Large Animal Review - Year 26, Number 2, April 2020 by E.V. Soc. Cons. a r.l.

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Poste Italiane spa - Spedizione in A.P. - D.L. 353/2003 (conv. in L. 27/02/2004 N. 46) art. 1, comma 1, DCB Piacenza - Concessionaria esclusiva per la pubblicità: E.V. Soc. Cons. a r.l. - Cremona

02/20

Bimonthly, Year 26, Number 2, April 2020

LAR Large Animal Review

ISSN: 1124-4593

LARGE ANIMAL REVIEW is ranked in Citation Index (SciSearch®) Journal Citation Reports/Science Edition and CAB ABSTRACTS

ORIGINAL ARTICLES BOVINE • Metabolic adaptation in first week after calving and early prediction of ketosis type I and II in dairy cows • Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration, health, growth performance and meat quality OVINE • The effect of final weight on slaughtering and carcass quality characteristics of lambs in concentrate-based or pasturebased production systems • Enzootic pneumonia in sheep: ewe and lamb immune response after Mannheimia haemolytica vaccine administration under field condition in Italy • Infezione da Ovine herpesvirus tipo 2 in allevamenti ovini in Umbria EQUINE • Cuboni Reaction: non-invasive late pregnancy test in Martina Franca jennies REVIEWS BOVINE • Genomics of subacute ruminal acidosis CASE REPORTS CAMELID • A case of epiploic foramen entrapment of jejunal intestinal tract in an alpaca (Vicugna pacos) cria

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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INDEX

ORIGINAL ARTICLES

Anno 26, numero 2, Aprile 2020 Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IF (2017/2018): 0.26

BILJANA DELIĆ, BRANISLAVA BELIĆ, MARKO CINCOVIĆ, RADOJICA DJOKOVĆ, IVANA LAKIĆ

Editor in chief: Massimo Morgante

Technical Editor: Enrico Fiore

CARLO ANGELO SGOIFO ROSSI, SILVIA GROSSI, RICCARDO COMPIANI, GIANLUCA BALDI, MARIA AGOVINO, LUCIANA ROSSI 57

OVINE

LARGE ANIMAL REVIEW è una rivista bimestrale pubblicata per favorire l’aggiornamento dei veterinari che si dedicano alla prevenzione e alla cura delle malattie degli animali da reddito e alla qualità e salubrità dei prodotti derivati.

Direttore Responsabile Antonio Manfredi

The effect of final weight on slaughtering and carcass quality characteristics of lambs in concentrate-based or pasture-based production systems BULENT EKIZ, ALPER YILMAZ, HULYA YALCINTAN, OMUR KOCAK, MUSTAFA OZCAN

Enzootic pneumonia in sheep: ewe and lamb immune response after Mannheimia haemolytica vaccine administration under field condition in Italy

Consiglio direttivo SIVAR 2020-2023 Mario Facchi (Presidente) Daniele Gallo (Presidente Senior) Alberto Ferrero (Vice-Presidente) Michela Conterbia (Segretario) Vito Loconte (Tesoriere) Alessandro Federici (Consigliere) Osvaldo Parolin (Consigliere) Chiara Musella (Consigliere) Mattia Bottacini (Consigliere) Giuseppe Argiolas (Consigliere) Edizioni SCIVAC Palazzo Trecchi - 26100 Cremona Tel. 0372/460440 Iscrizione registro stampa del Tribunale di Cremona n. 299 del 25/9/1995

Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration, health, growth performance and meat quality

Editorial Board 2019-2021: Anna Rita Attili - Roberto Bardini Francesca Bonelli - Marta Brscic Marco Colombo - Vincenzo Cuteri Antonella Dalle Zotte - Enrico Fiore Giovanni Franzo - Matteo Gianesella Elisabetta Giudice - Paolo Moroni Davide Ranucci - Antonia Ricci Giuseppe Stradaioli - Erminio Trevisi Managing Editor: Matteo Gianesella

BOVINE Metabolic adaptation in first week after calving and early prediction of ketosis type I and II in dairy cows

CRISTINA PESCA, KATIA FORTI, ANDREA FELICI, ELEONORA SCOCCIA, CLAUDIO FORTE, PIETRO ANTENUCCI, SABINA MUNTONI, LUCIA ANZALONE, ANTONELLA DI PAOLO, SILVIA CROTTI 73

Infezione da Ovine herpesvirus tipo 2 in allevamenti ovini in Umbria ORIANA RAFFAELE, EMILIA DEL ROSSI, LORENZO CASTELLI, STEFANO PIGNANI, STEFANO PETRINI, MARIA TERESA MANDARA, MARIA LUISA MARENZONI 79

EQUINE Cuboni Reaction: non-invasive late pregnancy test in Martina Franca jennies

Stampa Press Point - Via Cagnola, 35 20081 Abbiategrasso (MI) - Tel. 02/9462323

ROBERTA BUCCI, BRUNELLA ANNA GIANGASPERO, MICHELA D’ANGELO, DOMENICO ROBBE, PATRIZIA PONZIO, ANNA CHIARA MANETTA, LORELLA DI GIUSEPPE, IPPOLITO DE AMICIS 87

Spedizione Poste Italiane SPA - Spedizione in A.P. D.L. 353/2003 (Conv. in L. 27/02/2004 N. 46) Art. 1, Comma 1, DCB Piacenza

REVIEWS

Concessionaria esclusiva per la pubblicità E.V. Soc. Cons. a r.l. Palazzo Trecchi - 26100 Cremona Ufficio Pubblicità: Paola Orioli Tel. 0372/403539 - E-mail: info@sivarnet.it Prezzo di copertina: € 10,00. La rivista è inviata a tutti i veterinari interessati ai settori degli animali da reddito con il versamento di € 52,00 per l’Italia; € 62,00 per l’Estero. Servizio abbonamenti: Tel. 0372/403507. Ai Soci SIVAR in regola con il pagamento della quota associativa, la rivista è inviata gratuitamente in quanto la quota è comprensiva dell’abbonamento alla rivista stessa.

BOVINE Genomics of subacute ruminal acidosis MUHAMMAD IRFAN MALIK, MUHAMMAD AFZAL RASHID

CASE REPORTS CAMELID

A case of epiploic foramen entrapment of jejunal intestinal tract in an alpaca (Vicugna pacos) cria LAKAMY SYLLA, MARTINA CROCIATI, DOMENICO CAIVANO, VASILICA FLORY PETRESCU, LORENZO PISELLO, CALOGERO STELLETTA 99

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IL PORTALE DEL VETERINARIO

DI FIDUCIA

Il portale del Veterinario di Fiducia è una piattaforma multimediale rivolta a Medici Veterinari che svolgono attività clinica e manageriale negli allevamenti italiani. È gestito dalla SIVAR (Società Italiana Veterinari per Animali da Reddito - Federata ANMVI) che ne è anche proprietaria. Tutti i dati vengono trattati ai sensi della normativa sulla privacy. Il portale contiene: – notizie – materiali didattici – DES (Database Epidemiologico Sanitario) – DDD (Database per il Monitoraggio degli Antibiotici) – forum di discussione

DES

DDD

DEFINED DAlLY DOSE Software sperimentale che consente di calcolare le quantità di antibiotici somministrati sui propri allevamenti

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➜ DATABASE EPIDEMIOLOGICO SANITARIO Raccoglie dati sanitari ed epidemiologici di cui dispone solo il veterinario d’azienda

AREA LIBERA DEL PORTALE Alcune sezioni e funzioni (es. notizie e materiali didattici sono di libero accesso e non richiedono l’inserimento di credenziali (né password né username EGO). AREA RISERVATA DEL PORTALE Alcune sezioni e funzioni sono accessibili solo utilizzando il proprio Codice Ego (username e password) dopo averne richiesto l’attivazione alla casella: vetdifiducia@anmvi.it oppure info@sivarnet.it. Sono in area riservata le seguenti funzionalità: – DES (Database Epidemiologico Sanitario) – DDD (Database per il Monitoraggio degli Antibiotici) – forum di discussione – alcuni materiali didattci

Se sei un Socio SIVAR in regola con la quota annuale, puoi richiedere di essere abilitato scrivendo a: vetdifiducia@anmvi.it oppure info@sivarnet.it

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B. Delić et al. Large Animal Review 2020; 26: 51-55

Metabolic adaptation in first week after calving and early prediction of ketosis type I and II in dairy cows

BILJANA DELIĆ1, BRANISLAVA BELIĆ1, MARKO CINCOVIĆ1, RADOJICA DJOKOVĆ2, IVANA LAKIĆ1 1

Department of Veterinary Medicine, Laboratory of pathophysiology, Faculty of Agriculture, University of Novi Sad, Trg Dositeja Obradovića 8, Novi Sad, Serbia 2 Department of Animal Science, Faculty of Agronomy, Čačak, University of Kragujevac, Cara Dušana 34, Čačak, Serbia

SUMMARY The aim of this study is to determine differences in metabolic adaptation in first week after calving between healthy and cows that develop ketosis type I (diagnosed 3-6 weeks after calving) or II (diagnosed 2-3 weeks after calving) after calving. Experiment included 50 healthy cows, 50 ketosis I type cows and 50 ketosis II cows. Animals were selected retrospectively form 435 Holstein cows that were constantly monitored. Monitoring is regular procedures during all season. Blood samples were taken in first week after calving. Concentration of ketone body was measured every two day by test stripes from the end of 1st until the end of 6th week. Any color change in test strip (5 mg/dL, trace, or higher) was indicator of ketosis in cows. Cows were clinically evaluated to determine any clinical symptoms (reduced appetite, rumen atony, behavioral changes). In cows with ketosis type I were noted higher concentrations of nonesterified fatty acid (NEFA) and lower concentrations of glucose and insulin compared to control group. Higher concentrations of beta hydroxybutyrate (BHB), tumor necorsis factor alpha (TNF-α) and total bilirubin were noted in cows with ketosis type II compared to control group. Value of revised quantitative insulin sensitivity check index (RQUICKI) was lower, and aspartate aminotransferase (AST) was higher in blood of ketosis II cows compared to ketosis I cows. Prediction of ketosis type I is significant by logistic regression model which include insulin, NEFA and glucose as independent predictor (area under ROC curve, AUC=0.78, p<0.05). Possibility of ketosis I development increases with NEFA increase and decrease of glucose and insulin concentrations. Prediction of ketosis type II development is significant by logistic regression model which include BHB, TNF-α and total bilirubin where increase of these parameters indicates higher possibility of ketosis II development (AUC=0.87, p<0.01). Differentiation of ketosis type I and II is significant by logistic regression model which include value of RQUICKI and AST (AUC=0.74, p<0.05), so with increasing of AST and decreasing of RQUICKI in first week of lactation increase risk for type II ketosis. Percentage of variation in the metabolic parameters that is predictable from the BHB in first week after calving was significantly higher in ketotic (10.9-18.5%) then in healthy cows (2.5-9.1%). Cows in ketosis type I and II show different metabolic adaptations in first week after calving. These differences allow prediction of development a exact type of ketosis. Metabolic adaptation in function of ketogenesis was developed in first week after calving, early before manifestation of ketosis.

KEY WORDS Cows, ketosis, inflammation, insulin resistance, liver function.

