/g of the tertiary form and N-oxides of echivulgarine, vulgarine, uplandicine, echiuplatine, 3’-O-acetylechimidine and leptanthine, which are characteristic of the plant Echium vulgare (blueweed)(50). In Australia, a study analyzed honeys that came from plants that produce pyrrolizidine alkaloids (n= 29) and honeys that came from plants that do not produce pyrrolizidine alkaloids or whose floral origin was not associated with a specific source (n= 35). In honeys that came from alkaloid-producing plants, concentrations of pyrrolizidine alkaloids were found in a range of 0.033 to 2.27 μg/g, and 19 honeys without a specific floral source or from

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a source that does not produce pyrrolizidine alkaloids were positive and had concentrations between 0.003 and 0.8 μg/g, due to the mixing of honeys of different floral origins(51). The mixing and homogenization of honeys that come from different apiaries, carried out by some producers and marketers, causes pyrrolizidine alkaloids to be frequently found in commercial honeys, although not all honeys used in the process come from nectar from plants that produce these alkaloids. In a study carried out in Ireland, it was found that in 50 samples of honeys that were retailed in Ireland, 16 % were positive for pyrrolizidine alkaloids and had an average concentration of 1.26 μg/g of honey, amounts capable of causing chronic poisoning in humans(9). In Switzerland, a study analyzed 18 honey samples and 36 nectar samples collected from places where the alkaloid-producing plant Echium vulgare grows. Concentrations of up to 0.153 μg/g of pyrrolizidine alkaloids in honey and a range of 0.3-95.1 μg/g in nectar were found, it was also found that the most frequent alkaloid in both honey and nectar samples was echimidine(6). In Germany, a study conducted with 216 honey samples, obtained from European supermarkets as well as from online honey distributors, found that 19 samples contained pyrrolizidine alkaloids in concentrations of 0.02 to 0.12 μg/g. The method used in this study only detects pyrrolizidine alkaloids of the retronecine and heliotridine types, but it does not detect other types of alkaloids, so it is likely that the amount of pyrrolizidine alkaloids per sample was higher than that shown by the results(52). In another study that was developed in this country, in which 8,000 samples of imported honey were analyzed, positive samples for the presence of pyrrolizidine alkaloids were found, with an average concentration of 0.036 μg/g, with echimidine, lycopsamine and lycopsamine N-oxide being the pyrrolizidine alkaloids that were found most frequently(53). In Germany, in 2016, it was detected that honey imported from Mexico was contaminated with these alkaloids with concentrations of 0.46 μg/g, which caused it to be classified as a serious risk by the health authorities and caused it to be withdrawn from the market(54), while, in 2019, pollen imported from Spain containing pyrrolizidine alkaloids in a concentration of 0.786 μg/g was detected, which caused it to also be classified as a serious risk and to be withdrawn from the market(54).

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Conclusions In some countries, there are regulations that determine the concentration limit of pyrrolizidine alkaloids in certain foods and in some of these countries, limits on the daily consumption of these alkaloids have also been established for humans. Despite of the scientific evidence that indicates that honey may be a source of pyrrolizidine alkaloids, and that chronic poisoning is of greatest concern, because of honey consumption, there are no reports of poisoning by pyrrolizidine alkaloid through honey consumption in humans. It is possible that this is because pyrrolizidine alkaloids are also toxic to honeybees, so they cannot consume honey with high concentrations of these compounds. It is important to consider that honeybee colonies produce honey for their own consumption; therefore, honeybees avoid collecting nectar with high levels of pyrrolizidine alkaloids to protect themselves from their toxic effects. However, it is important to have specific regulations that establish the maximum permitted limits of these compounds in honey, as well as specific acceptance or rejection criteria regarding pyrrolizidine alkaloids for the national and international commercialization of honey. It is very important to establish the maximum tolerable limits of pyrrolizidine alkaloids in honey, since it is a natural food consumed by humans as obtained from the hive and the presence of this type of compounds is a cause of loss of safety and quality in honey. Honey production is an important activity in Mexico, in which the export of this product has a very important role from the economic point of view, so any impediment to its international commercialization must be taken into serious account. There are recent cases in which Mexican honeys were withdrawn from the German market because the concentration of pyrrolizidine alkaloids was classified as a severe risk to the health of consumers. Information on the presence of pyrrolizidine alkaloids in Mexican honeys is very limited, so studies are required to define the types and concentrations of pyrrolizidine alkaloids that can be found in Mexican honeys according to their floral origin or region where they are produced, in order to avoid or reduce their effects on human health and their possible impact on the export of this product. Literature cited: 1. Alqarni AS, Owayss AA, Mahmoud AA, Hannan M. Mineral content and physical properties of local and imported honeys in Saudi Arabia. J Saudi Chem Soc 2012;5:618–625. 2. Escuredo O, Dobre I, Fernández-González M, Seijo MC. Contribution of botanical origin and sugar composition of honeys on the crystallization phenomenon. Food Chem 2014;149:84–90.

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3. Tornuk F, Karaman S, Ozturk I, Toker OS, Tastemur B, Sagdic O, et al. Quality characterization of artisanal and retail Turkish blossom honeys: Determination of physicochemical, microbiological, bioactive properties and aroma profile. Ind Crops Prod 2013;46:124–131. 4. Croteau R, Kutchan TM, Lewis NG. Natural products (Secondary metabolites). In: Buchanan B, Gruissem W, Jones R editors. Biochemistry and molecular biology of plants. Maryland, USA: Am Soc Plant Physiol; 2000:1250-1318. 5. Sepúlveda-Jiménez G, Porta DH, Rocha SM. La participación de los metabolitos secundarios en la defensa de las plantas. Rev Mex Fitopatol 2003;21(3):355-363. 6. Lucchetti M, Glauser G, Kilchenmann V, Dübecke A, Beckh G, Praz C, et al. Pyrrolizidine alkaloids from Echium vulgare in honey originate primarily from floral nectar. J Agric Food Chem 2016;64(25):5267-5273. 7. Deinzer ML, Thompson PA, Burgett DM, Isaacson DL. Pyrrolizidine alkaloids: Their occurrence in honey from tansy ragwort (Senecio jacobaea L.). Science 1977;795:497-499. 8. Crews C, Startin JR, Clarke PA. Determination of pyrrolizidine alkaloids in honey from selected sites by solidphase extraction and HPLC-MS. Food Addit Contamin 1997;14:419-428. 9. Griffin C, Danaher M, Elliot C, Kennedy D, Furey A. Detection of pyrrolizidine alkaloids in commercial honey using liquid chromatography – ion trap mass spectrometry. Food chem 2013;136:1577-1583. 10. FAO. Organización de las Naciones Unidas para la Alimentación y la Agricultura. Food and agriculture data. 2020. http://www.fao.org/faostat/en/#data/QA Consultado 1 Ago, 2020. 11. SIAP Servicio de Información Agroalimentaria y Pesquera. Anuario estadístico de producción ganadera 2019. https://nube.siap.gob.mx/cierre_pecuario/ Consultado 7 Ago, 2020. 12. Baker HG, Baker I. Studies of nectar-constitution and pollinator–plant coevolution. In: Gilbert LE, Raven PH, editors. Coevolution of Animals and Plants. Austin, Texas, USA: University of Texas Press; 1975:100–140. 13. Facchini PJ. Alkaloid biosynthesis in plants: Biochemistry, cell biology, molecular regulation, and metabolic engineering applications. Annual Review of Plant Physiology and Plant Molecular Biology 2001;52:29-66.

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14. Hartmann T, Witte L. Chemistry, biology and chemoecology of the pyrrolizidine alkaloids. In: Pelletier SW editor. Alkaloids: Chemical and biological perspectives. 1rst ed. Oxford: Pergamon Press; 1995:155–233. 15. Robins DJ. The pyrrolizidine alkaloids. Prog Chem Org Nat Prod 1982;(41):115-203. 16. Edgar JA, Roeder E, Molyneux R. Honey from plants containing pyrrolizidine alkaloids: a potential threat to health. J Agric Food Chem 2002;50(10):2719-2730. 17. El-Shazly A, Wink M. Diversity of pyrrolizidine alkaloids in the boraginaceae structures, distribution, and biological properties. Divers 2014;6:188-282. 18. Reinhard A, Janke M, von der Ohe W, Kempf M, Theuring C, Hartmann T, et al. Feeding deterrence and detrimental effects of pyrrolizidine alkaloids fed to honey honeybees (Apis mellifera). J Chem Ecol 2009;35:1086–1095. 19. Mattocks AR, White IN. The conversion of pyrrolizidine alkaloids to dihydropyrrolizine derivatives by rat-liver microsomes in vitro. Chem Biol Interact 1971;3:383-396. 20. Johnson AE, Molyneux RJ, Merrill GB. Chemistry of toxic range plants. Variation in pyrrolizidine alkaloid content of Senecio, Amsinckia, and Crotalaria species. J Agric Food Chem 1985;33:50–55. 21. Wang Y, Fu P, Chou M. Metabolic activation of the tumorigenic pyrrolizidine alkaloid, retrorsine, leading to DNA adduct formation in vivo. Int J Environ Res Public Health 2005;2(1):74–79. 22. Stegelmeier B, Edgar J, Colegate S. Gardner D, Schoch T, Coulombe R et al. Pyrrolizidine alkaloid plants, metabolism and toxicity. J Nat Toxins 1999;8:95–116. 23. Kempf M, Reinhard A, Beuerle T. Pyrrolizidine alkaloids (PAs) in honey and pollenlegal regulation of PA levels in food and animal feed required. Mol Nutr Food Res 2010;54:158–168. 24. Mattocks, AR. Chemistry and Toxicology of Pyrrolizidine Alkaloids. Academic Press, London 1986. 25. Allsopp M. Biocontrol of bloublommetjies. S Afric Honeybee J 1993;65(2):32-36. 26. Williams L, Chou MW, Yan J, Young JF, Chan PC, Doerge DR. Toxicokinetics of riddelliine, a carcinogenic pyrrolizidine alkaloid, and metabolites in rats and mice. Toxicol Appl Pharmacol 2002;182:98–104.

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27. Yang YC, Yan J, Doerge DR, Chan PC, Fu PP, Chou MW. Metabolic activation of the tumorigenic pyrrolizidine alkaloid, riddelliine, leading to DNA adduct formation in vivo. Chem Res Toxicol 2001;14:101–109. 28. Wink M, Schimmer O. Modes of action of defensive secondary metabolites. In: Wink M editor. Functions of plant secondary metabolites and their exploitation in biotechnology. Sheffield, England: Sheffield Academic Press; 1999:17-134. 29. Clark AM. The mutagenic activity of some pyrrolizidine alkaloids in Drosophila. Z Vererbungslehre 1960;91:74–80. 30. Candrian U, Lüthy J, Graf U, Schlatter C. Mutagenetic activity of the pyrrolizidine alkaloids seneciphylline and senkirkine in Drosophila and their transfer to rat milk. Food Chem Toxic 1984;22(3):223-225. 31. Frei H, Lüthy J, Brauchli J, Zweifel U, Würgler FE, Schlatter C. Structure/activity relationships of the genotoxic potencies of sixteen pyrrolizidine alkaloids assayed for the induction of somatic mutation and recombination in wing cells of Drosophila melanogaster. Chem-Biol Inter 1992;83:1–22. 32. Chan PC, Haseman JK, Prejean JD, Nyska A. Toxicity and carcinogenicity of riddelliine in rats and mice. Toxicol Lett 2003;144:295-311. 33. Boppré M. The ecological context of pyrrolizidine alkaloids in food, feed and forage: an overview. Food Addit Contam 2011;85(3):260–281. 34. Wiedenfeld H, Roeder E. Pyrrolizidine alkaloids: structure and toxicity. Dtsch Apoth Ztg 1984;124:2116–2122. 35. Fu P, Xia Q, Lin G, Chou M. Pyrrolizidine alkaloids–genotoxicity, metabolism, enzymes, metabolic activation and mechanisms. Drug Metab Rev 2004;36:1–55. 36. IPCS. International Programme on Chemical Safety. Pyrrolizidine Alkaloids. Environmental Health Criteria 80. OMS. Geneva, 1988. 37. Stuart KL, Bros B. Veno-occlusive disease of the liver. Q J Med 1957;26: 291–315. 38. Newberne PM, Rogers AE. Nutrition, monocrotaline and aflatoxin BI in liver carcinogenesis. Plant Foods Man 1973;1:23-31. 39. Hartmann T. Plant-derived secondary metabolites as defensive chemicals in herbivorous insects: a case study in chemical ecology. Planta 2004;219:1-4.

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40. Bundesgesundheitsamt, Bekanntmachung uber die Zulas- . sung und Registrierung von Arzneimitteln, Bundesanzeiger 1992;111- 4805. 41.

BfR. Bundesinstitut für Risikobewertung. Analytik und Toxizität von Pyrrolizidinalkaloiden sowie eine Einschätzung des gesundheitlichen Risikos durch deren Vorkommen in Honig. Stellungnahme 2011;038.

