RMCP Vol. 11, Num. 2 (2020): April-June [english version] by Revista Mexicana de Ciencias Pecuarias

Edición Bilingüe Bilingual Edition

Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 11 Núm 2, pp. 311-604, ABRIL-JUNIO-2020

ISSN: 2448-6698

Rev. Mex. Cienc. Pecu. Vol. 11 Núm. 2, pp. 311-604, ABRIL-JUNIO-2020

Ganado caprino en Piedras Negras, Coahuila. Fotografía: INIFAP, Concurso de fotografía 2010.

REVISTA MEXICANA DE CIENCIAS PECUARIAS Volumen 11 Número 2, AbrilJunio 2020. 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 04-2016-060913393200-203. 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 mayo de 2020.

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, México Dra. Maria Cristina Schneider, PAHO, Estados Unidos Dra. Elisa Margarita Rubí Chávez, UNAM, México 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. Sergio D. Rodríguez Camarillo, INIFAP, México Dr. Martin Talavera Rojas, Universidad Autónoma del Estado de México, 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 Dra. Silvia Elena Buntinx Dios, Facultad de Medicina Veterinaria y Zootecnia, UNAM, México Dr. José Armando Partida de la Peña, INIFAP, México Dr. José Luis Romano Muñoz, 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. Fernando Cervantes Escoto, Universidad Autónoma Chapingo, México Dr. Adolfo Guadalupe Álvarez Macías, Universidad Autónoma Metropolitana Xochimilco, México Dr. Alfredo Cesín Vargas, UNAM, México.

TIPOGRAFÍA Y FORMATO Nora del Rocío Alfaro Gómez 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.

total por publicar es de $ 5,600.00 más IVA por manuscrito ya editado. Se publica en formato digital en acceso abierto, por lo que se autoriza la reproducción total o parcial del contenido de los artículos si se cita la fuente. El envío de los trabajos de debe realizar directamente en el sitio oficial de la revista. Correspondencia adicional deberá dirigirse al Editor Adjunto a la siguiente dirección: Calle 36 No. 215 x 67 y 69 Colonia Montes de Amé, C.P. 97115 Mérida, Yucatán, México. Tel/Fax +52 (999) 941-5030. Correo electrónico (C-ele): rodriguez_oscar@prodigy.net.mx.

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.

La correspondencia relativa a suscripciones, asuntos de intercambio o distribución de números impresos anteriores, deberá dirigirse al Editor en Jefe de la Revista Mexicana de Ciencias Pecuarias, CENID Salud Animal e Inocuidad, Km 15.5 Carretera México-Toluca, Col. Palo Alto, D.F. C.P. 05110, México; Tel: +52(55) 3871-8700 ext. 80316; garcia.arturo@inifap.gob.mx o arias.alfonso@inifap.gob.mx. Inscrita en la base de datos de 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), indizada en el “Journal Citation Report” Science Edition del ISI (http://thomsonreuters. com/) y en los Índices SCOPUS y EMBASE de Elsevier (www.elsevier.com)

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

VISITE NUESTRA PÁGINA EN INTERNET Artículos completos desde 1963 a la fecha y Notas al autor en: http://cienciaspecuarias.inifap.gob.mx Revista Mexicana de Ciencias Pecuarias is an open access peer-reviewed and refereed scientific and technical journal, which publishes results of research carried out in any scientific or academic institution, especially related to different areas of veterinary medicine and animal production. Papers on disciplines different from those shown in Editorial Committee can be accepted, if related to livestock research.

Part of, or whole articles published in this Journal may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying or otherwise, provided the source is properly acknowledged. Manuscripts should be submitted directly in the official web site. Additional information may be mailed to Associate Editor, Revista Mexicana de Ciencias Pecuarias, Calle 36 No. 215 x 67 y 69 Colonia Montes de Amé, C.P. 97115 Mérida, Yucatán, México. Tel/Fax +52 (999) 941-5030. E-mail: rodriguez_oscar@prodigy.net.mx.

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For subscriptions, exchange or distribution of previous printed issues, please contact: Editor-in-Chief of Revista Mexicana de Ciencias Pecuarias, CENID Salud Animal e Inocuidad, Km 15.5 Carretera México-Toluca, Col. Palo Alto, D.F. C.P. 05110, México; Tel: +52(55) 3871-8700 ext. 80316; garcia.arturo@inifap.gob.mx or arias.alfonso@inifap.gob.mx. Registered in the EBSCO Host database. The Latin American and the Caribbean Spain and Portugal Scientific Journals Network (RedALyC) (www.redalyc.org). The Iberoamerican Network of free access Veterinary Scientific Journals (www.veterinaria.org/ revistas/ revivec). Thomson Reuter´s “Journal Citation Report” Science Edition (http://thomsonreuters.com/). Elsevier´s SCOPUS and EMBASE (www.elsevier.com) and the Essential Electronic Agricultural Library (www.teeal.org).

All contributions will be carefully refereed for academic relevance and quality. Submission of an article is understood to imply that the research described is unique and unpublished. Rev. Mex. Cien. Pecu. is published quarterly in original lenguage Spanish or English. Total fee charges are US $ 325.00 per article in both printed languages.

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. 11 No. 2

ABRIL-JUNIO-2020

CONTENIDO ARTÍCULOS

Pág. Preliminary study of ivermectin residues in bovine livers in the Bogota Savanna Estudio preliminar de residuos de ivermectina en hígado de bovinos en la Sabana de Bogotá Carmen Teresa Celis-Giraldo, Diego Ordóñez, Leonardo Roa, Sergio Andrei Cuervo-Escobar, Dajane Garzón-Rodríguez, Milena Alarcón-Caballero, Luisa Fernanda Merchán……………………………………311

Eficacia de la ivermectina para el control de nematodos gastrointestinales en burros (Equus asinus) en el altiplano mexicano Ivermectin effectiveness for gastrointestinal nematode control in donkeys (Equus asinus) in the Mexican High Plateau Guadalupe Galicia-Velázquez, Arturo Villarreal-Nieto, Cristina Guerrero-Molina, Cintli Martínez-Ortizde-Montellano……………………………………………………………………………………………………….…..…326

Artemisia cina 30 CH homeopathic treatment against Haemonchus contortus Artemisia cina 30 CH como tratamiento homeopático contra el Haemonchus contortus Rosa Isabel Higuera-Piedrahita, María Eugenia López-Arellano, Raquel López-Arellano, César CuencaVerde, Jorge Alfredo Cuéllar-Ordac .................................................................................................. 342

Inclusión de harina de Tithonia diversifolia en raciones para gallinas ponedoras de primer ciclo y su efecto sobre la pigmentación de yema de huevo Tithonia diversifolia meal in diets for first-cycle laying hens and its effect on egg yolk color

María Elena Carranco-Jáuregui, Vilma Barrita-Ramírez, Benjamín Fuente-Martínez, Ernesto ÁvilaGonzález, Leonor Sanginés-García.................................................................................................... 355

Efecto de un complejo multienzimático y un probiótico en gallinas de postura alimentadas con dietas sorgo-soya-canola Effect of a multienzyme complex and a probiotic in laying hens fed sorghum-soybeanrapeseed diets Pedro Juárez Morales, Arturo Cortes Cuevas, José Arce Menocal, Juan Carlos Del Río García, Gabriela Gómez Verduzco, Ernesto Avila González ......................................................................................... 369

Tendencias genéticas y fenotípicas para pico productivo, rendimiento lechero y persistencia de lactación en la raza Murciano-Granadina Phenotypic and genetic trends for peak yield, milk yield, and lactation persistency in the Murciano-Granadina breed Judith Carmen Miranda Alejo, José Manuel León Jurado, Camillo Pieramati, Mayra Mercedes Gómez Carpio, Jesús Valdés Hernández, Cecilio José Barba Capote ............................................................. 380

III

Calidad seminal de ovinos de pelo suplementados con Moringa oleifera (Moringaceae) y Trichanthera gigantea (Acanthaceae) Semen quality of hair sheep supplemented with Moringa oleifera (Moringaceae) and Trichanthera gigantea (Acanthaceae) Marco A. Ramírez-Bautista, Julio P. Ramón-Ugalde, Edgar Aguilar-Urquizo, William Cetzal-Ix, Roberto Sanginés-García, Álvaro E. Domínguez-Rebolledo, Ángel T. Piñeiro-Vázquez……………………….……393

Use of a glycogenic precursor during the prepartum period and its effects upon metabolic indicators and reproductive parameters in dairy cows Uso de un precursor glucogénico en el preparto y su efecto sobre indicadores de energía y parámetros reproductivos en vacas lecheras Carlos Leyva Orasma, Jesus Jaime Benitez-Rivas, Juan Luis Morales Cruz, Cesar Alberto MezaHerrera, Oscar Ángel-García, Fernando Arellano-Rodríguez, Guadalupe Calderón-Leyva, Dalia Ivette Carrillo-Moreno, Francisco Veliz Deras……………………………………………………………………….…….408

Relationship of the compositional content and sanitary quality of Holstein cows’ milk of the high tropic of Nariño Relación entre la calidad composicional y sanitaria de la leche de bovinos Holstein del trópico alto de Nariño Henry Armando Jurado-Gámez, Carlo Eugenio Solarte-Portilla, Álvaro Javier Burgos-Arcos, Aldemar González-Rodríguez, Carol Rosero-Galindo ...................................................................................... 421

Caracterización de Aspergillus flavus y cuantificación de aflatoxinas en pienso y leche cruda de vacas en Aguascalientes, México Characterization of Aspergillus flavus and quantification of aflatoxins in feed and raw milk of cows in Aguascalientes, Mexico Erika Janet Rangel-Muñoz, Arturo Gerardo Valdivia-Flores, Onésimo Moreno-Rico, Sanjuana Hernández-Delgado, Carlos Cruz-Vázquez, María Carolina de-Luna-López, Teódulo Quezada-Tristán, Raúl Ortiz-Martínez, Netzahualcóyotl Máyek-Pérez ......................................................................... 435

Comparación de la castración quirúrgica al nacimiento versus inmunocastration sobre las características de la canal y carne en machos Holstein Comparison of surgical castration at birth versus immunocastration on carcass and meat traits in growing Holstein males Jorge A. Cervantes-Cazares, Cristina Pérez-Linares, Fernando Figueroa-Saavedra, Alma R. TamayoSosa, Alberto Barreras-Serrano, Francisco G. Ríos-Rincón, Eduardo Sánchez-López, Issa C. GarcíaReynoso, Pedro Mendoza Peraza, Angelina León Villanueva, Luis A. García-Vega ........................... 455

Ascosferosis en abejas melíferas y su relación con factores ambientales en Jalisco, México Ascospherosis in honey bees and its relationship to environmental factors in Jalisco, Mexico José María Tapia-González, Gustavo Alcazar-Oceguera, José Octavio Macías-Macías, Francisca Contreras-Escareño, José Carlos Tapia-Rivera, Tatiana Petukhova, Ernesto Guzmán-Novoa .......... 468

Desarrollo y validación de un patrón visual para la evaluación del color de la carne de bovino en México Development and validation of a visual pattern for evaluating beef meat color in Mexico Sara Salinas Labra, María Salud Rubio Lozano, Diego Braña Varela, Rubén Danilo Méndez Medina, Enrique Jesús Delgado Suárez .......................................................................................................... 479

REVISIONES DE LITERATURA

Bolos intrarruminales con liberación controlada de minerales traza. Revisión Trace mineral controlled-release intraruminal boluses. Review Misael León-Cruz, Efrén Ramírez-Bribiesca, Raquel López-Arellano, Leonor Miranda-Jiménez, Gabriela Rodríguez-Patiño, Víctor M. Díaz-Sánchez, Alma L. Revilla-Vázquez................................................ 498

Implicaciones, tendencias y perspectivas del transporte de larga distancia en el ganado bovino. Revisión Implications, trends, and prospects for long-distance transport in cattle. Review Marcela Valadez Noriega, Genaro Cvabodni Miranda de la Lama ..................................................... 517

NOTAS DE INVESTIGACIÓN

Growth, viability, and post-acidification of Lactobacillus plantarum in bovine transition milk Crecimiento, viabilidad y post-acidificación de Lactobacillus plantarum en la leche de transición bovina Hugo Calixto Fonseca, Eduardo Robson Duarte, Lívia Caroliny Almeida Santos Souza, Emanuelly Gomes Alves Mariano, Ana Clarissa dos Santos Pires, Tatiana Santos Lima, Maximiliano Soares Pinto .......................................................................................................................................................... 539

Caracterización de la leche y queso artesanal de la región de Ojos Negros, Baja California, México Characterization of the milk and artisanal cheese of the region of Ojos Negros, Baja California, Mexico Laura E. Silva-Paz, Gerardo E. Medina-Basulto, Gilberto López-Valencia, Martin F. Montaño-Gómez, Rafael Villa-Angulo, José C. Herrera Ramírez, Ana L. González-Silva, Francisco Monge-Navarro, Sergio A. Cueto-González, Gerardo Felipe-García ........................................................................................ 553

Factores asociados al decomiso de hígados positivos a Fasciola sp en una zona endémica del sureste de México Factors associated with the seizure of livers positive to Fasciola sp in an endemic area of southeastern Mexico Nadia Florencia Ojeda-Robertos, Roberto González-Garduño, Santiago Cornelio-Cruz, Jorge Alonso Peralta-Torres, Carlos Luna-Palomera, Carlos Machain-Williams, Heliot Zarza, Oswaldo Margarito Torres-Chablé, Enrique Reyes-Novelo, Carlos Baak-Baak, Alfonso Chay-Canul................................ 565

Análisis genético del desarrollo en peso vivo y tasa de gestación en primer parto en bovinos Brahman de Venezuela Genetic analysis of live weight and pregnancy rate at first calving in Brahman cattle from Venezuela Alejandro-Palacios-Espinosa, Omar-Verde, Narciso-Ysac-Ávila-Serrano, Alberto-Menéndez-Buxadera .......................................................................................................................................................... 576

Pedigree analysis of Santa Inês sheep and inbreeding effects on performance traits Análisis de pedigrí de las ovejas Santa Inês y los efectos de la endogamia en los rasgos de rendimiento Ana Carla Borges Barbosa, Gabrieli de Souza Romano, Jonatan Mikhail Del Solar Velarde, José Bento Sterman Ferraz, Victor Breno Pedrosa, Luís Fernando Batista Pinto ................................................ 590

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.

bibliográficas una extensión máxima de 30 cuartillas y 5 cuadros. 6.

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.

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

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

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

VII

de investigaciones. El texto del Artículo científico se divide en secciones que llevan estos encabezamientos:

Procure abstenerse de utilizar los resúmenes como referencias; las “observaciones inéditas” y las “comunicaciones personales” no deben usarse como referencias, aunque pueden insertarse en el texto (entre paréntesis).

Introducción Materiales y Métodos Resultados Discusión Conclusiones e implicaciones Literatura citada

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.

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.

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.

VIII

Revistas

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.

XI)

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.

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.”). 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.

Sólo número sin indicar volumen.

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.

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 ofthe use of artificial insemination in developing countries. World Anim Rev 1993;(74-75):26-35.

Tesis. 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.

No se indica el autor. 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.

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

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

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.

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.

DL50 dosis letal 50% g gramo (s) 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

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. 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

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

caloría (s) centímetro (s) grado centígrado (s)

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.

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 â&#x20AC;&#x153;Guide for submit articles in the Web site of the Revista Mexicana de Ciencias Pecuariasâ&#x20AC;?. 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.

contain the following sections, and each one should begin on a separate page.

Title page Abstract Text Acknowledgments and conflict of interest Literature cited 7.

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.

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.

Manuscripts of all three type of articles published in Revista Mexicana de Ciencias Pecuarias should

Introduction Materials and Methods Results Discussion Conclusions and implications Literature cited 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.

b) Technical Notes. They should be brief and be

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 names(s), the number of the edition, the country, the printing house and the year.

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 i n the opinion of the editors. The text will contain the same information presented in the sections of the research article but without section titles.

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.

Organization, as author

XII

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.

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.

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.

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.

Organization as author

XIII

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.

m Âľl Âľm mg ml mm min ng

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 Â°C DL50 g ha h i.m. i.v. J kg km L log Mcal MJ

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) mega joule (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.

XIV

https://doi.org/10.22319/rmcp.v11i2.4992 Article

Preliminary study of ivermectin residues in bovine livers in the Bogota Savanna

Carmen Teresa Celis-Giraldo a* Diego Ordóñez a Leonardo Roa a Sergio Andrei Cuervo-Escobar b Dajane Garzón-Rodríguez a, Milena Alarcón-Caballero a Luisa Fernanda Merchán a

Universidad de Ciencias Aplicadas y Ambientales U.D.C.A. Facultad de Ciencias Agropecuarias. Bogotá, Colombia. b Universidad de Ciencias Aplicadas y Ambientales U.D.C.A. Facultad de Ciencias. Bogotá, Colombia.

*Corresponding author: ccelis@udca.edu.co

Abstract: The present study tended to determine the ivermectin residues in cattle livers using the competitive ELISA technique and correlate gender and age variables with residual presence. Also, it was described the histopathological findings in analyzed samples. A total of 90 livers of randomly selected cattle were sampled in a type II slaughterhouse located in the Bogota Savanna. The samples were analyzed for ivermectin residues using the competitive ELISA technique and a histopathological evaluation was performed using the H&E technique. Only the 22 % (20/90) of the analyzed samples presented ivermectin residues. Of the positive individuals, the majority came from Cogua 35 % (7/20), Zipaquira 30 % (6/20) and Sopo 20 % (4/20). The breeds with residues presence corresponded to Half-Blood 35 % (7/20), Zebu 25 % (5/20), Normande 20 % (4/20) and Jersey x Holstein 15 % (3/20). Eighty five, 85 % (17/20) of the individuals were older than 1.5 yr. In regard to the gender variable, the majority of animals were males 65 %

311

Rev Mex Cienc Pecu 2020;11(2):311-325

(13/20). Of the evaluated animals 3 % (3/90) exceeded the maximum residue limit (> 100 ppb). No association was found between the presence of residues and the gender and age variables (P>0.05). The majority of histopathological changes were mild or moderate, with alterations in architecture and inflammatory changes standing out. It was found association between the presence of residue and variables microcirculatory alteration, inflammatory alteration and changes similar to the cell death (P<0.05). As a conclusion, the competitive ELISA test used in this study served as a screening method for the detection ivermectin residues in the analyzed samples. Key words: Antiparasitic drugs, Ivermectin, ELISA, Liver, Macrocyclic lactones, Bovine.

Received: 17/07/2018 Accepted: 03/04/02019

Introduction Parasitic diseases are one of the most important causes of economic losses for the livestock industry in tropical and subtropical production systems. These diseases have a huge prevalence impact related to epidemiological chain factors such as the presence of vectors and environmental changes(1). Ivermectin is a popular macrocyclic lactone since its introduction to the market(2,3). This drug is a mixture of two homologs in a ratio of 80 % 22,23-dihydroavermectin B1a (H2B1A) and no more than 20 % of 22,23dihydroavermectin B1b (H2B1b)(4). The maximum residue limits (MRL) in the European community have been determined to utilize the B1A portion as a marker(5). The outstanding use in veterinary medicine is derived from the spectrum activity for the control of nematodes and arthropods, generating a remarked interest for research development. Additionally, the macrocyclic lactones group has also been used in agriculture for pest control and in human medicine mainly for onchocerciasis treatment(6,7). From a pharmacokinetic point of view, studies have shown that the main organ for Ivermectin residues detection is the liver in almost all the evaluated species(8,9,10). Therefore, due to the huge usage in the field, consumption of viscera and possible toxicity, this organ was chosen as the matrix to carry out this study. For detecting residues, several methodologies have been developed, liquid chromatography (HPLC) and mass spectrometry are the most recommended due to their high sensitivity and selectivity, however, they also present certain disadvantages such as complex extraction process and expensive equipment is needed(11). Another semi-quantitative-methodologies such as 312

Rev Mex Cienc Pecu 2020;11(2):311-325

Competitive ELISA are used as screening method in several matrices. Some advantages are related to a low cost, fastness with a relative good sensitivity and specificity in comparison to HPLC(4,12). The purpose of this investigation corresponded to determine the presence of ivermectin residues in bovine livers from the Bogota, D.C. savanna using the competitive ELISA technique, correlating the presence of residues presence with gender and age variables. This study also describes the histopathological changes of the evaluated samples.

Material and methods Study location

Samples were obtained from ZipaquiraÂ´s slaughterhouse, which has an average of 200 animals slaughtered daily. This slaughterhouse is classified as type II, according to INVIMA (Decree 1036 of 1991) and the Ministry of Health and is also certified by ISO 9001: 2008 and NTCGP1000: 2009 normativity.

Sample size

Considering an error of 10 % and a confidence level of 99 %, the number of analyzed livers corresponded to 89. The animals were chosen randomly in the suspension area and subsequently identified. Once they were in the evisceration area, approximately 100 g of liver were taken in sterile bags for the ELISA test. Each sample was marked, duly labeled, refrigerated, transported and later stored at -20 Â°C until their analysis. At the same time, data corresponding to origin, breed, gender, and age of the evaluated animals were obtained.

In the standardization phase the liver of a bovine was included as a negative control. This animal certainly did not receive any previous treatment with ivermectin. Prior authorization from the Bioethics Committee of the U.D.C.A, an 8-d-old calf was euthanized using EuthanexÂŽ (60 mg/kg, I.V) and liver samples were taken for analysis. The pregnant cow belonged to the university and did not receive antiparasitic treatment with macrocyclic lactones during pregnancy.

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Rev Mex Cienc Pecu 2020;11(2):311-325

Procedure

Competitive ELISA test was used for residue detection and quantification. This test uses rabbit polyclonal antibodies against ivermectin and the analysis process for each sample was carried out considering the kit provider information(13), briefly the amount of active ingredient in the samples was expressed as equivalents of ivermectin (ng/ml). In the case of the liver, the values that were obtained from the calibration curve were multiplied by a factor of 5 in order to express the concentration in ng/g. The equivalents of ivermectin corresponded to the maximum percentage of absorbance of each extract read from the calibration curve of the seven standards (Table 1). The determination coefficients obtained in each calibration curve were considered to calculate the specific residues concentration. Table 1: Concentration of the ivermectin in the standard solutions Standard ng/ml

0 --

1 1.25

2 2.5

3 5

4 10

5 25

6 50

Each sample was thawed and homogenized with a food processor. The obtained material was subjected to an extraction process and therefore residue quantification based on the kit supplier parameters. Samples were considered as residually positives when they exceeded the kitâ&#x20AC;&#x2122;s reported limit of detection (LOD) of 8 ppb. The maximum residue limit (MRL) for the liver corresponded to 100 ppb, thus considering the reported values in Resolution 1382 of 2013 of the Ministry of Health and Social Protection. The samples were analyzed in duplicate at the U.D.C.A microbiology laboratory.

Histopathological analysis

In addition to the sample taken for ELISA analysis, 5 g of liver were obtained for histopathological evaluation, conserving them in 10% formaldehyde. Samples were processed by hematoxylin and eosin technique in the U.D.C.A pathology laboratory. Three zones of the hepatic lobe were evaluated as follows, zone 1, located at a further distance from the centrilobular vein, specifically in the periphery of the lobule. Zone 2 corresponded to the middle of the periphery and centrilobular vein, and the zone 3, it was considered as closest to the centrilobular vein. In aforementioned areas, the following variables were evaluated: Alterations in architecture: vacuolation, lobular inversion, canalicular hyperplasia and parenchymal fibrosis. Microcirculatory alterations: in this case, centrilobular congestion, generalized congestion, focal hemorrhage, and edema were considered. 314

Rev Mex Cienc Pecu 2020;11(2):311-325

Inflammatory alterations: presence of polymorphonuclear, mononuclear and mixed cells. An evaluation of the lobular areas was done either. Changes similar to cell death: apoptosis and necrosis were considered. For each of the variables, the degree of the lesion was classified as follows: (0) apparently normal, (1) mild, (2) moderate and (3) severe(14).

Statistical analysis

This study was observational with simple random sampling. The data was recorded in Microsoft Office Excel 2016 spreadsheet and analyzed with STATA MP14 statistical package. A descriptive analysis of the origin, breed, gender and age variables was carried out. To analyze the effect of gender, age and histopathological variables with ivermectin concentration, a Chi2 (P<0.05) association test was performed.

Results The coefficients of determination obtained to calculate residue concentration were 97 % on average. Most individuals presented levels lower than 8 ppb (78 %), 19 % between 8 and 100 ppb and only 3 % presented residues over 100 ppb, exceeding the MRL (Figure 1). Figure 1: Distribution of ivermectin residues in the evaluated population

The population without residues (LOD <8 ppb) was mostly originated from Zipaquira 44 % (28/70), followed by Cogua 26 % (18/70), and to Sopo and Chiquinquira respectively corresponded the remaining 10 % (7/70) of the evaluated animals. For the 315

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breed variable, the highest proportion of animals were Normande 34 % (24/70), followed by Holstein 28 % (20/70) and Half-Blood 23 % (16/70). In the case of the age variable, only 13 % (9/70) were animals under 1.5 yr old and 87 % were older than 1.5 yr old. For the gender variable, it was observed that 59 % (41/70) of the sampled animals were females and 41 % (21/70) were males. The individuals with residues prescense were mostly originated from Cogua 35 % (7/20), Zipaquira 30 % (6/20), Sopo 20 % (4/20). For La Vega, Bogota and Guasca, only one individual was positive respectively. The breeds in which residues were found corresponded to Half-Blood 35 % (7/20), Zebu 25 % (5/20), Normande 20 % (4/20) and Jersey x Holstein 15 % (3/20); for Holstein there was only one individual. 85 % (17/20) of the animals were older than 1.5 yr old and the remaining 15 % (3/20) were less than 1.5 yr old. In terms of the gender variable, they were molstly males 65 % (13/20) and 35 % (7/20) were females. Only 3 % (3/90) of samples exceeded the MRL. All of the positive animals were male, two samples came from Sopo and one from Cogua and two of them were older than 1.5 yr old. These animals were Half-Blooded, Holstein and Jersey x Holstein breeds (Table 2). When performing the Chi2 association test between variables age (P=0.84), gender (P=0.06) and ivermectin concentration, no association was found. Table 2: Descriptive statistics of the population with residues presence Samples % Concentration (â&#x2030;Ľ 8 ppb) with residue Samples Samples Variable \ Total with >MRL* population residues Range Average SE Gender: Males 13\42 31 8.40-154.72 58.50 14.71 3 Females 7\48 15 8.32-26.92 16.30 2.38 0 Origin: Cogua 7\25 28 8.86-154.72 37.86 19.58 1 Sopo 4\11 36 8.32-140.25 86.05 31.92 2 Zipaquira 6\34 18 8.40-87.65 32.75 10.50 0 Age: < 1.5 yr old 3\12 25 8.86-154.72 60.98 46.96 1 â&#x2030;Ľ 1.5 yr old 17\78 22 8.32-140.25 40.68 10.11 2 Breed ** Half-Blood 7\23 30 8.32-136.40 33.74 17.45 1 Zebu 5\12 42 18.28-87.65 34.67 13.32 0 Normande 6\28 14 8.40-59.22 31.43 8.87 0 Jersey x Holstein 3\4 75 15.12-140.25 61.53 39.56 1 *MRL= Maximum Residue Limit. **Only one individual of Holstein breed exceeded the MRL with 154.72 ppb.

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For the histopathological analysis, 86 samples were evaluated, and as it was mentioned before, 20 livers presented ivermectin residues and 66 livers where considered as negative. According to lesion severity and the different variables analyzed, the data was recorded in Table 3. It is worth mentioning that no lobular inversion and lesions compatible with necrosis were observed in the analyzed samples. The majority of changes observed were mild in both cases. For the samples without residues, the main changes observed corresponded to the inflammatory level, with a mild presence of monocytes stood out (28/66), moderate (6/66) and severe (3/66). Mild changes in vacuolization (41/66), hyperplasia (15/66), moderate hyperplasia (22/66), mild fibrosis (27/66) and moderate fibrosis (21/66) were also described. We found association between variables microcirculatory alteration, inflammatory alteration and similar to the cell death (P<0.05) (Table 3).

Table 3: Histopathological findings in the studied population

Inflamma tory alteration

Microcirculatory alteration

Architecture alteration

Severity Degree Normal Vacuolization Mild Moderate Normal Hyperplasia Mild Moderate Normal Mild Fibrosis Moderate Severe Normal Lobulillar congestion Mild Moderate Normal Mild Generalized congestion Moderate Severe Normal Mild Focal hemorrhage Moderate Normal Edema Mild Moderate Normal Mild Polymorphonuclear Moderate 317

LOD < 8ppb

LOD â&#x2030;Ľ 8ppb

21 41 4 29 15 22 18 27 21 0 49 17 0 64 1 0 1 66 0 0 66 0 0 66 0 0

10 7 3 11 8 1 6 11 2 1 2 11 7 7 9 4 0 16 1 3 15 6 1 5 13 2

P value 0.318

0.447

0.708

0.000*

0.006*

0.000*

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Monocytes

Mixed

Similar to the cell death

Zone 3

Apoptosis

Normal Mild Moderate Severe Normal Mild

29 28 6 3 66 0

3 15 2 0 7 12

Moderate

Normal Mild Normal Mild Moderate

33 33 66 0 0

17 3 12 7 1

0.027*

0.000* 0.003*

0.000*

P<0.05.

Animals with residues evidence (20) presented alterations on architectural variables as follows: 10 samples had vacuolar changes (7 mild and 3 moderate); 9 had canalicular hyperplasia (8 mild, 1 moderate) and 14 had portal fibrosis (11 mild, 2 moderate and 1 severe) (Figure 2). Eighteen (18) samples that contained ivermectin residues presented centrilobular congestion (11 mild, 7 moderate); 13 samples presented generalized congestion and sinusoidal dilatation (10 mild, 4 moderate) and 4 hemorrhagic foci (1 mild, 3 moderate) (Figure 3). Figure 2: Bovine liver without ivermectin residues

A. Normal architecture in the hepatocytes (400X) and B. the portal area (100X). Architectural changes in the hepatic parenchyma of cattle with residues. C. Vacuolar changes, note the presence of intracytoplasmic vacuoles (arrow tip) (400x). D. Mild fibrosis of the portal area (arrow) (100X).

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Figure 3: Microcirculatory changes in the hepatic parenchyma of cattle positive for ivermectin residues. Mild congestion and sinusoidal dilatation were evident

In relation to the inflammatory infiltrate, 13 of the 20 animals presented mixed infiltration, predominantly by mononuclear cells (15 mild, 1 moderate) in centrilobular distribution. Comparatively, in both groups, a wide range of changes were found associated to microcirculatory, architectural, inflammatory and cell death (apoptosis) processes (Figure 4). In relation to the last alteration, only eight of the analyzed samples showed changes associated with similar to cell death, characterized by a multifocal distribution in the positive tissues with ivermectin residues (Table 3). Figure 4: Changes compatible with apoptosis, bovine liver with ivermectin residues obtained from a slaughterhouse. Cells with related changes with cellular death (arrows)

Discussion Ivermectin is an active compound that has been used for almost 20 yr in numerous countries and different animal production systems. As a result of its usage, control testing 319

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on food products derived from livestock is carried out in order to harmonize the commercialization processes. Several studies have been done reporting different methodologies for this purpose, such as the competitive ELISA technique. This method was developed before(4), using rabbit produced polyclonal antiserum and the limit of detection (LOD) was 1.6 Îźg/kg and the antibodies presented a cross-reaction with doramectin but not with moxidectin. In that study, negative liver (1.6 ng/g), 100 ng/g ivermectin fortified liver and 0.5 mg/kg topically treated liver samples from animals slaughtered on day 7, 14, 21 and 28 were analyzed. In this case, ivermectin levels on d 7 post-treatment were 52.7 ng/g decreasing to 4.1 ng/g by d 28. The data was confirmed by another complementary technique such as high-performance liquid chromatography (HPLC). The range of both tests was 1 to 58 ng/g with a close correlation (r = 0.99). In a study carried out by other authors(12) where an ELISA was developed for the detection of multiple avermectins (abamectin, eprinomectin, and ivermectin) the modified avermectin 4C-O-succinoylavermectin was conjugated with bovine albumin and ovoalbumin as immunogens for polyclonal antibodies the preparation. The LOD for this test corresponded to 1.06 ng/ml for all three avermectins. The percentage of recovery ranged from 53.8 % to 80.6 % and it was similar to previous reports using HPLC, converting this technique as a rapid method for screening avermectins in bovine liver. In the present study the LOD of 8 ppb (reported by the manufacturer), 70 samples were under, 17 between 8 and 100 ppb and only 3 exceeded 100 ppb. It is important to bear in mind that in this case no additional confirmatory tests were performed, however considering the obtained data it would be relevant to expand this kind of approach in other production systems with a greater usage of this active compound, situation that has been reported in other country areas. Turning to the MRL, only 3 % of the samples exceeded the maximum value reported by Colombian authorities (>100 ppb), this could be considered as a low level. In Colombia, a study determining residuality of organophosphorus, carbamates and ivermectin insecticides in raw milk from farms in tropical areas. Of 609 samples analyzed, 37.44 % contained organophosphate levels exceeding the MRL, with Magdalena Region containing almost all the positive samples. Although, 180 milk samples were analyzed for ivermectin detection and quantification but any sample exceeded the MRL(15). Data obtained in this study were similar to the observed in other countries as Mexico, where evaluated the presence of ivermectin residues due to the increased use of this molecule on the farms. In this case, 234 liver samples were taken during the course of a month and analyzed by HPLC. Only one sample was not within parameters (149 Îźg/kg), situation that was similar to another previously reported by a control scheme for this area(16). The aforementioned data differ from another reported in Brazil. Due to residuality problems during 2010 several detection techniques were developed in muscle matrices, allowing the establishment of the MRL and pertinent measures were taken to exercise drug control within the production systems(17). Some of the strategies used corresponded to education campaigns for producers in order to prevent parasitic disease by management 320

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and medication use, considering several factors that can lead to exceed the residues maximum permitted levels, such as product formulation, active ingredient physicochemical properties, overdose, administration method, product use in nonrecommended species, heterogeneous animal lots, and finally non-compliance with withdrawal times(2,11). In terms of the epidemiological characteristics, no association was observed between gender and age variables with the residues presence. Despite that fact, there was a greater presence of residues in males than females. Considering this, reported pharmacokinetic data show that there are differences related to gender and plasma disposition of the active ingredient. These characteristics have been studied in various animal species and specifically for cattle, found differences related to gender in terms of plasma concentration when they evaluated ivermectin and doramectin, bioavailability was 10% higher in heifers than in steers for both molecules(18). This difference was associated with the amount of adipose tissue in males and females, considering the characteristics of the active ingredient such as the high liposolubility of the endectocide agent that promotes its storage in this tissue and favors its persistence(9). Despite that residues were observed in females, the percentage found was very low (15%), possibly the evaluated animals were in nonlactating (dry) period, driving to their discard and slaughter, but any case exceeded the limits and most relevant data was found in males. Even so, it is important to bear in mind that the only avermectin approved for dairy farming is eprinomectin and that other factors, such as hormonal influence can affect drug metabolism in terms of its transformation and elimination(15,19). The histopathological evaluation of this study allowed a wide range of hepatic parenchymal alteration findings, even in animals with and without residues. When the microcirculatory changes were evaluated, some animals with residues presented a greater degree of the congestive changes and hemorrhagic foci severity, suggesting a greater susceptibility to develop disorders associated with alterations of the vascular network of the liver parenchyma; however, the number of animals that developed these kind of disorders does not turn out to be significant. Numerous chemical substances suffer part of their biotransformation in the hepatic tissue, this also means that a major part of these compounds could generate cellular alterations of the parenchyma, leading to possible disorders associated to liver function alterations such as protein synthesis, carbohydrate transformation and lipid metabolism. Included amongst these are pharmaceuticals, bacterial toxins, and toxic plants. In this sense, the unique or concomitant effect of this type of compounds on the analyzed samples cannot be ruled out. A study conducted in rats for the evaluation of the effect of massive and long-lasting treatments with ivermectin among other molecules against various parasites, showed some clinical alterations compatible with hepatotoxicity and anatomopathological 321

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changes revealed architecture distortion, hepatocellular necrosis, and Kupffer cell hyperplasia(20). In hyperacute cases of intoxication in goats, sudden death is reported without systemic pathological alterations(21). In humans, a case report showed the hepatotoxic potential of ivermectin after administration in a patient with filarial parasitism, finding intralobular inflammatory infiltrate and accumulation of ceroid pigment, perivenular necrosis, necrosis and apoptosis(22). According to the international guidelines, the acceptable daily intake established for humans is 10 μg/kg (600 μg/person/d)(5), levels that were not observed in the analyzed samples. It is worth noting that comparatively with these reports, the present study also evidenced changes similar to apoptosis only in animals with residues presence, however further studies like imnunohistochemistry should be conducted in order to clarify the association between residues and hepatic lesion development. The ivermectin poisoning has been associated with overdosing or with the presence of a P-glycoprotein mutation that promotes the development of neurological symptoms in several animal species. The human intoxication has been associated with mild to moderate effects of a Mazzotti reaction and encephalopathy linked to microfilaricidal therapy or macrocyclic lactones exposure(23).

Conclusions and implications In conclusion, the competitive ELISA test used in this study served as a screening method to analyze ivermectin residues in bovine livers. Only 3 % of the samples exceeded the MRL and no correlation was found between the presence of residues with gender and age variables (P>0.05). The majority of the histopathological changes were mild or moderate, with alterations in architecture and inflammatory changes standing out. Exist statistical association between the presence of residue of ivermectin and variables microcirculatory alteration, inflammatory alteration and changes similar to the cell death (P<0.05).

Acknowledgments The authors thank specially to Zipaquira slaughterhouse and Universidad de Ciencias Aplicadas y Ambientales (U.D.C.A) for all the support in the execution of this project, as well as to Basic Farm® company for the all the provided guidance. Also to Professor Juan de Jesús Vargas for his support in the statistical analysis.

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Conflict of interest The authors claim no conflict of interest in the presentation of this document.

Literature cited: 1. Singh NK, Singh H, Jyoti, Haque M, Rath SS. Prevalence of parasitic infections in cattle of Ludhiana distric, Punjab. J Parasit Dis 2012;36(2):256-259. 2. Danaher M, Radeck W, Kolar L, Keegan J, Cerkvenik-Flajs V. Recent developments in the analysis of avermectin and milbemycin residues in food safety and the environment. Curr Pharm Biotechnol 2012;13:936-951. 3. Ộmura S. Ivermectin:25 years and still going strong. Inter J Anti Agen 2008;31:91-98. 4. Crooks SRH, Baxter AG, Traynor IM, Elliot C, McCaughey WJ. Detection of ivermectin residues in bovine liver using an enzyme immunoassay. Analyst 1998;123:355-358. 5. EMEA. Ivermectin (All mammalian food producing species). Committee for Medicinal Products for Veterinary Use. European Medicines Agency. http://www.ema.europa.eu/docs/en_GB/document_library/Maximum_Residue_Li mits_-_Report/2014/05/WC500167329.pdf. Accessed May 15, 2018. 6. Wolstenholme AJ, Rogers AT. Glutamate-gated chloride channels and the mode of action of the avermectin/milbembycin anthelmintics. J Parasitol 2005;131:S85-S95. 7. Ộmura S, Crump A. Ivermectin: panacea for resource-poor communities? CellPress. Trends Parasitol 2014;30(9):445-455. 8. Escribano M, San Andrés MI, de Lucas JJ, Gonzáles-Canga A. Ivermectin residue depletion in Food producing species and its presence in animal foodstuffs with a view to human safety. Curr Pharm Biotecnhno l2012;13:987-998. 9. McKellar QA, Gokbulut C. Pharmacokinetic features of the antiparasitic macrocyclic lactones. Curr Pharm Biotechno 2012;13:888-911. 10. Lee Chiu S-H, Buhs R, Sestokas E, Taub R, Jacob T. Determination of ivermectin residue in animal tissues by High-Performance Liquid Chromatography-Reverse isotope dilution assay. J Agr Food Chem 1985;33:99-102.

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11. Danaher M, Howells LC, Crooks SRH, Cerkvenik-Flajs V, O´Keeffe M. Review of methodology for the determination of macrocyclic lactone residues in biological matrices. J Chromato B 2006;844:175-203. 12. Shi W, He J, Jiang H, Hou X, Yang J, Shen J. Determination of multiresidue of avermectins in bovine liver by an indirect competitive ELISA. J Agr Food Chem 2006;54:6143-6146. 13. EuroProxima. Ivermectin ELISA, 5141IVER. http://europroxima.com/products/contaminants-andresidues/anthelmintics/ivermectin-elisa/; www.europroxima.com. Accessed May 15, 2016. 14. Lopez Panqueva RP. Useful algorithms for histopathological diagnosis of liver disease based on patterns of liver damage. Rev Colomb Gastroenterol 2016;31:443457. 15. González Reina A, Palomares Velosa JE, Parra JL, Silva Sakzuk J, Abuabara Y, Mojica JE et al. Determination of organophosphorus, carbamate insecticides and ivermectin residues in raw milk from cattle farms in the Colombian low tropics. Abstract 10. Rev Colomb Cienc Pecu 2011;24:552-557. 16. Solis Rivera C, Wilcock A, Arellano Chavez S, Morales Loredo A, Mcewen S. Prevalence of ivermectin residues in cattle slaughtered in federally inspected abattoirs in Nuevo Leon, Mexico. Food Protect Trends 2011;31(4):212-215. 17. Rübensam G, Barreto F, Barcellos Hoff R, Mara Pizzolato T. Determination of avermectin and milbemycin residues in bovine muscle by liquid chromatographytandem mass spectrometry and fluorescence detection using solvent extraction and low temperature cleanup. Food Control 2013;29:55-60. 18. Toutain PL, Upson DW, Terhune TN, McKensie ME. Comparative pharmacokinetics of doramectin and ivermectin in cattle. Vet Parasitol 1997;72:3-8. 19. Mugford CA, Kedderis GL. Sex-dependent metabolism of xenobiotics. Drug Meta Rev 1998;30(3):441-498. 20. Idowu ET, Alimba CG, Olowu EA, Otubanjo AO. Artenether-lumefantrine treatment combined with albendazole and ivermectin induced genotoxicity and hepatotoxicity through oxidative stress in Wistar rats. EJBAS 2015;2:110-119

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21. Mohsen G, Pourjafar M, Badiei K, Habibi H. Ivermectin toxicity in a goat herd. Toxico Lett 2011;211S:43-216. 22. Veit O, Beck B, Steuerwald M, Hatz C. First case of ivermectin-induced severe hepatitis. Case report. Trans R Soc Trop Med Hyg 2006;100:795-797. 23. Yang CC. Acute human toxicity of macrocyclic lactones. Curr Pharm Biotechnol 2012;13:999-1003.

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https://doi.org/10.22319/rmcp.v11i2.5100 Article

Ivermectin effectiveness for gastrointestinal nematode control in donkeys (Equus asinus) in the Mexican High Plateau

Guadalupe Galicia-Velázquez a Arturo Villarreal-Nieto a Cristina Guerrero-Molina a Cintli Martínez-Ortiz-de-Montellano a*

Universidad Nacional Autónoma de México, Facultad de Medicina Veterinaria y Zootecnia, Circuito Exterior, Ciudad Universitaria, Av. Universidad 3000. 04510, Ciudad de México, México.

*Corresponding author: cintli@unam.mx

Abstract: The donkey has been used as a working animal for centuries, and 96 % of the world population of this species is in developing countries. Gastrointestinal nematodes with anthelmintic resistance (AHR) are the most serious parasitic problem in equidae. This study analyzes the phenomenon of AHR to ivermectin (IVM) in donkeys, and economic thresholds, with an evaluation of the practices by the owners through surveys, are considered. Based on 53 donkeys from the Mexican High Plateau, the experiment was divided into two stages: 1) economic thresholds were determined for 53 animals, and the experimental groups were formed. 2) the IVM efficacy test was performed, and two experimental groups (n= 30) were established: the treated group and the control group, without treatment. The economic threshold of eggs per gram of feces was 600, and the threshold of body condition (BC) of 91 % of the animals was acceptable (2.5 to 3.5). At a higher BC, the egg discharge obtained was lower (P<0.05). Of the 100 larvae identified, 63 % were cyathostomidae, and the rest were large strongyles. In this nematode population, IVM efficacy was 100 %. Eighty, 80% of the surveyed owners admit that they use as the only strategy the treatment provided by

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volunteer Veterinarians, which consists of 1% IVM. This antiparasitic is still a valuable resource and must be used properly in order to prevent AHR. This is the first step toward targeted selective deworming in equidae in Mexico. Key words: Donkeys, Equus asinus, Cyathostomidae, Nematodes, Ivermectin, Anthelmintic resistance.

Received: 05/10/2018 Accepted: 05/04/2019

Introduction The donkey (Equus asinus) has been used as a working animal for 5,000 yr(1) and more than 96 % of the world population of this species is found in developing countries. In Mexico, it is estimated that there are around 3.3 million donkeys(2). In the Mexican High Plateau, they are mainly used for agricultural activities(2). In some cases, these donkeys suffer from poor nutrition, as they are fed with agricultural debris supplemented with low-quality grains or commercial concentrates (1-2). Donkeys are also hosts to a large number of parasites whose life cycles are similar to that of the parasites present in horses; therefore, these can act as a significant reservoir for the infection of other equidae(3). The most important parasites in these animals are certain helminths such as Anoplocephala perfoliata or Parascaris equorum, but the ones with a greater impact due to the clinical implications of the larval migration and the hypobiosis that they exhibit(4,5) are those of the Strongylida family, where the gastrointestinal nematodes (GIN) of the subfamily Cyathostominae, also known as small strongyles, are located, and those of the subfamily Strongylidae (Strongylus spp.), known as large strongyllids(6,7). These gastrointestinal nematodoses in donkeys are perhaps one of the greatest challenges in clinical management, since donkeys with significantly high helminth loads may be apparently healthy and rarely exhibit clinical signs, unlike horses(8). It is known that, between horses and donkeys, there are great differences in behavior and physiology, and specifically in the mechanism through which they metabolize certain drugs and, in turn, respond differently to the pathologies that affect them(9). This is not reflected in the pharmaceutical industry, since donkeys have a limited economic impact compared to horses(9). Therefore, due to underdosing, there is the possibility of finding donkeys with chemical-resistant GIN, as has been demonstrated in other domestic species(10,11). In domestic animals worldwide, GIN and their anthelmintic resistance (AHR) represent health, economic and productive problems(12,13). The phenomenon of AHR is widely studied in Mexico and Latin America, especially in ruminants(12,14) and other equidae(5). Several studies carried out in some ecological zones in the high plateau and the central parts of the country measure the 327

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antiparasitic effect of ivermectin (IVM) in equines(15); however, their efficacy in donkeys has never been measured. In the Mexican High Plateau region, veterinary doctors in the field have provided, at least twice a year for more than 10 yr, free deworming to donkeys with 1% oral IVM (personal communication Prado-Ortiz, O.), since the development of the AHR of the parasite population is imminent. Due to these practices, it is relevant to carry out studies that reveal the current panorama of the AHR in the region. At present, in order to address AHR, it must first and foremost know relevant aspects of the epidemiology of the disease at the regional, local and particular levels (11,16,17). For this purpose, it is necessary to establish certain criteria that are characteristic of economic thresholds, such as the distribution of the elimination of eggs per gram of faeces (EPG), the body condition (BC) values, and the identification of the genus of nematodes existing in the ecological study area, as well as to detect potential practices that may indicate treatment failures and cause AHR(18,19). This study analyzes the phenomenon of AHR in donkeys and considers these thresholds based on the evaluation of owner practices. This contributes to the planning of different strategies to delay AHR and prolong the effectiveness of a drug, with a view to achieving Integrated Parasitic Control (IPC) in donkeys in the future.

Material and methods The study was carried out in four communities located in the Mexican High Plateau: Aljibes and San Pedro, in the municipality of Tecozautla, Hidalgo, and Santa Rosa Xajay and Vaquerias, in the municipality of San Juan del RĂo, QuerĂŠtaro.

Animals Donkeys from the above communities were selected because these receive continuous support from volunteer Veterinarian (VET), who provided information on the owners of the donkeys and data on their treatment protocols. The last anthelmintic treatment applied consisted of 1% IVM applied orally, six months prior to the experiment. It is worth mentioning that the donkeys of these communities have been cared for by these doctors for more than 10 yr and they have never been administered any other family of anthelmintics or drugs.

Experimental design Fifty-three (53) donkeys (Equus asinus) of either sex were selected within an age range of 3 mo to 25 yr. The experiment was divided into two stages. In the 1st stage, all 53 animals allowed the determination of the economic thresholds (explained below) in the ecological study area and the formation of the 2nd stage experimental groups on the pre-treatment day.

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In the 2nd stage, two experimental groups of 15 animals each were established: a treatment group (Tg) and a control group, without treatment (Cg). Following the methodology suggested by the World Association for the Advancement Veterinary Parasitology (WAAVP) (20-22), the animals were selected at random, provided that they had a minimum of 150 EPG. The ranges exhibited by each group were 150 to 2,850 HPG for the Cg, and 150 to 2,150 EPG for the Tg. On day zero, the Tg was administered 1% IVM orally, at the recommended dose of 0.2 mg/kg body weight(21). In order to calculate the individual dose of the drug, the approximate body weight was obtained using “The Donkey Sanctuary” weight estimator(23,24). Fecal samples were collected from both groups on days 7 and 14 posttreatment(21).

Economic thresholds Eggs per gram of feces (EPG) In order to determine the EPG number, stool samples were obtained directly from the donkeys' rectum(21,22). The samples were kept at 4 ºC until they were processed, using the modified McMaster technique(20-22) at the Research Laboratory of the Department of Parasitology of the Faculty of Veterinary Medicine (FMVZ, Spanish acronym) of UNAM. Body condition (BC) The BC was obtained from the visual estimation of the animal, on a scale of 1 to 5, in increments of 0.5, using The Donkey Sanctuary's Body Condition Score Chart(23).

Identification of genera of gastrointestinal nematodes Stool cultures were performed in order to obtain the infecting larvae (L3), using the culture technique described by Figueroa et al(25) and the larval migration technique with the Baermann device, with an incubation period of 10 d(25). Subsequently, the L3 were collected and washed using the GIN larval cleaning technique by density gradients with 40% sucrose(25,26). Finally, 100 L3 of the cultures from the pre-treatment day and from the 7th and 14th post-treatment days were identified (25,26).

Fecal egg count reduction test (FECRT) The percentage of efficacy of the drug was determined using the FECRT, performed according to the guidelines of the WAAVP(21,22,27). McMaster tests were performed on d 1 pre-treatment for the Tg (n= 15) on d 7 and 14 post-treatment for the Cg (n= 15).

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Donkey owner surveys

An exploratory survey(28,29) was conducted with 25 donkey owners. The questionnaire included questions about the medical care provided by the doctors, specifically deworming, as well as the type of confinement and feed given to their animals, with the aim of detecting management failures and causes that may generate AHR. In the event that the owner provided the complementary treatment to that applied by the VET, the survey considered issues such as frequency criteria, application procedures, and application of other anthelmintics (29).

Statistical analysis

The correlation data of BC and EPG were analyzed with the RTM statistical package(30). The FECRT values were calculated using the RESOTM software(31) according to the formula: Efficacy (%) = (Cg pretreatment EPG - Tg post-treatment EPG / Cg pretreatment EPG) x100(20-22). A parasite population is considered susceptible to an AH when the efficacy percentage is higher than 95% and the lower limit of the 95% confidence interval is above 90%. AHR is considered suspicious when a population meets only one of the two criteria(2022,31) .

Results Economic thresholds

Eggs per gram of feces (EPG) In the 1st stage, 98.1 % (n= 52) of the stool samples were positive for strongylid-type eggs. The EPG elimination range at this stage ranged from 0 to 3,000 EPG, as shown in Figure 1. Based on the distribution and on the estimated median value of 350 EPG and third quartile value of 600 EPG, it was determined that the economic threshold for donkeys in the Mexican High Plateau is 600 HPG.

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Figure 1: Distribution of egg elimination per gram of feces (EPG) in donkeys of the Mexican High Plateau

Green bars= animals with <350 EPG; Orange bars= animals with 350 to 600 EPG; Red bars= animals with >600 EPG.

Body condition (BC) The BC of most of the animals (91 %) was acceptable (2.5 to 3.5); three animals exhibited values of 4.5 (overweight), and two, of 1.5 (poor), according to the reference estimate of condition and weight (2.3). In addition, a highly significant negative correlation (P= 0.01) was observed; thus, there is evidence to assume that at higher BC the egg discharge obtained is lower (P<0.05), with a 95% confidence interval. (0.07 to 0.56). The determination coefficient was 0.1154 (Figure 2). Therefore, for each increase of 0.5 in BC, the egg discharge is reduced by 0.33 %. However, this decrease in egg removal may vary within a range of 0.07 to 0.56 %. Figure 2: Correlation between the elimination of eggs per gram of feces (EPG) and body condition (BC) evaluated in donkeys of the Mexican High Plateau

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Identification of gastrointestinal nematode Genera

One hundred (100) infective larvae were identified, of which 63 % were observed to be cyathostomidae (Figure 3). Small strongyles included species of the genera Poteriostomum, Gyalocephalus, and Oesophagodontus. The most frequent species among the larger strongyles was Strongylus edentatus, followed by S. equinus and S. vulgaris. Figure 3: Distribution of genera of gastrointestinal nematodes present in donkeys of the Mexican High Plateau

Test to determine the effectiveness of ivermectin FECRT At the 2nd stage of the experiment, the results of the FECRT test indicated that the oral administration of 1% IVM had an efficacy of 99 % in the Tg on d 7 post-treatment, and of 100 % on d 14, with a 95% confidence interval; therefore, this parasite population is considered to be susceptible to treatment with 1% IVM. In contrast, the elimination range for the Cg was 100 to 850 EPG on d 14.

Donkey owner surveys The average age of the animals, most of which were males (79 %) was 11.05 yr. Only 40 % of the animals coexist in pens, some of them in common accommodations in each community, during the dry season, when they do not carry out daily agricultural activities, although in times of sowing and harvesting, these animals are mostly housed in pens as a nocturnal enclosure, after the workday. In the case of grazing animals, the owner comments that the animals graze on land of his own property, used exclusively for planting such 332

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products as corn and beans. Eighty, 80 % admit to using parasite control as the only strategy that is provided by the volunteer medical service. The remaining 20 % did not know the name of the commercial product or the active ingredient, and they administer the product without knowing the weight of the animal or the appropriate dose. It should be noted that the owners indicate that the treatment is applied during the period prior to the rains, or up to twice a year (Table 1). Table1: Responses to the survey on deworming practices carried out on donkey owners in the Mexican High Plateau Question

(n)

What kind of management do you give Grazing to your animals? Night confinement Pen Where do they graze? Exclusive plot Shared plot Number of animals/pen Average

36 24 40 36 20 NA

9 6 10 9 5 1.3

In addition to the services provided by Yes VET, do you deworm your animals? No What product do you use? Does not remember “Paste” (does not know the name) Do you check the expiration date? Yes No Which is the dose of the product you Does not know/ Does not regularly apply? remember By “cm”/ The whole product Route of administration Oral Intramuscular Subcutaneous Does not know Who applies the product? ZVD Owner Other Weighing Yes No Frequency As recommended by the VET Semestral

20 80 8 12

5 20 2 3

4 16 12

1 4 3

20 0 0 0 8 12 4 0 20 0

5 0 0 0 2 3 1 0 5 0

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When do you practice this management?

Annual Previous to breeding time Before birth Before the rainy season After the rainy season Other

8 0 0 20 0 0

2 0 0 5 0 0

Discussion At present, no studies of donkeys of the Mexican High Plateau exist showing the population dynamics measured in the seasonal variation of EPG elimination, like those previously performed in Argentina(32), and including the identification of parasitic species in the area; nor are there studies on the economic threshold of EPG typical of the ecological study area indicating whether the animal is a candidate for deworming or not, like for other species(33,34), or on the potential relationship between thresholds such as the EPG and the BC of donkeys in the ecological study area. Parasitic diagnosis in donkeys is poor, and therefore the establishment of thresholds has not been a priority. In previous works(2), a median of 600 EPG was observed, and specifically in a tropical ecological zone, a median of 1,000 EPG was determined, confirming the differences that exist between each zone. These variations speak, above all, of the epidemiology of parasites; hence the importance of defining economic thresholds for EPG in particular. In order to determine the economic threshold criteria for EPG, this study identified the median of the distribution as 350 EPG, which means that 50 % of the animals are routinely dewormed. This involves an expensive and unnecessary deworming program, as some do not require the medication. The challenge here begins with the so-called phenomenon of superdispersion, in regard to which several authors(35,36) agree that only 20 to 25 % of the animal population are infected with parasites (as they are great eliminators) and are likely candidates for treatment. Also estimated was the value of the third quartile (75 %), which was 600 EPG, considered the economic threshold of EPG in donkeys of the Mexican High Plateau. This would indicate that only 25 % of this population would be a candidate for treatment, as long as they presented other criteria of economic thresholds that reflect an apparent parasitosis. Another aspect to be assessed by this study of the dispersion of the GIN is that the donkeys represented by the green and orange bars indicate the GIN-resistant population, and those animals represented by the red bars that do not exhibit clinical signs or depression rare the GIN-resilient population. These two resistant and resilient populations are most probably the relevant and redeemable refuge population in the face of the phenomenon of AHR, which are crucial concepts for understanding the phenomenon of

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parasitism and its hosts(37). Considering also the ecological zone, the elimination phenomenon observed in the Cg stands out, which after 14 d reduced the EPG value to a range of 100 to 850 when before the treatment it was 150 to 2,850. One hypothesis with regard to this involves the role of endemic plants with bioactive components against GIN, studied in other species(38), as well as the hypobiosis phenomenon, discussed below. Cyathostomidae have gained importance due to the encystment of larvae in the intestinal submucosa and of hypobiotic larvae(39-41). At the same time, it is known that in countries where in the autumn and winter period, when this study was carried out, is marked, the larvae that may exist in the intestinal mucosa are undetectable(21,39); therefore, knowledge of the annual dynamics of the parasite population may provide a better overview of the existing nematodoses and allow the development of strategies for their treatment. It is worth mentioning that the BC obtained in 91 % of the donkeys has acceptable values of 2.5 to 3.5, which indicates that the animals, despite their environmental conditions, express the rusticity that characterizes them and withstand parasitosis(3) , a phenomenon that helps reaffirm why not all donkeys require treatment under fixed schemes. On the other hand, when the correlation was made between the values of the BC and the EPG, it was observed that there is an apparent reduction of 0.33 % of the EPG when the BC increases by 0.5. This study defined that the EPG, the BC and their close relationship are valuable thresholds in the management of gastrointestinal nematodosis in donkeys. It is important to note that, although there are studies where a relationship between the BC and the parasite load in donkeys is not observed(2,3,23). Yoseph et al(40) suggest the measurement of this economic threshold, as long as it is met with a high-quality, forage-based diet and proper management of an anthelmintic treatment. As parasitoses are one of the diseases that go unnoticed by the owner, several authors(41) mention the importance of reestablishing the association between doctor and producer/owner for the design of parasite management and control strategies, together with the growing concern about AHR in Mexico, due to the constant and indiscriminate use of AH. This happens mainly with IVM. In this study, the effectiveness of IVM was found to be 100 %. However, there are studies(42,43) in which it is mentioned that, since the tests for the effectiveness of AH in equines have not yet been completely standardized, there may be a margin where the validation of a resistant population requires additional tests. Other authors(44) also highlight the importance of developing comparative tests such as the LMIT (larval migration inhibition test), in which the sensitivity to IVM of the population of cyathostomidae present in equines can be known. As a complement to this study, the assessment of the ERP (egg reappearance period) after treatment is suggested, since, although the FECRT indicates that the parasite population is 100 % sensitive to the assessed AH, it has been observed that this threshold 335

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may expose a latent resistance process. In equines, the observed ERP was 8 to 13 wk for IVM. At The Donkey Sanctuary, in the UK, this ERP in donkeys that have had little exposure to the drug is 6 wk(44) and has probably diminished due to the development of "juvenile" specimens that were not eliminated during the treatment(45). The ineffectiveness of the drug against hypobiotic larvae may trigger AHR, due to their constant exposure to IVM; therefore, the selection pressure of the parasite is increased, resulting in a low ERP, and therefore resistant species in the next generation(44). Further studies are required in order to verify this. It is not possible to speak of the effectiveness of a drug or AHR if the practices that owners or veterinarians carry out with respect to deworming are not evaluated in parallel. In this study, probable causes of AHR and potential failures in deworming were detected through surveys. The entire donkey population was treated, including seemingly healthy individuals. This increases the selection pressure of the parasites, making subsequent generations resistant and reducing the existence of refuge hosts within the population; for this reason, reserving such a valuable resource as IVM for those animals that actually require treatment prevents the generation of new species without sensitivity to the drug. This same AH was dosed without knowing the exact body weight of the animal and was utilized as a systematic or suppressive treatment (e. g. twice a year). These actions involve the risk that parasites may acquire AHR without efficient elimination or control and transmit it genetically to their offspring. A clear example is the reduced action of the IVM against hypobiotic larvae, where the selection pressure on the parasite is increased. The owner's ignorance regarding the applied products and the adequate doses, or the aim of management and its correct implementation, as mentioned above, may result in unremarked treatments and in drug underdose or overdosing. Thus, the implementation of new parasite control tools, which focus on reducing the selection pressure of parasites for various AH, has become one of the main requirements of parasite management(41). In response to this need, different tools are being integrated into a management and control strategy. IPC aims at slowing the growth of parasitic populations with a high proportion of individuals genetically resistant to one or more AH(46). The scheme proposes to integrate various principles based on the parasitic population dynamics of a known herd or stable. This scheme includes the TST(16), which is perceived as viable in this region.

Conclusions and implications Although the development of resistance to IVM is not yet present, it has been observed that these communities are at a critical point; therefore, the correction of failures in anthelmintic treatment and the prevention of probable causes of AHR can help doctors, together with the

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owners, to take care of a unique resource such as this AH through the correct and rational use of new molecules available in the market, the evaluation and the obtainment of their own thresholds, and the development of tools for their implementation, setting the precedent of awareness of the correct use of AHs, and thus improving the quality of the welfare of donkeys.

Acknowledgements To Veterinarian Omar Prado Ortiz for the valuable technical help received during the development of this research.

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caprine). Vet Parasitol 1995;58:181-213. 22. Geurden T, van Doorn D, Claerebout E, Kooyman F, De Keermaecker S, Vercruysse J et al. Decreased strongyle egg re-appearance period after treatment with ivermectin and moxidectin in horses in Belgium, Italy and The Netherlands. Vet Parasitol 2014;204(34):291-296. 23. The Donkey Sanctuary. Condition scoring and weight estimation. Devon, Reino Unido: The Donkey Sanctuary. 2013. 24. The Donkey Sanctuary. Condition scoring and weight estimation. Devon, Reino Unido: The Donkey Sanctuary 2014; https://www.thedonkeysanctuary.org.uk/sites/sanctuary/files/document/1421423234830-donkey_health_and_welfare.pdf. Consultado Mar 30, 2017. 25. Figueroa JA et al. Examen coproparasitoscópico. En: Rodríguez Vivas RI, editor. Técnicas para el diagnóstico de parásitos con importancia en salud pública y veterinaria. 1ª. ed. México: AMPAVE. Consejo Técnico Consultivo Nacional de Sanidad Animal. 2015:101-118. 26. Zajac MA, Conboy GA. Veterinary Clinical Parasitology. 8a ed. USA: Am Assoc Vet Parasitol. 2012. 27. Cabaret J, Berrag B. Faecal egg count reduction test for assessing anthelmintic efficacy: average versus individually based estimations. Vet Parasitol 2004;121:105-113 28. López Romo H. La metodología de encuesta en Galindo Caceres LJ, coordinador. Técnicas de investigación en sociedad, cultura y comunicación. México: Logman; 1998:83-93. 29. Alegría López MA, Rodríguez Vivas RI, Torres Acosta JFJ, Ojeda Chi MM, Rosado Aguilar JA. Use of Ivermectin as endoparasitide in tropical cattle herds generates resistance in gastrointestinal nematodes and the tick Rhipicephalus microplus (Acari: Ixodidae). J Med Entomol 2015;52:214-221. 30. R Development Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; 2018. ISBN 3-900051-07-0, URL http://www.R-project.org. 31. Rodríguez Vivas RI [editor]. Técnicas para el diagnóstico de parásitos con importancia en salud pública y veterinaria. 1a Ed. México: AMPAVE / Consejo Técnico Consultivo Nacional de Sanidad Animal. 2015:355-403. 32. Fusé L, Saumell CA, Iglesias L. Variación estacional del parasitismo interno en equinos: Fenómeno de hipobiosis de los pequeños estrongílidos (Cyathostominae) en Tandil, 339

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Buenos Aires, Argentina. Rev Med Vet 2013;94(3):62-72. 33. Martinez Ortiz-de-Montellano C, Cervantes Morali J, Figueroa Castillo JA, Torres Acosta JFJ. Distribution of gastrointestinal nematodes egg counts in sheep from Mexico Center south and its implications of parasitic integrated control. Int Conf World Assoc Advanc Vet Parasitol. Kuala Lumpur, Malasia; 2017. 34. Martínez-de-Castro Dubernard A, Dominguez Hernández Y, Quiroz Rocha G, Saldaña Hernandez N, Ojeda Robertos N et al. Importance of egg per gram treshold determination in goats from Mexican plateau: To deworm or not to deworm?. Int Conf World Assoc Advanc Vet Parasitol. Kuala Lumpur, Malasia; 2017. 35. Sréter T, Molnár V, Kassai T. The distribution of nematode egg counts and larval counts in grazing sheep and their implications for parasite control. Int J Parasitol 1994; 24(1):103-108. 36. Hoste H, Le Frieleux Y, Pommaret A. Distribution and repeatability of feacal egg counts and blood parameters in dairy goats naturally infected with gastrointestinal nematodes. Res in Vet Sci 2011;70(1):57-60. 37. Van-Houtert MFJ, Sykes AR. Implications of nutriction for the ability of ruminants to withstand gastrointestinal nematode infections. Int J Parasitol 1996;26(11):1151-1167. 38. Peachey LE, Pinchbeck GL, Matthews JB, Burden FA, Mulugueta G, Scantlebury CE et al. An evidence-based approach to the evaluation of ethnoveterinary medicines against strongyle nematodes of equids. Vet Parasitol 2015;210:40-52. 39. Zachary JF. Pathologic basis of Veterinary diseases. 6th ed. USA; 20017. 40. Yoseph S, Smith DG, Teklu F, Firew T, Betere Y. Seasonal variation in the parasite burden and body condition of working donkeys in East Shewa and West Shewa Regions of Ethiopia. Trop Anim Health Prod 2005;37(1):35-45. 41. Statford CH, McGorum BC, Pickles KJ, Matthews JB. An update on cyathostomins: Anthelmintic resistance and diagnostic tools. Eq Vet J 2011;43:133-139. 42. Molento MB, Nielsen K, Kaplan RM. Resistance to avermectin/milbemycin anthelmintics in equine cyathostomins – Current situation. Vet Parasitol 2012;185:1624. 43. Mejía ME, Fernández Igartúa BM, Schmidt EE, Cabaret J. Multispecies and multiple anthelmintic resistance on cattle nematodes in a farm in Argentina: the beginning of high resistance?. Vet Res 2003;34:461-467. 44. McArthur CL, Handel IG, Robison A, Hodgkinson JE, Bronsvoort B, Burden F. et al.