INTRODUCTION Ketosis is significant metabolic disorder of cows that is developed as a consequence of negative energetic balance and it is presented with higher concentrations of ketone bodies in blood, milk and urine. Subclinical ketosis may be diagnosed when blood serum BHB concentrations are above 1.2 mmol/L, and clinical ketosis with blood BHB level above 2.6 mmol/L as gold standard test1. Basal metabolic adaptations in cows that develop ketosis are: higher concentrations of BHB, higher concentrations of NEFA, lower concentrations of glucose, higher concentrations of liver enzymes and bilirubin, disturbances in macro and micro elements, greater values of inflammatory markers, oxidative stress and insulin resistance. It can be followed

Corresponding Author: Marko R. Cincović (mcincovic@gmail.com).

with abomasal dislocation, mastitis, metritis, laminitis and behavioral changes2-4. On pathohysiologic aspect, ketosis can be divided into ketosis type I and II5-7. Type I is developed 3-6 weeks after calving while type II develops 1-3 weeks after calving and it is tightly related to fatty liver. Ketosis I develops as a consequence of underfeeding and increase in milk production, while ketosis II is developed as a consequence of homeorhetic processes in early lactation. Use of some biochemical markers from blood and milk can be predicted development of ketosis in cows. Milk parameters may be also used for objective ketosis diagnosis8-10. Chuang et al.11 have been determined differences in values of some laboratory parameters in blood between ketosis I and II measured from moment of calving until fourth week of lactation. Early prediction of ketosis in the first week after calving may be possible. Classification of cows according to lipolysis, ketogenesis or activity of anabolic hormones in first week after calving shows metabolic differences between the classes for seve-

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Metabolic adaptation in first week after calving and early prediction of ketosis type I and II in dairy cows

ral consecutive weeks after calving. Kessel et al.12 noted that retrospectively classified cows with high BHB concentration (over 1 mmol/L) have higher concentrations of BHB, NEFA, AST, GGT and lower concentrations of IGF-I in first 14 weeks after calving. Cincović et al.13 founded that intensive lipolysis and ketogenesis in first week before and after calving cause lower concentrations of glucose, total proteins, cholesterol and urea and higher concentrations of bilirubin and AST compared to cows in control group. Cows loaded with metabolic stress (high catabolic and low anabolic blood indicators) showed significant difference in metabolic adaptation in relation to control group: higher values of STH, BHB, higher values of bilirubin, AST, ALT, GGT, AP and MDA and lower levels of glucose, total proteins, albumin and body condition14. The aim of this study is to determine differences in metabolic adaptations in first week after calving in ketotic cows type I and II and to determine possibility of prediction of ketosis development and type of ketosis with metabolic parameters in first week after calving. The aim is, also, to determine if there is any correlation of metabolic parameters and BHB and to determine differences in function of type of ketosis in cows in first week after calving.

termined values were read on reader RT-2100 C (Rayto, PRC). Insulin resistance indices were calculated by standard methodology16: RQUICKI = 1/[log (conc.t0 glucose mg/dL) + log (conc.t0 insulin mmol/L)+log (conc.t0 NEFA mmol/L)]. Statistics: Differences in concentrations of metabolic parameters and indexes of insulin resistance between groups were determined by ANOVA analysis and post hock LSD-test. Possibility of ketosis diagnosis was determined by multiple logistic regressions in which model was included metabolic parameters that were showed significant differences in LSD-test. Odds ratio (OR) and area under ROC curve (AUC) were determined for every model. Model that questions possibility of differential diagnosis of ketosis type I and II was made with metabolic parameters that are different in ketosis I and II by multiple logistic regressions. Early changes in metabolism in function of ketogenesis were determined by coefficient of correlation and determination (percentage of variation) in linear model between BHB and other metabolic parameters concentrations in first week after calving. Using the Fisher rto-z transformation, will be calculate a value of z that can be applied to assess the significance of the difference between two correlation coefficients (healthy vs. ketosis type I; healthy vs. ketosis type II and ketosis type I vs. ketosis type II).

MATERIALS AND METHODS RESULTS Animals and model: 150 animals were included in experiment: 50 healthy, 50 cows in type I ketosis and 50 cows in type II ketosis. Animals were selected retrospectively form 435 Holstein cows that were constantly monitored. Monitoring is regular procedures during all season. Cows were from second to fourth parity. During the early lactation the daily ration consisted of 4 kg alfalfa hay, 15 kg maize silage (30%DM), 8 kg alfalfa haylage, 4 kg ear maize silage (68%DM), 2 kg dry sugar beet pulp, 2 kg extruded soybean meal, and 4.5 kg concentrate (18%CP), NEL 153 MJ. Energy balance was evaluated according to body weight, offered meal and average milk production using NRC [2001] standards15. Blood samples were taken in first week after calving. From the end of the 1st to the end of the 6th week concentration of ketone body in urine was measured every two day by test stripes (Ketostix, Bayer, GER). Any color change in test strip (5 mg/dL, trace, or higher) was indicator of ketosis in cows. Cows were clinically evaluated to determine any clinical symptoms (reduced appetite, rumen atony, behavioral changes). Healthy cows did not show clinical symptoms of ketosis and urinary ketone bodies were not diagnosed in those cows. In cows with clinical symptoms and moderate or large color change on Ketostix was determined blood BHB concentration. Concentration of BHB >1.2 mmol/L in blood was cut-off value for final diagnosis of ketosis1. Based on the time when ketosis occurred, cows were classified into ketosis type I (diagnosed 3-6 weeks after calving), ketosis type II cows (diagnosed 2-3 weeks after calving) and healthy cows. Blood sample analyses: In first week after calving concentrations of BHB, NEFA, glucose, total proteins, albumins, bilirubin, AST, GGT, cholesterol, triglycerides were determined by biochemical colorimetric kits (Biosystem, SP ans Randox, UK). Concentrations of observed parameters were determined by the Chemray analyzer (Rayto, PRC). Insulin concentration was determined by ELISA method (Cusabio, PRC) just like TNF-α concentration (Cloud-Clone Corp., USA). De-

In first week after calving differences in metabolic adaptations in cows with different type of ketosis were found (Table 1). Higher concentrations of BHB, TNF-α and total bilirubin were founded in cows that developed ketosis type II in comparison to control group. Higher concentrations of NEFA and lower concentrations of glucose and insulin were noted in cows that developed ketosis type I compared to control group during first week after calving. Determination of ketosis type I or II by metabolic parameters can be done by lower RQUICKI index and higher AST levels in blood of cows that develop ketosis type II. Prediction of ketosis type I is significant by logistic regression model which include insulin, NEFA and glucose as independent predictor (area under ROC curve, AUC=0.78, p<0.05). Possibility of ketosis I development increases with NEFA increase and decrease of glucose and insulin concentrations. Prediction of ketosis type II development is significant by logistic regression model which include BHB, TNF-α and total bilirubin where increase of these parameters indicates higher possibility of ketosis II development (AUC=0.87, p<0.01). Differentiation of ketosis type I and II is significant by logistic regression model which include value of RQUICKI and AST (AUC=0.74, p<0.05), so with increasing of AST and decreasing of RQUICKI in first week of lactation increase risk for type II ketosis. Results are presented in Table 2, Figure 1 and 2. Linear correlation analysis between BHB and metabolic parameters was analysed in first week after calving. Percentage of variation in the metabolic parameters that is predictable from the BHB in first week after calving was significantly higher in ketotic (10.9-18.5%) then in healthy cows (2.59.1%). We found statistically significant difference in coefficient correlation between healthy and ketotic cows. Those results indicated that metabolic pathways are changed in function of ketogenesis in first week after calving and before ketosis manifestation.

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B. Delić et al. Large Animal Review 2020; 26: 51-55

DISCUSSION Ketosis development in cows and its early prediction is researched with different models. All models showed that great significance in ketosis prediction have values of NEFA, AST, glucose and other parameters which is in compliance with

our results. Asl et al.8 founded that cut-off point for NEFA concentrations of 0.26 mmol/L can be used during early lactation for diagnosis of subclinical ketosis in first 8 weeks after calving. Sun et al.9 founded that thresholds were more than 0.76 mmol/L for NEFA, more than 104 U/L for AST, less than 140 U/L for cholinesterase and more than 3.3 µmol/L

Table 1 - Blood parameters in healthy cows and cows with ketosis type I and II. Parameter

Healthy control

Ketosis I

Ketosis II

Significance

BHB (mmol/L)

0.52±0.19a

0.71±0.16b

0.89±0.14b

* ** ** * **

NEFA (mmol/L)

0.49±0.18

0.71±0.2

0.54±0.19ab

Glucose (mmol/L)

2.51±0.35a

2.13±0.36b

2.4±0.32a

T. bilirubin (µmol/L)

5.6±2.1

AST (IU/L)

94.5±20.5ab

8.9±2.4b

6.2±2.9

89±19.1a

119.3±21.2b

GGT (IU/L)

33.5±2.5

32.4±1.7

34.3±3.2a

Albumin (g/L)

33.6±2.1a

32.4±1.7a

31.2±2.6a

T. Protein (g/L)

62±4.4

59±3.9

58±3.8

Cholesterol (mmol/L)

3.01±0.29a

2.92±0.18a

2.86±0.24a

NS NS

Triglycerides (mmol/L)

0.14±0.014

0.14±0.017

0.13±0.016

TNF-α (ng/mL)

0.78±0.18a

0.83±0.19ab

1.01±0.17b

Insulin (µIU/L)

4.5±1.4a

3.1±1.5b

4.3±1.6ab

RQUICKI

0.51±0.015ab

0.52±0.016a

0.50±0.014b

** * **

NS: p > 0.05; *: p < 0.05; **: p < 0.01. Means followed by different letters differ significantly at p < 0.05.

Table 2 - Combination of biomarkers in evaluation of ketosis I and II development. Estimated

% of explained variance

Model significance

Ketosis type I from Healthy control Insulin

-0.2

1.1-1.6

NEFA

0.14

1.5-2.8

Glucose

-0.78

0.9-1.8

76%

82%

***

71%

Ketosis type II from Healthy control BHB

0.21

1.8-3.1

TNF-α

0.52

1.9-2.8

T. bilirubin

1.3

1.5-2.6 Ketosis type I from Ketosis type II

RQUICKI AST

-0.092

1.05-1.7

8.9

1.2-1.8

*: p < 0.05; **: p < 0.01; ***: p < 0.001.

Figure 1 - Prediction of ketosis I and II development and prediction of ketosis.