42. COT. Committee on toxicity of chemicals in food, consumer products and the environment. Annual Report. 2008. 43. RIVM Rijksinst;ituut voor Volksgezondheid en Milieu, RIKILT Institute of Food Safety, Risicobeoordeling inzake de Aanwezigheid van Pyrrolzidine Alkaloiden in Honing, Wageningen, Netherlands. 2007. 44. Van der Zee M. Advies inzake pyrrolizidine alkaloiden in kruidenpreparaten, , RIVM, Bilthoven. RIVM/S1R, report 09685A00. 2005. 45. FDA. Food and Drug Administration. Advises dietary supplement manufacturers to remove comfrey products from the market. 2001. 46. Comisión del Codex alimentarius. Documento Debate sobre los Alcaloides de Pirrolizidina. 2011. 47. ANZFA. Australia New Zealand Food Authority. Pyrrolizidine alkaloids in food a toxicological review and risk assessment. Technical report series no. 2. 2001. 48. ANZJFSC. Australia and New Zealand Joint Food Standards Code. Prohibited and restricted plants and fungi. Standard 1.4.4: Issue 67. 2015. 49. FSANZ. Food Standards Australia New Zealand. Consumers advised to limit consumption of Paterson’s Curse/Salvation Jane honey, Fact Sheet, 9. Canberra, Australia. 2004. 50. Betteridge K, Cao Yu, Colegate S. Improved method for extraction and LC-MS analysis of pyrrolizidine alkaloids and their N-oxides in honey: Application to Echium vulgare Honeys. J Agric Food Chem 2005;53:1894−1902. 51. Beales K, Betteridge K, Colegate S, Edgar J. Solid-phase extraction and LC−MS analysis of pyrrolizidine alkaloids in honeys. J Agric Food Chem 2004;52:6664−6672. 52. Kempf M, Beuerle T, Bühringer M, Denner M, Trost D, von der Ohe K, et al. Pyrrolizidine alkaloids in honey: Risk analysis by gas chromatography-mass spectrometry. Mol Nutr Food Res 2008;52:1193–1200.

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53. 54.

Raezke K. Pyrrolizidine alkaloids in honey. FEEDM General Meeting. 2010. European Commission. Rapid Alert System for Food and Feed. https://webgate.ec.europa.eu/rasffwindow/portal/?event=searchResultList&StartRo w=301. Consultado 20 Ago, 2020.

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https://doi.org/10.22319/rmcp.v13i3.5878 Review

Effects of phytoestrogens on the reproductive physiology of productive species. Review

Miguel Morales Ramírez a Dinorah Vargas Estradab Iván Juárez Rodríguez c Juan José Pérez-Rivero d Alonso Sierra Reséndiz b Héctor Fabián Flores González e José Luis Cerbón Gutiérrez f Sheila Irais Peña-Corona*g

Universidad Autónoma Metropolitana. Unidad Iztapalapa, Departamento de Biología de la Reproducción, Ciudad de México, México. b

Universidad Nacional Autónoma de México (UNAM). Facultad de Medicina Veterinaria y Zootecnia, Departamento de Fisiología y Farmacología, Ciudad de México, México. c

UNAM. Facultad de Medicina Veterinaria y Zootecnia, Departamento de Medicina Preventiva y Salud Pública, Ciudad de México, México. d

Universidad Autónoma Metropolitana. Unidad Xochimilco, Departamento de Producción Agrícola y Animal, Ciudad de México, México. e

UNAM. Facultad de Medicina Veterinaria y Zootecnia, Departamento de Genética y Bioestadística, Ciudad de México, México. f

UNAM. Facultad de Medicina Veterinaria y Zootecnia, Departamento de Reproducción, Ciudad de México, México. g

UNAM. Facultad de Química, Departamento de Biología, Ciudad de México, México.

*Corresponding author: sheila.ipc@live.com

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Abstract: Phytoestrogens (PEs) are chemical compounds from the secondary metabolism of some plants, they have a potential effect on the reproductive parameters of domestic animals, acting as agonists or antagonists of estrogen receptors. The objective of this review is to know the effects produced by a diet rich in PEs on the reproductive physiology of slaughter animals. A systematic review was carried out in two databases using keywords related to the effects produced by the intake of PEs in the diet on the reproduction of animals for slaughter, only controlled studies developed in vivo were considered. Contradictory results were found, on the one hand, the intake of a high content of polyphenolic compounds from different fodders, in the bovine female, was related to the decrease in fertility, presence of abortions and ovarian cysts; on the other hand, the intake of the high content of PEs induced an increase in the semen quality of the males of the species: cattle, sheep and leporids, so these effects can be attributed to the concentration, the type of PEs, sex, species and even the breed of the animal. Key words: Phytoestrogens, Slaughter animals, Reproductive effects, Coumestans, Isoflavones, Lignans.

Received: 25/11/2020 Accepted: 04/08/2021

Introduction In livestock activities, the selection of feed represents one of the main production costs and is one of the most technically complex aspects for the search for nutritional satisfaction in the different stages of animal production (breeding, rearing, gestation, lactation, weaning, growth and fattening), considering their physiological state, age and breed(1-4). Generally, the feed of slaughter animals (SAs) is based on grains of sorghum and corn, since they provide a rich source of energy, in addition to other feeds that provide protein, such as soybeans, canola, alfalfa and clover(2-4). It is essential to highlight that it has been suggested that changes be made in the feed or forage source in the event of alterations in the reproduction of SAs(4). Plant-based feeds are considered a rich source of phytoestrogens (PEs), nonsteroidal polyphenolic compounds derived from plant metabolism with conformational structure

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similar to 17-β estradiol (E2)(3,5). When ingested by animals, they can act as selective modulators of estrogen receptors (ERs) and act as endocrine disruptors in an agonistic or antagonistic manner, depending on the dose ingested(5,6), thus interfering with the synthesis, secretion, transport and metabolism of reproductive hormones, during embryonic development and in adult life(6-11). Nearly 100 PEs have been recognized; these are categorized, according to their chemical structure, into four classes: isoflavonoids (genistein, daidzein, formononetin); flavonoids (naringenin, kaempferol); coumestans (coumestrol “COU”), sativol, COU diacetate, 4methoxycoumestrol) and lignans (enterolactone and enterodiol)(12,13). Soybeans are the most abundant source of isoflavones, as it is one of the feeds with the highest content of genistein and daidzein, while alfalfa and clover contain a high amount of coumarins. It is worth mentioning that PEs are naturally present in plants, as glycosides, which are hydrolyzed to aglycone (which is the active form) catalyzed by enzymes present in the digestive system(14,15). Through several studies, it has been shown that the intake of PEs causes reproductive alterations in animals of both sexes, as well as temporary infertility syndromes(16,17). However, there is also evidence that PEs favor reproduction because they increase the concentration, motility and volume of spermatozoa necessary for fertilization(18-20). Due to the economic and productive importance that reproductive alterations represent in the context of animal production, the objective of this work is to know the potential effect that feeds rich in PEs have on the reproduction of SAs.

Method This document was developed in accordance with the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses)(21) statement.

Literature search

A systematic review was conducted on the Web (Figure 1); Google Scholar and PubMed were used as specialized information search engines with the aim of identifying studies that explored the effects of PEs present in the diet on the reproductive physiology of SAs, which have been published in blind peer-reviewed journals, extended abstracts in specialty

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congresses, and in graduate theses between 1996 and 2019. The keywords were established according to the PICO (Participants, Interventions, Comparisons and Outcomes) principle: P as slaughter animals (cattle, sheep, goats, equines, pigs, leporids and poultry); I as feeding with a diet rich in PEs; C as control groups, or groups of animals with low phytoestrogen diet, or reproductive variables of the same experimental group reported before or after exposure to the high-phytoestrogen diet; and R as reproductive variables in males and females. The statement of the topic of the review question was developed as: Do phytoestrogens present in the diet produce adverse effects on reproductive variables in SAs? According to the keywords, Google Scholar was first searched and, subsequently, PubMed, dated until June 2020. Generally, the following words and Boolean operators were used, in order to identify the studies available on the web: {‘Reproduction’ AND ‘Animals’ AND ‘Phytoestrogens’ AND/OR ‘Fertility’ AND/OR ‘Feed’ AND/OR ‘Isoflavones’ AND/OR ‘Coumestans’, AND/OR ‘Lingans’ AND ‘Cows’ OR ‘Heifers’ OR ‘Bulls’ OR ‘Ewes’ OR ‘Small Rumninant’ OR ‘Sheep’ OR ‘Ovine’ OR ‘Goat’ OR ‘Nanny-goat’ OR ‘Equine’ OR ‘Mare’ OR ‘Horses’ OR ‘Porcine’ OR ‘Sows’ OR ‘Pigs’ OR ‘Leporids’ OR ‘Rabbits’ OR ‘Hens’ OR ‘Rooster’ OR ‘Chickens’} {‘Reproducción’, AND ‘Animales’, AND ‘Fitoestrógenos’ AND/OR ‘Fertilidad’ AND/OR ‘Alimentación’ AND/OR ‘Isoflavonas’ AND/OR ‘Coumestanos’ AND/OR ‘Lignanos’ AND ‘Vacas’ OR ‘Vaquillas’ OR ‘Toros’ OR ‘Pequeños rumiantes’ OR ‘Ovejas’ OR ‘Ovinos’ OR ‘Cabras’ OR ‘Equinos’ OR ‘Yeguas’ OR ‘Caballos’ OR ‘Porcinos’ OR ‘Cerdas’ OR ‘Lepóridos’ OR ‘Conejos’ OR ‘Aves’ OR ‘Gallinas’ OR ‘Gallos’ OR ‘Pollos’}. All the articles were searched in English and Spanish, were initially examined by reading their abstracts. The full texts of the documents included preliminarily were reviewed again to select the material to be used. Figure 1 shows a flowchart that details the selection of studies.

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Figure 1: Flowchart of study search progress

Inclusion and exclusion criteria The studies considered were those on controlled experiments in vivo, in SAs fed a diet rich or supplemented with PEs (soybeans, clover, alfalfa) for more than seven days, and that included the determination of the amount of PEs ingested or sufficient information to calculate it, in that case, the amount of PEs in the diet (mg/kg of DM [dry matter]) was obtained from the database of the United States Department of Agriculture(22,23); likewise, to obtain the amount of daily feed, the ratio of the animals’ weight and the amount of feed consumed containing PEs (reported in the article) was obtained. Those studies in which the administration of PEs was not related to feeding, or was administered orally through an excipient, or when reproductive parameters were not assessed, were excluded. Data extracted from eligible studies were recorded as follows: species, breed, daily diet, main effects on reproductive variables, exposure time in days, PEs present in the diet and amount of PEs (mg/kg of DM).

Risk of bias analysis The included papers were evaluated by two examiners in order to clarify emerging doubts, a third party was consulted to identify the presence of bias of randomization, blinding, results reporting and others (Table 1). To represent the bias analysis, a color scale was proposed: if the study met the described criterion, the green color was used; if the study was unclear or there was not enough information for the evaluation of bias, the yellow color was used. 807

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Table 1: Summary of bias analysis of each included study

Reference

The random selection of the animal sample is clearly described

The researchers who selected the sample did NOT know to which treatment it would be assigned

Blinding of evaluators to the assigned treatment was ensured

The study The is free of results other are sources of complete bias

García et al., 2018(24) Woclawek-Potocka et al., 2005(25) Hashem et al., 2016(26) Rodríguez et al., 2013(27) Piotrowska et al., 2006(28) Yurrita et al., 2017(29) Cantero et al., 1996(30) Pace et al., 2006(31) Hashem et al., 2018(32) Pace et al., 2011(33) Aragadvay-Yungán et al., 2018(2) Sierra et al., 2015(34) Domínguez et al., 2014(19) Ferreira-Dias et al., 2013(35) Gentao et al., 1999(36) Yuan et al., 2012(37) Cardoso et al., 2007(38) Cardoso et al., 2009(39) Yousef et al., 2004(18) Hashem et al., 2008(40) Saleh et al., 2019(41) Ni et al., 2007(42) Lu et al., 2017(43) Wistedt et al., 2012(44) Arija et al., 2006(45) Heng et al., 2017(46) The green color indicates low risk of bias, the yellow color indicates that it is not clear or there is not enough information for the evaluation of the criterion, according to the authors’ judgment.

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Results In the systematic search, 26 specialized documents were finally collected: six on cattle (one article in Spanish and three in English, a master’s thesis in Spanish and a doctoral thesis in English); seven on sheep (one in Spanish and four in English, a master’s thesis in Spanish, a conference abstract in Spanish); one on equines in English; two on pigs in English; four on leporids in English and six on poultry in English (Table 2). All articles were published between 1996 and 2019.