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https://doi.org/10.22319/rmcp.v11i2.4946 Article

Artemisia cina 30 CH homeopathic treatment against Haemonchus contortus

Rosa Isabel Higuera-Piedrahita a* María Eugenia López-Arellano b Raquel López-Arellano c César Cuenca-Verde c Jorge Alfredo Cuéllar-Ordaz c

Universidad Nacional Autónoma de México. Programa de Maestría y Doctorado en Ciencias de la Producción y de la Salud Animal. Carr. Cuautitlán-Teoloyucan Km 2.5, Col. San Sebastián Xhala. Cuautitlán, Estado de México, México. b

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), Centro Nacional de Investigación Disciplinaria en Salud Animal e Inocuidad (CENID-SAI). Jiutepec, Morelos, México. c

Universidad Nacional Autónoma de México. Facultad de Estudios Superiores Cuautitlán. Cuautitlán, Estado de México, México.

*Corresponding author: rositah_10@hotmail.com

Abstract: The anthelmintic resistance problem is widely recognized in sheep production. Therefore, new methods of control against gastrointestinal nematodes (GIN) need to be integrated. The aim of this work was to assess the toxicity of A. cina 30 CH as a homeopathic product against Haemonchus contortus in in vitro and in vivo assays. A. cina 30 CH was obtained from a commercial laboratory, and confirmation of artemisinin as a key ingredient was performed with mass spectrophotometry. The A. cina 30 CH and the artemisinin pure reagent were used for the inhibition of egg hatching (IEH) and for the inhibition of larval migration of H. 342

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contortus L3 (ILM). In addition, three groups of 10 naturally infected lambs with GIN were treated with A. cina 30 CH and albendazole, and 10 were used as control. The parasitic infection was monitored at 0, 7, 14 and 28 d postreatment (PT) to determine the number of eggs per gram (epg) and FAMACHA index. The in vitro data showed 100 % IEH and 64.7 % ILM by A. cina 30 CH, and nonlethal activity was observed with the artemisinin pure reagent. The toxicity of A. cina 30 CH against H. contortus in infected lambs was observed after 7 d of infection. Administration of the A. cina 30 CH yielded a 69 % reduction in the epg at 28 d PT, similar to the albendazole (P<0.05). In conclusion, A. cina 30 CH had the ability to IEH and ILM of H. contortus in in vitro assays and reduced the number of eggs of H. contortus, which is the primary parasitic nematode in grazing lambs, thereby reducing infection. Key words: Artemisia cina 30 CH, Artemisinin, Haemonchus, Lambs.

Received: 14/06/2018 Accepted: 04/03/2019

Introduction Gastrointestinal nematodes (GIN), primarily H. contortus, which is the most prevalent nematode in tropical regions, are among the primary pathogens that reduce animal production(1). For a long time, anthelmintic drugs have been used as the main traditional method of control, and only one is on the market(2). However, the inadequate use of this drug has caused worldwide resistance problems in various ruminant species(3,4). The high prevalence and the fast dispersion of anthelmintic resistance have increased in domestic ruminants, which show resistance to multiple anthelmintic drugs in certain regions (5). In Mexico, diverse reporting on GIN has occurred, and other strategies of control are under study(6). The use of different methods of control have been denoted Parasite Integral Control (PIC)(7). The strategy of most studies is to focus on the control of highly pathogenic nematodes, such as H. contortus and Teladorsagia, in small ruminants because of their blood-feeding habits. Paddock rotation, selection of resistant breeds, biological control (i.e., nematophagous fungi and predatory nematodes)(8), selective deworming, vaccines and derivatives of the traditional herbolary (i.e., homeopathic products) are considered in the PIC(9). However, more studies

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are needed of alternative methods to reduce the epg and the adult nematodes during husbandry procedures(10). Homeopathic compounds are substances of different origins, such as vegetable or mineral, that have therapeutic effects. The homeopathic products are prepared following the instructions of the Homeopathic Pharmacopeia(10,11). For instance, the homeopathic products obtained from plants are acquired as ethanolic extracts (generally), and they are diluted in 99 parts alcohols until the desired concentration (decimal and centesimal) below the Avogadro number 6.02214*1023 is reached. In this way, the homeopathic drugs are obtained with low inversion and easy extraction and represent a safe method of control(12). Recently, several reports regarding the possible use of homeopathic products with a nematicidal effect have provided a new opportunity to integrate A. cina as a novel method of control. A. cina is a plant that belongs to the Asteraceae family and contains artemisinin as the active metabolite(13). This plant has shown anthelmintic and antimalarial properties(14). For instance, A. cina appears to have a potential therapeutic effect against parasites, but further study is required to determine if A. cina can be used as a homeopathic or natural product as a possible anthelmintic against GIN. A. cina took is conformided by the 30 centesimal hannemaniana (CH) concentration as reported by the Mexican Homeopathic Pharmacopoeia (concentration: 10-60M), which is suggested to be administered in ruminants. The aim of this study was to determine the antiparasitic efficacy of a homeopathic product based on A. cina 30 CH in in vitro and in vivo assays against a natural infection of small ruminants with GIN.

Material and methods Locality

The analysis by mass spectrometry was carried out in laboratory 5 of Unidad de Investigación Multidisciplinaria, and the in vitro analysis carried out in laboratory 3 and 5 of the Unidad de Investigación Multidisciplinaria of Facultad de Estudios Superiores Cuautitlán (FESC), UNAM, in Cuautitlan Municipality, State of México and the in vivo assay on a farm in Mixquiahuala Municipality, Hidalgo State at 2,100 m asl with a semi-dry climate, an annual temperature of 16.6° C and rainfall of 500 mm(15).

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Identification of artemisinin in A. cina 30 CH

The artemisinin molecules were identified from A. cina 30 CH commercial products (Millenium Lab, México). Ultra-performance liquid chromatography with mass spectrometry (UPLC/MS) was used with a reversed-phase column in positive mode. All samples were performed according to the following conditions: 70 cone velocity, Sm (Mn 2*0.75) and UPLC/MS reading from 200 to 300 m/z laboratory 5 of Unidad de Investigación Multidisciplinaria. Concentration of A. cina 30CH was 10-60M.

Parasites

Faeces positive for parasitic nematode eggs were collected from a donor lamb previously infected with 5,000 eggs of H. contortus L3, a strain isolated and maintained in the FESC, UNAM. The quantitative McMaster technique was used to determine the number of epg, and coproculture techniques were performed to collect H. contortus L3 at 21 d post-infection (PI). Larvae were kept at -20 oC until used (the larvae recovered from the larval culture were cryopreserved in glycerol; for bioassays, the larvae were thawed at room temperature, and 95 % motility was verified).

Bioassays

Two different in vitro assays were performed to determine the inhibition of egg hatching (IEH) and the inhibition of larval migration (ILM)(16). All techniques were performed using 100 eggs or infective stages of larvae (L3) of H. contortus. For each assay, three replicates were prepared, and five treatments were applied as follows: 1) 20 µL A. cina 30 CH (1060 M); 2) 100 µL of distilled water (DW, control); 3) 50 mg/mL albendazole (ABZ, control) (Sigma-Aldrich, St Louis, Missouri, USA), solubilized with 0.1 mg/mL dimethyl sulfoxide (DMSO); 4) 20 µL of ethanol; and 5) 1 mg/mL artemisinin (Sigma-Aldrich, St Louis, Missouri, USA). The IEH was performed in ELISA plates that were incubated at 28 °C for 48 h. The IEH reading was conducted using Lugol’s iodine solution, which was added to each well after incubation. The total volume of each ELISA well was read to count the number of active H. contortus L1 and IEH per well with a microscope under 10× magnification (Olympus, model CK-2, Japan). 345

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The ILM received similar treatment as the IEH, except that the ABZ was replaced by levamisole (300 mg/mL). Larvae were also read using Lugol´s solution after incubation. The total volume per well was read to determine the ILM.

In vivo assays

Lambs

Thirty Suffolk breed lambs, 16 males and 14 females, 3 mo of age and at 20 d post-weaning, were naturally infected with GIN. All lambs were kept in semi-stabled conditions, grazing in paddocks during the day and kept inside at night. Lambs were fed commercial concentrate and water ad libitum. No anthelmintic treatment was applied to any lamb before the present study. All lambs were positive for GIN eggs, which was confirmed by McMaster and coproculture techniques.

Experimental design

Prior to the treatments, the lambs were randomized into three groups of 10 each with the support of the Statgraphics Centurion XV. Treatments were designed as follows: group A received 1 mL per 5 kg of body weight (BW) by oral route of A. cina 30 CH (Millenium Laboratories, México) as a single dose, concentration of A. cina 30CH was 10-60M. (1); group B was orally treated with ABZ at 7.5 mg/kg of BW; and group C, without treatment, was used as the control. Faecal and haematological samples were collected at 1 d pretreatment (0 day) and at 7, 14 and 28 d post-treatment (PT). In addition, eye mucosa colour was observing using the FAMACHA index.

Statistical analysis

The means of H. contortus eggs and larvae L3 were compared between treatments and control groups using ANOVA analysis, complementary with Tukey’s test to identify the differences between treatments, using Statgraphics Centurion XV software. The number of epg was transformed to log10 epg + 10 to stabilize the variance, and the least significant difference (LSD) test was applied using Statgraphics Centurion XV software with a completely 346

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randomized design that considered repeated measurements over time and treatment. Differences with P<0.05 were considered to be significant.

Ethics note

The management of the lambs was performed according to the Guideline of the Institutional Committee for the Care and Use of Experimental Animals of the Facultad de Estudios Superiores Cuautitlรกn-UNAM (CICUAE- FESC- UNAM) and authorized under Protocol No. DC- 2014-14.

Results Identification of artemisinin in A. cina 30 CH

The mass spectrophotometry analysis showed artemisinin molecules in the A. cina 30 CH commercial products used in the present study. The A. cina 30 CH chromatographic analysis was performed to compare the profile of the commercial products to the artemisinin pure reagent. Figure 1, a-b showed artemisinin molecules corresponding to A. cina 30 CH and the artemisinin pure reagent with 279.20 m/z. Figure 1a-b: Mass spectrophotometry analysis of pure reagent (a) and A. cina 30 CH (b), showing similarities between 283 and 290 m/z a

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In vitro assays

IEH. Data obtained from the evaluation of A. cina 30 CH showed 100 % IEH of H. contortus A. cina after 48 h; this result was followed by the ABZ treatment, with 93 %. In contrast, no IEH of H. contortus was observed with the 80 % ethanol, the artemisinin pure reagent and the DW treatments did not show effect (Figure 2). Figure 2: Inhibition of egg hatch assays against Haemonchus contortus eggs exposed with Artemisia cina 30 CH and albendazole treatments and controls (artemisinin, water and methanol)

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ILM. The A. cina 30 CH showed 65.7 % inhibition of migration of the H. contortus infective larvae. Different results were observed with the artemisinin pure reagent used at 0.1 and 1 mg/mL with DW and ethanol, with all treated groups showing 100 % larval migration, indicating that no inhibition was observed in the control groups. The levamisol used as the anthelmintic showed lethal efficacy of 100 % against larvae; therefore, no migration was observed (Table 1). Table 1: Percentage of inhibition of larval migration (ILM) against L3 Haemonchus contortus (XÂąSE) Treatments

Migration (%) 35.0 + 8.1 0 92.0 + 12.4 86.6 + 11.5

Artemisia cina 30 CH Levamisole (300 mg/mL) Water Ethanol 80%

Artemisia cina nematicide efficacy

Natural infection with GIN on grazing lambs showed two main GIN species: H. contortus (75%) and T. circumcincta (25 %). Infected lambs for all groups showed a mean of approximately 2,000 epg prior to treatment (d- 0). Through the following periods, significant differences in the reduction in epg were observed at 7 and 14 d PT (P<0.05) (Figure 3). In addition, significant differences were also observed between groups A (A. cina 30 CH) and B (ABZ) in comparison to group C (control, P<0.05) at 7 d. Figure 3: Egg per gram observed at -7, 0, 7, 14 and 28 d postreatment of lambs infected naturally with gastrointestinal nematodes *(P<0.05)

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FAMACHA Index Card. For all groups, the FAMACHA card index was determined to be 3.0 to 5.0 in the in vivo study. The FAMACHA values were variable for all groups. Important differences were observed at 14 and 28 d PT for the A. cina 30 CH and ABZ treatments (Table 2). Table 2: FAMACHA index of lambs naturally infected with gastrointestinal nematodes and receiving Artemisia cina 30 CH or albendazole Days post-treatment Treatment 0 7 14 28 p Artemisia cina 30 3.0 + 0.13a 4.0 + 0.44aA 3.0 + 0.52bB 2.0 + 0.31bA 0.44 CH Albendazole

5.0 + 0.26ÂŞ

3.0 + 0.21bB

2.0 + 0.30cA

1.0 + 0.22cA

0.19

Control

3.0 + 0.25b

2.0 + 0.29aB

1.0 + 0.20aA

3.0 + 0.71bB

0.62

Equal lower case letters have no statistical significance and different lower case letters have statistical difference within the group (P<0.05). Equal capital letters have no statistical significance and different capital letters have statistical difference between groups (P<0.05).

Discussion A. cina has chemical compounds, such as the terpenoids, that provide insecticidal activity against reproductive capacity and cause antioxidative stress in the pathogens(17,18,19). In recent years, important results identified artemisinin as having a possible anthelmintic effect(20). For instance, Akkari et al(21), reported a lethal dose (LD) of Artemisia campestris of 0.8 mg/mL against H. contortus when using an ethanolic extract. In the present study, A. cina 30 CH showed 100 % of H. contortus IEH. In addition, A. cina 30 CH showed efficacy for decreasing the larval migration, and these results were like those reported by others(22). Bashtar et al.(22) described the ethanolic extract of A. cina with efficacy against the cestode Moniezia. In addition, the present study had 64.7 % ILM of H. contortus using A. cina 30 CH. It was reported a reduction in the larval rate in rats infected with the nematode Trichinella spiralis when the rats were treated with A. cina 30 CH, Podophyllum 0 and Santoninun 30 CH (homeopathic products) in 68.14 %, 84.10 % and 81.20 % respectively(23). Conversely, artemisinin pure reagent was used as control, and no inhibition of egg or larval migration was observed. These results suggest that the absence of activity might have been produced by the chemical conformation of A. cina 30 CH, and a solvent that used compounds with hydrogens and phenyl rings, thereby enabling a fast change in conformation. Regarding the natural infection with GIN and the A. cina 30 CH treatments applied after 2 wk, the ABZ and A. cina 30 CH showed significant differences (P<0.05) in reductions in the 350

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number of epg. These findings were like those reported by Bashtar et al(22), who found a reduction of proglottids of Moniezia sp. in animals treated with A. cina. However, more studies are required to confirm the possible anthelmintic effect of A. cina 30 CH against nematode stages using artemisinin from native plants. Treatments with A. cina 30 CH and ABZ against natural infection improved the FAMACHA values caused by the blood-feeding habit of H. contortus at 7 and 14 d after administration (P<0.05). Similar results were found by Cala et al(1), with artemisinin supported as a possible nematicide metabolite after infection. Demeler et al(24) showed the anaemia caused by H. contortus infection in lambs treated with ABZ showed nematicidal efficacy. A review carried out by Kerboeuf et al(25) suggests the activity of flavonoids on the structure and cell target is similar to the antioxidant effect caused by artemisinin. Although the A. cina anthelmintic mechanism of action is unknown, determination of this mechanism is needed for its application to nematodes infecting hosts. It was demonstrated the stability of artemisinin in the rumen, which was detectable in blood samples at 33 mg of artemisinin/kg of body weight(21). The study of A. cina 30 CH showed participation of the drug as an anthelmintic, and it should be considered as a possible method for use in the control of parasitic nematodes.

Conclusions and implications The A. cina 30 CH had anthelmintic efficacy against H. contortus egg hatching during natural infection. The FAMACHA index suggest reduction of nematode activity after treatment with A. cina 30 CH and ABZ. In addition, this product demonstrated inhibition of egg hatching and larval migration, which indicates its possible anthelmintic effect. To optimize the use of this homeopathic compound, the mechanism of its action must be determined.

Acknowledgments This study was supported by PAPIIT IN226217, named “Efecto antihelmíntico del extracto etanólico de Artemisia cina, semilla de papaya (Carica papaya) y taninos condensados sobre el nemátodos hematófago Haemonchus contortus.” Miss Higuera-Piedrahita R.I. was partially supported by a CONACYT grant, México. The strain of Haemonchus contortus used was isolated and maintained in laboratory 1 and 3 of the Multidisciplinary Research Unit of the Facultad de Estudios Superiores Cuautitlán, UNAM.

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Conflict of interest statement

None of the authors declare a conflict of interest.

Literature cited: 1. Peter JW, Chandrawathani P. Haemonchus contortus: parasite problem No. 1 from tropics - Polar Circle. Problems and prospects for control based on epidemiology. Trop Biomedicine 2005;22:131–137. 2. Medina-Pérez P, Ojeda-Robertos N, Reyes-García M, Cámara-Sarmiento R, TorresAcosta J. Evaluation of a targeted selective treatment scheme to control gastrointestinal nematodes of hair sheep under hot humid tropical conditions. Small Ruminant Res 2015;127:86-91. 3. Van der Brom R, Moll L, Kappert C, Vellema P. Haemonchus contortus resistance to monepantel in sheep. Vet Parasitol 2015;209:278-280. 4. Geurden T, Chartier C, Fanke J, di Regalbono AF, Traversa D, et al. Anthelmintic resistance to ivermectin and moxidectin in gastrointestinal nematodes of cattle in Europe. Int J Parasitol: Drugs and drug resistance 2015;5:163–171. 5. Crook E, O´Brien D, Howell S, Storey B, Whitley N, Burke J, Kaplan R. Prevalence of anthelmintic resistance on sheep and goat farms in the mid-Atlantic region and comparison of in vivo and in vitro detection methods. Small Ruminant Res 2016;143:8996. 6. Sales N, Love S. Resistance of Haemonchus contortus sp. to monepantel and reduced efficacy of a derquantel/abamectin combination confirmed in sheep in NSW, Australia. Vet Parasitol 2016;228:193-196. 7. González-Garduño R, López-Arellano M, Ojeda-Robertos N, Liébano-Hernández E, Mendoza de Gives P. Diagnóstico in vitro y en campo de resistencia antihelmíntica en nematodos gastrointestinales de pequeños rumiantes. Arch Med Vet 2014;46:399-405. 8. Torres-Acosta J, Hoste H. Alternative or improved methods to limit gastro-intestinal parasitism in grazing sheep and goats. Small Ruminant Res 2008;77:159-173. 9. Delgado C, Rosegrant M, Steinfeld H, Ehui S, Courbois C. Livestock to 2020: The next food revolution. IFPRI, FAO/UN, ILRI Discussion Paper 1999;28-72.

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10. Coop RL, Kyriazakis I. Influence of host nutrition on the development and consequences of nematode parasitism in ruminants. Trends in Parasitol 2001;17: 325-330. 11. Farmacopea homeopática Mexicana. 2007. www.ismhei.com.mx/.../Farmacopea%20Homeopatica%20Dr%20Osvaldo_Martinez.p . Accesed Nov 10, 2017. 12. Meier L, Torgerson P, Hertzberg H. Vaccination of goats against Haemonchus contortus with the gut membrane proteins H11/H-gal-GP. Vet Parasitol 2016;229:15- 21. 13. Zhonglei W, Liyan Y, Xiuwei Y, Xiaohua Z. Advances in the chemical synthesis of artemisinin. Synthetic Communications: An Int J Rapid Communic Synthetic Organic Chemistry 2014;44(14):1987-2003. 14. Montalvo X, López M, Vázquez V, Liébano E, Mendoza P. Resistencia antihelmíntica de nematodos gastroentéricos en ovinos a febendazol e ivermectina en la región noroeste del estado de Tlaxcala. Téc Pecu Méx 2006;44(1):81-90. 15. Cavalcanti A. Efeito do medicamento homeopático sulphur sobre nematódeos gastrintestinais, resistentes a invermectina, de cordeiros infectados naturalmente. [tesis maestría]. Salvador, Brasil: Universidad Federal de Bahia; 2008. 16. Zacharias F, Guimaraes F, Araújo R, Almeida M, Ayres M, Bavia M, Mendonca-Lima M, Effect of homeopathic medicines on helminth parasitism and resistance of Haemonchus contortus infected sheep. Homeopathy 2008;97:145-151. 17. Saitbaeva I, Sidyakin G. Artemisin from Artemisia cina. Chem Nat Comp 1971;7(1):113. 18. Bhakuni R, Jain D, Sharma R, Kumar S. Secondary metabolites of Artemisia annua and their biological activity. Current Sci 2011;80(11):35-48. 19. Ayuntamiento de Mixquiahuala de Juárez. Enciclopedia de los municipios y delegaciones de México. Mixquiahuala de Juárez, Estado de Hidalgo 2013. http://mixquiahuala.hidalgo.gob.mx/ . consultado 30 Nov, 2017. 20. Coles G, Borgsteede F, Geerts S, Klei T, Taylor M, Waller P. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) methods for the detection of anthelmintic resistance in nematodes of veterinary importance. Vet Parasitol 1992;44:35-44. 21. Akkari H, Rtibi K, B´chir F, Rekik M, Aziz M, Gharbi M. In vitro evidence that the pastoral Artemisia campestris species exerts an anthelmintic effect on Haemonchus contortus from sheep. Vet Res Comun 2014;38:249-255.

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22. Bashtar A, Hassanein M, Abdel-Ghaffar F, Al-Rasheid K, Hassan S, Mehlhorn H, et al. Studies on monieziasis of sheep I. prevalence and antihelmintic effects of some plant extracts, a light and electron microscopic study. Parasitol Res 2011;108:177-186. 23. Sukul N, Ghosh S, Sinhababu S. Reduction in the number of infective Trichinella spiralis larvae in mice by use of homeopathic drugs. Forsch Komplementarmed Klass Naturheilkd. 2005;12(4):202-205. 24. Demeler J, Kleinschmidt N, Kuttler U, Koopmann R, Von Samson G. Evaluation of the egg hatch assay and the larval migration inhibition assay to detect anthelmintic resistance in cattle parasitic nematodes on farms. Parasitol Int 2012;61:614-618. 25. Kerboeuf D, Riou M, Guegnard F. Flavonoids and related compounds in parasitic disease control. Mini Rev Med Chem 2008;8:116-128.

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https://doi.org/10.22319/rmcp.v11i2.5090 Article

Tithonia diversifolia meal in diets for first-cycle laying hens and its effect on egg yolk color

María Elena Carranco-Jáuregui a Vilma Barrita-Ramírez b Benjamín Fuente-Martínez c* Ernesto Ávila-González c Leonor Sanginés-García a

Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Departamento de Nutrición Animal Dr. Fernando Pérez-Gil Romo, Vasco de Quiroga No. 15, Col. Belisario Domínguez Sección XVI, Alcaldía Tlalpan 14000, Ciudad de México. México. b

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

Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia. Centro de Enseñanza, Investigación y Extensión en Producción Avícola, , Ciudad de México. México.

*Corresponding author: benjaminfuente@yahoo.com.mx

Abstract: Consumers in Mexico expect egg yolks to have a certain color. Attaining this color in intensive production systems requires addition of natural pigments to laying hen diets. Feed is the largest input cost in egg production and added pigments increase costs. Use of alternative natural pigment sources, such as tree marigold Tithonia diversifolia, can help to control costs. An evaluation was done of how addition of T. diversifolia meal (TDM), as a 355

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yellow pigment source, to diets for first-cycle laying hens affected productive variables and egg yolk color. Over a six-week experimental period meal made from T. diversifolia leaves and petioles was added to poultry diets at four percentages (1.77, 5, 10 and 15 %). A total of 240 chickens were distributed in five treatments (Control, and 1.77 %, 5 %, 1 0% and 15 % TDM) tested in two trials: Trial 1 (weeks 1-3), no red pigment added; Trial 2 (weeks 4-6), red pigment added. Measured variables were laying percentage, egg weight and mass, and feed intake and conversion. At the end of each trial, 20 eggs/treatment were collected for quantification of xanthophylls by HPLC, and measurement of yolk color based on DSM fan colors and reflectance colorimetry. The design was completely random and differences between means were identified with a Tukey test. Egg weight and feed conversion did not differ between treatments (P>0.05). Laying percentage and egg mass in the 10 and 15% TDM treatments, and feed intake in the 15% TDM treatment were lower than in the Control (P<0.05). Yolk color was most intense in the 10 and 15% TDM treatments in both trials. Tithonia diversifolia meal is a promising alternative natural source of yellow pigment in laying hen diets at up to a 10% inclusion level, and does not affect productive variables. Key words: Tithonia diversifolia, Laying hens, Eggs, Productive variables, Pigment.

Received: 02/10/2018 Accepted: 20/03/2019

Introduction Mexicoâ&#x20AC;&#x2122;s poultry industry currently produces more than 5 million tons of products (e.g. eggs, chicken and turkey) annually to meet national demand(1). This represents 63.8 % of the countryâ&#x20AC;&#x2122;s total annual livestock production, eggs alone accounting for 29 %. Projected egg production for 2018 is 2.806 million tons. Feed costs are 60 to 70 % of the input costs in poultry production(1). Eggs are an excellent food due to their high biological value, ease of handling, various preparation options, especially in combination with other foods, and their accessible cost. Consumers expect egg yolks to have a certain color. In intensive production systems laying hens are not exposed to natural pigments through their feed, meaning these must be added for egg yolks to have the expected color. This represents a substantial increase in input costs and affects final product price(2,3). Natural source yellow and red pigments, including carotenoids, are added to feed for laying hens(2,3). Research is ongoing into new sources of

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natural pigments that provide carotenoids, are easy to use, and have minimal impact on production costs. Tithonia diversifolia, known as tree marigold among many other common names, is native to Central America and Mexico. Its almost 15,000 species can be found worldwide in tropical and subtropical areas(4). It grows quickly, even in unfavorable conditions such as roadsides, and multiplies easily. In soils this plant is known to improve nutrient recycling, prevent erosion, reduce the effects of animal trampling and produce high biomass productivity without agrochemical inputs(5,6). It is a multipurpose plant that can be used as a living fence, green manure, grazing forage in silvopastoral livestock systems, and cut forage in poultry and ruminant systems. Tithonia diversifolia has reported values of 23.0 g/100 g dry matter, 14.8-28.7 g/100 g crude protein, 21.4 g/100 g ash, and 78.6 g/100 g organic matter(7). Of note is its high carotenoid content, suggesting its use as a pigment source in egg production systems; indeed its inclusion in feed for laying hens at 15% results in good egg yolk color(8). The present study objective was to evaluate the effect of different inclusion levels of Tithonia diversifolia meal in diets for first-cycle laying hens on yolk color and egg quality.

Material and methods Study area, sampling and pigment quantification

Tithonia diversifolia was collected at the Veterinary Medicine and Zootechny Academic Unit, Autonomous University of Nayarit, in Compostela, Nayarit, Mexico. Regional climate is tropical, with summers much rainier than winters, a 22.4 °C average annual temperature and 1,060 mm approximate average annual rainfall(9). Leaves and petioles of T. diversifolia were harvested manually after 60 d regrowth (644.5 kg/fresh). All foreign matter was removed from the material, which was pre-dried in shade at the harvest site. The pre-dried leaves and petioles were stored in black plastic bags and transported to the Dr. Fernando Pérez-Gil Romo Department of Animal Nutrition, Salvador Zubirán National Institute of Medical Sciences and Nutrition. Here the material was oven dried at 60 °C/24 h. Once dried it was ground in a hammer mill with a 1 mm mesh to produce Tithonia diversifolia meal (TDM) and stored for later analysis.

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Pigment content in the TDM was quantified by high-resolution HPLC (Industrias VEPINSA, S.A. de C.V.; Research and Development Office)(10).

Diets and experimental design

All feed trials were done at the Poultry Production Teaching, Research and Extension Center (Centro de Enseñanza, Investigación y Extensión en Producción Avícola - CEIEPAv) of the Faculty of Veterinary Medicine and Zootechny (FMVZ), National Autonomous University of Mexico (Universidad Nacional Autónoma de México – UNAM). All experimental procedures involving animals were reviewed and approved by the Institutional Subcommittee on Experimental Animal Care and Use (FMVZ, UNAM), and met applicable federal regulations(11). Experimental animals were 240 Bovans White line laying hens. The diets were formulated to meet animal nutritional needs by production phase using the Allix2 ver. 5.37.1 software. Five treatments were tested using a completely random design with four replicates per treatment and twelve birds per replicate: 1) Control, 15 ppm yellow pigment; 2) 1.77% TDM + 15 ppm total xanthophylls; 3) 5% TDM + 42.5 ppm total xanthophylls; 4) 10% TDM + 85 ppm total xanthophylls; and 5) 15% TDM + 127.5 ppm total xanthophylls. Water and feed were freely available. Two trials were run: weeks 1-3, no red pigment in diets; and weeks 46, red pigment included in diets (Tables 1 and 2).

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Table 1: Experimental diets in first trial (weeks 1-3) using Tithonia diversifolia meal and no red pigment (kg) Tithonia diversifolia meal (%) Ingredients Control 1.77 5 10 15 Sorghum Soy paste Calcium carbonate TDM Calcium phosphate Salt Vit + min premix1 DL-Methionine (84%) Vegetable oil L-Lysine HCl Choline chloride (60%) Yellow pigment2 Antioxidant3 Bambermycin Phytase4 Total Calculated analysis Metabolizable energy, kcal/kg Crude protein, % Total Met + Cys, % Total lysine, % Total threonine, % Total tryptophan, % Crude fiber, % Total calcium, % Available phosphorous, % Sodium, %

660.500 221.390 101.791 0.000 4.568 3.026 2.400 2.289 1.482 1.179 0.500 0.500 0.150 0.125 0.100 1000

647.950 213.960 100.435 17.700 4.659 3.033 2.400 2.327 5.552 1.209 0.500 0.000 0.150 0.125 0.100 1000

621.800 202.402 98.000 50.000 4.553 3.046 2.400 2.401 13.277 1.246 0.500 0.000 0.150 0.125 0.100 1000

539.921 222.920 94.033 100.000 4.361 3.057 2.400 2.042 30.391 0.000 0.500 0.000 0.150 0.125 0.100 1000

456.946 242.914 90.112 150.000 4.183 3.068 2.400 1.704 47.798 0.000 0.500 0.000 0.150 0.125 0.100 1000

2800

17.400 0.730 0.860 0.622 0.205 2.446 4.100 0.420 0.180

17.400 0.730 0.860 0.623 0.199 2.583 4.100 0.420 0.180

17.400 0.730 0.860 0.625 0.189 2.831 4.100 0.420 0.180

18.970 0.730 0.866 0.691 0.196 3.331 4.100 0.420 0.180

20.480 0.730 0.967 0.754 0.201 3.824 4.100 0.420 0.180

Content per kilogram: Vit. A, 4.0 MUI; Vit. D3, 666,666.7 UI; Vit. E, 10,000.0 UI; Rovimix HyD 5 kg: Vit. K3, 1.67 g; Vit. B1, 0.83 g; Vit. B2, 2.33 g; Vit. B6, 1.17 g; Vit. B12, 6.666.67 mg; niacin, 10 g; D-pantothenic acid, 3.33 g; folic acid, 0.33 g; biotin, 33.33 mg; choline, 100 g; Fe, 20 g; Zn, 26.67 g; Mg, 36.67 g; Cu, 5 g; I, 0.33 g; Se, 0.1 g. 2 Florafil 93 Powder (Vepinsa): 30 g/kg (minimum) total xanthophylls. 3 BHA, 1.2%; BHT, 9.0%; Ethoxyquin, 4.8%; chelating agents, 10.0%. 4 Quantum Blue 5000 FTU/kg derived from E. coli.

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Table 2: Experimental diets in second trial (weeks 4-6) using Tithonia diversifolia meal, with added red pigment (kg). Tithonia diversifolia meal (%) Ingredients Control 1.77 5 10 15 Sorghum 660.090 647.550 621.400 539.721 456.936 Soy paste 221.000 213.860 202.002 222.720 242.414 Calcium carbonate 101.791 100.435 98.000 94.033 90.112 TDM 0.000 17.700 50.000 100.000 150.000 Calcium phosphate 4.568 4.559 4.553 4.361 4.183 Salt 3.026 3.033 3.046 3.057 3.068 1 Vit + min premix 2.400 2.400 2.400 2.400 2.400 DL-Methionine (84%) 2.289 2.327 2.401 2.042 1.704 Vegetable oil 1.482 5.252 13.277 29.991 47.508 L-Lysine HCl 1.179 1.209 1.246 0.000 0.000 Choline chloride (60%) 0.500 0.500 0.500 0.500 0.500 2 Red pigment 0.800 0.800 0.800 0.800 0.800 3 Yellow pigment 0.500 0.000 0.000 0.000 0.000 4 Antioxidant 0.150 0.150 0.150 0.150 0.150 Bambermycin 0.125 0.125 0.125 0.125 0.125 5 Phytase 0.100 0.100 0.100 0.100 0.100 Total 1000 1000 1000 1000 1000 Calculated analysis Metabolizable energy, 2800 2800 2800 2800 2800 kcal/kg Crude protein, % 17.400 17.400 17.400 18.970 20.480 Total Met + Cys, % 0.730 0.730 0.730 0.730 0.730 Total lysine, % 0.860 0.860 0.860 0.866 0.967 Total threonine, % 0.622 0.623 0.625 0.691 0.754 Total tryptophan, % 0.205 0.199 0.189 0.196 0.201 Crude fiber, % 2.446 2.583 2.831 3.331 3.824 Total calcium, % 4.100 4.100 4.100 4.100 4.100 Available phosphorous, % 0.420 0.420 0.420 0.420 0.420 Sodium, % 0.180 0.180 0.180 0.180 0.180 1

Content per kilogram: Vit. A, 4.0 MUI; Vit. D3, 666,666.7 UI; Vit. E, 10,000.0 UI; Rovimix HyD 5 kg: Vit. K3, 1.67 g; Vit. B1, 0.83 g; Vit. B2, 2.33 g; Vit. B6, 1.17 g; Vit. B12, 6.666.67 mg; niacin, 10 g; D-pantothenic acid, 3.33 g; folic acid, 0.33 g; biotin, 33.33 mg; choline, 100 g; Fe, 20 g; Zn, 26.67 g; Mg, 36.67 g; Cu, 5 g; I, 0.33 g; Se, 0.1 g. 2 Avired 5 g/kg (minimum) xanthophylls from Capsicum annum. 3 Florafil 93 Powder (Vepinsa): 30 g/kg (minimum) total xanthophylls. 4 BHA, 1.2%; BHT, 9.0%; Ethoxyquin, 4.8%; chelating agents, 10.0%.

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Quantum Blue 5000 FTU/kg derived from E. coli.

During the six-week experimental period weekly records were kept of laying percentage, egg weight and mass, and feed intake and feed conversion. At the end of trial 1 (week 3) and trial 2 (week 6), 20 eggs were collected per treatment and yolk color measured with a TSS QCC Yolk Color automated device (Technical Service and Supplies, Inc., England, UK). Readings were transformed to absolute values of the DSM color fan, which range from 1 (light yellow) to 15 (yellow-orange). At the same time, eight eggs per trial were collected and egg yolk pigments quantified by HPLC(10). Yolk color was also measured by refraction colorimetry applying a three-dimensional definition scale based on the CIE system, which quantifies luminosity (L*), yellow hue (b*) and red hue (a*).

Statistical analysis

A completely random design was used with the model(12): Yij = Âľ + Ti + ei(j) i = 1, 2, 3, 4 and 5

j = 1, 2, 3 and 4

Where: Yij = Response variable (laying percentage, feed intake/bird/day (g), egg weight (g), egg mass/day (g), feed conversion (kg:kg), yolk color and pigment quantification; Âľ = General mean; Ti = Effect of i-th treatment; ei(j) = Experimental error. Differences between the means were identified with a Tukey test using a 0.05 significance level, and applied with the SPSS for Windows ver. 21.0 software package. Box-Cox transformations were applied to create variance homogeneity by transforming the pigment quantification variables (i.e. total carotenoids and lutein)(12): Total carotenoids = Lutein =

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Results Pigment quantification of the TDM showed the majority component to be lutein (50.67 %), followed by zeaxanthin (0.65 %) and total carotenoids (0.92 %) (Table 3). Neither egg weight nor feed conversion were affected (P>0.05) by TDM inclusion levels during the sixweek (42-d) experimental period (Table 4). Laying percentage was lower in the 15% TDM treatment (89.8 %) versus the Control and the other inclusion levels. Feed intake/d decreased by an average of 5 g in the 15% TDM treatment versus the other treatments (P<0.05). Egg mass was lowest (P<0.05) in the 15% TDM treatment (52.5 g), higher in the 10% TDM treatment (55.1 g), and did not differ between the 1.77% TDM (55.9 g) and 5% TDM treatments (56.3 g) and the Control (56 g).

Table 3: Tithonia diversifolia meal pigment composition Pigments (g/kg) Wet base Total carotenoids 0.85 Carotene esters 28.10 Î˛-cryptoxanthin 5.90 Trans-lutein 46.70 Trans-zeaxanthin 0.60 Epoxy-trans-lutein 1.80

Dry base 0.92 30.49 6.40 50.67 0.65 1.95

Mean of n = 3.

Table 4: Productive variables, color, pigments and colorimetric values in egg yolk at different levels of Tithonia diversifolia meal inclusion Tithonia diversifolia meal (%) Control 1.77 5 10 15 SME 1 Productive variables Laying, % 94.8a 94.3a 94.8a 92.7ab 89.8b 0.52 Egg weight, g 59.1 59.2 59.4 59.5 58.5 0.24 Feed intake, 105a 105a 106a 105a 100b 0.64 bird/day/g Feed conversion, 1.876 1.892 1.895 1.910 1.908 0.011 kg:kg Egg mass, bird/day/g 56.0a 55.9a 56.3a 55.1ab 52.5b 0.44

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Color2 Yolk color, no red pigment Yolk color, red pigment Pigments (μg/100g)3 Total carotenoids Lutein Zeaxanthin Capsanthin Colorimetric values2 L* a* b*

10a

0.12

11a

10b

11a

0.78

235b 152b 16.5 5.5

142c 87c 9.75 5.25

269ab 173ab 13.25 5.25

367ab 245ab 13.0 6.5

440a 291a 11.75 5.5

31.66 21.06 0.92 0.49

67.38ab -2.66bc 49.49ab

69.40a -4.29c 44.01c

66.02ab -2.71bc 47.47bc

65.21b -1.37b 49.28abc

64.30b 0.44a 5.53a

0.92 0.42 1.30

n=48 birds/treatment. n=20 eggs/ treatment. 3 n=3 samples/ treatment. SME= Standard mean error. abc Different letter superscripts in the same row indicate significant difference (P<0.05). 2

Yolk color based on DSM values, with no added red pigment (Figure 1 A), was lowest in the 1.77% TDM treatment (DSM= 8) and highest in the 10 and 15% TDM treatments (DSM= 10). When red pigment was added to the diet the values followed a similar pattern to when no red pigment was added, with the lowest value (DSM= 8) in the 1.77% TDM treatment (value 8) and higher values in the other treatments and the Control (DSM= 11) (P<0.05) (Figure 1 B). Total carotenoids content was lowest in the 1.77% TDM treatment (142 μg/100 g) and then increased from the Control (235 μg) to the 5% TDM treatment (269 μg), the 10% (367 μg) and the 15% (440 μg). Lutein values were also lowest in the 1.77% TDM treatment (87 μg/100 g) and highest (P<0.05) in the 15% TDM treatment (291 μg). No differences between treatments were observed for zeaxanthin and capsanthin contents.

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Figure 1: Egg yolks from treatments with increasing amounts of Tithonia diversifolia meal

B A= without added red pigment; B= added red pigment.

Discussion Studies of how inclusion of natural ingredients in livestock forages affects animal productive behavior and product quality traits have been done largely in ruminants, particularly in agrosilvopastoral systems. Very little research of this type has been done in monogastric species such as chickens since this type of digestive system does not degrade high fiber diets, although fiber can be used in poultry diets(13). There is therefore only a very limited literature on the use of T. diversifolia in poultry systems and even less comparing it to ruminant systems. In the present results laying percentage, feed intake and egg mass were better in the 5% TDM treatment, which did not differ from the control. Feed conversion was better in the 10% TDM treatment. These data generally coincide with those in a study of the effects of T. diversifolia foliage meal inclusion (5, 10, 15 and 20% TDM, and a commercial feed control) in laying hens in which egg production did not differ between treatments, feed intake was lowest in the 20% TDM (96.3 g/bird/d) compared to the control (107 g/bird/d), and feed conversion was best in the 15% TDM(8). This response may have been due to the negative effects of high fiber content and the presence of antinutritional factors in the 20% TDM treatment, which could compromise absorption of nutrients, mainly of amino acids(14,15).

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Tannins are a common antinutritional factor which can undermine feed palatability by imparting bitter flavors, but can also form complexes with proteins, starches and digestive enzymes, consequently reducing feed nutritional value, and negatively influencing animal growth, feed digestibility and protein and amino acids availability. Lower T. diversifolia inclusion levels do not seem to negatively affect productive performance in poultry; for example, 2 % inclusion of T. diversifolia leaf meal in laying hen diets is reported to improve egg mass and feed conversion(16). Poultry do not synthesize pigments that will add color to skin or egg yolk, which therefore need to be added to diets. The present data on the pigment contents of the studied TDM can function as a standard for its use as a source of xanthophylls for improving egg yolk color. Other natural sources of carotene pigments include alfalfa leaf meal (396 mg/kg), yellow corn (22 mg/kg), dehydrated Ladino clover meal (490 mg/kg), chili meal (187 mg/kg) and Tagetes erecta (Mexican marigold flower) (8,000 to 10,000 mg/kg), which has the highest xanthophylls content (80 to 90 % lutein). When ingested by laying hens xanthophylls enter the blood stream and are deposited in the skin, fatty tissue, the liver and egg yolks; carotenes are incorporated in smaller quantities and are transformed into vitamin A(17). In a study of laying hens fed diets including 5, 10, 15 and 20% TDM, the T. diversifolia was found to be the only source of yellow pigments (lutein and zeaxanthin) in egg yolks(8). Some variation is to be expected between different studies due to study conditions such as the age and element of the plant used as pigment source, animal assay conditions, climate, temperature, etc. Relative to the treatments without red pigment (Trial 1), addition of red pigment to the feed formulation (Trial 2) in the present study notably increased yolk red hue in the Control, 5, 10 and 15% TDM treatments, although not in the 1.77% TDM treatment. This behavior may have been due to the combination of yellow and red colors in the treatments, which resulted in an orange color. Diet yellow pigment content increased as TDM inclusion level increased. Because red pigment was added in comparable amounts to the yellow pigments yolk color differed minimally between treatments, which is why the 1.77% TDM treatment had the least orange color of the red pigment treatments. The two 1.77% TDM treatments (no red pigment, red pigment) had the lowest color values since they were formulated based on an adjusted total carotenoids content of 15 ppm, the same proportion as in the Control, considering total carotenoids as the pigment. The egg yolk pigment content results were therefore directly proportional to TDM inclusion level, with increases in lutein corresponding to approximately 50 % of total carotenoids. In the egg yolk colorimetry values the yellow hue (b*) was higher in the Control and 15% TDM treatments, confirming the majority presence of lutein in the TDM.

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Conclusions and implications The leaves and petioles of Tithonia diversifolia constitute a valid source of natural pigments. They can be included in laying hen diets at up to 10% to improve egg yolk color, without negatively affecting productive parameters. Combination of a natural red pigment (canthaxanthin) with the yellow pigments in the Tithonia diversifolia meal produced egg yolks with a stronger orange color.

Acknowledgements and conflicts of interest The research reported here was financed by the CONACyT, the Sistema Nacional de Becas and the Dirección General de Estudios de Posgrado of the Universidad Nacional Autónoma de México. This study forms part of the Masters in Science degree of Vilma Barrita Ramírez. Thanks are due Gustavo Rodríguez and Dr. Manuel Quiróz of Empresas VEPINSA S.A. de C.V. for their support with laboratory analyses. The authors declare no conflict of interest in the research reported here, and that all authors approved the final manuscript.

Literature cited: 1.

UNA. Unión Nacional de Avicultores: Compendio de Indicadores Económicos del Sector Avícola. Dirección de Estudios Económicos. 2018. http://www.una.org.mx. Consultado 16 Abr, 2018.

Martínez PM, Cortés CA, Ávila GE. Evaluación de tres niveles de pigmento de flor de cempasúchil (Tagetes erecta) sobre la pigmentación de la piel en pollos de engorda. Téc Pecu Méx 2004;42(1):105-111.

Cuca GM, Ávila GE, Pro MA. Alimentación de las aves. Universidad Autónoma de Chapingo. Dirección de patronato universitario. Departamento de Zootecnia. Texcoco, México; 2009.

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CONABIO. Comisión Nacional para el Conocimiento y uso de la Biodiversidad. Catalogo taxonómico de especies en México. http:www.conabio.gob.mx. 2010. Consultado 20 Feb, 2015.

Olabode OS, Ogunyemi S, Akanbi WB, Adesina GO, Babajide PA. Evaluation of Tithonia diversifolia (Hemsl.) Gray for soil improvement. World J Agr Sci 2007;3(4):503-507.

Murgueitio E, Rosales M, Gómez ME. Experiencias sobre la utilización de la Tithonia diversifolia (Hemsl.) A. Grayen Colombia y Panamá. Memorias. VIII Taller Internacional Silvopastoril “Los árboles y arbustos en la ganadería”. [CD-ROM]. EEPF “Indio Hatuey”. Matanzas, Cuba. 2009.

Rosales M. In vitro assessment of the nutritive value of mixtures of leaves from tropical fodder trees [tesis doctorado]. Oxford, UK: Oxford University; 1996.

Odunsi AA, Farinu GO, Akinola JO. Influence of dietary wild sunflower (Tithonia diversifolia) leaf meal on layers’ performance and egg quality. Niger J Anim Prod 1998;23(1-2):28-32.

INEGI. Instituto Nacional de Estadística y Geografía. 2018. http://www.inegi.org.mx. Consultado 16 Abr, 2018.

10. USP 29/NF 24. The United State Pharmacopeia/The National Formulary, United States Pharmacopeia Convention Inc., Rockville, USA: USP 29/NF 24. 2012. 11. NOM. Norma Oficial Mexicana 062-ZOO. Especificaciones técnicas para la producción, cuidado y uso de los animales de laboratorio. Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación, México. 1999. 12. Kuehl RO. Diseño de experimentos. Principios estadísticos de diseño y análisis de investigación. Thomson Editores, SA. de CV. 2ª ed. The University of Arizona, USA. 2001. 13. Sarria P. Forrajes arbóreos en la alimentación de monogástricos. Seminario “Agroforestería para la Producción Animal en América Latina”. Memorias de Conferencias. Universidad Nacional de Colombia, Sede Medellín. 2000. 14. Lezcano Y, Soca M, Ojeda F, Roque E, Fontes D, Santana H, Martínez J, Cubillas N. Caracterización bromatológica de Tithonia diversifolia (Hemsl.) A. Gray en dos etapas de su ciclo fisiológico. Pastos y Forrajes 2012;35(3):275-282.

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15. Rodríguez B, Savón L, Vázquez Y, Ruíz TE, Herrera M. Evaluación de la harina de forraje de Tithonia diversifolia para la alimentación de gallinas ponedoras. Livestock Res Rural Develop 2019; http://www.Irr.org/Irrd30/3/brod30056.html/ 16. Yalçin S, Özsoy B, Erol H. Yeast culture supplementation to laying hen diets containing Soybean meal or Sunflower seed meal and its effect on performance, egg quality traits and blood chemistry. J Appl Poultry Res 2008;15(2):229-236. 17. Cuca M, Pino JA, Mendoza C. El uso de pigmentos en la alimentación de las aves. Tec Pecu Méx 1963;2:39-42.

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https://doi.org/10.22319/rmcp.v11i2.4843 Article

Effect of a multienzyme complex and a probiotic in laying hens fed sorghum-soybean-canola diets

Pedro Juárez Morales a Arturo Cortes Cuevas a* José Arce Menocal b Juan Carlos Del Río García c Gabriela Gómez Verduzco a Ernesto Avila González a

Universidad Nacional Autónoma de México, Facultad de Medicina Veterinaria y Zootecnia, Centro de Enseñanza, Investigación y Extensión en Producción Avícola. Manuel M. López s/n Col. Zapotitlán, 13209, Tláhuac, Ciudad de México, México. b

Universidad Michoacana de San Nicolás de Hidalgo, Facultad de Medicina Veterinaria y Zootecnia. Morelia, Michoacán, México. c

Universidad Nacional Autónoma de México, Facultad de Estudios Superiores Cuautitlán. Estado de México, México.

*Corresponding author: cortesca68@gmail.com

Abstract: This study aimed to evaluate the productive parameters and the concentrations of serum intestinal secretory IgA, cholesterol, LDL, and HDL of laying hens fed sorghum + soybean + canola diets with lower content of nutrients and added with a multienzyme complex (proteases, amylases, and xylanases) and a probiotic (Bacillus subtilis). A total of 180 Bovans White laying hens, between 42 and 54 wk of age, were randomly assigned to three treatments: 1) control diet; 2) low metabolizable energy diet (50 kcal/kg and 2 % of protein

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and methionine and lysine amino acids) + enzymes; 3) diet number two + probiotic. Productive performance results showed a significant difference (P<0.05) in egg weight between both treatments; treatment 3, added with the enzymes and probiotic, increased egg weight, compared to treatment 2. Humoral immunity, cholesterol, LDL, and HDL variables showed no statistical differences (P> 0.05) between treatments. This study demonstrates that low-nutrient (ME, protein, and lysine and methionine amino acids) diets added with a multienzyme complex and probiotic allow similar results in productive parameters compared to the control diet, and without changing intestinal immunity and levels of cholesterol, high- and low-density lipoproteins in Bovans White hens. Key words: Enzymes, Bacillus subtilis, Laying hen, Immunity, Cholesterol.

Received: 05/04/2018 Accepted: 26/06/2019

Introduction In the last decade, the use of exogenous enzymes in poultry diets has increased in order to enhance energy and protein digestibility(1,2). Non-starch polysaccharides (NSP) are the major components of fiber in traditional ingredients; previous studies indicate that sorghum contains around 6.5 % and soybean meal 3.3 % of these polysaccharides(3). Cereal grains contain arabinoxylans and glucans, while soybean and canola contain arabinans, arabinogalactans, galactans, mannans, and pectins(4,5). The NSP of cell walls, such as soluble and insoluble arabinoxylans, are degraded by xylanases, releasing nutrients encapsulated within the cell wall and improving access to endogenous enzymes(6,7). In the food industry, xylanases and Î˛-glucanases(8) are widely used to degrade NSP and reduce the loss of endogenous amino acids(9). Products with multienzyme activity have shown that the combination of xylanases, amylases, and proteases enhances digestibility in poultry diets(10,11,12). Amerah et al(9) reported that corn-soybean meal diets supplemented with proteases, amylases, and xylanases improved the ileal digestibility of protein, energy, as well as nitrogen retention in broiler chickens, which translated into higher productive performances. The addition of beneficial bacteria, such as those of the Bacillus genus, to poultry diets, is an alternative to the use of antibiotics for growth promotion(13,14,15). 370

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Recent studies of a commercial product based on three Bacillus strains have shown positive effect in corn-soybean diets(16). Since commercial poultry diets already include exogenous enzymes and probiotics, there is little information about how they interact with each other; enzymes are associated with an intestinal probiotic effect in chickens(12,17). They also modulate the immune response and suppress the inflammatory immune reactions in the intestinal walls(18). Moreover, a previous study reported that, in laying hens, the addition of probiotics improves feed conversion and egg quality (decrease in yolk cholesterol level, increase in shell thickness and egg weight), and decreases blood cholesterol levels(19). This study aimed to evaluate, in Bovans White hens, the effect of the addition of a multienzyme complex (xylanases, proteases, and amylases) alone or combined with a probiotic (Bacillus subtilis) in sorghum-soybean-canola diets with low energy and protein content, on production parameters, production of intestinal secretory IgA, and serum levels of cholesterol, LDL, HDL.

Material and methods The experiment was carried out in the facilities of the Center for Teaching, Research and Extension in Poultry Production (CEIEPA) of the Faculty of Veterinary Medicine and Animal Husbandry of the Universidad Nacional AutĂłnoma de MĂŠxico (UNAM). A total of 180 Bovans White 42-wk-old laying hens were randomly assigned to three treatments with five replicates of 12 hens each, which were distributed as follows: 1) control diet; 2) low metabolizable energy and protein diet (50 kcal/kg) with limiting methionine and lysine amino acids (2 % of control diet) + enzymes; 3) diet number two + probiotic. Diets are shown in Table 1. The positive control diet complies with the recommendations for the productive stage of the Bovans White strain and another control diet with low metabolizable energy (ME), protein, and essential amino acids. The reduction of these nutrients considerably reduced the amount of soybean meal and vegetable oil in the diet. Both were commercial diets and included phytase. The low-nutrient diets were supplemented with two commercial products: a multienzyme complex (Axtra XAPR 101 TPT, Dupont, Animal Nutrition) at a rate of 250 g/t of feed 371

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containing xylanases (20,000 U/g derived from Trichoderma longibrachiatum), proteases (40,000 U/g derived from Bacillus subtilis), and amylases (2,000 U/g derived from Bacillus licheniformis); and a probiotic containing three strains of B. subtilis (Enviva PRO 201 GTR, Dupont, Animal Nutrition 3E+08 CFU/g) at a rate of 250 g/t of feed. Table 1: Composition and calculated analysis of basal sorghum-soybean meal-canola meal diets added with a multienzyme complex and a probiotic in 42- to 54-wk-old laying hens Ingredients Sorghum Soybean meal Canola Vegetable oil Calcium orthophosphate Calcium carbonate Salt DL-Methionine 99 % L-Lysine HCl 78 % Vitamins and minerals * Bacitracin MD 10 % Choline chloride 60 % Capsicum red pigment ** Larvicide BHT Antioxidant Apo-ester 10 % Phytase Total Metabolizable energy, kcal/kg Crude protein, % Total lysine. % Total Met+Cyst, % Total calcium, % Available phosphorus, % Sodium, %

Control + (kg) Control - (kg) 653.780 675.850 148.520 136.950 58.160 58.160 13.720 3.000 9.190 9.200 105.530 105.560 4.320 4.320 1.340 1.310 0.470 0.670 2.400 2.400 0.500 0.500 0.500 0.500 0.800 0.800 0.500 0.500 0.150 0.150 0.050 0.050 0.045 0.045 1000 1000 Calculated analysis 2800 2750 15.79 15.41*** 0.73 0.71*** 0.67 0.66*** 4.25 4.25 0.46 0.46 0.18 0.18

* Vit A, 3000 000 IU; Vit D3, 750 000 IU; Vit E, 6 000 IU; Vit K3, 1.0 g; niacin, 25 g; biotin, 0.063 g; choline chloride, 250 g; selenium, 0.2 g; cobalt, 0.1 g; iodine, 0.3 g; copper, 10 g; zinc, 50 g; iron, 100 g; manganese, 100 g; excipient qs 1.000.00 g. **Capsicum red pigment (Avired powder) plant-based colorant, 5 g/kg ***2 % reduction compared to the control diet.

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Birds were housed in a naturally ventilated broiler house, in cages with three chickens, for 12 wk. Artificial and natural light were provided for a total of 16 h daily. The experimental laying hens were fed sorghum-soybean-canola diets in the form of flour, at a rate of 105 g/bird/d; with free access to water. Weekly, during the 12 wk of the study, productive data were recorded and summarized; egg weight, feed consumption per bird per day, and conversion ratio. At the end of the study, 20 eggs per treatment were used to determine shell thickness manually using a micrometer without considering internal membranes; Haugh units and yolk color were determined using the QCM+ automated system from Technical Services and Supplies INC (TSS). The intestinal antibody response was evaluated in five hens per treatment; animals were selected and processed in the slaughterhouse following the Official Mexican Standard NOM-033-ZOO-1995 for Humane slaughter of domestic and wild animals. The intestinal lumen of 10 cm ileal samples were washed three times with 10 ml of cold and sterile isotonic saline solution (ISS), the washing solution was collected and frozen at -20 Â°C until its subsequent evaluation with the ELISA test following the procedure previously described by GĂłmez(20). At 54 wk of age, blood samples were collected from 30 hens (10 hens per treatment), each sample was centrifuged at 3,000 rpm/10 min to obtain the serum and determine the levels of cholesterol, LDL, and HDL in the Pathology Department of the Faculty of Veterinary Medicine and Animal Husbandry, UNAM. Results were transformed from mmol/dL to mg/dl using the conversion factor 0.0259. Productive variables, egg yolk cholesterol, serum cholesterol, LDL, and HDL data were subjected to an analysis of variance based on a completely randomized design, and the differences between treatments were compared by Tukey test using the statistical software IBM SPSS Statistics v. 19(21).

Results Table 2 shows the data obtained in 84 days of experimentation. Laying percentage, feed intake, and feed conversion results were similar (P>0.05) between treatments. However, egg weight was higher (P<0.05) in hens fed with the control diet (T1) and the reduced diet with enzymes + probiotic (T3).

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Table 2: Productive variables of 42- to 54-week-old Bovans hens fed sorghum-soybeancanola diets Laying Egg weight Feed Feed Diets (%) (g) intake/bird/d conversion (g) (g/g) 1) Control diet 92.2±1.9 60.0±0.4a 104±0.5 1.88±0.04 2) Low-nutrient diet + enzymes 92.8±1.7 58.9±0.7b 104±0.8 1.90±0.03 3) Diet 2 + probiotic 91.5±1.7 59.2±0.4ab 104±0.8 1.91±0.03 a,b

Values ± standard error Values with different letters are statistically different (P<0.05).

Table 3 shows data obtained during the experiment on egg quality. The results of Haugh units, shell thickness, and DSM yolk color fan were similar between treatments (P>0.05). Table 3: Quality variables of eggs laid by 42-wk-old hens fed sorghum-soybean mealcanola meal diets Diets Shell thickness Haugh Yolk color (DSM (µm) units fan) 1) Control diet 337±7.7 90.8±2.8 9.4±2.8 2) Low-nutrient diet + enzymes 352±7.2 92.2±2.5 9.2±0.4 3) Diet 2 + probiotic 347±9.2 91.2±1.8 9.4±0.5 Values ± standard error. (P>0.05).

Table 4 shows the average results of the analyses of serum cholesterol, LDL, HDL, and the production of intestinal secretory IgA. There were no statistical differences between treatments (P>0.05). Table 4: Changes in serum cholesterol, LDH, HDL, and secretory IgA content in 42 to 54 wk-old Bovans hens fed sorghum-soybean meal-canola meal diets Diets Cholesterol LDL HDL Secretory (mg/dL) (mg/dL) (mg/dL) IgA (%) 1) Control diet 100.2±26.2 13.9±10.8 30.0±2.5 40.9±30.4 2) Low-nutrient diet + enzymes 109.7±15.7 17.3±2.3 37.0±5.2 68.7±34.0 3) Diet 2 + probiotic 122.9±21.1 16.2±0.9 35.7±5.0 63.0±27.9 Values ± standard error. (P>0.05).

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Discussion These results are similar to those reported by Wena et al(22), who evaluated an enzyme cocktail in corn-soybean diets with lower nutrient content, and found that the nutritional value of the feed was improved; for this reason, enzymes are used in the food industry to reduce formulation costs without affecting productive behavior(8); similarly, in this study, nutrient reduced diets, based on the nutritional recommendations for the Bovans White strain, had a lower content of soybean meal and oil. Sobczak and Kozlowski(23) evaluated the effect of adding Bacillus subtilis on egg production without significant changes in egg weight, laying percentage, feed intake, and feed conversion. Other studies also show that the use of probiotics in hen diets has no influence on productive performance(24). Higher egg weights were observed in diets without nutrient reduction, which probably suggests that the reduction of 50 kcal of ME in sorghum-soybean-canola+enzymes diets did not provide a sufficient amount of ME for the multienzyme complex. However, the diets supplemented with the enzymes and probiotic increased egg weight, probably due to the promotion of a more favorable microbiome and enhanced intestinal health caused by Bacillus subtilis. Amerah et al(9) also demonstrated that amylases, xylanases, and proteases, alone or combined, increase non-starchy polysaccharides digestibility. However, a different study(16) evaluated the productive performance of broilers fed diets supplemented with phytase, alone or combined with xylanases, amylases, proteases, and Bacillus amyloliqueciens as a probiotic; there were no significant positive effects. Although the combination did increase the apparent ileal digestibility of sugars and fat, with an increase in ME; it also reduced the pathogenic bacteria populations. A previous study reports that corn-soybean-canola meal diets supplemented with carbohydrases and proteases increase weight gain and feed conversion; it also enhances protein digestibility and metabolizable energy(25); these results differ from findings of this study since these productive parameters were not improved. The cholesterol, HDL, and LDH data showed no significant difference in this study; however, Salma et al(26) used different concentrations of a Rhodobacter capsulatus probiotic in hens fed corn-soybean diets and reported that the diets with a higher concentration of the probiotic increased serum high-density lipoproteins (HDL), cholesterol, and the atherogenic index. Recently, a study reported(3) that the addition of 0, 250, 450, and 900 U/kg of xylanases derived from the fermentation of Bacillus subtilis to corn-wheat-soybean meal diets fed to Hy-Line Brown hens, did not improve the productive parameters; however, there was an effect on 375

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shell thickness and Haugh units; which does not agree with the results of the present study.

Conclusions and implications The use of a multienzyme complex of amylases, proteases, and xylanases plus a Bacillus subtilis spore-based probiotic in sorghum-soybean-canola diets with the nutritional recommendations for the Bovans White strain, allows reducing the ME in 50 kcal/kg, as well as the protein and essential amino acids, lysine and methionine, in 2 %, with no detrimental effect on the productive performance of 42- to 54 wk-old Bovans White hens. The enzyme complex and Bacillus subtilis probiotic did not affect the intestinal secretory IgA or serum cholesterol, HDL, and LDH values.

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Amerah AM, Romero LF, Awati A, Ravindran V. Effect of exogenous xylanase, amylase, and protease as single or combined activities on nutrient digestibility and growth performance of broilers fed corn-soy diets. Poult Sci 2017;96(4):807-816.