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Metabolic adaptation in first week after calving and early prediction of ketosis type I and II in dairy cows

Figure 2 - RQUICKI and AST in cows in ketosis I and II.

for total bilirubin. Cao et al.17 confirmed significance of glucose, AST and NEFA levels in evaluation of healthy and ketotic cows (GLU >3.04 mmol/L, AST <100 U/L, and NEFA <0.82 mmol/L). In listed experiments cut-off values are calculated on the moment of ketosis diagnosis no matter the type of ketosis. Unlike these studies, our experiment examined possibility of ketosis prediction based on values of blood parameters in first week after calving. Comparison of AUC value in our and mentioned studies showed that our model (that considers values of parameters in first week after calving) with similar efficiency determines ketotic cows just like model that includes single blood parameters (measured in the moment of ketosis diagnosis). Parity effect on metabolic parameters was not significant in our research. The lack of influence of parity is in compliance with the results found by Fiore et al.18. Cows in ketosis type I have higher concentrations of BHB and NEFA and lower concentration of insulin and glucose compared to control group. Ketosis type I is developed 3-6 weeks after calving as a consequence of reduced food intake. Experiment of Bjerre-Harpøth et al.19 showed significant increase of NEFA and reduction of insulin and glucose in cows that had food restriction in early lactation. Similar metabolic changes were found in our trial. More recent results show that decreased dry matter intake in the prepartum period is a significant predictor of ketosis in cows, with the additional finding that cows in ketosis had lower dry matter intake du-

ring postpartal period20. Our results are consistent with characteristics of ketosis I that are described in classification of the authors cited6-8. Cows in ketosis type II have higher concentration of BHB, total bilirubin and TNF-α compared to healthy cows and/or ketosis type I type group. Oetzel7 showed that fatty liver is the main characteristic of ketosis II type. Mostafavi et al.21 noted that concentrations of BHB, NEFA, AST and NEFA/cholesterol relation are very specific in evaluation of fat liver development in cows. The increase in AST and NEFA concentrations during the postpartum period is considered a suitable indicator of ketosis associated to hepatic steatosis or liver damage22,23. Recent studies showed that cows in ketosis have higher concentrations of TNF-α24. Aplication of ketoprofen as non-steroid anti-inflammatory drug in cows during early lactation decrease sign of liver inflammation and AST activity in blood25. Concentration of TNF-α was higher in monocyte cell culture, during cultivation with BHB26. High expression of TNF-α in the liver is upregulated by interleukin-8 (IL-8) and IL-1β secreted from placenta, which is liver-related metabolic disorder mechanism27. Excessive inflammation in periparturient period is in basis of ketosis in dairy cows28. Comparison of ketosis type showed that cows that developed ketosis type II had greater activity of AST and lover value of RQUICKI index compared to ketosis type I group of cows. Higher degree of insulin resistance (reflected in lower RQUICKI index) with higher AST level indicated development of ketosis II type. Chuang et al.11 founded higher concentrations of BHB, NEFA, glucose, bilirubin, AST and ALT in ketosis II type compared to ketosis type I. Xu et al.29 noted lower RQUICKI index in ketotic cows compared to control group. Djoković et al.3 showed that insulin resistance indexes that are measured in basal conditions significantly affect dynamical changes in concentrations of insulin, glucose and NEFA in ketotic cows. Possibility of early prediction of ketosis development and type of ketosis in cows by metabolic markers in first week after calving was proved in this research. In model of logistic regression given markers showed higher risk for ketosis development and question of correlation of these markers with

Table 3 - Coefficient of determination of significant ketosis predictors with BHB in first week after calving. Type 2

Healthy control

Ketosis type I

Ketosis type II

TNF-α

2.5%

15.2%

11.3%

T. bilirubin

6.4%

14.5%

12.6%

Insulin

8.4%

14.6%

16.4%

NEFA

9.1%

15.4%

18.5%

Glucose

2.4%

13.1%

15.7%

RQUICKI

6.3%

11.3%

13.8%

AST

5.2%

10.9%

11.7%

NS: p > 0.05; *: p < 0.05; **: p < 0.01.

Ketosis type I: Healthy control

Ketosis type II: Healthy control

Ketosis type I: Ketosis type II

** ** ** * ** ** **

* ** ** ** ** ** **

NS NS NS NS NS NS NS

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B. Delić et al. Large Animal Review 2020; 26: 51-55

BHB had appeared. Healthy cows have lower degree of commonly explained variation between BHB and parameters in healthy then in ketotic cows. That was founded in this research and indicates early adaptation of metabolism in ketosis function in cows. Results of Djoković et al.30 showed that in early lactation correlation of BHB with insulin and other indicators of functional status of liver is not in function of energetic balance in cows that indicates importance of BHB in metabolic adaptations of cows in early lactation.

10.

11.

12.

13.

CONCLUSIONS Cows in ketosis type I and II show different metabolic adaptations in first week after calving. These differences allow prediction of development a exact type of ketosis. Ketosis type I is determined by values of glucose, insulin and NEFA (lipolysis and insulin secretion). Ketosis type II is determined by values of BHB, TNF-α and bilirubin (ketogenesis, inflammation and functional status of liver). Differentiation of ketosis type I and II is possible by RQUICKI and AST (insulin resistance and hepatocytes damage). Proportion of the variation of metabolic parameters that is predictable from the BHB was higher in ketotic than in healthy cows. Metabolic adaptation in function of ketogenesis was developed in first week after calving, early before manifestation of ketosis.

14.

15. 16. 17.

18.

19.

20.

ACKNOWLEDGEMENT This research is supported by projects “Influence and clinical evaluation of blood serum tumor necrosis factor alpha (TNF-α) in inflammatory response of ruminants and dogs” - Provincial secretariat of high education and science, Vojvodina, Serbia.

21.

22.

23.

References 1. Benedet A., Manuelian C.L., Zidi A., Penasa M., De Marchi M. (2019). Invited review: β-hydroxybutyrate concentration in blood and milk and its associations with cow performance. Animal, 1-14. 2. Berge A.C., Vertenten G. (2014). 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, 97: 2145-2154. 3. Djoković R., Dosković V., Cincović M., Belić B., Fratrić N., Jašović B., Lalović M. (2017). Estimation of Insulin Resistance in Healthy and Ketotic Cows during an Intravenous Glucose Tolerance Test. Pak Vet J, 37: 387-392. 4. Rodriguez-Jimenez S., Haerr K.J., Trevisi E., Loor J.J., Cardoso F.C., Osorio J.S. (2018). Prepartal standing behavior as a parameter for early detection of postpartal subclinical ketosis associated with inflammation and liver function biomarkers in peripartal dairy cows. J Dairy Sci, 101: 8224-8235. 5. Holtenius P., Holtenius K. (1996). New aspects of ketone bodies in energy metabolism of dairy cows: a review. J Vet Med Series A, 43: 579-587. 6. Herdt TH. (2000). Ruminant adaptation to negative energy balance: Influences on the etiology of ketosis and fatty liver. Vet Clin North Am Food Anim Pract, 16: 215-230. 7. Oetzel G.R. (2007). Herd-level ketosis-diagnosis and risk factors. In Proceedings of the 40th annual conference of bovine practitioners, Vancouver, Canada. pp. 67-91. 8. Asl A.N., Nazifi S., Rowshan Ghasrodashti A., Olyaee A. (2011). Prevalence of subclinical ketosis in dairy cattle in the Southwestern Iran and detection of cutoff point for NEFA and glucose concentrations for diagnosis of subclinical ketosis. Prev.Vet. Med., 100: 38-43. 9. Sun Y., Wang B., Shu S., Zhang H., Xu C., Wu L., Xia C. (2015). Critical thresholds of liver function parameters for ketosis prediction in dairy

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cows using receiver operating characteristic (ROC) analysis. Veterinary Quarterly, 35: 159-164. Kayano M., Kataoka T. (2015). Screening for ketosis using multiple logistic regression based on milk yield and composition. J Vet Med Sci, 14-0691. Chuang X., Tai-yu S., Yuan Y., Hong-jiang Y., Cheng X, Hong-you Z. (2016). Blood clinicopathological differences between type I and II ketosis in dairy cows. Ind J Anim Res, 50: 753-758. Kessel S., Stroehl M., Meyer H.H.D., Hiss S., Sauerwein H., Schwarz F.J., Bruckmaier R.M. (2008). Individual variability in physiological adaptation to metabolic stress during early lactation in dairy cows kept under equal conditions. J Anim Sci, 86: 2903-2912. Cincović M.R., Belić B., Radojičić B., Hristov S., Ðoković R. (2012). Influence of lipolysis and ketogenesis to metabolic and hematological parameters in dairy cows during periparturient period. Acta veterinaria (Beograd). 62: 429-444. Belić B., Cincović M., Lakić I., Ðoković R., Petrović M., Ježek J., Starič J. (2018). Metabolic Status of Dairy Cows Grouped by Anabolic and Catabolic Indicators of Metabolic Stress in Early Lactation. Acta SciVet, 46: 9. National Research Council. (2001). Nutrient requirements of dairy cattle: 2001. National Academies Press. Holtenius P., Holtenius K. (2007). A model to estimate insulin sensitivity in dairy cows. Acta Vet Scand, 49: 29-31. Cao Y., Zhang J., Yang W., Xia C., Zhang H.Y., Wang Y.H., Xu C. (2017). Predictive value of plasma parameters in the risk of postpartum ketosis in dairy cows. J Vet Res, 61: 91-95. Fiore E., Piccione G., Arfuso F., Zumbo A., Gianesella, M. (2017). Metabolic changes in dairy cows at different lactation class during the transition period. Large Anim Rev, 23: 45-48. Bjerre-Harpøth V., Friggens N.C., Thorup V.M., Larsen T., Damgaard B.M., Ingvartsen K.L., Moyes K.M. (2012). Metabolic and production profiles of dairy cows in response to decreased nutrient density to increase physiological imbalance at different stages of lactation. J Dairy Sci, 95: 2362-2380. Pérez-Báez J., Risco C.A., Chebel R.C., Gomes G.C., Greco L.F., Tao S., Thompson I.M., do Amaral B.C., Zenobi M.G., Martinez N., Staples C.R., Dahl G.E., Hernández J.A., Santos J.E.P., Galvão K.N. (2019). Association of dry matter intake and energy balance prepartum and postpartum with health disorders postpartum: Part I. Calving disorders and metritis. J Dairy Sci, 102: 9138-9150. Mostafavi M., Seifi H.A., Mohri M., Jamshidi A. (2013). Optimal thresholds of metabolic indicators of hepatic lipidosis in dairy cows. Rev Méd Vét, 164: 564-571. Fiore E., Barberio A., Morgante M., Rizzo M., Giudice E., Piccione G., Marcello L., Gianesella M. (2015). Glucose infusion response to some biochemical parameters in dairy cows during the transition period. Anim Sci Pap Rep, 33: 129-136. Fiore E., Perillo L., Piccione G., Gianesella M., Bedin S., Armato L., Giudice E., Morgante M. (2016). Effect of combined acetylmethionine, cyanocobalamin and α-lipoic acid on hepatic metabolism in high-yielding dairy cow. J Dairy Res, 83: 438-441. Zhang G., Hailemariam D., Dervishi E., Goldansaz S.A., Deng Q., Dunn S.M., Ametaj B.N. (2016). Dairy cows affected by ketosis show alterations in innate immunity and lipid and carbohydrate metabolism during the dry off period and postpartum. Res Vet Sci, 107: 246-256. Kovačević Z., Belić B., Cincović M.R., Stojanac N., Stevančević O., Erdeljan M., Davidov I., Stojanovic, D. (2019). Effects of Ketoprofen Administration on Relation between Acute Phase Proteins and Metabolic Parameters in Cows during Early Lactation. Acta Sci Vet, 47: 1663. DOI: 10.22456/1679-9216.91667. Jain S.K., Kannan K., Lim G., McVie R., Bocchini J.A. (2002). Hyperketonemia increases tumor necrosis factor-α secretion in cultured U937 monocytes and type 1 diabetic patients and is apparently mediated by oxidative stress and cAMP deficiency. Diabetes, 51: 2287-2293. Loor J.J., Dann H.M., Everts R.E., Oliveira R., Green C.A., Guretzky N.A.J., Rodriguez-Zas S.L., Lewin H.A., Drackley J.K. (2005). Temporal gene expression profiling of liver from periparturient dairy cows reveals complex adaptive mechanisms in hepatic function. Physiol. Genomics., 23: 217-226. Mezzetti M., Minuti A., Piccioli-Cappelli F., Amadori M., Bionaz M., Trevisi, E. (2019). The role of altered immune function during the dry period in promoting the development of subclinical ketosis in early lactation. J Dairy Sci, 102: 9241-9258. Xu C., Shu S., Xia C., Wang B., Zhang H., Jun B. (2014). Investigation on the Relationship of Insulin Resistance and Ketosis in Dairy Cows. J Veterinar Sci Technol, 5: 162. doi:10.4172/2157-7579.1000162. Djoković R., Cincović M., Belić B., Toholj B., Davidov I., Hristovska, T. (2015). Relationship between blood metabolic hormones, metabolites and energy balance in Simmental dairy cows during peripartum period and lactation. Pak Vet J, 35: 163-167.