Cattle

In a study conducted on 11-month-old Bradford heifers (n=15 per group) fed, six months before their first service, with 0.8 % of soybeans, calculated based on live weight (LW), (feed that was considered by the authors to be high in PEs when compared to the usual feeding of 0.3 % LW), a percentage of gestation (%G) of 93 % and an abortion were observed, compared with the control group, where a 100 % success rate in pregnancy and no cases of abortion were reported; the authors mention that it is not possible to affirm that feeding with 0.8 % LW was the cause of abortion, and that this feeding did not affect reproductive parameters(24). In another study, was evaluated the %G in Holstein/Polish heifers (75 and 25 %) that were constantly fed 2.5 kg of soybeans, it was observed that the pregnancy rate was not significantly different compared to the control group, although equol and p-ethyl-phenol (compounds derived from the metabolism of PEs) were detected in the serum of the heifers(25). On the other hand, it has been reported that, in Holstein heifers fed clover for five months, the %G decreased (61.5 %), compared to heifers fed corn silage (92.3 %); the percentage of heifers that did not become pregnant after several inseminations was higher (38.46 %) in the group of cows fed clover compared to its control (7.7 %)(26) (Table 2). In adult Holstein/Polish cows (75 and 25 %) that were fed 2.5 kg of soybeans from a lactation period prior to mating, they had a %G of 60 % compared to the control group, which showed 100 % gestation(25). In the lactation stage of Holstein cows fed for 60 d with alfalfa or red clover meal, alterations in the concentrations of E2, progesterone (P4) and luteinizing hormone (LH) were reported. A significant decrease in E2 was documented in the groups of cows fed alfalfa (2.32 ± 0.12 pg/mL) or red clover (2.25 ± 0.67 pg/mL) compared to control cows (4.24 ± 0.31 pg/mL); the concentration of P4 decreased at the end of the supplementation period in cows fed alfalfa (1.586 ± 0.27 ng/mL) or red clover (0.988 ± 0.3 ng/mL) compared to control animals (2.82 ± 0.34 ng/mL); LH also decreased in cows in the groups fed alfalfa (3.82 ± 0.22 IU/mL) or red clover (3.7 ± 0.26 IU/mL) compared to control

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cows (6.66 ± 0.39 IU/mL)(27). In another study, it was observed that the concentrations of P4 decreased throughout the estrous cycle in Holstein/Polish black cows when they were fed 2.5 kg of soybeans for 21 d, compared to the control group, this suggests that PEs contained in soybeans can alter the function of the corpus luteum (CL), it is worth mentioning that the effects began to be observed between d 15 and 18 after its intake(28) (Table 2). In the only available study conducted on Angus bulls, with a diet with 10 % of soybeans from weaning to pre-puberty, an improvement in scrotal growth and semen quality in adulthood was observed(29). The documented differences between sexes, derived from the effects produced by PEs, are biologically plausible, given that PEs are known xenoestrogens that alter the endocrine system depending on the availability of ERs and target organs of both sexes. In the studies reviewed, the age of exposure to PEs and the type of PEs determine their biological effect, observing a greater harmful effect in adult cows kept with feed rich in PEs than in heifers.

Small ruminants

In Manchega ewes that consumed alfalfa ad libitum for 10 mo and that contained COU at the rate of 25 ppm in autumn, 30 ppm in winter and 17 ppm in spring, it was found that 43 % of them showed alterations in the genital tract: cysts or microcysts in the endometrium accompanied by petechiae and ecchymosis in the uterine mucosa, increased glandular activity and paraovarian cysts(30). There are studies in which no harmful effects on the reproductive parameters of ewes and males of the Comisana breed are documented, it has even been suggested that prolonged administration of subterranean clover, with low formononetin content (less than 10 % of the total isoflavones on a dry basis), induces a significant improvement in the weight gain of animals and, in males, good carcass and meat characteristics(31). In another study conducted on pregnant Rahmani ewes, which were fed for two months prior to calving and until the induction of the next heat (3.5 mo postpartum) with 849.4 g/kg of DM of Trifolium alexandrinum, no significant differences between groups in ovarian activity and ovulation in induced heat were documented, but females fed Trifolium alexandrinum showed shorter estrus duration (20 h) compared to ewes fed corn (34 h), in which P4 concentrations in the luteal phase of the induced estrus were significantly higher, compared to treated ewes(32). In another work conducted on female lambs of the Sarda and Comisana breeds, the effects of feeding ad libitum with alfalfa or subterranean clover were evaluated, in these animals, no alterations were observed in the development of the reproductive system, fertility, fecundity, reproductive performance and calving interval, although animals fed clover had a higher

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weight at puberty, the authors suggest that some clover varieties do not negatively affect the reproduction of ewes and appear to improve the growth rate of animals(33). In Creole rams fed 1.1 kg/d of alfalfa contaminated with Pseudopeziza medicaginis at 10, 40 and 70 % for 45 d, a significant decrease in sperm concentration was documented in the rams fed alfalfa contaminated with the fungus at 40 % and 70 %, compared to the group with 10 %(2). It should be mentioned that fungal infestations caused by Pseudopeziza medicaginis increase the synthesis of phytoestrogenic substances such as coumarins and isoflavones(2). Other studies do not report differences between sperm characteristics, for example, in a study with hybrid Hampshire/Suffolk sheep, which were fed about 1 kg of alfalfa (2.5-6.5 mg of COU/100 g of alfalfa(22,23)) or 200 g of extruded soybeans (57 mg of genistein/100 g of extruded soybeans; 31 mg of daidzein/100 g of extruded soybeans(22,23)), daily for 90 d, no alterations were reported in sperm volume, color, motility and concentration, evaluated in fresh or cryopreserved semen(34). Likewise, in three-month-old hybrid Katahdin/Pelibuey lambs fed 23 % of alfalfa for 90 d, no differences were observed in sperm volume, sperm membrane integrity, total or progressive motility and acrosome status(19) (Table 2). In sheep, contradictions were found regarding the deleterious or beneficial effects attributed to the intake of PEs on reproductive variables.

Equines

The effects of PEs due to feeding on equines are poorly described. Conjugate and free (active) forms of COU and its metabolites have been identified in the plasma of mares fed for 14 d with alfalfa pellets in increasing concentrations of up to 1 kg/d, during the time of the experiment, all mares cycled, on day zero half of the mares were in the follicular phase, and the other half in the luteal phase, on d 13 and 14 of the experiment, all the mares had the luteal phase(35) (Table 2). The results suggest that PEs affect the length of the estrous cycle and can prolong luteal function in the mare, due to the induction of the persistence of CL(35).

Pigs

In pregnant sows fed a regular diet supplemented with 0.005 mg of daidzein/kg of LW, in the peripartum period (from 30 ds prepartum to d 7 postpartum), they showed an increase in the weight of the litter compared to the control group, which did not consume daidzein; in addition, the production of milk, colostrum proteins and growth hormone increased by about 12 % in those sows fed that isoflavone(36). In minipigs fed low concentration of isoflavones

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(250 ppm), no negative effects on reproduction were observed; in contrast, when the concentration increased (500 ppm), the testicular index ((bilateral testicular weight/total body weight) x 100 %) LH and testosterone (T4) decreased; in addition, there was an increase in apoptotic germ cells, indicating testicular peroxidation(37) (Table 2). In sows, feeding supplemented with daidzein favors the weight of the offspring at calving and milk production, in the case of males, the effect of PEs in the diet depends on the amount administered, low amounts of PEs favor testicular function.

Leporids

In a study conducted on pregnant does fed a diet of 18 % of soybean meal (13 mg/kg of isoflavones/body weight) during the stages of gestation, lactation and until the offspring were 33 weeks old, it was observed that males were earlier in the time of onset of puberty compared to the control group, no significant differences were reported in the morphology of the reproductive organs, semen quality and sexual behavior(38). In another study, the effect of exposure to a commercial rabbit diet with 18 % of soybean meal during perinatal period (intrauterine and lactation) on the morphology of the reproductive organs of males at 26 wk of age was evaluated. No alterations were observed in the reproductive tract of the male progeny(39). In 7-mo-old adult New Zealand male rabbits fed 30 % of Berseem (Trifolium alexandrinum) hay and supplemented with 2.5-5 mg of isoflavones/kg of body weight every third day for 13 wk, improvements in semen and libido characteristics were documented, since there was an increase in sperm volume, concentration and percentage of sperm motility(18). When the effect of a diet rich in soybeans (80 g of soybeans/kg of feed) and flaxseed (100 g of flaxseed/kg of feed) in adult rabbits was evaluated, increases in the occurrence of abnormalities in spermatozoa, decrease in libido and in the process of steroidogenesis were observed, however, when they were crossed with untreated females, pregnancy rate, litter size and fertility were not affected(40) (Table 2). The results of the previous studies suggest that the administration of commercial feed, with a concentration of up to 18 % of soybeans, to pregnant does, does not produce, in the offspring, alterations in the morphology of the reproductive organs in F1 males. Nor do diets with percentages less than 80 g of soybean meal or 100 g of flaxseed/kg of feed cause effects on the reproductive organs of males, the volume of the ejaculate or fertility. No studies evaluating the effects of PEs directly on the reproductive physiology of females were found (Table 2).

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Poultry

In a study conducted on 65-wk-old Bovans Brown laying hens(41) fed diets supplemented with 1 g of flax seeds or fenugreek/kg of feed (rich in PEs, 0.20 mg of isoflavones/kg of DM) for 6 wk, an increase in the concentrations of E2 and LH, weight and an improvement in shell thickness at the end of the egg laying cycle were observed, both with the supplementation of the seeds separately and with their combination (flax and fenugreek)(41). The increase in eggshell thickness documented in the previous study was also present in another, in which 445-d-old ISA laying hens were fed a diet supplemented with 10 mg of daidzein/kg of feed(42). In addition, in this last study, a significant increase in the proportion of oviduct weight to body weight was reported(42). There were no alterations in the width and length of the egg, nor in the serum concentrations of E2(42). On the other hand, it has been observed that in 44-wk-old Rugao laying hens fed supplemented diets from 60 to 248 mg of daidzein/kg of feed for 12 wk, it did not generate significant differences in egg quality or fertility, although an increase in the hatchability of egg laying was observed(43). Wistedt et al(44) also reported the absence of effects on reproductive variables in 15-wk-old Lohmann Selected Leghorn (LSL) and Lohmann Brown (LB) laying hens on the morphology and size of ovaries and oviducts after being supplemented with 50 mg of daidzein/kg of feed. A difference in the sensitivity of the breeds to daidzein was observed, since the eggshells were thicker in LB hens than in LSL hens(44). In 1-d-old Cobb broilers, fed 100, 200 or 300 mg of extruded or raw beans/kg of feed for 21 d, a decrease in T4 and androstenedione concentrations was observed with raw bean feeding, in contrast, the administration of extruded beans increased the same variables(45). Finally, in 70-d-old young breeding roosters fed a commercial product added with isoflavones, it was observed that, with 5 mg/kg, testicle weight, concentration of gonadotropin-releasing hormone (GnRH) and expression of the mRNA of the StAR enzyme(46) were increased (Table 2). The results of the studies suggest that PEs cause favorable effects on reproductive variables of male and female poultry.

Bias

In none of the studies included in the present review, the evaluators were blinded to the assigned treatment, nor in the selection of the sample. In contrast, complete results were identified in all the papers, and they were free from other sources of bias. More than half of the included studies used some randomization technique for their experimental units(18,27,30,31,36,37,38,39,40,41,42,43,45,46).