10. Cowieson AJ, Ravindran V. Effect of exogenous enzymes in maize-based diets varying in nutrient density for young broilers: Growth performance and digestibility of energy, minerals and amino acids. Br Poult Sci 2008;49(1):37-44. 11. Tang D, Hao S, Liu G, Nian F, Ru Y. Effects of maize source and complex enzymes on performance and nutrient utilization of broilers. Asian-Austral J Anim Sci 2014;27(6):1755-1762. 12. Romero LF, Sands JS, Indrakumar SE, Plumstead PW, Dalsgaard S, Ravindran V. Contribution of protein, starch, and fat to the apparent ileal digestible energy of cornand wheat-based diets in response to exogenous xylanase and amylase without or with protease. Poult Sci 2014;93(1):1-13. 13. Rinttilä T. Apajalahti J. Intestinal microbiota and metabolites Implications for broiler chicken health and performance. J Appl Poult Res 2013; 22(5):647–658. 14. Latorre JD, Hernandez X, Kallapura G, Mencini A. Evaluation of germination, distribution, and persistence of Bacillus subtilis spores through the gastrointestinal tract of chickens. Poult Sci 2014;93(8):1793–1800. 15. Ribeiro LFT, Albino HS, Rostagno SLT, Barreto MI, Hannas D, Harrington FA, et al. Effects of the dietary supplementation of Bacillus subtilis levels on performance, egg quality and excreta moisture of layers. Anim Feed Sci Technol 2014;195(1):142-146. 377

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16. Wealleans AL, Walsh MC, Romero LF, Ravindran V. Comparative effects of two multi-enzyme combinations and a Bacillus probiotic on growth performance, digestibility of energy and nutrients, disappearance of non-starch polysaccharides, and gut microflora in broiler chickens. Poult Sci 2017;96(4):4287-4297. 17. Romero LF, Parsons CM, Utterback PL, Plumstead PW, Ravindran V. Comparative effects of dietary carbohydrates without or with protease on the ileal digestibility of energy and amino acids and AME. Anim Feed Sci Technol 2017;181(1):35-44. 18. Li L, Xu CL, Ji C, Ma Q, Hao K, Jin ZY, Li K. Effects of a dried Bacillus subtilis culture on egg quality. Poult Sci 2006;85(2):364–368. 19. Sohail HK, Muhammad A, Nasir M. Effects of supplementation of multi-enzyme and multi-species probiotic on production performance, egg quality, cholesterol level and immune system in laying hens. J Appl Anim Res 2011;39(3):386-398. 20. Gómez VGG. Modulación nutricional de la inmunidad en pollo de engorda mediante el empleo de un estimulante (paredes de levaduras de Saccharomyces cerevisiae) [tesis doctorado]. México, CDMX: Universidad Nacional Autónoma de México; 2009. 21. IBM®SPSS®STATISTICS VER. 19.0.0©COPYRIGHT SPSS Inc. 1989 2010.: 22. Wena LC, Wanga YM, Zhoua Z, Jiangb Y, Wanga T. Effect of enzyme preparation on egg production, nutrient retention, digestive enzyme activities and pancreatic enzyme messenger RNA expression of late-phase laying hens. Anim Feed Sci Technol 2012;172(1):180–186. 23. Sobczak A, Kozlowski K. The effect of a probiotic preparation containing Bacillus subtilis ATCC PTA-6737 on egg production and physiological parameters of laying hens. Ann Anim Sci 2015;15(4):711–723. 24. Panda AK, Reddy MR, Rama Rao SV, Praharaj NK. Production performance, serum/yolk cholesterol and immune competence of White Leghorn layers as influenced by dietary supplementation with probiotic. Trop Anim Health Prod 2003;35(1):85–94. 25. Toghyani M, Wu SB, Pérez-Maldonado RA, Iji PA, Swick RA. Performance, nutrient utilization, and energy partitioning in broiler chickens offered high canola meal diets 378

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supplemented with multicomponent carbohydrate and mono-component protease. Poult Sci 2017;96(12):3960-3972. 26. Salma U, Miah AG, Tareq KM, Maki T, Tsujii H. Effect of dietary Rhodobacter capsulatus on egg-yolk cholesterol in laying hen performance. Poult Sci 2007;86(7):714-719.

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https://doi.org/10.22319/rmcp.v11i2.5152 Article

Phenotypic and genetic trends for peak yield, milk yield, and lactation persistency in the Murciano-Granadina breed

Judith Carmen Miranda Alejo a,b José Manuel León Jurado b,e Camillo Pieramati c Mayra Mercedes Gómez Carpio b* Jesús Valdés Hernández d Cecilio José Barba Capote a

Universidad de Córdoba. Departamento de Producción Animal, Campus de Rabanales. Edif. de Producción Animal, Córdoba 14071, España. b

Universidad de Córdoba. Departamento de Genética. Campus de Rabanales. Córdoba, España. c

Università Degli Studi di Perugia. Dipartimento di Medicina Veterinaria. Perugia, Italy.

Universidad Autónoma de Barcelona. Departamento de Ciencia Animal y de los Alimentos. Centre for Research in Agricultural Genomics, Barcelona, España e

Delegación de Agricultura y Medio Ambiente. Centro Agropecuario Provincial. Diputación de Córdoba, Córdoba. España

* Corresponding author: mayragomezcarpio@gmail.com

Abstract: This study evaluated the genetic (TG) and phenotypic (TF) trends for the peak yield (PP), milk yield (RL), and lactation persistency (P) traits of the Murciano-Granadina (MG) goat lactation curve obtained from 80,872 lactations of 85,404 goats (historical records from 1990-2012). The biomodelling of lactation curves allowed us to estimate the PP, RL, and P traits using the Spline model in the “R” software. The genetic values (VGs) were obtained using the univariate animal model with repeated observations, employing the 380

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MTDFREML package. The TG and TF were estimated via the least-squares regression of the average of VGs and known yield information according to the year of birth. While calculating the TG and TF, linear regression coefficients (b) were obtained, where the b values for PP, RL, and P were +0.00071, +0.00698; +0.00114, +0.01117; and +0.00002, -0.00076, respectively. The TG and TF for the PP and RL behaved similarly following an upward trend line with increasing and decreasing intervals. The TG for P showed seasonal variations while the TF followed a downward trend with more consistent points in its trajectory; supporting the idea that high yields are detrimental to the P. These results inform the breeders about the behavior of these traits and provide the basis for the incorporation of the lactation persistency as a selection criteria in the genetic program of the MG breed. Key words: Breeding program, Goat, Milk, Genetic value.

Received: 19/11/2018 Accepted: 19/03/2019

Introduction Goats are relevant worldwide, especially in developing countries, where they are bred with multifunction criteria(1). However, dairy goat breeds are especially important in the southern European livestock, and even though their census is low compared to the total global population, France and Spain have achieved considerable yield increases and more extended lactation periods. This productive efficiency responds to the need to provide large quantities of milk to the high-quality goat-cheese industry, linked to deep-rooted cultural traditions in some European countries, which continue to offer an optimistic perspective for the goat sector(2). The Murciano-Granadina (MG) breed is part of a select group of specialized dairy goats, becoming one of the leading dairy breeds of Spain, both in census (104,000 breeding females included in the Breed Registry) and yields (584.4 kg of milk per lactation)(3). This information positions the MG breed in the first order, next to the Florida and MalagueĂąa breeds, due to its high potential yield compared to other native Spanish breeds like the Guadarrama, Majorera, Palmera, Payoya, and TinerfeĂąa goats. The MG breed was officially recognized in the seventies, and since then, there are references on the Scheme for the Genetic-Functional Assessment of Reproductive Males (Resolution of March 28, 1979, of the Directorate General for Agricultural Products(4)). The current animal genealogical and functional records integrate a robust database, which allows breeders to perform genetic evaluations.

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The current MG selection program has been consolidated after 29 yr of selection, emphasizing traits like milk yield, fat, protein, and dry extract yield. In this case, the estimation of genetic trends (TG) is important to evaluate the efficacy of the applied breeding schemes and to provide the breeders with information that will allow them to develop more efficient selection programs(5). Also, the evaluation of TG and phenotypic trends (TF) improves the understanding and helps transmit the selection effect compared to previous generations(6,7), as well as to contrast the obtained results depending on the proposed scheme, allowing to correct any deviation from the expected. In reference studies performed in dairy species, researchers have generally focused on the TG and TF for milk yield (RL) and composition traits; however, the genetic behavior of other traits related to the lactation curve has not been reported (8). Furthermore, this is the first study of the TGs for lactation curve parameters, peak yield (PP) and persistency (P) in particular, in MG goats. This study aims to justify the consolidation of these traits for their inclusion as selection criteria in the MG breeding program. Therefore, this study aimed to evaluate the TG and TF for the PP, RL, and P traits in the Murciano-Granadina (MG) breed.

Material and methods Data and editing procedures

The genealogy and functional information used in this study belong to the historical archives of the MG goat official breeding program. The original database included 180,872 lactations, adjusted to 210 d (A4 method of ICAR (9)), from 85,404 goats from 229 livestock farms, encompassing the birth years of 1990-2012. During the exploratory analysis, the database was edited and standardized to eliminate the data considered anomalous: repeated data, lactations with less than six controls, daily yields exceeding 10 kg of milk or below 0.2 kg, and null values; based on the standardization carried out by the MG goat breeding program.

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Genetic analysis and statistical models

The biomodelling of individualized lactation curves allowed to estimate the PP, RL, and P traits using the Spline model for its best fit for the breed, R2, and flexibility(10). Also, for the model resolution, it was used the statistical software “R“ version 3.2.3(11). The P values of lactation are mainly expressed as dimensionless measures(12). The individual values of the traits (PP, RL y P) in the lactation curves were analyzed using an animal model with repeated observations and the univariate option (single trait). The single trait model used is written in matrix notation (13). y = Xb + Zaa + Zpp + e Where: y= is the phenotypic information vector of the analyzed PP, RL, and P traits. b= vector of the fixed effects (contemporary group of herd-year-season (HYS-unified), lactation number, birth type, and age (covariable)). a= vector of the additive random effect of the animal. p= vector of the permanent environmental effect. e= vector of residual effects for the analyzed traits. X, Za, and Zp: incidence matrices (known) of fixed effects (X) and random effects (Za and Zp). The variance components for all the random effects were estimated with the Multiple Trait Derivate Free Restricted Maximum Likelihood (MTDFREML) program(14) and adjusting the univariate animal model (described previously). To evaluate the logic estimations, we used a convergence criterium of Var [-2log(L)] < 1x10-9 (where L represents the likelihood function). The genetic values (VG) for the traits of interest were estimated using the best linear unbiased prediction method (BLUP) (15). To estimate the TG and TF, the estimated VGs were adjusted in a fixed-effects model with the year of birth as the only fixed effect. Alternatively, for the traits of interest, we also estimated the environmental trends (TA) using the predictions from the linear regression coefficients (b) obtained from the average of the VGs expressed by year of birth and known yield information in that year. These procedures were carried out with the statistical program “R”(11).

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Results The descriptive statistics for the analyzed traits revealed a typical mean and deviation of 1.05 ± 0.32 and 1.21 ± 0.35 kg of milk for RL and PP, respectively, and 1.03 ± 0.35 for P. Figures 1-6 show the TG and TF regarding the year of birth for the analyzed traits with their respective linear regression coefficient (b). These results suggest dynamic variations for the MG goat population, detecting a rebound stationary point in 1999, which coincides with the start of the modern selection scheme based on the BLUP evaluations. Subsequently, oscillations around the X-axis for these three traits reveal that for 22 year, the PP and RL traits showed an upward trend behavior and similar magnitudes; meanwhile, the P trait showed a stationary to a downward trend.

Figure 1: Peak yield genetic trend

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Figure 2: Yield genetic trend

Figure 3: Persistency genetic trait

Figure 4: Peak yield phenotypic trend

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Figure 5: Yield phenotypic trend

Figure 6: Persistency phenotypic trend

The TGs for PP and RL over the analyzed years (1990 to 2012) showed an irregular behavior with increasing and decreasing intervals (Figures 1 and 2) but following an upward trend line with coincident peaks in the years 1992, 1996, 2004, and 2009. For these same traits, the TFs (Figures 4 and 5), also show upward trend lines with regular and consistent fluctuations in the increasing/decreasing intervals (coincident prominent peaks in the years of 1992 and 1998). The TG for the P trait shows an irregular behavior with increases, decreases, and prominent peaks in the years of 1993, 1995, 2005, 2010, and 2012, unlike the behavior of the other traits that show a stationary trend line (Figure 3). 386

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Particularly, we observed a positive TG for PP and RL and irregular deviations from linearity (Figure 1 and 2). The increase for these treats was significant (P<0.05) with a b of 0.00071 kg/year and 0.00114 kg/year for PP and RL, respectively. Similarly, the TG for P was also positive with prominent peaks (1993, 1995, 2005, 2010, and 2012) and notorious decreases (1992, 1998, 2006 to 2009), showing irregular fluctuation between them with a no-significant increase (P>0.05) and a b of 0.0000219 units/year with a stationary trend line (Figure 3). Although the TF direction for PP and RL shows an upward trend line, the fluctuations of the increasing/decreasing intervals are also regular and consistent due to the proximity between the points (prominent coincident peaks in 1992 and 1998) and the trend line (Figures 4 and 5). Moreover, the TF for P (Figure 6) decreased when the fluctuations of the increasing and decreasing intervals were regular and consistent in time, with peaks in the years of 1991, 1993, 2002, and 2012. Consequently, the TFs were positive for PP and RL showing a significant (P<0.05) increase in the TF average for these traits, where b was 0.0069821 kg/year and 0.0111697 kg/year, respectively (Figures 4 and 5). However, although the TF for P was negative with regular fluctuations and a significant (P<0.05) decrease with a b value of -0.0007629 units/year (Figure 6), there is also a slight increase in this trend since the year of 2009, which stabilizes during 2010, with an increase in 2011. The TA analysis for P suggests a negative influence, although not significant (P>0.05 with b of -0.00000293 units/year).

Discussion The TGs monitor and evaluate the selection program efficiency(16). The TG evaluation of dairy traits indicates the direction of the breed selection vector, as well as the rate of genetic improvement due to the application of the breeding program(17). It is also essential to provide information to the breeders for them to develop more efficient selection programs(5). In this study, the direct estimation of the TG indicated that there was a significant and positive genetic improvement in all the studied traits, except for the P trait, which indicates that selection was effective, both in the period based in bulk selection (19901999) and in familiar information (1999-2012). Therefore, this information could be useful to evaluate previous efforts for the improvement and preservation of the genetic potential of these traits and to determine the future strategies and work in the population of MG goats. It is important to consider that this is the first study in which the PP, RL, and P traits are evaluated as a group, RL being a trait routinely used for breed selection, while PP and P are candidate traits for possible inclusion in the selection scheme. Our results show that, 387

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in the 22 analyzed years, the PP and RL traits showed a similar and upward trend behavior, while the P trait showed a stationary to a downward trend, which supports the idea that high yields are detrimental to the P. The results show that both the TG and TF for the three traits (PP, RL, and P) have an irregular behavior with fluctuating increasing and decreasing intervals, most noticeable in the years before 1999; until that year, the selection was based on the phenotype (bulk selection). However, since 1999, when the annual mean VG trends for the PP and RL traits present a more constant-positive evolution, consistent with the year when the work guidelines for the selection scheme of dairy goat stallions of the MG breed were established(4), based on family information and BLUP estimations, according to the guidelines published in the Resolution of May 12, 1999, of the Ministry of Agriculture, Fisheries, and Food. Additionally, the reference points of the mean VGs are closer to the trend line, with prominent peaks in the years of 2004 and 2009, and noticeable decreases in the years of 2005, 2006, 2011, and 2012; according to MURCIGRAN (4), they could be related to the incorporation of animals to the selection schemes from livestock farms outside the selective nucleus in the mentioned years. The TGs for PP and RL are parallel, prominent peaks are observed in years before 1999; the most noticeable decrease occurred in 2012. In this regard, in December 2011 it was officially agreed to merge the associations of CAPRIGRAN and ACRIMUR in MURCIGRAN(4), leading to the management of a single Breed Book, and therefore the data and records thereof, which would justify the noticeable decreases between 2011 and 2012 in both the TG and TF. Even though the TG for P has prominent peaks (1993, 1995, 2005, 2010, and 2012) and notorious decreases (1992, 1998, 2006 to 2009) in addition to alternating and irregular between them (sic), it shows a positive seasonal trend line. Meanwhile, the TF is a discrete decreasing trend line with less prominent peaks in the years 1991, 1993, 2002, and 2012. This information supports the idea of paying attention to the P trait and its group behavior with others because of its direct impact on the lactation curves. Lengthening the P would allow for flattening the declining part of the lactation curve, or at least mitigate the crucial peaks that affect the immune system (the antagonistic relationship between milk production and disease resistance traits) promoting a more efficient lactation with considerable benefits, such as avoiding animal health risks and associated costs(18,19). In this regard, specific studies in dairy goats conclude that animal selection using P as a criterium and linked to the peak yield value is possible without changing the amount of total milk(20), and therefore genetically modify the lactation curve(19). Thus, early estimation of P in lactations in progress can represent a useful tool for both the breeding and management strategies(21), especially in the implementation of strategies to reduce the production of greenhouse gases derived from the fight for climate change mitigation(22). 388

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The TG estimations evaluate the results of the program and inform the breeders about the selection decisions made, allowing the necessary adjustments to optimize the genetic progress of each population(23); therefore, the results of this evaluation allow to propose the incorporation on the P trait to the breeding program. Finally, in general, the phenotypes and the breeding values for the PP and RL traits increased in the period of study; however, although the P trait was genetically stationary, its TF was decreasing, probably due to environmental factors, such as the change of management. Studies referred to in this regard indicate that the environment does not directly modify the genetic constitution of the individual. However, it does determine the extent of its expression and the genetic potential of animals, which will express to the extent that environmental conditions allow(24).

Conclusions and implications The global analysis of the TG and TF for the analyzed traits demonstrated irregular dynamic variations with increasing and decreasing intervals, as well as peaks in determined points throughout the study. The genetic and phenotypic behaviors of these three traits indicate that, in 22 years, the PP and RL traits showed a similar upward trend and magnitude, while the P trait was stationary to decreasing. The latter supports the idea that high yields are detrimental to the P. In all cases, important events in the breeding program, like the federation of associations or the introduction of family selections, left their mark on trends. These findings will allow to inform breeders about the behavior of these traits and consider incorporating the P in the selection scheme.

Acknowledgements and conflict of interests Authors thank the Spanish Federation of Goat Breeders, Murciano-Granadina (MURCIGRAN) breed. A special thanks to MsC. BolĂvar Samuel Sosa Madrid for his valuable contributions to the manuscript. There is no conflict of interest.

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Adewumi OO, Oluwatosin BO, Tona GO, Williams TJ, Olajide OO. Milk yield and milk composition of Kalahari Red goat and the performance of their kids in the humid zone. Arch Zootec 2017; 66:587-592.

FAO. Producción y productos lácteos: pequeños rumiantes. 2018. http://www.fao.org/agriculture/dairy-gateway/produccion/pequenos-rumiantes/#3. Consultada: 25 Feb, 2018.

Delgado JV, Landi V, Barba CJ, Fernández J, Gómez MM, Camacho ME, et al. Murciano-Granadina Goat: A Spanish local breed ready for the challenges of the twenty-first century. Simões J, Gutiérrez C. editors. In: Sustainable goat production in adverse environments: Volume II. Springer International Publishing; 2017:205– 219.

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Khojastehkey M, Aslaminejad AA. Study of the environmental, genetic and phenotypic trends for pelt traits and body weight traits in Zandi sheep. J Appl Anim Res 2013;41:356–361.

Kuthu ZH, Javed K, Babar ME, Sattar A, Abdullah M. Estimation of genetic parameters for pre-weaning growth traits in Teddy goats. J Anim Plant Sci 2017;27:1408–1414.

Filho KE, Silva L, Alves R, Figuereido GR. Tendência genética na raça Gir. Pesqui Agropecu Bras 2000;35:787–791.

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ICAR. International Committee for Animal Recording Rome, Italy;1990. http://www.icar.org/

10. León JM, Macciotta NPP, Gama LT, Barba C, Delgado JV. Characterization of the lactation curve in Murciano-Granadina dairy goats. Small Ruminant Res 2012;107:76–84.

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11. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. R-project; 2014. https://www.r-project.org/. 12. Macciotta NPP, Dimauro C, Steri R, Cappio-Borlino A. Mathematical modelling of goat lactation curves. In: Antonello Cannas G, Pulina A, editors. Dairy goats feeding and nutrition. Sassari: CAB International; 2008:31–46. 13. Mrode RA. Linear models for the prediction of animal breeding values. 3th ed. Hulbert S, Povey L, editors. Malta; 2014. 14. Boldman KG, Kriese LA, Van Vleck LD, Van Tassel CP, Kachman SD. A manual for use of MTDFREML. A set of programs to obtain estimates of variances and covariances (DRAFT). Lincoln, NE: USDA. ARS. 1995. 15. Henderson CR. A simple method for computing the inverse of a numerator Relationship matrix used in prediction of breeding values. Biometrics 1976;32:6983. 16. Montaldo H, Almanza A, Juárez A. Genetic group, age and season effects on lactation curve shape in goats. Small Ruminant Res 1997; 24:195–202. 17. Bosso NA, Cissé MF, van der Waaij EH, Fall A, van Arendonk JAM. Genetic and phenotypic parameters of body weight in West African Dwarf goat and Djallonké sheep. Small Ruminant Res 2007; 67:271–278. 18. Capuco AV, Ellis SE, Hale SA, Long E, Erdman RA, Zhao X and Paape MJ. Lactation persistency: insights from mammary cell proliferation studies. J Anim Sci 2003;81 Suppl 3:1831. 19. Jakobsen JH. Genetic correlations between the shape of the lactation curve and disease resistance in dairy cattle [Doctoral tesis]. Copenhague, Denmark: The Royal Veterinary and Agricultural University; 2000 http://www.forskningsdatabasen.dk/ en/catalog/ 20. Pala A, Savas T. Persistency within and between lactations in morning, evening and daily test day milk in dairy goats. Arch Anim Breed 2005;48:396–403. 21. Macciotta NPP, Dimauro C, Rassu SPG, Steri R, Pulina G. The mathematical description of lactation curves in dairy cattle. Ital J Anim Sci 2011;10(4): e51.

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22. Broucek J. Methane abatement strategies based on genetics and dietary manipulation of rumiants: a review. Arch Zootec 2018;67:448-458. 23. Torres-Vázquez JA, Valencia M, Castillo H, Montaldo HH. Tendencias genéticas y fenotípicas para características de producción y composición de la leche en cabras Saanen de México. Rev Mex Cienc Pecu 2010;1:337–348. 24. Cerón-Muñoz MF, Tonhati1 H, Costa C, Benavides F. Genotype and environment interaction in Colombian Holstein cattle. Arch Latinoam Prod Anim 2001;9:72–78.

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https://doi.org/10.22319/rmcp.v11i2.5010 Article

Semen quality of hair sheep supplemented with Moringa oleifera (Moringaceae) and Trichanthera gigantea (Acanthaceae)

Marco A. Ramírez-Bautista a,b Julio P. Ramón-Ugalde a Edgar Aguilar-Urquizo a William Cetzal-Ix b* Roberto Sanginés-García a Álvaro E. Domínguez-Rebolledo c Ángel T. Piñeiro-Vázquez a

Tecnológico Nacional de México, Instituto Tecnológico de Conkal, División de Estudios de Posgrado e Investigación, Ave. Tecnológico s/n Conkal, 97345, Yucatán, México. b

Tecnológico Nacional de México, Instituto Tecnológico de Chiná, Campeche, México.

Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias, Campo Experimental Mocochá, Yucatán, México.

*Corresponding author: rolito22@hotmail.com

Abstract: It was assessed the effect of the inclusion of Moringa oleifera Lam. and Trichanthera gigantea (Bonpl.) Nees in the diet of hair (Pelibuey) sheep on the quality of their semen. During 90 d, the diets of 15 sheep (24 kg ± 3.95) were divided into three treatments: T1: integral diet with 40 % of M. oleifera + Taiwan grass (Pennisetum purpureum Schumach.), T2: integral diet with 40 % of T. gigantea + Taiwan grass, and T3: 40 % commercial feed + Taiwan grass. The daily weight gain (DWG), carcass yield (CY), testicular development

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(DT) determined by the diameter (AE) and the scrotal circumference (SC) of these sheep were determined, and the volume, concentration, motility (CASA), viability (SYBR-14/IP), mitochondrial activity (J-C1) and acrosomal integrity (FITC-PSA) of their sperm samples were assessed. No differences were found (P>0.05) in the GDP and RC. Differences were found (P<0.05) in DT, T1 (AE= 48.84 ± 5.99 mm, CE=$9.29 ± 1.13 cm) and T3 (AE= 48.83 ± 4.34 mm, C = 26.62 ± 1.27 cm) exhibited higher values than T2 (44.57 ± 5.59 AE= mm, CE= 25.42 ± 1.50 cm); as for viability, T2 (62.90 ± 6.10 %) and T1 (54.00 ± 6.61 %) had higher percentages than T3 (24.45 ± 7.56 %); in motility, T1 (93.9 ± 2.1 %) and T2 (88.6 ± 1.9 %) had a higher percentage than T3 (71.9 ± 4.0 %). The inclusion of M. oleifera and T. gigantea in the diet allows to obtain a GDP, RC and DT similar to those of commercial feed and increases the number of viable sperm cells by more than 20 %; it also improves certain parameters of motility, thereby improving the reproductive potential of the stallions. Keywords: Viable sperm cells, motility, Pennisetum purpureum, channel performance.

Received: 03/08/2018 Accepted: 06/05/2019

Introduction The high demand for sheep meat forces producers to improve all its processes aimed at reducing production costs, to increase the production and reproduction parameters in order to increase the productivity per unit area, to improve animal welfare, and to propose a sustainable approach to ensure the permanence of the system through time(1). In tropical areas, the atmosphere is a determining factor for livestock production, and in particular for sheep production, which must deal, among other factors, with a marked seasonality in the production of forage, forcing producers to implement supplementation strategies (mainly with commercial feed) in order to maintain optimal levels of production; this has generated a dependency on the feed, as well as a considerable increase in production costs(2). Within this context, the use of tree species with fodder potential has become relevant in animal feed(3,4,5), because its implementation allows to generate strategies of partial replacement of grains in the diet(6). However, the use of tree species with fodder potential is conditioned by their high nutrient content, tolerance to pruning, steady production of biomass and content of secondary compounds that may affect the animals that consume them(7,8). This is one of the main reasons that limit their use for animal feed(9), since it can 394

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generate estrogenic effects that inhibit, among others, the growth and maintenance of the reproductive system. In the reproductive system of males, the affectations by presence of secondary compounds can be: an increased number of immature sperm with a significant decrease in motility, a reduction of the lumen of seminiferous tubules, an increase in the apoptotic index of germ cells, prostatic atrophy, and affectation of spermatogenesis(10,11). Within this context, various species such as Moringa oleifera Lam. (Moringaceae) and Trichanthera gigantea (Bonpl.) Nees (Acanthaceae) are alternative dietary supplements(8). The presence of secondary metabolites has been reported in these; for example, García(12) recorded the presence of phenols, flavonoids and tannins that precipitate the protein and condensed tannins; on the other hand, Balercia et al(13), Rajimakers et al(14) and Ghasi et al(15) observed in M. oleifera and T. gigantea, the presence of riboflavin, niacin, folic acid, pyridoxine, protein, vitamins A, B, C, and E, as well as amino acids and high levels of βcarotene; consumption of these antioxidants helps to improve semen quality by eliminating free radicals found in the seminal plasma. For this reason, it is necessary to study the effect that these plants may generate in the animals that consume them. Therefore, the objective was to evaluate the effect of the inclusion of M. oleifera and T. gigantea in the diet of Pelibuey sheep on the seminal quality.

Material and methods The study was carried out in the agricultural and livestock production and research area of the Technological Institute of Conkal, Yucatán, Mexico (21°05' N and 89°32' W), which is located at an altitude of 7 meters above sea level and has an AW0 climate, with an annual precipitation of 900 mm and an average temperature of 29 ºC.

Treatments, animals and management

Fifteen (15) male pubertal Pelibuey sheep (aged 6 ± 0.5 mo) with an average weight of 24 kg ± 3.95 were utilized and distributed in a fully random way in three treatments (n=5): T1= integral diet with 40 % M. oleifera + Taiwan grass (Pennisetum purpureum Schumach.). T2= integral diet with 40% Trichanthera gigantea + Taiwan grass. T3= integral diet + Taiwan grass. The amount of feed offered was 4.7 % of the live weight during the 90 days of the experiment (the consumption was adjusted every seven days). Table 1 shows the contents of the percentage composition of the experimental diets, and Table 2, the proximate analysis. 395

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Table 1: Percentage composition of the experimental diets Component Moringa Trichanthera Molasses Minerals Canola Corn Sorghum Taiwan grass

Diets (%) T1 16 0 4.24 1.2 3.2 8.28 7.08 60.0

T2 0 16 3.72 1.08 5.88 8 5.32 60.0

T3 0 0 7 3 21 5 4 60.0

T1: intergral diet with 40% M. oleifera + Taiwan grass (Pennisetum purpureum Schumach.), T2: integral diet with 40% T. gigantea + Taiwan grass, and T3: commercial feed + Taiwan grass.

Table 2: Proximal chemical analysis of the offered diets Diets Neutral Acid Dry Crude and detergent Detergent matter protein grass fiber Fiber T1 90.83 13.81 20.22 16.18 T2 91.54 14.01 26.27 14.21 T3 98.82 23.16 22.83 13.47 Taiwan grass 90.96 8.00 67.26 42.21

Ashes 1.01 0.95 0.98 0.97

T1: intergral diet with 40 % M. oleifera + Taiwan grass (Pennisetum purpureum Schumach.), T2: integral diet with 40% T. gigantea + Taiwan grass, and T3: commercial feed + Taiwan grass

Development of lambs

To determine the development of lambs, the animals were weighed every seven days; prior to each weighing, the animals underwent a period of 18 h of fasting. They were weighed on a T31P (OHAUS) electronic platform scale with a capacity for 300 kg, with a margin of error of 0.1 kg. The daily weight gain was determined through the analysis of these weights(16).

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Testicular development

The measurement of the scrotal circumference was taken with a plastic measuring tape, while the testicular diameter was registered using a TRUPER brand digital caliper (17).

Semen quality

For the assessment of the semen quality, after the 70-days experimental period (at age 8 ± 0.7 mo), six semen samples per animal (two per week) were collected in each treatment using an estrogenized female dummy and the artificial vagina method with a rubber sleeve (Liner). Warm water at 45 ⁰C and air were introduced by blowing through a valve in order to obtain the temperature and pressure required to stimulate ejaculation(18). Once the ejaculates were obtained, they were transported to the laboratory in isothermal conditions (35 to 37 ⁰C) for evaluation. At the laboratory, the samples were placed in a double boiler at 37 ⁰C for immediate evaluation. The following were determined in the collected samples: 1) The volume of the ejaculate, with a graduated pickup tube. 2) The sperm concentration was determined with the CASA system (ISAS®v1 (Proiser R&D, Valencia, Spain) by diluting 5 µl of pure semen in 995 µl of distilled water (1:200); 9 µl of the diluted sample were then placed in a Bürker chamber for analysis. Sperm motility was evaluated by depositing 5 µl of diluted sample at a concentration of 30 x 106 spermatozoa/ml on a Makler® counting chamber (Sefi Medical Instruments, Haifa, Israel), and four recordings were made of each sample; the dilution was performed with the commercial diluent (Triladyl ® + distilled water + 20 % egg yolk). The parameters evaluated for this variable were: percentage of total motility (TM), percentage of progressive motility (PM), curvilinear speed (CLS, µm/s), rectilinear speed (RLS, µm/s), average speed (AS, µm/s), linearity index (LIN), straightness index (STR), oscillation index (WOB), average amplitude of lateral displacement of the head (ALH), sperm head beat frequency (HBF). 3) The sperm viability was assessed by staining with the Fluorochrome SYBR-14/IP (Live/Dead® kit L-7011, InvitrogenTM); 1 µL of SYBR-14 (10 µM) was mixed with 1 µl IP (12 µM) in 100 µl of diluted sperm sample and kept for 10 min at 37 ⁰C; 5 µl of the stained sample were subsequently placed on a glass slide preheated at 37 ⁰C for evaluation in a (CX31, Olympus, Tokyo, Japan) fluorescence microscope with a wavelength of 488 nm. 397

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4) The mitochondrial activity was determined using the J-C1 fluorochrome (153 µM, Molecular Probes® T-3168, InvitrogenTM).1 µl of the fluorochrome was mixed with 100 µl of diluted sperm sample and kept for 10 min at 37 ⁰C; 5 µl of the stained sample were subsequently placed on a glass slide preheated to 37 ⁰C for evaluation under a fluorescence microscope with a wavelength of 488 nm. 5) The integrity of the acrosomes was evaluated with the FITC-PSA fluorochrome (100µg/mL, L-0770, Sigma-AldrichTM), adding 5 µl of the fluorophore in 100 µl of diluted sperm sample and was kept for 30 min at 37 ⁰C; 5 µl of the incubated sample were subsequently placed on a slide and were evaluated in a fluorescence microscope with a wavelength of 485 nm. The data obtained were analyzed using the PROC GLM procedure of the statistical package SAS 9.4 for Windows (Inst. Inc. Cary, NC, USA); a repeated means variance analysis (ANOVA) a Tukey mean comparison test were performed in order to evaluate the effect of diet on the sperm quality.

Results and discussion Productive behavior

The daily weight gains of the studied animals did not exhibit any differences (P˂0.05) between treatments. These gains are greater than those recorded by Madera-Solis et al(19), who with different levels of inclusion of Leucaena leucocephala (Lam.) de Wit found daily weight gains of 25, 41.67, 50, and 75 g in Pelibuey sheep. For their part, Rivers et al(20) reported profits of 54, 87 and 56 g/d when assessing the use of Morus sp. and Gliricidia sepium Kunth ex Walp. as substitutes for concentrated feed for growing lambs. Daily revenues of 45.5, 96.3, 124.5 and 106.4 g with different levels of inclusion of hay from Hibiscus rosa-sinensis L. are also reported in growing sheep(4). The profits obtained in this study are less than those mentioned by others(16), which recorded daily gains of 229 ± 10 g/d for Pelibuey-Katadin animals housed in elevated cages and fed a commercial concentrate with a 16 % crude protein content and Cynodon nlemfuensis Vanderyst hay. When assessing the performance of the carcass, no differences were found (P˂0.05) between treatments: 50.79, 49.44 and 46.94 % for treatments T1, T2 and T3, respectively; these values are similar to those registered by Magaña-Monforte et al(16), who obtained a yield of 398

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49.1 ± 0.58 with crossbred animals of the Pelibuey-Katahdin breeds. The yields of this study were lower than those found in another research(21), which cites a carcass output of 52.8 % for whole males, 56.64 % for castrated males, and 55.87 % for females. The proper nutritional balance present in the assessed diets generated an adequate, similar productive response in the study animals, compared to other researchers performed under similar conditions. These results in the evaluated rations (Table 2) can be attributed to the adequate level of protein and the improved nutritional balance(22); this stimulates an increase in the level of efficiency of nutrients produced by greater microbial activity in the rumen(23) and ensuring metabolic stability(24), positively affecting the various productive parameters evaluated in the present study (daily weight gains and carcass output).

Testicular development

Testicular volume is an indicator of the fertilizing ability of the male and of the highest fertility; also a smaller number of abnormal spermatozoa has been observed in animals with a larger scrotal circumference. In the study animals, the testicular development determined by means of the increase in scrotal circumference and diameter exhibited differences (P<0.05) between the treatments evaluated for both variables (Table 3). For the variable scrotal circumference, treatment T3 had a higher mean than T2, while T1 did not differ (p>0.05) from the other two treatments. For the scrotal diameter variable, there was no difference between treatments T1 and T3; however, both were larger than with T2. Table 3: Scrotal circumference, scrotal diameter, and hair sheep seminal volume and concentration supplemented with M. oleifera and T. gigantea (mean ± standard error)

Treatments

Scrotal circumference (cm)

Scrotal diameter (mm)

26.48±0.33ab

48.84±1.05a

25.42±0.33b

44.57±1.05b

26.62±0.33ª

48.83±1.05a

Volume (ml) 0.70 0.05a 0.61 0.05a 0.63 0.06a

Concentration ± ± ±

1882.63 ± 202.66a x 106 1841.92 ± 182.34a x 106 1014.49 ± 482.44a x 106

T1: integral diet with 40% M. oleifera + grass (Pennisetum purpureum Taiwan), T2: integral diet with 40 % T. gigantea + Taiwan grass, and T3: commercial feed + Taiwan grass. a,b ( ) different letters between columns indicate significant differences (P<0.05).

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The means for the scrotal circumference were: 26.4 ± 0.33, 25.4 ± 0.33 and 26.6 ± 0.33 cm for treatments one two and three, respectively (Table 3); they are lower than those recorded by Luna et al.(25), who found averages of 28.4 cm in sheep supplemented with animal fat and palm oil; as well as those reported in other studies(26,27), of 29.4 and 36.5 cm, respectively. The averages of scrotal diameter were lower than those recorded by Ballin et al.(26). Testicular development is determined by various factors, mainly age, race, and nutrition(27); in this regard the study animals, with an average age of 7±0.5 months, received a diet with adequate levels of protein and nutrients required for meeting the nutritional requirements according to their physiological stage, which is reflected in the weight gain and in the obtained testicular development.

Seminal evaluation

The variables of the ejaculate volume and sperm concentration did not exhibit any differences (P>0.05) between treatments (Table 3). The volume for the three treatments lies within the range considered normal, between 0.5 and 2.0 ml, and this volume varies, depending on the age, size, body condition of the animal, frequency of collection, and dexterity of the operator(28). Despite this, the values reported in this experiment were lower than those reported by Mansano et al(29), who obtained volumes of 1.3 ± 0.4 ml; Carrillo and Hernández(30) obtained 1.41 ± 0.11 ml; for their part Córdova-Izquierdo et al(31) obtained 1.11 ml; however, these two studies were performed with mature males unlike the pubertal males (7 ± 0.5 mo of age) used in the present study. The low ejaculate volume values obtained in this study, contain a greater concentration of spermatozoa per ml. Therefore, these values are higher than those recorded in other studies, which indicate ranges of 206.4 to 711.89 × 106(30,31), but similar to those found by Domínguez et al(32), of 2.19 ± 0.04 and 108 ± 8.0 × 106 spermatozoa/ml, when assessing the semen quality of F1 Katahdin × Pelibuey lambs supplemented with alfalfa. In contrast, the concentration values obtained here are lower than those found by Quintero et al(33), who obtained 2,628.2 × 106 when assessing hair sheep aged 2 to 5 yr There were no differences (P<0.05), in total motility, curvaceous speed, and AD, where treatments T1 and T2 were better than T3; and in the variables AS and RLS, T1 exhibited the best results with respect to T3; however, no differences were observed between T1 and T2. For the rest of the analyzed variables (PM, LIN, IR, OI, and BF), no differences were found between experimental groups (Table 4).

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Table 4: Seminal motility parameters of hair sheep supplemented with M. oleifera and T. gigantea (mean ± standard error) Treatments Variables T1 T2 T3 a a MT, % 93.9 ± 2.1 88.6 ± 1.9 71.9 ± 4.0b AS, µm/s 100.4 ± 3.7a 88.6 ± 3.2ab 73.0 ± 6.8b CLS, µm/s 157.8 ± 5.0a 146.6 ± 4.4a 117.3 ± 9.2b RLS, µm/s 70.3 ± 3.1a 61.3 ± 2.7ab 54.6 ± 5.8b AD, µm 4.8 ± 0.1a 4.6 ± 0.1a 3.7 ± 0.3b PM, % 30.79±2.64ª 29.23±2.32ª 30.18±4.87ª IR, % 64.72±1.31ª 64.98±1.15ª 69.62±2.42ª a WOB, % 61.96±1.63ª 59.85±1.43 60.78±3.01ª a HBF 10.32±0.25ª 10.57±0.22 9.89±0.47ª a LIN, % 42.27±1.9ª 40.85±1.67 44.35±3.5a T1= 40% of M. oleifera + Taiwan, T2= 40% of T. gigantea + Taiwan, T3= commercial feed + Taiwan. MT= total motility; AS= average speed; CLS= curvilinear; speed; RLS= rectilinear speed; AD= amplitude of displacement; PM= progressive motility; STR= straightness index; WOB= oscillation index; HBF= sperm head beat frequency; LIN= linearity index. a,b Values with different letters in the same row are different (P<0.05).

The results for the motility variable are higher than those recorded by Mansano et al(29), of 52 %, but similar to those found in another research(32), which registered an 80 % total motility and over 20 % progressive motility; however, the results of this study are lower than those recorded by Quintero et al(33), who obtained 90.69 % progressive motility. In the analysis of the plasma membrane viability variable, differences (P<0.05) were found between the assessed treatments (T1: 54 %, T2: 63 % and T3: 21 %), T2 being the treatment that exhibited the highest percentage of viability, in contrast with T3, which obtained the lowest percentages (Figure 1).

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Figure 1: Feasibility, mitochondrial activity and integrity of the acrosome of hair sheep supplemented with Moringa oleifera and Trichanthera gigantea

T1: integral diet with 30% M. oleifera + Taiwan grass (Pennisetum purpureum Schumach.), T2: integral diet with 30% T. gigantea + Taiwan grass, and T3: commercial feed + Taiwan grass.

The mitochondrial activity and acrosome integrity variables did not reveal any differences (P>0.05) between treatments, while T1 exhibited the highest means for the mitochondrial activity variable, T3 showed the highest percentages for the mitochondrial activity variable (Figure 1). The integrity of the plasma and acrosome membranes and mitochondrial activity are major factors for fertilization, as an intact plasma membrane indicates that the spermatozoon is alive; an intact acrosomal membrane allows penetration of the oocyte, and the mitochondrial activity ensures the presence of energy required to move the spermatozoon (Figure 2). For this reason, it is essential to include it in the assessments on semen quality(31). The results observed in this study agree with those recorded by Cordova-Izquierdo et al(31) who obtained 68.5 % of the integrity of the plasma membrane by assessing sheep of the Suffolk race. They also match the results of Rubio-Guillen et al(34), who recorded 42 % when evaluating West African sheep. However, the percentages obtained in this study are lower than those found in other researches(30,32,33) but are consistent with the values estimated by DomĂnguez et al(32) for acrosomal integrity.

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Figure 2: Sperm cells from sheep fed with Trichanthera gigantea or Moringa oleifera

A. Living (green) and dead (red or orange) sperm heads stained with SYBER-14/IP fluorochromes; viability of the acrosome located in the apical region using the fluorochrome FITC-PSA (colorless: viable; green: corrupted). B. Mitochondrial activity located in the middle region of the tail stained with the fluorophore JC1, with activity (yellow or orange) and without activity (green).

The parameters recorded in the assessment of the semen quality as well as of the testicular development may be attributed to the adequate nutritional balance of the assessed diets, as sperm production and quality, and testicular size, are directly influenced by nutrition. An increase in the nutritional level increases the frequency of pulses of LH and increases levels of FSH(35), hormones that promote spermatogenesis and androgen secretion(36). The animals assessed in the present study are pubescent, animals, and animals in this condition have a greater amount of immature, dead or defective spermatozoa, which in turn produce high amounts of free radicals that affect those spermatozoa that are in good conditions, since free radicals cause a cascade of lipid peroxidation affecting mainly the unsaturated fatty acids of the plasma membrane of the sperm, causing loss of sperm function and cell apoptosis, and therefore are one of the leading causes of death of sperm cells(37). It has been observed that the inclusion of antioxidants in the diet improves the percentage of live sperm cells because antioxidants eliminate the free radicals that are found in the seminal plasma and confer protection to the spermatozoa(13,14); Priyadarshani and Varma(38) observed a higher percentage of live cells when they used M. oleifera in the diet of mice. This result is similar to what has been observed in the present study and may be ascribed to the contribution of antioxidants (beta-carotene) contained in M. oleifera and T. gigantea(15). The secondary compounds present in both assessed species may be related to the values found in the present study; therefore, further studies determining the presence and concentration of these would contribute greater evidence in this regard.

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Conclusions and implications The replacement of 40 % of commercial feed by M. oleifera or T. gigantea in the diet of Pelibuey sheep promotes adequate testicular development, and improves the sperm viability by more than 20 %, as well as certain parameters of motility —such as total motility, average speed, curvilinear speed, rectilinear speed, and the average amplitude of lateral motility of the sperm head—, whereby the reproductive potential of the stallions may be improved.

Acknowledgments This work was funded partly by the CONACYT Basic Science Project 164592 and by project 5304.14-P of the National Technological Institute of Mexico. We are grateful for the anonymous reviewers’ suggestions and comments on the manuscript.

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https://doi.org/10.22319/rmcp.v11i2.5685 Article

Use of a glycogenic precursor during the prepartum period and its effects upon metabolic indicators and reproductive parameters in dairy cows

Carlos Leyva Orasma a Jesus Jaime Benitez-Rivas b Juan Luis Morales Cruz c Cesar Alberto Meza-Herrera d Oscar Ángel-Garcíaa Fernando Arellano-Rodríguez c Guadalupe Calderón-Leyva c Dalia Ivette Carrillo-Moreno c Francisco Veliz Deras a*

Universidad Autónoma Agraria Antonio Narro. Departamento de Ciencias Médico Veterinarias, Torreón, Coahuila, México. b

Universidad Autónoma Agraria Antonio Narro. Posgrado en Ciencias en Producción Agropecuaria, Torreón, Coahuila, México. c

Universidad Autónoma Agraria Antonio Narro. Departamento de Producción Animal, Torreón, Coahuila, México. d

Universidad Autónoma Chapingo. Unidad Regional Universitaria de Zonas Áridas, Bermejillo, Durango, México.

* Corresponding author: velizderas@gmail.com

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Abstract: The aim was to evaluate if 1-2 propanodiol plus calcium propionate (glycogenic precursor) supplementation during the transition period in high yielding dairy cows reduces metabolic and reproductive dysfunctions during early lactation. Cows (n= 202) were divided into two homogeneous groups regarding number of lactations and body condition score. 1) Treated group (GG; n= 112) received 60 g/cow/d for15 d of a glycogenic precursor during the transition period. 2) Control group (GC; n= 90) received no treatment. Postpartum levels of beta hydroxybutyrate (BHB) (GG= 0.9 ± 0.2 mmol/L vs GC =1.3 ± 0.2 mmol/L; P<0.05), and non-esterified fatty acids (NEFA) (GG= 0.6 ± 0.1 mEq/L vs GC = 0.8 ± 0.1 mEq/L; P<0.05) were higher in the GC-group. Similarly, GC-cows had a higher percentage of retained placenta (23 % vs 13 %; P≤0.06) subclinical ketosis (GG= 10 %, GC= 56 %; P<0.05), and mastitis (GG= 8 %, GC= 16 %; P<0.05). Metritis, dystocia, abortions, clinical ketosis, hypocalcemia and ruminal acidosis showed no differences between groups. Administration of a glycogenic precursor during the transition period demonstrated a positive effect upon BHB and NEFA blood levels during early lactation, with parallel decreases of subclinical ketosis and retained placenta; this could be an alternative to enhance the dairy herd reproductive efficiency. Key words: Beta hydroxybutyrate, NEFA, Subclinical ketosis, Postpartum.

Received: 27/08/2018 Accepted: 11/03/2019

Introduction During the first weeks of lactation, cows with a high milk production face a negative energy balance (NEB), because of the high energy usage promoted by an inadequate adaptation during the transition period and the low intake of dry matter during the beginning of the lactation(1,2). This situation promotes the mobilization of hepatic lipids, increasing the plasmatic levels of non-esterified fatty acids (NEFA) and beta hydroxybutyrate (BHB), which are considered as sensitive markers of NEB(3,4). Such physiologic and metabolic scenario may cause hepatic lesions leading to a high probability of suffering other disorders related to metabolic dysfunction such as ketosis, mastitis, metritis, hypocalcemia, abomasal 409

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displacement and retained placenta(2). In fact, at the onset of lactation, BHB concentrations higher than 1.2 mmol/L are related with the presence of subclinical ketosis(5), and that levels of NEFA from 0.7 to 1.0 mEg/L are able to predict postpartum health problems(6). Considering all the processes that take place during the transition period and with the aim of maintaining normoglycemia, several metabolic alternatives have been used in order to maintain glycaemia to fulfill the requirements of the peripheral tissues(7). The last throughout the process of glycogenesis, which allows the production of glucose starting from the glucose precursors as propionate or propylene glycol(8-11). Based on the previous findings, the aim of this study was to evaluate the use of a glycogenic precursor during the transition period upon the blood levels of NEB (NEFA and BHB) and the incidence of metabolic and reproductive diseases at the beginning of lactation in high yielding dairy cows.

Material and methods Study site, animals and management

The research took place from August to September 2015, in the Comarca Lagunera (25° 44 36 N, 103° 10 15 W; 1,111 m asl). This northern region of Mexico is characterized by an extremely hot-dry climate, with temperatures ranging from 23 ºC to 43 ºC in summer and 2 ºC to 9 ºC in winter, annual mean rainfall of 240 mm and relative humidity of 29 to 83 %. The study was conducted in a commercial dairy herd with a stock of 1,600 Holstein cows, managed under an intensive production system in open pens. The cows were fed with a completely mixed diet (50 % forage and 50 % concentrate, DM basis 1.62 Mcal/Kg NEL, 18 % CP), formulated in order to fulfill the nutritional requirements of cows in lactation with a milk production > 33 kg milk/d(12). The cows were fed ad libitum with a daily fed leftover of 10 % of the feed offered in four times (06:0, 1000, 12:0 & 16:0 h).

Health and reproductive management

All cows received four intramammary infusions of 375 mg of cephalexin (Rilexine®, Virbac, Mexico), during the dry period. Also, cows received a prolonged released cake consisting in micro minerals and vitamins A, D and E (Megabric®, Neolite, Laboratory, France). Routine 410

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control of mastitis included disinfection of nipples before and after milking, the California mastitis test and a regular somatic cell count. Cows included in this study were subjected to a reproductive management of fresh cows from d 0 to 10; in order to achieve an adequate uterine involution, a 25 mg PGF2Îą injection was administered on d 35 and 47 postpartum. Besides, every cow was vaccinated according to the vaccine preventive program of the herd, mainly focused against diseases like bovine viral diarrhea, infectious bovine rhinotracheitis, bovine respiratory syncytial virus, parainfluenza type 3 and leptospirosis (5 varieties).

Experimental design and response variables

Cows (n= 202) with no reproductive issues were selected and divided into two homogenous groups regarding the number of previous lactations (3.2 Âą 1.17) and body condition score (3.3 Âą 0.5; 1-5 scale). While the average milk production at 305 d was 12,200 Âą 147 kg the average number of inseminations was 3.7 Âą 2.4 (1-10 inseminations range). A first group of cows (n= 112; GG; 41.1 Âą 0.7 L per day) received a daily administration of 60 g/cow of a glycogenic precursor (1-2 propanediol and calcium propionate, LipofeedÂŽ, Mexico) during the first 15 d of the transition period, which was added to the diet, while the second group (n= 90; GC; 40.1 Âą 0.7 L per day) received no treatment.

Fatty acids and beta hydroxybutyric acid

Concentrations (mean Âą SE) of non-esterified fatty acids (NEFA) in serum was determined by taking a coccygeal blood sample with vacuum tubes (BD VacutainerÂŽ) at 7, 14 and 21 d postpartum; the obtained samples were identified and refrigerated until they arrived at the laboratory where they were centrifuged (450 gđ?&#x2018;Ľ 20 min), afterwards, they were frozen and stored at -20 ÂşC until analyzed. In vitro quantitative determination of serum NEFA was performed with an automatic analyzer (Randox RX MonzaÂŽ, USA), while the determination of concentrations (mean Âą SE) of beta-hydrybutyric acid (BHB) at 7, 14 and 21 d postpartum was done with a portable BHB-meter (Precision Xtra system testsÂŽ), which consists of the use of reagent strips to quantify the ketone bodies in blood(3,13).

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Metabolic diseases

The percentage of cows with clinical ketosis was calculated, it was diagnosed based on a low milk yield and decreased appetite and the percentage of subclinical ketosis was determined with reagent strips during the first 21 d in milk. In addition, their relationship with other clinical diseases during the first 35 d postpartum were also evaluated(14). The occurrence of clinical mastitis was detected at milking by determining heat and inflammation by touching the udder and by evaluating consistency changes of the milk (watery-bloody, secretions and blood clots); this was daily performed during the first 3 wk postpartum(15).

Reproductive diseases

Percentage of cows with retained placenta (RP) was quantified in both groups, and was defined as the failure to expel fetal membranes within the first 24 h after parturition(16) and diagnosed by observing the presence of fetal membranes protruding from the vulva for more than 24 h after parturition. Metritis was defined as the inflammation that involved all the uterine wall(17). This was confirmed by evaluating, by rectal palpation, the size of the uterus in relation to the moment of parturition, the uterine wall thickness and the presence of liquid in one or both uterine horns. Abortion was defined as fetal death and expulsion between 50 and 260 d of pregnancy.

Statistical analyses

Blood concentrations of BHB and NEFA were analyzed with the GLM procedure and mean comparison was performed by a Student-T. Percentage of cows showing reproductive (retained placenta, metritis, abortion and dystocia) and metabolic (clinical and subclinical ketosis, hypocalcemia, mastitis and ruminal acidosis) diseases, was calculated and compared by a X2 test. All analyses were performed with the SAS program, considering a significance level of P<0.05 and of P>0.05 and Pâ&#x2030;¤0.10 for statistical tendency.

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Results BHB and NEFA concentrations

The results for concentrations of BHB and NEFA for both groups at 7, 14 and 21 d postpartum are shown in Figure 1. Levels of BHB during the first 3 wk of lactation were higher in the GC-cows (1.3 Âą 0.2 vs 0.9 Âą 0.2 mmol/L; P<0.05). No differences were found neither in time nor treatment at d 7 postpartum. Nonetheless, a difference was found between time and treatment at 14 and 21 d postpartum (P<0.05). Moreover, NEFA levels were higher for GC cows (0.8 Âą 0.1 vs 0.6 Âą 0.1 mEq/L; P<0.05). A treatment đ?&#x2018;Ľ time interaction effect occurred at 7, 14 and 21 d postpartum (P<0.05). Figure 1: Postpartum blood levels of non-esterified fatty acids (NEFA) and BHB (mean Âą SE) in high yielding dairy cows treated (GG) or non-treated (GC) with a glucose precursor for 15 d during the transition period

*Significant difference (P<0.05).

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Reproductive and metabolic dysfunction incidence

Table 1 concentrates the percentage for reproductive and metabolic diseases in both experimental groups. The percentage of retained placenta for GG cows was 13 % (15/112) vs 23 % (21/90) for the GC-cows, (Pâ&#x2030;Ľ0.06). No differences between experimental groups (P>0.05) occurred for percentages of metritis, dystocia or abortion. In relation to metabolic diseases, a higher percentage (P<0.05) of subclinical ketosis was observed in the GC-cows (56 %; 50/90 vs 10 %; 12/112) as well as for mastitis (18 %; 16/90 vs 8 %; 9/112, respectively). No differences were observed for the remaining metabolic diseases between experimental groups (P>0.05). Table 1: Percentage of postpartum reproductive and metabolic diseases of dairy cows treated (GG) or no treated (GC) with a glucose precursor for 15 d during the transition period (%) P value

Groups Variables GG (n= 112)

GC (n =90)

Retained placenta

13 (15/112)

23 (21/90)

0.067

Metritis

5 (6/112)

11 (10/90)

0.132

Dystocia

7 (8/112)

11 (10/90)

0.325

Abortion

1 (1/112)

2 (2/90)

0.438

Clinical ketosis

1 (1/112)

3 (3/90)

0.216

Subclinical ketosis

10 (12/112

56 (50/90)

0.000

Hypocalcemia

1 (1/112)

1 (1/90)

0.864

Mastitis

8 (9/112)

18 (16/90)

0.037

Ruminal acidosis

6 (10/112)

10 (9/90)

0.467

Reproductive diseases:

Metabolic diseases:

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Discussion The results demonstrate that cows treated with a glycogenic precursor during the transition period depicted a lower NEB regarding the non-treated cows. In fact, females facing NEB have higher BHB levels(18); high yielding dairy cows during the beginning of lactation increase their glucose requirements for lactose production and, with the lack of this, the animal mobilizes the glycogenic substrates that originate ketone bodies(19). The last could probably be due because such glycogenic precursor is an important substrate for gluconeogenesis, which may have stimulated the hepatic glycogen dynamic that is necessary to satisfy the requirements of hepatic glucose during the transition period(19). In fact, some studies have demonstrated that the use of propionate(10,11), or propylene glycol(8,9), stimulate glucose synthesis in dairy cows at the beginning of lactation(20). The above probably helped to reduce the blood BHB levels in the GG-cows; it has been stated that glycogenic precursors decrease ketonic bodies concentration(20). On the other hand, the administration of a glycogenic precursor decreased the blood NEFA concentrations in the GG-cows (0.6 Âą 0.1 mEq/L) compared to GC-cows (0.8 Âą 0.01 mEq/L), suggesting that the GG-cows were probably on a less intense NEB regarding the control-cows(6). Certainly, at the beginning of lactation cows face a NEB, due to the high milk yield which parallels a decrease in feed consumption, causing an increased lipid mobilization from body fat to the liver in order to make available the required glucose levels essential to compensate the observed energy deficit during the NEB(1,6,21). Therefore, those cows treated with the glycogenic precursor during the transition period decreased the incidence of females facing metabolic disturbances. In fact, the GG-cows had less subclinical ketosis than the control cows (10 % vs 56 %). McArt et al(18) indicate that the mean level of BHB in blood to assume the presence of a subclinical ketosis is > 1.2 mmol/L, which was near the average for the non-treated group (1.3 mmol/L). Indeed, more than 50 % of the cows in the GC showed subclinical ketosis, which is a risk factor for the health of the animals, and increases the incidence of reproductive diseases during the cool period. In addition, subclinical ketosis also increases the return-to-estrus interval, as well as, rises the rate of waste of the herd while it decreases milk production, causing economic losses(3,4,14). This was probably due because of the administration of the glycogenic precursor which reduced, in turn, the circulating BHB concentrations (0.9 mmol/L in the GG-cows), suggesting failures in the adaptation ability during the transition period(22). Certainly, a high milk yielding cow has a high glucose demand for lactose production. Yet, since there is a lack of blood glucose, cows try to compensate such metabolic challenge by mobilizing lipids from liver. Such physiological and metabolic scenario probably provoked that the control 415

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cows generated an increased serum BHB level which, in turn, provoked a high percentage of subclinical ketosis(6,19,20). The last, may also promoted an increased risk of diseases like fat liver and ketosis(6,10,20). On the other hand, the GG-cows had 50 % less clinical mastitis than the control-cows. The last probably was due to the fact that control-cows had blood BHB concentrations â&#x2030;Ľ 0.6 mmol/L from the pre-partum to the beginning of lactation, which has been reported as a factor that increases the probability of cows presenting pre-partum diseases and a consequent decrease in milk production(6,23). This scenario probably occurred because ketosis has been associated as a factor that increases the risk of clinical mastitis(14,24). Moreover, NEFA increases (â&#x2030;Ľ 0.8 mEq/L) is another risk factor associated to other diseases like metritis, mastitis and clinical ketosis(4,6). Certainly, any unbalance of both energy status and health status, decreases the immune compensatory response of cows and make them susceptible to poorly counteract any health compromising insult(25). Studies have demonstrated that high concentrations of NEFA are related with metabolic and inflammatory diseases that induce inflammation and affect the immune system(26,27). On the other hand, the reduced percentage of cows with retained placenta, in comparison with the control cows, could be explained because of the treated cows had a better energetic metabolism which improved the functionality of their immune system and, in consequence, promoted less incidence of inflammation processes(28). The low NEFA concentrations of the GG-cows, it is known to increase the blood lipid content while a higher serum NEFA concentration, and it is associated with a higher incidence of peri-parturient diseases (i.e. retained placenta, abomasal displacement) as well as the predisposition to inflammatory diseases (i.e mastitis, metritis)(29,30) the last may have increased the percentage of retained placenta incidence(22,31). Furthermore, a higher concentration of blood BHB, generates a higher risk of cows presenting reproductive diseases(3,4).

Conclusions and implications Cows treated with a glycogenic precursor during the transition period reduced the impact of the negative energy balance and, in consequence, diminished some health problems such as retained placentas, subclinical ketosis and mastitis at the beginning of lactation. The results indicate also that such supplementation strategy could be an interesting alternative to positively influence the energetic balance during the transition period of dairy cows while improving their health status. All of these metabolic and health responses should contribute to improve the reproductive efficiency of high yielding dairy cows. 416

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Acknowledgments To the National Council of Science and Technology (CONACYT) for the grant awarded to opt for the Master of Science degree. To the students of the Graduate Program in Agricultural Production Sciences for the technical support during the realization of the present investigation.

Literature cited: 1.

Duffield TF, Sandals D, Leslie KE, Lissemore K, McBride BW, Lumsden JH, et al. Effect of prepartum administration of monensin in a controlled-release capsule on postpartum energy indicators in lactating dairy cows. J Dairy Sci 1998;81(9):2354-2361.

González FD, Muiño R, Pereira V, Campos R, Benedito JL. Relationship among blood indicators of lipomobilization and hepatic function during early lactation in highyielding dairy cows. J Vet Sci 2011; 2(3):251-255.

Oetzel GR. Monitoring and testing dairy herds for metabolic disease. Vet Clin North Food Anim Pract 2004;20(3):651-674.

Suthar VS, Canelas-Raposo J, Deniz A, Heuwieser W. Prevalence of subclinical ketosis and relationships with postpartum diseases in European dairy cows. J Dairy Sci 2013;96(5):2925-2938.

Schallenberger GR, Cardoso F, De Souza GF, Reyes CLJ, Gonzalez F. Administration of early post-partum orlan drench in dairy cows: effect on metabolic profile. Rev Med Vet Zoot 2015;62(3):10-17.

McArt JA, Nydam DV, Oetzel GR, Overton TR, Ospina PA. Elevated non-esterified fatty acids and β-hydroxybutyrate and their association with transition dairy cow performance. Vet J 2013;98(3):560-570.

Patton RS, Sorenson CE, Hippen AR. Effects of dietary glucogenic precursors and fat on feed intake and carbohydrate status of transition dairy cows. J Dairy Sci 2004;87(7):2122-2129.

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Butler ST, Pelton SH, Butler WR. Energy balance, metabolic status, and the first postpartum ovarian follicle wave in cows administered propylene glycol. J Dairy Sci 2006;89(8):2938-2951.

Lien TF, Chang LB, Horng YM, Wu CP. Effects of propylene glycol on milk production, serum metabolites and reproductive performance during the transition period of dairy cows. Asian-Australas J Anim Sci 2010;23(3):372-378.

10. Reynolds CK, Aikman PC, Lupoli B, Humphries DJ, Beever DE. Splanchnic metabolism of dairy cows during the transition from late gestation through early lactation. J Dairy Sci 2003;86(4):1201-1217. 11. Larsen M, Kristensen NB. Precursors for liver gluconeogenesis in periparturient dairy cows. Animal 2013;7(10):1640-1650. 12. National Reseach Coucil. Nutrient Requirements of Dairy Cattle: Seventh Revised Edition. 2001. 13. Iwersen M, Falkenberg U, Voigtsberger R, Forderung D, Heuwieser W. Evaluation of an electronic cow side test to detect subclinical ketosis in dairy cows. J Dairy Sci 2009;92(6):2618-2624. 14. Berge AC, Vertenten G. A field study to determine the prevalence, dairy herd management systems, and fresh cow clinical conditions associated with ketosis in western European dairy herds. J Dairy Sci 2014;97(4):2145-2154. 15. Mellado M, Antonio-Chirino E, Meza-Herrera C, Veliz FG, Arevalo JR, Mellado J, et al. Effect of lactation number, year, and season of initiation of lactation on milk yield of cows hormonally induced into lactation and treated with recombinant bovine somatotropin. J Dairy Sci 2011;94(9):4524-4530. 16. Dervishi E, Zhang G, Hailemariam D, Dunn SM, Ametaj BN. Occurrence of retained placenta is preceded by an inflammatory state and alterations of energy metabolism in transition dairy cows. J Anim Sci Biotechnol 2016;7(1):26. 17. Sheldon IM, Lewis GS, LeBlanc S, Gilbert RO. Defining postpartum uterine disease in cattle. Theriogenology 2006;65(8):1516-1530. 18. McArt JAA, Nydam DV, Oetzel GR. Epidemiology of subclinical ketosis in early lactation dairy cattle. J Dairy Sci 2012;95(9):5056-5066.