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C.A. Sgoifo Rossi et al. Large Animal Review 2020; 26: 57-64

Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration, health, growth performance and meat quality

CARLO ANGELO SGOIFO ROSSI1, SILVIA GROSSI1, RICCARDO COMPIANI1, GIANLUCA BALDI1, MARIA AGOVINO2, LUCIANA ROSSI1 1

University of Milan, Faculty of Veterinary Medicine, Department of Veterinary Science for Health, Animal Production and Food Safety, Milan 20133, Italy 2 European ruminant manager, Alltech, Casalecchio di Reno 40033, Italy

SUMMARY The effects of different mineral sources (organic vs inorganic) on beef cattle serum Se, Zn, Cu, Mn concentration, health, growth performance and meat quality (hot carcass weight, dressing percentage, conformation score, fattening score and pH) were assessed in 156 intact Charolaise males. The aim of the study was to investigate a possible ameliorative action of the organic sources of those minerals compared to the inorganic ones, essential for animal welfare and productivity. The animals were allotted to three treatment groups, receiving different sources (organic or inorganic) of minerals at different levels. H-In group received a mineral supplementation from an inorganic source according to the average mineral inclusion recommended by the European beef system. The TRT group received an organic source of minerals at the doses recommended by the producer (TRT, Total Replacement Technology, Alltech). The In group received minerals from an inorganic source, but following the same doses employed in the TRT group. Growth performance and health status were monitored throughout the fattening period (186 days). Blood samples were evaluated in relation to mineral serum concentrations, antioxidant status and immunity reactions. After slaughtering, carcass characteristics and meat quality of Longissimus dorsi samples were evaluated. Growth performance and health status were improved in the TRT group (P<0.05). The incidence of Bovine Respiratory Disease was also lower in this group. The animals in the TRT group had a better immune response (P<0.05) due to the higher circulating mineral concentrations which positively affected the immune function. In addition, carcass characteristics were positively affected in the TRT group because of the enhanced myogenesis (P<0.05). Meat quality was improved in the TRT group due to the higher level of antioxidants in the meat (P<0.05). The results suggest that organic sources have a strong impact on animalsâ&#x20AC;&#x2122; metabolism and immune function, which led to an improvement in growth performance, health and antioxidant status together with carcass and meat quality.

KEY WORDS Cattle, minerals, performance, health, meat quality.

INTRODUCTION Intensive beef cattle production is mainly based on fattening weaned young cattle, which have been moved from the farms of origin. In Europe, these farms are often located in other countries, which are specialized in extensive suckler cow breeding. In Italy young cattle are mainly imported from France. This procedure causes several stressors other than weaning, such as long-distance transport, group mixing, feed and water restrictions. Young cattle also have to adapt to a new environment and feeding conditions in the fattening units1. All these factors promote immunosuppression which can lead to pathogen colonization and proliferation, and cause various diseases. Bovine respiratory disease (BRD) is currently the most common one. In stressful situations, se-

Corresponding Author: Silvia Grossi (silvia.grossi1994@libero.it).

veral nutritive substances such as minerals become essential and supplementation is recommended1. Mineral supplementation can help cattle recover quickly from transport fever and reactivate ruminal activity, thereby limiting oxidative stress and promoting the reactivity of their immune system2. Supplementation of the main micro-minerals is also required in the fattening period in order to satisfy the animalsâ&#x20AC;&#x2122; requirements and optimize ruminal and metabolic functions; consequently, rumen efficiency and growth performance improve. Several events can lead to pro-inflammatory conditions in fattening cattle, particularly when the diet is characterized by energy levels that enhance the risk of sub-clinical acidosis and connected pathologies3,4. Mineral supplementation can limit these problems and help the cattle recover quickly, reducing the negative effect of stress on animal performance and growth. Trace minerals such as zinc (Zn), copper (Cu), manganese (Mn) and cobalt (Co), as well as selenium (Se), play an important role in stress, health and growth responses in cattle5. Normally, they are required in very small

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Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration

amounts. However, under stressful situations and in farms with high production standards, they are needed in increasing amounts. Consequently, supplementing the diets with these minerals is necessary to avoid deficiencies. The type of mineral sources used also has different biological effects. Minerals can in fact be provided as inorganic mineral salts or in organic forms such as chelates or enriched yeast. Due to the higher bioavailability of minerals, supplementing organic complexed minerals, such as Zn, Se, Cu, Mn, Co, may be of further benefit based on their enhanced absorption, retention and biological activity compared to inorganic sources6. The hypothesis was that the use of organic mineral will lead to an improvement in growth performance and health status of highly stressed cattle in contrast to inorganic supplementation at different levels, due to the higher bioavailability of organic minerals and their immune and antioxidant abilities, since little is known about the effects of organic vs inorganic sources of several basic minerals such as selenium, zinc, copper and manganese. The aim of the trial was to evaluate the effects of different dietary mineral sources (organic or inorganic) and doses on absorption, health, growth performance and meat quality.

Table 1 - Mineral and vitamin supplementation. Nutrient

H-In

TRT

Mn, mg/d

550

180

Cu, mg/d

250

140

Zn, mg/d

1000

280

Se, mg/d

TRT and IN Almost in line with NCR requirements and according to manufacturing recommendations. H-In comes from an average of several mineral mixes used in Europe and with levels of trace minerals similar to that recommended by NRC for stressed animals.

neral inclusion recommended by the European beef system. Animals in the TRT group received an organic source of minerals at the doses recommended by the producer (TRT, Total Replacement Technology, Alltech). Animals in the inorganic (In) group received minerals from an inorganic source at the same doses employed in the TRT group the same minerals as the inorganic source but following the same amount as the TRT group.

PARAMETERS MATERIAL AND METHODS Nutritional value of the diets Animals and Farm The study was carried out in an intensive beef fattening unit in northern Italy and involved 156 intact Charolaise males. The animals were monitored from their arrival from France throughout the entire fattening period of 186 days. Upon arrival, animals were submitted to a typical prophylaxis protocol: vaccination against respiratory diseases, antibiotic treatment if necessary, and treatment against endo- and ecto-parasites.

Samples of the experimental diets (100 g/each) were collected every two weeks, pooled and analysed in relation to dry matter, crude protein, ether extract, starch and ash, according to the AOAC (1990) methods6. Neutral detergent fibre was determined according to Van Soest et al. (1991)8. The net energy content of the diets, was calculated using the reference values for all feed ingredients reported by NRC (2016)2.

Minerals titration in TMR Animal assignment to treatment groups Three trucks of animals arrived simultaneously. The animals from each truck were randomly allotted to the three different treatment groups in order to avoid the bias due to the batch effect. Each truck was considered as a block, the random assignment to one of the three treatment groups was performed according to the order of that the animals went into the weighing chute.

Feed macro- and trace mineral concentrations (Zn, Se, Mn, Cu) were determined by standard wet chemistry techniques using the titration procedure. The titration analysis consists in an inductively coupled plasma emission spectroscopy. The selenium concentration was determined using fluoro-metric methods, according to AOAC (1990)6.

Growth performance and health Housing and feeding The cattle were group-fed and housed in pens of 5-6 animals each on a slatted floor. The animals from the three experimental groups were submitted to the same nutritional management and diet. During the entire fattening period two diets were administered, based on the same feedstuffs: silomais, corn meal, brewers, wheat straw, wheat bran, sunflower meal, rice bran, coconut cake, DDGS (distillers dried grains solubles) and rape cake. The first diet was formulated for the adaptation phase (0,68 Mcal/kg; 13,20 CP %DM; 34,73 Starch %DM; 4,40 EE %DM) and the second for the fattening phase (0,89 Mcal/kg; 14,66CP %DM; 46,68 Starch %DM; 4,69 EE %DM). The diets differed only for the mineral premixes employed as reported in Table 1. Animals in the H-In group received a mineral supplementation from an inorganic source according to the average mi-

On day 0 (arrival), and at days 21, 54 and 186, before the morning feeding, the cattle were individually weighed and the average daily gain (ADG) was calculated. Once a week the average daily feed intake (ADFI) was calculated: before further feed administration, the feed intake was recorded by weighing the TMR offered and the residue in the manger 24h later. The pen feed conversion ratio (FRC) was calculated. Animals were inspected twice a day by the farm veterinary staff for sickness, including BRD symptoms. Animals were considered affected by BRD if the rectal temperature was â&#x2030;Ľ40.0Â°C and if both the depression and respiratory character scores differed from the normal health status - score 0 of Baggot et al., 20119. The animals diagnosed as affected by BRD were treated appropriately and registered. The mortality was also recorded.

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Blood parameters Blood samples were collected before the morning feeding from 14 animals on days 0, 21 and 105 to evaluate the immune status. Blood samples were collected by jugular venipuncture. Immunity reaction was evaluated by a BHV-1 (Bovine Herpesvirus-1) serum neutralization test, performed according to OIE (2012)10. The neutralizing antibody titre was measured against the homologous vaccine virus. Antibodies were titrated by the constant virus-varying serum dilution from ½ onward. The data of the neutralizing antibody titres were transformed as log(1/x), where x was the serum dilution. Serum bactericidal activity was performed according to Amadori et al. (1997)11. Blood samples were also collected on days 0, 21, 105 and 186 to evaluate mineral (Se, Zn, Cu and Mn) serum concentrations and antioxidant status. Blood samples were collected by jugular venipuncture into 10 ml EDTA tubes and 10 ml glass tubes and immediately cooled in ice. Two tubes were collected for each animal, one was used for serum extraction and one for the evaluation of the antioxidant condition. Serum samples were obtained by centrifugation at 3000 rpm for 10 min at 4°C and stored at -20°C. Mineral concentrations were determined in serum samples using an inductively coupled plasma source. The antioxidant condition was evaluated using glutathione peroxidase (GPx activity) titration. GPx activity was measured indirectly by a coupled reaction with glutathione reductase (GR), using the glutathione peroxidase assay kit12.

concentration of minerals (Se, Zn, Cu, Mn) was then determined by flame atomic absorption spectrometry, using a spectrometer. Drip loss analysis was carried out following Honikel (1998)14. After the meat samples were cut from the carcass, they were immediately weighed and placed in the netting and then suspended in an inflated watertight nylon bag. The meat samples were stored at chill temperatures (4°C). In the following seven days, the samples were taken out of the bags, dried by a paper towel and then weighed again. The drip loss was expressed as a percentage of the initial weight. Every day the instrumental colour parameter, such as L (lightness), a (redness), b (yellowness) were measured for seven days, using a tristimulus colorimeter. Before each measurement, the colorimeter was standardized. Meat cooking loss was estimated by the Shilling method15. The samples were weighed and cooked, achieving a temperature of 75°C inside. The samples were then weighed again. Cooking loss was expressed as a percentage of the initial weight. Thawing loss was also assessed, evaluating the difference in weight before and after thawing. The thawing loss was expressed as a percentage of the initial weight. The tenderness of the samples was also examined, using the WarnerBratzler Shear Force procedure, which measures the force required to cut a piece of meat, according to the AMSA guidelines (1995)16. The shelf life of the different samples was also evaluated.