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Discussion In ruminants, isoflavones are metabolized in the rumen, which generates estrogenic or nonestrogenic compounds. Secoisolariciresinol and matairesinol are the precursors of COU, enterodiol and enterolactone; biochanin A and genistein can be broken down into p-ethylphenol (non-estrogenic); formononetin is metabolized to daidzein and finally to equol, a more estrogenic compound that is absorbed through the rumen wall(47,48). The main effects produced by PEs in cattle include hormonal alterations, decrease in %G and increase in abortions, this depends on the reproductive stage, the time of consumption and the type of PEs involved. Early embryonic mortality and the increase in the abortion rate can be explained by the ability of PEs to inhibit the secretion of P4 stimulated by LH(49,50). In cows, the release of prostaglandins (PG) with luteolytic action is regulated by E2 and P4(51). PEs and their active metabolites alter the PGE2-F2α ratio, which leads to the nonphysiological production of luteolytic agents during gestation and the estrous cycle(25). In gestation, the balance between PGE2-F2α is crucial for the maintenance and function of CL, the recognition of pregnancy, embryo implantation and development, so the stimulation of PGF2α production can lead to interfere with this balance and embryonic development(52). PEs as agonists, in non-pregnant animals, reduce the length of the estrous cycle, since during luteolysis and ovulation, stimulation of PGF2α secretion can accelerate positive feedback between PGF2α and oxytocin(51,52). On the other hand, PEs, as antagonists, induce alterations in follicular development and therefore, the absence of estrus(53,54). In addition, a possible positive relationship between the concentration of isoflavones in blood plasma and the incidence of silent heat in dairy cattle fed soybeans has been reported(55). Being structurally similar to E2, PEs act as agonists or antagonists of ERs(7,56). The biological effects attributed to PEs occur with concentrations around 1,000 times higher than endogenous E2 concentrations (1-10 nM). This is based on the fact that Woclawek-Potocka et al reported 1.6 ± 0.3 μM of p-ethyl phenol and 1.2 ± 0.28 μM of equol(25); Piotrowska et al reported 1.28 ± 0.10 μM of equol and 6.24 ± 0.30 μM of p-ethyl phenol(28); Zdunczyk et al reported daidzein, genistein, equol and p-ethyl-phenol in a range of 0.1-3.6 μmol/L in cows that showed reproductive alterations(55). In another study, it was calculated that the intake of 66.8 mg/kg of COU (present in alfalfa) produced plasma concentrations of 13 ng/mL of this compound, which also turns out to be 1,000 times higher than the concentration of E2 during estrus(25). Even considering that the biological activity of COU is 160 times lower compared to that of E2, the amount would be equivalent to six times the effective concentration of E2 in estrus, and therefore, sufficient to induce estrogenic changes similar to those found in the cow during this phase of the estrous cycle(57). The above data suggest that cows that are continuously exposed to a diet that includes PEs may show reproductive alterations, contrary

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to what is reported in males, in which feeding with 10 % of soybeans as a source of protein improves sperm formation and concentration(29). In sheep, the effects of PEs on reproductive variables are contradictory. In some studies, it has been suggested that these compounds do not cause alterations as in other species(19,20); in others, their deleterious effect is evident, even the “Clover Syndrome” has been described, which consists of infertility, prolapse of the uterus and dystocia(58,59). The main effects reported in females are morphological alterations in reproductive organs and increased activity of the endometrial glands, which leads to quantitative and qualitative changes in cervical mucus, which can hinder fertilization(30,60). It is reported that PEs sensitize the cervix to estrogenic action, in addition to occupying ERs in the cervix, they also stimulate the appearance of new binding sites(61). In addition, PEs alter the secretion of the folliclestimulating hormone (FSH)(62). In ewes, the number of recruitable follicles depends on FSH concentrations(63), therefore, PEs are likely to interfere with follicular recruitment. It has been reported that the factors related to the absence of effects caused by PEs in some studies are seasonality, the dose of PE ingested and the species and conditions of the plant used, since it has been documented that, in a single plant species, more than one type of PEs can be found at different concentrations; for example; under normal conditions, the concentrations of COU in alfalfa are 1 to 2 mg/kg and in circumstances of defense of the plant, the concentration of that PE can increase up to 100 mg/kg(64). The concentration of PEs also depends on the presentation and organ of the plant, for example: the isoflavone content in raw green soybeans is 48.9 mg/100 g of DM; that of extruded soybeans is 91 mg/100 g of DM and textured soybeans contain 172.6 mg/100 g of DM(22,23). With respect to flavonoids, mature soybeans contain 37.41 mg/100 g of DM and green soybeans contain 1.23 mg/100 g of DM(22,23). The transition of photoperiods contributes to the regulation of estrous behavior, ovulation, ERs availability and concentration of endogenous steroids(32). In sheep, it has been suggested that prolonged ingestion of subterranean clover with PE levels below 0.3 % or ~10 mg/g DM of alfalfa does not produce infertility or reproductive disorders, it even significantly increases body growth(33), probably because PEs also stimulate growth hormone(42,65). It has been suggested that the differences in deleterious effects on reproduction may be due to the type of isoflavones administered, the difference in the number of ERs and the type of metabolism between the different species(66). In male sheep, contradictory results were also found, Aragadvay et al(2) describe reproductive alterations with the feeding of alfalfa contaminated with Pseudopeziza medicaginis at 40 and 70 %; other studies do not report harmful effects(19,34). The reason why this difference in results exists is not described yet, probably due to the difference in the sensitivity of steroidogenic compounds between breeds of the same species, as reported in other animal models(67), it is even likely that male sheep are less

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susceptible to the effect of phytoestrogens(20) or that some plant varieties do not negatively affect the reproduction of ewes(20,33). According to the criteria established in this review, no published studies related to goats were found. However, it has been reported that goats constantly fed 30 % of dehydrated alfalfa had an incidence of 20 % of rectal or vaginal prolapses in the last month of gestation(68). In a case report of mares fed 5-8 kg/d of mixture of alfalfa and clover hay for at least 5 mo, uterine edema, absence of ovulation and accumulation of uterine fluid and return to normal ovarian cyclicity within 2 to 3 wk after withdrawal of the feed rich in PEs, were observed(35). The mechanism of absorption of PEs is not fully described; in this species, digestion is very fast and feed can pass through the stomach and small intestine within the first 5 h(69). This was confirmed in a study in which active forms of COU reached their highest level between 1 and 3.5 h after ingestion(35). The amount of PEs in the pasture depends on the season of the year, since a decrease in COU and its metabolites was found when evaluated from November to March and it is known that its synthesis increases under adverse conditions for plants(35). It is worth mentioning that the dehydration of alfalfa to produce pellets could reduce estrogenic activity(70). Therefore, the exposure time, the presentation of PEs and the season of the year are fundamental for the effects to occur. The effects of PEs, present in the diet of pigs, on reproductive variables are few, in contrast to the amount of data on estrogenic effects of feed contaminated with mycotoxins, or works in which the effect of isoflavones on meat quality and growth is studied(71-73). Daidzein and genistein are the main isoflavones contained in soybean meal, which is the basic protein ingredient in the diet of pigs, and it is known that both represent 88 % of the isoflavones circulating in the blood(73). One of the effects of feeding sows with diets containing PEs in the last third of gestation is an increase in the weight of animals, this may be due to the positive effect produced by PEs on growth hormone concentrations(74). In males, the effect is much more evident, since, in the fetal stage, the concentrations of circulating estrogens are low, and the presence of PEs would stimulate growth due to a change in metabolism, proliferation and differentiation of skeletal muscle(75). The increase in milk production and better milk quality in sows fed isoflavones could be due to their agonist effect on insulin-like growth factor-1 (IGF-1) and prolactin reported in periparturient sows(76) or to a better balance of antioxidants(77). In males, a positive effect on reproduction has been identified when low concentrations of PEs are administered(37). This has been attributed to the binding of isoflavones to ERs in the hypothalamus, pituitary gland and testicles, thus stimulating spermatogenesis, sperm maturation and gonadal growth(6). As for leporids, it is reported that females are more sensitive to the effect of environmental estrogens(38). In male rabbits, intrauterine and lactational administration of PEs do not induce deleterious effects on semen production or sexual behavior, it has even been suggested that 816

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soybean meal can be used as part of the normal diet in pregnant females without reproductive damage to the progeny(38,39). The beneficial influence of isoflavones on semen parameters has been attributed to their antioxidant effect because they reduce the production of hydrogen peroxide and protect spermatozoa against oxidative damage(18). In addition, in adult males, estrogen plays a major role in the prevention of the apoptosis in germ cells through its paracrine or autocrine actions in the testicles(32). The type of PEs consumed by rabbits also influences the final effect, for example, lignans suppress T4 synthesis more strongly during spermatogenesis and libido, compared to isoflavones(32). In poultry, as in other domestic animals, PEs can act as agonists/antagonists, depending on the dose, tissue type, ER subtype and presence of endogenous hormones, therefore they are considered selective modulators of ERs(78). In poultry, the effects produced by the consumption of PEs are related to the genetic component, since lines of LB and LSL hens showed different sensitivity to these compounds(44); this could be due to the fact that there is a difference in the location and expression of ERs ß in uterine capillaries between genetic lines, which could influence the obtaining of greater weight of the oviduct(44). In bird males, it was observed that a diet rich in PEs promotes testicular growth due to the increase in hormonal secretion, probably due to the regulatory participation of isoflavones in the feedback mechanism of the hypothalamic-pituitary-gonadal axis(79), which in turn regulates the secretion of FSH, LH and T4 that promotes the growth and maturation of Sertoli and Leydig cells. The administered dose also has an important role, it is documented that genistein has partial agonist effects in roosters at doses of 50-200 mmol/kg, and acts as an antagonist at doses of 400-500 mol/kg(78); in addition, PEs have the ability to inhibit the activity of steroidogenic enzymes, and influence the viability of sex hormones through the regulation of their binding proteins; they alter brain centers related to sexual behavior as they cross the blood-brain barrier and bind to ER α and ß(80). Finally, the metabolism of dietary PEs is predominantly determined by gastrointestinal bacterial metabolism and depends on the reproductive stage, for example, during gestation, genistein has the potential to influence fetal metabolism and growth; in the colon, genistein can be metabolized to dihydrogenysteine or 6’-hydroxy-O-desmethylangolensine, while daidzein can be reduced to dihydrodaidzein and converted into O-desmethylangolensin or equol, these metabolites can be absorbed or metabolized into phenols in the colon lumen(81). After the consumption of PEs, these are deconjugated by the intestinal bacterial flora, reabsorbed, re-conjugated in the liver and excreted in the urine(25,61). The demethylation of these compounds occurs in the intestine by acetogenic bacteria and in the liver(81). Therefore, the intestine microbiome present in each species will influence the final effect of PEs on productive and reproductive variables.

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Conclusions Based on the papers reviewed and discussed, it is concluded that phytoestrogens cause alterations in the reproductive physiology of slaughter animals considering four factors: 1) The plant species consumed; 2) The season of the year in which the plant species is consumed; 3) The particularities of the animals (metabolism, age, breed, species and sex) and 4) The processing conditions of the plant. Future studies should be carried out to elucidate the endocrine mechanisms of the actions of PEs in the reproduction of animals. It is necessary to reevaluate the ingredients that make up the feeds of the different productive animals. Literature cited: 1. Meléndez P, Bartolomé J. Advances on nutrition and fertility in dairy cattle: Review. Rev Mex Cienc Pecu 2017;8(4):407-417. 2. Aragadvay-Yungán R, Novillo-Rueda M, Núñez-Torres O, Rosero-Peña Herrera M, Lozada-Salcedo E. Calidad seminal de carneros alimentados con dietas que contienen alfalfa (Medicago sativa) contaminada con Pseudopeziza medicaginis. Rev Ecua Invest Agrope 2018;2(1):14-19. 3. Pérez-Rivero JJ, Setién A, Martínez-Maya JJ, Pérez-Martínez M, Serrano H. Los fitoestrógenos y el efecto de su consumo en diferentes órganos y sistemas de animales domésticos. Agric Tec 2007;67(3):325-331. 4. Magnusson U. Environmental endocrine disruptors in farm animal reproduction: research and reality. Reprod Domest Anim 2012;47(Suppl 4):333-337. 5. Di Gioia F, Petropoulos SA. Phytoestrogens, phytosteroids and saponins in vegetables: Biosynthesis, functions, health effects and practical applications. Adv Food Nutr Res 2019;90:351-421. 6. Adams NR. Detection of the effects of phytoestrogens on sheep and cattle. J Anim Sci 1995;73(5):1509-1515. 7. Almstrup K, Fernández MF, Petersen JH, Olea N, Skakkebæk NE, Leffers H. Dual effects of phytoestrogens result in u-shaped dose-response curves. Environ Health Perspect 2002;110(8):743-748. 8. Cederroth CR, Zimmermann C, Nef S. Soy, phytoestrogens and their impact on reproductive health. Mol Cell Endocrinol 2012;355(2):192-200.

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9. Ropero AB, Alonso-Magdalena P, Ripoll C, Fuentes E, Nadal A. Rapid endocrine disruption: environmental estrogen actions triggered outside the nucleus. J Steroid Biochem Mol Biol 2006;102(1-5):163-169. 10. Shanle EK, Xu W. Endocrine disrupting chemicals targeting estrogen receptor signaling: identification and mechanisms of action. Chem Res Toxicol 2011;24(1):6-19. 11. Whitten PL, Patisaul HB. Cross-species and interassay comparisons of phytoestrogen action. Environ Health Perspect 2001;109(Suppl 1):5-20. 12. Nilsson S, Makela S, Treuter E, Tujague M, Thomsen J, Andersson Gr, et al. Mechanisms of Estrogen Action. Phys Rev 2001;81(4):1535-1565. 13. Wocławek-Potocka I, Mannelli C, Boruszewska D, Kowalczyk-Zieba I, Waśniewski T, Skarżyński DJ. Diverse effects of phytoestrogens on the reproductive performance: cow as a model. Int J Endocrinol 2013;2013:650984. 14. Bonilla CA. Isoflavonas en ginecología, terapia no convencional. Rev Colomb de Obstet y Ginecol 2004;55(3):209-217. 15. Wang H, Murphy PA. Isoflavone Content in Commercial Soybean Foods. J Agric Food Chem 1994;42(8):1666-1673. 16. Peña-Corona S, León P, Mendieta E, Villanueva M, Salame A, Vargas D, et al. Effect of a single application of coumestrol and/or dimethyl sulfoxide, on sex hormone levels and vaginal cytology of anestrus bitches. Vet Méx 2019;6(1):1-15. 17. Pérez-Rivero JJ, Martínez-Maya JJ, Pérez-Martínez M, Aguilar-Setién Á, Serrano H. Efecto del coumestrol sobre la producción espermática y la conducta de exploración olfatoria de perros estimulados con moco vaginal estral. Vet Méx 2009;40(1):9-16. 18. Yousef MI, Esmail AM, Baghdadi HH. Effect of isoflavones on reproductive performance, testosterone levels, lipid peroxidation, and seminal plasma biochemistry of male rabbits. J Environ Sci Health, Part B 2004;39(5-6):819-833. 19. Domínguez-Rebolledo AE, Alcaraz-Romero A, Cantón-Castillo JG, Loeza-Concha H, Ramón-Ugalde J. Efecto de la Alfalfa (Medicago Sativa L.) en la dieta sobre la calidad de los espermatozoides epididimarios de ovinos Katahdin con Pelibuey. Reunión Científica Tecnológica, Forestal y Agropecuaria Tabasco 2014 y III Simposio internacional en producción agroalimentaria tropical. Tabasco: Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias 2014:186-189. 20. Valderrabano J. Alteraciones reproductivas asociadas al consumo de fitoestrógenos. Investigación Agraria Producción y Sanidad Animal 1992;7(2):115-124.