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19. Aschenbach JR, Kristensen NB, Donkin SS, Hammon HM, Penner GB. Gluconeogenesis in dairy cows: the secret of making sweet milk from sour dough. IUBMB life 2010;62(12):869-877. 20. Bjerre-Harpøth V, Storm AC, Vestergaard M, Larsen M, Larsen T. Effect of postpartum propylene glycol allocation to over-conditioned Holstein cows on concentrations of milk metabolites. J Dairy Res 2016;83(2):156-164. 21. Tóthová C, Nagy O, Kováč G. Relationship between some variables of protein profile and indicators of lipomobilization in dairy cows after calving. Archiv Tierzucht 2014;57(1):1-9. 22. Duffield TF, LeBlanc SJ. Interpretation of serum metabolic parameters around the transition period. In: Southwest Nutrition and Management Conference; 2009:106-114. 23. Chapinal N, Carson ME, LeBlanc SJ, Leslie KE, Godden S, Capel M, et al. The association of serum metabolites in the transition period with milk production and earlylactation reproductive performance. J Dairy Sci 2012;95(3):1301-1309. 24. Leslie KE, Duffield TF, Schukken YH, LeBlanc SJ. The influence of negative energy balance on udder health. In: Proc National Mastitis Council Regional Meeting; 2000:2533. 25. Berry DP, Lee JM, Macdonald KA, Stafford K, Matthews L, Roche JR. Associations among body condition score, body weight, somatic cell count, and clinical mastitis in seasonally calving dairy cattle. J Dairy Sci 2007;90(2):637-648. 26. Zhang WY, Schwartz E, Wang Y, Attrep J, Li Z, Reaven P. Elevated concentrations of nonesterified fatty acids increase monocyte expression of CD11b and adhesion to endothelial cells. Arterioscler Thromb Vasc Biol 2006;26(3):514-519. 27. Yaqoob P, Calder PC. Fatty acids and immune function: new insights into mechanisms. Br J Nut 2007;98(S1):41-45. 28. Roche JR, Berry DP. Periparturient climatic, animal, and management factors influencing the incidence of milk fever in grazing systems. J Dairy Sci 2006;89(7):27752783. 29. Dyk PB, Emery RS, Liesman JL, Bucholtz HF, Vandehaar MJ. Prepartum non-esterified fatty acids in plasma are higher in cows developing periparturient health problems. J Dairy Sci 1995;78(1):264. 419

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30. Sordillo LM, Contreras GA, Aitken SL. Metabolic factors affecting the inflammatory response of periparturient dairy cows. Anim Health Res Rev 2009;10(1):53-63. 31. LeBlanc SJ, Leslie KE, Duffield TF. Metabolic predictors of displaced abomasum in dairy cattle. J Dairy Sci 2005;88(1):159-170.

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https://doi.org/10.22319/rmcp.v11i2.5118 Article

Relationship of the compositional content and sanitary quality of Holstein cows’ milk of the high tropic of Nariño

Henry Armando Jurado-Gámez a* Carlo Eugenio Solarte-Portilla a Álvaro Javier Burgos-Arcos b Aldemar González-Rodríguez b Carol Rosero-Galindo b

Universidad de Nariño. Departamento de Producción y Procesamiento Animal, Programa de Zootecnia., Cll 18 N° 50-02 Torobajo, Municipio de Pasto, Nariño, Colombia. b

Universidad de Nariño. Colombia.

* Corresponding author: henryjugam@gmail.com

Abstract: Agricultural production seeks to obtain high quality, safe products for human consumption- a great concern for the dairy chain. The present investigation seeks to identify the correlation between the compositional and sanitary quality of raw milk (SCC/mL). The investigation was carried out in three districts of the Department of Nariño, Colombia. For this purpose, sampling and information collection was executed throughout the years 2016 and 2017. To determine the relationship between composition and sanitary quality, an analysis was made of the principal components of the milk, and a design of mixed models was creating using selected variables. The analysis showed that there is a relationship between the compositional variables, and the mixed model indicated that there is a significant relationship between the somatic cell count and the milk quality of the region. It was concluded that a somatic cell count above 500,000 CFU/mL has negative effects on protein, casein, and milk production. Key words: Food safety, Animal health, Public health.

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Received: 17/11/2018 Accepted: 29/04/2019

Introduction Agricultural production worldwide requires high quality, safe products for human consumption, a constant search which concerns all the constituents of the dairy chain. This process begins on the farm and must be enforced in order to guarantee the best conditions for obtaining a product of optimum quality(1). In Colombia, specialized dairy industries are located in high tropical areas such as the Altiplano CundĂboyacense, the NariĂąo Altiplano and the north and northeast highlands of Antioquia. These systems are characterized by the presence of Bos taurus, intensive use of production factors (land, capital and labor), use of fertilizers, irrigation, rotation of pastures, use of food supplements and two daily milking. The improvement of the hygienic quality of milk is carried out through a simple process, showing rapid results that begin with the improvement of milking practices in order to avoid milk contamination, while maintaining perfect hygienization of the milk canteens or storage tanks(2). This type of livestock activity must adhere to Decree 616 of 2006 of Colombian regulations, which outlines the requirements that must be met by bovine, buffalo, and goat milk destined for human consumption, in order to protect life, health, and security and prevent practices that may mislead, confuse, or deceive consumers(2). In this regard, Benbrook et al(3) defines high-quality milk as having an excellent composition (fat, protein, lactose, vitamins and minerals), low microbial counts (hygienic), is free of pathogens, and has no physical-chemical contaminants. Quality milk is an indispensable requirement for good quality products, and the herd is the first condition in achieving good products. According to Ministry of Agriculture Resolution 000017 of 2012(4), the hygienic quality of milk refers to the hygiene level of the process through which milk is obtained and handled. In this order of ideas, the somatic cell count per milliliter (SCC/mL) in most cases can be associated with diseases such as mastitis, an inflammatory reaction of the mammary gland, which produces physical and chemical alterations in milk, an increase in the number of somatic cells due to the presence of pathogenic microorganisms, and changes such as the loss of functionality(1).

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For this reason, the somatic cell count (SCC) is one of the most influential parameters in determining the udder health and milk quality. SCC in milk increases in direct proportion to the severity of the infectious disease. In a milk that does not contain subclinical mastitis, the SCC is low (<100,000 SCC/ mL). The increase in SCC depends on the pathogen that causes mastitis(5). High SCC is associated with inflammation of the udder, which leads to bacteriological problems in milk, an alteration in its composition, and changes in the characteristics of dairy products when compared to normal values(6). However, in addition to its immune function in the udder and protective functions in milk, it has recently been shown that SCC have a positive influence on the composition and technological properties of dairy products, which influence the final quality of the dairy products through its endogenous enzymes(7). In Nariño, as is the case with the rest of Latin America, there is little information on the compositional and sanitary quality of the production and commercialization of raw milk. Along with the above, the lack of responsibility of the producers of milk for the quality of their product (in spite of the Colombian law that establishes health and safety regulations) increases the uncertainty about the quality variables of the products produced in the region. Based on the above, the objective of the present investigation was to evaluate the compositional and hygienic quality of the raw milk received from the different districts evaluated in the Department of Nariño, as well as to observe the relation of the sanitary quality on its compositional profile.

Material and methods Location

Information from 87 farms specialized in milk production was evaluated. The farms are distributed among three districts belonging to the Department of Nariño; 39 from the district of Guachucal, 24 farms from Pasto, and 24 from Pupiales. These geographical areas are located between 2,527 and 3,180 m asl, with an average annual temperature between 8 and 12 ºC(8).

Selection of samples

The selected farms managed a standardized record system for milking (twice a day) information and a routine management program of the CMT test. A total of 11,293 samples from 1,659 lactations of Holsteins from the districts of Guachucal, Pasto, and Pupiales were evaluated. Various variables were determined, including: precipitation (mm/month), milk production (kg),

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density (g/mL), day of lactation, calving interval, age of the cow (years), delivery number, kilograms of fat, protein, casein, of total solids, and somatic cell count (SCC/mL). The samples were taken at the time of milking, and the identification, conservation, and transport of the samples was carried out according to the protocol established by CORPOLAC. Sampling was done every 3 wk on each farm. The samples were analyzed at a compositional and sanitary level in the laboratories of the MEGA (Mejoramiento Genético Animal) research group located in the University of Nariño, Torobajo. The milk samples were analyzed in a MilkoScanᵀᴹ FT1, determining the compositional profile of the milk according to the protocol established by the manufacturer of the equipment. A 100 mL sample of raw cow's milk was used for this process (SGC-PR-04 daily procedure for the management of the MilkoScan FT1 equipment). The analysis of the sanitary profile was created using EkoMilk Scan® equipment, according to the protocol established by the manufacturer (SGC-PR-05 daily procedure for the operation of EKOMILSCAN equipment- SGC-FT-02 EKOMILKSCAN technical data). The somatic cell count was performed using a PortaSCC® test.

Statistical analysis

The data was evaluated through descriptive statistics. The relationship between variables was determined by Pearson's correlation after the standardization of the variables to ensure a better fit of the results. The values of fat, protein, total solids, and casein were transformed into kilograms using the formula proposed in resolution 000017 of 2012 by the Ministry of Agriculture and Rural Development (MADR)(4): Value kg value % ∗ milk density ∗ 10 For production, which is expressed in liters, the density was used to convert the value to kilos Kg milk density ∗ volume (l) The somatic cell count variable was transformed to somatic cell score by the formula proposed by Ali and Shook(9) in order to correct the normality of the variable somatic cell score (SCR): SCR ((log 2 (SCC/100.000)) + 3 The relationship between the variables was determined through a principal component analysis (PCA) with rotation (varimax method) in order to identify the related groups. With the variables selected through the PCA, a mixed model was created, where the farm and the animal nested within the farm were used as random factors and the precipitation with the dry and rainy season levels and the somatic cell count as fixed factors. The SCC was categorized into four levels, as follows: <2’000,000, 201,000 to 5000,00, 501,000 to 999,000 and >1’000,000. Protein, fat, casein, total solids and production was considered dependent variable(10). Statistical analyzes were performed with the statistical package SPSS(11). 424

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Results The mean and standard error of the mean of the variables can be seen in Table 1 and the correlation in Tables 2, 3 and 4. The means for the three districts show that the density was the same. Precipitation, total solids, day of lactation, calving interval, age, and number of deliveries showed similar results. On the contrary, the data collected for production, fat, protein and casein varied, with Pupiales showing the highest values, followed by Pasto and finally Guachucal. Table 1: Descriptive statistics of production and compositional parameters Guachucal Pasto Pupiales Mean SEM Mean SEM Mean SEM Precipitation, mm/mo 101.691 0.5511 99.749 0.9630 101.980 1.4078 Production, kg 16.592 0.0730 19.621 0.1366 20.673 0.1951 Density, g/mL 1.031 0.0001 1.031 0.0002 1.031 0.0001 Day lactation, day 182.427 1.2939 173.892 1.9738 163.128 2.9465 Calving interval, day 452.668 1.3196 431.900 1.9461 431.901 3.0784 Year 5.754 0.0258 5.635 0.0467 5.593 0.0653 Delivery number 2.918 0.0195 3.034 0.0376 3.020 0.0482 Fat, kg 0.653 0.0030 0.755 0.0054 0.775 0.0075 Protein, kg 0.560 0.0023 0.640 0.0042 0.693 0.0064 Total solid, kg 2.133 0.0092 2.130 0.0220 2.585 0.0253 Casein, kg 0.426 0.0018 0.831 0.0153 0.557 0.0056 SCC, SCC/mL 3.313 0.0104 3.209 0.0141 3.350 0.0261 SEM= standard error of the mean.

The correlation shows that the compositional variables and production are highly related and this set of variables are negatively related to the days of lactation. There is also a highly significant relationship (P<0.01) between day of lactation and calving interval, and also between the number of births and age. Finally, the somatic cell count shows a significant relationship (P<0.05) with production, protein, and casein. The other variables show low correlation.

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Pr1

Pr2

Table 2: Matrix of correlation Guachucal Dl Ci Y Dn F Pr3

Pr1 Pr2 D Dl Ci

1 -0.02 0.017 0.004 0.02

0.01

1 -0.009 1 -.458* 0.031 -.291* 0.129 0.068 0.171* 0.111 -0.116

-0.02

.852**

0.019

.949**

0.063

-.335*

-.231* 0.059 0.137 .864** 1

.974**

0.036

-.418*

-.278* 0.046 0.132 .934** .967**

.962**

0.053

-.342*

-.236* 0.063 0.142 .861** .996**

.971** 1

0.016 Ts 0.019 C 0.027 SCC 0.012 Pr3

1 .694**

0.129

0.156* 1

SCC

0.015 0.059 .922** 1 -.229* 0.055 0.132 1 0.357*

-0.048 0.135 0.254*

0.123 0.145 0.128

1 * 0.029 0.386 0.054 0.394*

Pr1= precipitation, Pr2= production, D= density, DL= day of lactation, Ci= calving interval, Y= years, Dn= delivery number, F= fat, Pr3= protein, Ts= total solids, C= casein, SCC= somatic cell count.

Table 3: Matrix of correlation Pasto Pr1

Pr2

Pr1

Pr2

0.007

-0.011 -0.023

Pr3

-0.009 -0.455

0.054

0.036

-0.240* 0.081

1 0.566* *

-0.012 -0.017

-0.047 0.180

o.020

0.073

-0.088 0.045

0.007

0.830** -0.061 -0.381* -0.224 -0.040

0.019 1

Pr3

0.017

0.944** 0.048

0.022 0.839** 1

0.011

SCC

1 *

0.012

0.970

0.907

SCC -0.008 -0.234

0.002 0.042

0.174

0.105

0.910** 1

-0.352* -0.220 -0.048 -0.439

-0.324

-0.006 0.125

-0.257 -0.054 -0.205 -0.029 0.102

0.131

0.026 0.922** 0.962 0.032 0.785

0.106 -0.117

0.942 -0.206

0.891 *

-0.112 -0.213* 1

Pr1= Precipitation, Pr2= production, D= density, Dl= day of lactation, Ci= calving interval, Y= years, Dn= delivery number, F= fat, Pr3= protein, Ts= total solids, C= casein, SCC= somatic cell count.

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Table 4: Matrix of correlation Pupiales Pr1

Pr2

Pr1

Pr2

0.021

0.106

0.063

-0.046 -0.389

-0.013 -0.253

-0.020 -0.040

-0.010 0.070

0.080

0.024 0.165

-0.185

0.786** 0.076 **

0.022

0.949

0.081

0.947** 0.124

-0.125 0.659

Pr3

SCC

1 *

SCC -0.001 -0.183

0.651** 1

-0.180* 0.159*

Pr3 C

1 *

0.126 0.038 -0.055

0.002

0.179*

0.069

0.871** 1

-0.312* -0.264* -0.001 -0.263

-0.203

-0.064

-0.344* -0.272* -0.087 -0.230 0.069

-0.085 0.070

0.047 0.790** 1 0.030 0.884** 0.942** 1 0.142 0.512*

0.122 0.232

0.075 1

0.134 -0.071

0.670** 0.509 -0.189

-0.106 -0.207* 1

Pr1= Precipitation, Pr2= production, D= density, Dl= day of lactation, Ci= calving interval, Y= years, Dn= delivery number, F= fat, Pr3= protein, Ts= total solids, C= casein, SCC= somatic cell count.

The results of the analysis of the principal components can be seen in Tables 5 and 6. For the three districts, four components were analyzed, representing 79.97, 77.04, and 75.53 % of the variability explained, for the Guachucal, Pasto and Pupiales respectively. As in the correlation analysis, the results show that production, fat, protein, casein and total solids are highly related and represent the first axis (compositional). Both age and number of parturition constitute the second axis, while the third axis is formed by the variables day of lactation and calving interval. The relationship found for these last two axes, however, may be a consequence of time. Table 5: Results of the variance observed in the axes used Extraction Initial Guachucal Pasto Pupiales Precipitation 1.000 0.856 0.881 0.856 Production 1.000 0.953 0.946 0.935 Density 1.000 0.361 0.313 0.473 Day lactation 1.000 0.772 0.704 0.741 Calving interval 1.000 0.810 0.738 0.820 Year 1.000 0.928 0.935 0.901 Delivery number 1.000 0.910 0.937 0.868 Fat 1.000 0.853 0.823 0.785 Protein 1.000 0.965 0.959 0.939 Total solid 1.000 0.991 0.980 0.930 Casein 1.000 0.969 0.894 0.624 SCC 1.000 0.223 0.133 0.172

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Table 6: Variability of the components Rotated Component Matrices Guachucal 1 2

Precipitation

.062

-.032

.086

Pasto 1 2

.918

.029

.052

-.043

Pupiales 1 2

.936

-.026

0.086

-.091

.916

-.036

-.168

.019

Production

.956

.054

-.188

.008

.956

.026

-.180

.003

0.951

Density

.138

-.368

.398

.219

.096

-.214

.399

-.316

0.172

-.311

.397

.435

0.091

.812

-.070

Day lactation

-.323

.090

.806

-.095

-.327

.087

.767

.041

-.263

Calving interval

-.168

.084

.879

-.043

-.126

.098

.833

.139

-.136

0.123

.887

.007

0.939

.116

-.063

Year

.050

.954

.122

-.010

-.007

.958

.133

-.006

0.026

Delivery number

.128

.945

.025

.009

.056

.966

.019

.032

0.114

0.924

-.021

-.042

-.002

-.167

.121

Fat

.917

.034

-.102

-.004

.893

-.007

-.162

.023

0.862

Protein

.980

.026

-.064

.006

.975

-.028

-.080

-.008

0.964

-.064

-.056

.032

Total solid

.985

.021

-.141

.013

.975

-.020

-.169

.002

0.938

-.087

-.158

.132

Casein

.981

.031

-.075

-.002

.943

-.014

-.064

-.011

0.745

0.106

.012

-.238

-.226

-.071

0.012

0.046

.248

SCC

-.184

.089

.036

-.345

-.152

.196

.062

The fourth axis is represented by precipitation. It should be noted that the somatic cell count is not well represented in some of the four components evaluated, however, it is observed that there is a contribution to the components one and four in the three districts, indicating some degree of relationship between these components. The results of the mixed model analysis can be seen in Table 7. The variables fat and total solids were not affected by season and SCC (P>0.05). In the case of production, protein and casein, the results showed that cell count do have significant influence (P<0.05).

Parameter

Production Fat Protein Total solids Casein

0 1 0 1 0 1 0 1 0 1

Table 7: Mixed model coefficients SCC (range) P-value â&#x2030;¤200 201-500 501-999 â&#x2030;Ľ 1000 Ss 18.02 16.92 15.93 14.84 0.755 17.97 17.05 15.76 14.21 0.684 0.672 0.673 0.672 0.315 0.692 0.683 0.685 0.681 0.597 0.577 0.559 0.541 0.757 0.591 0.573 0.555 0.545 2.216 2.122 2.197 2.181 0.248 2.168 2.131 2.148 2.105 0.530 0.483 0.446 0.424 0.103 0.543 0.494 0.450 0.429

SCC

Ss*SCC

0.048

0.444

0.123

0.355

0.011

0.827

0.148

0.328

0.022

0.201

Ss= season (1: dry, 0: rainy); SCC= somatic cell count; Ss*SCC= interaction effect.

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Discussion The interpretation of the results was made considering current Colombian regulations, which revolves around Decree 616(12) and Resolution 000017(4), responsible for guaranteeing the safety of human milk consumption and the compositional and sanitary quality of milk. Based on the information provided by the statistical analysis, it can be established that the compositional quality is closely related to milk production. It was also observed that the SCC does not affect the compositional quality of the same, and that there is no evidence to support this premise. In this regard, various authors found values of 24.28 kg of milk, which is higher than that found in the three districts(13). Manterola(14) reported an average production of 20 kg/d/cow of milk, and points out that age is a minor factor if the replacement rate is normal, though it does have a greater effect on the volume of production and thereby on the content of total solids. This was proven through the high correlation between production and the compositional parameters of the milk. Various authors also mention that the milk production of a cow is the result of the relationship of the environment and the inheritance(15). Precipitation, however, did not show significant relationships with these variables, as is observed in the diagram of the two components of PCA, the precipitation is very close to the cutoff point of the two coordinate axes. The Ministry of Social Protection, through decree 616, has established that the density of raw milk at 15 ÂşC ranges between 1.030 and 1.033 g/cm3. In this sense, the milk density of the samples evaluated fall within the regulatory framework. Other authors found an average value of 1.032 g/cm3 in the milk samples evaluated, and concluded that milk from healthy animals compared to that of animals with subclinical mastitis do not show variation in the density value(2). In animals with mastitis, however, the reflected density is affected by values lower than 1.029 g/ml. A study indicates that the composition of milk determines nutritional and industrial quality, which directly affects the profitability and competitiveness of milk production systems (16). Composition depends on the availability of blood precursors that reach the mammary gland, which can be manipulated through nutrition to vary milk components, though this factor was not evaluated in the present investigation. It was found that the raw milk received from the three districts complies with the parameters established by Decree 616 of 2006 regarding fat. The average value of the districts surpasses that reported by other authors with an average of 0.577 kg fat, implying an optimal value of milk fat for Holstein dairy(17). On the other hand, Gallego-Castro et al(13) reported values of 0.84 kg of milk protein for Holstein and Manterola(14) reported 0.90 kg of milk per cow per day. A studyâ&#x20AC;&#x2122;s suggest that variations in the production of milk fat within a group of cows fed in similar 429

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conditions, depends on the individual metabolic capacity of each animal(18). It should, however, be taken into account that the values observed in the districts of Pasto and Pupiales demonstrate higher levels of fat per day cow compared to the district of Guachucal. These differences may be the result of the handling of the herds in the area, though the present study cannot corroborate this hypothesis, as management variables could not be included in the analysis. On the other hand, biochemical adaptations of lipid metabolism depended directly on the stage of lactation of the cows. High values in milk fat during early lactation (5.49 %) suggest a lipid mobilization from body fat deposits, a factor that is not observed in the present investigation. Protein values comply with the parameters established by Decree 616 of 2006. Other authors report lower protein content per day cow, with an average value of 0.451 kg of protein(17). In this regard, other articles found values of 0.67 and 0.7 kg of protein(13,14), values close to those found in the present investigation. Various authors state that the protein concentration of milk does not present outstanding changes with nutritional manipulation(19). However, the effect of soybean meal on nitrogen use and protein production in Holstein cows has been evaluated, reaching conclusions that milk and protein yield do not show an increase with a supplementation level of soybean meal higher than 16.5 %. As an alternative to nutritional manipulation, the effect of genetic variants and haplotypes on the protein composition of milk has been studied. In a study conducted with 1,912 Holstein cows, the authors indicated that the genotypes β-CN and κ-CN haplotype A2B, were associated with protein yield and protein/ L of milk concentration respectively(20). The authors mentioned that selection of these genotypes and haplotypes would result in cows that produce milk better suited for cheese production. In a separate investigation, the author suggested that knowledge of genetic variability could be useful when altering the composition of milk protein, since the estimation of the genetic parameters of the six main milk proteins determined by capillary electrophoresis in zone are highly related(21). According to García et al(19), this information suggests the possibility of modifying the protein composition of cow's milk through selective breeding, which in turn offers the opportunity to satisfy the new consumer demands. Casein results reported an average value of 0.454 kg, which in contrast to that found by other authors with values of 2.4 %, presents a desirable and superior casein value of within milk production(22). Recent research claims that casein constitutes about 78 % of milk proteins, and precipitate when the milk is acidified to a pH of 4.6(19). They also state that casein is mainly linked to calcium phosphate Ca3(PO4)2 in a solid and spongy structure called casein micelle, an important component for cheese making. The treatment of milk with the chymosin enzyme of the rennet of suckling calves produces the destabilization of micelle, as the κ-casein (κ-CN) loses its hydrophilic region by proteolysis in the caseinomacropeptide segment, facilitating the addition of the para-κ-CN fragment(23). As this protein component is fundamentally hydrophobic, the casein content directly influences the coagulation time of all cheeses, and therefore quality and yield(24).

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For total solids, it was found that the raw milk received from the three districts meets the parameters established by Decree 616 of 2006, indicating excellent milk quality(4,12). Similarly, other authors reported values of 1.351 kg of total solids, a value that is lower than that found in this study(25). A national somatic cell standard was not adopted, as it does not exist in Colombian legislation. For Decree 616 of 2006 and Resolution 00017 of 2012 of the MADR, the SCC benefits are voluntary and discretionary for the companies that wish to improve this aspect of the quality of the milk. Even so, companies like Colanta report that values below 400,000 SCC/ mL and up to 200,000 SCC/mL are rewarded with $USD 0.007 per liter. Additionally, if the values are below 200,000 SCC/mL the incentive increases to $0.01. If they are above 1,000,000 SCC/ mL, the milk is not received, and a deduction is made(24). Currently, one quarter of the mammary gland is considered healthy; that which does not show any external pathological changes, when the milk is free of pathogenic microorganisms, and has a somatic cell level of <100,000 CFU/mL(26). The results of the mixed model indicate that counts higher than 500,000 CFU/mL affect the compositional quality of milk, decreasing production as well as protein and casein contents the milk. In this regard, other studies found similar results in Canadian Holstein cows, demonstrating that subclinical mastitis affects the compositional quality of milk(27).

Conclusions and implications Somatic cell count affects protein, casein, and production variables in the specialized milk systems of Guachucal, Pasto and Pupiales.

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https://doi.org/10.22319/rmcp.v11i2.5686 Article

Characterization of Aspergillus flavus and quantification of aflatoxins in feed and raw milk of cows in Aguascalientes, Mexico

Erika Janet Rangel-Muñoz a Arturo Gerardo Valdivia-Flores a* Onésimo Moreno-Rico b Sanjuana Hernández-Delgado c Carlos Cruz-Vázquez d María Carolina de-Luna-López a Teódulo Quezada-Tristán a Raúl Ortiz-Martínez a Netzahualcóyotl Máyek-Pérez e

Universidad Autónoma de Aguascalientes, Centro de Ciencias Agropecuarias, Av. Universidad 940, Col Cd. Universitaria, 20131, Aguascalientes, México. b

Universidad Autónoma de Aguascalientes. Centro de Ciencias Básicas. Aguascalientes, Aguascalientes, México. c

Instituto Politécnico Nacional. Centro de Biotecnología Genómica. Reynosa, Tamaulipas, México. d

Instituto Tecnológico El Llano, Municipio de El Llano, Aguascalientes, México.

Universidad México Americana del Norte A. C. Coordinación de Investigación. Reynosa, Tamaulipas, México.

Corresponding author: avaldiv@correo.uaa.mx

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Abstract: Contamination of agricultural and livestock products with aflatoxins (AF) is distributed worldwide. AFs are toxic, carcinogenic, and immunosuppressive; however, in Mexico, there is little information about Aspergillus flavus, the main fungus that produces them. The objective was to characterize the molecular and morphological, aflatoxigenic isolates of A. flavus and quantify the AFs in the feed and in the milk of Holstein cows in Aguascalientes (Mexico). A dairy production unit (2,749 cows) was selected for reasons of convenience, and monthly samples of food ingredients and total mixed ration (n= 267), raw milk (n= 288), and agricultural soil (n =40) were collected during 24 months and were cultivated (in PDA) using the pour plate technique with serial dilutions. The fungi were characterized using SEM, TLC and vapors of ammonium in coconut agar; the genes of calmodulin and a regulator of the biosynthetic pathway of AF, as well as the region of the internal spacer of the transcript, were sequenced. AFs were quantified in feed with HPLC and in milk, using ELISA. A total of 283 fungal isolates were characterized molecularly; of which 88 proved to be Aspergillus spp. Five of these were A. flavus with an aflatoxigenic capacity, and one was non-aflatoxigenic. 99.3 % of the samples of feed and 39.9 % of the milk samples exhibited detectable levels of AF (14.8 and 0,021 Âľg/kg). The cows ate daily 621 Âľg of AF and eliminated 0.09 % as AFM1 in milk. This suggests that the occurrence of aflatoxigenic A. flavus in the feed of dairy cows leads to a widespread contamination of the diets and food chain with AF. Key words: Aflatoxins, A. flavus, Dairy foods, Calmodulin gene, Regulatory gene of aflatoxin.

Received: 04/09/2018 Accepted: 29/04/2019

Introduction

Mexican bovine dairy is developed especially in a biogeographic province known as the Mexican Central High Plateau(1). Within this region, dairy production units (DPU) face insufficiency issues in the production, economic profitability and safety of the products (2). Among the food safety problems, the presence of aflatoxins (AF) has been highlighted because they cause a strong economic impact associated with the contamination of agricultural crops, a deterioration in the health of animals, a decline in productivity, and contamination of food of animal origin(3). AF have hepatotoxic, nephrotoxic and

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immunosuppressive properties and are considered as the most powerful known natural carcinogens(4). AF have been quantified in feed, milk and milk products intended for food for the Mexican human population(5-7). This suggests that the presence of AFs in the feed of cows is a common problem in the DPUs that leads to contamination of the milk with the metabolites of AF. When dairy cows consume agricultural products contaminated with AF, the enzymatic mechanisms bioactivate the mycotoxin through the formation of an epoxide that reacts with the cellular structures and nucleic acids, damaging their integrity(4). The reactive epoxide can also be neutralized by conjugation with glutathione or its excretion as a metabolite that is eliminated in the milk mainly as aflatoxin M1 (AFM1); furthermore, AFM1 is considered as a toxic and carcinogenic agent for humans(4). AF are secondary metabolites of several filamentous fungi of the genus Aspergillus, which are distributed worldwide and contaminate a large variety of agricultural products, particularly cereals(8). Also A. flavus has been identified in Mexico, both in agricultural soil and in corn grains(9,10). Asexual reproduction of Aspergillus species has been described previously(11,12), and so has its phylogeny, morphology and biological cycle(8,13). Although Aspergillus flavus is considered to be the species with the greatest capacity for production of aflatoxins(14), other strains of A. flavus without aflatoxygenic capacity, as well as other species of Aspergillus, produce AFs (A. parasiticus, A. nomius, A. pseudonomiu, A.arachidicola, A. bombycis, A. minisclerotigenes, A. pseudotamarii, and A. Togoensis)(15). Aflatoxygenic capacity is expressed mainly under conditions of environmental stress(16,17), as long as its genotype includes the information involved in the metabolic production chain of AF(18,19). A strategy for the molecular identification of the different species of the genus Aspergillus is the use of the gene of calmodulin (CaM), the fragments corresponding to the region of the internal transcribed spacers (ITS) and the regulatory gene of the aflatoxin (AfIR) biosynthetic pathway(8,20). The objective of this work was to characterize the molecular and morphological, aflatoxigenic isolates of Aspergillus flavus obtained from dairy production units of the Central High Plateau of Mexico.

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Material and methods

Study area

The present study was performed with a descriptive, longitudinal and non-experimental design. A dairy production unit was selected for convenience, using the non-probabilistic method, and was followed for 24 mo. The DPU was located in the Central High Plateau of Mexico (21°48'N, 102°03' W; 1986-2008 m asl), with a dry temperate and a semi-warm semidry climate, with summer rains, average temperature of 18.4 º C, an annual rainfall of 518.4 mm, and a maximum altitude of 2,300 m asl. During the observation period, the DPU had an average of 2,749 Holstein cows housed in confinement within open-air pens enclosed with metal fences, shaded areas and freely accessible feeders. The cows were milked with automated equipment, obtaining an average daily production of 28.1 L per cow. The milk production is exported to agro-industrial plants in the region. Diets were developed as a total mixed ration (TMR) in a feed mill, where the corn silage and the concentrate were blended in mixer wagons. The TMR release is formulated to meet the nutritional requirements of dairy cows. The corn for silage is obtained directly from the agricultural areas of the UPL, while the protein-energy concentrate was purchased from the Local Association of Dairy Farmers of Aguascalientes, using as main ingredients canola, soybean, corn, rolled, sorghum, soybean meal, corn distilled dry grains, cottonseed, alfalfa and oats hay, as well as premixed vitamins and minerals.

Sample collection and handling

A total of 288 samples (1.0 kg) were obtained: total mixed ration (TMR), concentrated feed and corn silage were dried up in a forced air circulation oven (OF-22G-TECH, JEOI Lab Companion, Corea). The samples were pulverized (500-800 µm) in a continuously operating universal mill (MF series 10 Basic, IKA®-Werke, Germany) and stored in cooling system (4-5 °C) until processing (<7 d). During the (morning and evening) milking, a total of 288 samples of raw milk were obtained directly from the collector tank (500 ml) for each production batch (high, medium and low). The samples were transported in refrigeration and were preserved in freezing (-20 °C) until processing (<7 d).

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Five agricultural plots were sampled with the help of a soil punch, choosing four sampling points on the surface of each plot of land and taking five 100 g sub-samples at each point, at a depth of 3-30 cm. The five sub-samples were gathered in a zip lock bag. Finally, they were screened (500-800 µ) and kept in refrigeration until processing.

Quantification of aflatoxins

Food samples were analyzed according to the official method 990.33 of the AOAC(21), using solid phase columns (SPE; SupelcleanTM LC-18 SPE tube, Sigma-Aldrich, USA). The eluate extracted from the samples derivatized with trifluoroacetic acid was analyzed by HPLC (detection limit 2 µg/kg) with a fluorescence detector (binary Pump Vary Pro Star; FP 2020 detector, Varian Associates Inc., Victoria, Australia), a C18 column, and a column guard (LC-18 LC-18; Thermo Fisher Scientific, Massachusetts, USA). The quantification data were obtained using the Galaxie software (version 1.9.302.530), and AF concentrations were calculated using standard curves of purified AFs (Sigma-Aldrich, St. Louis, MO, USA). The AFM1 was quantified in raw milk with the competitive ELISA technique using a commercial kit (Ridascreen fast® aflatoxin M1 R-1121, R-Biopharm, Germany; detection range 0.005-0.08 µg/kg). The samples were homogenized and centrifuged according to the manufacturer's instructions. The absorbance was measured at 450 nm in an ELISA microplate reader (BioTek Instruments, Inc., USA). The results were interpreted upon the basis of the calibration curve made with purified 1 AFM (Sigma-Aldrich, St. Louis, MO, USA).

Fungal Isolation and characterization

The fungi were isolated using the pour plate technique with four serial dilutions in sterile peptone water(22); the samples were seeded in a potato, dextrose and agar (PDA), malt extract with rose Bengal and Czapeck medium, and incubated (28 °C) in the darkness during 7 days. The colonies were isolated and purified, and the microscopic morphological characteristics consistent with the description of the genus were identified(13). Isolates consistent with A. flavus(8,13) were submitted to the process of fixating with glutaraldehyde (2%), gradual alcohol dehydration, and conditioning with a critical point dryer (Samdri Tousimis Research 795, Rockville, Meryland) and a metalizer (Desk II, Denton Vacuum, USA) were digitized in a scanning electron microscope (JEOL JSM-5900 LV,JEOL, USA) and conducted 10 439

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measurements of each structure (stipe, gallbladder, spore, esclerocio and fialide), and using a JEOL software (Scanning Electron Microscope). The morphological structures of the isolates identified were compared against known strains of A. flavus, known as AF-36, AFCuatitlan (AF-C) and AF-Tamaulipas, (AF-T)(14.23). The strains obtained in this study were recorded at the NCBI (National Center for Biotechnology Information) with the respective access codes. The aflatoxigenic capacity of the isolates was characterized by thin-layer chromatography (TLC)(24); silica gel plates without fluorescence indicator were utilized (Z265829, SigmaAldrich, USA) activated in a high temperature oven (OF-22G-TECH, JEOI Lab Companion, Corea). The plates with purified standards of AFs (6636-50MG, Sigma Aldrich, USA) were placed inside of a chromatographic chamber with mobile phase chloroform - acetone isopropanol (85:10:5, v/v/v) for an hour and a half. Eventually, the dry plates were visualized in a transilluminator. The technique ammonium vapors in coconut agar technique was also used according to the methodology described above(25). Monosporic isolates were inoculated in sterile coconut agar and were left to incubate in the dark (30 °C, 5-7 d). Subsequently, ammonium hydroxide (200 µl; J.T. Baker, Mexico) at 25 % was added to the cover of the Petri dishes, and the distribution and the intensity of the color was observed. The presence of color was taken as indicative of AF production.

Molecular analysis

The genomic DNA of monosporic cultures of Aspergillus spp. was extracted according to previously standardized methods(26). The quality of the obtained DNA was displayed with electrophoresis (45 min to 85 volts) in agarose gel (1%) with a TAE 1X damper and was quantified by comparing against known concentrations of DNA of phage λ (Thermo Fisher Scientific, MA USA). The DNA visualization was performed using a device for photoanalysis (Bio-Rad Gel Image®- Doctm XR, CA USA) with the Quantity One software version 4.6.7. An amplification of a fragment corresponding to the region of internal spacers of the transcript (ITS; ITS1-5.8S-ITS2 RNAr) and ITS4 (5´-TCCTCCGCTTATTGATATG -3´) was carried out in accordance with the previously described protocols(27,28); the gene of calmodulin (CaM) was amplified with the primers CMDA7F (5'GCCAAAATCTTCATCCGTAG-3') and CDMA8R (5'-ATTTCGTTCAGAATGCCAGG3') and the regulatory gene of the aflatoxin biosynthetic pathway (aflR) was amplified using the primers aflR-F (5'-GGGATAGCTGTACGAGTTGTGCCAG aflR-3') and aflR-R (5'TGGKGCCGACTCGAGGAAYGGGT-3') of Eurofins Genomics, Louisville KY, USA. The polymerase enzyme Go-Taq (Promega, Madison, WI USA) and a model 9700 thermal 440

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cycler (Applied Biosystems) were utilized in the amplification. The PCR products obtained (ITS, calmodulin and aflR) were separated by electrophoresis in (1%) agarose gel and they were observed using as intercalating agents SYBR® Gold and Orange DNA Loading Dye (Thermo Fisher Scientific, MA USA); the resulting bands were observed in an image analysis device (BIO-RAD IMAGE® GEL Molecular- DOCTM XR CA, USA) with the Quantity One software (version 4.6.7). Molecular weight marker ladders (Axygen Biosciences, CA, USA) were included. PCR products were purified with the reagent ExoSAP-IT® PCR Product Cleanup (Afflymetrix, Thermo Fisher Scientific Inc. Santa Clara, California, USA). The purified PCR products were sequenced in forward and reverse chains with the dideoxy method(29). The samples were injected into a sequencer (ABI 3730XL Genetic Analyzers), and the resulting sequences were recorded in an electropherogram. The electropherograms were visualized with the Chromas Lite software and were compared with the records of the NCBI using BLAST (Basic Local Alignment Search Tool).

Statistical analysis

The quantitative data of the total mixed ration, milk production, AF concentration in feed and milk, and of the measurements of the structure of each isolate were subjected to a oneway variance analysis (ANOVA), considering as separate factors the time of year and the level of milk production (high, medium or low) in which the cows were classified in batches. The differences between the means and the 95 % confidence intervals were determined using the honestly significant difference (HSD) Tukey’s test; P<0.05 was considered significant for all statistical analyses.

Results

Frequency of Aspergillus spp

It was identified a total of 283 fungal isolates; 56.8 % came from samples of feed for dairy cows, and the rest, from the agricultural land (Table 1). A total of 88 isolates (31.1 %) exhibited morphological features corresponding to those described for the genus Aspergillus. Figure 1 shows the conidiophores and conidia, and the characteristic metulae. Isolates were also found with a morphology compatible with the genera Penicillium, Fusarium, Rhizopus,

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Mucor, Cladosporium, Richoderma, Alternaria, Curvularia and Bipolaris. The proportions of each gender are shown in Table 1. Table 1: Fungal genera identified in monthly samples * of a dairy production unit in the Central High Plateau of Mexico Soil

Gender Samples Aspergillus Penicillium Fusarium Rhizopus Mucor Cladosporium Trichoderma Alternaria Curvularia Bipolaris Total

Corn silage

Feed concentrate

Total mixed ration

Total

No.

40 30 23 20 15 15 5 10 4 0 0 122

-24.6 18.9 16.4 12.3 12.3 4.1 8.2 3.3 0.0 0.0 100

96 16 3 3 4 6 4 0 2 0 0 38

-42.1 7.9 7.9 10.5 15.8 10.5 0.0 5.3 0.0 0.0 100

96 19 4 11 7 4 10 0 1 2 0 58

-32.8 6.9 19.0 12.1 6.9 17.2 0.0 1.7 3.4 0.0 100

96 23 9 2 8 9 10 0 2 1 1 65

-35.4 13.8 3.1 12.3 13.8 15.4 0.0 3.1 1.5 1.5 100

328 88 39 36 34 34 29 10 9 3 1 283

-31.1 13.8 12.7 12.0 12.0 10.2 3.5 3.2 1.1 0.4 100

*Sample of food ingredients: 24 months, in quadruplicate. Soil samples: 5 plots, in quadruplicate, 2 seasons.

Figure 1: Aspergillus morphological structures

Panels: A) Conidiophore: Cc = conidial head, Ee = Stipe, Pe = foot cell, Mo = mycelium. B) conidial head uniseriate portions: Co = Conidium or spore; Fe = Phyalid, Va = Vesicle C) Biseriate conidial head: Co = Conidium, Fe = Phyalid , Va = Vesicle, i = Metula.

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Only six isolates exhibited a morphology consistent with A. flavus and came from the feed concentrate (AC1, AC2 and AC3) and corn silage (EM1, EM2, EM3); when cultivated in PDA, they exhibited an olive green hue with a whitish periphery. Most of the isolates (5/6) exhibited the presence of sclerotia of dark brown color; only one did not manifest these structures. At a microscopic level, conidiophores of A. flavus exhibited irradiate and uniseriate conidial heads, the stipe with rough walls, the spherical vesicle, globe-shaped conidia with irregularities in their surface, and a whitish, septate mycelium (Figure 2).

Figure 2: Morphological structures of Aspergillus flavus

Panels: A) Cc = conidial head, Ee = stipe. B) Va = vesicle. C) Conidiophore. Fe = phyalids, Co = conidium. D) Cc = conidium, Fe = phyalid. E) Mo = mycelium, So = septum. F) Eo = Sclerotium.

The six isolates of A. flavus from feed exhibited significant differences in the morphometry of their structures when compared against the control strains (AF-C and AF-T; Table 2). These strains presented conidial heads, with rough radiated stipe and spherical vesicles, while AF-C only exhibited (uniseriate) phyalids and (biseriate) AF-T phyalids metulae; the conidia were globe-shaped with irregularities on the surface. Four strains isolated were classified as L (long sclerotium, >400 Âľm), one as S (short sclerotium, <400 Âľm), and one as without sclerotium.

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Table 2: Comparison of the dimensions of the morphological structures of Aspergillus flavus isolates obtained from corn silage (CS) and whole feed (WF) for dairy cows, as well as of the Cuautitlan (C) and Tamaulipas (T) strains Isolate

Stipe Mean

Vesicle

Mean

Spore UL

Mean

Sclerotium

LL UL

Mean

Fialide Mean

LL UL

AF-C

237

183 291

33.6

27.5 39.7

3.3

3.1 3.4

418

353 483

5.9

4.9 6.9

AF-T

646

564 727

76.6

67.7 85.5

2.8

2.7 2.9

453

361 545

6.4

4.9 7.8

AF-AC1

313

241 386

54.3

46.3 62.2

3.1

3.0 3.3

592

516 667

5.1

4.4 5.8

AF-AC2

373

292 454

58.2

49.3 67.0

3.0

2.8 3.1

428

352 503

5.1

4.3 5.8

AF-AC3

441

347 534

47.8

37.5 58.0

3.1

2.9 3.3

6.6

5.5 7.7

AF-EM1

331

255 407

50.5

42.1 58.8

3.5

3.4 3.7

4.1 5.5

AF-EM2

218

115 320

38.8

27.6 50.1

3.2

AF-EM3

320

239 401

47.3

38.4 56.2

3.6

268

193 344

4.8

3.1 3.4

672

597 748

5.4

4.1 6.6

3.5 3.8

561

505 617

6.9

6.0 7.9

-- Without sclerotium- LL, UL, lower and upper limit of the confidence intervals. abcd Means of columns with different letters show significant statistical differences (P<0.05).

The isolates identified as A. flavus were analyzed using TLC and ammonium vapors in coconut agar techniques in order to identify the presence of aflatoxin (Figure 3). Five isolates (AF-AC1, AF-AC2, AF-AC3, AF-EM2 and AF-M3) had a positive reaction to the presence of AF, while the other (AF-EM1) produced no aflatoxins, with both techniques. These results coincided with the amplified aflR gene. Figure 3: Intensity of color in a coconut agar medium in contact with ammonia vapors of Aspergillus flavus isolates after 4 d of growth

Panels: A) AF-36, non-aflatoxigenic (AF-). B) AF-Cuatitlรกn, and C) AF-Tamaulipas, aflatoxigenic (AF+). D) AF-EM1, AF- isolate from corn silage. E) AF-AC2, AF+ isolate from concentrated feed. F) AF-EM3, AF+ isolate from corn silage.

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Molecular analysis

Of the 88 isolates morphologically identified as Aspergillus spp., 49 % were amplified for the calmodulin gene, and 31 % were amplified for ITS (Figure 4). The analysis of the sequences obtained showed that the species were A. oryzae (45.5 %), A. niger (10.2 %), A. ochraceus (3.4 %), A. pseudodeflectus (4.5 %), A. ustus (10.2 %), A. flavus (6.8 %), A. versicolor (5.6 %), A. nidulans (5.6 %), A. sublatus (4.5 %) and A. Sydowii (3.4 %).

Figure 4: Electrophoresis in an agarose gel at 1% of the PCR products amplified from the calmodulin gene (Panel A), internal transcribed spacer of the rDNA region 5.8S-ITS2 (Panel B), and regulatory gene of the aflatoxin biosynthetic pathway, aflR (Panel C)

Rails: MWM) Molecular weight marker (100pb). Controls: Non-aflatoxigenic A. flavus (AF-36) and aflatoxigenic fungi (Cuautitlan, AF-C; Tamaulipas, AF-T). A. flavus isolates of corn silage (EM1, EM2 and EM3) and feed concentrate (AC1, AC2 and AC3) intended for dairy cows

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The five local isolates identified as aflatoxigenic A. flavus amplified a fragment of 796 pb for the aflR gene. The analysis of the sequences of the control strains (AF-36, AF-C and AFT) and the six isolates of A. flavus (AF-AC1, AF-AC2, AF-AC3, AF-EM1, AF-EM2 and AFEM3) have been shown to have an identity percentage of over 90% with the isolates of A. flavus registered in the database of the NCBI (Table 3).

Table 3: Identity of the isolates of Aspergillus flavus obtained from diets for dairy cows in Aguascalientes, Mexico, compared to existing scripts at the National Center for Biotechnology Information (NCBI)a ID of the Isolate

Origin

Primerb

Access code

Coincidence (%)

Control- AF-36

Control

ITS1

LN482513.1

ITS4

KX550912.1

CMDA7F

AY974341.1

CMDA8R ITS1 ITS4 AFLRF AFLRR

AY974340.1 LC105688.1 KT254587.1 FN398160.1 L32576.1

96 100 99 100 100

ITS1

KX015990.1

100

ITS4 AFLRF AFLRR

KF221065.1 XM_002379905.1 AF441435.2

99 100 99

ITS1

KF434090.1

100

ITS4 AFLRF AFLRR

KF434090.1 AF441434.1 XM_002379905.1

99 100 99

CMDA7F

AY974341.1

ITS1 ITS4 AFLRF AFLRR

KM285408.1 EF409812.1 XM_002379905.1 FN398160.1

100 95 100 93

CMDA7F

XM_002374071.1

CMDA8R ITS1 ITS4 AFLRF AFLRR

XM_002379905.1 KF434090.1 KT356196.1 AF441435.2 AF441434.1

99 99 97 99 99

CMDA7F

AY510451.1

CMDA8R ITS1 ITS4 ITS1

XM_002374071.1 HQ010119.1 KX641192.1 KT356196.1

100 99 99 98

Control+ C (AHY255)

Control+ T (AHY256)

AC1 (AHY257)

AC2 (AHY258)

AC3 (AHY259)

EM1 (AHY195)

Corn (Cuatitlan)

Corn (Tamaulipas)

Feed concentrate

Corn silage

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LN482513.1

100

EM2 (AHY203)

Corn silage

AFLRF AFLRR

MH752566.1 KY769955.1

100 100

EM3 (AHY204)

Corn silage

CMDA7F CMDA8R ITS1 ITS4 AFLRF AFLRR

XM_002374071.1 XM_002374071.1 KX572367.1 KT356196.1 L32577.1 MH752564.1

100 100 99 99 100 100

NCBI (National Center for Biotechnology Information): https://blast.ncbi.nlm.nih.gov/ B CMDA = calmodulin; ITS = internal transcribed spacer; AFLR = regulator of the biosynthetic pathway; R = Reverse; F = forward.

Aflatoxins detected in feed and in milk

Of the 288 samples obtained from food, 286 (99.3 %) exhibited detectable levels of AFs (18.5 ± 3.7 µg/kg); of which 10.4 % exceeded the maximum limits allowed by the Mexican law (20 µg/kg). The presence of AFM1 (0.021 µg/kg) was detected in 39.9% of the 183 samples of raw milk, and 12.0% exceeded the maximum permissible limit used as a standard by the agro-industry which acquired the raw milk (0,050 µg/kg). The highest incidence of AF in feed occurred in summer and autumn (P<0.01), compared to winter and spring (17.4a ± 3.2 and 14.8ab ± 1.6 vs 8.1 bc ± 0.5 and 5.9c ± 0.5 µg/kg, respectively). In turn, the concentration of AFM1 was directly correlated with the concentration of AFs in the TMR in a double squared model (P<0.01; R2= 30.5 %). Also the presence of AFM1 in milk was significantly correlated with the level of milk production and the consumption of TMR; on average, the cows consumed daily 621 µg of aflatoxins in 42 kg of TMR; had a daily production of 26.2 L of milk with a total load of 0.603 µg of AFM1 ―which represented a great capacity of dairy cows to metabolize the AF―, and eliminated only a fraction (0.09 %; Table 4); in general, cows with a high production were exposed to the ingestion of larger amounts of AFs (658 µg/cow/day), and the presence and elimination of AFM1 in the milk of highly productive cows was greater than in cows with a medium and low production (0.22 µg/cow/d).

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Table 4: Average exposure (± SE) of dairy cows to the natural contamination by aflatoxins in the total mixed ration (TMR) and average elimination of AFM1 per productive batch Cows

Milk production

TRM

AF ingestion

(No)

(Kg/cow/day)

(µg/cow/day)

High

1760

32.0a

44.7a

658a

± 97.0

0.024a

Means

606

20.2b

36.4c

546a

± 83.4

Low

388

14.8c

38.4b

568a

Total

2754

28.0

42.0

621

Batch

AFM1 elimination (µg/kg)

(µg/cow/day)

(%)

± 0.005

0.72

0.11

0.022a

± 0.005

0.44

0.08

± 82.6

0.015b

± 0.004

0.22

0.04

± 92.0

0.021

± 0.005

0.58

0.09

Different letters indicate significant differences between production batches P<0.05.

Discussion In this two-year longitudinal study, the presence of Aspergillus flavus in the feed for dairy cows in the Central High Plateau of Mexico was proven. These isolates exhibited morphological, toxicological and molecular features which are consistent with the strains that have the capacity to produce aflatoxins. Furthermore, the accumulation of AFs in the feed and in the milk produced by cows was corroborated. Although information about the existence in Mexico of indigenous A. flavus populations with and without the ability to produce aflatoxins was already available, in general terms, this study adds for the first time the molecular, toxicological and morphometric characterization of A. flavus to the quantification of aflatoxins in the dairy, which is relevant for livestock production and public health.

Frequency of Aspergillus spp. and A. flavus The fungal genus Aspergilus had the highest occurrence at the DPU (31 %), followed by Penicillium (13.8 %) and Fusarium (12.7 %). These genera have already been identified in Mexico in stored yellow corn(9) and in corn hybrids intended for human and animal consumption(30). It has been suggested that the frequent presence of micotoxigenic fungi and their toxins in corn is due to improper handling of the crop and to ineffective implementation of strategies for the control of infections(5,31). In this study, A. flavus was isolated from the feed for dairy cows at a ratio of 6.8% of the total identified Aspergillus spp. (6/88). This species was not identified in agricultural soil intended for the alternating production of fodder maize and oats. In Mexico, A. flavus has been 448

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identified in commercial corn kernels(32) from soil for crops of grain corn(10). This suggests that fungal populations of this species are distributed across the agricultural areas of Mexico. In this study, the morphometric analysis showed that one-third of the isolates of A. flavus contained abundant short sclerotia (< 400 Âľm) and were therefore classified as S strains, while the rest were classified as L strains, as they had few but larger sclerotia (>400 Âľm). The importance of this morphology and its association with the aflatoxigenicity of S strains has already been pointed out; on the other hand, L strains contain both toxigenic and atoxigenic isolates(33,34).

Molecular analysis All of the 25 sequences obtained from the isolates identified morphologically as A. flavus exhibited a large percentage of identity (> 90%) with registered sequences of A. flavus strains. Amplified fragments were also obtained for the 468 pb CaM gene, for the 796 pb aflP gene, and for the ITS with a range of 600-800 pb (ITS1-5.8S-ITS2). It has been pointed out that the rDNA region of the internal transcribed spacers is the official DNA bar code for fungi because it is the most frequently sequenced marker and is a useful tool for the description of A. flavus species(8). It has also been said that the CaM gene is able to distinguish between almost all species of Aspergillus(8). On the other hand, the aflR gene is necessary for the transcription of most genes involved in the activation of enzymatic reactions that are necessary for the formation of aflatoxins(35-37). Based on the above, this work integrates molecular identification to the morphological identification of six isolates of A. flavus obtained from the dairy production chain in the Central High Plateau region of Mexico.

Aflatoxigenic capacity The six isolates identified as A. flavus were evaluated with TLC and vapors of ammonium in coconut agar in order to identify their capacity to produce aflatoxins; five were classified as aflatoxigenic fungi, and one was classified as a non-producer of aflatoxins. These results evidenced a behavior comparable to those obtained in other studies conducted in Mexico(10) in which A. flavus isolates were identified as genetically related to A. flavus AF-36(32). This non-aflatoxigenic strain has been employed in the development of biological control strategies to reduce aflatoxin production of aflatoxigenic strains in cotton-producing fields(15). The above suggests that the main aflatoxigenic A. flavus populations are distributed worldwide across agricultural ecosystems, and that they coexist with strains that have no capacity to produce aflatoxins. 449

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Aflatoxins detected in feed and in milk A total of 99.3 % (286/288) of the samples of feed for dairy cows had detectable levels of AFs (18.5 ± 3.7 µg/kg), of which 10.4% exceeded the maximum limits allowed by Mexican law (20 µg/kg); furthermore, 39.9 % (73/183) of the raw milk samples exhibited the presence of AFM1. Comparable proportions of AF contamination have been detected in balanced feeds produced in Mexico(5), in the total mixed rations of stables in Mexico(38), and in samples of feed of Asian origin(39). The incidence of AFM1 has been reported in raw bovine milk in Mexico(6,7); also, incidence of AFM1 has been identified in milk for human consumption in various countries(3,40-42). This suggests that the aflatoxin contamination of food for human and animal consumption is a global public health issue. This study showed an association between the level of milk production of cows with the amount and concentration of AFM1 eliminated in the milk, in such a way that highly productive cows consumed a larger amount of feed and were exposed to larger amounts of aflatoxins in their feed; therefore, the total amount was larger, and the concentration of AFM1 in raw milk was higher (P<0.05). It has been pointed out that the AFM1 elimination rate in milk is influenced by the amount of feed consumed per day and the amount of milk produced per day by each animal, as well as by the stage of lactation(43).

Conclusions and implications This study proved morphologically, toxicologically and molecularly the occurrence of A. flavus in corn silage and in the total mixed ration, which led to the aflatoxin contamination of almost all diets of dairy cows, as well as to the residual presence of AFM1 in raw milk. Dairy cows were able to metabolize and eliminate more than 99 % of aflatoxin present in their diet through routes other than milk.

Acknowledgments Financial support was provided by the National for Science and Technology Council of Mexico (Consejo Nacional de Ciencia y Tecnología; Basic Science Project: 178546 and Grant No. 514818) and the Autonomous University of Aguascalientes (Universidad Autónoma de Aguascalientes) (PIP project / SA 15-1). Also, thanks for the technical support of Araceli Adabache Ortiz and Marcelo Silva Briano.

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Conflicts of Interest

The authors declare that there is no potential conflict of interests with respect to the present research or to the authorship and/or to publication of this article.

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https://doi.org/10.22319/rmcp.v11i2.4885 Article

Comparison of surgical castration at birth versus immunocastration on carcass and meat traits in growing Holstein males

Jorge A. Cervantes-Cazares a Cristina Pérez-Linares a* Fernando Figueroa-Saavedra a Alma R. Tamayo-Sosa a Alberto Barreras-Serrano a Francisco G. Ríos-Rincón b Eduardo Sánchez-López a Issa C. García-Reynoso a Pedro Mendoza Peraza a Angelina León Villanueva a Luis A. García-Vega c

Universidad Autónoma de Baja California, Instituto de Investigaciones en Ciencias Veterinarias, A. Obregón y J. Carrillo s/n Col. Nueva, Mexicali, Baja California, 21100, México. b

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

Ganadera Mexicali, S.A. de C.V, Baja California, México.

*Corresponding author: cristinapl@yahoo.com

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Abstract: Castration of male cattle affects carcass and meat traits. An evaluation was done of the effect of surgical castration at birth and immunocastration on carcass and meat traits in 7-8-mo-old male Holstein calves (average weight= 240.8 kg). Animals in the surgical castration treatment were castrated 24 h after birth, while those in the immunocastration treatment were administered doses of the Bopriva vaccine at 1, 21, 101 and 181 d of growth. Live weight was recorded in both groups at 21, 101 and 181 d, and carcass and meat traits were quantified after slaughter. The surgically-castrated animals exhibited higher average weight (P<0.05), and higher weight at slaughter. Cold carcass weight, hot carcass weight, ribeye area and subcutaneous fat thickness were all higher in the surgically-castrated animals (P<0.05). No differences between treatments were found in meat pH and sheer force (P>0.05), but the b*, C* and H* values were higher in the IC animals (P<0.05). Castration at birth resulted in better average carcass weight and meat traits than immunocastration, but animal welfare must be considered when using surgical castration. Key words: Inmunocastration, Holstein males, Carcass evaluation.

Received: 07/05/2018 Accepted: 11/04/2019

Introduction Castration is a common management tool in beef production. It provides benefits such as reduction of aggressive and sexual behavior which facilitates safer handling. This in turn promotes better carcass quality due to subcutaneous fat deposition and less carcass damage from mounting or aggression in the finishing pen; all these favor animal welfare(1-5). Surgical castration is the norm but requires additional work and cost, causes prolonged pain in the animal(6,7), and can lead to infections and bleeding(8) or death in some cases(9). Immunocastration is a non-surgical technique intended to preserve animal welfare when in intensive finishing pens(10). Immunocastrated males produce an antibody against GnRh that consequently reduces testosterone concentrations(11) and physical activity(12). Holstein males have been increasingly included in growth pens as a production alternative. This breed differs from traditional meat-producing breeds in its friendly and playful temperament, but can become very aggressive if not castrated(13). Immunocastration has been 456

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tested in different breeds, with different vaccination programs, diets and implant programs(1417) . Immunological castration programs need to be evaluated in different production systems (breeds, diets, implants); for instance, Holstein beef cattle in commercial production where final weights greater than 550 kg are required for slaughter. The present study objective was to compare the effects of surgical castration at birth vs immunocastration on carcass and meat traits in growing Holstein males.

Material and methods Geographical location

The study was done in the city of Mexicali, Baja California, Mexico (32°32’00” N; 115°12’41” W). The region has a dry desert climate with an average temperature of 34.7 °C (-5 °C winter; 50 °C summer), average rainfall of 37 mm, and average relative humidity of 50 %(18).

Study design

The experimental animals were 720 Holstein males of the same origin, 7 to 8 mo’ age on arrival at the growth facility, with a 240 kg average live weight. Two treatments were applied: surgically-castrated (SC) males (T1) and immunocastrated (IC) males (T2). The animals were randomly assigned to the treatments, with 90 animals per pen and four pens per treatment. Those in the T1 treatment were castrated 24 h after birth at a dairy farm.

Handling after reception at growth facility

Twenty-four hours after arriving at the growth facility the animals were vaccinated, wormed, and an implant applied (contains trenbolone acetate, estradiol and tylosin). The animals in T2 were immunocastrated by subcutaneously administering 1 ml Bopriva® (Laboratorios Zoetis, Salud Animal, Mexico) at four times after arrival: 24 h (d 1), and on d 21, 101 and 181 of the experiment). The SC animals were administered 1 ml saline solution on the same days. Live weight (LW) was recorded for each animal on days 1, 21, 101 and 181 of the experiment and before slaughter. The animals were fed twice a day, following a six-diet 457

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program widely used in northern Mexico which consists of wheat hay, Sudan grass, tallow, DDGS (dry distiller’s grains with solubles) and a mineral premix.

Serum testosterone levels

Ten animals were randomly selected from each pen to measure serum testosterone levels; each was identified with an additional earring. Blood samples were taken on the same days as Bopriva application. A final sample was taken at slaughter at the bleeding station in the slaughterhouse production line. Approximately 5 ml blood were extracted from the coccygeal vein. The samples were centrifuged at 3,500 rpm (TRIAC centrifuge, Clay Adams, Model 0200, New Jersey, USA) to obtain the serum and stored at -20 °C until analysis. Testosterone concentration was measured using the ELISA (Bovine) Testosterone Kit (Abnova Corporation, Taipei City, Taiwan), following manufacturer instructions.

Slaughter

The animals were killed at 242 d after attaining a 607.85 ± 12.89 kg average weight. On the day of slaughter the animals were herded by a wrangler on horseback about 1.5 km to the slaughterhouse. Here they were kept in rest pens with access to water for approximately 5 h. They were slaughtered in a Federal Inspection Type (Tipo Inspeccion Federal - TIF) slaughterhouse, following established procedures (NOM-033-SAG/ZOO-2014).

Carcass evaluation

The carcasses from both treatments were stored at 2 °C for 24 h. Sample cuts were made between rib 12 and 13 to collect carcass data. A total of 120 carcasses per treatment (T1 and T2) were made available by the slaughterhouse for carcass trait data collection: hot carcass weight (HCW); cold carcass weight (CCW); back fat thickness (BFT); kidney, pelvic and heart fat (KPH); marbling; ribeye area (REA), pH and color (L*, a*, b*, C* and H*). Back fat deposition was measured in mm using a metric ruler(19). Ribeye area was evaluated using a plastic template following the Iowa State University method. Estimated KPH was determined subjectively and expressed as a percentage of HCW, and marbling was estimated with a seven-level scale (traces, light, small, modest, moderate, slightly abundant and moderately abundant)(20). 458

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Meat quality

Meat samples (approx. 1,000 g each) were taken 48 hours after slaughter from the Longissimus dorsi muscle of ten randomly selected carcasses from each pen in both treatments (total samples 80). These were vacuum-packed, refrigerated and sent to the Animal Quality Products Laboratory of the Veterinary Science Research Institute of the Autonomous University of Baja California. The pH was measured with a potentiometer (Hannah Instruments, Inc., pH 101). Color values (L*, a*, b*, C*, H*) were measured on the surface of the Longissimus dorsi muscle cut using a spectrophotometer (Minolta CM-2002, Minolta Camera, Co., Ltd., Japan). The specular component included (SCI) mode was used with illuminant D65, and a 10° observer; L* is the brightness index, a* is red intensity and b* is yellow intensity. Shear force (SF) was quantified using previously cooked pieces of meat extracted perpendicular to the muscle fibers with a 1 cm diameter punch and placed in a texturometer (Lloyd Instruments, England) equipped with Warner-Bratzler blades. All measurements were done in triplicate.

Statistical analyses

Total variation was analyzed with a linear model: Yij =µ+ τi + ξij with i = 1, 2 and j = 1, 2, . . ., r Where: Yij are meat pH, color and SF values as response variables; µ is the general mean, τi is the fixed effect of treatment (surgically-castrated vs. immunocastrated); ξij is the residual random effect [ξij ~ NI(0, σ2e )]. When the treatments were a source of significant variation (P≤0.05), a Tukey was applied to compare the treatments’ mean values; this was done with the GLM procedure in the SAS statistics package (SAS Inst. Inc., Cary, NC)(21). Mean serum testosterone levels were compared between treatments and over time (days 1, 21, 101 and 181, and at bleeding) with a mixed linear model: 459

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Yijk = Âľ+ Ď&#x201E;i +Ak(i) + Dj + (Ď&#x201E;D)i j + Îžijk with i = 1, 2 ; j = 1, 2, ..., 5, and k = 1, 2, â&#x20AC;Ś, r Where: Yijk is testosterone concentration of the k-th animal taken at j-th time and belonging to the ith treatment, as a response variable; Âľ is the general mean, Ď&#x201E;i is the fixed effect of treatment; Ak(i) is the random effect of animal within treatment [Ak(i) ~ NI(0, Ď&#x192;2a )], Dj is the fixed effect of time, in days, (Ď&#x201E;D)i j is the effect of the treatment Ă&#x2014; time interaction; Îžijk is the residual random effect [Îžij ~ NI(0, đ?&#x153;&#x17D;đ?&#x2018;&#x2019;2 )]. The analysis was run using the MIXED procedure with the REPEATED label in the SAS package. Analysis of repeated records, including correlations between records of the same animal and heterogeneous variances between records in time was done by evaluating the covariance structures: unstructured (US); compound-symmetry (CS); and first-order autoregressive (1AR). This was done based on the Akaike and de Schwartz criteria, choosing that with the lowest values for these two indicators. In this case the selected covariance structure was unstructured (UN). When the treatment x time interaction was a source of variation (Pâ&#x2030;¤0.05) a Tukey-Kramer procedure was applied to compare the least squares means between treatments for each time increment(22).