Statistical analysis Carcass characteristics and meat quality The main carcass characteristics (hot carcass weight, dressing percentage, conformation score, fattening score and pH) were also evaluated at the slaughterhouse. Hot carcass weight (HCW) was obtained after the removal of the head, hide, intestinal tract and intestinal organs. Dressing percentage is the percentage of the animals that ends up as a carcass, and was computed from the following formula: Dress % = (HCW ÷ live weight) × 100% The carcasses were classified according to the official carcass classification scheme, which includes carcass conformation and fattening score. The carcass conformation score was evaluated using the SEUROP classification. The fattening score was evaluated using a 1 to 5 scale (1 - very low, 5 - very high), according to the EU Beef Carcass Classification Scheme. The pH was measured 24h post-mortem. In addition, meat quality was evaluated in 10 longissimus dorsi samples per experimental group. 2,5 cm thick steaks were cut from the ribeye (between the 12th thoracic and 5th lumbar vertebra) to evaluate centesimal composition, meat mineral continent, drip loss, instrumental colour, thawing loss, cooking loss, tenderness and shelf life. The samples were examined for the centesimal composition: moisture, ash, lipid and protein composition were determined, according to official methods6. Before analysing the meat mineral content, the muscle samples were digested using an ETHOS 900 microwave digestion system. The sample digestion procedure was performed according to the NF EN 13805 standard “Foodstuffs Determination of trace elements -Pressure digestion”13. The

Data were analyzed with the GLM (General Linear Model) procedure of SAS. Student “t” and Tukey tests were used to compare the means of each group. The level of significance to indicate differences stated in the ANOVA model were P<0.05; levels of P≤0.1 were considered a tendency. The d0 values were used as a covariate in the statistical analysis. Weight at d0 was set as a covariate for growth performance and hot carcass weight; meat samples weight at d0 was set as a covariate for meat weight and drip loss.

RESULTS AND DISCUSSIONS Mineral titration in TMR Mineral titration in the different experimental TMR diets, in all the different experimental diets, are reported in Table 2. All the mineral concentrations were higher in the H-IN group, both in the adaptation and the fattening diets. In fact, the mineral quantities supplemented were higher in the H-In group.

Growth performance and health condition The cattle growth performances are reported in Table 3. Average live weight was higher for the TRT group compared to the inorganic (In) group starting from d21 until the end of the fattening cycle. TRT fed cattle showed a higher live weight compared to H-In at d105 and d186. At d186 a statistical difference was also found between H-In and In live weights. During the critical moment of the adaptation phase, the administration of minerals from an organic source led to a better ADG compared to animals fed with the same amount of minerals but from an inorganic source (ADG d0-21 TRT vs

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Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration

Table 2 - Mineral titration in TMR. Mineral concentrations

H-In adaptation

H-In fattening

TRT adaptation

TRT fattening

IN adaptation

IN fattening

Se mg/kg DM

0.79

0.54

0.62

0.49

0.65

0.46

Zn mg/kg DM

184.24

124.52

82.39

60.12

80.94

62.45

Cu mg/kg DM

43.66

28.64

26.52

21.08

27.88

19.16

Mn mg/kg DM

111.39

68.17

57.39

34.12

58.3

36.28

Table 3 - Growth performance.

nÂ°

Table 4 - Health status.

H-In

TRT

H-In

Weight, kg d0

430.48

d21

456.19

d105

d186

576.16

704.12

458.04

586.22

714.87

13.46 (7)

5.77 (3)

19.23a (10)

BRD relapses, % (n)

9.61a (5)

0.00b (0)

Lameness, % (n)

1.92 (2)

1.92 (1)

Mortality, % (n)

5.77 (3)

0.00 (0)

572.60

a, b, c

BRD first pull, % (n)

454.80

TRT

on the same raw: P<0.05.

693.54

ADG, kg/d 0-21

1.224

1.312a

1.158b

21-105

1.445b

1.544a

1.419b

105-186

1.530a,x

1.578a,y

1.476b

0-186

1.473b

1.531a

1.416c

0-21

6.86

7.36a,x

6.57b

AFI, kg/d

21-105

10.24

10.29

10.20

105-186

11.62

0-186

10.44

10.52

10.39

FCR, kg/d 0-21

5.60

5.61

5.67

21-105

7.09

6.66

7.19

105-186

7.59

7.36

7.87

0-186

7.09

6.87

7.34

a, b, c

on the same row: P<0.05. x, y, z on the same row: P<0.1.

such animals may return to normal health quite rapidly, for several days they may reduce their feed consumption and consequently their weight gain. In fact, there is a strong relation between health condition, feed intake and ADG. This delay in growth in the first part of the fattening cycle is rarely compensated in the following weeks a statistical difference in the overall ADG was in fact found between TRT and the inorganic mineral source at the two levels examined in this study. These results are in line with Richeson and Kegley who found that highly stressed, newly received cattle treated with supplemental trace minerals had a reduced influence of BRD and an improvement in ADG18,19. As reported in Table 4, no difference was detected regarding the incidence of lameness which was generally low and in line with the average prevalence in this farming system in well-managed beef cattle farms4. On the other hand, a higher mortality was found in H-In group though even with no statistical difference. Two out of the three animals died because they failed to recover from BRD after several relapses and one because of enterotoxaemia.

Blood parameters IN; P<0.05). The same growth results were achieved only by increasing the level of inorganic supplementation. These findings could be related to the improvement in ruminal and immunity functions promoted by a higher bioavailability of some basic minerals administered such as selenium, zinc, copper and manganese17. In fact, the animals in the TRT group showed a better health status during adaptation. Data in Table 4 reports how animals supplemented with minerals from the inorganic source showed a higher incidence of respiratory disease (BRD first pull: In 19.23% vs TRT 5.77%; P<0.05), while those supplemented with a high level of inorganic minerals presented a higher incidence of BRD relapses (BRD relapses: H-In 9.61% vs TRT 0%; P<0.05). The higher incidence of BRD in the inorganic (In) group influenced the weight gain in the adaptation phase because of a reduction in feed intake as reported in Table 3. During the adaptation phase, several animals incurred mild undetected forms of BRD but recovered spontaneously and efficiently through the natural immune defense. Although

The better health condition shown by TRT group animals was confirmed by a better immune reaction after vaccination. Micronutrients such as Se, Zn, Cu, Mn are involved in maintaining the correct functionality of the immune system. Supplementation with these nutrients can improve the functionality of the immune system and also the production of antibodies. Arthington and Havenga found that the use of an injectable solution of trace minerals (Cu, Zn, Mn, and Se) at the same time as the vaccination, enhanced the antibody response to Bovine Herpes Virus 120. In their study, the animals were vaccinated on arrival against Bovine Herpes Virus 1 of Infectious Bovine Rhinotracheitis among other pathogens. Since in France no animals are vaccinated and the virus has almost been eradicated, this parameter is a good index to evaluate the immune reaction. In our study, as expected, on arrival the animals presented no serum antibody (Figure 1). At d21, the TRT animals showed a higher presence of antibodies compared to the In group, and a trend for a difference was also found at d105. The activity and functionality of the

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C.A. Sgoifo Rossi et al. Large Animal Review 2020; 26: 57-64

Figure 1 - Specific immunity serum indicator. a, b: P<0.05 - x, y: P<0.1.

immune system, under different mineral supplementations, was evaluated with the seroneutralization test, as reported in Figure 1. This test is used to quantify the titer of neutralizing antibodies for a virus, in this case for BHV-1. The graph shows an improvement in antibody production in the TRT group, which may reflect a higher lymphocyte B activity, due to a higher GSH-Px activity. This result could be due to the higher bioavailability of organic minerals, especially of Se, supplemented in this group. These results agree with the findings of Nicholson et al. and Arthur et al.21,22. Indeed, selenium supplementation improves the antioxidant status and especially gluthatione peroxidase activity. Organic selenium sources have better effects on the antioxidant status. Calves supplemented with organic sources of selenium (Se-yeast) have been shown to have better in vitro macrophage phagocytosis compared with Na selenite supplementation23. As reported by Arthur et al. (2003)20, Se improves the vitality and activity of the immune cells, including lymphocyte B. The better immune reaction after vaccination is likely due to the higher serum availability of minerals which are essential for the immune system. In fact, as reported in Table 5, starting from d21, the circulating mineral concentration of selenium, zinc, copper and manganese was higher in the TRT group compared to the In and H-In groups. These findings are in line with other studies. Chirase found that calves supplemented with Zn-methionine tend to recover from diseases more rapidly than calves supplemented with ZnO, due to the higher effect of the organic source used24. Also, in steers challenged with infectious bovine rhinotracheitis virus, supplementation with Zn-methionins has been shown to increase the feed intake and lower the rectal temperature, compared with the control. These are signs of a better immune function25. Copper levels are also involved in the immune response to bovine herpes virus 1. Arthington found that copper deficiency altered the acute-phase protein response in cattle in

Table 5 - Mineral bioavailability.

nÂ°

H-In

TRT

Se, g/L d0

31.36

30.46

31.15

d21

26.48

30.65

27.46b

d105

31.83

33.15a

31.05b

d186

31.39

32.59a

30.14b

Zn, mol/L d0

11.58

11.77

11.79

d21

13.57b

15.46a

12.63b

d105

13.41y

14.83x

13.44y

d186

13.31

14.55

13.26b

7.08

7.28

7.14

d21

8.54b

9.88a

d105

d186

Cu, mol/L

7.39c

9.05

11.06

8.88b

9.11b

11.54a

8.63b

Mn, g/L d0

2.57

2.50

2.52

d21

2.92

3.34

2.79b

d105

3.53b

3.93a

3.03c

d186

3.58b

4.08a

3.18c

GSH-Px, U/g Hg

a, b, c x, y, z

176.57

176.43

174.57

d21

174.86

183.19

175.57b

d105

164.27b

177.57a,x

170.68y

d186

168.71b,z

177.93a,x

173.50y

on the same row: P<0.05. on the same row: P<0.1.

Sgoifo Rossi_imp_ok 24/04/20 13:40 Pagina 62

Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration

terms of infection20. Copper is a component of ceruloplasmin, one of the acute phase proteins. The average serum level was higher with the same trend also throughout the fattening cycle. Serum minerals levels of animals supplemented with high levels of inorganic minerals never reached those of the TRT group but were higher compared to the In group for Cu at d21 and Mn at d105 and d186. The body’s ability to react to oxidative stress was higher in the TRT group. As reported in Table 5, glutathione peroxidase activity (GSH-Px) was higher in the TRT group starting from d21, with a similar trend to the observed serum selenium concentration. This effect could be attributed to the supplementation with an organic form of selenium, which is more available and has a greater effect on the oxidant status. In fact, selenium is a component of glutathione peroxidase, an important water-soluble antioxidant. This data agrees with Juniper et al., who found higher blood glutathione peroxidase and higher glutathione peroxidase activities in beef cattle fed with organic Se, compared to those fed sodium selenites. Selenium deficiency affects the neutrophil bactericidal activity26. A deficient Se status is associated with a decline in the immune response against infectious diseases, because of this decreased neutrophil bactericidal activity17.