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21. Moher D, Liberati A, Tetzlaff J, Altman DG, PRISMA Group. Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement. PLoS Med 2009;6(7):e1000097. 22. Bhagwat S, Haytowitz DB, Holden JM. USDA database for the isoflavone content of selected foods, release 2.0. Maryland: US Depart of Agricult 2008;15. 23. Bhagwat S, Haytowitz DB, Holden JM. USDA database for the flavonoid content of selected foods, Release 3.1. US Depart of Agricult: Beltsville, MD, USA. 2014. 24. García DC, Martín AA, Vella MA, Nasca JA, Roldan Olarte EM. Evaluación de la alimentación con distintos niveles de inclusión de soja en la recría de vaquillonas. Vet Arg 2018;35(357)1-9. 25. Woclawek-Potocka I, Acosta TJ, Korzekwa A, Bah MM, Shibaya M, Okuda K, et al. Phytoestrogens Modulate Prostaglandin Production in Bovine Endometrium: Cell Type Specificity and Intracellular Mechanisms. Exp Biol Med 2005;230(5):326-333. 26. Hashem NM, El-Azrak KM, Sallam SMA. Hormonal concentrations and reproductive performance of Holstein heifers fed Trifolium alexandrinum as a phytoestrogenic roughage. Anim Reprod Sci 2016;170:121-127. 27. Rodríguez MCE. Determinación del contenido de fitoestrógenos presentes en la alfalfa (Medicago sativa) y el trébol rojo (Trifolium pratense) y evaluación de su efecto sobre el perfil hormonal en vacas Holstein en la Granja Tungüavita Paipa-Boyacá [tesis maestría]. Colombia, Tunja: Universidad Pedagógica y Tecnológica de Colombia; 2013. 28. Piotrowska KK, Woclawek-Potocka I, Bah MM, Piskula MK, Pilawsk W, Bober A, et al. Phytoestrogens and their metabolites inhibit the sensitivity of the bovine corpus luteum to luteotropic factors. J Reprod Dev 2006;52(1) 33-41. 29. Yurrita SC. Evaluation of dietary phytoestrogen exposure on growth, semen parameters, and reproductive anatomy development of growing Angus bulls [doctoral thesis]. USA, Texas: Angelo State University; 2017. 30. Cantero A, Sancha JL, Flores JM, Rodriguez A, Gonzalez T. Histopathological changes in the reproductive organs of Manchego ewes grazing on lucerne. J Vet Med Seri A 1996;43(6):325-330. 31. Pace V, Carbone K, Spirito F, Iacurto M, Terzano M, Verna M, et al. The effects of subterranean clover phytoestrogens on sheep growth, reproduction and carcass characteristics. Meat Sci 2006;74(4):616-622.

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32. Hashem NM, El-Azrak KM, El-Din ANM, Sallam SM, Taha TA, Salem MH. Effects of Trifolium alexandrinum phytoestrogens on oestrous behaviour, ovarian activity and reproductive performance of ewes during the non-breeding season. Anim Reprod Sci 2018;196:1-8. 33. Pace V, Conto G, Carfi F, Chiariotti A, Catillo G. Short-and long-term effects of low estrogenic subterranean clover on ewe reproductive performance. Small Ruminant Res 2011;97(1-3):94-100. 34. Sierra SLA. Evaluación del efecto de los fitoestrógenos presentes en la alfalfa (Medicago sativa) sobre la calidad del semen ovino fresco y criopreservado [tesis de maestria]. Colombia, Tunja: Univesidad Pedagógica y Tecnológica de Colombia; 2015. 35. Ferreira-Dias G, Botelho M, Zagrajczuk A, Rebordão MR, Galvão AM, Bravo PP, et al. Coumestrol and its metabolite in mares' plasma after ingestion of phytoestrogen-rich plants: potent endocrine disruptors inducing infertility. Theriogenology 2013;80(6):684692. 36. Gentao L, Yuanlin Z, Weihua C, Jie C, Zhengkang H. Effect of daidzein fed to pregnant sows on milk production and the levels of hormones in colostrum. Nanjing Agric Univ 1999;22(1):69-72. 37. Yuan X, Zhang B, Li L, Xiao C, Fan J, Geng M, et al. Effects of soybean isoflavones on reproductive parameters in Chinese mini-pig boars. J Anim Sci Biotechnol 2012;3(1):31. 38. Cardoso JR, Báo SN. Effects of chronic exposure to soy meal containing diet or soy derived isoflavones supplement on semen production and reproductive system of male rabbits. Anim Reprod Sci 2007;97(3-4):237-245. 39. Cardoso J, Bao S. Morphology of reproductive organs, semen quality and sexual behaviour of the male rabbit exposed to a soy-containing diet and soy-derived isoflavones during gestation and lactation. Reprod Dom Anim 2009;44(6):937-942. 40. Hashem NM, Abo-Elsoud MA, El-Din ANM, Kamel KI, Hassan GA. Prolonged exposure of dietary phytoestrogens on semen characteristics and reproductive performance of rabbit bucks. Dom Anim Endocrinol 2018;64:84-92. 41. Saleh AA, Ahmed EAM, Ebeid TA. The impact of phytoestrogen source supplementation on reproductive performance, plasma profile, yolk fatty acids and antioxidative status in aged laying hens. Reprod Dom Anim 2019;54(6):846-854.

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42. Ni Y, Zhu Q, Zhou Z, Grossmann R, Chen J, Zhao R. Effect of dietary daidzein on egg production, shell quality, and gene expression of ER-α, GH-R, and IGF-IR in shell glands of laying hens. J Agri Food Chemis 2007;55(17):6997-7001. 43. Lu J, Qu L, Shen MM, Li SM, Dou TC, Hu YP, et al. Safety evaluation of daidzein in laying hens: Effects on laying performance, hatchability, egg quality, clinical blood parameters, and organ development. Poult Sci 2017;96(7):2098-2103. 44. Wistedt A, Ridderstråle Y, Wall H, Holm L. Effects of phytoestrogen supplementation in the feed on the shell gland of laying hens at the end of the laying period. Anim Reprod Sci 2012;133(3-4):205-213. 45. Arija I, Centeno C, Viveros A, Brenes A, Marzo F, Illera JC, et al. Nutritional evaluation of raw and extruded kidney bean (Phaseolus vulgaris L. var. Pinto) in chicken diets. Poult Sci 2006;85(4):635-644. 46. Heng D, Zhang T, Tian Y, Yu S, Liu W, Xu K, et al. Effects of dietary soybean isoflavones (SI) on reproduction in the young breeder rooster. Anim Reprod Sci 2017;177:124-131. 47. Cepeda VA. Disociación de respuestas estrogénicas en presencia de un extracto alcohólico de planta chilena, evaluadas en útero de rata pre-púber [tesis pregrado]. Chile, Santiago: Universidad de Chile; 2008. 48. Cox RI, Braden AW. The metabolism and physiological effects of phyto-oestrogens in livestock. Proc Aust Soc Anim Prod 1974;10:122. 49. McGarvey C, Cates PS, Brooks AN, Swanson IA, Milligan SR, Coen CW, et al. Phytoestrogens and gonadotropin-releasing hormone pulse generator activity and pituitary luteinizing hormone release in the rat. Endocrinology 2001;142(3):1202-1208. 50. Hughes CL, Kaldas RS, Weisinger AS, McCants CE, Basham KB. Acute and subacute effects of naturally occurring estrogens on luteinizing hormone secretion in the ovariectomized rat: Part 1. Reprod Toxicol 1991;5(2):127-132. 51. Goff AK. Steroid hormone modulation of prostaglandin secretion in the ruminant endometrium during the estrous cycle. Biol Reprod 2004;71(1):11-16. 52. McCracken JA, Custer EE, Lamsa JC. Luteolysis: a neuroendocrine-mediated event. Physiol Rev 1999;79(2):263-323. 53. Dubey RK, Rosselli M, Imthurn B, Keller PJ, Jackson EK. Vascular effects of environmental oestrogens: implications for reproductive and vascular health. Hum Reprod Update 2000;6(4):351-363.

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54. Rosselli M, Reinhart K, Imthurn B, Keller PJ, Dubey RK. Cellular and biochemical mechanisms by which environmental oestrogens influence reproductive function. Hum Reprod Update 2000;6(4):332-350. 55. Zdunczyk S, Piskula M, Janowski T, Baranski W, Ras M. Concentrations of isoflavones in blood plasma of dairy cows with different incidence of silent heat. Bull Vet Inst Pulawy 2005;49:189-191. 56. Beato M. Gene regulation by steroid hormones. Cell 1989;56(3):335–344. 57. Romero RCM, Tarrago CMR, Muñoz MR, Arista RR, Rosado GA. Síndrome estrogénico en vacas lecheras por consumo de alfalfas con grandes cantidades de coumestrol. Vet Méx 1997;28(1):25-30. 58. Mustonen E, Taponen S, Andersson M, Sukura A, Katila T, Taponen J. Fertility and growth of nulliparous ewes after feeding red clover silage with high phyto-oestrogen concentrations. Animal 2014;8(10):1699-1705. 59. Bennetts HW, Underwood EJ, Shier FL. A specific breeding problem of sheep on subterranean clover pastures in Western Australia. Aust Vet J 1946;22(1):2-12. 60. Adams NR. Permanent infertility in ewes exposed to plant oestrogens. Aust Veter J 1990;67(6):197-201. 61. Tang BY, Adams NR. Changes in oestradiol-17β binding in the hypothalami and pituitary glands of persistently infertile ewes previously exposed to oestrogenic subterranean clover: evidence of alterations to oestradiol receptors. J Endocrinol 1978;78(2):171-177. 62. Arispe SA, Adams B, Adams TE. Effect of phytoestrogens on basal and GnRH-induced gonadotropin secretion. J Endocrinol 2013;219(3):243-250. 63. Driancourt MA, Gibson WR, Cahill LP. Follicular dynamics throughout the oestrous cycle in sheep. A review. Reprod Nutr Dev 1985;25(1A):1-15. 64. Cortés-Sánchez ADJ, León-Sánchez JR, Jiménez-González FJ, Díaz-Ramírez M, Villanueva-Carvajal A. Alimentos funcionales, alfalfa y fitoestrógenos. Revista Mutis 2016;6(1):28-40. 65. Misztal T, Wañkowska M, Górski K, Romanowicz K. Central estrogen-like effect of genistein on growth hormone secretion in the ewe. Acta Neurobiol Exp (Wars) 2007;67(4):411-419. 66. Lundh T. Metabolism of estrogenic isoflavones in domestic animals. Proc Soc Exp Biol Med 1995;208(1):33-39.