Results and discussion Weight gain

Animal weight was higher in T1 (surgically-castrated) (P<0.05) starting with the second vaccination (21 d), and remained so until slaughter (Table 1).

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Table 1: Mean values Âą standard error (SE) of animal weight (kg) by treatment on vaccination days until slaughter Growth day 1 21 101 181 Slaughter

T1 Surgicallycastrated 243.25 278.30 394.94 520.80 620.74

T2 Immunocastrated

P> t

238.39 269.70 379.53 509.52 594.95

2.50 2.49 2.51 2.54 6.90

0.052 NS 0.006* 0.001* 0.001* 0.002*

The trend observed in the present results differs from previous studies. For example, in a study of live weight in beef cattle, at 280 days males immunocastrated with Bopriva were heavier (P<0.05) than those surgically castrated at 91 days growth(10). In a study comparing males immunocastrated with Bopriva to other males surgically castrated between fifteen and seventeen days post-vaccination in the first group, weight did not differ (P>0.05) up to slaughter(9). One notable difference in the present study is that the animals in T1 were castrated at birth, meaning recovery time due to infection and weight loss had no impact at seven monthsâ&#x20AC;&#x2122; age when the animals were placed in the growth pens.

Serum testosterone concentrations

Testosterone concentrations in both treatments were below 1 ng/ml on each vaccination day until slaughter, confirming the effectiveness of vaccination in suppressing serum testosterone concentrations in cattle(4,9). The present results also support previous reports that seven months is the optimal age for immunization against GnRH and generates maximum antibody production in Bos taurus males(22).

Carcass and meat quality

Both HCW and CCW were higher (P<0.05) in the T1 animals (Table 2), which differs from the lack of difference in CCW reported elsewhere(14). Values for BFT and REA differed (P<0.05) between treatments, with the highest values in T1 carcasses. No differences were observed in KPH between treatments (P>0.05). In a previous study using male Nellore breed carcasses no differences (P>0.05) were apparent in BFT and REA between SC and IC animals(14). Results for BFT more in agreement with the present results have been reported 461

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in SC and IC Nellore and Nellore x Angus males(15). Previous reports of REA values are lower than those observed in the present study(15): castrated = 81.06 ± 1.78 cm2 vs. immunocastrated = 83.61 ± 1.73 cm2. These discrepancies may respond to breed since, for example, Holstein cattle are reported to have a larger and longer body structure and a longer growth period, allowing them to develop larger carcasses than beef cattle breeds(23). Table 2: Mean values ± standard error (SE) of carcass quality variables by treatment Growth day HCW, kg CCW, kg BFT, cm REA, cm2 KPH, %

T1 Surgically-castrated 376.60a 374.87a 0.65a 91.41a 1.49a

T2 Immunocastrated 362.61b 361.38b 0.55b 86.83b 1.60a

P> f

4.14 4.09 0.33 1.54 0.09

0.0009 0.0011 0.0042 0.0048 0.2473

WCW = warm carcass weight; CCW = cold carcass weight; BFT = back fat thickness; REA = ribeye area; KPH = kidney, pelvic and heart fat. a,b Different letter superscripts in the same row indicate significant difference (P<0.05).

Previous studies comparing SC and IC males in Holstein x Cebu(24) and Nellore cattle(10,14), found no differences (P>0.05) in BFT and REA values. No differences in CCW, BFT and REA values (P>0.05) were also observed in a study of Holstein males in which the animals were slaughtered at a lower average weight: 477 kg (SC) and 486 kg (IC)(9). Weight at slaughter was notably higher in the present results: 600+ kg (T1) and 594 kg (T2). The differences between the present carcass quality trait values and those in previous studies(10,14,15) may be due to surgical castration schedules; in previous studies it was done some days before or contemporaneously with Bopriva application while in the present study it was done 24 h after birth, meaning the animals had fully recovered when placed in growth pens at seven months’ age. Marbling category frequency did not differ (P>0.05) between the treatments, with the largest number of carcasses in both treatments classified in the “slight” and “small” marbling categories (Table 3). These results agree with previous comparisons between carcasses from IC and SC males(25). In another study no differences (P>0.05) were observed in intramuscular fat percentage between the carcasses of SC and IC Holstein males(9).

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Table 3: Marbling classification in male Holstein carcasses by treatment T1 T2 Marbling SurgicallyImmunocastrated Pr > X2 classification castrated (n= 126) (n= 126) Traces 3 0 -Light 67 66 0.9309 Small 46 50 0.6831 Modest 9 0 -Moderate 1 10 -No differences (P>0.05) between treatments observed in the Light and Small categories in a test of a proportional equality hypothesis.

Meat pH values did not differ (P>0.05) between the treatments (Table 4), with values in a normal range of 5.5 to 5.8(26). These results coincide with the 5.57 pH at 24 h reported for Holstein cattle carcasses(27). Table 4: Mean values ± standard error (SE) for meat physicochemical variables

Variables pH L* a* b* C* H* SF (N) a,b

T1 SurgicallyCastrated 5.54a 32.14ª 11.97ª 7.93b 14.63b 33.23b 52.17ª

T2 Immunocastrated

P> f

5.56a 32.74ª 10.70b 12.86ª 16.87ª 49.28ª 56.38a

0.04 0.43 0.33 0.47 0.48 1.21 0.24

0.7163 0.1731 0.0002 0.0001 0.0001 0.0001 0.0919

SE = standard error; SF = shear force. Different letter superscripts in the same row indicate significant difference (P<0.05).

Most of the color values (a*, b*, C* and H*) differed (P<0.05) between treatments, the L* value being the one exception (P>0.05). No difference in L* values (P>0.05) has also been reported in previous comparisons of meat from SC and IM male cattle(10,15). In addition, a lack of difference for the L*, a* and b* values was observed between the carcasses of SC males (L* = 33.9; a* = 17.1; b * = 2.6) and IC males (L* = 34.0; a * = 16.9; b* = 2.4)(9). Although meat pH was normal in the present results, the color values observed here are similar to those reported for DFD meat (L* = 34.8; a* = 18.8; b* = 6.7)(28), suggesting that the animals had been exposed to stressors prior to slaughter. 463

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Shear force (SF) did not differ (P<0.05) between treatments. Based on established criteria(29), meat tenderness was intermediate (tender: 22.26-35.10N; intermediate: 40.01-52.95N; hard: 57.85-70.60N). The same has been reported elsewhere(10,16), with no differences in SF between meat from SC and IC animals. Indeed, another study found no differences in SF due to sexual condition (SC males: 56.60 ± 0.36N; IC males: 53.37 ± 0.35N; whole males: 48.85 ± 0.35N)(15). Even in males slaughtered at eleven months of age SF did not differ between SC (51.9N) and IC males (52.9N)(9).

Conclusions and implications After more than 200 d growth, surgical castration 24 h after birth in Holstein males resulted in heavier animals with better carcass traits than males immunocastrated with Bopriva. Further research is needed to assess post castration impacts on animal welfare.

Acknowledgements The authors thank the personnel of Ganadera Mexicali S.A. de C.V. and TIF Slaughterhouse No. 511 for their assistance, as well as Dr. Priscila Castro Osuna for her help as technical advisor for Laboratorio Zoetis.

Literature cited: 1.

Bouissou MF, Boissy A, Neindre PL, Veissier I. The social behaviour of cattle. In: Keeling LJ, Gonyou HW editors. Social behaviour in farm animals. Oxford, UK: CABI Publishing; 2001;113-274.

Price EO, Adams TE, Huxsoll CC, Borgwardt RE. Aggressive behavior is reduced in bulls actively immunized against gonadotropin-releasing hormone. J Animal Sci 2003;81(2):411-415.

Stookey JM, Watts JM. Production practices and wellbeing: Beef cattle. Oxford. UK.: Blackwell Publishing; 2004.

Amatayakul-Chantler S, Jackson JA, Stegner J, King V, Rubio LMS, Howard R, Lopez E, Walker J. Inmunocastration of Bos indicus x Brown Swiss bulls in feedlot with

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gonadotropin-releasing vaccine Bopriva provides improved performance and meat quality. J Anim Sci 2012;90:3718-3728. 5.

Marti S, Devant M, Amatayakul-Chandler S, Jackson JA, Lopez E, Janzen ED, Schwartzkoppf-Genswein KS. Effect of anti-gonadotropin-releasing facto vaccine and band castrtion on indicators of welfare in beef cattle. J Anim Sc 2015;93:1581-1591.

Bonneau M, Enright W. Immunocastration in cattle and pigs. Livest Prod Sci 1995;42:193-200.

Mach N, Bach A, Realini C, Font-Furnols M, Velarde A, Devant M. Burdizzo prepubertal castration effects on performance, behavior, carcass characteristics, and meat quality of Holstein bulls fed high-concentrate diets. J Meat Sci 2009;81:329-334.

Turner AS, McIiwraith CW. Thecniques in large animal surgery. Philadelphia, PA. Lea and Febiger; 1989.

Marti S, Jackson JA, Slootmans N, Lopez E, Hodge A, Pérez-Juan M, Devant M, Amatayakul-Chantler S. Effects on performance and meat quality of Holstein bulls fed high concentrate diets without implants following immunological castration. J Meat Sci 2017;126:36-42.

10. Amatayakul-Chantler S, Hoe F, Jackson JA, Roça RDO, Stegner JE, King V, Howard R, Lopez E, Walker J. Effects on performance and carcass and meat quality attributes following immunocastration with the gonadotropin releasing factor vaccine Bopriva or surgical castration of Bos indicus bulls raised on pasture in Brazil. Meat Sci 2013;95:7884. 11. Thompson DL. Immunization against GnRH in male species (comparative aspects). J Anim Sci 2000;60:459-469. 12. Jannet F, Gerig T, Tschuor AC, Amatayakul-Chantler S, Howard R, Bollwein H, Thun R. Vaccination against gonadotropin-releasing factor (GNRF) with BOPRIVA® significantly decreases testicular development, serum testosterone levels and physical activity y pubertal Bulls. Theriogenology 2012;78:182-188. 13. Glenn CD, McMurphy CP. Feeding Holstein steers from start to finish. Vet Clin Food Anim 2007;23:281-297. 14. Ribeiro EL, Hernandez JA, Zanella EL, Shimokomaki M, Prudêncio-Ferreira SH, Youssef E, Reeves JJ. Growth and carcass characteristics of pasture fed LHRH immunocastrated, castrated and intact Bos indicus bulls. J Meat Sci 2004;68:285-290.

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15. Giuliana ZM, Faria MH, Roça RO, Santos CT, Suman SP, Faitarone ABG, et al. Immunocastration improves carcass traits and beef color attributes in Nellore. J Meat Sci 2014;96:884-891. 16. Miguel GZ, Roca RO, Suman SP, Faria MH, Santos CT, Resende FD, et al. Immunocastration and surgical castration improves color attibutes of beef from Nellore males. J Meat Sci 2014;96:462-463. 17. Andreo N, Bridi AM, Soares AL, Prohmann PEF, Peres LM, Tarsitano MA, De Lima GB, Takabayashi AA. Fatty acid profile of beef from immunocastrated (BOPRIVA®) Nellore bulls. Meat Sci 2016;117:12-17. 18. García E. Modiﬁcaciones al sistema de clasiﬁcación climática de Koppen (para adaptarlo a las condiciones de la República Mexicana. México, DF. Instituto de Geografía, UNAM;1981. 19. Hale DS, Goodson K, Savell JW. USDA beef quality and yield grades. USA: USDA; 2013.URL: http://meat.tamu.edu/beefgrading/.pdf. Consultado 10 Abr, 2017. 20. AMSA, American Meat Science Association. Meat evaluation handbook. USA. American Meat Science Association; 2001. 21. SAS. SAS software release 9.4. STAT 14.1. Cary, NC, USA: SAS Institute. Inc. 2015. 22. Steel RG, Torrie J. Bioestadistica: principios y procedimientos. México, D.F. McGrawHill Interamericana;1985. 23. Adams TE, Daley CA, Adams BM, Sakurai H. Testis function and feedlot performance of bulls actively immunized against gonadotropin-releasing hormone: Effect of age immunization. J Anim Sci 1996;71:950-954. 24. Duff GC, McMurphy CP. Feeding Holstein steers from start to finish. Veterinary Clinics of North America: Food Anim Practice 2007;23(2):281-297. 25. De Freitas VM, Leão KM, de Araujo Neto FR, Marques TC, Ferreira RM, Garcia LL F, de Oliveira EB. Effects of surgical castration, immunocastration and homeopathy on the performance, carcass characteristics and behaviour of feedlot-finished crossbred bulls. Semina: Ciências Agrárias 2015;36(3):1725-1734. 26. Mariño G, Vilca M, Ramos D. Evaluación del pH en canales de toros Holstein (Bos taurus) y Nelore (Bos indicus). Rev Investig Vet Perú 2005;16:90-95. 27. Silva JA, Patarata L, Martins C. Influence of ultimate pH on bovine meat tenderness during ageing. J Meat Sci 1999;52:453-459.

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28. Wulf DM, Emnett RS, Leheska JM, Moeller SJ. Relationships among glycolytic potential, dark cutting (dark, firm, and dry) beef, and cooked beef palatability. J Anim Sci 2002;80:1895-1903. 29. Boleman SJ, Boleman SL, Miller RK, Taylor JF, Cross HR, Wheeler TL, Johnson DD. Consumer evaluation of beef of known categories of tenderness. J Anim Sci 1997;75:1521-1524.

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https://doi.org/10.22319/rmcp.v11i2.4926 Article

Ascospherosis in honey bees and its relationship to environmental factors in Jalisco, Mexico

José María Tapia-González a Gustavo Alcazar-Oceguera a José Octavio Macías-Macías a* Francisca Contreras-Escareño b José Carlos Tapia-Rivera a Tatiana Petukhova a,c Ernesto Guzmán-Novoa a,d

Universidad de Guadalajara. Centro Universitario del Sur. Departamento de Ciencias Económicas Administrativas y Departamento de Ciencias de la Naturaleza. Centro de Investigaciones en Abejas (CIABE). Av. Enrique Arreola Silva no. 883. CP 49000. Cd. Guzmán, Jalisco. México. b

Universidad de Guadalajara. Departamento de Producción Agrícola. Centro de Investigaciones en Abejas (CIABE). Centro Universitario de la Costa Sur. México. c

University of Guelph. Department of Population Medicine. Guelph, Ontario, Canada.

University of Guelph. School of Environmental Sciences. Guelph, Ontario, Canada.

*Corresponding author: joseoc@cusur.udg.mx

Abstract: Ascospherosis or chalkbrood is an infectious disease of honey bees (Apis mellifera) caused by the fungus Ascosphaera apis that results in the death of larvae. In severe cases it can cause a decrease

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of the population of adult bees and in the productivity of the colony. This is the first study performed in Mexico with the objective of determining the prevalence of A. apis in honeybee colonies in a beekeeping region. It was carried out in nine municipalities of southern Jalisco, distributed in two climatic zones (sub-humid and subhumid temperate warm), and the relationship of the fungus with factors such as height above sea level, precipitations and temperature was analyzed. Samples of bees were collected from 365 breeding colonies, of which 74.1 % were proven chalkbrood-positive by microscopic analysis. 75.6 % of the colonies in the warm area and 72.2 % of those of the temperate area were A. apis-positive and were not significantly different in terms of the prevalence of chalkbrood (P>0.05). The logistic regression analysis indicated that the increase in rainfall also significantly increases the number of colonies infected with A. apis (odds= 3.53; P<0.01). There was a significant correlation between the rainfall and the proportion of ascospherosis-positive cases (r= 0.87, P= 0.003). The other studied factors had no significant relationship with cases of ascospherosis. Due to the high percentages of colonies infected with A. apis found in this study, further research should be considered in order to determine the causes of the high prevalence of this fungus in honey bee colonies in the state of Jalisco. Key words: Ascosphaera apis, Apis mellifera, Environmental effects, Jalisco.

Received: 05/06/2018 Accepted: 18/04/2019

Introduction Ascospherosis or chalkbrood is a fungal disease of honeybees (Apis mellifera L.) caused by the fungus Ascosphaera apis(1). The fungus that causes chalkbrood affects mainly the larvae but also the pupae of bees. The two main routes of infection are digestive, through consumption of food contaminated with spores of the fungus, or else, the cuticle of the offspring, through the germinative tube coming out of the spores. Both forms of infection produce mycelia that penetrate the body of the larvae, causing their death and giving them the characteristic of mummies(2). There are predisposing factors that favor the occurrence of ascospherosis outbreaks, such as excess moisture inside the hive, low temperatures or excessive manipulation of the colonies(3). Empirical observations suggest that chalkbrood increases with the stress caused by the frequent transportation of hives in order to pollinate agricultural crops, as a consequence of the exposure to the pesticides used on the pollination sites, and due to the new pathogens that affect bees. All of these factors depress the immune system of the bees and favor the development of other pathogens, including

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A. apis(4). Mortality from chalkbrood in the offspring of bees is generally low, but there are times when it exceeds 30 %(5). The disease has been reported in almost all countries in the world, including Mexico(5-9). It is important to highlight that the prevalence of ascospherosis in certain countries increased considerably, to the extent that it has been considered a threat almost as serious as infestation with Varroa destructor(10). Ascospherosis has been little studied in Mexico, where it has not been given the required importance. However, in Yucatán, Medina and Mejía(11) found an association between ascospherosis and the collapse of bee colonies. Other than isolated studies carried out in Yucatán, it may be said that there are no data that allow knowing how prevalent and generalized chalkbrood is in Mexico, as no epizootiological study has been carried out to determine its prevalence in the country or in a particular state of the country. Furthermore, the situation of ascospherosis and the relationships that it may have to geographical and climate variables in apiaries of Mexico are unknown; therefore, the objective of the present study was to determine the presence of the disease in honey bee colonies and its relationship with geographic and climate variables in the municipalities of the south-southeast region of the state of Jalisco, Mexico.

Material and methods The study was carried out in nine municipalities located in two climate areas considered to be temperate and warm (temperate subhumid and warm subhumid climate, respectively) in order to determine the prevalence of A. apis and it there is a relationship between the climate and the prevalence of the fungus in honey bee colonies. The municipalities are located in western Mexico (19º 24’, 21º 14’ N; 101º 59’, 104º 5’ W). The Gómez Farías, Zapotlán el Grande, Tapalpa and Unión de Guadalupe municipalities have a temperate subhumid climate, and the Tecalitlán, Tamazula, Zacoalco de Torres, Sayula and Cocula municipalities have a warm subhumid climate(12). The municipalities with a warm subhumid climate have a mean annual temperature of 21 °C. This climate does not exhibit a clear-cut winter temperature change(13). Rainfalls occur from June to September, with a mean precipitation of 801 mm, and the average altitude is 1,495 m asl(12). Municipalities with a temperate subhumid climate have a well-defined winter temperature change; the mean annual temperature is 14.4 °C, and the mean annual precipitation is 1,117 mm; the average altitude is 1,711 m asl(13). Samples were collected from 365 honey bee colonies in 145 apiaries in both geographical areas. The sample size was estimated based on 4,950 beehives in the selected regions of the state of Jalisco. 365 (13.56 %) of these beehives were selected for analysis. The calculation was made using the formula of transversal studies and a disproportionate stratified random sample with a 95 % confidence interval and an 80 % statistical power. The result was 21 samples per municipality including 15% due to losses, for a total of 160 beehives. However,

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the decision was made to take a total of 365 beehives to obtain a greater accuracy in the analysis (Table 1).

Table 1: Number of apiaries and colonies sampled for diagnose of Acosphaera apis by municipality and climate area in southern Jalisco Municipality Cocula Sayula Tamazula Tecalitlán Zacoalco de Torres Gómez Farías Tapalpa Unión de Guadalupe Zapotlán el Grande Total

Apiaries

Colonies

17 21 36 21 14 6 12 8 10 145

41 52 93 54 39 15 37 20 14 365

Climate area Warm subhumid Warm subhumid Warm subhumid Warm subhumid Warm subhumid Temperate subhumid Temperate subhumid Temperate subhumid Temperate subhumid

Samples of the honeycomb were obtained from the central frames of the brood chamber where the open brood of the bees was located, and in certain cases signs of ascospherosis (mummification of the brood) were observed. A 10 x 10 cm piece of honeycomb of each assessed colony was cut and wrapped in a paper sheet and kept in a cardboard box in order to be transported to the laboratory where the diagnose was carried out. The altitude of each apiary was recorded using a GPS (Sportrack-color, Magellan, USA), while the data of the environmental temperature (ºC) and the rainfall (mm), were taken from the records of the National Commission of Water for each municipality(14). The brood contained in the samples of honeycombs were processed at the Laboratory of Microbiology of the University Center of the South (CUSur) of the University of Guadalajara, where lactophenol cotton blue was utilized to diagnose the fungi. The utilized slides were cleaned with an ethyl ether solution; once they were dry, a drop of physiological salt solution was added, in which a small fragment of a larva suspected to be infected with chalkbrood fungus was placed. The larval sample was extended with a toothpick until it absorbed the drop of water; then a drop of lactophenol cotton blue was deposited on the larva and covered with a slide. Subsequently, A. apis mycelia and sporocysts were sought under the dry weak objective of a microscope (40 X). The presence of A. apis was determined based on the finding of sporocysts that are typical of the fungus

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in the analyzed samples(15), as well as on the percentage of beehives with ascospherosis in each of the municipalities. In the statistical analysis, the response variable was the number of colonies of bees infected with the A. apis fungus between different municipalities. The predictors (explanatory variables) included in the analysis were: environmental temperature, rainfall, and height above the sea level. The explanatory variables were standardized in order to reduce multicolinearity issues. The statistical analysis was performed using the following techniques. The proportions of infected colonies between municipalities and between areas were compared using the Chi-square tests with Bonferroni adjustments for identifying significant differences. A multiple logistic regression analysis was performed according to the criteria of Hosmer and Lemeshow(16) in order to identify factors associated with the fungal infection; this analysis allowed calculating the ratio of likelihood (odds) of finding ascospherosis-positive colonies and its corresponding 95% confidence interval (CI). A Pearson’s correlation was also carried out between the proportion of ascosphorosis-positive cases and the factors. The statistical analyses were performed using the R package, version 3.3.2(17).

Results Of the 365 assessed samples, 74.1 % were found to be A. apis-positive. The municipalities with the highest percentages of positive samples were Gómez Farías and Zacoalco de Torres, with 93.3 and 92.3 %, respectively, while those that exhibited lowest percentages, were Zapotlán el Grande, Unión de Guadalupe and Tecalitlán, with 57.1, 60.0 and 61.1 %, respectively (Table 2). Table 2: Mean percentages of Ascosphaera apis-positive honey bee colonies in nine municipalities of two climate areas of southern Jalisco Municipality Cocula Sayula Zacoalco Tamazula Tecalitlán Tapalpa Gómez Farías Unión de Gpe. Zapotlán el Grande Total

Mean percentage 82.93 73.08 92.31 68.82 61.11 78.38 93.33 60.00 57.14 74.10

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Climate area Warm subhumid Warm subhumid Warm subhumid Warm subhumid Warm subhumid Temperate subhumid Temperate subhumid Temperate subhumid Temperate subhumid

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Apiaries with 100 % A. apis-positive colonies were found in the municipalities of Gómez Farías, Cocula, Sayula, Tamazula, Tapalpa, Tecalitlán, Zapotlán el Grande and Zacoalco de Torres; however, in Unión de Guadalupe, the highest percentage of positive colonies in an apiary was 60 %. The distribution of the percentage of colonies infected among the nine municipalities was examined by means of a box plot (Figure 1). The figure shows that the median of the percentage of infected colonies was 71 %. Half of all the sampled colonies exhibited ascospherosis prevalence values ranging between 57 and 82 %.

Figure 1: Box plot depicting the distribution of the percentage of bee colonies infected with the fungus Ascophaera apis

The values shown here include the minimum percentage, in quartile 1 (25 % of the data); the mean (50 % of the data); the maximum percentage, in quartile 3 (75 % of the data), and the range between quartiles.

A total of 36 comparisons between the proportions of bee colonies infected with A. apis in the nine municipalities. When the critical area was adjusted for multiple comparisons, the differences between proportions were not significant (P>0,05). As for the areas, the results show that the prevalence of ascospherosis between the warm (75.6 %) and the temperate (72.2 %) areas did not differ significantly (x2= 0.86, n= 209, P= 0.35). The results of the logistical regression analysis are shown in Table 3. The equation of the complete logistic regression model (the model including all the predictors) was the following:

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log odds = 1.04141 â&#x2C6;&#x2019; 0.03434 Temperature + 1.2188 Precipitation â&#x2C6;&#x2019; 0.02706 Height above the sea level Table 3: Predictors of infection of honey bees by the fungus Ascosphaera apis in the logistic regression analysis (complete model) Variables Temperature Precipitation Height above the sea level

Probability (Odds ) *0.97 *3.38

95 % CI (0.69 - 1.37) (1.73 - 7.48)

*0.97

(0.64 - 1.50)

p 0.84 <0.01 0.89

*Ascosphaera apis colonies.

The analysis showed that precipitation was significantly associated to infection of bee colonies with the fungus. The increase in the rainfall also increased the number of colonies infected with A. apis significantly (odds= 3.35; P<0.01; Figure 2). Likewise, a significant correlation was found between the rainfalls and the proportion of ascospherosis-positive cases (r= 0.87, n= 9, P= 0.003). The other studied factors had no significant relationship with cases of chalkbrood. Figure 2: Relationship between the rainfalls and the proportion of positive cases of infection by the fungus Ascosphaera apis in nine municipalities evaluated in Jalisco, Mexico

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Discussion This is the first study carried out in Mexico with the purpose of determining the prevalence of A. apis in honey bee colonies in several beekeeping regions of a state. In a similar study carried out in the semi-desertic region of the Highlands of Jalisco, Mexico, during the months of July to October (the rainy season), in which 42 colonies distributed along 12 km only within this region were monitored, 67.1 % of the colonies were found to be A. apis-positive(18). The study of the semidesertic region of the Highlands of Jalisco is based on a very small sample (42 colonies) of a restricted area of the state of Jalisco, whereas the present study included 365 colonies in the two regions with the largest production of honey in the state of Jalisco. However, it is worth noting that, although the study of the Highlands of Jalisco was performed during the rainy season, the percentage of positive samples was similar to the percentage found in the present study, performed during the dry season (74.1 %). This reinforces the hypothesis that the prevalence of chalkbrood in the bee colonies of the state of Jalisco is high at different times of the year. The environmental temperature influences the temperature and moisture of the beehive, but the bees create their own inner microclimate of moisture and temperature(19); therefore, it is difficult to determine the impact of the external environmental climate during the rainy season and during the dry season. However, other researches mention that a reduction of the inner temperature of the colony from 35 to 30 °C increases the prevalence of A. apis(20). In regard to the moisture, other studies agree with the findings of this study in the sense that the rainfalls are related to a high prevalence of chalkbrood. For example, in a recent research, relative moistures of 85 to 90 % and inner temperatures of 25 to 30 °C in A. mellifera colonies have been determined to favor the occurrence and proliferation of A. apis spores(21). On the other hand, colonies that keep the brood nest relatively dry and warm (> 35 °C) significantly limit infection by A. apis. Bramford and Heath(22) also determined that the temperature and the internal moisture of the brood nest are factors that predispose for the presence of A. apis in the colony. These authors assessed the cooling of the colony at 25 °C, which resulted in 95 % of chalkbrood mummies, while mummification diminished to 43 % with 30 °C, and to 29 % with 35 °C. As for the relative moisture, when it surpassed 85 % at a temperature of 30 °C, mummification increased by 7.75 % in relation only to the effect of the temperature at 30 °C. Likewise, with 68 % moisture at a temperature of 30 °C, the mummification of larvae of the colony increased only by 0.95 %. Clearly, low temperatures and a high level of moisture favor the occurrence of ascospherosis. Other researchers have found that other factors, such as stamped wax and pollen. contribute to the transmission and development of the chalkbrood disease in bee colonies(5,23). Also, a reduction in the proportion of adult bees to the brood contributes to the prevalence of the disease (24). Some of

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these factors may be contributing to the high prevalence of chalkbrood in Jalisco. However, only further studies will make it possible to determine whether these or other factors are responsible for this high prevalence of the disease.

Conclusions and implications 74.1 % of the 365 assessed samples were found to be A. apis-positive; this constitutes a high prevalence of the fungus in bee colonies of southern Jalisco. The prevalence of A. apis was particularly high in certain municipalities, where the average of positive samples reached over 90 %, and there were no differences in prevalence between the areas. The climate factor which had a significant association with the presence of A. apis and which most probably promoted a higher occurrence of the fungus in the bee colonies was rainfall. The altitude and environmental temperature had no significant effects on the prevalence of A. apis in this study. Due to the high percentages of A. apis in the assessed colonies, further studies must be considered in order to determine the causes (other than rainfall) of the high prevalence of A. apis in the bee colonies of southern Jalisco.

Acknowledgments and conflicts of interest The authors are grateful to the beekeepers of the Jalisco delegation of the Mexican Federation of Beekepers for the facilities provided for the realization of this study. All the researchers who participated in this paper declare that they have no conflict of interests.

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16. Hosmer DW, Lemeshow S. Special topics. Applied Logistic Regression. Second ed. USA: John Wiley & Sons; 2005. 17. R Core, Team R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org/. 2013. 18. Álvarez-Ramírez AL, Jiménez-González E, Ortiz-Muñoz E, Ruíz-Garcia I, Orozco-Hernández R. Influencia de las condiciones ambientales en la presentación de Ascosferosis (Ascosphaera apis) o cría de cal en Apis mellifera (abeja). Aba Vet 2017;7(3):37-46. 19. Mizue O, Hidetoshi I, Toshifumi K, Tadaaki A, Ryuichi O, Etsuro I. Control of hive environment by honeybee (Apis mellifera) in Japan. 6th Int Conf on Methods and Techniques in Behavioral Res. The Netherlands. 2009;41:782-786. 20. Vojvodic SA, Jensen AB, Markussen B, Eilenberg J, Boomsma JJ. Genetic variation in virulence among chalkbrood strains infecting honey bees. PLoS one 2011;6(9):e25035. 21. Yoder JA, Nelson BW, Main RL, Lorenz AL, Jajack AJ, Aronstein KA. Water activity of the bee fungal pathogen Ascosphaera apis in relation to colony conditions. Apidologie 2017;48(2):159-167. 22. Bamford S, Heath LAF. The effects of temperature and pH on the germination of spores of the chalkbrood fungus, Ascosphaera apis. J Apic Res 1989;28(1):36-40. 23. Flores JM, Spivak M, Gutiérrez I. Spores of Ascosphaera apis contained in wax foundation can infect honey bee brood. Vet Microbiol 2005;108(1-2):141-144. 24. Koenig JP, Boush GM, Erickson EH. Effects of spore introduction and ratio of adult bees to brood on chalkbrood disease in honeybee colonies. J Apic Res 2015;26(3):191-195.

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https://doi.org/10.22319/rmcp.v11i2.5181 Article

Development and validation of a visual pattern for evaluating beef meat color in Mexico

Sara Salinas Labra a María Salud Rubio Lozano b Diego Braña Varela c Rubén Danilo Méndez Medina b Enrique Jesús Delgado Suárez b*

Zoetis México.

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

Elanco Animal Health.

Corresponding author: enriquedelgado.suarez@gmail.com

Abstract: This study aimed to develop a visual scale for beef color evaluation. A total of 1,165 loins were analyzed 24 h postmortem in four slaughterhouses in Mexico. In each sample, it was determined color using a visual pattern and a spectrophotometer (CIELAB scale), taking a photograph of each loin. Seven categories were identified using the visual method (from very light red to very dark red), and the instrumental color variables (L*, a*, b*, C*, and h*) were used to create prediction models for the visual categories. The scale was constructed using L* as the sole predictor, as this model explained > 90 % of the observed variation. The pattern was illustrated with photos of the samples with an L* value within the 95 % confidence interval of the mean in each category, from very light red (48.1 <L*<48.8) to very dark red (32.7 <L*<33.4). The total color difference between the categories fluctuated between 2.8 and 5.5, which suggests that these are distinguishable with the naked eye. A trained sensory 479

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panel and a consumer panel, through tests, validated the scale. Trained panelists correctly rated the samples in 92.6 % of the evaluations. In meat with dark-cutting (DC) appearance, the trained panelists had 100 % hits, and the consumer panelists 85.3 %. The proposed visual pattern is supported by instrumental measurements and proved to be technically feasible for the evaluation of color in beef by trained personnel and consumers. Key words: Beef, Bovine, Quality, Color, Visual, Instrumental, Pattern.

Received: 05/12/2018 Accepted: 30/07/2019

Introduction Fresh meat color is among the main quality attributes that influence the consumer buying decision(1,2). It is a fact that meat color defects cause significant economic losses, as they lead to a discount on meat prices(3-5). Therefore, color is one of the main attributes used to evaluate the quality of carcasses and meat in countries that are big traders, like the United States, Japan, Canada, and Australia(6-9). These assessment schemes are carried out using visual scales, which are highly correlated with the consumer buying decision(10). Furthermore, the early detection of these color defects allows the segregation of meat with an undesirable appearance and redirect it to manufacturing processes in which this attribute is less important(7). Different countries develop visual patterns for meat color evaluation because this attribute is a multifactorial phenomenon. For example, factors like breed, production system, diet, and pre- and post-slaughter handling, which are different in each country, are relevant sources of meat color variation(11,12). Therefore, the use of these tools by the meat industry must follow scientific evidence originated from local herds. The latter is especially important in cattle, a species that usually presents a high variation in its quality attributes(13). In Mexico, beef cattle production is among the most economically important livestock activities(14). Furthermore, beef is highly popular in the country, with an annual per capita consumption of 17.40 kg(15), however, there are no scientifically supported tools for the segregation of meat according to color. The Mexican standard for carcass classification includes color as one of the quality determining attributes(16). However, it proposes the use of a solid color scale from the Pantone system of just three levels, which are cherry-red, deep 480

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red, and dark red meat color. This scale is not representative of the whole range of hues that beef can have, nor does it describe the appearance associated with quality defects, like darkcutting beef (DC). Moreover, this method does not consider that meat has an irregular surface, with muscular fibers in different directions, connective tissue, and intramuscular fat. Therefore, the use of photographic patterns is considered a better alternative for the subjective evaluation of meat color(17). Despite some current private initiatives and several regional studies(18-22), so far, a visual pattern has not been developed to serve as a reference for the national industry. This situation represents a commercial disadvantage for local producers, who can receive economic penalties according to subjective evaluations from customers. Therefore, this study aims to develop and validate a visual pattern for beef meat color evaluation at an industrial scale in Mexico. This research hope to contribute to market organization and to improve communication between the different links of the value chain, as well as to generate a subjective evaluation method for meat color, based on scientific evidence, that will eliminate the deficiencies in the system considered in current regulations.

Material and methods Sampling scheme

The slaughterhouses that participated in the study were selected using a non-probability sampling plan, based on the following criteria: 1) companies located in one of the three ecological-livestock areas of Mexico (arid and semiarid, wet tropics, and dry tropics); 2) carcass halves are cut between the twelfth and thirteenth ribs; 3) the company is a Federal Inspection Type (TIF) slaughterhouse, and 4) availability of working areas with technical lighting and space conditions, access to primary cuts, and cutting area with a refrigeration temperature of 6-8 ÂşC. According to these criteria, four TIF slaughterhouses were selected, located in Baja California, Sinaloa, QuerĂŠtaro, and Tabasco states, in which a total of 1,165 samples were analyzed (Figure 1). Before color evaluation, meat was exposed to oxygen for 30 min; this created a much slower working rate compared to the process flow. Therefore, it was not possible to analyze all the carcasses processed in one day. The goal was to take at least 170 carcasses per slaughterhouse, which was the minimum sample size determined, considering a confidence level of 95 %, an accuracy of 0.5 units in the instrumental color variables, and a variance of 10.69, estimated from preliminary tests. Thus, using the equation đ?&#x2018;&#x203A; = 481

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, it

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was obtained a sample size of 164, which was rounded to 170. However, in order to obtain the highest possible variation, in each establishment, all the loins that could be collected in an 8-hour shift for 3 or 4 days were analyzed. Therefore, the actual sample size per slaughterhouse fluctuated between 172 and 405, according to the slaughter volume in each establishment. Figure 1: Number of loin samples analyzed in TIF slaughterhouses of four Mexican states between November 2012 and July 2013

The population of animals from which the data were obtained was made up, in about 80%, of non-castrated entire males, the remaining percentage were females. As the research was carried out on slaughterhouses, it was not always possible to know the age at slaughter. However, in a sample of around 300 animals in which this data was available, 86 % were 24mo-old or less. Overall, this was the age expected for the studied sample, considering previous reports that document the preponderance of young bulls in the beef cattle slaughter population in Mexico(23).

Color measurement

Color measurements, both visual and instrumental, were carried out following the American Meat Science Association guidelines(24). Readings were made at 24 h postmortem in the cutting and deboning area of each TIF slaughterhouse, with a controlled room temperature of 6 to 8 ยบC. The complete loins (Longissimus dorsi muscle), recently separated from the carcass and with a temperature not higher than 3 ยบC, were collected. The selected loins were left to rest for 30 min on a stainless-steel table, with the loin eye area between the twelfth and thirteenth ribs exposed to air, to allow optimal oxygenation of myoglobin before the readings. The visual color evaluation was performed under standardized conditions. As a light source, it was used a photographic quality Osram incandescent lamp with an intensity of 150 foot 482

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candles (1,614 luxes) and a color temperature of 3,200 ºK, placed at a 45° angle concerning the surface of the chop. A professional photographer took high-resolution photos of each loin chop evaluated; for this purpose, was used a 12-megapixel Nikon D300S camera with a Sigma 24-70 f. 2.8 zoom lens. Photographs were taken against a black background to eliminate color differences associated with the meat surface. The photographs function as illustrative images of the different hues of the meat, which could be included in the visual color scale that was being developed. Subsequently, using the United States eight-level color pattern for beef (Figure 2)(9), an experienced researcher performed the visual color evaluation in order to have a preliminary reference about the number of possible visual categories in the studied sample. Figure 2: Visual scale for color evaluation in beef carcasses developed in the United States of America(9)

For instrumental measurements, was used a Hunter MiniScan EZ 4500L spectrophotometer with a 45/0 geometry and a port size opening of 25 mm (Hunter Associates Laboratory, Inc, Reston, Virginia, United States). The instrument was configured as follows: A/10° illuminant/observer combination and specular component excluded. Furthermore, the instrument was remotely operated using the program OnColor QC Lite, version 6 (CyberChrome, Inc., New Paltz, New York, United States) for computer data capture. Calibrations were performed before starting measurements and after every 100 readings or after 1 hour (whichever occurred first), using the black trap and white tile supplied by the manufacturer. From each loin chop, four readings were obtained, and their average was used to calculate the lightness (L*), redness (a*), yellowness (b*), hue (h*), and Chroma (C*) of each loin chop in the CIELAB scale. Along with these data, we also collected the spectral curves, which constitute the color footprint and were necessary for the professional impression of the scale.

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Data analysis and visual scale conformation For the statistical analysis was used the Statgraphics XV Centurion software for Windows (Statpoint, Inc., The Plains, Virginia, United States). The means of the color instrumental values were compared between the quality categories visually assigned. For this, a one-way analysis of variance (ANOVA) was performed using the Generalized Linear Model (GLM) procedure. As a result of the different number of observations per level, when significant differences were found, the means were discriminated using the Bonferroni multiple comparison procedure. Different prediction models were tested to construct the scale, by means of the GLM procedure, using the visual category as the dependent variable and different combinations of the instrumental variables and their interactions as explicative variables (See Table S1 in Supplementary Information). Among the generated models, the one that explained the highest percentage of the observed variation between visual categories was chosen, which turned out to be the one that uses L* as the only explicative variable. Therefore, 95 % confidence intervals of L* were constructed within each visual category. To illustrate the scale, we selected the photographs that corresponded to L* loin chop values within the confidence interval of the mean in each category. Lastly, it was necessary to determine if the categories could be differentiated visually. For this, it was calculated the total color difference (â&#x2C6;&#x2020;đ??¸*ab ), which is the sum of the modular 2

differences of L*, a*, and b* between two samples: (â&#x2C6;&#x2020;đ??¸*ab = â&#x2C6;&#x161;Î&#x201D;L*2 + Î&#x201D;a*2 + Î&#x201D;b* ). The greater the â&#x2C6;&#x2020;đ??¸*ab , the easier it is to visually distinguish the color difference between two samples. However, usually, samples with â&#x2C6;&#x2020;đ??¸*ab values >2 units are considered easily distinguishable(25).

Preliminary validation experiment

In order to validate the visual scale, we evaluated the newly developed visual standards in an industrial setting, under the conditions described below. The visual scale was tested in a slaughterhouse not included in the initial sampling. For validation, a trained sensory panel was formed, with six people familiar with the proposed color pattern. Panelists were selected using the Farnsworth-Munsell test (http://www.color-blindness.com/farnsworth-munsell100-hue-color-vision-test/#prettyPhoto), which allows dismissing people with color appreciation deficiencies. Selected panelists were required to have a color vision deficiency score of â&#x20AC;&#x153;noneâ&#x20AC;? or â&#x20AC;&#x153;mildâ&#x20AC;? (0 to 70 points). The initial group was formed by 20 candidates 484

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selected according to the generally accepted guidelines for the planning and selection of trained judges(26). As a result of the selection process, the sensory panel ended up with six members. Each panelist participated in six evaluation sessions, in which they evaluated six meat samples (2 of normal-colored meat (N), 2 with moderate DC, and 2 with extreme DC), for a total of 36 measurements per judge. For the evaluations, the judges received instructions on how to use a structured 10-cm-long scale, ranging from very light red (L* = 50) to very dark red (L* = 30): Very dark red (L*=30)

Very light red (L*=50)

According to the proposed visual scale, extreme DC meat has L* values between 30 and 34; moderate DC between 35-37; and N meat, 38 or more. Judges were asked to mark the scale with a cross to indicate the color that corresponded to the evaluated sample. The position of the cross was measured with a ruler to obtain the estimated L* value. The data were analyzed by a three-factor analysis of variance (judge, sample, session, and their interactions); this corroborated that the only source of significant variation in the evaluations was the sample. Finally, a correlation analysis was performed between the L* values estimated from the color evaluation made by the judges and the real L* values determined with the spectrophotometer. In order to validate the proposed visual scale for beef color evaluation, it was necessary to obtain a significant discriminatory power of the judges, a significant high-magnitude correlation between the real L* values of the samples and the L* values estimated by the judges, as well as a positive hit rate > 80% when assigning a color category to each sample. In previous studies with orange juice and wine, the visual differences of the samples were validated with a much lower hit percentage of the sensory panel (e.g., >50 %)(27,28). However, the present study looked for a higher hit rate in order to determine if the visual scale worked for most people, which is why color evaluations were also conducted with an untrained panel (n = 6). Consumer panelists were asked to indicate if they observed any difference between N and DC meat samples. The latter meant to corroborate the reliability of the scale when used by people without color evaluation training. Samples were selected and displayed under the same conditions previously described for the trained panel. However, the untrained panel was only presented with pairs of samples, and panelists were asked to choose the sample they preferred based on color.

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Results Definition of visual categories and their relation with instrumental measurements

Visual evaluation allowed to identify, in the studied sample, seven of the eight categories represented in the North American visual standards because none of the samples had an appearance similar to category 5 (slightly dark red). Figure 3 shows the distribution of samples in each of the seven identified classes Figure 3: Distribution of samples (n = 1,165) in each of the visual categories identified using the North American pattern for beef color evaluation (the description of each category is the same as in Figure 2).

The meat proportion associated with quality defects (categories 7 and 8 represent different degrees of DC appearance) was relatively low (7 %). In contrast, more than 70 % of the samples corresponded to the categories 2-4, an appearance generally associated with normal quality meat. Moreover, the ANOVA was significant (P<0.0001) for all the instrumental color variables (Table 1). However, L* was the only variable with significant differences between the means across all the categories. Furthermore, the ANOVA of L* was the only one with a high coefficient of determination (R2=0.9171), while in the other variables, it ranged from 0.2744 to 0.4284.

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Table 1: Least square means of the instrumental color variables in meat of each of the visual categories identified in the sample Visual categories Variable 1 2 3 4 6 7 8 SEď&#x201A;ą1 R2 n 30 62 283 264 314 144 68 a b c d e f L* 48.8 46.7 44.4 42.1 40.1 37.7 34.8g 0.94*** 0.9171 a* 27.4a 27.8a 27.5a 27.1ab 26.3b 25.1c 22.6d 2.04*** 0.2744 b* 20.1a 20.2a 19.1b 18.3c 17.3d 16.0e 13.5f 2.12*** 0.3522 C* 34.1a 34.3a 33.4ab 32.7b 31.5c 29.8d 26.3e 2.21*** 0.3082 h* 36.3a 35.9a 34.7b 33.9c 33.2d 32.4e 30.7f 1.41*** 0.4284 1

a,b,c,d,e,f,g

Standard error of the estimator. Means with different letters in a same row indicate statistical difference (P<0.05). ***P<0.0001.

Although other prediction models were tested (see Table S1 in supplementary information), none of them were better than the one obtained with L* as the only explicative variable and, hence, were discarded. Therefore, 95 % confidence intervals of the mean value of L* were used as a criterion to select the representative photos from each of the visual categories that form the developed color scale (Figure 4). An important feature of the latter is that meat appearance in some adjacent categories is very similar. For example, categories 1 and 2 represent light meat. Similarly, categories 3 and 4 are representative of the bright cherry-red hue, which is usually the most attractive to consumers at retail. Therefore, at first, we considered merging both pairs of categories into one. However, the â&#x2C6;&#x2020;đ??¸*ab between these pairs was of almost three CIELAB units, which means that they are clearly differentiable from each other with the naked eye. Therefore, instead of merging them, it was used a denomination that would allow the user to identify that both categories are associated with similar hue meat. Hence, categories 1 and 2 were renamed as 1A and 1B, respectively, while 3 and 4 were denominated 2A and 2B. These adjustments forced the name modification of the remaining categories (5, 6, and 7) into 3, 4, and 5; their appearance is clearly differentiable from each other, with a much higher â&#x2C6;&#x2020;đ??¸*ab between them (3.3 to 5.5 CIELAB units).

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Figure 4: Visual scale for evaluating beef meat color in Mexico

L* values correspond to the 95% confidence interval of the mean for each category. The numbers at the bottom indicate the total color differences (â&#x2C6;&#x2020;đ??¸*ab ) between adjacent categories.

Preliminary validation of the descriptive scale under industrial conditions The first validation criterion for the color scale was for the sensory panel to detect the differences between the samples. Judges, both individually and as a group, showed sufficient discriminatory power (significant ANOVA, P<0.0001); therefore, results were satisfactory. Furthermore, the sample was the only factor that significantly influenced (P=0.0002) the score of the judges (see Tables S2 to S4 in supplementary information). The correlation between the real L* values of the samples and the values assigned by the judges (Figure 5), which was the second validation criterion, was significant and of high magnitude (r=0.9338, P<0.0001). These results correspond to the percentage of correct answers by the judges, third and last validation criterion of the scale; judges, on average, correctly assigned the samples to the visual category of the pattern in 92.6 % of the evaluations. The scale showed even better performance in the detection of DC; the judges correctly assigned samples with this condition in 100 % of the evaluations. Furthermore, the scale also allowed to distinguish the different degrees of the DC defect; trained judges confused moderate DC with extreme DC in only 5 % of the evaluations. Finally, the evaluation performed by the untrained panel corroborated the relevance of the categories that describe the DC defect, since in 85.3 % of the evaluations performed by untrained panelists, the color of N meat was preferred over the color of DC meat. Overall, the results of the sensory tests demonstrate the technical viability of the proposed color scale since both trained personnel and consumers were able to use it to dictate the appearance of meat correctly.

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Figure 5: Regression analysis between the L* estimated by the trained sensory panel and the real L* of the samples measured using the spectrophotometer (n=108)

The model includes the bilateral prediction limits. ***P<0.0001.

Discussion This research presents the first visual pattern for evaluating beef meat color in Mexico, scientifically supported by data obtained from domestic cattle. The developed scale contains categories that describe light, cherry-red, deep red, dark red, and very dark red-looking beef. These hues are visually differentiable and, also, show a high correlation with instrumental color variables, particularly with L*. The latter coincides with results from previous studies, in which L* was the variable best related to the visual appearance of the meat(29,30). Some studies performed in Mexico have used Chroma (C*<30) as one of the criteria to identify DC meat(3). Although the instrument and measurement conditions in this study were different, C* values of less than 30 were also observed in meat with dark-cutting appearance. However, C* only explained about 30 % of the differences between the different levels of the scale, while for L*, the coefficient of determination was greater than 90 %. The high correlation between L* and visual appearance observed in this study suggests that instrumental measurements can either guide evaluators who use the visual scale or substitute the use of the latter in companies with instrumental technology to measure color. Moreover, the categories included in the scale can be associated with specific commercial advantages or quality defects. For example, the meat represented in categories 1A and 1B 489

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has an appearance similar to that described in pale, soft, and exudative (PSE) meat(31). This defect is associated with impaired functional properties, especially with a reduced water holding capacity. However, the PSE phenomenon occurs very rarely in cattle, and it usually only occurs when the carcasses are subjected to very slow chilling(32). Nonetheless, 46 units (3.9 %) of the analyzed sample had an extremely pale appearance, and another 150 (12.9 %) were classified as moderately pale; probably due to slow chilling of carcasses or excessive muscularity that limits or delays heat loss and promotes a faster drop in muscle pH, conditions that could be common in many TIF slaughterhouses in Mexico. However, recent studies suggest that Mexican consumers perceive the light red color as a quality indicator in beef(33), which implies that fresh meat with this appearance must not suffer price penalties. The following two categories (2A and 2B) represent the typical appearance (bright cherryred) that consumers look for in fresh beef(4). Apparently, a good part of the beef produced in the country meets this demand, since 60 % of the samples analyzed presented these hues. Therefore, the identification of this type of meat helps to exploit to the maximum the competitive advantages offered by its favorable appearance for retail sales. Moreover, category 3 describes the meat that is in the limit of acceptable quality. Its appearance is slightly darker, determined by the lowest L* values, which puts it at a disadvantage concerning cherry-red, which is associated with younger animals. As animals grow old, the lightness of meat decreases, which results in a darker appearance(34). However, category 3 keeps a relatively safe total color difference (3-5 CIELAB units) regarding the categories that describe the DC defect (4 and 5). Finally, categories 4 and 5 represent the DC defect, which leads to millionaire losses to the industry and is, by far, on a global scale, the most important quality defect in beef(7). Although different degrees of the DC condition have been described (e.g., classic, light, and atypical), recent studies have shown that all share an undesirable dark appearance and deteriorated quality attributes(5,35), for which they deserve economic penalization. In Mexico, according to estimations, the value of DC carcasses decreases by approximately 85 USD(3). Although the proportion of units with DC appearance in the analyzed sample is low (7 %), the economic impact of this rate, if it occurred at a national scale, can represent millions of dollars, considering that the annual slaughter is 4 million heads, only on TIF slaughterhouses (36). Therefore, the description of DC meat on the proposed scales is highly relevant, since it provides the basis for the early detection of this defect, offering the possibility of segregating the defective meat and, also, managing the slaughter-associated processes to reduce its incidence.

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The calculation of â&#x2C6;&#x2020;đ??¸*ab showed that the categories represented on the scale can be easily differentiated visually. Some studies have suggested that the human eye can perceive color differences from values of â&#x2C6;&#x2020;đ??¸*ab > 1(25), a much lower figure than those observed in this work among the visual categories proposed (2.8-5.5). Moreover, validation proved the relevance of the followed scale developing strategy, with a high correlation between the trained sensory panel and the predictive variable used (L*); this undoubtedly contributed to more than 90 % of the trained judges correctly assigning the visual category to the evaluated samples. Similarly, the categories associated with DC, which is the primary quality defect in beef, are easily identified by both trained people and consumers. The latter opens the possibility of using the visual scale routinely in the industrial environment, as an alternative to instrumental measurements, which requires economic investments that are beyond the reach of most of the industry. Despite what has been analyzed so far, it should be noted that the present study does not propose the use of the visual pattern as the only criterion to determine meat quality. Although it is widely documented that color is a key factor affecting consumer choices, it is known that it does not correlate well with tenderness or palatability of meat(4). Hence, to evaluate the quality of carcasses, it will be necessary to complement the color evaluation with that of other quality attributes (e.g., final pH, physiological maturity of the animals, marbling, among others)(7). Furthermore, the scale herein is based on measurements made at 24 h postmortem. Therefore, its application across segments of the distribution chain could not be entirely consistent, since storage temperature, type of packaging, muscle biochemistry, among other factors, can modify the color of meat and its stability(11,37). Moreover, although it is possible to opt for the exclusive use of instrumental measurement for color evaluation, certain precautions must be taken. First, the typical L* values reported for each category were measured with a spectrophotometer whose configuration (port size opening, geometry, illuminant, among others) may be different from that of other equipment. Therefore, the use of different instruments or configurations may vary the results. Despite the latter, the developed scale can be very useful to estimate, from the slaughterhouses, whether the appearance of the meat could have a positive or negative impact on the consumer buying decision. Furthermore, its use can facilitate more efficient communication through the use of an objective technical descriptor, when marketing meat. In particular, the scale proved to work very well for the identification of meat with a darkcutting appearance, the early detection of which, on the slaughterhouses, is of great economic importance.

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Conclusions and implications The descriptive scale developed in this study provides representative illustrations of beef appearance in Mexico, as well as the L* intervals associated with each one of them. The concurrence of visual and instrumental criteria in the tool allows its versatile implementation, either with sensory panels, with instrumental measurements, or by combining both. The scale is conceived as a tool for color evaluation on the slaughterhouse at 24 h postmortem and has shown excellent performance for the detection of meat with a dark-cutting appearance in validation tests performed under industrial conditions. In companies that perform carcass evaluation, this tool could be included as an additional criterion to define their quality.

Acknowledgments This study was carried out with resources from the SAGARPA-CONACYT sector fund, project 109127. The authors thank Professor Melvin C. Hunt for his technical assistance in reviewing and developing the work methodology used in this study.

Literature cited: 1. Wulf D, Wise W. Measuring muscle color on beef carcass using the L*a*b* color space. J Anim Sci 1999;77(9):2418-2427. 2. Mancini RA, Hunt MC. Current research in meat color. Meat Sci 2005;71(1):100-21. 3. Leyva-García IA, Figuerosa-Saavedra F, Sánchez-López E, Pérez-Linares C, BarrerasSerrano A. Impacto económico de la presencia de carne DFD en una planta de sacrificio Tipo Inspección Federal (TIF). Arch Med Vet 2012;44(1):39-42. 4. Troy DJ, Kerry JP. Consumer perception and the role of science in the meat industry. Meat Sci 2010;86(1):214-226. 5. Prieto N, Lopez-Campos O, Suman SP, Uttaro B, Rodas-Gonzalez A, Aalhus JL. Exploring innovative possibilities of recovering the value of dark-cutting beef in the Canadian grading system. Meat Sci 2018;137(1):77-84. 6. Aus-Meat. Handbook of Australian meat, 7th ed. Australia: South Brisbane; 2005.

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7. Aalhus JL, López-Campos Ó, Prieto N, Rodas-González A, Dugan MER, Uttaro B, Juárez M. Review: Canadian beef grading – Opportunities to identify carcass and meat quality traits valued by consumers. Can J Anim Sci 2014;94(4):545-556. 8.

JMGA. Japan Meat Grading Association. Beef carcass grading http://wagyu.org/breed-info/meat-grading/. Accessed May 5, 2018.

standard.

Tatum D. Beef grading. National Cattlemen’s Beef Association. https://www.beefresearch.org/CMDocs/BeefResearch/PE_Fact_Sheets/Beef_Grading. pdf. Accessed May 4, 2018.

10. Carpenter CE, Cornforth DP, Whittier D. Consumer preferences for beef color and packaging did not affect eating satisfaction. Meat Sci 2001;57(4):359-363. 11. King DA, Shackelford SD, Wheeler TL. Relative contributions of animal and muscle effects to variation in beef lean color stability. J Anim Sci 2011;89(5):1434-51. 12. King DA, Shackelford SD, Kuehn LA, Kemp CM, Rodriguez AB, Thallman RM, Wheeler TL. Contribution of genetic influences to animal-to-animal variation in myoglobin content and beef lean color stability. J Anim Sci 2010;88(3):1160-1167. 13. Chávez A, Pérez E, Rubio MS, Méndez RD, Delgado EJ, Díaz D. Chemical composition and cooking properties of beef forequarter muscles of Mexican cattle from different genotypes. Meat Sci 2012;91(2):160-164. 14. SAGARPA. ACUERDO por el que se dan a conocer las Reglas de Operación del Programa de Fomento Ganadero de la Secretaría de Agricultura, Ganadería, Desarrollo Rural, Pesca y Alimentación. http://dof.gob.mx/nota_detalle.php?codigo=5327093&fecha=18/12/2013. Consultado 4 May, 2018. 15. FAOSTAT. Food Balance Sheets. Food and Agricultural Organization of the United Nations, Statistics Division. http://faostat3.fao.org/download/FB/FBS/E. Accessed May 6, 2018 16. SCFI. NMX-FF- 078-SCFI-2002. Productos pecuarios - carne de bovino en canal clasificación (cancela a la NMX-FF-078-1991). http://www.economianmx.gob.mx/normas/nmx/2002/nmx-ff-078-scfi-2002.pdf. Consultado 5 May, 2018. 17. Girolami A, Napolitano F, Faraone D, Braghieri A. Measurement of meat color using a computer vision system. Meat Sci 2013;93(1):111-118. 18. Pérez LC, Figueroa SF, Barreras SA. Relationship between management factors and the occurrence of DFD meat in cattle. J Anim Vet Adv 2006;5(7):578-581.

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19. Peréz LC, Figueroa-Saavedra F, Barreras-Serrano A. Management factors associated to DFD meat in bovine on desertic climate. Arch Zoot 2008;57(220):545-547. 20. Torrescano UG, Sánchez EA, Vásquez PM, Paz PR, Pardo GD. Characterization of bovine carcasses and meat from animals fattened in Central Sonora. Rev Mex Cienc Pecu 2010;1(2):157-168. 21. Miranda-de la Lama GC, Leyva IG, Barreras-Serrano A, Perez-Linares C, SánchezLópez E, et al. Assessment of cattle welfare at a commercial slaughter plant in the northwest of Mexico. Trop Anim Health Prod 2012;44(3):497-504. 22. Zorrilla-Rios JM, Lancaster PA, Goad CL, Horn GW, Hilton GG, Galindo JG. Quality evaluation of beef carcasses produced under tropical conditions of México. J Anim Sci 2013;91(1):477-482. 23. Mendez RD, Meza CO, Berruecos JM, Garces P, Delgado EJ, Rubio MS. A survey of beef carcass quality and quantity attributes in Mexico. J Anim Sci 2009;87(11):37823790. 24. AMSA. Meat color measurement guidelines. American Meat Science Association. http://www.meatscience.org/publications-resources/printed-publications/amsa-meatcolor-measurement-guidelines. Accessed May 5, 2018. 25. Abril M, Campo MM, Onenc A, Sanudo C, Alberti P, Negueruela AI. Beef colour evolution as a function of ultimate pH. Meat Sci 2001;58(1):69-78. 26. Costell E, Durán L. El análisis sensorial en el control de calidad de los alimentos. III. Planificación, selección de jueces y diseño estadístico. Rev Agroquím Tecnol Aliment 1981;21(4):454-470. 27. Fernández-Vázquez R, Stinco CM, Hernanz D, Heredia FJ, Vicario IM. Colour training and colour differences thresholds in orange juice. Food Qual Prefer 2013;30(2):320-327. 28. Martínez JA, Melgosa M, Pérez MM, Hita E, Negueruela AI. Note. Visual and instrumental color evaluation in red wines. Food Sci Technol Int 2001;7(5):439-444. 29. Holman BWB, Mao Y, Coombs CEO, van de Ven RJ, Hopkins DL. Relationship between colorimetric (instrumental) evaluation and consumer-defined beef colour acceptability. Meat Sci 2016;121(1):104-106. 30. Goñi V, Indurain G, Hernández B, Beriain MJ. Measuring muscle color in beef using an instrumental method versus visual color scales. J Muscle Foods 2008;19(2):209-221. 31. Adsitey F, Nurul H. Pale soft exudative (PSE) and dark firm dry (DFD) meats: causes and measures to reduce these incidences - a mini review. Int J Food Res 2011;18:11-20. 494

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32. Aalhus JL, Best DR, Murray AC, Jones SDM. A comparison of the quality characteristics of pale, soft and exudative beef and pork. J Muscle Foods 1998;9:267-280. 33. Ngapo TM, Brana Varela D, Rubio Lozano MS. Mexican consumers at the point of meat purchase. Beef choice. Meat Sci 2017;134:34-43. 34. Gagaoua M, Picard B, Monteils V. Associations among animal, carcass, muscle characteristics, and fresh meat color traits in Charolais cattle. Meat Sci 2018;140:145156. 35. Holdstock J, Aalhus JL, Uttaro BA, Lopez-Campos O, Larsen IL, Bruce HL. The impact of ultimate pH on muscle characteristics and sensory attributes of the longissimus thoracis within the dark cutting (Canada B4) beef carcass grade. Meat Sci 2014;98(4):842-849. 36. SIAP. Resumen estatal pecuario, sector porcino. Producciรณn, precio, valor y peso de ganado en pie 2014. http://www.siap.gob.mx/ganaderia-resumen-estatal-pecuario/. Consultado 5 May, 2018. 37. de Huidobro FR, Miguel E, Blazquez B, Onega E. A comparison between two methods (Warner-Bratzler and texture profile analysis) for testing either raw meat or cooked meat. Meat Sci 2005;69(3):527-36.

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(Supplementary information) Table S1: Visual category prediction models, tested in a step-by-step selection strategy, using the visual category as a dependent variable, and the instrumental color variables and their interactions as explicative variables (n= 1,165) in the Generalized Linear Model procedure of Statgraphics XV Centurion Explicative variables included in the model SEď&#x201A;ą1 P R2 L* 0.40 <0.0001 0.9145 a* 1.22 <0.0001 0.2134 b* 1.13 <0.0001 0.3269 C* 1.18 <0.0001 0.2651 h* 1.05 <0.0001 0.4172 L*, a*, b*, C*, h* 0.40 <0.0001 0.9168 L*, a*, b*, C* 0.40 <0.0001 0.9153 L, a*, b* (L* x a* x b*) 0.40 <0.0001 0.9163 L*, a*, (L* x a*) 0.40 <0.0001 0.9165 L*, b*, (L* x b*) 0.40 <0.0001 0.9159 L*, C*, (L* x C*) 0.40 <0.0001 0.9160 L*, h*, (L* x h*) 0.40 <0.0001 0.9156 1

Standard error of the estimator.

Table S2: F value and statistical significance level (P) of the ANOVA performed with the color grading data emitted by each trained judge (n= 36) in meat samples with light red, normal, moderate DC, and extreme DC appearances Judge FANOVA P 1 193.3 <0.0001 2 139.1 <0.0001 3 138.3 <0.0001 4 116.5 <0.0001 5 103.9 <0.0001 6 91.9 <0.0001

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Table S3: ANOVA performed with the color grading data emitted by trained sensory panel in meat samples with light red, normal, moderate DC, and extreme DC appearances Source Sum of squares DF1 Mean square F ratio P value Between groups 141.022 3 47.0074 639.52 0.0000 Intra-groups 7.64444 104 0.0735043 Total (Cor.) 148.667 107 1

Degrees of freedom.

Table S4: Multifactorial ANOVA1 for color grading Effect F ratio P value Judge 0.95 0.4491 Sample 7.04 0.0002 Session 0.89 0.4919 Judge x sample 0.86 0.6126 Judge x session 0.68 0.8701 Sample x session 1.03 0.4254 Judge x sample x session 0.92 0.9860

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https://doi.org/10.22319/rmcp.v11i2.5349 Review

Trace mineral controlled-release intraruminal boluses. Review

Misael León-Cruz a Efrén Ramírez-Bribiesca a* Raquel López-Arellano b Leonor Miranda-Jiménez a Gabriela Rodríguez-Patiño b Víctor M. Díaz-Sánchez b Alma L. Revilla-Vázquez b

Colegio de Postgraduados. Programa de Ganadería. Campus Montecillo. Km. 36.5 Carr. México-Texcoco. 56230, Estado de México. México. b Universidad

Nacional Autónoma de México, FES Cuautitlán, Cuautitlán Izcalli, Estado de

México. * Corresponding author: efrenrb@colpos.mx

Abstract: Trace minerals are essential nutrients for sustaining life, growth, and reproduction. In ruminants, mineral deficiencies affect the physiologic and metabolic functions that often trigger diseases. The design and use of controlled-release intraruminal boluses (CRIRB) are an alternative to correct the microelement deficiencies in the organism. This review aims to highlight the available information on the different types of trace mineral CRIRB, as well as the manufacturing methods that include hot-melt extrusion, melt granulation, and direct melting techniques. Furthermore, this review describes the effects of CRIRB related to health, productive, and reproductive parameters in ruminants.