Table 6 - Carcass characteristics.

n°

The results of slaughtering performance and meat quality seem to be related to animals’ live performance, health status and blood parameters. As reported in Table 6, animals’ hot carcass weight was statistically different with the same ADG trend but better results were found in the TRT group. More carcasses from the inorganic (In) group were assigned to a lower conformation category compared to the H-In and TRT groups. These findings are closely related to animal health. In fact, several authors have reported a worsening in slaughtering performance for animals with BRD27. Regarding the fattening score, the results are in contrast with Gardner et al. 199928, who reported a lower cover fat in animals with BRD. In the present study, the higher bioavailability of minerals may have improved both the metabolic and ruminal activities, enhancing myogenesis rather than adipogenesis, in agreement with the observed improved ADG. In the present experimental conditions, supplementation with organic minerals seemed to lead to leaner carcasses than the other two groups. Meat mineral contents were consistent with the different levels of bioavailability of minerals with higher levels of Se, Zn, Cu and Mn in the TRT group compared to the inorganic supplemented animals, as reported in Table 7. H-In samples did not obtain the mineral levels of the TRT group but were higher compared to the inorganic group. The absence of an effect of mineral sources on meat centesimal composition is consistent with other studies on fattening cattle29,30. The potential different antioxidant action from the different supplemented minerals influenced the meat characteristics. TRT supplementation statistically improved the meat drip loss, color and overall shelf life. These findings are a consequence of the improved stability of cellular membranes due to higher antioxidant properties. This increase in muscular antioxidant ability is consistent with the findings of several authors30. During ruminal de-

TRT

Hot carcass weight, kg 419.46b

426.33a

411.39c

Dressing percentage, % 59.57

59.63

59.32

Conformation, % (n°) S

0.00 (0)

93.88a (46)

94.23a (49)

76.92b (40)

4.08 (2)

5.77 (3)

23.08a (12)

2.04 (1)

0.00 (0)

Fattening score, % (n°) 5

0.00 (0)

18.37a,x (9)

5.77b (3)

28.84a,y (15)

b,x

81.63

Carcass characteristics and meat quality

H-In

(40)

0.00 (0)

94.23 (49)

71.15b,y (37)

0.00 (0)

a, b, c

on the same row: P<0.05. x, y, z on the same row: P<0.1.

Table 7 - Meat chemical composition.

n°

H-In

TRT

5.71

5.74

pH 5.74

Centesimal composition Humidity, %

73.35

73.05

73.44

Fat, %

2.52

2.59

Protein, %

23.14

23.39

23.01

Ash, %

0.96

0.98

0.97

Meat mineral content Se, mg/100 g DM

0.092b

0.133a

0.069c

Zn, mg/100 g DM

16.41b

17.95a

11.90c

Cu, mg/100 g DM

0.29

0.32

0.19b

Mn, µ/100g DM

29.44b

31.95a

20.99c

a, b, c

on the same row: P<0.05.

gradation and intestinal digestion, organic minerals are submitted to different chemical processes from inorganic minerals, leading to higher absorption and storage at several tissue levels. The increase in muscular antioxidant activity might have reduced the extent of enzymatic and myofibrillar protein oxidation, thus promoting higher membrane stability. This results in better water holding with a lower drip loss in TRT meat samples, as reported in Table 8. This hypothesis is supported by Rowe et al.31, who reported that dietary antioxidant supplementation in beef (vitamin E) reduced protein oxidation, and positively promoted post mortem pro-

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C.A. Sgoifo Rossi et al. Large Animal Review 2020; 26: 57-64 Table 8 - Drip loss.

Table 9 - Meat color. H-In

TRT

H-In

TRT

nÂ°

135.32

45.68b

49.42a

42.00c

132.78b

133.49a

132.64b

45.01b

48.86a

42.56c

131.02

131.96

130.91

43.05

47.98

40.53c

128.50b

129.81a

128.68

38.69b

46.97a

35.62c

125.05b

126.27a

126.04

38.80b

45.75a

35.69c

34.32b

40.54a

32.93b

34.60

32.62b

Meat weight, g

Lightness, L*

Drip loss, %

a, b, c

1.93

1.43

2.15

d1-3

1.32

1.17

1.36

d3-5

2.01

1.72

1.90

23.88b,y

25.71a

25.20x

d5-7

2.72a

2.77a

2.10b

23.69

23.10b

24.79a

d1-7

20.85

21.72

17.53b

19.80

21.11a

17.57b

17.32b

20.35a

15.40c

12.69b

16.71a

11.21b

13.52

11.70c

13.71b

14.78a,x

13.97y

13.50b

14.46a,x

13.80y

7.99

7.10

Redness, a*

7.51

on the same row: P<0.05.

teolysis. In our study, higher oxidant stability led to a better meat color from the first day after cutting and in the following seven days, as reported in Table 9, with a better lightness, redness and yellowness index. An improvement in lightness due to the supplementation of organic selenium was also reported by Cozzi et al.30, who found an increased L* value after 6 and 11 days of vacuum packaged ageing. Taylor et al. (2008)29 also found that meat from cattle fed a selenium-enriched diet had a higher Se content and tended to have a higher average a* and b* during 12 days on the shelf, compared with animals fed an unenriched diet. In addition, in our study TRT meat samples showed a better overall shelf life of more than one day compared to In and H-In groups (Table 10). Several statistical differences were also found between the H-In and In groups. H-In meat samples were characterized by better lightness from d1 to d5, redness at d1, d3, d5 and d7, yellowness at d5 and d7 and shelf life. These findings demonstrate that the mineral supplementation level also improves meat quality. Some improvements with a high inorganic mineral supplementation are possible compared to a low supplementation but not as high as the level achieved with the organic supplementation. No statistical differences were observed regarding thawing loss, cooking loss and tenderness as reported in Table 10.

a, b, c

12.37

13.72

11.73b

11.77b

13.65a

11.88b

9.11b

13.23a

6.82c

7.74b

10.92a

6.48b

10.67

5.97c

H-In

TRT

6.10a

4.54b,y

7.42

on the same row: P<0.05.

Table 10 - Meat quality.

nÂ°

Shelf life, d b,x

5.00

Thawing loss, % 1.72

1.62

1.72

Cooking loss, % 34.29

33.79

33.89

WBSH, kg/cm

a, b, c x, y, z

Under our study conditions, the supplementation of minerals from an organic source to beef cattle, led to better animal health conditions and growth performance that probably relates to the high bioavailability of essential cofactors of basic metabolic and immune processes. These conditions also led to an improvement in meat quality. The administration of minerals from inorganic sources can lead to good but not excellent farming results only if administered at high concentrations.

16.87

Yellowness, b*

3.36

CONCLUSIONS

39.70

3.32

3.31

on the same row: P<0.05. on the same row: P<0.1.

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Effects of different mineral supplementation programs on beef cattle serum Se, Zn, Cu, Mn concentration

3. Sgoifo Rossi C.A., Compiani R., 2016. Ruminal acidosis of beef cattle and related diseases. Large Animal Review 22(6), 273-279, Record Number: 20173048552. 4. Compiani R., Sgoifo Rossi C.A., Baldi G., Desrochers A., 2014. Dealing with lameness in Italian beef cattle rearing. Large Animal Review 20, 239-247 - ISSN 1124-4593. 5. Spears J.W., 2000. Micronutrients and immune function in cattle. Proceedings of the Nutrition Society 59, 587-594, https://doi.org/10.1017/ S0029665100000835. 6. Marquez R.S., Cooke R.F., Rodriques M.C., Cappeloza B.I., Larson C.K., Moriel P., Bohnert D.W., 2016. Effects of organic or inorganic Co, Cu, Mn, and Zn supplementation to late gestating beef cows on productive and physiological responses of the offsprings. J. Anim. Sci. 94, 1215-1226, https://doi.org/10.2527/jas.2015-0036. 7. AOAC, 1990. Official methods of analysis of the AOAC, 15th ed. Methods 932.06, 925.09, 985.29, 923.03. Association of official analytical chemists. Arlington, VA, USA. 8. Van Soest P.J., Robertson J.B., Lewis B.A., 1991. Methods for dietary fiber, neutral detergent fiber and nonstarch polysaccharides in relation to animal nutrition. J. Dairy Sci. 74, 3583-3597, 10.3168/jds.S00220302(91)78551-2. 9. Baggott D., Casartelli A., Fraisse F., Manavella C., Marteau R., Rehbein S., Wiedemann M., Yoon S., 2011. Demonstration of the metaphylactic use of gamithromycin against bacterial pathogens associated with bovine respiratory disease in a multicentre farm trial. Vet. Rec. 168(9), 241. https://doi.org/10.1136/vr.c6776. 10. OIE, 2012. Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. Seventh Edition. 11. Amadori M., Archetti I.L., Frasnelli M., Bagni M., Olzi E., Caronna G., Lanteri M., 1997. An immunological approach to the evaluation of welfare in Holstein Frisian Cattle. J. Vet. Med. Serb. B44, 321-327, https://doi.org/10.1111/j.1439-0450.1997.tb00982.x. 12. Cigliano L., Strazzullo M., Rossetti C., Grazioli G., Auriemma G., Sarubbi F., Iannuzzi I., Spagnuolo M.S., 2014. Characterization of blood redox status of eary and mid-lactating dairy cows. Czech I. Anim. Sci. 59 (4), 170-181, https://doi.org/10.17221/7341-CJAS. 13. Millour S., Noe L., Kadar A., Chekri R., Vastel C., GuĂŠrin T., 2011.Simultaneous analysis of 21 elements in foodstuffs by ICP-MS after closed-vessel microwave digestion: Method validation. Journal of Food Composition and Analysis 24, 111-120, https://doi.org/10.1016/j.jfca. 2010.04.002. 14. Honikel K.O., 1998. Reference methods for the assessment of physical characteristics of meat. Meat Sci. 49, 447-457, https://doi.org/10.1016/ S0309-1740(98)00034-5. 15. Schilling E., 1996. Muskelstructure und Fleischqualitat. Tierzucht und zuchtsbiologie 2, 219-243. 16. AMSA, 1995. Research Guidelines for Cookery, Sensory Evaluation, and Instrumental Tenderness Measurements of Fresh Meat. Amer. Meat Sci. Assoc., Chicago, IL. 17. Enjalbert F., Lebreton P., Salat O., 2006. Effects of copper, zinc and selenium status on performance and health in commercial and dairy beef herds: retrospective study. J. Anim. Physiol. Anim. Nutr. 90, 459-466, https://doi.org/10.1111/j.1439-0396.2006.00627.x. 18. Richeson J.T., Kegley E.B., 2011. Effect of supplemental trace minerals

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B. Ekiz et al. Large Animal Review 2020; 26: 67-72

The effect of final weight on slaughtering and carcass quality characteristics of lambs in concentrate-based or pasture-based production systems

BULENT EKIZ, ALPER YILMAZ, HULYA YALCINTAN, OMUR KOCAK, MUSTAFA OZCAN Department of Animal Breeding and Husbandry, Istanbul University-CerrahpasĚ§a, Faculty of Veterinary Medicine, 34320 Avcilar, Istanbul, Turkey

SUMMARY In lamb finishing for meat production, determination of optimal slaughter weight is an important issue for the provision of a profitable and high-quality lamb production. Aim of the study was to investigate the effect of final weight (LOW: 25-26 kg, MEDIUM: 30-31 kg and HIGH: 35-36 kg) on the slaughtering and carcass quality characteristics of Kivircik lambs, which were finished with concentrate-based diet (CON) or on pasture (PAS). CON lambs (n = 27) were weaned at 76-d and then finished on concentrate feed and alfalfa hay until slaughter in sheepfold. PAS lambs (n = 25) were on native pasture in the day-time with their mothers and allowed to suck their mothers until the slaughter. In the CON system, commercial and real dressing percentages were lower in the LOW group compared to MEDIUM and HIGH groups. In MEDIUM and HIGH weight groups, CON system yielded higher commercial dressing than PAS system. CON lambs had higher liver percentage than PAS lambs in all final weight groups. LOW lambs had lower values for carcass measures and indices, except chest roundness index than HIGH lambs. CON lambs a had higher buttock width and circumference, carcass width, carcass compactness and chest roundness index in all final weight groups. Lambs from LOW group in PAS system had a higher yellowness than that in CON system. In conclusion, CON system can be preferred to produce a higher amount of lamb meat in a shorter time. Final weight of 35-36 kg might be preferred in both production systems to increase lamb production, without any adverse effect on carcass quality.