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67. Takahashi O, Oishi S. Testicular toxicity of dietarily or parenterally administered bisphenol A in rats and mice. Food Chem Toxicol 2003;41(7):1035-1044. 68. Gutiérrez J, Castañón J. Prolapsos rectales y vaginales en cabras atribuibles al exceso de alfalfa deshidratada. En: Producción ovina y caprina. Jornadas de la Sociedad Española de Ovinotecnia y Caprinotecnia de la Universidad de Castilla-La Mancha. Castilla. 1994:239-242. 69. Van Weyenberg S, Sales J, Janssens G. Passage rate of digesta through the equine gastrointestinal tract: A review. Livestock Sci 2006;99(1):3-12. 70. Bickoff EM, Booth AN, Livingston AL, Hendrickson AP. Observations on the effect of drying on estrogenic activity of alfalfa samples of varying maturity. J Anim Sci 1960;19(3):745-753. 71. Gatta D, Russo C, Giuliotti L, Mannari C, Picciarelli P, Lombardi L, et al. Influence of partial replacement of soya bean meal by faba beans or peas in heavy pigs’ diet on meat quality, residual anti-nutritional factors and phytoestrogen content. Arch Anim Nutr 2013;67(3):235-247. 72. Groot MJ. Phyto-estrogenic activity of protein-rich feeds for pigs. RIKILT-Institute of Food Safety 2004. https://edepot.wur.nl/29220. Accessed Mar 8, 2021. 73. Kuhn G, Hennig U, Kalbe C, Rehfeldt C, Ren M, Moors S, et al. Growth performance, carcass characteristics and bioavailability of isoflavones in pigs fed soy bean based diets. Arch Anim Nutr 2004;58(4):265-276. 74. Liu G, Chen J, Han Z. Effects of isoflavonic phytoestrogen daiazein to lactating sows. Anim Husb Vet Med 1997;29:5-7. 75. Ren MQ, Kuhn G, Wegner J, Nurnberg G, Chen J, Ender K. Feeding daidzein to late pregnant sows influences the estrogen receptor beta and type 1 insulin-like growth factor receptor mRNA expression in newborn piglets. J Endocrinol 2001;170(1):129-135. 76. Farmer C, Robertson P, Xiao C, Rehfeldt C, Kalbe C. Exogenous genistein in late gestation: effects on fetal development and sow and piglet performance. Animal. 2016;10(9):1423-1430. 77. Hu YJ, Gao KG, Zheng CT, Wu ZJ, Yang XF, Wang L, et al. Effect of dietary supplementation with glycitein during late pregnancy and lactation on antioxidative indices and performance of primiparous sows. J Anim Sci 2015;93(5):2246-2254. 78. Ratna, WN. Inhibition of estrogenic stimulation of gene expression by genistein. Life Sci 2002;71(8):865–877.

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79. Nicholls J, Lasley BL, Nakajima ST, Setchell KD, Schneeman BO. Effects of soy consumption on gonadotropin secretion and acute pituitary responses to gonadotropinreleasing hormone in women. J Nutr 2002;132(4):708-714. 80. Wu N, Xu W, Cao GY, Yang YF, Yang XB, Yang XW. The blood-brain barrier permeability of lignans and malabaricones from the seeds of Myristica fragrans in the MDCK-pHaMDR cell monolayer model. Molecules 2016;21(2):134. 81. Kelly GE, Nelson C, Waring MA, Joannou GE, Reeder AY. Metabolites of dietary (soya) isoflavones in human urine. Clin Chim Acta 1993;223(1-2):9-22.

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Table 2: Summary of the main effects of phytoestrogens (PEs) on productive species Species

Condition and breed

Daily intake

Holstein/Polish cows

2.5 kg of soybeans

Holstein cows

~5 kg of alfalfa

Holstein cows Holstein/Polish cows Cattle

Bradford heifers

Decrease in PR

2.5 kg soybeans

Holstein heifers

~7 kg of clover

Manchega ewes Sheep Rahmani ewes

300

Increase in E2, decrease in P4 60 and LH ~5 kg of red Increase in E2, decrease in P4 60 clover and LH 2.5 kg of Decrease in P4 21 soybeans 2.22 of kg Decrease in PR, increase in 300 soybeans abortions, not significant

Holstein/Polish heifers

Angus male calves

Exposure (days)

Main effects

~800 g of soybean meal Alfalfa pasture ad libitum 800 g of subterranean clover

No difference in PR

Decrease in PR and P4 Increase in E2

150

Increase in sperm concentration

360

Endometrial cysts

300

and

paraovarian

Shorter estrus duration

826

180

Amount of PEs according to the species and presentation of the plant (mg/kg of DM)

Authors and year

Woclawek1900 of GEN and DAI Potocka et 2005(25) Rodríguez et ~25-65 of COU 2013(27) ~100 of GEN and 110 of Rodríguez et DAI 2013(27) Piotrowska et 1900 of GEN and DAI 2006(28) ~570 of GEN and 310 of García et DAI 2018(24) Woclawek1900 of GEN and DAI Potocka et 2005(25) 6.60 of GEN, 8.05 of DAI, Hashem et 2.85 of FOR and 282.5 of 2016(26) BI ~135 of GEN and 6.32 of Yurrita et DAI 2017(29) Cantero et 17-30 of COU 1996(30) 6.60 of GEN, 8.05 of DAI, Hashem 2.85 of FOR and 265 of BI 2018(32)

al., al., al., al., al.,

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Subterranean clover pasture ad libitum Alfalfa pasture Sarda female lambs ad libitum Subterranean Sarda female lambs clover pasture ad libitum Subterranean Comisana female clover pasture lambs ad libitum Comisana lambs

Creole rams

Equines Pigs

female

~1 kg of alfalfa

Hampshire/Suffolk 1 kg of alfalfa rams meal 200 g of Hampshire/Suffolk extruded rams soybeans Katahdin/Pelibuey ~230 g of alfalfa male lambs Subterranean Comisana male clover pasture lambs ad libitum 1 kg of alfalfa Lusitano mares pellets Large White/Erhualian ~1.5 mg of DAI pregnant sows

797 of GEN, DAI, FOR Pace et al., 2006(31) and BI

No alterations in reproductive 60 system, fertility and fecundity

900-10210 of GEN, BI and Pace et al., 2011(33) FOR

No alterations in reproductive 60 system, fertility and fecundity

900-10210 of GEN, BI and Pace et al., 2011(33) FOR

No alterations in reproductive 600 parameters

810-880 of GEN, BI and Pace et al., 2011(33) FOR

Sperm concentration inverse to 45 the dose

~25-65 of COU

No alterations in sperm parameters

~25-65 of COU

No alterations in sperm parameters

~570 of GEN and 310 of Sierra et (34) DAI 2015

No alterations in sperm parameters or morphology

~25-65 of COU

No effects on reproduction

797 of GEN, DAI, FOR Pace et al., 2006(31) and BI

No effects on reproduction

AragadvayYungán et 2018(2) Sierra et 2015(34)

~25-65 of COU

Ferreira-Dias al., 2013(35)

Increase in milk production

Gentao 1999(36)

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al., al.,

Domínguez et al., 2014(19)

High concentrations of COU in 14 serum 37

al.,

et al.,

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Minipig boars

New Zealand pregnant does New Zealand bucks Leporids

New Zealand bucks Line V bucks Line V bucks Bovans Brown hens Bovans Brown hens

Poultry

Bovans Brown hens ISA hens Rugao hens

At low doses, increase in ~6.75–27 mg of testicular index; at high doses, soybean decrease in testicular index, LH isoflavones and T4 No effect on reproductive ~20 g of organs, semen quality and soybean meal sexual behavior of F1 ~32 g of No alterations in reproductive soybean meal organs Improvement in the 7.13-14.25 mg characteristics of semen and of ISO* libido ~15.44 g of It does not affect semen fertility, soybeans decrease in T4 ~19.3 g of It does not affect semen fertility, flaxseed decrease in T4 ~120 mg of Increase in LH, FSH, E2 flax ~131 mg of Increase in LH, E2 fenugreek ~128 mg of flax and 128 Increase in LH, FSH, E2 mg of fenugreek ~1.2 mg of Increase in oviduct weight DAI ~5.4-22.57 mg No effect on fertility of DAI

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Yuan et (37) 2012

al.,

125-500 of ISO

~256

~135 of GEN and 632 of Cardoso et al., DAI 2007(38)

~135 of GEN and 632 of Cardoso et al., DAI 2009(39)

84 84

240.4 of DAI and 131 of GEN 368 of SECOI and 52.8 of DAI

Yousef et 2004(18)

Hashem 2018(40) Hashem 2018(40) Saleh 2019(41) ~0.1 of DAI and 0.1 of Saleh GEN 2019(41)

al.,

et al., et al., et

al.,

~0.1 of DAI and 0.1 of Saleh et GEN 2019(41)

al.,

Ni et al., 2007(42)

Lu et al., 2017(43)

42 42

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Lohmann Brown hens Selected Leghorn hens Young breeders

Cobb chickens

No changes in morphology and ~5 mg of DAI size of ovaries and oviducts 84 high sensitivity to DAI No changes in morphology and ~5 mg of DAI 84 size of ovaries and oviducts ~0.75 mg of Increase in testicle weight, 63 ISO GnRH ~2.38-7.14 mg Increase in T4 and of extruded 21 androstenedione beans ~2.38-7.14 mg Decrease in T4 and 21 of raw beans androstenedione

Wistedt 2012(44)

al.,

Wistedt et 2012(44) Heng et (46) 2017

al., al.,

Arija et 2006(45)

al.,

Arija et (45) 2006

al.,

BI= Biochanin A; COU= Coumestrol; DAI= Daidzein; E2= 17-ß estradiol; FOR= Formononetin; F1= Filial generation 1; GEN= Genistein; GLY= Glycitein; GnRH= Gonadotropin-releasing hormone; ISO= Isoflavones; LH= Luteinizing hormone; MCOU= Methoxycoumestrol; PEP= Para-ethyl-phenol; P4= Progesterone; SECOI= Secoisolariciresinol; T4= Testosterone; PR= Pregnancy rate; -= Not applicable; *= Every 3 days; ~= approximate calculation.

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https://doi.org/10.22319/rmcp.v13i3.5730 Technical note

Genetic structure and environmental aptitude of sideoats grama [Bouteloua curtipendula (Michx.) Torr.] populations in Chihuahua, Mexico

Alan Álvarez-Holguína Carlos Raúl Morales-Nieto b* Raúl Corrales-Lerma a Jesús Alejandro Prieto-Amparán a Ireyli Zuluami Iracheta-Lara a Nathalie Socorro Hernández-Quiroz a

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP). Campo Experimental La Campana. Carretera Chihuahua-Ojinaga km. 33.3, 32190. Aldama, Chihuahua, México. b

Universidad Autónoma de Chihuahua, Facultad de Zootecnia y Ecología. Chihuahua, México.

* Corresponding author: cnieto@uach.mx

Abstract: Research has increasingly centered on selecting outstanding grass genotypes for grasslands restoration, although most focuses on agronomic characteristics. Little importance has been given genotype genetic structure and environmental adaptation. An analysis was done of the genetic structure and environmental suitability of sideoats grama (Bouteloua curtipendula) populations in Chihuahua, Mexico. Fifty-one populations were evaluated through AFLP markers and analysis of their genetic structure. In a novel approach, the MaxEnt algorithm, commonly used only at the species level, was used to design models to quantify

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environmental aptitude of the groups generated by the genetic analysis. The STRUCTURE analysis divided the B. curtipendula populations into two different genetic groups (AMOVA; P<0.0001). Most (89 %) of the Group 1 populations are in the state’s semi-arid region while most (90 %) of the Group 2 populations are in the arid region. The MaxEnt results showed the two genetic groups to have different environmental aptitude. The climatic niche of Group 1 is mainly located in the state’s center and south, while that of Group 2 is in the center, west and northeast. Restoration programs involving B. curtipendula would benefit most from using local ecoregion-specific genotypes in areas for which they have the highest environmental aptitude. Key words: AFLP; Climatic niche; MaxEnt; STRUCTURE.