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Keywords: Intraruminal boluses, Release mechanisms, Mineral concentration, Release kinetics.

Received: 24/04/2019 Accepted: 21/05/2019

Introduction Trace mineral deficiencies in pasture and forage from various livestock regions are common around the world and in Mexico. The low mineral concentration in the soils and the antagonism between minerals difficult their availability for plants and animals(1). Trace minerals like copper (Cu), zinc (Zn), cobalt (Co), selenium (Se), and iodine (I) are deficient in ruminants(2-5) and impair productive and reproductive parameters(1). Faced with this problem, the food and veterinary pharmaceutical industries have developed products for trace mineral supplementation in the form of mineral premixes, blocks, and injectable solutions; however, frequent dosages are required, and consumption is variable when freely accessible(6,7). Prolonged-release intraruminal boluses are devices designed for oral administration and must remain in the reticulorumen for periods of three months or up to one year(8,9), considered the most indicated method to correct trace mineral deficiencies in grazing ruminants(6,10,11). The design of CRIRBs follows basic criteria: dimensions, release system geometry, and density. These characteristics determine the mineral release mechanism; therefore, materials (excipients) with different mechanisms for dosage rate control have been used(8,9). This review describes the different types of trace mineral CRIRBs, as well as their manufacturing methods, including hot-melt extrusion, melt granulation, and direct melting techniques. Furthermore, this review discusses the effects of CRIRBs on ruminant health, productive, and reproductive parameters.

Controlled-release intraruminal boluses The CRIRBs are solid devices that can release trace minerals, drugs, growth promoters, and nutrients in the reticulorumen of ruminants(8,9). These devices must meet specific 499

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characteristics to remain in the reticulorumen and release the correct immediate or controlled dose. Their design considers three essential criteria(8,9,12,13): Dimensions. The bolus diameter must be of approximately 25 mm with a variable length of 40-100 mm. Release system geometry. Usually, boluses have a cylindrical or spherical shape, with round tips and a smooth or capsule-shaped surface, for oral administration. However, there are designs with collapsible wings, compressible sheets, and rings that expand to avoid regurgitation and remain in the reticulorumen during treatment. Density. Must be greater than 2.0 g cm-3 to ensure the bolus remains in the reticulorumen. The addition of densifying agents like iron (Fe) or soluble glass to the bolus formulation achieves the required density.

Compressed boluses

This type of bolus consists of a compressed matrix that contains drugs or trace minerals and a polymeric membrane coating(14). Compressed boluses were designed to dissolve or disintegrate by the mechanic action of the rumen(8); therefore, the release of drugs or trace minerals occurs through the erosion or diffusion of the bolus during predetermined periods(14). Densities higher than 2.0 g cm-3 were determined to be sufficient to ensure bolus retention in the reticulorumen and prevent regurgitation(15). For example, in Australia, an erodible bolus based on cobaltic oxide (CO3O4) and other diluents was developed to supply Co to grazing ruminants(16). Boluses contained between 30 to 90 % Co3O4, with a variable weight of 5.5 to 30 g, and were efficient for supplying Co for more than a year(16,17). Although the boluses had a specific weight between 3.5 and 4.1(17), they presented regurgitation problems, as well as the formation of a superficial calcium phosphate layer that prevented their correct dissolution(7,17). To decrease the incrustation of calcium phosphate in the bolus, the authors decided to administer a steel screw that, when making contact with the bolus would disintegrate the calcium phosphates(17,18). This problem also occurred in Se pellet boluses formulated with 5 or 10 % of Se and 90 or 95 % of Fe, with an approximate weight of 10 g, a 12-mm diameter, and 15 mm long(19). However, some formulations were not very effective, confirming that the Se particle size used in the manufacture of the pellets was the limiting factor in the long-term release of Se(18,20). Similarly, Zn pellet boluses were developed based on the mixture of 5 g of Zn and 5 g of Fe filing. The boluses showed release rates of 10 mg of Zinc per day(21).

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Several authors have designed compressed boluses to supply Se to goats(22) and sheep(23,24). For example, one study demonstrated the effectiveness of 10 g boluses to correct Se deficiency in sheep. The bolus was manufactured with 5.23 % of sodium selenite, 68.77 % of Fe, 25 % of cutin, and 1 % of magnesium stearate(24). However, with this supplementation strategy, regurgitation problems arose; therefore, the design must consider the geometric shape and density of the bolus. To solve this problem, they designed a bolus with polymeric wings that expand upon contact with the ruminal fluid; this prevents the regurgitation of the bolus. The device contains inside a spring that acts on a plunger that, in turn, exerts pressure on the erodible matrix that may contain drugs or trace minerals. The release occurs when the matrix makes contact with the external environment through a portal at one end of the bolus(8). Using a different approach, Evrard et al(23). designed a bolus for the treatment of coccidiosis in lambs. The bolus consisted of a matrix with 30 % of sodium sulfamethazine, 54.4 % of reduced Fe, 15 % of hydrogenated castor oil, and 0.5 % of magnesium stearate. The average weight of the bolus was 18.5 g, and the density was 2.3 g cm-3. The release of sulfamethazine occurs by diffusion and erosion of the matrix. Furthermore, in this study, the authors determined that the dose regimen of 800 mg kg-1 of live weight and compression forces higher than 2,160 kg cm-2 were enough to maintain the release rate and the plasmatic concentrations of sulfamethazine (>25 Âľg ml-1) for up to 100 h, as long as the mechanical strength of the boluses is 33.5 Âą 1.2 kg(23). The boluses formulated with sodium selenite and sulfamethazine have an average weight of 20.13 g, a density of 2.0 g cm-3, a length of 52.05 mm, and a width of 21.22 mm. These boluses were efficient in controlling coccidiosis and maintaining the correct amount of sulfamethazine and selenium in goat kids(22). Different polymers, insoluble in ruminal fluids, have been used in the manufacture of boluses as a coating for matrices containing drugs or trace minerals. Polymers form a brittle membrane that allows matrix erosion and the release of trace minerals or drugs(25). An example of this approach is the All-TraceÂŽ bolus composed of a 30 g compressed mixture of inorganic salts (copper oxide, sodium selenite, cobalt sulfate, potassium iodide, manganese sulfate, zinc oxide (ZnO), zinc sulfate) and vitamins A, D3, and E. At one end, the bolus has an 18 g counterweight that ensures the bolus remains in the reticulorumen; at the end of its shelf life, it dissolves entirely without leaving residues. An inert polymer resin coats the bolus, except for the upper end that will be in contact with the ruminal environment, and controls the release of trace minerals for approximately 240 d(26,27).

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Extruded boluses

This type of bolus is obtained by extrusion of the trace mineral and polymeric excipient formulation(14); the process is described in the section of Bolus Manufacturing Methods. This bolus was developed in New Zealand for the treatment of facial eczema. The bolus consists of an extruded ZnO matrix coated with a waxy material and impermeable to ruminal fluid, except for one end which, when in contact with the ruminal fluid, erodes to release the microelement. As the matrix erodes, the waxy coating disintegrates; this ensures the constant exposure of a part of the matrix to the ruminal environment(14,28,29).

Soluble glass boluses

Soluble glass boluses (SGBs) were designed to supply Cu, Zn, Co, Se, and I to grazing ruminants(6,11,30-35). Their composition consists of phosphorus pentoxide (P2O5), sodium oxide (Na2O), and calcium oxide (CaO) as glass forming and modifying oxides. This composition allows a release rate in the reticulorumen not higher than 25 mg cm -2 day-1(36), making possible its administration to ruminants to release trace minerals for more than a year(30,31). The release of microelements occurs by glass diffusion or dissolution, and to a greater extent by erosion(8,13,30,36,37). Sheep boluses have a variable weight of 30 to 35 g, a length of 40 to 50 mm, a diameter of 14 to 19 mm, and densities ranging from 2.7 to 4.0 g cm-3; while cattle boluses are bigger, with weights of 100 g, diameters of 24 to 26 mm, and lengths of 80 mm(36). Other aspects to consider in the bolus design, to achieve the release rate aforementioned, are the particle size and the pH of the harboring environment(37). The SGBs present multiple advantages; these include less calcium phosphate formation in the bolus(1), which facilitates its dissolution, improved plasmatic profiles(30,31,35), higher productive and reproductive indexes(32,35,38,39), as well as economic benefits(6,11). However, during the SGB manufacturing process, it is not possible to include materials (excipients or drugs) sensitive or unstable to the high temperatures required for glass formation. Still, pressure-mediated sintering processes can incorporate drugs or other components into the glass(37).

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Magnesium boluses

Magnesium (Mg) boluses consist of a cylinder formed by an alloy of Mg, aluminum, and Cu (86, 12, and 2 %, respectively), containing dispersed Fe in the bolus matrix to increase density. Sheep boluses weight around 35 g and erode due to electrochemical influences in the rumen, releasing Mg during approximately three weeks(40). A different example of a magnesium bolus consists of two cylindrical halves linked with a rubber to facilitate its administration. When the bolus reaches the rumen, it opens to avoid regurgitation and releases Mg, by electrolytic action, at an approximate rate of 2 g per device during three months(41).

Capsules with copper oxide wire Copper oxide (CuO) wires have been used to correct Cu deficiencies in sheep(21,42,43) and cattle(44). They measure between 3 and 12 mm in length, 0.5 to 1 mm in diameter, and have a specific gravity of 6.1 to 6.4 (42,45,46), they are generally covered by a mixture of cupric and cuprous oxide(21) or contained in gelatin capsules(42,46). CuO wires enter the reticulorumen and then flow to the abomasum, where hydrochloric acid dissolves them and release Cu ions, absorbed in normal biochemical processes(41,46). The administration of CuO wire capsules increases the concentration of hepatic Cu during 6 to 12 months; this is due to the relative inertia of the particles in the reticulorumen and the retention in the abomasum(21).

CRIRB manufacturing methods Hot-melt extrusion

This technique is used to produce pharmaceutical products like tablets, capsules, films, and implants for oral, transdermal, and transmucosal drug administration(47,48). The process consists of pumping raw materials through a screw that rotates at temperatures from 30 up to 250 Â°C inside a die; this results in a homogeneous mixture of the active compounds and binding agents (thermoplastics and polymers). The equipment commonly used is called an extruder; it consists of a barrel that contains bands that heat, soften, and compress the

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mixture of chemical compounds. Finally, the extrudate is led to the die to give it the required shape and dimensions(47,49).

Melt granulation

Melt granulation is a technique based on the use of solid binding agents that melt at temperatures between 50 to 80 Â°C; this allows it to be used for the formulation of drugs or trace minerals sensitive to humidity, avoiding the use of aqueous or organic solvents(50). The granulation process can consist of a single step; for this, the binding agent is added with the rest of the components of the mixture to a high-speed mixer granulator. The binding agent melts because of the heat generated with the air stream during the mixing, kneading, and drying phase. Finally, the granulate results from the union of the molten binding agent and the powder particles, once dry, the granulate must be sieved to obtain the desired granule size(51). Depending on the pharmaceutical purpose, the granules can be encapsulated for immediate drug release, or compressed to form controlled-release intraruminal boluses.

Direct melting

This technique consists of melting the mixture of active components with the excipients at temperatures between 500 to 1,100 Â°C. The melt is poured into molds to form a final product, for example, glass rods, glass tubes, glass discs, granules, and controlled-release monolithic boluses. Generally, highly soluble glass-forming components are used, such as glass-forming oxides (vitrifying) and modifying oxides (fluxes and stabilizers) that allow the correct formation of the glass network(36,37).

Design of boluses with trace minerals The present research group proposes an inexpensive and straightforward procedure to prepare trace mineral controlled-release intraruminal boluses. For example, in the design of selenium boluses, the procedure consists of adding amounts of sodium selenite, 13 %, cutin, 32 %; Fe, 54 %, and magnesium stearate, 1 % (Figure 1). Lamb boluses can have an average weight of 8 g, a hardness of 21.17 kp, and a density of 2.08 g cm-3. The

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recommended density values for retention in the rumen should be greater than 2.0 g cm3(8,9,15) . The excipient used to form the matrix was suitable for releasing Se. Figure 1: Diagram of the elaboration process of selenium intraruminal boluses by the melt granulation method

Trace mineral supplementation with CRIRB Cu, Zn, Co, Se, and I are trace minerals that have been used to produce CRIRB(6,11,21,24,30). There are several types of commercial boluses with different design, shape, size, weight, concentration of trace minerals (Table 1), and release rates. The latter is essential in the design of boluses since it indicates the daily release of the mineral and the duration of the bolus. In the United Kingdom, a SGB was developed to correct Cu, Co, and Se sheep deficiencies, the bolus was designed to have a release rate of 2.53 mg cm -2 d-1 in the rumen and supply 11 mg of Cu, 0.5 mg of Co, and 0.21 mg of Se, for up to 6 mo(30,31). The Cu, Co, and Se requirements in a sheep that consumes 1.5 kg of dry matter (DM) are 10.5, 0.3, and 0.3 ppm, respectively. Zn deficiency in sheep has been treated with a SGB that contains 15.2 % of Zn, 0.5 % of Co, and 0.15 % of Se (32,39). The daily requirement is 20 to 33 mg of Zn kg-1 of DM(52). According to the bolus manufacturer, the Zn release during 180 days is 505

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approximately 28 mg d-1(32), considering the previous example, the sheep would demand a consumption between 30 and 49 mg of Zn, with a deficiency of 2 and 21.5 mg, which can be neutralized by the diet contribution. A different study reported a dissolution rate of 326 mg d-1, equivalent to daily releases of 49.3 mg of Zn, 1.7 mg of Co, and 0.5 mg of Se; thus, the bolus met the daily requirement of sheep(11). However, the dissolution rate depends on the rumen conditions, such as pH, type of feed, bolus accommodation site, ruminal contractions, as well as on the abrasion effects of other materials in the reticulorumen(8,11,13). Another type of bolus designed for sheep presented an average rumen release rate of 103.55 mg d-1, equivalent to the daily supply of 23.01 mg of Zn, 0.535 mg of Co, and 0.258 mg of Se(53). The soluble glass technology has allowed the design of boluses of 100 g with 13.4 % of Cu, 0.5 % of Co, and 0.30 % of Se for grazing cattle; according to the manufacturer, two boluses liberated 156, 5.9, and 3.4 mg d-1 of Cu, Co, and Se, respectively(33). The requirement of a 500 kg bovine that consumes 10 kg of DM is 100, 2.5, and 3 mg d-1 of Cu, Co, and Se, respectively(54); therefore, the SGBs are effective in treating trace mineral deficiencies(55,56,57). The reported daily release of two commercial All-TraceÂŽ boluses with trace minerals and vitamins was 138 mg of Cu, 113 mg of Zn, 71 mg of Mn, 2.1 mg of I, 2.0 mg of Co, 2.0 mg of Se, 4,644 IU of vitamin A, 929 IU of vitamin D, and 9 IU of vitamin E during 8 mo(58); these boluses were designed for cattle with more than 150 kg of LW. While the daily release of two boluses for growing calves was 60 mg of Cu, 1.0 mg of Co, 0.6 mg of Se, 36.8 mg of Mn, 53.3 mg of Zn, 1.25 mg of I, 3033 IU of vitamin A, 607 IU of vitamin D, and 9.1 IU of vitamin E(26). Approximately half the weight of the bolus matrix (30 g) is released in the first 6 wk; after that, the release rate decreases until the seventh month(27). Table 1: Trace mineral concentration in different types of controlled-release intraruminal boluses Product Cosecure*(34,38) Zincosel(11,32,35,53) Cosecure*(33,55) All-Trace(58) smAll-Trace(26,27) Tracesure(59) Ferrobloc(60) CRIRB+ (61) CRIRB++ (62)

Copper (mg) 4356-4900 / 13400 16200 5300-5427 / 333 3944 /

Zinc (mg) / 3764-5040 / 13320 4700-4797 7 36 4366 /

Cobalt (mg) 165-220.5 94.1-171.6 500 236 90 1000 60 95 582

Selenium (mg) 49.5-92.75 43.29-49.5 300 251 50-54 1000 8 45 148

Iodine (mg) / / / 497 100-225 6800 24 330 2908

*SGB for sheep; ** SGB for cattle; +CRIRB for sheep; ++CRIRB for cattle.

506

Manganese (mg) / / / 8280 3200-3312 / 160 3013 11853

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Some studies show that the supply of trace minerals using CRIRBs increases the blood concentration of minerals(30,31,35), improves productive and reproductive (32,35,38,39) parameters , and promotes the humoral immune response in ruminants(11). Studies have confirmed the effectiveness of SGBs in supplying Cu, Co, and Se to correct and prevent their deficiency in grazing sheep for up to a year(30,31). Furthermore, they increased the content of Se in the fetus and newborn lambs(38). The tablets with 25 % CuO, equivalent to 19.3 % of Cu in the active matrix, increased Cu concentrations in blood from 10.4 to 14.0 Âľmol L-1 and in the liver from 120 to 684 mg kg-1 of DM in growing sheep(45). Female lambs in a semi-intensive (semi-stable) system were administered 5 g tablets with 1 and 4.6 % of Se, which were effective for up to 90 days; however, a higher concentration of Se in the blood (182.01 ng g-1) was found in the lambs that received the bolus with 4.6 % of Se(10). Tablets made of cement-based tile adhesive with 5 and 10 % of Se have been reported to maintain adequate concentrations of Se in the blood (148.49 and 158.48 ng g-1) for up to 120 days(63). In a different study, the predicted Se release from a bolus with 5.23 % sodium selenite was 0.177 mg d-1. Boluses increase the Se blood content in sheep after 30 d of being administered(24). In Afshari sheep, Abdollahi et al.(60) observed an increase in Cu, Se, and I the mating day and at 90 and 100 d of gestation, using a CRIRB with various minerals (Ca, Mg, Na, Cu, Mn, I, Fe, Co, Zn, and Se). A study suggested that Se, I, Fe, Zn, and Mn are essential for embryo survival and fetus development(64). Overall, the administration of a CRIRB with several trace minerals to pregnant sheep increases the concentrations of Zn, Cu, Co, and Se in newborn lambs(61). SGB with Cu, Zn, Co, and Se have been widely used to correct trace mineral deficiencies in semi-intensive lambs(6,32,39), grazing sheep(34), backyard goats(65,66), growing camels(55,67), and grazing cattle(30,68,69).

Effect of CRIRB on the productive and reproductive parameters There are few published studies about the effects of CRIRB on the productive and reproductive parameters of ruminants. In a study performed in weaned Holstein-Friesian calves fed a balanced diet and administered boluses with several trace minerals and vitamins, the daily weight gain (0.59 kg) of the calves treated with the bolus was higher than that of the animals that did not receive the bolus (0.53 kg)(26). Similar results were observed with Cu injections (25 mg of Cu mL-1) and boluses of 20 g with CuO micropellets. The authors found no difference in daily weight gain (564.2 g d -1) in Aberdeen Angus and Hereford breed calves; however, they suggested CuO boluses as an alternative to prevent Cu deficiencies(70). The lambs treated with a SGB containing Zn, Co, 507

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and Se presented an improved daily weight gain (153.22 g) compared to the control group (136.61 g), with significant differences during the last fattening stage(11). Moreover, a study reported that CRIRBs with several minerals improve the performance and nutritional quality of the colostrum (6.70 % of protein, 6.92 % of fat, and 0.59 % of inorganic matter) from Najdi breed sheep; which increases the health and growth of lambs until weaning(61). Aliarabi et al(53) reported birth weights of 4.63 kg and daily weight gains of 0.243 kg in lambs born from females treated with a bolus containing 20 % Zn, 0.50 % Co, and 0.23 % Se. Trace mineral and vitamin supplementation through CRIRBs increased milk production with 8.19 kg day-1 cow-1(62). Sheep treated with a trace mineral and vitamin CRIRB showed twinning rates of 65.5 %, while sheep injected with Cu (12.5 mg of Cu) had 44.8 % of twin births. To confirm the effect of the bolus, the authors administered a CRIRB to sheep with Cu and Se deficiencies, improving twinning rates by 59 %, while in the control group, 11 % of female sheep were sterile(27). In another study, the authors obtained 80 % of multiple births due to the administration of two CRIRB to sheep before synchronization(60). Grazing dairy cattle supplemented with two SGBs containing 13.4 % of Cu showed a lower number of services to conception (2.5 Âą 0.3 to 1.7 Âą 0.2), and the calving interval decreased from 407 to 371 days(68). Supplementation of Zn, Co, and Se through SGBs has improved the motility and proportion of live sperm cells, as well as the sperm membrane integrity(35,39). Zn participates in the catabolism of the lipids located in the intermediate part of sperm cells to generate the energy required for sperm motility(71). Cu, Zn, and Se have antioxidant properties and can reduce reactive oxygen species and avoid the damage to sperm cells(72,73). Another study evaluated the effect of a bolus with 500 mg of Se on the semen quality of Hampshire and Suffolk sheep; boluses increased the activity of glutathione peroxidase (GSH-Px), motility, number of live sperm cells, and stallion sperm viability(74).

Effect of CRIRB on animal health Se deficiency is a severe problem that affects animal production. Especially in sheep, the mortality rate of lambs during the perinatal and neonatal stages is 62 %(75), signs of white muscle disease, or nutritional muscular dystrophy are usually evident(4). In a study with sheep supplemented with a SGB before gestation, the incidence of white muscle disease signs decreased in born lambs(31). The bolus released 0.21 mg of Se day-1, enough to be transferred to the placenta or colostrum received by the lambs in their first hours of life(31,56). The Se concentration in milk from cows supplemented with a SGB was higher 508

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(0.0658 Âľg g-1) than the control group (0.0374 Âľg g-1) for 7 mo(56). In a different study, lambs born from sheep treated with a SGB containing 20 % of Zn, 0.50 % of Co, and 0.23 % of Se showed no incidence of white muscle disease, while lambs born from untreated sheep achieved a mortality rate of 10.3 % and signs of white muscle disease by 18 %(53). Regarding the role of trace minerals in the immune function, some reports indicate that Zn boluses cause a positive effect in the humoral immune response of lambs against the keyhole limpet hemocyanin (KLH) antigen. However, it seems that the joint action of Zn, Co, and Se in the bolus improved the immune response(11). In camels, a similar effect was found in the humoral immune response as a result of the intravenous injection of 2 mL of a 20 % suspension of sheep red blood cells. Furthermore, a study reported that the SGB improved the cell-mediated immunity, increasing the thickness of skin folds 24 h after the injection of phytohemagglutinin (PHA)(67). In goat kids, treatment with a bolus of sodium selenite and an injection of Se (0.25 mg kg-1 of LW) improved the humoral immune response to immunization with a bacterin-toxoid (Toxo Bac Neumonias)(76). Munday et al(28) developed a Zn bolus to protect lambs against facial eczema. The bolus consists of a 43 g core of ZnO covered by a waterproof coating, except for one end which, when in contact with the ruminal fluid, erodes to release a Zn dose of 20 mg kg-1 d-1. The boluses with Zn concentrations of 54, 81, and 108 g protected sheep against the sporidesmin produced by the fungus Pithomyces chartarum(77). Calves treated with ZnO boluses and exposed to sporidesmin showed a lower gamma-glutamyl transferase activity in serum than the control group; this demonstrated the effectiveness of boluses in reducing the incidence and severity of facial eczema(29).

Conclusions Technological innovations used in the manufacturing processes of veterinary pharmaceutical products can contribute to improving mineral supplementation methods. CRIRB are the most effective method to supply trace minerals for up to a year. The controlled-release mechanisms of the intraruminal boluses are practical to correct and prevent the trace mineral deficiencies in grazing ruminants. Several studies demonstrate that CRIRB improve productive and reproductive parameters, as well as the humoral immune activity of ruminants. The use of CRIRB decreases animal handling and stress, as well as economic costs. However, in Latin America, boluses are not a common method for trace mineral supplementation; therefore, further research is necessary for the development of CRIRB based on the deficiency profile of grazing soils and forages. 509

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38. Zervas G, Telfer SB, Carlos G, Anderson P. The effect of soluble-glass boluses containing copper, cobalt and selenium on the blood composition of ewes. Anim Feed Sci Technol 1988;(21):23-29. 39. Kendall NR, Green A, McMullen S, Rodway RG. The effect of a zinc, cobalt, and selenium bolus on ram semen quality and trace element status. In: Roussel AM, et al, editors. Trace elements in man and animals. Boston, MA. Springer, 2002. 40. Sakkinen H, Eloranta E, Vahtila S, Puukka M, Timisjarvi J, Saarela S, et al. Effects of magnesium oxide and magnesium alloy rumen boluses on plasma and urinary magnesium and calcium concentrations in reindeer (Rangifer tarandus tarandus). Small Riminant Res 2004;(54):69-79. 41. Vandamme TF, Ellis KJ. Issues and challenges in developing ruminal drug delivery systems. Adv Drug Deliv Rev 2004;(56):1415-1436. 42. Dewey DW. An effective method for the administration of trace amounts of copper to ruminants. Search 1977;(8):326-327. 43. Langlands JP, Donald GE, Bowles JE, Smith AJ. Trace element nutrition of grazing ruminants. 3. Copper oxide powder as a copper supplement. Austr J Agr Res 1989;(40):187-193. 44. Burke JM, Miller JE. Control of Haemonchus contortus in goats with a sustainedrelease multi-trace element/vitamin ruminal bolus containing copper. Vet Parasitology 2006;(142):132-137. 45. Parkins JJ, Hemingway RG, Lawson DC, Ritchie NS. The effectiveness of copper oxide powder as a component of a sustained-release multi-trace element and vitamin rumen bolus system for cattle. Br Vet J 1994;(150):547-553. 46. Huerta BM, Amándola MR, Martínez HPA, García MJG, Sánchez RC, Domínguez VI, et al. Suplementos minerales para rumiantes. Postgrado de Producción Animal, Departamento de Zootecnia. Universidad Autónoma de Chapingo; 2010. 47. Patil H, Tiwari RV, Repka MA. Hot-melt extrusion: from theory to application in pharmaceutical formulation. AAPS Pharm Sci Tech 2016;(17):20-42. 48. Repka MA, Bandari S, Raman KV, Vo AQ, McFall H, Pimparade MB, et al. Melt extrusión with poorly soluble drugs-an integrated review. Int J Pharm 2018;(535):6885. 49. Crowley MM, Zhang F, Repka MA, Thumma S, Upadhye SB, Kumar BS, et al. Pharmaceutical applications of hot-melt extrusión: Part I. Drug develop and Pharm 2007;(33):909-926. 513

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50. Ochoa DL, Igartua OM, Hernández MRM, Gascón RA, Pedraz MJL. Granulación por fusión en mezcladores granuladores de alta velocidad. VITAE, Rev Fac Quím Farm 2006;(13):40-47. 51. Rodríguez GA. Validación del proceso de fabricación de bolos intrarruminales de selenio inorgánico. [tesis licenciatura] Cuautitlán Izcalli, Edo. de México: Facultad de Estudios Superiores Cuautitlán, Universidad Nacional Autónoma de México; 2006. 52. NRC. National Research Council. Nutrient requirements of small ruminants: sheep, goats, cervids and new world camelids. Washington, DC, USA: National Academy Press; 2007. 53. Aliarabi H, Fadayifar A, Alimohamady R, Hossein DA. The effect of maternal supplementation of zinc, selenium, and cobalt as slow-release ruminal bolus in late pregnancy on some blood metabolites and performance of ewes and their lambs. Biol Trace Elem Res 2019;(187):403-410. 54. NRC. National Research Council. Nutrient Requirements of Beef Cattle. 7th rev. ed. Washington, DC, USA: National Academy Press, 2000. 55. Liu ZP, Xiong GL. The effect of a copper, selenium and cobalt soluble glass bolus on the trace element status of Bactrian camels. J Anim Feed Sci 2007;(Suppl 1):313-317. 56. Hidiroglou M, Proulx J, Jolette J. Effect of intraruminally administered, selenium soluble-glass boluse on selenium status in cows and their calves. J Anim Sci 1987;(65):815-820. 57. Hidiroglou M, Proulx J. Evaluation of a long-acting selenium and copper preparation for intraruminal adminitration to cattle. Ann Rech Vet 1988;(19):187-191. 58. Baumgurtel KL, Judson GJ. Evaluation of a sustained release bolus to supply trace elements and vitamins beef cattle. Anim Prod Austr 1998;(22):133-136. 59. Rose M, Pearson S, Cratchley T. Effect of iodine, selenium and cobalt rumen boluses given to dry dairy cows on the immunoglobulin and thyroid hormone status of calves. Anim Sci J 2012;(83):543-548. 60. Abdollahi E, Kohram H, Shahir MH, Nemati MH. The influence of a slow-release multi-trace element ruminal bolus on trace element status, number of ovarian follicles and pregnancy outcomes in synchronized Afshari ewes. Iranian J Vet Res 2015;16(1):63-68. 61. Abdelrahman MM, Aljumaah RS, Ullah KR. Effects of prepartum sustained-release trace elements ruminal bolus on performance, calustrum composition and blood metabolites in Najdi ewes. Environ Sci Pollut Res 2017;(24):9675-9680. 514

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62. Pulgar AR, Vera VR, Serrano CE. Efectos de la utilización de bolos intraruminales a base de oligoelementos, sobre indicadores reproductivos y productivos en vacas lecheras de alta producción. Sitio Arg Prod Anim 2013;51-52. 63. Gutiérrez OC, Spross SAK, Rosiles MR, Ducoing WA, Ortiz HA. Selenio sanguíneo y fecal en ovinos a partir de comprimidos inorgánicos intrarruminales. Vet Mex 2005;36(3):313-324. 64. Hostetler CE, Kincaid RL, Mirando MA. The role of essential trace elements in embryonic and fetal development in livestock. Vet J 2003;(166):125-139. 65. Serra AB, Serra SD, Nakamura K, Orden EA, Cruz LC, Fujihara T. Effect of selenium in soluble glass bolus on selenium content of milk and blood of goats. Biol Trace Elem Res 1996;(55):207-212. 66. Hayashida M, Orden EA, Cruz EM, Cruz LC, Fujihara T. Effects of intraruminal soluble glass bolus on blood selenium and plasma mineral level of grazing does under Backyard conditions in selected áreas in Nueva Ecija, Philippines. Asian-Aust J Anim Sci 2003;16(2):187-197. 67. Alhidary IA, Abdelrahman MM, Harron RM. Effects of a long-acting trace mineral rumen bolus supplement on growth performance, metabolic profiles, and trace mineral status of growing camels. Trop Anim Health Prod 2016;(48):763-768. 68. Moeini MM, Telfer SB, Sanjabi MR. The effect of Cosecure® supplementation on the copper status and fertility of grazing Holstein-Friesian dairy cattle. Acta Vet Scand 2003;(Suppl 98):257. 69. Sprinkle JE, Cuneo SP, Frederick HM, Enns RM, Schefer DW, Carstens GE, et al. Effect of a long-acting, trace mineral, reticulorumen bolus a range cow productivity and trace mineral profiles. J Anim Sci 2006;(84):1439-1453. 70. Pechin GH, Sánchez LO, Cseh S. Evaluación de dos formas de administración (bolos de liberación lenta vs. EDTA Cu inyectable) en la prevención de la deficiencia de cobre en bovinos para carne. Ciencia Vet 2006;8(1):5-15. 71. Roy B, Baghel RPS, Mohanty TK, Mondal G. Zinc and male reproduction in domestic animals: A review. Indian J Anim Nutr 2013;(30):339-350. 72. Suttle NF. Mineral nutrition of livestock. 4th ed. London, UK. CABI Publishing. United Kingdom; 2010. 73. Zhao CY, Tan SX, Xiao XY, Qiu XS, Pan JQ, Tang ZX. Effects of dietary zinc oxide nanoparticles on growth performance and antioxidative status in broilers. Biol Trace Elem Res 2014;(160): 361-377. 515

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https://doi.org/10.22319/rmcp.v11i2.4767 Review

Implications, trends, and prospects for long-distance transport in cattle. Review

Marcela Valadez Noriega a* Genaro Cvabodni Miranda de la Lama b

Universidad Nacional Autónoma de México. Facultad de Medicina Veterinaria y Zootecnia. Coyoacán, Ciudad de México, México. b

Universidad Autónoma Metropolitana. Unidad Lerma. Departamento de Ciencias de la Alimentación. Estado de México, México.

* Corresponding author: mvz.mvaladez@outlook.com

Abstract: The growth of international trade and population has increased the demand for animal protein in developing and emerging countries, which has led to a considerable increase in the number of animals bred, transported, and processed worldwide. As a result, transport distance and duration have increased, which has driven specific improvements in livestock infrastructures, such as trucks with greater autonomy and load capacity, adapted to the biological needs of animals; reduction of operating costs; and liberalization of animal health restrictions that facilitate international trade. In this review, was conduct an integrated, detailed, and updated analysis of long-distance transport. Considering that the current trend is to increase transport duration, logistical scales, and mixed transportation, it is necessary to develop evaluation and decision-making systems with tools and protocols that minimize the biological cost in cattle. Key words: Animal welfare, Long-distance transport, Bos indicus, Bos taurus, Meat quality.

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Received: 14/02/2018 Accepted: 24/07/2019

Introduction Transport is an inevitable stage in the life of a production animal with various purposes, such as breeding, fattening, sale, slaughter, reproduction, and entertainment(1). Several studies indicate that transport is a strange, invasive, aversive, and very physically demanding procedure for animals(2); including unfamiliar stimuli such as sound, visual, and olfactory cues; social mix; vibration; temperature variations; risk of injury; spatial restriction; fasting, and limited access to water(3). The direct effect of transport has implications for animal welfare and health, as well as for meat quality(4). Currently, the growing interest in food safety and quality in meat production chains seeks to incorporate sustainable production commitments and promote animal welfare in the search for a new concept of quality(5,6). The modern globalization and the increasing demand for animal protein have considerably increased the number of animals bred, transported, and processed for slaughter worldwide(7). The development of more complete and efficient supply chains facilitates international trade, thanks to improvements in livestock infrastructure, such as more autonomous trucks and specialized designs, reduction of operating costs, and liberalization of animal health restrictions(8). In this context, long-distance transport is a strategic element of the livestock industry. In some countries, due to climatic conditions, internal production is limited, and the importation of live cattle is necessary to supply the meat markets. In others, the breeding and fattening centers are distant from each other, due to feed availability and climatic conditions; here animals are born and bred in grazing zones, due to the availability of lowcost forage, and are sent for completion to intensive fattening centers. Other countries prefer to import live cattle since, for religious reasons, animals must be alive at the time of Kosher or Halal slaughter(9,10). Other cross-border livestock flows take place due to the attraction of added value, such as the certification offered by the United States Department of Agriculture (USDA) through the labeling of â&#x20AC;&#x153;improved beef,â&#x20AC;? which promotes the export of live cattle from Canada for slaughter in the United States of America (USA), with very long journeys resulting in high losses(11). Additionally, the specialization by species of many slaughterhouses located at strategic points near the marketing channels has increased the travel distance from the farm to these slaughterhouses(12).

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It is crucial to point out the economic importance of live animal exportation, which provides many direct and indirect jobs in the transport, logistics, and storage sectors. However, it is highly likely that large-scale regional planning would allow for the redistribution of slaughter centers close to production sites, in such a way that transport duration would be reduced, in addition to seeking the gradual replacement of the export of live cattle to meat export(13). In Latin America, due to the geography, commercial terms, and distribution of livestock production centers, long-distance transport is the rule rather than the exception. Regulations in these countries tend to be much laxer in terms of distance compared to European regulations. This review evaluates long-distance transport of cattle from the perspective of different countries with very particular situations given by different factors such as the geographical location of the country; pre-transport processes; vehicle design, loading density, and microenvironment; as well as current research on the risks associated with the driver and the effects that each of these factors may have on the beef chain.

Typology of long-distance transport

Long-distance transport includes repopulation, living, and slaughter transport. Historically, cattle for immediate slaughter has dominated the trade, but in the new century, there has been a rapid growth in the number of â&#x20AC;&#x153;half fattenedâ&#x20AC;? cattle for further value-adding before slaughter through fattening and finishing, including males in the dairy sector. Therefore, it is increasingly common to transport cattle several times during their lives(14), estimating 296 million head of beef cattle transported worldwide during 2005, some of them transported more than once(11). The main transport reasons include sale for herd repopulation, change of owner, search for cheaper or more abundant sources of supply (pasture and water), breeding or replacement of livestock for reproduction, the supply of intensive fattening units, auctions, livestock shows and fairs(1). The inappropriate grouping and management of livestock, primarily those extracted from extensive systems, results in animals with high levels of stress at the beginning of transport. During prolonged groupings, it is advisable to give the animals time to recover in the pre-loading pens. It is important to emphasize that animals with little human contact or aggressive temperament will be more susceptible to stress and have a higher chance of injuring handlers due to an excessive response or fear-induced aggression(9,15). Although there are not enough data, it has been proposed that previous experience may affect the fear response of animals and may be responsible for the variable results reported in transport studies(15). However, other studies have shown that more docile cattle lose less weight during transport and tend to recover faster once they continue with their production cycle(16).

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In the past, cattle transport for reproductive purposes was required for breeding; however, with new biotechnologies, this transport became impractical. The starting point was artificial insemination in domestic species, which originated in 1779, while embryo transfer was reported as another successful technique in 1890(17), and in 1973, in vitro fertilization(18). Worldwide, more than 750,000 embryos are produced annually from superovulated donors, and more than 450,000 embryos are produced using in vitro techniques(17). Although these technologies were developed for breeding purposes, the number of livestock transported for reproductive purposes must have decreased considerably from the 18th century, when artificial insemination began. Moreover, exhibitions, fairs, and actions continue to require the physical presence of livestock, which demands constant transport. At the international level, there are efforts to eliminate this practice using the internet or television-based systems(19), an example of this are auctions in Europe, the USA, and recently in Argentina, Brazil, and Colombia; in these auctions, animal transport only occurs once you have a secured buyer. However, this field has not yet been studied, and the available information is scarce, so it represents an important area of study as part of the current livestock industry, where the use of technology facilitates the commercialization of livestock. There is a tendency to decrease or disappear unnecessary animal handling. Repopulation transport includes movements between countries, between farms of the same country, or within the same property(20). For example, Mexico is the largest trading partner of the USA introducing live animals, the trade consists of animals with a minimum of blood from zebu breeds to supply fattening units or feedlots from that country(21). Moreover, the supply and domestic consumption of Mexico depends on the cattle from the southeast tropical and subtropical regions and Central American countries(22), this supply consists of the long-distance transport of animals, of which there is still little information. Further research in repopulation transport, especially in animal health and welfare repercussions is required; this will provide the competent authorities with the background information required to establish rules and regulations on the conditions before, during, and after transport, in addition to considering aspects such as the ideal state of an animal to be transported, maximum transport duration, and water and food restriction depending on the region(23).

International regulatory trends

Livestock transport is an important concern of governments, animal protection organizations, and consumers in general, due to the perception of an absence of welfare in this link of the chain, as well as the possible consequences on the quality and product 520

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safety(24). A bad image during transport or accident management creates a negative perception of the transport activities(25). There are well-intentioned regulations with possible negative consequences for animals; regulations on cattle transport do not always consider fundamental aspects for their welfare(26). For example, under Canadian regulation, livestock can be deprived of water for up to 57 h. Animals may also be deprived of food for up to 81 h during transport to a federal slaughterhouse(11). The European Community has the most demanding legislation in the world regarding the transport of cattle in terms of animal welfare; it establishes a maximum duration of 14 hours of travel, followed by an hour of rest to drink water, being able to continue with another 14 h travel. This sequence can be repeated when the animals have been unloaded, fed, provided with water, and rested for at least 24 h(27,28). Despite the latter, more than a million citizens of the European Community requested, demanded, a general transport duration limit of 8 h. The European Parliament adopted a statement that upholds an 8-hour limit for cattle transport(3). The World Organization for Animal Health (OIE) developed guidelines for the welfare of livestock during transport; however, the signatory countries and the livestock export sector are not obliged to comply with them(29). With very opposite scenarios, countries like South Africa, Kenya, and some European countries have welldeveloped legislation on the welfare and transport of livestock. In contrast, Central and South American countries have a weak legislative framework, with a low level of compliance where knowledge of the legislation is absent, even among stakeholders(30).

Stress factors associated with long-distance transport

Factors associated with the pre-transport process

Transport-related activities begin with the grouping of animals; in some countries, the grouping can begin 48 hours before loading, since livestock is dispersed over large territorial extensions. The number and duration of various handling practices before loading; such as mixing of animals, food and water deprivation; represent a challenge that predisposes animals to dehydration and energy expenditure(31). Animals in detrimental conditions lack the same capacity to withstand long-distance transport. There are guidelines, similar to those used in Europe, to determine if an animal is fit for transport. An animal can travel if: it walks normally, carrying its weight evenly in its four legs; it is healthy, without visible disease or injury that could harm it during transport; it can stay with the group during loading and unloading; it has at least one functional eye, 521

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and it is not on late pregnancy(15). Loading is more stressful for animals than the unloading process; however, the physical integrity risks of the animal are similar in both cases (32). Animal loading, as well as the early stages of transport, cause high levels of stress; after this period, animals can adapt to transport conditions; however, after 12 hours, animals get tired and compromise their health, which is why transport should be interrupted(33,34). Stressors will initiate a series of reactions in the organism, with the activation of the sympathetic-adrenomedullary system and the hypothalamic-pituitary-adrenal axis, causing an increase in the levels of catecholamines and glucocorticoids(35), in addition to marked effects on the immune system, clearly visible in animals transported for repopulation purposes. Other repercussions can manifest several weeks after travel, such as lack of growth, low weight gain, and mortality, especially in young or recently weaned animals(36). Most research on the effects of transport and its regulations has focused on transport duration; for example, in Canada, the maximum transport time is 52 hours before arriving at destination; in the USA transport should not take longer than 28 hours, and in the European Union the maximum transport time is 30 hours. However, few studies have focused on the total time in which animals are confined in vehicles, waiting before departure, transport time, type of road, number and duration of stops, waiting for unloading, among others(37,38). Factors such as the cost of transportation; truck specifications and design; loading density, vibrations, and movement; microclimate conditions; climatic and geographic conditions; route planning; factors associated with the driver and risk of accidents must be considered as a whole within transport logistics(8).

Transport design

Livestock transport vehicles must be designed, built, and maintained in order to protect the animals from inclement weather, extreme temperatures, adverse changes in climatic conditions, and injury. Overall, there are four types of specialized vehicles: small trucks (â&#x2030;¤3 t), individual units (>13 m long), semitrailers, and double semitrailers(3). For a vehicle to guarantee greater animal comfort during transport, it is recommended to include drinking and ventilation systems, species-customized ramps, roof, non-slip flooring, lateral walls that prevent any part of the animal from leaving the truck, removable partitions to separate smaller and easier to handle groups, lateral inspection doors, and temperature control(39). However, the design of the truck and its impact on welfare has been poorly studied(40). In Central and South America, trucks can be articulated or not, generally without a roof, with metal or wooden structures(13). In Latin American countries, there are laws that try to protect animals by avoiding animal cruelty and unnecessary suffering. There is also legislation on the transport of animals for consumption in most countries. However, it deals 522

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mainly with sanitary and public health requirements (vehicle cleaning, antemortem animal health inspection, and postmortem meat inspection), instead of animal welfare, such as the case of Paraguay, Peru, Colombia, Ecuador, Argentina, Venezuela, and Uruguay(13). In Brazil, there is no specific legislation regulating the transport of farm animals, although most of the government agencies and large slaughterhouse companies are aware of the OIE recommendations. In Mexico, the Official Mexican Standard â&#x20AC;&#x153;NOM-051-ZOO-1995 for Humane practices in animal transportâ&#x20AC;? covers different animal species, but is not up-to-date and has weak and not very specific stipulations in terms of transport design. In North America, including Mexico, livestock is generally transported in pot-belly trailer trucks(41); these vehicles have an aluminum cover and five compartments: compartment 1 (nose), compartment 2 (belly), compartment 3 (back), compartment 4 (deck), and compartment 5 (doghouse)(42). In Europe, single or semitrailer trucks are the most common(43). The choice of vehicle will generally depend on the type and quantity of livestock, the specific demands of the market, the duration of transport, and the geographical region(3).

Loading density

From an economic point of view, loading density can increase or decrease operating costs per unit(44). The space required per animal during transport can be represented in three ways: (m2/100 kg), (kg/m2), and by the amount of surface used by each one (m2/animal). Other studies(45) concluded that the Space Allowance (SA=m2/animal), and an allometric coefficient that includes live weight of the animal (k=ED/PV0.6667), was a better indicator of available space for comparisons between studies in homogeneous weight batches. The area per animal is proportional to its surface area; a 400 kg cow should be transported in an area of 1.16 m2(45). Drivers or unit operators must be careful about the space availability in their trucks and know the characteristics of the species to be transported (horned or hornless; waste, fattening, dairy, recently weaned animals, among others); this, together with climate limitations, allows defining or altering the recommended loading density(46). When the loading density is of fewer animals per square meter, animals have more room to lie down, but if the way of driving or the road conditions are poor, it will be easier for the animals to lose their balance(47). A study reported that with a density of 170 kg/m2 (below the 360 kg/m2 recommended by the Farm Animal Welfare Council of the USDA), animals tend to lie down during transport(48). Eldridge and Winfield(49)examined the effects of different densities on long-distance transport, and although there were no effects in the ultimate pH (pHu) in beef, the incidence of bruises was higher with the lower and higher densities.

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Vibration, movement, and livestock exhaustion

During transport, animals are exposed to vertical, lateral, and horizontal vibrations. Unpaved roads or roads with strong wind currents transmit a more significant amount of vibrations, animalâ&#x20AC;&#x2122;s sensitivity increases after long standing periods(50), causing fatigue and displacement of their gravity center, which leads to falls and injuries(51). Additionally, animals make a more considerable effort seeking for a place to lean on the truck during braking(52). Long-distance transport is so physiologically demanding that it tends to affect the neutrophil/lymphocyte (N/L) ratio, which increases the probability of opportunistic infections(53). Gebresenbet et al(50)placed vibration sensors in a truck with an air suspension system and observed that the highest vibration level on animals was 2.27 Âą 0.33 m/s2 when driving in gravel roads at 70 km/h. Horizontal and lateral vibrations were lower on animals located perpendicular to the road direction. Avoiding rough, gravel, or dirt roads can reduce the exposure to vibrations, as well as using a truck that is serviced and operated by trained drivers. The pre-transport stages produce additional energy expenditure in order to meet transport demands; however, the long periods of fasting to which the animals are subjected will have negative effects on the muscle glycogen concentration, leading to a high pHu, which will result in Dark, Firm, and Dry (DFD) meat(34,54,55). Recent reports describe the Fatigue Cattle Syndrome (FCS), animals that develop mobility problems shortly after reaching a slaughterhouse, similar to that reported in pigs. Cattle present clinical signs of tachypnea and respiratory distress, animals may also present lameness, stiff gait, or supine position in the absence of evidence indicating injury or illness, in addition to elevated concentrations of lactate and creatine kinase (CK)(56).

Temperature, microclimate, and ventilation

Theoretical estimates indicate that in a typical trailer with a recommended density for 500 kg cattle, the heat produced inside would be 13,400 watts, which is why a ventilation system is required(4). There are two ventilation systems: passive ventilation (openings) and active ventilation (fans). Passive ventilation is given by openings throughout the truck and depends on the movement and speed of the truck(57). Active ventilation is controlled by sensors and uses extractor fans in air inlets and outlets(7).

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The microclimate inside the truck (temperature, relative humidity, and temperature and humidity index) is affected by the macroclimate, loading density, and airflow, as well as by animal respiration, transpiration, and secretions. Microclimate has a broad and potential impact on animal welfare, especially in adverse environmental conditions. Long-distance transport increases the probability of exposing animals to different climatic regions(58). For example, during the transport between Canada and the USA, temperature ranges from -42 to 45 °C(37). In extreme climatic conditions, the temperature inside the truck varies greatly, so the driver must be careful to open or close the ventilation openings. In warm climates, ventilation is hampered by air density, and the use of temperature and humidity recording devices is recommended so that the driver can make decisions during transport(36). The driver must also avoid stopping the truck for long periods since the internal temperature increases rapidly due to the external temperature, lack of ventilation, and the temperature emitted by the cattle. Under these temperature conditions, in trips longer than a day, cattle suffer an important weight loss(59). In cold climates, the incidence of post-transport morbidity and in-transit injuries caused by the freezing of sensitive body parts may increase(46). In these climates, straw bedding is recommended to improve animal comfort and to maintain a warmer temperature. High humidity conditions should be avoided during cold or hot climates since it has detrimental effects on the thermoregulatory capacity of animals(60,61). Depending on the increase in body temperature, the upper critical point for sheep and cattle is around 24-26 °C. Most mammals die when body temperature reaches 42-45 °C, which is above the normal body temperature by about 3 to 6 °C (62). The accumulation of ammonia represents a risk in high densities and poor ventilation conditions since it correlates with temperature and air humidity(62).

Risk factors associated with the driver

The driver’s ability to control the truck affects the quality of driving. Acceleration, braking, cornering, and driving techniques affect the ability of animals to maintain a stable posture, increasing excitability, reactivity, and injury(63). Moreover, the leading causes of road accidents during livestock transport in Spain, the USA, and Mexico are related to fatigue and poor decision-making by the driver, which results from long working hours, poor route design, and changes in sleep cycles(25). An analysis of articulated truck failures and accidents identified that the most common form of driver-associated accidents is related to an error in decision-making while driving(64). For example, the number of accidents during livestock transport in Mexico is unknown; drivers in this country frequently travel at high speeds, which affects the ability of the driver to deal with truck control in curves and other obstacles that may arise on the road(11).

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Other factors included the age of the driver, due to the combination of experience and good health, the ideal age to drive trucks is between 28 and 54 yr old, drivers under the age of 27 obtained higher accident/fatality ranges, which increases again in drivers older than 63 yr(65). Alcohol consumption, fatigue, and chronic health problems such as being overweight or obese, are other factors associated with the driver(66,67). In a study performed in Spain, most accidents involved pig (57 %), cattle (30 %), poultry (8 %), and sheep (5 %) transport(25); while in another study performed in the USA and Canada, Woods and Grandin(68) found that cattle (56 %) and pigs (27 %) were the most affected species. Of these accidents, 59 % occurred between 2400 and 0900 h, most of them were overturns, similar to what was observed in a study carried out in Mexico, where overturns were the most common type of accident (58.8 %) in long-distance transport of cattle(67); these were retrospective studies based on the analysis of newspaper reports, news, and driver surveys(67,68). In this type of accident, the surviving animals are usually stunned and disoriented; they can also suffer pain, states of fear and anxiety, which complicates their handling and increases the risk of secondary accidents(3). Therefore, driver training should be a priority in the logistics chain, covering aspects of animal behavior and welfare, as well as factors related to the mechanical operation of their trucks(46). The livestock industry must take action to reduce fatigue and, therefore, the risk of accidents, which results in the loss of human and animal lives, besides significant economic losses in the logistics chain of animal transport. The only effective strategy to prevent fatigue accumulation is an ergonomic interaction of the vehicle design, besides ensuring that drivers consistently get good quality and adequate sleep(66).

Routes and geography

Geographic conditions have a strong influence on livestock production systems and on the opportunities to commercialize it. In some cases, the geographic location of a country allows or hinders international exchanges and requires a variety of different types of transport(69). In countries like Chile, it is not possible to reduce livestock transport duration due to its unique geography and few adequate routes, with transport durations of up to 63 h(36). Brazil is another example, with long transport periods due to its territorial extension(6) and the global trend of reduction and specialization of slaughterhouses(12). Brazilâ&#x20AC;&#x2122;s road network system is over 1.6 million km long, and transport conditions vary depending on geographic characteristics. Roads are usually unpaved and in poor condition, a situation that worsens especially in the rainy season, increasing transport duration, the number of broken trucks, broken bridges, and road accidents(13).

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Impacts on welfare and productivity

The effect of long-duration transport on livestock is an important economic and animal health issue. This type of transport, as occurs in the Chilean Patagonia, causes significant loss of body weight(36), prolonging the physical recovery of livestock at the final destination.

Live weight loss, mobility, and mortality

Live weight loss in cattle is probably the most significant economic effect of transport. In a study with a group of heifers transported 518 km (8 h), with a maximum ambient temperature of 32.2 Â°C, live weight decreased by 6 % after transport(48). Weight loss is the most notorious effect at first, but a primary factor is the recovery time elapsed before starting to generate weight gain in animals transferred to fattening centers. Loerch and Fluharty(70) reported that a feed and water deprivation period above 72 h in addition to an eight-hour transport reduces the total ruminal protozoa. Moreover, animals subjected to long-distance transport can suffer dehydration, especially in warm-dry or very cold climates, when the airflow inside the truck is high. Providing small amounts of nutrients or electrolytes with correct tonicity immediately before and after transport, reduces tissue dehydration and the catabolism of muscle proteins, glycogen, and lipids, as well as reducing acid-base and electrolyte imbalances(71). Another concern is the risk of disease or death due to the variable climatic conditions and toxin exposure, among other factors(5). During transport, animals can get sick or die; these effects can occur several weeks after arriving at the destination. One of the most important diseases in cattle from intensive systems is the bovine respiratory disease. This disease usually affects young cattle, although it also increases due to the transport process to fattening units. In the USA, the bovine respiratory disease affects 14.4 % of the cattle that enter the fattening units(72). The immune response of transported cattle is usually suppressed by the high concentrations of cortisol associated with stress(73), so the disease usually manifests days or weeks after arrival. The injuries suffered during transport must be specially cared for; it is crucial to attend open wounds and keep in observation the animals that present difficulties in moving. Injured animals should receive anti-inflammatory and pain-relieving treatments to facilitate their recovery. The Fatigue Cattle Syndrome should be considered in the case of animals 527

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with mobility problems immediately after transport(56); this syndrome is accentuated in Bos indicus cattle, which is more temperamental and tends to lie down and "surrender" in trucks with high populations(1), presenting greater difficulty in adapting to confinement conditions. However, there is little information about this syndrome, and studies do not show consistent information, further information is needed. Transport mortality is a reflection of a severe welfare problem; this includes animals dead on arrival (DOA) and animals without apparent injuries (Non-ambulatory, non injured - NANI) that die at a later time(74). Mortality records during transport in commercial and experimental conditions have shown that mortality increases with high or very low temperatures, long-distance transport, or in the transport of very young animals(75,76). Transport mortality is variable and depends on different factors. Animals that lose 10 % of their body weight during transport are more likely to die or become non-ambulatory animals. Mortality also increases with lower space availability in the transport vehicle(37,38).

Bruising

Injuries and damage to the carcass caused by improper transport practices, or long-distance transport will affect the severity of bruising and, therefore, the quality of the carcass and meat. Based on the current market requirements, animal transport must avoid this type of damage. A carcass with less damage suggests better welfare conditions and, therefore, a higher ethical quality of the product. In the canal, bruising can be associated with different factors. During an experiment, the amount of bruising was higher in females than in males; also, the most severe injuries were found in old cows and not in heifers, which could be because most of these cows have a lower body condition (less muscle and subcutaneous fat). In this study, transport-related bruises were observed in a â&#x20AC;&#x153;dark redâ&#x20AC;? color and therefore considered to be less than or equal to 24 h in age, bruises were found on the sides, around the hip bones (ischial tuberosity), probably due to the contact that the cattle have with the sidewalls of the vehicle; bruising increased with higher animal densities(31). In males, the mating behavior and headbutts, which are common behaviors in beef cattle, are related to an increase in bruising, especially in holding pens(77); in this study, the appearance of this behavior could not be related to bruising.

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Strategies to reduce stress on long-distance transport

The production and transport of live cattle will continue to be stimulated by the growing human population(78), but with the integration of activities within the logistics chain, it would be possible to obtain the following advantages: 1) distance and time reduction through route optimization; 2) improve animal welfare; 3) expand the market area for producers; 4) decrease operating costs and increase competitiveness; 5) reduce carbon dioxide (CO2) emissions; 6) improve traceability for authorities and consumers; 7) narrow the participation between producers, distributors, merchants, and consumers; 8) promote the exchange of knowledge, experience, and information(79). Important members of the industry have begun to introduce their own policies to reduce stress and improve the welfare conditions of animals for slaughter.

Pharmacology applied to transport

Several studies have allowed the implementation of strategies that improve the conditions of animals during long-distance transport. Different studies recommend the use of some ingredients and drugs such as dexamethasone to support the treatment of some problems associated with transport(34,80). There is evidence that Mg can reduce the effects of preslaughter stress and improve the quality of meat because it suppresses neuromuscular stimulation(81) and, when added to the diet, results in the attenuation of the secretion of glucocorticoids and catecholamines(82). Tryptophan (Trp) is the precursor of serotonin, which regulates numerous biological functions, including temperature, pain sensitivity; feeding, sexual, and aggressive behavior(83); although its effects during transport have not been studied, preparations containing tryptophan are marketed worldwide as calming agents to treat excitable horses(84). It is important to administrate a fluid and electrolyte therapy during and after transport(1), a study demonstrated that providing cattle with electrolytes before slaughter improves carcass yield, without affecting the pHu, color, or water holding capacity(85); electrolytes also reduce dehydration and weight loss associated with transport(86). Moreover, allostatic modulators (AM), which contain substances such as ascorbic acid, acetoxybenzoic acid, sodium chloride, and potassium chloride, have been shown to mitigate the stress caused by the capture and handling of cattle during transport. A diet supplemented with 10 g of an AM, fed before slaughter for 30 d, showed anti-inflammatory properties, decreased stress levels determined by physiological parameters, and increased meat color stability at 24 and 28 d post-mortem(87). 529

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Conclusions Distance in transport is a strategic component of the global food and agriculture, and meat production economy. However, it is necessary to develop guidelines and technologies in terms of handling, operation, and logistics aimed to improve the welfare and health conditions of cattle. The impact of stress on biological functions, behavior, and suffering of animals has been underestimated in the past. Nowadays, it is important to integrate animal welfare into a broad concept of quality in animal production. Therefore, it is essential to invest in improvements aimed to establish logistics programs that have animal welfare as the axis of an operational quality program, in addition to legislation that regulates longdistance travel based on scientific evidence, and vehicle designs that adjust to different climatic conditions, as well as to the characteristics and behavior of each species.

Acknowledgments To the Consejo Nacional de Ciencia y Tecnología (CONACYT), for the financing of project number 259327, within the Call for Basic Scientific Research of 2015.

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39. Pezzaioli. Camiones y carrocerías para el transporte de ganado – 2016. http://www.pezzaioli.es. Consultado: 20 Ene, 2017. 40. Mitchell MA, Kettlewell PJ. Engineering and design of vehicles for long distance transport of livestock (ruminants, pigs and poultry). Vet Italiana 2008;(44):201–213. 41. Weschenfelder AV, Torrey S, Devillers N, Crowec T, Bassols A, Saco Y, Piñeiro M, Saucier L, Faucitano L. Effects of trailer design on animal welfare parameters and carcass and meat quality of three Pietrain crosses being transported over a short distance. Livestock Sci 2013;(157):234–244. 42. Schwartzkopf-Genswein K, Grandin T. Cattle transport by road - Livestock handling and transport. Fourth ed. Wallinford, UK: CABI; 2014. 43. Miranda-de la Lama GC, Villarroel M, Liste G, Escós J, María GA. Critical points in the pre-slaughter logistic chain of lambs in Spain that may compromise the animal's welfare. Small Ruminant Res 2010;(90):174–178. 44. Sánchez M, Vieira C, De la Fuente J, Pérez MC, Lauzurica-Gomez S, González de Chavarri E, DíazMT. Effect of season and stocking density during transport on carcass and meat quality of suckling lambs. Spanish J Agr Res 2013;(11):394-404. 45. Patherick CJ, Phillips JC. Space allowances for confined livestock and their determination from allometric principles. Appl Anim Behaviour Sci 2009;(117):1–12. 46. Schwartzkopf-Genswein K, Haley DB, Church S, Woods J, O’byrne T. An education and training programme for livestock transporters in Canada. Vet Italiana 2008;(44): 273–283. 47. Tarrant PV, Kenny FJ, Harrington D. The effect of stocking density during 4 hour transport to slaughter on behaviour, blood constituents and carcass bruising in Friesian steers. Meat Sci 1988;(24):209–222. 48. Theurer EM, White JB, Anderson ED, Miesner DM, Mosier AD, Coetzee FJ, Amrine ED. Effect of transportation during periods of high ambient temperature on physiologic and behavioral indices of beef heifers. Am J Vet Res 2013;(74):481–490. 49. Eldridge GA, Winfield CG. The behaviour and bruising of cattle during transport at different space allowances. Australian J Exper Agr 1988;(28):695–698. 50. Gebresenbet G, Aradom S, Bulitta FS, Hjerpe E. Vibration levels and frequencies on vehicle and animals during transport. Biosyst Engineering 2011;(110):10–19.

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51. Miranda-de la Lama GC, Monge P, Villarroel M, Olleta JL, Garcia-Belenguer S, María GA. Effects of road type during transport on lamb welfare and meat quality in dry hot climates. Trop Anim Health Prod 2011;(43):915–922. 52. Broom DM. Causes of poor welfare and welfare assessment. In: Grandin T editor. Livestock handling and transport. Publisher city. 2007:29-40. 53. Ekiz B, Ekiz EE, Kocak O, Yalcintan H, Yilmaz A. Effect of pre-slaughter management regarding transportation and time in lairage on certain stress parameters, carcass and meat quality characteristics in Kivircik lambs. Meat Sci 2012;(90):967976. 54. Bourguet C, Deiss V, Gobert M, Durand D, Boissy A, Terlouw EMC. Characterizing the emotional reactivity of cows to understand and predict their stress reactions to the slaughter procedure. Appl Anim Behaviour Sci 2010;(125): 9–21. 55. Van De Water G, Verjans F, Geers R. The effect of short distance transport under commercial conditions on the physiology of slaughter calves; pH and colourprofiles of veal. Livestock Prod Sci 2003;(82):171–179. 56. Thomson UD, Loneragan GH, Henningson NJ, Ensley S, Bawa B. Description of a novel fatigue syndrome of finished feedlot cattle following transportation. J Am Vet Med Assoc 2015;(247):66–72. 57. Gilkeson CA, Thompson HM, Wilson MCT, Gaskell PH. Quantifying passive ventilation within small livestock trailers using computational fluid dynamics. Comput Electron in Agr 2016;(124):84-99. 58. Phillips CJC. The welfare of livestock during sea transport. Appleby MC, Cussen VA, Garcés L, Lambert LA, Turner J. Long distance transport and welfare of farm animals. Wallingford, UK, CABI 2008:137–154. 59. Gregory NG. Animal welfare and meat science. CAB International, Wallingford, UK, 1998. 60. Fisher AD, Stewart M, Duganzich DM, Tacon J, Matthews LR. The effects of stationary periods and external temperature and humidity on thermal stress conditions within sheep transport vehicles. N Z Vet J 2005;(53):6-9. 61. Silanikove N. Effects of heat stress on the welfare of extensively managed domestic ruminants. Livestock Prod Sci 2000;(67):1–18.

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62. Pines MK, Phillips CJC. Accumulation of ammonia and other potentially noxious gases on live export shipments from Australia to the Middle East. J Environment Monitoring 2011;(13):2798–2807. 63. Cockram MS, Baxter EM, Smith LA, Bell S, Howard CM, Prescot RJ, Mitchell MA. Effect of driver behaviour, driving events and road type on the stability and resting behaviour of sheep in transit. Animal Sci 2004;(79):165–176. 64. Iversen H, Rundmo T. Personality, risky driving and accident involvement among Norwegian drivers. Personality and individual Differences 2002;(33):1251-1263. 65. Häkkänen H, Summala H. Fatal traffic accidents among trailer truck drivers and accident causes as viewed by other truck drivers. Accident Analysis & Prevention, 2001;(33):187–196. 66. Darwent D, Roach G, Dawson D. How well do truck drivers sleep in cabin sleeper berths? Applied Ergonomics 2012;(43):442–446. 67. Valadez-Noriega M, Estévez-Moreno LX, Rayas-Amor AA, Rubio-Lozano MS, Galindo F, Miranda-de la Lama GC. Livestock hauliers’ attitudes, knowledge and current practices towards animal welfare, occupational wellbeing and transport risk factors: A Mexican survey. Preventive Vet Med 2018;160:76-84. 68. Woods J, Grandin T. Fatigue: a major cause of commercial livestock truck accidents. Vet Italiana 2008;(44):259–262. 69. Rahman PJ, Brooke PD, Collins LM. Asia. Appleby MC, Cussen VA, Garcés L, Lambert LA, Turner J. Long distance transport and welfare of farm animals. Wallingford, UK, CABI 2008:288–318. 70. Loerch SC, Fluharty FL. Physiological changes and digestive capabilities of newly received feedlot cattle. J Anim Sci 1999;(77):1113-1119. 71. Schaefer AL, Dubeski PL, Aalhus JL, Tong AKW. Role of nutrition in reducing antemortem stress and meat quality aberrations. J Anim Sci 2001;(79):E91-E101. 72. Fike K, Spire MF. Transportation of cattle. Vet Clin North Am Food Anim Pract 2006;(22):305–320. 73. McEwen BS, Biron CA, Brunson KW. The role of adrenocorticoids as modulators of immune function in health and disease: neural, endocrine and immune interactions. Brain Res Rev 1997;(23):79–133.