KEY WORDS Carcass quality, fat colour, lamb, production system.

INTRODUCTION Most of the sheep farms in Turkey are small-scale family farms as in other the Middle East and some of the Mediterranean countries. Sheep farming is usually conducted in rural areas by low-income villagers. Lamb sales constitute an important part of the income generated in these enterprises1. Moreover, lamb and sheep meat are consumed with pleasure in Middle Eastern countries and it also makes an important contribution to meeting the animal-based food needs of low-income people2. Feed cost forms an important part of a livestock enterpriseâ&#x20AC;&#x2122;s expenditures. If the pasture areas are sufficient and pasture conditions are favourable, grazing on pasture is usually the cheapest way for lamb feeding. Therefore, in the traditional production systems, lambs are kept in the sheepfold until 7590 days of age and then finished at natural pastures in the daytime by supplementing with roughage at nights1. However, the amount of milk sold is reduced as the lamb sucks their mothers in this production system. On the other hand, pasture areas have gradually decreased, and uncontrolled and excessive grazing has caused the quality of existing pastures to decline in recent years. As a result, natural pastures are no longer sufficient to meet the nutritional requirements

Corresponding Author: Bulent Ekiz (bekiz@istanbul.edu.tr).

of lambs for finishing nowadays. These adversities led sheep farmers to search for alternative systems for lamb finishing. Nowadays, many sheep farmers prefer the concentratesbased lamb finishing system, in which lambs are weaned at 75-90 days of age and fed with concentrates until slaughter1. Slaughtering characteristics and carcass quality of lambs may vary due to production systems3,4,5,6. Final or slaughter weight may also influence these characteristics7,8,9,10. Therefore, the deciding the appropriate production system and the final weight of lambs in commercial lamb production is of great importance in obtaining high-quality lamb carcasses. Many sheep breeds are bred in Turkey, and the majority of these breeds are fat-tailed. However, the most preferred sheep breed by consumers is Kivircik, which is a thin-tailed breed. Kivircik and its crossbreds are also bred in Greece, Bulgaria and some Balkan countries11. The aim of the current study was to determine the growth rate, slaughtering and carcass quality traits of Kivircik lambs at three final weights (LOW: 25-26 kg, MEDIUM: 30-31 kg and HIGH: 35-36 kg) in pasture-based and concentrate-based production systems.

MATERIALS AND METHODS Handling, feeding and housing procedures applied in the current study were approved by the local Ethics Committee of Istanbul University Faculty of Veterinary Medicine (No: 05-109).

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The effect of final weight on slaughtering and carcass quality characteristics of lambs in concentrate-based

Animal material and production systems The study was conducted at sheep farm of the Veterinary Faculty. The first week after birth the lambs were fed only with dam’s milk. Thereafter, the lambs were given alfalfa hay in addition to their mother’s milk until 76-day of age. Fiftyfour male lambs born as a single in early February were selected for the animal material of the study when the average lamb age attained about 76 days (76.5 ± 0.6 days). These lambs were randomly divided into two production systems with 27 lambs in each group. Afterwards, lambs of each production system were randomly allocated into LOW (25-26 kg), MEDIUM (30-31 kg) and HIGH (35-36 kg) final weight groups. Handling, housing and feeding procedures in production systems were: Concentrate-based (CON) lamb production

The lambs, which selected for the CON system, were weaned at 76-day immediately after grouping. Afterwards, three separate pens (5.0 × 3.2 m each) were built in the sheepfold for LOW, MEDIUM and HIGH final weight groups, and lambs were placed in these pens. During the whole finishing period, lambs had free access to the concentrate feed, alfalfa hay and fresh clean water. Concentrate feed consumptions of each weight group was determined at weekly basis. Mean daily concentrate consumption was 0.73 kg, 0.92 kg and 1.01 kg in LOW, MEDIUM and HIGH final weight groups, respectively. Pasture-based (PAS) lamb production

PAS lambs were not weaned until slaughter. These lambs were grazed with their dams in the natural pasture during the day and fed free access with alfalfa hay in the sheepfold during the night. Composition of pasture (on dry matter basis) was: 52% Gramineae (Lolium spp. and Festuca spp.), 22% Leguminosae (Vicia spp., Medicago spp. and Trifolium spp.) and 26% other families (mainly Conium spp., Viola spp., Geranium spp., Plantago spp. and Rumex spp.)1. The composition of the alfalfa hay, concentrate feed and pasture are given in Table 1. Table 1 - Chemical composition of concentrate feed, alfalfa hay and pasture used in the study. Concentrate feed3

Alfalfa hay

Pasture

Dry matter (%)

90.30

92.14

37.95

Crude protein (%)

16.90

15.85

11.5

Ether extract1 (%)

3.14

2.10

5.46

Crude cellulose1 (%)

14.65

26.50

24.4

Ash (%)

6.92

8.49

10.43

Neutral detergent fibre1 (%)

29.41

46.16

42.67

Acid detergent fibre1 (%)

10.30

33.92

35.97

Metabolizable energy2, (MJ ME/kg DM)

11.81

8.67

9.21

Chemical composition

As a percentage of dry matter. Calculated values. 3 Concentrate feed contained: wheat bran 40%, barley 15.5%, corn 15.3%, soybean meal 8%, sunflower meal 6%, DDGS (dried distillers grains with solubles) 5%, molasses 4%, Safflower meal 3%, CaCO3 2.5%, NaCl 0.5%, vitamin/mineral premix 0.2%. 2

Two lambs in HIGH group of PAS system were excluded from the study since they had laminitis and therefore could not graze on the pasture. Hence, 25 lambs in PAS system and 27 lambs in CON system were slaughtered in the study. All lambs were weighed every Wednesday morning before feeding. Lambs reaching the target weight in weekly weighings were transferred to Istanbul University experimental slaughterhouse for slaughtering.

Slaughter procedures and determination of carcass characteristics Lambs were kept overnight at lairage unit of the slaughterhouse with free access to water but not feed. After recording the pre-slaughter live weight, the lambs were electrically stunned and then slaughtered. Afterwards, head, skin, feet, lungs and trachea, liver, heart, spleen, testicles and gastro-intestinal tract were removed and weights of these tissues were recorded. After the removal of these tissues, hot carcass weight was recorded. Afterwards, the carcasses were kept in cold storage (4oC) for 24 h. At 24 h post-mortem, colour variables of subcutaneous fat were measured by using a colorimeter (Minolta CR-400) from the tail root. Afterwards, following carcass measurements were also determined on the intact carcass as reported by Ekiz et al.12: buttock width, buttock circumference, leg length, carcass length, carcass width, chest depth and chest circumference. Next, carcasses were split along the vertebral column into right and left parts. Left parts of the carcasses were used to measure hind limb length and internal carcass length12. Once the carcass measurements have been determined, hind limb compactness, carcass compactness and chest roundness index were calculated as reported by Ekiz et al.12. After all carcass measurements were recorded, the right sides of carcasses were separated into ribs, thoracic and pelvic limbs, neck and flank joints at 24 h post-mortem13.

Statistical analyses The effects of production system, final weight group and their interaction on slaughtering and carcass characteristics were analysed using the General Linear Model (GLM) procedures in SPSS 13.0 programme. Moreover, one-way ANOVA and Duncan’s multiple range test were applied to compare the final weight groups for each production system. The comparison of PAS and CON systems for each final weight group was performed by independent sample t-test.

RESULTS As planned in the experimental design, initial ages and weights of lambs in six sub-groups (2 production system × 3 final weight groups) were similar (Table 2). Finish duration was lower in lambs of CON system in both LOW and HIGH weight groups than lambs of the PAS system. In the CON system, commercial and real dressing percentages were lower in the LOW group compared to MEDIUM and HIGH groups. However, differences among final weight groups in terms of commercial and real dressing percentages were not significant in the PAS system. In MEDIUM and HIGH weight groups, CON system yielded higher commercial dressing than PAS system, but such a difference was

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B. Ekiz et al. Large Animal Review 2020; 26: 67-72

Table 2 - Performance traits and slaughtering characteristics of lambs in different final weights1 due to production systems. Characteristics

Concentrate-based system

Pasture-based system

P-Value

SEM

LOW

MEDIUM

HIGH

LOW

MEDIUM

HIGH

WG×PS

Initial age, days Initial weight, kg Finishing duration, days

76.67 17.37 43.78 c,x

76.11 17.93 75.67 b

76.78 17.91 94.67 a,x

77.89 17.49 60.33 f,y

75.56 17.80 85.00 e

77.43 17.91 107.57 d,y

1.539 0.230 2.555

0.914 0.573 <0.001

0.887 0.878 0.015

0.970 0.912 0.840

Pre-slaughter weight, kg Hot carcass weight, kg Commercial dressing2, % Real dressing3, %

24.09 c 11.56 c 48.03 b 54.69 b

28.46 b 14.26 b,x 50.10 a,x 56.11 a

33.26 a 16.82 a,x 50.57 a,x 56.37 a,x

24.11 f 11.24 f 46.68 53.87

27.98 e 13.19 e,y 47.05 y 54.24

32.47 d 15.07 d,y 46.46 y 54.20 y

0.161 0.131 0.346 0.272

<0.001 <0.001 0.267 0.261

0.071 <0.001 <0.001 0.005

0.173 0.035 0.272 0.571

Non-carcass parts, % Head Feet Skin Lungs and trachea Liver Heart Spleen Empty stomachs Empty intestines Gastro-intestinal content