Received: 13/07/2020 Accepted: 10/01/2022

Degradation of grasslands results in significant loss of the products and services that these ecosystems provide to humans, such as livestock fodder, water capture, soil retention and carbon sequestration(1). Some of the main problems affecting grasslands are fragmentation, expansion of invasive species, conversion to agricultural uses, and human population growth, among others(2). Grassland rehabilitation is becoming more frequent, and in recent years research has focused on selecting the grass species genotypes most apt for this application(3,4,5). However, genetic material selection has concentrated mainly on agronomic traits, with minimal importance given genetic structure and potential adaptation to climatic conditions. A grass species’ genetic structure is largely determined by its adaptation to environmental conditions within its distribution area. For this reason, understanding population genetic structure can help delimit genetic type distributions and their potential use in ecological restoration programs(6,7). In northern Mexico, one of the species most used in grasslands restoration is sideoats grama [Bouteloua curtipendula (Michx.) Torr.] because it adapts to a wide range of climates and has excellent forage value. Due to its potential, recent research has emphasized selection of outstanding B. curtipendula genotypes for use in restoring grasslands in the state of Chihuahua, Mexico(8,9,10). Identifying ideal genotypes requires analysis of the genetic structure of B. curtipendula populations in the state and of their environmental aptitude. Mathematical models, such as the maximum entropy method applied in the MaxEnt software(11), are an effective way of estimating areas with potential environmental suitability

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for the distribution of a species. Applying these models via MaxEnt provides advantages such as that only presence data is required, reliable results are produced with limited data, continuous and/or categorical environmental data are used, and are these graphically displayed via maps, facilitating interpretation. MaxEnt also employs a Jackknife test to identify the relevance of each environmental variable in species distribution(11,12). Models generated with this program have been used widely to estimate climatic niche for flora and fauna species(13,14). It has been used to identify areas with environmental aptitude with potential B. curtipendula distribution in Mexico and the United States(15,16), a prerequisite for ecosystem rehabilitation(17). However, MaxEnt has only been applied at the species level. It has not been used to estimate areas with environmental suitability for genetic types within B. curtipendula, which could make ecosystem restoration programs more efficient. The present study objective was to evaluate B. curtipendula genetic structure in the state of Chihuahua, Mexico, by applying MaxEnt models at the genetic group level and identifying those areas with environmental suitability for the resulting genetic groups. Sampling was done in 51 populations, distributed in 29 municipalities of the state of Chihuahua, in northern Mexico (32° and 25° N; -103° and -109° W). To include as much genetic diversity as possible, sampling sites were located in the state’s arid and semi-arid ecoregions (Figure 1). Figure 1. Geographic location of collected genetic material, corresponding to 51 sideoats grama (Bouteloua curtipendula) populations in Chihuahua, Mexico

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The genetic structure of B. curtipendula was analyzed using Amplified Fragment Length Polymorphisms (AFLP) molecular markers. Plant leaves were collected at each site and DNA extracted from them based on the method proposed by Doyle & Doyle(18). The AFLP analysis was done using the method proposed by Vos et al.(19). First, 2 µl diluted DNA were digested by the EcoRI and MseI restriction enzymes and the digested DNA fragments ligated with EcoRI and MseI adapters. For pre-amplification, an extra nucleotide was added to the primers (EcoRI + A and MseI + A). Selective amplification was done using four combinations of fluorescence-labeled primers: MseI + CTG - EcoRI + AAG, MseI + CTG - EcoRI + ACT, MseI + CAG - EcoRI + AGG, MseI + CAG - EcoRI + AAC. The polymerase chain reaction (PCR) was done in a thermal cycler (Applied Biosystems Veriti 2720), with the following program: one cycle of 94 °C for 30 sec, 65 °C for 30 sec, 72 °C for 1 min; 12 cycles of 94 °C for 30 sec, 65 °C for 30 sec, 72 °C for 1 min; and 23 cycles of 94 °C for 30 sec, 56 °C for 30 sec, 72 °C for 1 min. The selective amplification products (2 µL) were mixed with 8 µl formamide and 1 µL Eco 700 GeneScan label (Applied Biosystems). Separation of the amplified fragments was done in a LI-COR DNA analyzer, loading 0.8 µL sample per well. Fluorescence-marked oligos or primers at different wavelengths (700 nm and 800 nm) were used. With the band pattern produced by the AFLP analysis, a binary band presence (1) / absence (0) matrix was constructed. A genetic structure analysis, based on the Bayesian clustering algorithm, was applied to the binary data using the STRUCTURE version 2.3.4 software(20,21). The STRUCTURE program was run 30 times for each K number of genetic clusters and analyzed from K=1 to K=10. Ten thousand (10,000) Markov-Monte Carlo chain (CMMC) repetitions and 100,000 burn-in periods were done in each run. This analysis was performed using a correlated allele frequency and admixture model. The optimal number of K clusters was considered to be that which attained the highest value for the average posterior probability (LK) and ΔK, according to the criteria proposed by Evanno et al(22). The LK and ΔK values were obtained from the Structure Harvester website(23). An analysis of the association between climatic zones and the distribution of the B. curtipendula populations in the genetic groups was done with a χ2 test of independence (α=0.05). An analysis of molecular variance (AMOVA) was applied to compare the groups formed in the genetic structure analysis(24), using the GenAIEx ver. 6 software(25). Using the F (ΦST) statistics produced with the AMOVA, the inter-group gene flow index was calculated with the formula Nm= [0.25 (1- ΦST)/(ΦST)](26). The genetic data were also analyzed using the Monmonier algorithm to detect possible eco-geographical barriers affecting interpopulational gene flow. This analysis was run with the Barrier ver. 2.2 software(27), in which the Bootstrap values of each barrier were calculated with 100 Dice coefficient distance matrices.

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For the genetic groups formed by the STRUCTURE analysis, the GenAIEx ver. 6 software(25) was used to calculate diversity statistics, polymorphic loci percentages, the average number of alleles per locus, the number of effective alleles, and the Shannon information (I) and Nei diversity (He) indices. Both these indices were estimated based on the assumption that each locus represents a pair of alleles when the presence or absence of an AFLP fragment is identified in a band. Diversity statistics for each population were compared using the Wilcoxon test with the Bonferroni correction (α=0.05). Genetic group environmental fitness was identified using the MaxEnt algorithm in the MaxEnt ver. 3.3.3 software(11). For each genetic group, the model was run separately with the coordinates of the genetically analyzed populations. Of the total data set, 75 % was used to test the models and the remaining 25 % to validate the models using the bootstrap test with 50 replicates. The generated environmental suitability models were evaluated using receiver operating characteristic (ROC) curve and area under the curve (AUC) analysis. The AUC score is useful for measuring model performance; the higher (closer to 1) the AUC value the better the model estimates species presence probability. A total of 22 climatic variables were used as predictors to model the potential distribution of B. curtipendula. Nineteen were bioclimatic variables: annual mean temperature (Bio1); mean diurnal range (Bio2); isothermality (Bio3); temperature seasonality (Bio4); max temperature of warmest month (Bio5); min temperature of coldest month (Bio6); temperature annual range (Bio7); mean temperature of wettest quarter (Bio8); mean temperature of driest quarter (Bio9); mean temperature of warmest quarter (Bio10); mean temperature of coldest quarter (Bio11); annual precipitation (Bio12); precipitation of wettest month (Bio13); precipitation of driest month (Bio14); precipitation seasonality (Bio15); precipitation of wettest quarter (Bio16); precipitation of driest quarter (Bio17); precipitation of warmest quarter (Bio18); and precipitation of coldest quarter (Bio19)(28). Three additional variables were included: average annual solar radiation (Rad); average annual evapotranspiration (Vapr); and average annual wind speed (Wind). These variables were obtained from the WorldClim database (https://www.worldclim.org) and limited to the geographic space of the state of Chihuahua using the ArcMap ver. 10.3 software. Climate data are interpolation estimates for 1950-2000, with a 30 arc-seconds spatial resolution. Applying MaxEnt generated a logistic map showing potential B. curtipendula distribution within a 0 (inadequate) to 1 (optimal) value range. The genetic structure analysis (STRUCTURE) divided the sampled B. curtipendula populations into two genetic groups since K=2 produced the highest ΔK and LK values (Figure 2).

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400 350 300 250 200 150 100 50 0

-5000 -4000

ΔK

Figure 2. Delta K (ΔK) and average posterior probability (LK) values for the genetic structure of 51 sideoats grama (Bouteloua curtipendula) populations in Chihuahua, Mexico (grupos = groups).

-3000 -2000 -1000 0

K groups

Values range from K=1 to K=10; K=2 is the optimum number of groups.

Group genetic divergence appears to have been generated by adaptations to Chihuahua’s climatic conditions since there was a high association between the state’s ecoregions and group formation (χ2 = 32.9; P<0.0001). Most (89 %) of the Group 1 populations are in the semi-arid region and 11 % are in the arid region. In contrast, 90 % of the Group 2 populations are in the arid region and only 10 % in the semi-arid region (Figure 3B). These results agree with those of the BARRIER analysis, which identified genetic barriers coinciding with the border between the arid and semi-arid regions (Figure 3C). Figure 3. Genetic structure of 51 sideoats grama (Bouteloua curtipendula) populations in Chihuahua

A) STRUCTURE analysis using K= 2 run and 186 AFLP fragments; the colors represent the proportion of probability of belonging to each genetic group. B) Group structure in geographic context of state’s climatic zones. C) Genetic barriers identified by BARRIER analysis; yellow lines represent barriers and numbers are Bootstrap values (1000 bootstraps). Pie charts represent percentage of populations in each genetic group within each region. 835

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The two groups generated by the STRUCTURE analysis were different (P<0.0001) according to the AMOVA. However, these differences only explained 7 % of overall variation, indicating the presence of substantial intragroup genetic variability. In grasslands it is common for intergroup differences to explain small proportions of overall variation due to generally high interpopulation genetic flow(29,30). The low interpopulation genetic differentiation (7 %) identified by the AMOVA apparently contrasts with the marked association between the state’s climatic zones and group formation. This can occur because formation of a single locus can be closely linked to adaptation to climatic conditions. For example, a study of Bouteloua gracilis populations identified a close relationship between the frequency of a single locus and precipitation in the driest quarter of the year (R2=0.84) and precipitation seasonality (R2= 0.77)(7). Compared to previous research using AFLP markers, the observed intergroup genetic exchange (Fst= 3.41) can be considered high. A study of Stipa pulcherrima grass distributed throughout Europe and Asia found a 0.76 gene flow among 30 populations(31), while one of Microlaena stipoides found an Fst of 0.02 among 85 Australian populations(29). The STRUCTURE analysis groups exhibited differences (P<0.05) in the evaluated diversity parameters. Group 2, mainly distributed in the arid region, had the highest values (P<0.05) in all the diversity parameters (Table 1). This coincides with the higher genetic diversity levels observed in Festuca ovina in arid zones(32). In this study a positive correlation was identified between the Nei genetic diversity index and mean annual temperature (r= 0.56), while a negative correlation (r= -0.60) was found between this index and mean annual precipitation. Similar results have been reported for Dactylis glomerata(33), and many studies indicate that plant populations tend to have greater diversity in adverse environments(34,35). Plant populations in extremely arid environments tend to develop greater genetic diversity as a mechanism of adaptation to drought(36). Based on the Shannon information index (I), both STRUCTURE groups presented high intragroup genetic diversity (Group 1= 0.302, Group 2= 0.427). By comparison, an evaluation of 56 accessions of Panicum virgatum produced an I value of 0.317(37), and one of 281 cultivars of Pennisetum purpureum produced I values ranging from 0.12 to 0.34(38). Both these studies included large numbers of genotypes, further emphasizing that the present genetic diversity results for B. curtipendula are relatively high in both groups.

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Table 1. Diversity parameters for two genetic groups of sideoats grama (Bouteloua curtipendula) in Chihuahua, Mexico Polymorphic loci Average alleles per Number of Group I He (%) locus effective alleles

59.1

1.56b

1.34b

0.302b 0.211b

90.4

1.90a

1.43a

0.427a 0.280a

Different letter superscripts in the same column indicate statistical difference (P<0.05; Wilcoxon test with Bonferroni correction). I= Shannon information index; He= Nei genetic diversity.

The MaxEnt model was run using the coordinates of 20 populations for Group 1 and 32 for Group 2. Average AUC value for the Group 1 climatic niche was 0.91 (SD = 0.031) while that for Group 2 was 0.93 (SD = 0.015). Both values indicate the estimated environmental suitability of both genetic groups is highly reliable(11,39). The respective climatic niches of the groups clearly differ (Figure 4). Group 1 is distributed mainly in the center and south of the state (Figure 4A), while Group 2 is distributed in the center, west and northeast (Figure 4B). This suggests that the genetic groups diverged by evolutionary adaptation and that each genotype is adapted to regional climatic conditions. Restoration programs involving B. curtipendula would probably benefit most by using local genotypes from each ecoregion. However, grassland restoration programs carried out to date in Mexico have employed genetic material from outside the region, mainly due to low seed availability. Bouteloua curtipendula varieties from the United States have been used; for example, the El Reno variety had unfavorable performance due to its low adaptability to the Mexican climate. A comparison of the El Reno variety with 277 B. curtipendula genotypes from different states in Mexico found that more than half of the genotypes provided better productive potential than the El Reno variety(40). Other studies(4) have reported that, compared to native material, the El Reno variety has low establishment and forage production capacities. Local materials are most effective in grassland restoration programs since they guarantee a greater probability of success and preserve local genetic structure(41,42). This agrees with the present results, emphasizing that B. curtipendula revegetation programs in Chihuahua should use genotypes specific to each of the state’s ecoregions. The high diversity identified within each genetic group suggests the possibility of selecting outstanding genotypes for each ecoregion. Previous studies have addressed genotype selection(9,43), but focused mainly on productive characteristics and gave little weight to the environmental adaptation of each genotype. In contrast, the present results provide basic information on the potential environmental suitability of B. curtipendula populations in Chihuahua, which could be valuable in selection programs for productive genotypes.

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Figure 4. MaxEnt model maps for two sideoats grama (Bouteloua curtipendula) genetic groups identified by AFLP markers and STRUCTURE analysis

Group 1 (A) and Group 2 (B). Red color represents areas of greatest environmental aptitude and blue those of least aptitude.