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74. Sutherland AM, Erlandson K, Connor FJ, Salak-Johnson LJ, Matzat P, Smith FJ, McGlone JJ. Health of non-ambulatory, non-injured pigs at processing. Livestock Sci 2008;(116):237–245. 75. Knowles TG, Warriss PD, Brown SN, Edwards JE, Watkins PE, Phillips AJ. Effects on calves less than one month old of feeding or not feeding them during road transport of up to 24 hours. Vet Record 1997;(140):116–124. 76. Knowles TG, Warriss PD. Stress physiology of animals during transport. In: Grandin T. editor. Livestock Handling and Transport, CAB International, Wallingford, UK, 2000:385–407. 77. Warriss PD. The handling of cattle pre-slaughter and its effects on carcass and meat quality. Appl Anim Behaviour Sci 1990;(28):171–186. 78. Rahman SA. Middle East. Appleby MC, Cussen VA, Garcés L, Lambert LA, Turner J. Long distance transport and welfare of farm animals. Wallingford, UK, CABI 2008:387–409. 79. Bosona TG, Gebresenbet G. Cluster building and logistics network integration of local food supply chain. Biosyst Engineering 2001;(108):293–302. 80. Cook NJ, Veira D, Church JS, Schaefer AL. Dexamethasone reduces transportinduced weight losses in beef cattle. Canadian J Anim Sci 2009;(89):335–339. 81. Hubbard JI. Microphysiology of vertebrate neuromuscular transmission. Physiology Rev 1973;(53):674–723. 82. Kietzmann M, Jablonski H. On the blocking of stress by magnesium aspartatehydrochloride in the pig. PraktischeTierarzt, 1985;(66):328–335. 83. Leathwood PD. Tryptophan availability and serotonin synthesis. Proc Nutrition Soc 1987;(46):143–156. 84. Grimmett A, Sillence MN. Calmatives for the excitable horse: a review of Ltryptophan. The Vet J 2005;170:24-32. 85. Schaefer AL, Jones SDM, Tong AKW, Young BA. Effects of transport and electrolyte supplementation on ion concentration, carcass yield and quality in bulls. Canadian J Anim Sci 1990;(70):107–119. 86. Schaefer AL, Jones SD, Stanley RW. The use of electrolyte solutions for reducing transport stress. J Anim Sci 1997;(75):258–265.

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https://doi.org/10.22319/rmcp.v11i2.4912 Technical note

Growth, viability, and post-acidification of Lactobacillus plantarum in bovine transition milk

Hugo Calixto Fonseca a Eduardo Robson Duarte a* Lívia Caroliny Almeida Santos Souza a Emanuelly Gomes Alves Mariano a Ana Clarissa dos Santos Pires b Tatiana Santos Lima a Maximiliano Soares Pinto a

Instituto de Ciências Agrárias, Universidade Federal de Minas Gerais, Montes Claros, MG, Brasil. b

Universidade Federal de Viçosa, Campus Universitário, Viçosa. Departamento de Tecnologia de Alimentos. Grupo de Termodinâmica Molecular Aplicada, MG, Brasil.

* Corresponding author. duartevet@hotmail.com

Abstract: In this study, four milk substrates were analyzed to evaluate bovine Lactobacillus plantarum strain viability after 24 and 48 h of fermentation. In addition, cell viability, and post-acidification in transition milk fermented by these bacteria were assessed over a 60-d storage period at 4 and 25 °C. Significant reduction (30.9 %) of cell viability after 48 h of fermentation was observed for the formulation with whole milk. However, in fermented transition milk stored at 4 °C, cell viability and acidity were maintained at acceptable levels throughout the 60-d storage period. The viability of L. plantarum in fermented transition milk stored at 25 °C remained acceptable up to 50 d and minimum pH values were analyzed after 38 d of storage and maximum acidity levels after 56 d. Considering these results, transition milk may be preserved by L. plantarum fermentation as a substitute for milk in the artificial feed for calves as functional food. 539

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Key words: Animal health, Calvesâ&#x20AC;&#x2122; feeding, Fermentation, Food preservation, Probiotic.

Received: 23/05/2018 Accepted: 04/06/2019

The use of probiotics in animal feed promotes animal health, improves productivity(1), and therefore represents a possible strategy for controlling and preventing colonization of the gastrointestinal tract by pathogenic bacteria(2). Probiotic potential for lactic acid bacteria has been characterized, and species of the Lactobacillus genus have revealed beneficial effects in vitro and in vivo in the control of diarrhea in calves(3).

The major challenge presented by the growing demands for probiotics in the world market is that a probiotic strain must be cultivated in appropriate concentrations in a given product, and cell viability must be maintained throughout shelf life(4,5). Probioticcontaining foods have been evaluated as technologies for probiotic delivery, and the incorporation of these microorganisms into fermented milk has resulted in products with high cell viability and functionality(1,6).

Either fermented colostrum (first to third days post-partum) or transition milk (until seventh day post-partum) can be used as a milk substitute for artificial feeding, reducing costs and promoting healthy development of calves(7,8). These initial lactation secretions have been suggested as substrate for animal probiotic production(6), as it does not have commercial value, despite its high protein and vitamin content(9,10). In addition, immunoglobulin cells in fermented colostrum have the same viability as colostrum in natura and are capable of transferring passive immunity to newborn calves(11). Nevertheless, losses by putrefaction during fermentation have been registered. Those may be associated with the proliferation of pathogenic or spoilage microorganisms(7).

Dairy products are considered the best matrix carriers of lactic acid bacteria the main group of probiotic species(12). In one preliminary study, it was selected a Lactobacillus strain from the gastrointestinal tract of calf that presented inhibitory effects against Escherichia coli strains that cause calf diarrhea. Additionally, it was observed higher daily weight gains for young female calves fed with fermented milk containing the Lactobacillus sp. strain(13). Deeper analysis of this probiotic strain in transition milk and of adequate storage times would be of great relevance since it could reduce the costs of artificial feedings and improve health. 540

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In this study, it was evaluated the potential of four dairy substrates on the growth the Lactobacillus plantarum during two fermentation periods. In addition, assessed cell viability and post-acidification were evaluated, at two different temperatures, over a 60d storage period in transition milk fermented by this bacterium.

The bacteria strain analyzed was isolated from the feces of a 4-mo-old weaned 3/4 Holstein 1/4 Gyr calf. The bacterium was selected for having greater resistance to both acidic pH and bile salts in vitro, which are important probiotic characteristics, and for demonstrating a stronger antagonistic effect against two Escherichia coli strains that cause calves colibacillosis(13).

To perform molecular identification of these bacteria, DNA was extracted, amplified via the polymerase chain reaction (PCR) by usage of primers 27F (5′AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′), as described by Lane(14), and the 16S rRNA gene was sequenced(15) by MegaBACE® 1000 (GE Life Sciences, Chicago, USA) automatic sequencer at the Myleus Biotechnology laboratory (Belo Horizonte, Brazil). The 16S rRNA gene sequence was verified by SeqScanner Software® v1.0 (Applied Biosystems, Foster City, USA) and compared against the NCBI database by the BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/). The strain was recognized as Lactobacillus plantarum considering a 99 % similarity threshold. Additionally, the bacteria strain rendered identification scores higher than 2.0 when analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using MALDI-Biotyper v2.0 software(16).

Bacteria were preserved frozen (-18 °C) in tubes containing de Man, Rogosa and Sharpe (MRS) broth with 20% (m/m) glycerol. To activate microorganisms, 0.2 mL of the frozen cultures were added to 10 mL of MRS broth and incubated for 24 h at 37 °C. Subsequently, two successive inoculations were performed in test tubes containing 10 mL of reconstituted skim milk at a concentration of 10% solids-not-fat. For each inoculation, the tubes were incubated for 24 h at 37 °C.

Primarily, the viability of this bacteria was tested in two different fermentation periods times and four different substrate formulations: (1) reconstituted skimmed milk (RSM), which consisted of skimmed milk powder (Molico, Nestlé®) reconstituted in distilled water at a concentration of 10% (m/v) solids-not-fat, (2) transition milk (TM) from Holstein cows on the third day after calving, (3) whole milk (WM) from animals of the same breed and dairy farm as the TM formulation, and (4) a mixture of 50% WM and 50% TM (WTM). 541

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Each formulation (25 mL) was dispensed into test tubes, and 0.3% (m/v) sodium citrate was added to each tube as a stabilizer. The tubes were then autoclaved at 121 °C for 15 min. Subsequently, the tubes were cooled to room temperature (25 ± 2 °C), and 2% (v/v) of L. plantarum culture (8 log CFU·mL-1) was added to each formulation, as first concentrations were of 6.6 log CFU mL-1. The tubes were agitated and incubated in a BOD incubator at 37 °C for 24 and 48 h. The production flowchart for each formulation is shown in Figure 1.

Figure 1: Flowchart of the production process for four milk products, fermented by Lactobacillus plantarum, with probiotic potential

In a second trial, bacteria viability and post-acidification of fermented transition milk which were prepared as described above and were evaluated, though, incubated at 37 °C for only 24 h. After fermentation, samples were stored at 4 and 25 °C for microbiological and physicochemical analyses, as shown in Figure 2.

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Figure 2: Flowchart of the production process for transition milk, fermented by Lactobacillus plantarum, with probiotic potential

Viable bacterium counts for the four fermentations were performed immediately after the 24 h or 48 h of incubation. For the fermented transition milk stored at two different temperatures, viable cell counts were evaluated at 0, 10, 20, 30, 40, 50, and 60 d of storage.

Samples of the fermented substrates were serially diluted to 10-7 in sterile 0.1% (m/v) peptone water, and 1-mL aliquots were transferred to sterile Petri dishes. MRS agar medium (HiMedia, Mumbai, India) was then added, and the material was homogenized by pour plate method. Plating was performed twice for all analyses. The plates were incubated in at 37 Â°C for 72 h under aerobic conditions, and colony-forming units (CFUs) were counted. Results were expressed as log CFU per mL of fermented substrate.

The pH of fermented substrates was measured by a digital potentiometer with a combined glass electrode (Hanna brand, model pH21). The titratable acidity, expressed

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as % (m/v) of lactic acid, was determined by acid-base titration. Both analyses were performed after 0, 10, 20, 30, 40, 50, and 60 d of storage.

Data were analyzed by analysis of variance, and the significance of differences between means was assessed by the Tukey test at the 95% confidence level (P<0.05). Regression analyses were performed to describe cell viability, pH, and titratable acidity as a function of storage time at each storage temperature. For both experiments, a factorial scheme was used, with four replicates for each condition, and the experimental design was completely randomized. The analyses were carried out by the Statistical Analysis System v9.4 (SAS, 2014).

The viability of microorganisms in probiotic foods is the main determinant of the functionality of these products. In this study, the viable cell counts of the probiotic cultures were greater than 8.40 log CFUÂˇmL-1 (Table 1). There was no significant difference in the concentration of L. plantarum in the four dairy substrates after the same incubation period. Transition milk enabled viable growth of probiotic cells to the same concentrations as whole milk did, suggesting it should be chosen as a growth substrate since it has no commercial value for the dairy industry.

Table 1: Lactobacillus plantarum viable cell counts (log CFU.mL-1) in reconstituted skimmed milk (RSM), transition milk (TM), whole milk (WM), and 50% TM + 50% WM (WTM) after 24 and 48 h fermentation at 37 Â°C Substrate Fermentation time RSM TM WM WTM Aa Aa Aa 24 h 8.77 8.67 8.91 8.62Aa 48 h 8.58Aa 8.82Aa 8.40Ab 8.64Aa Different uppercase letters for values in the same row and different lowercase letters for values in the same column indicate significant differences (P<0.05). Coefficient of variation: 2.61 %.

For the WM formulation, there was a significant decrease (30.9 %) in viable cell counts between 24 h and 48 h of fermentation. In contrast, for the RSM, TM, and WTM formulations, the L. plantarum viable cell counts were similar after 24 h and 48 h of fermentation, indicating that only 24 h of fermentation is required for the microorganism to grow to high concentrations in these substrates.

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In another study, the growth of five probiotic strains in UHT milk supplemented with tryptone and fructose was studied, and all strains reached maximum viable cell counts of 8.7 to 9.2 log CFU·mL-1 after 6–16 h of incubation. However, three strains exhibited a decrease of 0.4 to 1.1 log CFU·mL-1 in viable cell count between 24 h and 72 h of incubation(17). The fermentation of six probiotic strains over a 48-h period at different temperatures was also evaluated having UHT milk as a substrate. A temperature of 37 °C and an incubation time of 12–24 h yielded the highest growth and viable cell counts (8.65 to 9.21 log CFU·mL-1) for all evaluated strains of Lactobacillus spp.(18).

The substrate the strain and its adaptation to the culture medium, strongly influence the rate of fermentation and the duration of the cell growth phase. Faster growth causes faster nutrient consumption and acid production, which has a negative environmental impact, leading to rapid progression to the decline phase. In general, the duration of fermentation is determined by pH; fermentation continues until the pH reaches 4.5–4.6. In the production of yogurt that have varied probiotic species, faster fermentation was reported in whole milk than in skimmed milk(19). In other study, the total fermentation time for whole milk varied between 16 and 31 h, depending on the species of Lactobacillus spp. evaluated(20).

In this study, all formulations yielded viable cell counts above 6 log CFU·g-1 (Table 1), which is the minimum viable cell count required for products of Lactobacillus spp. to function as probiotics(21). Manufacturers of probiotic cultures also recommend a minimum viable cell count of 6 log CFU·g-1 in milk fermented by these bacteria(22). The results of this study were similar to those reported by Coman et al(23), who reported viable cell counts above 8 log CFU·mL-1 of L. rhamnosus and Lactobacillus paracasei, individually or combined at the end of the fermentation of whole milk.

Lasting viability and stability during storage are considered fundamental prerequisites for probiotic products. We therefore measured cell viability, pH, and titratable acidity in fermented transition milk over a 60-day storage period at 4 °C and 25 °C. As expected, each parameter was dependent on storage temperature (Table 2). After 40 days’ storage, the viable cell counts of fermented transition milk stored at 25 °C were significantly lower than those of the product stored at 4 °C. Likewise, storage of the fermented transition milk at 25 °C yielded a significantly lower pH after 10 d and a significantly higher titratable acidity after 20 d, compared with storage at 4 °C.

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Table 2: Viable cell count, pH, and titratable acidity (expressed as % lactic acid) of transition milk fermented by Lactobacillus plantarum and stored for 60 d at 4 and 25 °C Viable cell count Lactic acid pH -1 (log CFU·mL ) (%) Time (days) Temperature Temperature Temperature 4 °C 25 °C 4 °C 25 °C 4 °C 25 °C Aa Aa Aa Aa Aa 0 8.89 8.89 5.70 5.70 0.37 0.37Ac 10 8.72Aa 8.56Aa 5.52Aab 5.01Bb 0.42Aa 0.73Ab 20 8.79Aa 8.02Aab 5.47Aab 4.78Bb 0.53Aa 0.90Bab 30 8.50Aa 7.63Abc 5.36Ab 4.85Bb 0.55Aa 0.98Bab 40 8.71Aa 7.63Bbc 5.36Ab 4.86Bb 0.53Aa 1.03Bab 50 8.67Aa 6.88Bc 5.43Aab 4.89Bb 0.48Aa 1.14Ba 60 8.48Aa 5.65Bd 5.37Ab 4.95Bb 0.55Aa 1.19Ba Different uppercase letters for values in the same row and different lowercase letters for values in the same column, for each parameter, indicate significant differences (P<0.05). Coefficients of variation: 4.47% (Viable cell count); 2.36% (pH); 19.41% (lactic acid).

The viable cell count was 8.48 log CFU·mL-1 after 60 d of storage at 4 °C and did not change significantly (P>0.05) over the storage period. However, when the product was stored at 25 °C, there was a significant reduction (P<0.05) in cell viability of more than 1 log cycle after 30 d of storage. However, the decrease was more pronounced after 60 d of storage, showing viable cell count of 5.65 log CFU·mL-1 at the end of the storage period (Table 2). The decrease in cell viability at this storage temperature could be justified by lower pH and higher acidity. Using regression analysis, we inferred that the viable cell count of the product remained within acceptable limits (>6.5 log CFU·mL-1) for more than 60 d under refrigeration and for up to 50 d at 25 °C (Table 2 and Figure 3). For a probiotic product, stability of the viable cell count over its shelf life is essential, and stability of the probiotic at room temperature is particularly relevant because it allows producers to save energy while storing the transition milk for artificial feeding of calves.

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Figure 3: Quadratic regression of Lactobacillus plantarum viability (---) and acidity (— ) of fermented transition milk as a function of storage time (up to 60 d) at 25 °C. R2: coefficient of determination; y: viability or acidity; t: storage time

Different storage times under refrigeration have been used previously to evaluate the viability of probiotic microorganisms in fermented products. In the development of a milk fermented by L. plantarum, there was a decrease of 1.2 log CFU·mL-1 in viable cell counts when the product was stored for 70 d at 10 °C, which was a satisfactory result(24). Analysis conducted with colostrum and transition milk silages showed that appropriately fermented samples had an average Lactobacillus spp. concentration of 5.15 log CFU·mL-1 after 33 days’ storage at 25 °C(7).

Probiotic fermented milk can be stored for several weeks with minimal viability loss if acidification can be minimized by refrigeration(25). In this study, the pH of the product decreased significantly after 30 days’ storage at 4 °C, reaching a value of 5.36 after that period (Table 2). When stored at 25 °C, the pH of the product dropped from 5.70 to 5.01 after 10 d of storage. According to the regression equations, the fermented transition milk would have reached their minimum pH values after 44 and 38 d when stored at 4 and 25 °C, respectively (Figure 4).

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Figure 4: Quadratic regression of the pH of the fermented transition milk as a function of storage time (up to 60 days) at 25 °C (---) and 4 °C (—). R2: coefficient of determination; y: pH; t: storage time

PH affects protein conformation, enzyme activity, and acid dissociation, and is therefore the most important parameter to characterize milk acidity and dairy products. Coman et al(23) showed that, after fermentation and storage for 4 wk at 4 °C, whole milk fermented by L. paracasei and L. rhamnosus reached minimum pH values of 5.60 and 4.31, respectively(26). Another fact that should be taken into consideration is nutrient availability; for example, milk fermented by L. plantarum showed pH values of 5.81 for the control sample (without added nutrients) and 3.82 for samples with added nutrients (amino acids, vitamins, minerals, and nucleotides) after 72 h of fermentation(27).

PH reduction causes a passive flow of protons into microbial cells, which actively export protons. The uncontrolled influx of protons should decrease cell internal pH, inhibiting the synthesis of cellular components and cell multiplication. Undissociated lactic acid can penetrate the cell membrane and contribute to acidification of bacterial cytoplasm(28).

Initial and final pH, along with other factors such as organic acid production and exposure to different temperatures during storage, can affect cell viability during fermentation. The growth of undesirable microorganisms is reduced in products with a pH below 5.0(26). In this study, the pH of the fermented transition milk at 25 °C remained below those levels from 20 to 50 d (Figure 4). 548

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Ferreira et al(29) observed a rapid decrease in pH when colostrum was fermented naturally at 32.5 °C; products with pH values below 4.5 were obtained after 35 d of fermentation. In previous studies, bovine colostrums, from the second milking after calving, presented a mean 5.41 for the pH after 33 days of fermentation at 25 °C(7). In this study, the titratable acidity of the fermented transition milk did not change significantly (P>0.05) over the storage period when stored under refrigeration. However, when the product was stored at 25 °C, the titratable acidity almost doubled after 10 d, increasing from 0.37 to 0.73 % of lactic acid; another significant increase in acidity occurred only after 50 d, reaching 1.14 %. Regression analysis indicated that the fermented transition milk stored at 25 °C reached maximum values of titratable acidity after 56 d (Figure 4). These results show that the stability and viability of the product could be influenced by acidity, since the strain used in this study is acid-sensitive. The strain of L. plantarum with probiotic potential evaluated showed satisfactory growth in each of the milk-based formulations tested, yielding high concentrations of viable cells (> 8 log CFU·mL-1). Transition milk of the third day after calving represents a useful substrate for the growth of this bacterium and can be stored for up to 50 d at room temperature. Therefore, fermentation of transition milk by the L. plantarum strain shows to be a viable method for production those probiotics.

Acknowledgments The authors wish to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Apoio à Pesquisa de Minas Gerais (FAPEMIG) for their financial support.

Conflict of interest statement

The authors have no financial or personal relationship with people or organizations that could inappropriately influence or bias the content of the paper.

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Azevedo RA De, Araújo L, Coelho SG, Emygdio D, Filho DF. Desempenho de bezerros alimentados com silagem de leite de transição. Pesqui Agropecuária Bras 2013;48(5):545–552.

10. Uruakpa FO, Ismond MAH, Akobundu ENT. Colostrum and its benefits: a review. Nutr Res 2002;22:755–767. 11. Saalfeld MH, Pereira DIB, Borchardt JL, Sturbelle RT, Rosa MC, Guedes MC, et al. Evaluation of the transfer of immunoglobulin from colostrum anaerobic fermentation (colostrum silage) to newborn calves. Anim Sci J 2014;85(11):963– 967. 12. Silva KF, Faria, BKA, Reis IMF, Costa MX, Soares ACM, Mariano EGA, et al. Desempenho de bezerras leiteiras suplementadas com Lactobacillus sp.1. In: SBZ 2015. Anais da SBZ 2015- Sociedade Brasileira de Zootecnia. Belo Horizontes: SBZ, 2015.

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13. Mattila-Sandholm T, Millärinem R, Crittenden R, Mogensen G, Fondén R, Saarela M. Technological challenges for future probiotic foods. Int Dairy J 2002;12(23):173–182. 14. Lane DJ. 16S/23S rRNA sequencing In: Stackebrandt E, Goodfellow M, editors. Nucleic acid techniques in bacterial systematics. Chichester: Wiley; 1991:115-175. 15. Sanger F, Coulson AR. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J Mol Biol 1975;94(3):441–448. 16. Farfour E, Leto J, Barritault M, Barberis C, Meyer J, Dauphin B, et al. Evaluation of the andromas matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of aerobically growing gram-positive bacilli. J Clin Microbiol 2012;50(8):2702–2707. 17. Ostlie HM, Helland MH, Wicklund T, Narvhus JA. Growth and metabolism of selected strains of probiotic bacteria in milk. Int J Food Microbiol 2003;87:17–27. 18. Ostlie HM, Treimo J, Narvhus JA. Effect of temperature on growth and metabolism of probiotic bacteria in milk. Int Dairy J 2005;15(10):989–997. 19. Espírito Santo AP do, Perego P, Converti A, Oliveira MN. Influence of milk type and addition of passion fruit peel powder on fermentation kinetics, texture profile and bacterial viability in probiotic yoghurts. LWT - Food Sci Technol 2012;47(2):393–399. 20. Lanciotti R, Patrignani F, Iucci L, Saracino P, Guerzoni ME. Potential of high pressure homogenization in the control and enhancement of proteolytic and fermentative activities of some Lactobacillus species. Food Chem 2007;102(2):542–550. 21. Champagne CP, Ross RP, Saarela M, Flemming K, Charalampopoulos D. International Journal of Food Microbiology Recommendations for the viability assessment of probiotics as concentrated cultures and in food matrices. Int J Food Microbiol 2011;149(3):185–193. 22. Sanders ME. Probiotics: considerations for human health. Nutr Rev 2003;61(3):91– 99. 23. Coman MM, Verdenelli MC, Cecchini C, Silvi S, Vasile A, Bahrim GE, et al. Effect of buckwheat flour and oat bran on growth and cell viability of the probiotic strains Lactobacillus rhamnosus IMC 501®, Lactobacillus paracasei IMC 502® and their combination SYNBIO®, in synbiotic fermented milk. Int J Food Microbiol 2013;167(2):261–268. 24. Souza AHP de, Costa GAN, Miglioranza LH da S, Furlaneto-Maia L, Oliveira AF. Microbiological, physical, chemical and sensory characteristics of milk fermented with Lactobacillus plantarum. Acta Sci Heal Sci 2013;35(1):125–131. 551

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25. Soto LP, Frizzo LS, Bertozzi E, Diaz A, Marti LE, Santina RD, et al. Milk evaluation as growth and cold preservation medium of a probiotic inoculum for young calves. J Anim Vet Adv 2009;8(7):1353–1560. 26. Donkor ON, Henriksson A, Vasiljevic T, Shah NP. Effect of acidification on the activity of probiotics in yoghurt during cold storage. Int Dairy J 2006;16(10):1181– 1189. 27. Ma C, Cheng G, Liu Z, Gong G, Chen Z. Determination of the essential nutrients required for milk fermentation by Lactobacillus plantarum. LWT - Food Sci Technol 2016;65:884–889. 28. Kashket ER. Bioenergetics of lactic acid bacteria: cytoplasmic pH and osmotolerance . FEMS Microbiol Rev 1987;46:233–244. 29. Ferreira LS, Silva JT, Paula MR de, Soares MC, Bittar CMM. Colostrum silage: fermentative, microbiological and nutritional dynamics of colostrum fermented under anaerobic conditions at different temperatures. Acta Sci Anim Sci 2013;35(4):395–401. doi: 10.4025/actascianimsci.v35i4.19870.

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https://doi.org/10.22319/rmcp.v11i2.5084 Technical note

Characterization of the milk and artisanal cheese of the region of Ojos Negros, Baja California, Mexico

Laura E. Silva-Paz a Gerardo E. Medina-Basulto a Gilberto López-Valencia a* Martin F. Montaño-Gómez a Rafael Villa-Angulo b José C. Herrera Ramírez a Ana L. González-Silva a Francisco Monge-Navarro a Sergio A. Cueto-González a Gerardo Felipe-García a

Universidad Autónoma de Baja California. Instituto de Investigaciones en Ciencias Veterinarias. Fracc. Laguna Campestre carretera a San Felipe km 3.5, Mexicali, Baja California. México. b

Universidad Autónoma de Baja California. Instituto de Ingeniería. México.

*Corresponding author: gilbertolopez@uabc.edu.mx

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Abstract: The community of Ojos Negros is located in the municipality of Ensenada, Baja California, Mexico. Since 1930, local residents make a greatly appreciated handmade cheese in the region; however, the raw milk and cheese have never been analyzed for the microbiological quality and hygiene of the final product. The objective of this study was to evaluate the microbiological, physical and chemical quality of the raw milk used to produce cheese, and the artisanal cheese produced in the 22 individual production units. Samples of cheese and milk were collected from dairy production units in order to perform microbiological tests. There were physical and chemical determinations of protein, fat and lactose, using a LACTOSCAN-S analyzer. The results of milk analysis showed protein (33.11 g/L) and fat (39.89 g/L) contents within the parameters of the regulations. For the microbiological quality of milk, the results of the aerobic mesophilic count showed a 64 % compliance with the regulations; however, the same aerobic mesophilic count in the cheese samples resulted in only 4 % compliance. No Salmonella spp. or Listeria monocytogenes were detected in any of the tested milk or cheese samples. Good sanitation and manufacturing practices should be incorporated in order to enhance the sanitary quality and hygiene standards for the production of artisanal cheese in the community of Ojos Negros. Key words: Ojos Negros, Artisanal cheese, Chemical composition, Microbiological quality.

Received: 28/09/2018 Accepted: 29/04/2019

In Mexico the elaboration of artisanal cheese by medium-size and small producers is estimated at approximately 25 % of the total cheese produced per year(1). The preparation and sale of artisanal cheeses constitutes one of the main sources of income for small-scale farmers despite the low profitability of their activity(2,3). The so-called â&#x20AC;&#x153;pressed cheese of Ojos Negrosâ&#x20AC;? has been produced in the traditional way since 1930 in the Ojos Negros region of the municipality of Ensenada, Baja California, Mexico. Currently, the production of this cheese (produced from unpasteurized milk) reaches 30 t per month, providing sustenance for approximately 65 families(4). However, the cheese producers face a new challenge because Mexican regulations stipulate that the milk used for cheese production must be pasteurized(5). In addition, sanitary practices must be implemented to ensure a safe product that does not pose a risk to the consumer(6). In 2010 the regional producers organized an association in order to seek technical assistance and succeeded in developing dairy production units; however, the microbiological

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quality of the product is questionable, given the use of raw milk without pasteurization, coupled with the absence of an appropriate system of good manufacturing practices(7). Therefore, the objective of this study was to perform a situational analysis of the microbiological and physicochemical quality of milk and cheese produced in the 22 cheese production units of that region. The study was carried out in 22 production units (PU) of artisanal cheese located in the town of Ojos Negros, in the municipality of Ensenada, Baja California, Mexico (31°45' and 32°04' N and 116°06' and 116°27' W). All the PU participate in the tuberculosis and brucellosis control program. The production system is semi-extensive (free grazing and stabling) and has adequate infrastructure and equipment for producing milk and cheeses at a household level. On average, each herd produces 450 L of milk intended for the manufacture of artisanal cheese. The producers are in the initial phase of implementing a program of good manufacturing practices and hygiene. The milk samples were collected from all the PUs on the same day according to the specifications of the NMX-F-718-COFOCA(8). Out of each PU, 100 ml of milk were obtained from the tank in order to determine its sanitary quality; the cheeses were sampled according to the guidelines of the NOM-109-SSA(9). A piece of whole cheese of approximately 2.5 kg, was collected only once from each PU. Samples of milk and cheese were transported in cooler at a temperature of between 7 and 10 °C to the Dairy Analysis Laboratory of Analysis of the Institute for Research on Veterinary Sciences (IICV) of the UABC for further processing. The microbiological analyses of the milk and cheese were performed according to the procedure described in Appendices B10, B16, B17 of the NOM-243-SSA1(5), requiring, for milk and cheese, 1 ml and 10 g of sample dissolved in 9 ml and 90 ml, respectively, of 1% buffered peptone water (Difco, New Jersey). The colony forming units (CFU) of each sample were counted by performing five dilutions placing 1 ml on plates in duplicate for aerobic mesophilic bacteria in Standard Count Agar (MCD Lab, Tlalnepantla, Mexico); plates with 25 to 250 colonies were selected for counting. In order to determine the presence of fecal coliform bacteria, those plates with a register of 30 to 300 colonies were selected, and 1 ml of each dilution was planted and analyzed after five days in order to count the fungi and yeasts. In order to perform the analysis for Salmonella, 10 ml of milk and 10 g of cheese were pre-enriched in 90 ml of peptone water (Difco, New Jersey) and after 24 h of incubation at 35 ± 2 °C, they were enriched in tetrathionate broth (Difco, New Jersey) and Rappapot Vassilidis base (Difco, New Jersey); the sample was subsequently enriched through planting in XLD agar (Difco, New Jersey), Hecktona (Difco, New Jersey) and Bright Green media (Difco, New Jersey), identifying the bacterium by means of TSI (BD Bioxon, Cuautitlan, State of Mexico), LIA (BD Bioxon, Cuautitlan, State of Mexico), Urea (Difco, New Jersey), and RMVP biochemical tests (Difco, New Jersey). 555

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For the analysis of Listeria monocyogenes, a 25 g sample of cheese was homogenized in 225 ml of UVM broth (Difco, New Jersey), incubated at 30 °C for 24 h, and then enriched in 10 ml of Fraser broth (Difco, New Jersey) for 24 h at 35 ± 2 °C. Plantings were made in Oxford agar plates (Difco, New Jersey); colonies with brown pigmentation with a halo were isolated for purification and identification in Brain Heart Infusion Agar (Difco, New Jersey), using Gram positive coccobacilli chain morphology, positive motility at 20-25 °C, negative oxidase test, and positive catalase and API-Listeria tests (bioMerieux, St. Louis, MO), respectively, for confirmation, according to the respective procedures of appendices B13 and B12 of the NOM243-SSA(5). The acidity in the milk was determined according to the procedure described in the NOM-155SCFI(10), and the pH was evaluated with a potentiometer (Hanna Instruments, Carrolton TX). The physicochemical determination of the percentage of protein, fat, and lactose in the milk was performed using the LACTOSCAN-S analyzer (Milk Analyzer LTD model LS 90, Bulgaria). The mean ± standard error was calculated for each one of the analyzed variables. Student's t-test was used to detect differences (P<0.05) between the averages of each parameter, compared to the desirable parameters of the NOM standards that apply. The results of the physicochemical and nutritional quality of milk used in the elaboration of artisanal cheese are presented in Table 1. No significant differences were observed (P<0.05) between the averages of protein detected for each class (A, B, and C) with respect to a reference parameter that indicates the NMX-F-700COFOCALEC normativity(11). With regard to the fat, the class exhibited no difference (P>0.05) between the desirable parameter of the NOM and the average value. However, when comparing the average for class C against the desirable parameter, significant differences (P<0.05) were identified. In this study, the figures for A protein classes (32.98 g/L) and fat (35.62 g/L) were better than those reported by Bernal(12) in small herds of the State of Mexico, and Oliszewski(13) in dairy herds of the rural region of the Las Trancas Basin in Argentina, with lower amounts of protein (30.55 g/L) and fat (34.0 g/L); however, both figures are considered to be indicative of a good quality milk. A possible explanation for these variations could be the nutritional management of the cows with respect to the rationing of nutrients, free-range grazing or genetic factors in the breeds of animals, as pointed out by De la Cruz(14).

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Table 1: Physicochemical quality and nutritional status of milk used in the elaboration of artisanal cheese in 22 PUs in the region of Ojos Negros, Mexico * Indicator Reference Values** g/L N (%) Mean (SD) Protein, g/L

Class A desirable ≥ 31a Class B minimum 30 -30.9 Class C minimum 28 to 28.9

14 (63) 6 (27) 2 (10)

32.98 b (0.287) 30.46 a (0.116) 28.10 b (0.100)

Fat, g/L

Class A desirable ≥ 32to Class B minimum 31 Class C minimum 30

10 (46) 1 (4) 11 (50)

35.62 a (3.949) 31.50 a 27.01 b (1.138)

Lactose, g/L

Desirable 47.5 To Not desirable < 44

15 (73) 7 (27)

47.74a (0.495) 42.80b (0.728)

Lactic acid, g/L

Not desirable ≥ 1.9 Desirable 1.3 - 1.8 to Not desirable ≤ 1.29

15 (68) 4 (18) 3 (14)

2.438b (0.076) 1.720 b (0.080) 1.153 b (0.016)

Not desirable ≥ 6.9 Desirable 6.5-6.8 to Not desirable ≤ 6.4

4 (18) 15 (68) 3 (14)

6.95 b (0.016) 6.70 a (0.045) 6.33 a (0.066)

SD= standard deviation. * NMX-F-700-COFOCALEC-2012. ab

Mean values of each variable with different letters differ significantly (P<0.05) from the parameters.

With regard to lactose, 73 % of the PUs exhibited a desirable level without differences (P>0.05) with respect to the desirable parameter of the NOM. 27 % of the PUs that were identified as having low levels of lactose; a possible explanation for this is that they were also identified with cases of mastitis, as this disease is known to lead to a reduction in the secretion of lactose in milk(15,16). As for lactic acid, only 18 % of the PU were found to have acceptable levels of compliance with the norm. In addition, 68 % of the PU exceeded the parameter of the norm (P<0.05). One possible explanation is that bacterial populations degrade lactose with storage time, progressively developing acidity in milk(17,18,19). On the other hand, 68 % of the PU had a desirable average pH, while 18 % of the PU exhibited an average pH > 6.95 (P<0.05) This can be partly explained because the milk is kept in storage at room temperature (between 15 to 32 °C) for more than 6 h; this leads to an increase of lactic microorganisms and, above all, of coliforms and, therefore, to increased acidity(19,20). The mean titratable acidity (1,720 g/L) and pH (6.70) observed in this study exhibited similar values to those reported for the Argentina region by Oliszewski(13), with an acidity of 1,726 g/L and a pH of 6.75.

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Table 2 shows the microbiological quality of milk used in the elaboration of artisanal cheese. 64 % of the PUs obtained desirable (50 %) to acceptable (14 %) mean values with respect to the NMX-F-700-COFOCALEC(11) for aerobic mesophiles. These figures are similar to those reported by Oliszewski (4.94 log CFU/ml)(13). With regard to the averages of the undesirable classes,(3,4) compared with the reference parameter, a significant difference (P<0.05) was detected. Similar results were obtained by Perkins(21) and De la Cruz(14), who pointed out that mesophilic counts of > 6 log are indicative of health issues in the herd. One possible explanation is that the herds do not have a secondary preventive medicine program to identify health issues, including subclinical mastitis, a situation that is reflected in high mesophilic levels. Table 2: Microbiological quality of milk used in the preparation of cheese at 22 artisanal PU in the region of Ojos Negros, Mexico Indicator Reference values (log10 CFU/ ml−1) N (%) Mean (SD) *Aerobic mesophilic count /ml

Salmonella spp 25g

≤100,000 UFC/ml ( ≤5.0 ) a 101,000-300.000 ( 5.1 – 5.47 ) ≥301,000 – 599,000 ( ≥5.48 – 5.77 ) ≥600,000 – 1,200,000 ( ≥5.78 ) ≤10 UFC/ml (≤1)a <11-100 UFC/ml ( 1.04-2.0 ) ≥101 UFC/ml ( ≥2.1 ) Absence

Listeria monocytogenes 25g

Absence

Class 1 Class 2 Class 3 Class 4 **Coliforms (CFU/ml)

11 (50) Desirable 3 (14) Acceptable 1 (4) Not desirable 7 (32) Not desirable ND Desirable 1 (5) Not desirable 21 (95) Not desirable 22 (100) 22 (100)

1.67 b (0.189) 5.11 a (0.110) 5.60 a 7.53 b (0.348)

2.00 b 5.589 b (0.384)

SD= standard deviation. NMX-F-700-COFOCALEC-2012. ** NOM-243-SSA1-2010. Test methods: Total coliforms <10 CFU (≤1 log10 CFU ml-1). ND= not detected. ab Mean values of each variable with different letters differ significantly (P<0.05) from the parameters. *

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In addition, 100 % (22) of the PU had an average of undesirable coliforms (P<0.05) above the norm (NOM-243-SSA1)(5). However, the results are lower than those reported by Oliszewski(13) with 5.64 log CFU/ml. The increase in mesophiles and fecal coliform bacteria does not necessarily indicate a direct fecal contamination in milk, but it denotes specific deficiencies in hygiene and milking routines without good management practices during the procurement and storage of milk(14,22,23). Another important aspect was that none of the PU identified Salmonella spp. or Listeria spp., which is a desirable result, since these bacteria represent a risk to the consumers’ health(24) . The microbiological quality of the cheese produced in the 22 PU is shown in Table 3 where we can see that only 18 % of the PUs exhibited similar counts (P>0.05) to the desirable parameter of aerobic mesophiles. The rest of the PU exhibited undesirable averages (P<0.05) of mesophiles above the norm (NOM-243-SSA1)(5). With regard to the coliforms, 100 % of the PU exhibited average figures above the parameter set by the regulations (P<0.05). The high incidence of coliforms and mesophiles in the cheeses of the PU indicates deficiencies in hygiene and manufacturing practices. One possible explanation is that the cheese is made with milk stored at an average temperature of 20 °C. Several studies show that the conservation temperature is an important factor that may damage its stability by increasing the number of altering microorganisms; thus, unless kept under control, these microorganisms may have a direct effect on the quality and useful life of the cheese(19,23,25,26). It is important to note that values above 6 log10 ≥ mesophiles and ≥5 log10 coliforms were also reported in other studies(27-30). Torres(31) mentions that the increase of aerobic mesophiles is considered a normal process in the first 30 d of elaboration of the cheese, because there are chemical reactions that accompany the multiplication of microorganisms during the coagulation and drainage of the serum, due to the presence of lactic acid bacteria (LAB). Pathogenic bacteria such as Salmonella spp and Listeria monocytogenes were not detected in pressed cheese in compliance with the Mexican regulations.

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Table 3: Microbiological quality of artisanal cheese of 22 PU in the region of Ojos Negros, Mexico Indicator Reference values2 (log10 CFU/ g−1) N (%) Mean (SD) Aerobic mesophilic count ≤100,000 UFC/ml 4 (18) 4.65 a (0.472) (≤5) a Desirable 101,000-2,430,000 (5.1 – 6.4)

3 (14) Not desirable

6.43 b (0.115)

>3,120,000 ( ≥6.5) <100 UFC (≤ 2.0) a

15 (68) Not desirable ND Desirable

7.26 b (0.420)

Listeria monocytogenes 25 g

Absence

*Molds and yeasts

500 UFC/g (2.7) a

22 (100) Not desirable 22(100) Desirable 22 (100) Desirable 1 (4) Desirable

5.20 b (0.182)

Salmonella spp 25 g

990 – ≥184,000 (3 – ≥5) Absence

>500 UFC/g ( >2.71)

21 (96) Not desirable

4.57 b (0.230)

Coliforms

2.70 a

SD= standard deviation. NOM-243- SSA1-2010 total coliforms <100 UFC (≤ 2.0 Log10 CFU ml −1) ab Mean values of each variable with different letters differ significantly (P<0.05) from the parameters. *

Conclusions and implications The results show that certain parameters of physicochemical quality, such as protein, fat and lactose, outweigh the desirable parameters established by the norms. Another important aspect was that none of the PUs identified Salmonella spp. or Listeria spp.—a desirable result, since these bacteria represent a risk to consumers’ health. Certain indicators of the sanitary quality of milk and cheese are above the parameters established by the regulations. Therefore, it is imperative that these PUs continue working on the implementation of a preventive medicine program that addresses the cases of infectious mastitis, and, above all, that they continue to train producers to improve the microbiological quality of milk and cheese and thus reduce those

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indicators that are outside the norm. The artisanal production of the Ojos Negros cheese has a remarkable cultural, gastronomic and economic relevance in the region; however, its processes will have to be improved in order to comply with the requirements established by the current legislation and thereby prevent public health issues that may affect the marketing of their products.

Acknowledgments This work is part of the requirements that must be met by the first author in order to obtain a doctorate in Agricultural Sciences at the Autonomous University of Baja California (Universidad Autónoma de Baja California). The authors wish to express their gratitude for the technical assistance in the samplings to Gabriela Venegas, Cristina Flores, Dalia Gómez, Carolina Trillo, Ramón Valenzuela, Martha Solorio, and Fernando Inzunza. This work was supported in part by the Secretary of Agricultural Development of the State of Baja California and by SAGARPA, through the Outreach project. We also thank the cheesemakers of the municipality of Ojos Negros in Ensenada, Baja California; for the support given to the Autonomous University of Baja California through the Single Union of University Workers, and the Milk Quality Laboratory of the Institute for Research in Veterinary Science, for providing facilities and equipment. The authors declare that they have no conflict of interest.

Literature cited: 1. González CAF, Yesecas C, Ortiz EAM, De la Rosa AM, Hernández MA, Vallejo CB. Invited review: Artisanal Mexican cheeses. J Dairy Sci 2016;99:3250-3262. 2. Alejo MK, Ortiz HM, Recino MBR, González CN, Jiménez VR. Tiempo de maduración y perfil microbiológico del queso de poro artesanal. Revista Iberoamericana de Ciencias 2015;2:15-24. 3. Yohan Y, Somin L, Kyoung HCh. Microbial benefits and risks of raw milk cheese. Food Control 2016;63:201-215. 4. Silva PL. Proyecto Extensionismo para Gestión de las BPM en la Producción Inocua de Quesos en Ojos Negros Real Castillo. Informe del Servicio 2013-FOFAEBC-UABC, Mexicali BC. 2014. 5. Secretaria de Salud, NOM-243-SSA-2010. Leche, formula láctea, producto lácteo combinado y derivados lácteos. Disposiciones y especificaciones sanitarias. Métodos de prueba. Comisión 561

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Federal para la Protección contra Riesgos http://www.cofepris.gob.mx/MJ/Paginas/Normas-Oficiales-Mexicanas. aspx. Sep 8, 2017. 6.

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Costa DMA, Sant´Ana AS, Cruz AG, Faria JF, Fernandes OC, Bona E. On the implementation of good manufacturing practices in a small processing unity of mozzarella cheese in Brazil. Food Control 2012;24:199-205.

7. Gastélum LL. Estudio para la detección de necesidades de infraestructura e equipamiento en las unidades de producción de leche en la Región de Ojos Negros. Gobierno del Estado de BC. Informe del Servicio 2010. 8. Consejo para el Fomento de la Calidad de la leche y sus derivados, A.C. (COFOCALEC), NMX-F-718-COFOCALEC-2006. Sistema Producto Leche - Alimentos - Lácteos - Guía para el muestreo de leche y productos lácteos. http://www.cofocalec.org.mx/catalogo/por_clave=2014. Consultado Sept 9, 2017. 9. PROY-NOM-109-SSA1-1994 Procedimientos para la toma, manejo y transporte de muestras de alimentos para su análisis microbiológico. http://legismex.mty.itesm.mx/normas/ssa1/ssa1109p.pdf=. Consultado Sep 8, 2017. 10.

Secretaria de Economía (MX), NOM-155-SCFI-2012. Leche-Denominaciones, especificaciones fisicoquímicas, información comercial y métodos de prueba. Available:https://www.sinec.gob.mx/SINEC/Vista/Normalizacion/BusquedaNormas.xhtml=. Consultado Sep 8, 2017.

11. Consejo para el Fomento de la Calidad de la leche y sus derivados, A.C. (COFOCALEC), NMX-F-700-COFOCALEC-2012. Sistema Producto Leche – Alimento – Lácteo – Leche cruda de vaca – Especificaciones fisicoquímicas, sanitarias y métodos de prueba. http:// www.cofocalec.org.mx/ catalogo/por clave=2014. Consultado Sep 9, 2017. 12. Bernal MLR, Rojas GMA, Vázquez FC, Espinoza OA, Estrada FJ, Castelán OO. Determinación de la calidad fisicoquímica de la leche cruda producida en sistemas campesinos en dos regiones del Estado de México. Vet Méx 2007;38:395–407. 13. Oliszewski R, Cisint JC, Medina CF. Caracterización composicional fisico-química y microbiológica de leche de vaca de la Cuenca de Trancas. RAPA 2016;36:31-39. 14. De la Cruz EG, Diaz PS, Bonifaz N. Gestión de la calidad de leche de pequeños y medianos ganaderos de Centros de acopio y queserías artesanales, para la mejora continua. Caso de estudio: Carchi, Ecuador. La Granja: Rev Cienc Vida 2018;27:124-136.

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15. Hess HD, Florez H, Lascano CE, Baquero LA, Becerra A, Ramos J. Fuentes de variación en la composición de la leche y niveles de urea en sangre y leche de vacas en sistemas de doble propósito en el trópico bajo de Colombia. Pasturas Tropicales 1999;21:33–42. 16. Magariños H. Producción higiénica de la leche cruda. Guatemala: Producción y Servicios Incorporados S.A; 2001. 17. Oliszewsky R, Cisint JC, Nuñez KM. Manufacturing characteristics and shelf life of Quesillo, and Argentinean traditional cheese. Food Control 2007;18:736-741. 18. Fuentes CG, Ruiz RRA, Sánchez GJI, Ávila RDN, Escutia SJ. Análisis microbiológico de la leche de origen orgánico. Atributos deseables para su transformación. Agricultura, Sociedad y Desarrollo 2013;10:419-432. 19. Castro CG, Martínez CFE, Martínez CAR, Espinoza OA. Caracterización de la microbiota nativa del queso Oaxaca tradicional en tres fases de elaboración. Rev Soc Venezolana Microbiol 2013;33:105-109. 20. Rojas AM, Montaño LP, Bastidas MJ. Producción de ácido láctico a partir del lactosuero utilizando Lactobacillus delbrueckii subsp. bulgaricus y Streptococcus thermophilus. Rev Colomb Quim 2015;44:5-10. 21. Perkins NR, Kelton DF, Hand KL, MacNaughton G, Berke O, Leslie KE. An analysis of the relationship between bulk tank milk quality and wash water quality of dairy farms in Ontario, Canada. J Dairy Sci 2009;92:3714-3722. 22. Brousett, MM, Torres JA, Chambi RA, Mamani VB, Gutiérrez SH. Calidad fisicoquímica, microbiológica y toxicológica de leche cruda en las cuencas ganaderas de la región PunoPeru. Scientia Agropecuaria 2015;6:165-176. 23. Yucel N, Huriye U. A turkey survey if hygiene indicator bacteria and Yersinia enterocolitica in raw milk and cheese samples. Food Control 2006;17:383-388. 24. Kousta M, Mataragas M, Skandamis P, Drosinos EH. Prevalence and sources of cheese contamination with pathogens at farm and processing levels. Food Control 2010;21:805-815. 25. Millogo V, Svennersten SK, Ouedraogo GA, Agenas S. Raw milk hygiene farms, processing units and local markets in Burkina Faso. Food Control 2010;21:1070-1074. 26. Cuevas GPF, Heredia CPY, Méndez RJI, Hernández MA, Reyes DR, Vallejo CB, González CAF. Artisanal Sonoran cheese (Cocido cheese): an exploration of its production process, chemical composition, and microbiological quality. J Sci Food Agric 2017;97:4459-4466.

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27. Di Cagno R, Banks J, Sheehan L, Fox PF, Brechany EY, Cosetti A, Gobbetti M. Comparison of the microbiological, compositional, biochemical, volatile profile and sensory characteristics of three Italian PDO ewes´ milk cheeses. Int Dairy J 2003;13:961-972. 28. Martínez A, Villoch A, Ribot A, Ponce P. Evaluación de la calidad e inocuidad de quesos frescos artesanales de tres regiones de una provincia de Cuba. Rev Salud Anim 2013;35:210213. 29. Chombo MP, Kirchmayr M, Gschaaedler, Lugo CE, Villanueva RS. Effects of controlling ripening conditions of the dynamics of the native microbial population of Mexican artisanal Cotija cheese assessed by PCR-DGGE. Food Sci Technol 2016;65:1153-1161. 30. Sánchez VJJ, Colín NV, López GF, Avilés NF, Castelán OOA, Estrada FJG. Diagnóstico de la calidad sanitaria en las queserías artesanales del municipio de Zacazonapan, Estado de México. Salud Pública de México 2016;58:461-467. 31. Torres LlMJ, Vallejo CB, Diaz CME, Mazorra MMA, Gonzalez CAF. Characterization of the natural microflora of artisanal Mexican Fresco cheese. Food Control 2006;17:683-690.

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https://doi.org/10.22319/rmcp.v11i2.5173 Technical note

Factors associated with the seizure of livers positive to Fasciola sp. in an endemic area of southeastern Mexico

Nadia Florencia Ojeda-Robertos a Roberto González-Garduño b Santiago Cornelio-Cruz a Jorge Alonso Peralta-Torres a Carlos Luna-Palomera a Carlos Machain-Williams c Heliot Zarza d Oswaldo Margarito Torres-Chablé a Enrique Reyes-Novelo c Carlos Baak-Baak c Alfonso Chay-Canul a*

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

Universidad Autónoma Chapingo, URUSSE. Teapa, Tabasco, México.

Universidad Autónoma de Yucatán. Centro Regional de Investigaciones “Dr Hideyo Noguchi” Mérida, Yucatán. c

Universidad Autónoma Metropolitana. Departamento de Ciencias Ambientales, CBS, Unidad Lerma, Estado de México, México.

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*Corresponding author: aljuch@hotmail.com

Abstract: This study aimed to determine the frequency for the seizure of livers with damages attributed to the presence of Fasciola sp. and associated risk factors. It was conducted a prospective observational study with daily visits, for 12 months, to a municipal slaughterhouse in the Sierra area in the state of Tabasco. Of the seized livers, 25.8 % tested positive for the parasite presence; seizure was the same in male and female animals (X2= 0.011, gl= 1, P<0.05). The highest proportion of seized livers was observed during the rainy season (9.36 %). This study concludes that the prevalence of fascioliasis in the Jalapa area has not decreased in recent years and is related to animal origin. Fascioliasis is a disease that must be monitored to detect the factors that allow it to remain in a geographic region, in order to establish and propose strategic control and preventive measures adapted to the particular conditions of endemic areas. Keywords: Seizure, Fasciola sp., Liver, Tabasco

Received: 03/12/2018 Accepted: 02/04/2019

Fascioliasis is a zoonotic parasitic disease caused by the presence of Fasciola sp. trematodes in the hepatic ducts of ruminants(1,2). It is an economically important disease, since it affects productive species such as cattle, sheep, horses, and hogs, in addition to wild animals(3). The economic costs associated with its presence are estimated to amount to 3 million dollars worldwide(4). In Mexico, losses ascend to 130 thousand dollars(5), in addition to being a parasitic disease included in the priority research list of neglected tropical diseases(1). In recent years, fascioliasis prevalence in domestic animals and humans has increased due to climate change, animal movement from one area to another, and the traffic of travelers and migrants(6,7). In Tabasco, one of the main agricultural activities is cattle farming, based on intensive or extensive grazing systems, which favor the presence and transmission of the disease in the ruminants of the region. The disease occurs when animals ingest infective metacercariae, which are cysts attached to the grass, hence the importance of this disease in the production system.

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Fascioliasis has been reported as common parasitism in animal populations, with state prevalence of 19.7 %(8), with variations according to the area and the characteristics of each region. Rangel and MartĂnez(8), identified areas of high, medium, and low prevalence, which classifies the state as an endemic area of the disease, along with Chiapas and Veracruz. In the absence of a sensitive and specific diagnostic technique for the detection of positive animals, one way to determine the true prevalence is by monitoring slaughterhouses; this can provide relevant epidemiological information at a relatively low cost(9). The analysis of this information can be used to determine the behavior and significance of the disease in a specific region(9). Sanitary inspection is a routine procedure during the slaughter of beef cattle for human consumption and is crucial for epidemiological studies that allow seeing the disease scenario. The presence of livers positive to Fasciola is a cause for immediate seizure; however, more remains to be known about the factors related to the presence of the disease. This study aimed to determine the prevalence in slaughterhouses and the factors associated with the seizure of livers in an endemic area of southern Mexico. The study was carried out in the State of Tabasco, on the municipal slaughterhouse of Jalapa, which is located in the Rio Grijalva region, Sierra subregion of the state. The area is characterized by having a tropical rainforest climate (Af) with year-round rainfall, according to the Koppen climate classification modified by GarcĂa(10). Mean temperature of 24.9 Â°C and annual mean precipitation of 3,711 mm are recorded(11). The bodies of water of the Jalapa municipality are made up of two large rivers, the Rio de la Sierra and Puente Grande, as well as streams and lagoons. This complex orographic system overflows during the rainy season, causing flooding in the area. It was conducted a prospective observational study for 12 mo (January to December of 2014), with daily visits to a municipal slaughterhouse. Inspections were carried out following the slaughtering procedures of the slaughterhouse, which adhere to the slaughter guidelines in NOM 033-ZOO-1995. After the slaughter and once the visceral package was removed from the carcass, organs were inspected following the zoosanitary standard NOM-194-SSA-2004, which specifies that the revision of the viscera intends to look for the presence of parasites, and these must be absent. Livers with injuries suggestive of the presence of the parasite were separated for further inspection. Seized livers were inspected for injuries and for adult parasites or migratory forms, for which incisions were made in the parenchymal tissue and classified as positive and negative in the presence of Fasciola. Positive livers met any of the following criteria: macroscopic injuries like abscesses and thickening of the walls of bile ducts, living or dead adult or developing

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parasites in hepatic ducts or the parenchyma. A liver was considered negative when no injuries or parasites were found. From each animal, regardless if they tested positive or not, it was recorded the slaughter date (day and month), sex (male or female), locality of origin of the introducer (locality), and liver seizure (positive or negative). The number and frequency of seized livers during the study period were calculated by month and by the season of the year. The frequency of seized livers was calculated using the equation to determine prevalence described by Thrusfield(12) and was expressed as the percentage of the total number of cattle slaughtered compared to the number of animals that entered the slaughterhouse. Data were grouped by sex, month of seizure, season of the year (rainy, dry, and norther), and by population origin to determine its association with the seizure of livers positive to Fasciola sp. The month of seizure data were grouped into three seasons: dry (March-June), rainy (July-October) and norther (November-February). The data grouped by sex were analyzed using a Chi-squared test in a 2 x 2 contingency table. It was used a Kruskal-Wallis test to analyze the data and detect differences between months. Association analyses were performed, through a correspondence test, for the season of the year and locality. 3 x 2 and 27 x 2 contingency tables were used, respectively. The analyses were performed using the SPSS statistical program, version 8. The number of animals that entered the municipal slaughterhouse during the year varied every month, between 42 to 108, for January and May, respectively. Still, and within this context, the number of seized positive livers was 278, which represent 25.8 % of the total seized (Table 1), which is within the range of prevalence reported for the State of Tabasco(8,13,14), a percentage that remains without apparent change from the first studies carried out on Tabasco slaughterhouses(8). Table 1: Number of slaughtered cattle and seized livers during a year in a municipal slaughterhouse of the southeastern area, Jalapa, State of Tabasco, Mexico

Cattle Females Males Total

n 1025 53 1078

Seized livers Positive Negative 264 761 14 39 278 800

Positive % 25.7 26.4 25.8

P value 0.005

In cattle, the risk factors associated with disease prevalence are those related with the environment, like temperature and humidity, and water resources such as rivers; the presence of the intermediate host and the factors associated to the definitive host such as age, breed, animal species, and the exploitation system and feeding management. Likewise, the farm location and 568

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the prevailing microclimatic conditions in the grazing area are of great importance in the epidemiology of the disease(15). In this study, regardless of the number of animals that entered the slaughterhouse, the probability of males being positive was the same for females, as there was no relation between the seizure of positive livers and sex (X2=0.011, gl=1, P<0.05). These results are similar to those of Ticona(16), who mentions that sex is not a risk factor for the disease in Peru, age was the risk factor more associated with the presence of the disease(17,18). A factor that can be confused with sex and age is the breeding production system. Usually, females are bred fundamentally under grazing conditions(19) and remain for a more extended period in the farms, while males are usually sold to be fattened under intensive systems. However, males are also susceptible to parasitism when managed under a grazing system. In the present study, the monthly variation in the seizure of positives ranged between 3.6 and 12.3 %. We could detect Fasciola sp. throughout the year with monthly variations (X2=51.918, gl=11, P=0.000). However, February and November were the months with the highest and lowest percentage of positive livers, 17.3 % and 3.6 %, respectively (Figure 1). The rainy season showed the highest percentage of positive livers with 9.36 % (June-October), followed by the dry season with 8.34 % (March-May), and the norther season with 8.07 % (November-February). These results show a relationship between the sampling period and seizure (X2=6.511, gl 2, P=0.039). The latter is similar to the reported by Feunmayor and Ojeda-Robertos(14,19), who determined a relationship between the time of the year and the presence of animals positive to Fasciola, both authors used the sedimentation technique to determine the prevalence in animals under grazing production systems.

Seizure (%)

Figure 1: Percentage of livers positive to Fasciola sp. by month and time of the year 20 18 16 14 12 10 8 6 4 2 0

n=1078

Jan

Feb

Windy

Mar

Apr

May

Jun

Dry

Jul

Aug Rainy

Month and Season

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Oct

Nov

Dec

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In this study, the detection of positive livers and postmortem hepatic damage show that the animals, at some point in their lives, were in contact with the infective stage of Fasciola sp. These animals acquired the disease when the external environmental conditions in the farm were optimal for the development of the exogenous infective stage of the parasite, which is confirmed by the presence of different developmental stages in the hepatic ducts. The number of parasitic developmental stages found in positive livers was not determined. The season of the year is directly related to the development and survival of the intermediate host and the parasitic infective stages(20). Fasciola hepatica possesses a complex life cycle that includes the presence of obligate hosts, the definitive (ruminants and humans), and the intermediate (Fossaria and Pseudosuccinea mollusks of the Lymnaeidae family). In both hosts, the elimination of the parasitic developmental stages allows the parasite to fulfill its life cycle. Eggs enter the environment in the feces of the definitive host, while the snail releases the cercariae, which form cysts in the aquatic vegetation, grass, or at the side of water bodies, transforming into metacercariae (infective stage)(21,22). The definitive host becomes infected by ingesting metacercariae-contaminated grass or water. The metacercariae mature until its juvenile stage, in which they can migrate through the intestinal wall, subsequently staying in the abdominal cavity, peritoneum, Glisson's capsule, and hepatic parenchyma; they later establish in the bile ducts, where metacercariae reach their adult stage and sexual maturity, which enables them to produce fertile eggs, which are eliminated in the feces to the environment(15). During the postmortem examination, the liver shows hypertrophy, bleedings, with different degrees of fibrosis, calcification and hyperplasia of bile ducts, as well as the presence of parasitic forms in the hepatic tissue, which are reason for seizure, regardless of the degree of liver damage(23). A study in northeastern Nigeria, in the African continent, determined the degree of damage of livers seized due to the presence of Fasciola, the results show that severe liver damage was the most frequent (55.3 %), followed by moderate damage, these damages include fibrosis to total liver damage and atrophy(24). In this study, is describe the degree of damage in positive livers, which could be a future research subject. Herein, the time of year with the largest number of seized livers was possibly related to the largest number of animals that came from a locality where the prevalence and probability of sending a positive animal to the slaughterhouse are higher since the local conditions favor the presence of infective stages (floodable soils, proximity to the river and water blankets).

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The origin of the total seizures (n= 1,078), regardless of the cause, were distributed in 27 towns belonging to the municipality of Jalapa. Of the total number of registered localities, seven contributed more than half of the total number of positives (89.2 %, 248/278), ten localities contributed the remaining 10.8 % (30/278), and in the remaining ten, no seizures were detected. The contribution of the seven localities varied from 16 to 45 positive livers, the second group of localities contributed 1 to 7 positive livers, and the remaining ten, zero. From each town, the number of animals received for slaughter varied and depended on the needs of the producers to send animals for slaughter. The population-adjusted prevalence was 16.19 % (Huapacal) for the highest and 5.40 % for the lowest (Figure 2); a relationship between the localities of origin and the presence of positive livers was determined (X2=59.621, gl 26 p=0.000). The origin of the animals is an important factor related to seizure, since the regional environmental conditions of the locality, as well as the breeding production system, are reflected in the number of positive animals, and it is highly likely that the animals from these regions have had greater contact with the infective stages of the parasite due to the particular conditions of the breeding region(24).

Figure 2: Frequency of seized positive livers by locality in the Jalapa municipal slaughterhouse, Tabasco, Mexico

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It is concluded that the prevalence of Fascioliasis in the Jalapa area, Tabasco area, has not decreased in recent years, despite traditional control measures, including the use of fasciolicides for the control of immature and adult stages, as well as the restriction to graze areas near rivers, to avoid ingestion of metacercariae. The prevalence of the disease is related to the location or origin of the animal. However, other factors influence the presence of the disease and must be studied, including the type and system of livestock production, the use of fasciolicides, and the microclimatic conditions of each breeding location. Fascioliasis, a disease that affects animals and humans, should be monitored in order to increase knowledge about its epidemiology and propose control and preventive measures, depending on the microclimatic conditions. Amid climate change, state and nationwide monitoring are recommended; this could provide information to better understand the ecology of fascioliasis and its presence in ruminant animals.

Acknowledgments To the PFI 2013-UJAT fund for financing the key project UJAT-2013-IA-10. In memory of the MVZ Intern Santiago Cornelio Cruz, who was a key and determining component in the great work of daily data collection on the slaughterhouse. To the MVZ Juan Felipe JimĂŠnez for his great support, willingness, and patience to help us on the slaughterhouse. To the Animal Parasitology Laboratory of the Agricultural Sciences Research Center - UJAT for the facilities granted during the liver inspections.

Literature cited: 1. World Health Organization. Research priorities for zoonosis and marginalized infections. Technical Reports Series 2013. 2. Arafa WM, Hassan AI, Snousi SAM, El-Dakhly, Holman PJ, Craig TM, Aboelhadid SM. Fasciola hepatica infections in cattle and the freshwater snail Galba truncatula from Dakhla Oasis, Egypt. J Helminthol 2017; doi: https://doi.org/10.1017/S0022149X17000086..