The variables that contributed most to the Group 1 environmental suitability model were precipitation seasonality (79.5 %) and precipitation in the coldest quarter (7.4 %). The Group 2 environmental suitability model was mainly influenced by precipitation in the coldest quarter (36.2 %), annual thermal oscillation (13.2 %), average annual solar radiation (8.1 %) and average temperature of the driest quarter (7.4 %; Table 2). These results agree with previous studies modeling the B. curtipendula climatic niche in Mexico and the United States. In one, the ecological descriptors of thermal oscillation and precipitation contributed most to potential B. curtipendula distribution in Mexico(16), while another found average annual temperature contributed most to potential distribution in the United States(15). A detailed analysis using response curves of the most influential variables showed Group 1 genotypes to have a higher probability of developing in areas with a 120+ precipitation seasonality coefficient and that they develop best in areas with 0 to 20 mm precipitation in the coldest quarter (December, January and February) (Figure 5). Group 2 genotypes also develop best in areas with 0 to 20 mm precipitation in the coldest quarter. In addition, they prefer areas with a 49 to 53 annual thermal oscillation, 18,500 to 19,000 w m-2 annual average solar radiation and a 6 to 10 °C average temperature of driest quarter. Group 1 genotypes apparently do not resist long periods of drought and needs occasional precipitation during the warmer months (June-October). Group 2 genotypes can resist less thermal oscillation, higher temperatures and greater solar radiation, but need precipitation concentrated in the

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warmer months. These response curve results highlight the evolutionary differences between the two genetic groups and the importance of using them in their source ecoregion. Table 2. Relative contribution (%) of environmental variables to MaxEnt model for two genetic groups of sideoats grama (Bouteloua curtipendula) in Chihuahua, Mexico Contribution Contribution ID Variable (%) for (%) for Group 1 Group 2 Wind

Average annual wind speed

0.6

6.7

Rad

Average annual solar radiation

2.7

8.1

2.2

1.1

13.2

Bio-3

Mean diurnal range (Mean of monthly (max temp - min temp) Isothermality (Bio2/Bio7) (×100)

Bio-9

Mean temperature of driest quarter

1.8

7.4

Bio-13

Precipitation of wettest month

0.1

4.3

Bio-14

Precipitation of driest month

0.3

4.3

Bio-15

Precipitation seasonality (coefficient of variation)

79.5

1.4

Bio-17

Precipitation of driest quarter

0.2

5.3

Bio-18

Precipitation of warmest quarter

0.8

3.4

Bio-19

Precipitation of coldest quarter

7.4

36.2

Bio-2

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Figure 5. Response curves for two genetic groups of sideoats grama (Bouteloua curtipendula) based on the variables with greatest influence on environmental aptitude.

In conclusion, the state of Chihuahua, Mexico, sideoats grama Bouteloua curtipendula populations are divided into two genetic groups. Their distributions are highly influenced by ecoregion climatic conditions such that each genetic group has a different climatic niche. Restoration programs involving Bouteloua curtipendula could benefit from using local genotypes from specific ecoregions in environmentally suitable areas. These genotypes could also be used in edaphoclimatic conditions similar to those of their point of origin. The genetic diversity identified within each gene pool provides an opportunity for developing outstanding genotypes for use in ecoregion-specific grassland restoration programs. However, climatological projections are still needed to consider how climate change may affect

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Bouteloua curtipendula genetic types and what this could mean for future grassland restoration projects.

Literature cited: 1.

Chaplot V, Dlamini P, Chivenge P. Potential of grassland rehabilitation through high density-short duration grazing to sequester atmospheric carbon. Geoderma 2016;271:10-17.

Barnosky AD, Matzke N, Tomiya S, Wogan GO, Swartz B, Quental TB, et al. Has the Earth’s sixth mass extinction already arrived? Nature 2011;471:51-57.

Morales NCR, Melgoza CA, Jurado GP, Martínez SM, Avendaño AC. Caracterización fenotípica y molecular de poblaciones de zacate punta blanca (Digitaria californica (Benth.) Henr.). Rev Mex Cienc Pecu 2012;3:171-184.

Sánchez-Arroyo JF, Wehenkel C, Carrete-Carreón FO, Murillo-Ortiz M, Herrera-Torres E, Quero-Carrillo R. Establishment atributes of Bouteloua curtipendula (Michx.) Torr. populations native to Mexico. Rev Fitotec Mex 2018;41:237-243.

Morales-Nieto CR, Álvarez-Holguín A, Villarreal-Guerrero F, Corrales-Lerma R, Pinedo-Álvarez A, Martínez-Salvador M. Phenotypic and genetic diversity of blue grama (Bouteloua gracilis) populations from Northern Mexico. Arid Land Res Manag 2020;34:83-98.

Mattioni C, Martin MA, Chiocchini F, Cherubini M, Gaudet M, Pollegioni, et al. Landscape genetics structure of European sweet chestnut (Castanea sativa Mill): indications for conservation priorities. Tree Genet Genomes 2017;13:1-14.

Tso KL, Allan GJ. Environmental variation shapes genetic variation in Bouteloua gracilis: Implications for restoration management of natural populations and cultivated varieties in the southwestern United States. Ecol Evol 2019;9:482-499.

Corrales LR, Morales NCR, Melgoza CA, Sierra TJS, Ortega GJÁ, Méndez ZG. Caracterización de variedades de pasto banderita [Bouteloua curtipendula (Michx.) Torr.] recomendadas para rehabilitación de pastizales. Rev Mex Cienc Pecu 2016;7:201211.

841

Rev Mex Cienc Pecu 2022;13(3):830-845

Morales NCR, Avendaño AC, Melgoza CA, Vega GK, Quero CA, Martínez SM. Caracterización morfológica y molecular de poblaciones de pasto banderita (Bouteloua curtipendula) en Chihuahua, México. Rev Mex Cienc Pecu 2016;7:455-469.

10. Álvarez-Holguín A, Morales-Nieto CR, Melgoza-Castillo A, Méndez-Zamora G. Germinación de genotipos de pasto banderita (Bouteloua curtipendula) bajo diferentes presiones osmóticas. ERA 2017;4:161-168. 11. Phillips SJ, Anderson RP, Schapire RE. Maximum entropy modeling of species geographic distributions. Ecol Model 2006;190:231-259. 12. Abdelaal M, Fois M, Fenu G, Bacchetta G. Using MaxEnt modeling to predict the potential distribution of the endemic plant Rosa arabica Crép. in Egypt. Ecol Inform 2019;50:68-75. 13. Cruz-Cárdenas G, Villaseñor JL, López-Mata L, Martínez-Meyer E, Ortiz E. Selección de predictores ambientales para el modelado de la distribución de especies en MaxEnt. Rev Chapingo Ser Cie 2014;20:187-201. 14. Tran VD, Vu TT, Tran QB, Nguyen TH, Ta TN, Ha TM et al. Predicting suitable distribution for an endemic, rare and threatened species (grey-shanked douc langur, Pygathrix cinerea Nadler, 1997) using MaxEnt model. Appl Ecol Env Res 2018;16:1275-1291. 15. Martinson EJ, Eddy ZB, Commerford JL, Blevins E, Rolfsmeier SJ, McLauchlan KK. Biogeographic distributions of selected North American grassland plant species. Phys Geogr 2011;32:583-602. 16. Martínez SJÁ, Duran PN, Ruiz CJA, González EDR, Mena MS. Áreas con aptitud ambiental para [Bouteloua curtipendula (Michx.) Torr.] en México por efecto del cambio climático. Rev Mex Cienc Pecu 2020;11:49-62. 17. Hufford KM, Mazer SJ. Plant ecotypes: genetic differentiation in the age of ecological restoration. Trends Ecol Evol 2003;18:147-155. 18. Doyle JJ. A rapid total DNA preparation procedure for fresh plant tissue. Focus 1990;12:13-15. 19. Vos P, Hogers R, Bleeker M, Reijans M, Lee TVD, Hornes M, et al. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res 1995;23:4407-4414.

842

Rev Mex Cienc Pecu 2022;13(3):830-845

20. Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Genetics 2000;155:945-959. 21. Falush D, Stephens M, Pritchard JK. Inference of population structure using multilocus genotype data: dominant markers and null alleles. Mol Ecol Notes 2007;7:574-578. 22. Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol 2005;14;2611-2620. 23. Earl DA, VonHoldt BM. STRUCTURE HARVESTER: a website and program for visualizing STRUCTURE output and implementing the Evanno method. Conserv Genet Resour 2012;4:359-361. 24. Excoffier L, Smouse PE, Quattro JM. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 1992;131:479-491. 25. Peakall ROD, Smouse PE. GENALEX 6: genetic analysis in Excel. Population genetic software for teaching and research. Mol Ecol Notes 2006;6:288-295. 26. Whitlock MC, Mccauley DE. Indirect measures of gene flow and migration: FST≠ 1/(4Nm+ 1). Heredity 1999;82:117-125. 27. Manni F, Guérard E, Heyer E. Geographic patterns of (genetic, morphologic, linguistic) variation: how barriers can be detected by using Monmonier's algorithm. Hum Biol 2004;2:173-190. 28. Hijmans RJ, Cameron SE, Parra JL, Jones PG, Jarvis A. Very high resolution interpolated climate surfaces for global land areas. Int J Climatol 2005;25:1965-1978. 29. Mitchell ML, Stodart BJ, Virgona JM. Genetic diversity within a population of Microlaena stipoides, as revealed by AFLP markers. Aust J Bot 2015;62:580-586. 30. Wu WD, Liu WH, Sun M, Zhou JQ, Liu W, Zhang CL, et al. Genetic diversity and structure of Elymus tangutorum accessions from western China as unraveled by AFLP markers. Hereditas 2019;156:8. 31. Durka W, Nossol C, Welk E, Ruprecht E, Wagner V, Wesche K, et al. Extreme genetic depauperation and differentiation of both populations and species in Eurasian feather grasses (Stipa). Plant Syst Evol 2013;299:259-269.

843

Rev Mex Cienc Pecu 2022;13(3):830-845

32. Zhang C, Zhang J, Fan Y, Sun M, Wu W, Zhao W, et al. Genetic structure and ecogeographical differentiation of wild sheep Fescue (Festuca ovina L.) in Xinjiang, Northwest China. Molecules 2017;22:1316. 33. Zhang C, Sun M, Zhang X, Chen S, Nie G, Peng Y, et al. AFLP-based genetic diversity of wild orchardgrass germplasm collections from Central Asia and Western China, and the relation to environmental factors. PloS One 2018;13:e0195273. 34. Reisch C, Anke A, Rohl M. Molecular variation within and between ten populations of Primula farinosa (Primulaceae) along an altitudinal gradient in the northern Alps. Basic Appl Ecol 2005;6:35–45. 35. Kiambi DK, Newbury HJ, Maxted N, Ford-Lloyd BV. Molecular genetic variation in the African wild rice Oryza longistaminata A. Chev. et Roehr. and its association with environmental variables. Afr J Biotechnol 2008;7:1446-1460. 36. Zhao NX, Gao YB, Wang JL, Ren AZ. Genetic diversity and population differentiation of the dominant species Stipa krylovii in the Inner Mongolia Steppe. Biochem Genet 2006;44:513-526. 37. Todd J, Wu YQ, Wang Z, Samuels T. Genetic diversity in tetraploid switchgrass revealed by AFLP marker polymorphisms. Genet Mol Res 2011;10:2976-2986. 38. Wanjala BW, Obonyo M, Wachira FN, Muchugi A, Mulaa M, Harvey J, et al. Genetic diversity in Napier grass (Pennisetum purpureum) cultivars: implications for breeding and conservation. AoB Plants 2013;5:plt022. 39. Merow C, Smith MJ, Silander Jr JA. A practical guide to MaxEnt for modeling species’ distributions: what it does, and why inputs and settings matter. Ecography 2013;36:1058-1069. 40. Morales NCR, Quero CA, Melgoza CA, Martínez SM, Jurado GP. Diversidad forrajera del pasto banderita [Bouteloua curtipendula (Michx.) Torr.], en poblaciones de zonas áridas y semiáridas de México. Téc Pecu Méx 2009;47:231-244. 41. Mijnsbrugge VK, Bischoff A, Smith B. A question of origin: where and how to collect seed for ecological restoration. Basic Appl Ecol 2010;11:300-311. 42. Smith SW, Ross K, Karlsson S, Bond B, Upson R, Davey A. Going native, going local: revegetating eroded soils on the Falkland Islands using native seeds and farmland waste. Restoration Ecology 2018;26:134-144.

844

Rev Mex Cienc Pecu 2022;13(3):830-845

43. Morales-Nieto CR, Corrales-Lerma R, Álvarez-Holguín A, Villarreal-Guerrero F, Santellano-Estrada E. Caracterización de poblaciones de pasto banderita (Bouteloua curtipendula) de México para seleccionar genotipos con potencial para producción de semilla. Rev Fitotec Mex 2017;40:309-316.

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Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 3, pp. 584-845, JULIO-SEPTIEMBRE-2022

ISSN: 2448-6698

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Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 3, pp. 584-845, JULIO-SEPTIEMBRE-2022

CONTENIDO CONTENTS

Rev. Mex. Cienc. Pecu. Vol. 13 Núm. 3, pp. 584-845, JULIO-SEPTIEMBRE-2022