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3. Pérez Creo A, Béjar JP, Díaz CM, López A, Prieto M, Viña V, Martinez, Diez-Baños P, Ubeira FM, Morrondo P. Fasciola hepatica in sheep from north-western Spain. Risk factor analysis using a capture ELISA (MM3 SERO). Small Ruminant Res 2016;45:103-106. 4. Food and Agricultural Organization of the United Nations. Diseases of domestic animals caused by liverflukes: epidemiology, diagnosis and control of Fasciola, Paramphistome, Dicroceoelium, Eurytrema and Schistosome infections of ruminants in developing countries. Rome: FAO. 1994. 5. Rodríguez-Vivas RI, Grisi L, Pérez de León AA, Silva Villela H, Torres-Acosta JFJ, Fragoso Sánchez H, et al. Evaluación del impacto económico potencial de los parásitos del ganado bovino en México. Rev Mex Cienc Pecu 2017;8:61-76. 6. Mas-Coma S, Esteban JG, Bargues MD. Epidemiology of human fascioliasis: a review and proposed new classification. Bulletin WHO 1999;77:340-346. 7. Ashrafi K, Bargues MD, O´Nell S, Mas-Coma S. Fascioliasis: A worldwide parasitic disease of importance in travel medicine. Travel Med Infect Dis 2014;12:636-649. 8.-Rangel Ruiz LJ, Martínez DE. Pérdidas económicas por decomiso de hígados y distribución geográfica de Fasciolosis bovina en el Estado de Tabasco. Vet Méx 1994;25:327-331. 9. Roberts JA. Suhardono S. Approaches to the control of fasciolosis in ruminants. International J Parasitol 1996;26:971-981. 10. García E. Modificaciones al sistema de clasificación climática de Koppen. 5ta ed. Instituto Nacional de Geografía. UNAM. 2004. 11. De Dios OO. El medio ambiente tropical. En: Ecofisiología de los bovinos en sistemas de producción del trópico húmedo. Colección José N. Rovirosa. Edit. Universidad Juárez Autónoma de Tabasco, México. 2001. 12. Thrusfield M. Epidemiología veterinaria. México: Acribia Ed. Zaragoza. 1990. 13. Rangel-Ruiz LJ, Izquierdo MR, Nogeira BG. Bovine fasciolosis in Tabasco, Mexico. Vet Parasitol 1999;81:119-127.

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14. Ojeda-Robertos NF, Medina-Reynes U, Garduza-Arias G, Rangel-Ruiz LJ. Dinámica de excreción de huevos de Fasciola hepática y Paramphistomum spp en ganado bovino de Tabasco. Ecosist Recur Agropec 2014;1:73-80. 15. Acha P, Szyres B. Zoonosis y enfermedades transmisibles comunes al hombre y a los animales. 3a ed. Washington: OPS.; 2003. 16. Ticona S, Daniel, Chávez V, Amanda Chavera C, Alfonso Casas V, Gina Li E. Prevalencia de Fasciola hepática en bovinos y ovinos de Vilcashuamán, Ayacucho. Rev Invest Vet Perú 2010;168-174. 17. Recalde-Reyes DP, Padilla Sanabria L, Giraldo Giraldo MI, Toro Segovia LJ, González MM, Castaño OJC. Prevalencia de Fasciola hepática en humanos y bovinos en el departamento del Quindío-Colombia 2012-2013. Infectio 2014;18:153-157. 18. Sandoval SE, Medina R, Alfonso PS. Prevalencia de la distomatosis hepática en 4 unidades Agroecológicas del Bajo Tocuyo, Estado Falcón. Vet Trop 1989;14:43-51. 19. Fuenmayor A, Simoes D, González R, Chirinos A. La Distomatosis hepática y su asociación con los factores de riesgo en los Municipios Mara y Páez del estado Zulia, Venezuela. Rev Cient FCV-LUZ, 2000;103:183-190. 20. López-Villacís IC, Artieda-Rojas JR, Mera-Andrade RI, Muñoz-Espinoza MS, Rivera-Guerra VE, Cuadrado-Guevara AC, et al. Fasciola hepática: aspectos relevantes en la salud animal. J Selva Andina Animal Sci 2017;4(2):137-146. 21. Rojo-Vázquez FA, Ferre-Pérez I. Parasitosis hepáticas: Fasciolosis. En: Parasitología Veterinaria. Cordero del Campillo M, Rojo VFA editores. Madrid, España: Mc Graw Hill. Interamericana 1999:260-272. 22. Carrada BT, Escamilla JR. Fasciolosis: revisión clínico-epidemiológica actualizada. Rev Mex Patol Clin 2005;52(2):83-96. 23. Mwabonimana MF, Kassuku AA, Ngowi HA, Mellau SB, Nonga HE, Karimuribo ED. Prevalence and economic significance of bovine fasciolosis in slaughtered cattle at Arusha abattoir, Tanzania. Tanzania Vet J 2009;26(2):68–74.

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24. Karshima NS, Bata SL. Bobbo AA. Prevalence, risk factors and losses associated with Fasciolosis in slaughtered cattle in Bauchi, North-Eastern, Nigeria. Alexandria J Vet Sci 2016; 50:87-93.

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https://doi.org/10.22319/rmcp.v11i2.4813 Technical note

Genetic analysis of live weight and pregnancy rate at first calving in Brahman cattle from Venezuela

Alejandro-Palacios-Espinosa a Omar-Verde b Narciso-Ysac-Ávila-Serrano c* Alberto-Menéndez-Buxadera d

Universidad Autónoma de Baja California Sur, Departamento de Ciencia Animal y Conservación del Hábitat, La Paz, B.C.S., México. b

Unidad Territorial Yaracuy. Ministerio del Poder Popular para Ciencia, Tecnología e Innovación. Venezuela. Universidad del Mar, Cuerpo Académico “Ciencias Agropecuarias”, Puerto Escondido, Oaxaca. México. c

Departamento de Genética, Universidad de Córdoba, España.

*Corresponding author: reval1997@hotmail.com

Abstract: The live weight (LW) of 2,777 animals (1,377 females and 1,400 males with 53,258 individual data between 30 and 600 days of age), born between February 2000 and June 2011, was analyzed using a random regression (RR) model to estimate the genetic components of (co)variance throughout the age-sex scale. The pregnancy rate (PR) and the LW adjusted to 548 days of age (WA548) were studied using a multitrait (MT) model, an increase in the heritability (h2) estimates for PR compared to the classical univariate model (0.08 ± 0.03 vs. 0.11 ± 0.02) was observed, increasing the accuracy of the genetic value (GV) for PR in 15.7 %. The genetic correlation (rg) between the PR and the WA548 was 0.31 ± 0.11. The RR showed that, through time, the LW could not be considered as an expression of the same 576

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trait in both sexes, as the rg were less than 0.60. The principal component analysis showed that there are important changes in the animal growth on the age scale represented in these data. A prominent dimorphism of genetic origin manifested, estimated as the difference between the male and female GVs in LW, which shows a positive relationship with the GVs of PR. Key words: Heritability, Genetic correlations, Multitrait model, Random regression model, Sexual dimorphism.

Received: 16/03/2018 Accepted: 21/03/2019

A general breeding program for the reproductive traits of the Brahman breed is carried out in the experimental station â&#x20AC;&#x153;La Cumacaâ&#x20AC;?, Facultad de Ciencias Veterinarias of the Universidad Central de Venezuela, this represents a valuable source of genes for this population(1). The methodology used for genetic evaluations has different crucial elements. First, the WA548, used to estimate the genetic values (GVs), may be biased, since it assumes growth is linear. The results published (2,3,4,5) for B. indicus show growth variations throughout the behavioral tests. Furthermore, the sex of the animal is generally considered a fixed effect in the model, which implicitly assumes that the (co)variance components are the same in both sexes. This approach can incorporate another biased source for the GVs estimation, decreasing their accuracy and thus, affecting the breeding program development(6,7,8). The exposed elements can affect the genetic correlations of the same trait between both sexes (rFM), which could manifest genotype-sex interaction effects. The LW has been generally expressed at a fixed point, although it seems quite reasonable to examine these relationships throughout the age. If the information is available, the (co)variance components can be estimated using multitrait (MT) models, or, preferably, random regression (RR) models. Previous studies have compared the MT and RR models for the LW in Bos indicus cattle(2,4); their results show the advantages of RR. However, this longitudinal approach has not examined the relationships between the sex of the animals. Therefore, more evidence is required, particularly when considering possible relationships between LW and female reproductive behavior (RB). The importance of RB in the beef cattle economy is well known. However, the seemingly low h2 of most of the reproductive traits(9,10,11) has been a limiting factor for its use as a direct selection criterion. As an alternative, previous studies have reported the scrotal circumference (SC) or the measured LW in young males, and its response correlated with the RB of females

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measured by services per pregnancy, days to calving, and the PR at the first service. These encouraging results(12,13) correspond to a fixed age, but the evolution of these trends throughout the age and until first calving remains unknown. This study aimed to estimate the heritabilities and genetic correlations between the WA548 and the PR of heifers in their first breeding season using a MT model; as well as the genetic (co)variance components of the LW of both sexes regarding age using a RR model; and to compare the genetic values for each ith age (GVi), based on the RR, with the MT-based GVs. The experimental station “La Cumaca”, located at 472 m asl., near San Felipe City, Yaracuy State, Venezuela, has an extension of 433 ha, with 300 ha cultivated with Guinea, Star, Swazzi, Pará, and Wire grasses. The annual mean precipitation is 1,650 mm, with a mean temperature of 24 to 31.9 °C, and a mean relative humidity of 84 %(14). It has a herd of pure, registered Brahman cattle, with approximately 180 cows in production. The LW adjusted to 548 d of age (WA548) of 3,120 animals, born between February 2000 and June 2011, was modified, eliminating records with pedigree inconsistencies, absence, or problems in the date of birth. Finally, there were 2,777 animal records available (1,377 females and 1,400 males). These animals were born from 984 mothers (729 in the data vector) and 107 sires (48 in the data vector). The pedigree file included 3,977 animals. A total of 94,752 individual LW records from 1,776 females and 1,864 males, born between February 1978 and June 2011, were used. These animals were born from 1,291 mothers (929 in the data vector) and 128 sires (58 in the data vector), and the pedigree file included 4,070 animals. These data were edited, eliminating those records with pedigree inconsistencies, absence or problems in the date of birth, and data recorded less than 30 or more than 570 d of age. Data outside the range of ± 3.2 standard deviations within a 30-d range age classes were removed. Finally, a total of 53,258 individual data were available from 1,737 females and 1,803 males. Several models were analyzed using the SAS GLM procedure(15). Table 1 shows some indicators of the studied data.

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Table 1: Live weight indicators of Brahman animals in the experimental station “La Cumaca”, Venezuela Female sample size

Females

Male sample size

Males

Birth weight Weaning weight Weight at 365 d Weight at 450 d Weight at 548 d

1,776 1,639 1,396 1,378 1,340

29.5 ± 4.5 165.8 ± 26.2 209.9 ± 29.8 235.6 ± 32.0 290.0 ± 34.1

1864 1630 1410 1392 1385

31.9 ± 4.9 177.3 ± 28.3 231.8 ± 34.9 271.2 ± 38.9 326.3 ± 42.2

Number of live weight records

25,781

25781

27477

Pregnancy rate

1,377

0.67 ± 0.37

Trait

There were completed three block analyses using the ASReml3 program(16): Block 0. Multivariate (MU) model for WA548 and PR.

y 1  X 1 y    0  2 

0  b 1   Z 1  X 2  b 2   0

0  a 1  e 1   Z 2  a 2  e 2 

Where: yi

is a vector that corresponds to the WA548 and the PR analyzed at the same time;

bi is a fixed-effects vector of the jkth combination of sex-year-month (with 275 levels for WA548 and 124 for PR); ai is a random correlated vector due to the genetic additive effect of the ith animal with data and its predecessors without records (4,070 levels) for WA548 and PR; eil

is a random residual vector correlated between trait 1 and 2;

X and Z are incidence matrices that connect the fixed and random effects with the vector of observations.

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This model assumes that: Var

 σ2  a1 σ a In which Gi =  21

σa

a i  G i e    0  i

0 R i 



12 A 2 σ a2   ,

where

2 σa 1

and

2 σa

represent the genetic variances for

 σ e2  1 σa σ both traits, and 12 their covariance. The residual (co)variance Ri =  e 21 2 σe 1

σ e 12   σ e22  includes

and e 2 , which represent the variances for both traits and e12 their covariance. A is the relationship matrix between all the animals and  is the product symbol. With these parameters, it was estimated the h2 for each trait (h2ai ) and the genetic correlations (rgi) between both traits, using linear functions of the corresponding components and classical equations(17). The GVs for each trait were estimated as a solution of the described model, and the accuracy (Accij) of such estimates according to:

1 Accij =

Pev * 100  i2

Where: Pev is the prediction error variance (individual value for each animal and study trait), and σ2i : is the genetic variance of the trait in the studied population. It was applied this same model in its univariate form in order to present similar parameters to the original program in the experimental station.

To analyze the LW at different ages, it was applied RR, using different models and without considering the maternal effects, which variated in the fitting order of the polynomial for random effects, as well as the estimates of the total or intrasexual (co)variance components of the animal. In total, two model blocks were made: Block 1 - Assumes that the (co)variance components are the same in both sexes. 3 1 1 y ijkl  fixed i  sex j   Φr b1r   Φr a kr   Φr p i r  Z1q m  R  Model 1 r0 r0 r0 3 2 1 y ijkl  fixed i  sex j   Φr b1r   Φr a kr   Φr p i r  Z1q m  R  Model 2 r0 r0 r0 3 3 1 y ijkl  fixed i  sex j   Φr b lr   Φr a kr   Φr p i r  Z1q m  R  Model 3 r0 r0 r0

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Block 2 - Assumes that the (co)variance components are not the same in both sexes.  3   1   1  y ijkl  fixedi  sex    Φ b      Φ a     Φ p   Z q  R  model 4 r 1r r kr r ir j 1 m :j r  0 : j r  0 : j r  0 : j  3   2   1  y ijkl  fixedi  sex j    Φr b1r     Φr akr     Φr pir   Z1q m  R: j  model 5 r  0 : j r  0 : j r  0 : j  3   3   1  y ijkl  fixedi  sex j    Φr b1r     Φr akr     Φr pir   Z1q m  R: j  model 6 r  0 : j r  0 : j r  0 : j

In both blocks, Yijkl represents the different LW estimates in the l akth animal, of the jth sex. The fixed effects (fixedi) were year-month of control with 674 levels: sex-age at calving with 18 levels, represented in all the models, so that the results can be compared by applying the LogL information criteria; BIC and AIC. The six models only differ in the fitting order of the Legendre polynomial ( Φi ) for random effects and the residual variance (R), considered homogeneous for block 1 and intra jth sex in block 2. The strategy applied in block 2 consisted in estimating the inter- and intrasexual (co)variance components of the animal. The Z1 incidence matrix contains the elements 1 or 0 to connect each observation with the random effects of maternal permanent environment (qm) with 91 levels. For both blocks, the population growth curve was modeled by a regression coefficient (b1) dividing the age by the live weight, using the Φi coefficients of third-order, the random genetic effects of r =1, 2, 3 orders, and the individual permanent effect (pi) of first-order, due to the repetitions of the same trait in the animals throughout the age scale. The expected variance components in both blocks were: y ~ N [0, (σ 2y  Φi * [G 0  (A  K G )] * Φ'  I pσ i2  I qσ m2 I n σ e2 ]

Block 1

2 y ~ N [0, (σ 2y  Φ i * [G 0  (A  K G: j )] * Φ'  Φ i * [P0  (Ip  K P: j )] * Φ'  I q σ m  I n σ e2: j ]

Block 2

Where A is the numerator of the relationship matrix between the animals with data and their ancestors without records (n= 4,070 total animals). Ip is the identity matrix for the random effects of the individual permanent environment (p=3540 dimension for block 1 with σ2i variance, ph= 1,737, and pm=1,803 levels for females and males, respectively in block 2, with variances included in the random regression matrix of individual permanent effect intra jth sex of the animal (KP:j). Iq is the incidence matrix for the maternal permanent environment (q dimension=1,291 mothers and σ2m variance). R is the residual error with In as incidence matrix (n=53,258 records in block 1 and σ2e variance, and nh= 25,781 and nm=27,477 for females and males in block 2 with σ2e:jvariance, respectively). G0 have a random regression

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matrix (KG) of (r+1)*(r+1) dimension, in the most complex models of block 2 the elements will be:

  σ2  σ hmo σ hmos  σ hos   K   K h   ho hm  2  σ hso σ hs   σ hmso σhdms    KG    2  σ mo σ σ mhso  σ mos   K mh   mho   K  m  σ  2    mhos σ mhs   σ mso σ ms    ASReml automatically produces the principal component analysis of this matrix, which facilitates the interpretation of the estimated (co)variances trajectories. Herein, KG is a symmetrical matrix that consists of four submatrices with the same (co)variance components for the genetic effects in females (Kh); males (Km), and their covariances (Khm), with their corresponding variances of the intercept (σ2ho and σ2mo); slope (σ2hs and σ2ms), and covariances (σ2hso , σ2mso, σ2mhs, and σ2hmos). In these cases, the subscripts o and s indicate intercept and slope, respectively. The described matrix applies to a fitting order of r=1. Therefore, each submatrix has a 2x2 dimension; for r = 2 it will be 3x3, and for r = 3 it will be 4x4, and the additional components will be the quadratic and cubic terms, respectively. For block 1, the KG matrix does not represent the estimates for each sex. For both blocks, manipulating the elements of these matrices, as well as the r-order Legendre polynomial coefficients ( Φi ), it is possible to estimate the (co)variance components throughout the age and for each sex (18): 2  Φ K Φ' σ 2  Φ K Φ' σ  Φi K hmΦ'j σ hi i h i ; mi i m i , and hmij .

Generally, the genetic parameters of h2 and rg can be determined using classical equations(17). The GVs of LW are determined for each sex using the best model solution where, for the kth animal it will have: VG i  ak Φ'i k

and where  i are the corresponding Legendre polynomial coefficients for each ith point on the age scale. In this model, each animal (total; female or male) will be assigned a genetic function (ak) linked to the effects of the intercept, slope, and other terms according to the fitting order of the chosen polynomial. Table 2 shows the genetic parameters obtained from block 0, where the correlations between the estimated GVs determined by MU and MT were incorporated.

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Table 2: (Co)variance components and heritability of live weight adjusted to 548 d and the pregnancy rate at first calving (Block 0 models) Genetic parameter

Live weight (kg)

Pregnancy rate (d)

Genetic variance-MU

369.9

0.017588

Genetic variance-MT

375.3

0.02335

Heritability-MU

0.337±0.11

0.084±0.03

Heritability-MT

0.349±0.10

0.109±0.02

Genetic correlation- Weight and pregnancy rate

0.309±0.11

Accuracy, % of the MU genetic value (GV)

63.5±12.1

34.2±8.9

Accuracy, % of the MT genetic value

64.1±12.1

39.6±9.8

0.996

0.8971

Correlation between MU x MT genetic values Correlation between MU pregnancy rate GV x MU weight GV Correlation between MU pregnancy rate GV x MT weight GV

0.286 0.570

For the WA548 there were no differences between MU and MT. For the PR, the MT increased the h2, improving the accuracy of the GVs. The rg between both traits was positive (rg =0.309), which indicates the absence of antagonism in the improvement of both traits. The correlations between the GVs, based on the models, were higher than 0.897, from which it is inferred that there will be no changes in the order of merit for both procedures. The MT model has additional advantages, manifested in a higher correlation with the GV for PR, as well as greater accuracy in the GV estimates for this last trait, whose h2 value was low. The fitness of the six models in blocks 1 and 2 was determined using the LogL, AIC, and BIC criteria, all three agreed that the third-order polynomial for the genetic effect is the best fit to the data. Block 2 models present better results, which demonstrate that there is a significant variation between sexes for the (co)variance genetic components. The LW h2 throughout the age in both sexes, as well as the genetic correlation between them is showed in Figure 1. The h2 trends show slight increases as age progresses, being higher for females. The rg reflect an inverse pattern with values ranging from 0.25 to 0.35. In contrast, the frequency distribution of the GVs for WA548, estimated according to block 0 models and RR, in Figure 2 shows an overlap of the three GVs estimates. The principal component analysis of the KG matrix for the chosen model 6 demonstrates that the first (vp1) and second (vp2) eigenvectors explained the 57 and 31 % of the genetic variation, respectively. The GVs of the best 200 animals in the MU (official current method) and the

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RR throughout age and for each sex are shown in Figure 3. This figure shows that in males the trend is positive, while in females we can find animals with negative GVs.

Figure 1: Heritability and genetic correlation estimates for live weight in females and males throughout the age scale in Brahman animals (model 6)

Figure 2: Frequency distribution of genetic values for weight at 548 days of age according to the current model and by means of random regression (model 6)

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Figure 3: Evolution of the genetic values of Brahman animals of each sex and throughout the age scale, chosen based on the current evaluation model

The merit evolution based on the year of birth of the animals is shown in Figure 4. The annual genetic progress was of 0.933 ± 0.021 kg/yr for the WA548, the principal trait in the applied breeding program; for the PR it was of 0.354 ± 0.010 %/yr.

Figure 4: Evolution of the genetic merit for live weight and pregnancy rate in Brahman animals in the experimental station “La Cumaca”

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The h2 values for the WA548 are similar to several references published in Venezuela for this type of animal(19,20), as well as with the results published for B. indicus in different Latin American countries(21). Genetic progress for the WA548 was lower than that published by other authors(20,22). The h2 estimates with the MU for PR were low, which is similar to most of the publications about reproductive traits(10,23). However, the h2 levels for PR with the MT increased, (h2=0.109 vs 0.087), which boosts the average accuracy of the GVs in 15.7 %. The rg levels between both traits suggest the absence of antagonism, which means that a selection process for LW and PR is possible, this approach has already been suggested in other studies(11,12,24). The greatest genetic variability (Figure 2) and different h2 and rg levels for the WA548 throughout the age-sex scales (Figure 1) indicate that the expression of this trait should not be considered as an expression of the same trait in both sexes. The latter agrees with other published results(7,8). The sexual dimorphism (SD), evident in these data, has been studied in detail in the evolutionary context of the populations, creating a debate about the importance of the heterogeneous variance between sexes and its effects in the specialization and adaptability of the populations(25), while there are previous statements about changes in SD as a correlated response to fertility selection. In relation to this last point of view, these results present a new approach, this study presents the GVs in each sex for the WA548M and WA548F (model 6 solution results, block 2), which makes possible to estimate a SD of genetic origin like SDg=GVWA548M - GVWPA548F, and these estimates of SDg can be related with the GVs of the same animals for the PR (MT model solution, block 0). The analysis results indicate that a quadratic equation (TG= 0.574 + 0.1045*SDg + 0.000765*SDg2 - 0 .0000244*SDg3, and R2= 96.1 %) was the best fit for the data, with an order increase of +1.2 % in PR for each 10 kg of SDg, with a maximum point when 40>SDg<60 with PR= +4.4 %. However, when -10>SDg<0, the PR was -1.4 %. These results are encouraging, but more research is required on this topic, which may have an important practical application in beef cattle production systems. This study detected a wide genetic variety in the LW and PR of Brahman animals. It is suggested to use MT models, which allow substantial increases in h2 values and the accuracy of the estimated GVs, particularly in the PR. The RR analysis indicated that the h2 and rg levels between the LW of females and males vary throughout the age scale, which means that they should not be considered as expressions of the same trait. Finally, this study identified an important genetic variability in sexual dimorphism, which is related to the PR, although this suggestion requires further investigation with a larger number of animals.

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Literature cited: 1.

Plasse D, Verde O, Arango J, Camaripano L, Fossi H, Romero R, Rodriguez C, Rumbos J. (Co)variance components, genetic parameters and annual trends for calf weights in a Brahman herd kept on floodable savanna. Genet Mol Res 2002;1(4):282-297.

Menéndez BA, Guerra D, Planas T, Ramos F, Fernández L. Parámetros genéticos del peso vivo de machos Cebú en prueba de comportamiento en condiciones de pastoreo de Cuba, mediante modelo animal univariado, multicaracteres y regresiones aleatorias. Rev Cubana Cienc Agric 2006;40(4):397-408.

Martínez C, Elzo M, Manrique C, Jiménez A. Genetic parameters and breeding values for live weight using random regression models in a Bos taurus-Bos indicus multibreed cattle population in Colombia. Rev Colomb Cienc Pecu 2012;25:548-565.

Nobre P, Misztal I, Tsuruta S, Bertrand J, Silva L, Lopes P. Analyses of growth curves of Nellore cattle by multiple-trait and random regression models. J Anim Sci 2003;81:918–926.

Lopes F, Ulhôa C, Paulini F, Corrêa M, Sayuri E, Barbosa R. Analysis of longitudinal data of Nellore cattle from performance test at pasture using random regression model. Springer Plus 2012;1:49.

Garrick D, Pollak E, Quaas R, Van-Vleck L. Variance heterogeneity in direct and maternal weight traits by sex and percent purebred for Simmental-sired calves. J Anim Sci 1989;67:2515-2528.

Rodriguez AF, Van-Vleck L, Cundiff L, Kachman S. Heterogeneity of variance by sire breed, sex, and dam breed in 200- and 365-day weights of beef cattle from a top cross experiment. J Anim Sci 1995;73:2579–2588.

Pegolo N, Albuquerque L, Lobo R, Oliveira H. Effects of sex and age on genotype × environment interaction for beef cattle body weight studied using reaction norm models. J Anim Sci 2011;89:3410–3425.

Donoghue K. Genetic evaluation of female reproductive performance. http://www.bifconference.com/bif2002/Baker_Essay_pdfs/Donoghue_02BIF.pdf(02/0 4/2006).

10. Cammack K, Thomas M, ENNS R. Review: Reproductive traits and their heritabilities in beef cattle. Prof Anim Sci 2009;25:517–528. 11. Berry D, Evans R. Genetics of reproductive performance in seasonal calving beef cows and its association with performance traits. J Anim Sci 2014;92:1412–1422.

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12. Mercadante M, Packer I, Razook A, Cyrillo J, Figueiredo L. Direct and correlated responses to selection for yearling weight on reproductive performance of Nelore cows. J Anim Sci 2003;81:376–3841. 13. Guerra D, González D, Rodríguez M, Ramos F. Relación entre el crecimiento y la reproducción en el ganado Cebú cubano. Ciencia y Tecnología Ganadera 2008;2 (3):147-152. 14. Ewel J, Madriz A. Zonas de vida de Venezuela. Ministerio de Agricultura y Cría. Dirección de Investigación. 1968. 15. SAS. SAS/STAT User´s Guide. Release 9.0. SAS. Inst. Inc., Cary, NC, USA. 2002. 16. Gilmour A, Gogel B, Cullis B, Thompson R. ASReml User Guide Release 3.0. VSN International Ltd, Hemel Hempstead, HP1 1ES, UK. 2009. 17. Falconer D, McKay F. Introduction to quantitative genetics. 4th ed. Burnt Mill, England. 1996. 18. Jamrozik J, Schaeffer L. Estimates of genetic parameters for a test day model with random regression for production of first lactation. J Dairy Sci 1997;80:762–770. 19. Plasse, D, Verde O, Fossi H, Romero R, Hoogesteijn R, Bastidas P, Bastardo J. (Co)variance components, genetic parameters and annual trends for calf weights in a pedigree Brahman herd under selection for three decades. J Anim Breed Genet 2002; 119:141–153. 20. Arias M, Romero R, Camaripano L, Arriaga L. Genetic and non-genetic parameters for growth traits in a registered Brahman herd. Rev Fac Cs Vet UCV 2013;54(2):78-88. 21. Giannotti J, Packer I, Zerlotti M. Meta-Análise para estimativas de herdabilidade para características de crescimento em bovinos de corte. Rev Bras Zootec 2005;34 (4):11731180. 22. Seprocebu, (http://www.seprocebu.com/GENETICO/. Consultado Sep 15, 2017. 23. Donoghue K. Genetic evaluation of female reproductive performance. Beef Improvement Federation (BIF) annual conference. July 10-13, 2002, in Omaha, Neb., USA. http://www.bifconference.com/bif2002/. Consultado 24 Abr, 2011. 24. Meyer K, Hammond K, Mackjnnon M, Pamel P. Estimates of covariances between reproduction and growth in Australian beef cattle. J Anim Sci 1991;69:3533-3543.

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25. Poissant J, Wilson A, Coltman D. Sex-specific genetic variance and the evolution of sexual dimorphism: A systematic review of cross-sex genetic correlations. Evolution. 2010;64(1):97â&#x20AC;&#x201C;107.

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https://doi.org/10.22319/rmcp.v11i2.4899 Technical note

Pedigree analysis of Santa Inês sheep and inbreeding effects on performance traits

Ana Carla Borges Barbosa a Gabrieli de Souza Romano a Jonatan Mikhail Del Solar Velarde a José Bento Sterman Ferraz b Víctor Breno Pedrosa c Luís Fernando Batista Pinto a*

Universidade Federal da Bahia, Escola de Medicina Veterinária e Zootecnia, Av. Adhemar de Barros, 500, Ondina, Salvador – BA, 40170-110 . Brazil. b

Universidade de São Paulo. Faculdade de Zootecnia e Engenharia de Alimentos. Brazil. c

Universidade Estadual de Ponta Grossa. Departamento de Zootecnia. Av. Brazil.

*Corresponding author: luisfbp@gmail.com

Abstract: Population parameters such as effective population size and coefficients of inbreeding are important information of a population but have rarely been studied in Santa Inês sheep. Therefore, this study aimed to estimate population parameters in a Santa Inês sheep flock and the inbreeding effect on performance traits. A dataset with 11,564 animals, born from 2003 to 2011, was recorded for weights at birth (BW1), 60 (BW60), 180 (BW180) and 270 (BW270) days of age, daily weight gain from birth to 60 d (DWG1), 60 to 180 d (DWG2), and 60 to 270 d (DWG3). Percentages of animals with known pedigrees decreased over generations, from 70 % in the first generation to less than 5 % in the third. The effective population size decreased from 665 in 2004 to 45 in 2010. The effective number founders and ancestors were 285 and 273, respectively. Furthermore, the average relatedness coefficient was 0.47 %. The highest frequency of inbred animals was concentrated between 0 and 10 % of the inbreeding coefficient and only 263 animals

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showed F>10 %. The inbreeding coefficient had its lowest value in 2004 (0.19 %) and a highest value in 2008 (2.86 %). Significant inbreeding effect was found for BW1 (0.0054 Âą 0.0015), DWG2 (-0.9837 Âą 0.3025), and DWG3 (-0.5628 Âą 0.2377), while the analysis of breeding values indicated significant inbreeding depression for all traits, except DWG1. Results suggested that inbreeding had a negative effect on growth traits. To avoid losses in these traits it is necessary to mate non-related sires and dams. Key words: Ancestors, Effective population size, Relatedness, Variability.

Received: 16/05/2018 Accepted: 23/03/2019

Every breeding program depends on the genetic variability in the population, but it is often neglected. One of the methods used to evaluate the impact of selection on the genetic variability is studying the population genetic structure, which can be done through pedigree analysis(1). The effective population size (Ne) is a parameter widely used to indicate risk of inbreeding depression or even extinction risk. In the last 50 yr, the effective population size of several sheep breeds has drastically decreased, leading in some cases to inbreeding depression(2,3,4). Moreover, several sheep breeds were extinct in the 20th century due to reduction of Ne. Of the 1,495 sheep breeds recorded up to december 1999, only 656 were not at risk of extinction until that time(5).

Another important genetic population parameter is the number of ancestors explaining the genetic variability of a breed, because many commercial breeds usually have a reduced number of sires in mating. There are previous studies reporting large differences between the total number of ancestors and the number of ancestors that explain 50 % of the genetic variability in different sheep breeds(6-9). Additionally, the ratio between the effective number of founders (fe) and the effective number of ancestors (fa) indicates whether a population is under bottleneck effect. Examples of strong bottleneck effect have been observed in some sheep breeds(1,9).

The coefficient of inbreeding expresses the probability that two alleles at one locus will be identical by descent(10). Inbreeding causes a reduction in individual genetic merit for some productive traits, possibly due to the increase in homozygous genotypes for deleterious recessive alleles or a reduction of heterozygous genotypes(10). However, the depressive effect is relatively minor at low levels of inbreeding. Therefore, monitoring of inbreeding is indicated for maintenance or reduction of inbreeding level of a population(11). An increase in the average inbreeding coefficient along generations has been observed in some sheep breeds(6,8), and the most efficient way to control long-term 591

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inbreeding is to use breeding flocks with low average relatedness (AR). Thus, AR is another important parameter in population genetics.

Estimates of population parameters have been rarely reported for Santa Inês sheep(12,13) and the only one study reported estimates of inbreeding effect on phenotypic traits(12). These authors estimated inbreeding effects only for body weight traits, but did not estimated such effects on breeding values. Thus, the inbreeding effect on many traits and their breeding values remains unknown for Santa Inês sheep. The present study aimed to evaluate the genetic population structure of Santa Inês flocks, through pedigree information, and to estimate the effect of inbreeding on growth traits as well as for estimates of breeding values.

Dataset An initial dataset was preliminary edited based on a file containing 11,781 animals with productive information and 12,322 animals in the pedigree, belonging to 16 different flocks. After consistency analysis, animals with missing productive information or without genetic connection between at least two different flocks were discarded. Records with errors or incomplete information or contemporary groups (CGs) with fewer than five animals with valid measurements were eliminated. As well as CGs in which the animals were the offspring of only one sire and the information was outside the acceptable range, i.e., 3 standard deviation above or below the mean of the trait, were also removed. Additionally, records were checked to ensure that: there were no duplicate records; no progeny was born before neither of their two parents; progeny only appeared as progeny, but not as sire and/or dam in the same record; sires only appeared as sires, but not as dams; dams only appeared as dams, but not as sires.

The final dataset included 11,564 animals in the pedigree, born from 2003 to 2011, which is maintained by the Sergipe State Association of Goat and Sheep Breeders (Associação Sergipana de Criadores de Caprinos e Ovinos - ASCCO). Traits recorded were weights at birth (BW1), 60 (BW60), 180 (BW180), and 270 (BW270) days of age. Daily weight gains were calculated from birth to 60 (DWG1), 60 to 180 (DWG2), and 60 to 270 (DWG3) days of age.

Population parameters The software ENDOG(14) was used to estimate inbreeding coefficient (F)(15), effective population size (Ne)(14) and average relatedness coefficient (AR). POPREP(16) was used to estimate effective number of founders (fe), ancestors (ƒa), effective number of founders genomes (fge) and number of generations traced (g). Additionally, it was estimated the genetic diversity loss average due to bottlenecks and genetic drift. A complete description of these parameters can be found in Gutiérrez and Goyache(14). 592

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Breeding value prediction and Inbreeding effect analysis All traits were tested for data normality applying Shapiro-Wilk test, at 5% significance level, using Statistical Analysis System(17), before estimating genetic parameters. Estimates of variance components and breeding values were obtained by restricted maximum likelihood (REML), with a multitrait animal model, using the software VCE6(18) (for variance components) and PEST(19) (for breeding values). In this analysis, the matrix model can be described as follows:

y = Xb + Za + Mm + e where: y is the vector of phenotypic values; b is the vector of fixed effects of contemporary group, and the covariates damâ&#x20AC;&#x2122;s age and animalâ&#x20AC;&#x2122;s age; X is the incidence matrix that relates the observations in y to fixed effects in b; a is the vector of direct additive random effect; Z is the incidence matrix that relates the observations in y to direct additive random effects in a; m is the vector of maternal additive random effect; M is the matrix that relates the observations in y to maternal additive effect in m; e is the vector of random residual term.

The maternal component Mm was adjusted only for the traits BW1, BW60 and DWG1. The dataset used in this study had a low number of calves per ewe. Thus, the permanent maternal effect and the litter environmental effect were tested but presented problems such as non-convergence or inconsistent estimates of parameters. Therefore, it was chose not to include those effects in the final model. In addition, the dataset showed a low number of inbred Dams and, therefore, it was not include this effect in the model. The assumptions of the models for analyzes could be simply represented as follows: đ?&#x2018;&#x2039;đ?&#x2018;? đ?&#x2018;Ś đ?&#x2018;&#x17D; đ??ş 0 đ?&#x2018;&#x17D; đ??¸ [ ] = [ ] ; đ?&#x2018;&#x2019; đ?&#x2018;&#x2030; [đ?&#x2018;&#x161;] = [đ??şđ?&#x2018;Ľđ?&#x2018;&#x20AC; 0 đ?&#x2018;&#x161; đ?&#x2018;&#x2019; 0 đ?&#x2018;&#x2019; 0

đ??şđ?&#x2018;Ľđ?&#x2018;&#x20AC; đ?&#x2018;&#x20AC; 0

0 0] đ?&#x2018;&#x2026;

The (co)variance matrix for additive genetic effects is G = G A, where A is the relationship matrix and G is the additive genetic (co)variance matrix. The (co)variance matrix for maternal genetic effects is M = M A, where M is the genetic maternal (co)variance matrix. R = I R0 is the residual (co)variance matrix between the seven traits. GxM is the covariance between genetic additive and maternal effects.

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The contemporary group (CG) consisted of animals from the same farm (45 levels), sex (male or female), birth type (single or twins), year (2003 to 2011), and season of birth (dry or rainy). Contemporary groups with less than three animals were removed from the analysis. Table 1 shows the number of CG per trait and descriptive statistics for all traits.

Table 1: Sample size (N), number of contemporary groups (CG), mean and standard deviation (SD) of the traits Traits1

Birth weight Weight at 60 d (weaning) Weight at 180 d Weight at 270 d Daily weight gain from birth to 60 d Daily weight gain from 60 to 180 d Daily weight gain from 60 to 270 d Dam Sire

10232 6277 4541 3328 5786 3229 1863 4742 391

CG 291 319 403 374 319 403 374 -------

Mean 3.63 15.94 31.9 39.7 171.73 149.43 69.17 -------

SD 0.80 5.77 11.16 14.5 64.87 67.84 26.83 -------

Weight and weight gain were measured in kilograms.

For testing inbreeding effect on the phenotypic values it was used the mixed model: đ?&#x2018;Śđ?&#x2018;&#x2013;đ?&#x2018;&#x2014; = đ?&#x153;&#x2021; + đ??śđ??şđ?&#x2018;&#x2013; + đ?&#x203A;źđ?&#x2018;&#x2013;đ?&#x2018;&#x2014; (đ??ź) + đ?&#x203A;˝đ?&#x2018;&#x2013;đ?&#x2018;&#x2014; (đ??ˇ) + đ?&#x203A;żđ?&#x2018;&#x2013;đ?&#x2018;&#x2014; (đ??´) + đ?&#x203A;žđ?&#x2018;&#x2013;đ?&#x2018;&#x2014; (đ??ş) + đ?&#x2018;&#x2019;đ?&#x2018;&#x2013;đ?&#x2018;&#x2014; where: đ?&#x2019;&#x161;đ?&#x2019;&#x160;đ?&#x2019;&#x2039; is the phenotypic value of trait; đ?&#x153;&#x2021; is the global mean; đ??śđ??şđ?&#x2018;&#x2013; is the fixed effect of contemporary group; đ?&#x153;śđ?&#x2019;&#x160;đ?&#x2019;&#x2039; (đ?&#x2018;°) is the fixed effect of covariate inbreeding coefficient level; đ?&#x153;ˇđ?&#x2019;&#x160;đ?&#x2019;&#x2039; (đ?&#x2018;Ť) is the fixed effect of covariate damâ&#x20AC;&#x2122;s age; đ?&#x153;šđ?&#x2019;&#x160;đ?&#x2019;&#x2039; (đ?&#x2018;¨) is the fixed effect of covariate animalâ&#x20AC;&#x2122;s age; đ?&#x153;¸đ?&#x2019;&#x160;đ?&#x2019;&#x2039; (đ?&#x2018;Ž) is the random effect of covariate breeding value; đ?&#x2019;&#x2020;đ?&#x2019;&#x160;đ?&#x2019;&#x2039; is the residual random term. For DWG2 and DWG3 both initial and final animalâ&#x20AC;&#x2122;s age were included in the model as fixed effect.

In addition, inbreeding effect on breeding values were also tested and the model was as follows: đ?&#x2018;Śđ?&#x2018;&#x2013;đ?&#x2018;&#x2014; = đ?&#x153;&#x2021; + đ?&#x203A;źđ?&#x2018;&#x2013;đ?&#x2018;&#x2014; (đ??ź) + đ?&#x2018;&#x2019;đ?&#x2018;&#x2013;đ?&#x2018;&#x2014; where: đ?&#x2018;Śđ?&#x2018;&#x2013;đ?&#x2018;&#x2014; is the breeding value of trait; đ?&#x153;&#x2021; is the global mean; đ?&#x203A;źđ?&#x2018;&#x2013;đ?&#x2018;&#x2014; (đ??ź) is the fixed effect of covariate inbreeding coefficient level; and đ?&#x2018;&#x2019;đ?&#x2018;&#x2013;đ?&#x2018;&#x2014; is the residual term. The mixed procedure of SAS software(17) was used to estimate inbreeding regression coefficients for all traits. Significance level to declare inbreeding effect was 5%.

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Pedigree completeness Percentages of animals with known pedigrees decreased with the passing of the generations, from over 70% in the first generation to less than 5% in the third (Figure 1). This result may be a consequence of ASCCO had been started the phenotypic and pedigree records recently (about 3-4 generations), which may explain the little-known ancestry of the animals studied here. Loss of information from one generation to another in the present study was higher than those reported in previous studies with Santa InĂŞs flocks(12,13). Pedrosa et al(12) found known ancestry from parents to great-grandparents of 77, 59.5, and 38.75 %, while Teixeira Neto et al(13) found 80.84, 73.78, and 67.75 %. Previous studies about other sheep breeds reported varied levels of pedigree completeness. High levels were reported for Bharat Merino sheep, with values of 91.01, 82.63, 74.91, 67.10, and 57.78 % for the first, second, third, fourth, and fifth generation, respectively(8). However, for other sheep breeds were reported pedigree completeness less than those observed in the present study, especially in the first generation back (11.88 % for sire and 69.38 % for dam) in Guilan sheep(20), and (57 % for sire and 15 % for dam) in Mehraban sheep(21).

Figure 1: Pedigree and level of identification of the ancestors up to the third generation

The number of equivalent generations is the parameter that best describes the quality of a pedigree and higher value for this parameter indicates a more completeness pedigree. In the present study, this value was low (Table 2) indicating that, even with a reasonable amount of information (11,564 individuals), the average relatedness was less in this Santa InĂŞs dataset. In previous studies with Santa InĂŞs, higher values were found, 2.26(12) and 4.67(13), due to higher pedigree completeness. A low number of equivalent generations is common in sheep breeds with early conservation and breeding programs(6), resulting in pedigrees with a low degree of depth and incomplete information. For this reason, a 595

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reduced number of equivalent generations was also reported in several sheep breeds(2,6,9,21).

Table 2: Genetic parameters of the gene origin for Santa InĂŞs Flocks in Northeast of Brazil Genetic parameters Value Reference population Number of ancestors Effective number of founders (fe) Number of founding animals Effective number of ancestors (fa) Number of ancestors explaining 50% Effective number of founders genomes (fge) Average of genetic diversity loss Inbreeding (F) Average relatedness coefficient (AR) Average number of equivalent generations Average number of complete generations Average number of maximum generations Inbreeding increment (Î&#x201D;F) in equivalent generations Inbreeding increment (Î&#x201D;F) in complete generations Inbreeding increment (Î&#x201D;F) in maximum generations Effective population size (Ne) in equivalent generations Effective population size (Ne) in complete generations Effective population size (Ne) in maximum generations Generation Interval Father-Son Generation Interval Father-Daughter Generation Interval Mother-Son Generation Interval Mother-Daughter

11,564 3,984 285 486.84 273 146 35.71 0.0094 1.40% 0.47% 0.94 0.83 1.09 0.95 0.97 0.73 52.62 51.28 68.83 3.46 3.33 3.40 3.28

Structure and genetic diversity The effective population size (Ne) changed with time (Figure 2), being highest in 2004 (665) and lowest in 2010 (45). The largest effective size was observed for the maximum generation (Table 2). The variation in effective population size (Ne) over time has also been observed in other sheep breeds. Values of Ne ranging from 41.8 to 31.3 in Morada Nova sheep(22), while values of Ne from 280.2 to 12.4 for SegureĂąa sheep has been Ě&#x2026;đ?&#x2018;&#x2019; ) is a better reference and reported(6). Therefore, the average effective population size (đ?&#x2018; it was close to 50 when calculated for complete and equivalent generations in the present study (Table 2) and higher than 60 for the maximum generations. According to FAO (23), the desired effective population size is about 50 animals per generation, to restrict a rate 596

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of inbreeding of 1 % per generation. Thus, the Ne values by year observed for the Santa Ě&#x2026;đ?&#x2018;&#x2019; (Table 2) indicate a risk situation, InĂŞs flocks in the present study (Figure 2), as well as đ?&#x2018; but inbreeding increases were less than 1 % for complete, equivalent and maximum generations (Table 2), which is in line with FAO recommendations to avoid the risk of extinction. The Ne decrease and simultaneous increase of F (Figure 2) may be due to the registration of animals without pedigree information or the intense use of some sires in the ASCCO farms, since the average breeding values for DWG3 tended to increase as Ne decreases and F increases (note similar line curves of DWG3 and F in the Figure 2).

Figure 2: Average inbreeding (F), effective population size (Ne) and average breeding values for DWG3 per year of birth

A reference population of 11,564 Santa InĂŞs sheep was evaluated, with 3,984 ancestors. In this population, the effective number of founders (Ć&#x2019;e) was 285, while the number of founding animals was 486.84. The number of ancestors explaining 50% of the genetic variation was 146 and the effective number of ancestors was 273. The effective number of founders genomes (fge) was 35.71. Therefore, some Santa InĂŞs sires were used more intensely, in detriment to others, which may have contributed to the loss of genetic variability. All ancestors would contribute in the same way throughout the generations, but for many sheep breeds the total number of ancestral is much larger that number of ancestral explaining 50 % of genetic variability(8,9,22).

Largest inbreeding increment was observed in equivalent generation (Table 2). The highest number of inbred animals was concentrated between 0 and 10 % of the inbreeding coefficient and just 263 animals showed F>10 %. Generation interval for each of the four parent-offspring pathways demonstrated an average of 3.37 (Table 2). Estimates of average inbreeding coefficient ranged from zero to 6.25% along the generation (Table 3) 597

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and average inbreeding was 1.40 % when only inbred animals were considered. The estimates of average relatedness coefficient ranged from 0.22 to 0.52 % along the generation (Table 3) and general average relatedness coefficient was 0.47 %. The inbreeding coefficient had its lowest value in 2004 (0.19 %) and a higher value in 2008 (2.86 %) (Figure 2).

Table 3: Number of animals (N) per generation with their respective average inbreeding coefficient (F) and average relationship coefficient (AR) Generations

F (%)

AR (%)

7,562

0.00

0.22

2,901

0.88

0.45

844

2.42

0.51

210

3.81

0.51

3.82

0.52

6.25

0.43

Ideally, fe equals fa, or the difference is always as low as possible. Ratios much higher than 1.0 indicate a strong bottleneck effect, which may be due to small number of sires used in mating. This ratio in the present study (1.04 %) suggests that the majority of ancestors were founders and an insignificant genetic bottleneck. Despite the good fe/fa ratio in the present study, fe and fa had values much lower than the reference population and ancestors (Table 2), indicating that the animals evaluated here have a narrow genetic origin. Another study with Santa InĂŞs reported a higher (1.35) fe/fa ratio(12), demonstrating genetic variability reduction caused by the imbalance between ancestral and founders and the higher bottleneck effect. For other sheep breeds, a fe/fa close to one was also reported, such as 1.0 in Morada Nova sheep(22), and 1.18 in Iran-black sheep(7), and 1.12 in SegureĂąa sheep(6). However, large values were also reported in Xalda sheep (2.02)(1) and in Kermani sheep (2.07)(9). Finally, an average of genetic diversity loss of 0.0094 was detected over the studied period, demonstrating that genetic drift was significant to result in loss of genetic diversity in this population.

Inbreeding values above 10% are associated with decreased performance in sheep(24). It was observed few animals (2.27 %) with consanguinity higher than 10%, and a maximum F value of 37.5 %, while several animals (97.73 %) were not inbreed or showed an inbreeding coefficient less than 10 %. These values are similar to those reported for other sheep breeds(8,25). In a study with Bharat Merino sheep, 97.62 % of animals were noninbreed or showed F< 10 %, and the highest individual inbreeding was 32.81 %(8). In Iranian Shal sheep, 93.72 % of animals to be non-inbred or F<10 %, with a maximum individual inbreeding of 31.25 %(25). It is noteworthy that low pedigree completeness may 598

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have underestimated individual inbreeding coefficients in the present study. Previous studies about Santa Inês sheep, with better pedigree completeness, found maximum inbreeding of 41.02 %(12) and 54.83 %(13), respectively; both studies have shown that the number of inbred animals increases significantly after the first years of pedigree control. The average inbreeding coefficient of the population (inbred and non-inbred animals) was 0.36 %, but average inbreeding coefficient for inbred animals was 1.41 %. Higher values of population average inbreeding 2.33 %(12) and 6.92 %(13) were reported for Santa Inês sheep. In these studies, average inbreeding coefficients were 10.74 %(12) and 12.57 %(13) when only inbred animals were used. The lowest value found in the present study may be due to low pedigree completeness, especially in the first years, which makes computing inbreeding coefficient difficult. Small average inbreeding coefficients were reported in other sheep breeds with low pedigree completeness. Previous studies reported average inbreeding coefficients of 0.15, 1.6 and 0.60 % for whole analyzed pedigree of the Guilan(20), Baluchi(2), and Segureña(6) sheep breeds, respectively.

The increase in inbreeding throughout the generations (Table 3) may be reflecting a better flock pedigree control and consequently higher database quality, because the inbreeding coefficient depends on the number of known generations. The inbreeding increment (ΔF) in the present study was low for all of the generations traced (Table 2), suggesting that the Santa Inês flocks under investigation were in good condition. An increase in the average inbreeding coefficient along generations was also observed in other sheep breeds(6,8). An increase from zero to 7.09 (from the initial to the fourth generation) in Segureña sheep was reported(6), while an increase from zero to 1.54 (from the initial to the sixth generation) was reported to Bharat Merino sheep(8). The low AR values obtained in the present study (Table 3), show that the flocks are in a good situation, increasing the probability of mating among unrelated individuals. Another Santa Inês dataset(12) also showed a low estimate for AR (0.73 %), evidencing the great variability of this breed.

Inbreeding effect on phenotype and breeding values For phenotypic values, the individual inbreeding had no effect (P>0.05) on BW60, BW180, BW270, and DWG1 (Table 4), but significant effects (P<0.05) were observed for BW1 (0.0054 ± 0.0015), DWG2 (-0.9837 ± 0.3025), and DWG3 (-0.5628 ± 0.2377). For breeding values, depression inbreeding effect were significant (P<0.05) for all traits, except DWG1. For the Santa Inês sheep, only one previous study(12) tested inbreeding effect on phenotypic traits, but they evaluated only BW1, BW60 and BW180. They observed a reduction of 34, 52, and 204 grams per %∆F (equivalent to a traditional inbreeding coefficient of 2.2 % when 2.26 generations in the pedigree are known) in the weight of Santa Inês for BW1, BW60, and BW180, respectively. The present analysis for record traits did not confirm these findings, because it was found a positive inbreeding effect on BW1, where each 1 % of inbreeding increased 5.4 g in this weight, and no significant effects were observed for BW60, BW180 and BW270 (Table 4). Several previous studies with other sheep breeds reported depressive inbreeding effect on birth 599

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weight(21,26,27) where each 1% of inbreeding resulted in decreases ranging from -0.7 g per 1% F in Polish Olkuska sheep(26) to -51 g per 1% F in Thalli sheep(27). For other pre and post-weaning body weights, there are many results indicating depressive effects in different sheep breeds as well. Depression-inbreeding effect for BW60, with values range from -33 to -48 g per 1% F were reported(27,28). However, studies with Iranian Shal sheep(25) and Segureña sheep(6) did not any significant inbreeding effect on body weight.

Table 4: Regression coefficients of the effects of inbreeding on the performance traits Phenotype value Trait

Estimate

BW at birth BW at 60 d BW at 180 d BW at 270 d DWG from birth to 60 d DWG from 60 to 180 d DWG from 60 to 270 d

0.0054 0.0252 -0.0568 -0.0623 -0.2318 -0.9837 -0.5628

Standard P-value Error 0.0015 0.0004 0.0132 0.0555 0.0299 0.0575 0.0430 0.1469 0.2200 0.2921 0.3025 0.0012 0.2377 0.0180

Breeding value Estimate -0.0049 -0.0162 -0.0347 -0.0448 -0.0461 -0.2846 -0.0524

Standard P-value Error 0.0006 <0.0001 0.0030 <0.0001 0.0104 0.0009 0.0131 0.0006 0.0573 0.4213 0.0868 0.0011 0.0212 0.0137

BW= body weight; DWG= daily weight gain.

In some previous studies, no-significant effects are many times attributed to the low level of inbreeding of the animals as consequence of low pedigree completeness. In the present study, the dataset had also low pedigree completeness (Figure 1); consequently, large number of animals (7,562) showed F close to zero. Another hypothesis for no-significant effect is the reduced number of animals showed both F>0 and phenotypic record. To avoid this second problem, we decided to evaluate the effect of inbreeding on the breeding values. The results (Table 4) indicated a significant inbreeding effect on breeding values of all body weights (BW1, BW60, BW180 and BW270). In addition, the regression coefficients were negative, which is more consistent with previous studies reported in sheep(26,27,28).

Regression coefficients for DWG2 and DWG3 were higher than those (−0.263 ± 0.116) reported for daily weight gain from 90 to 365 in Sandyno sheep(4), and lower than those found for daily weight gain from 90 to 180 (−1.810 ± 0.017) and from 90 to 365 (−1.345 ± 0.083) in Baluchi sheep(2). Finding effect for daily weight gain and no effect for body weight seems incoherent, but it is easy to explain. The number of animals with records for BW60, BW180 and BW270 was different from the number of animals with records for DWG2 and DWG3, because to calculate the daily weight gain we need the same animal to have four information (the initial and final BW, and the initial and final ages). 600

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It is not a reality for all animals in our dataset. When used breeding values this problem was resolved, because all animal has the estimates of breeding values for all traits. It is possible observed in Table 4 that this incoherence practically not existed when we estimated inbreeding effect on breeding values (Table 4), suggesting a more consistent result.

It could be observed that the evaluated population had a low pedigree completeness and small average inbreeding. This population presented a decrease in the effective population size over the generations and an increase of the endogamy, which can compromise its genetic variability. The inbreeding had significant effect on BW1, DWG2 and DWG3 when evaluated phenotypic records. When was evaluated breeding values, the inbreeding effect was significant for all traits, except for DWG1. Regression coefficients obtained for breeding values suggested a more consistent analysis, because they were negative and significant for both BW (all ages) and post-weaning DWG. On the other hand, positive and significant regression inbreeding coefficients was found only for BW1 (in phenotypic analysis), but it was not found similar results for this trait in breeding value analysis. In both analysis, the inbreeding effect on growth traits were mainly negative, which implies the need to avoid related mating on the studied flocks of Santa Inês sheep.

Acknowledgments The authors thank to ASCCO for the dataset provided; to FAPESB for the scholarship of Ana Carla Borges Barbosa; and to CNPQ for the Productivity Scholarship for José Bento Sterman Ferraz, Victor Breno Pedrosa, and Luís Fernando Batista Pinto.

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Revista Mexicana de Ciencias Pecuarias

Edición Bilingüe Bilingual Edition

Rev. Mex. Cienc. Pecu. Vol. 11 Núm 2, pp. 311-604, ABRIL-JUNIO-2020

ISSN: 2448-6698

CONTENIDO CONTENTS Pags. Preliminary study of ivermectin residues in bovine livers in the Bogota Savanna

Eficacia de la ivermectina para el control de nematodos gastrointestinales en burros (Equus asinus) en el altiplano mexicano

Ivermectin effectiveness for gastrointestinal nematode control in donkeys (Equus asinus) in the Mexican High Plateau Guadalupe Galicia-Velázquez, Arturo Villarreal-Nieto, Cris�na Guerrero-Molina, Cintli Mar�nez-Or�z-de-Montellano……………………………………………………………………………………………………………………….…....…326

Artemisia cina 30 CH homeopathic treatment against Haemonchus contortus

Artemisia cina 30 CH como tratamiento homeopático contra el Haemonchus contortus Rosa Isabel Higuera-Piedrahita, María Eugenia López-Arellano, Raquel López-Arellano, César Cuenca-Verde, Jorge Alfredo Cuéllar-Ordaz………………………………………………………………………………………………….342

Inclusión de harina de Tithonia diversifolia en raciones para gallinas ponedoras de primer ciclo y su efecto sobre la pigmentación de yema de huevo

Tithonia diversifolia meal in diets for ﬁrst-cycle laying hens and its eﬀect on egg yolk color María Elena Carranco-jauregui, Vilma Barrita-Ramírez, Benjamín Fuente-Mar�nez, Ernesto Ávila-González, Leonor Sanginés-García……………………………………………………………………………..………………………….355

Efecto de un complejo multienzimático y un probiótico en gallinas de postura alimentadas con dietas sorgo-soya-canola

Effect of a multienzyme complex and a probiotic in laying hens fed sorghum-soybean-rapeseed diets Pedro Juárez Morales, Arturo Cortes Cuevas, José Arce Menocal, Juan Carlos Del Río García, Gabriela Gómez Verduzco, Ernesto Avila González……………………………………………………………………………...…………369

Tendencias genéticas y fenotípicas para pico productivo, rendimiento lechero y persistencia de lactación en la raza Murciano-Granadina

Phenotypic and genetic trends for peak yield, milk yield, and lactation persistency in the Murciano-Granadina breed Judith Carmen Miranda Alejo, José Manuel León Jurado, Camillo Pierama�, Mayra Mercedes Gómez Carpio, Jesús Valdés Hernández, Cecilio José Barba Capote………………………………………………………………380

Calidad seminal de ovinos de pelo suplementados con Moringa oleifera (Moringaceae) y Trichanthera gigantea (Acanthaceae)

Semen quality of hair sheep supplemented with Moringa oleifera (Moringaceae) and Trichanthera gigantea (Acanthaceae) Marco A. Ramírez-Bau�sta, Julio P. Ramón-Ugalde, Edgar Aguilar-Urquizo, William Cetzal-Ix, Roberto Sanginés-García, Álvaro E. Domínguez-Rebolledo, Ángel T. Piñeiro-Vázquez…………………………….…....…393

Use of a glycogenic precursor during the prepartum period and its effects upon metabolic indicators and reproductive parameters in dairy cows

Uso de un precursor glucogénico en el preparto y su efecto sobre indicadores de energía y parámetros reproductivos en vacas lecheras Carlos Leyva Orasma, Jesus Jaime Benitez-Rivas, Juan Luis Morales Cruz, Cesar Alberto Meza-Herrera, Oscar Ángel-García, Fernando Arellano-Rodríguez, Guadalupe Calderón-Leyva, Dalia Ive�e Carrillo-Moreno, Francisco Veliz Deras…………………………………………………………………………………………….…….………………………………………………………………………………………408

Relationship of the compositional content and sanitary quality of Holstein cows’ milk of the high tropic of Nariño

Relación entre la calidad composicional y sanitaria de la leche de bovinos Holstein del trópico alto de Nariño Henry Armando Jurado-Gámez, Carlo Eugenio Solarte-Por�lla, Álvaro Javier Burgos-Arcos, Aldemar González-Rodríguez, Carol Rosero-Galindo…………………………………………………………………………………………421

Caracterización de Aspergillus flavus y cuantificación de aflatoxinas en pienso y leche cruda de vacas en Aguascalientes, México

Characterization of Aspergillus flavus and quantification of aflatoxins in feed and raw milk of cows in Aguascalientes, Mexico Erika Janet Rangel-Muñoz, Arturo Gerardo Valdivia-Flores, Onésimo Moreno-Rico, Sanjuana Hernández-Delgado, Carlos Cruz-Vázquez, María Carolina de-Luna-López, Teódulo Quezada-Tristán, Raúl Or�z-Mar�nez, Netzahualcóyotl Máyek-Pérez…………………………………………………………………………………………………………………………………………….435

Comparación de la castración quirúrgica al nacimiento versus inmunocastration sobre las características de la canal y carne en machos Holstein

Comparison of surgical castration at birth versus immunocastration on carcass and meat traits in growing Holstein males Jorge A. Cervantes-Cazares, Cris�na Pérez-Linares, Fernando Figueroa-Saavedra, Alma R. Tamayo-Sosa, Alberto Barreras-Serrano, Francisco G. Ríos-Rincón, Eduardo Sánchez-López, Issa C. García-Reynoso, Pedro Mendoza Peraza, Angelina León Villanueva, Luis A. García-Vega………………………………………………………………………………………455

Ascosferosis en abejas melíferas y su relación con factores ambientales en Jalisco, México

Ascospherosis in honey bees and its relationship to environmental factors in Jalisco, Mexico José María Tapia-González, Gustavo Alcazar-Oceguera, José Octavio Macías-Macías, Francisca Contreras-Escareño, José Carlos Tapia-Rivera, Ta�ana Petukhova, Ernesto Guzmán-Novoa…………………………468

Desarrollo y validación de un patrón visual para la evaluación del color de la carne de bovino en México

Development and validation of a visual pattern for evaluating beef meat color in Mexico Sara Salinas Labra, María Salud Rubio Lozano, Diego Braña Varela, Rubén Danilo Méndez Medina, Enrique Jesús Delgado Suárez………………………………………………………………………………………………………………479

REVISIONES DE LITERATURA Bolos intrarruminales con liberación controlada de minerales traza. Revisión

Trace mineral controlled-release intraruminal boluses. Review Misael León-Cruz, Efrén Ramírez-Bribiesca, Raquel López-Arellano, Leonor Miranda-Jiménez, Gabriela Rodríguez-Pa�ño, Víctor M. Díaz-Sánchez, Alma L. Revilla-Vázquez……………………………………………….498

Implicaciones, tendencias y perspectivas del transporte de larga distancia en el ganado bovino. Revisión

Implications, trends, and prospects for long-distance transport in cattle. Review Marcela Valadez Noriega, Genaro Cvabodni Miranda de la Lama…………………………………………………………………………………………………………………………………………………………………………………………………………………517

NOTAS DE INVESTIGACIÓN Growth, viability, and post-acidification of Lactobacillus plantarum in bovine transition milk

Crecimiento, viabilidad y post-acidificación de Lactobacillus plantarum en la leche de transición bovina Hugo Calixto Fonseca, Eduardo Robson Duarte, Lívia Caroliny Almeida Santos Souza, Emanuelly Gomes Alves Mariano, Ana Clarissa dos Santos Pires, Ta�ana Santos Lima, Maximiliano Soares Pinto........539

Caracterización de la leche y queso artesanal de la región de Ojos Negros, Baja California, México

Characterization of the milk and artisanal cheese of the region of Ojos Negros, Baja California, Mexico Laura E. Silva-Paz, Gerardo E. Medina-Basulto, Gilberto López-Valencia, Mar�n F. Montaño-Gómez, Rafael Villa-Angulo, José C. Herrera Ramírez, Ana L. González-Silva, Francisco Monge-Navarro, Sergio A. Cueto-González, Gerardo Felipe-García…………………………………………………………………………………………553

Factores asociados al decomiso de hígados positivos a Fasciola sp en una zona endémica del sureste de México

Factors associated with the seizure of livers positive to Fasciola sp in an endemic area of southeastern Mexico Nadia Florencia Ojeda-Robertos, Roberto González-Garduño, San�ago Cornelio-Cruz, Jorge Alonso Peralta-Torres, Carlos Luna-Palomera, Carlos Machain-Williams, Heliot Zarza, Oswaldo Margarito Torres-Chablé, Enrique Reyes-Novelo, Carlos Baak-Baak, Alfonso Chay-Canul……………………………………………………………….565

Análisis genético del desarrollo en peso vivo y tasa de gestación en primer parto en bovinos Brahman de Venezuela

Genetic analysis of live weight and pregnancy rate at first calving in Brahman cattle from Venezuela Alejandro-Palacios-Espinosa, Omar-Verde, Narciso-Ysac-Ávila-Serrano, Alberto-Menéndez-Buxadera…………………………………………………………………………………………………………………………………………………………576

Pedigree analysis of Santa Inês sheep and inbreeding effects on performance traits

Análisis de pedigrí de las ovejas Santa Inês y los efectos de la endogamia en los rasgos de rendimiento Ana Carla Borges Barbosa, Gabrieli de Souza Romano, Jonatan Mikhail Del Solar Velarde, José Bento Sterman Ferraz, Victor Breno Pedrosa, Luís Fernando Ba�sta Pinto…………………………………………………590

Revista Mexicana de Ciencias Pecuarias Rev. Mex. Cienc. Pecu. Vol. 11 Núm 2, pp. 311-604, ABRIL-JUNIO-2020

Estudio preliminar de residuos de ivermectina en hígado de bovinos en la Sabana de Bogotá Carmen Teresa Celis-Giraldo, Diego Ordóñez, Leonardo Roa, Sergio Andrei Cuervo-Escobar, Dajane Garzón-Rodríguez, Milena Alarcón-Caballero, Luisa Fernanda Merchán………………………………….....….…...311

Rev. Mex. Cienc. Pecu. Vol. 11 Núm. 2, pp. 311-604, ABRIL-JUNIO-2020