6.62 y 3.04 a,y 10.23 1.84 a 2.39 x 0.52 y 0.23 3.57 6.83 a 13.96

6.52 y 2.81 b,y 10.66 1.56 b,y 2.29 x 0.50 0.21 3.45 6.04 b 12.02 y

6.80 2.68 c,y 10.77 1.49 b,y 2.18 x 0.50 y 0.20 3.34 5.42 c 11.51 y

7.08 x 3.22 d,x 10.92 1.80 2.06 y 0.56 x 0.22 3.55 6.38 d 15.59

7.10 x 3.02 e,x 11.09 1.73 x 2.02 y 0.53 0.24 3.55 5.83 e 15.46 x

6.91 2.86 f,x 11.47 1.73 x 2.05 y 0.54 x 0.22 3.53 5.41 f 16.83 x

0.048 0.019 0.141 0.022 0.028 0.005 0.005 0.049 0.086 0.455

0.905 <0.001 0.297 <0.001 0.269 0.110 0.209 0.584 <0.001 0.635

0.001 <0.001 0.037 0.007 <0.001 <0.001 0.165 0.392 0.206 <0.001

0.140 0.962 0.903 0.210 0.340 0.850 0.245 0.687 0.572 0.271

Final weight groups: LOW = 25-26 kg, MEDIUM = 30-31 kg, HIGH = 35-36 kg. WG: Final weight group; PS: Production system. Mean values with different superscripts in the same line for concentrate-based system are significantly different (P < 0.05). d, e, f Mean values with different superscripts in the same line for pasture-based system are significantly different (P < 0.05). x, y Mean values for CON and PAS systems with different superscripts within the same final weight group are significantly different (P < 0.05). 2 (Hot dressing percentage / pre-slaughter weight) × 100. 3 (Hot dressing percentage / empty body weight) × 100. a, b, c

not observed in LOW weight group. A decrease in percentages of feet and empty intestines were determined with the increase in final weight in both CON and PAS systems. Lambs of CON system had higher liver percentage than PAS lambs in all final weight groups. In both CON and PAS systems, LOW lambs had lower mean values in terms of carcass measures and indices, except chest

roundness index than HIGH lambs (Table 3). MEDIUM group had also higher values than LOW group for the majority of carcass measures. Lambs in CON system a had higher buttock width and circumference, carcass width, carcass compactness and chest roundness index in all final weight groups compared with PAS lambs. Lightness and redness values of subcutaneous fat were not

Table 3 - Carcass measures and indices of lambs in different final weights1 due to production systems. Characteristics

Concentrate-based system LOW

Carcass length, cm

MEDIUM

65.32 55.06

Leg length, cm

19.44

Hind limb length, cm

26.94 b

Internal carcass length, cm

66.11 57.46

20.39

27.81 ab

28.58 a 20.94

a,x

60.51

a,x

20.98

a,x

56.10

19.84

27.02 e 18.10

e,y

53.56

f,y

17.73

e,y

68.93

58.42

20.78

28.13 de

72.13

0.557

0.002

0.091

0.251

60.09

0.212

<0.001

0.110

0.594

21.01

0.095

<0.001

0.120

0.794

28.46 d

0.182

0.005

0.800

0.886

19.80

d,y

0.107

<0.001

0.972

58.79

d,y

0.204

<0.001

0.520

19.64

d,y

19.02

0.146

<0.001

0.380

Chest depth, cm

23.60 c

24.64 b

26.40 a

23.87 e

25.24 d

26.07 d

0.099

<0.001

0.371

0.175

Chest circumference, cm

64.38 c

69.08 b

72.67 a

65.47 f

68.38 e

70.97 d

0.218

<0.001

0.322

0.039

217.30 e,y 236.85 d,y

1.969

<0.001

0.045

69.46 c

Chest roundness index

0.74 x

82.06

b,x

0.78 x

18.51

de,y

WG×PS

Carcass width, cm

Hind limb compactness, g/cm

55.80

e,y

b,x

203.34 c,x 241.33 b,x 272.65 a,x 192.55 f,y

18.96

de,y

54.99

20.68

a,x

20.90

64.64

P-Value

SEM

HIGH

c,x

Carcass compactness, g/cm

58.32

b,x

60.02

MEDIUM

19.20

Buttock circumference, cm

20.18

68.50

Pasture-based system LOW

c,x

Buttock width, cm

b,x

HIGH

90.81 a,x

68.40 e

73.47 e,y

83.49 d,y

0.922

<0.001

0.004

0.203

0.75 x

0.69 y

0.68 y

0.71 y

0.006

0.482

<0.001

0.116

Final weight groups: LOW = 25-26 kg, MEDIUM = 30-31 kg, HIGH = 35-36 kg. WG: Final weight group; PS: Production system. a, b, c Mean values with different superscripts in the same line for concentrate-based system are significantly different (P < 0.05). d, e, f Mean values with different superscripts in the same line for pasture-based system are significantly different (P < 0.05). x, y Mean values for CON and PAS systems with different superscripts within the same final weight group are significantly different (P < 0.05).

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The effect of final weight on slaughtering and carcass quality characteristics of lambs in concentrate-based

Table 4 - Fat colour characteristics and proportions of carcass joints of lambs in different final weights1 due to production systems. Characteristics

Concentrate-based system

Pasture-based system

LOW

MEDIUM

HIGH

LOW

MEDIUM

HIGH

Fat colour Lightness Redness Yellowness Chroma Hue

69.96 4.64 6.81 y 8.26 55.55 b

69.58 3.89 7.31 8.29 61.96 a

69.09 4.09 7.48 8.54 61.46 a

70.04 4.40 7.75 x 8.96 60.82

69.99 4.20 7.81 8.88 61.56

69.64 4.01 7.96 8.92 63.11

Proportions of carcass joints, % Pelvic limb Ribs Thoracic limb Neck Flank Tail

34.24 a 25.38 19.23 y 7.47 b,y 10.32 1.17

33.23 ab 26.41 18.69 y 8.12 ab 10.01 1.26

32.20 b,y 26.95 x 18.78 y 8.58 a 9.91 1.33 x

34.03 d 24.79 e 20.30 x 8.33 x 10.05 1.01

32.64 e 26.44 d 20.14 x 8.81 9.40 1.04

34.18 d,x 24.73 e,y 20.28 x 8.65 9.73 0.96 y

P-Value

SEM WG

WG×PS

0.148 0.100 0.119 0.129 0.572

0.230 0.089 0.315 0.890 0.010

0.246 0.980 0.010 0.036 0.063

0.801 0.486 0.667 0.888 0.123

0.146 0.198 0.086 0.116 0.144 0.037

0.003 0.024 0.228 0.043 0.360 0.748

0.186 0.023 <0.001 0.033 0.230 0.001

0.002 0.073 0.531 0.385 0.821 0.494

Final weight groups: LOW = 25-26 kg, MEDIUM = 30-31 kg, HIGH = 35-36 kg. WG: Final weight group; PS: Production system. a, b Mean values with different superscripts in the same line for concentrate-based system are significantly different (P < 0.05). d, e Mean values with different superscripts in the same line for pasture-based system are significantly different (P < 0.05). x, y Mean values for CON and PAS systems with different superscripts within the same final weight group are significantly different (P < 0.05).

influenced from neither by the weight group nor by the production system (Table 4). Lambs from LOW group in PAS system had a higher yellowness than that in CON system. In CON system, hue value was lower in LOW group compared to MEDIUM and HIGH groups. In CON system, HIGH lambs had lower pelvic limb proportion and higher neck proportion than LOW lambs. Proportion of thoracic limb was higher in PAS lambs than CON lambs in all final weight groups.

DISCUSSION In lamb finishing for meat production, determination of optimal slaughter weight is an important issue for the provision of a profitable and high-quality lamb production. In Turkey, pricing of lamb carcasses is usually based on carcass weight, so marketing of overweight lambs may be considered by sheep farmers. In LOW and HIGH weight groups, lambs of CON system reached the final weight earlier than the PAS lambs. This may be due to the discrepancy between the two production systems in terms of food resources. CON lambs were fed ad-libitum with concentrate and alfalfa hay, while pasture grass, alfalfa and mothers’ milk were main feed resources of PAS lambs. Supporting the current results, Ekiz et al.2 for Kivircik lambs and Borton et al.14 for Targhee × Hampshire lambs found lower growth rate in pasture lambs than lambs fattened with concentrates. On the other hand, if pasture conditions are favourable, studies found similar growth rate for grazing and stall lambs were also available6,15. In MEDIUM and HIGH weight groups, CON lambs had higher hot carcass weight than lambs reared under PAS system. One possible explanation for higher hot carcass weights of CON lambs might be their higher dressing percentages. The results that higher carcass weights in lambs finished with concentrates than pasture lambs were also observed for Targhee × Hampshire lambs14 and Kivircik lambs2, previously. In MEDIUM and HIGH weight groups, a higher commercial and real dressing percentages observed

in CON lambs might be due to the higher percentages of head, feet, lungs and trachea, and gastro-intestinal content in PAS lambs. In lambs of PAS system, commercial and real dressing percentages were not affected by final weight group. Similar results were reported for suckling lambs by Santos et al.16. On the other hand, in CON lambs, commercial and real dressing percentages were lower in lambs of the LOW group than that of MEDIUM and HIGH weight groups. MajdoubMathlouthi et al.17 also found an increase in dressing percentage with increasing slaughter weight in lambs fattened with concentrates. The authors explained this result by reduced gut content percentage with increasing slaughter weight. Oliveira et al.18 explained a higher dressing percentage in Sante Ines lambs, which slaughtered at heavier weights by higher deposition of muscle and fat in carcasses of these lambs. Borton et al.14 also found higher dressing percentage for concentrate-fed lambs than pasture lambs and attributed this result to a decrease in the size of gastrointestinal tract. Indeed, in the current study, there was a tendency in CON lambs to decrease in the percentage of gastrointestinal content with the increase in the final weight for lambs (P = 0.06). Feet percentage dropped as final weight increased in both CON and PAS systems. These results might be explained by the fact that bone is the most precocious tissue followed by lean, while fat tissue develops later6. In the current study, lambs from different final weight groups were slaughtered at 4-6 months of age, therefore weight gains of lambs at these ages might have been based on mainly an increase in muscle and fat tissues rather than bone tissues. The higher head, feet, lungs and trachea, and heart percentage in PAS lambs were expected because CON lambs reached to the target final weight about 13 days earlier than PAS lambs. In the previous study, where Kivircik lambs were slaughtered at a constant weight of 30 kg, the percentages of feet and lungs and trachea of the older ones were found to be higher2. CON lambs had higher liver percentages than PAS lambs in all final weight groups. McClure et al.19 also found higher

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B. Ekiz et al. Large Animal Review 2020; 26: 67-72

liver weight for lambs fed concentrate-based diet than lambs of grazing-based system. Similarly, Ekiz et al.2 reported a higher liver percentage for weaned lambs finished with concentrates compared with lambs of grazing-based systems. Kochewad et al.20 also found higher liver percentage for Deccani lambs finished with concentrates than lambs finished at pasture. When carcass measurements are evaluated, a significant increase in the majority of carcass measures and indices were observed with increased final weight group in both CON and PAS systems. Similar trend regarding carcass measurements were reported previously for several breeds7,10,16. Conformation and body/carcass measurements of healthy and well-fed animals are expected to rise with increasing age and weight until reaching the mature age7. Therefore, the current results regarding effects of final weight group on carcass measurements and indices might be evaluated as expected results. Moreover, carcass measures and indices determined for LOW and MEDIUM groups were comparable with the previous results by Ekiz et al.21 for Kivircik lambs slaughtered at 26.6 and 30.3 kg, respectively. Production system did not affect length measures (lengths of carcass, internal carcass, leg and hind limb), and depth and circumference of chest in all final weight groups. But, CON lambs had higher mean values regarding width measures (buttock width and carcass width) and buttock circumference in all final weight groups. Hence, a higher width and circumference measurements of CON lamb carcasses caused the objective carcass indices (carcass compactness, hind limb compactness and chest roundness index) in these lambs to be higher than in PAS lambs. Supporting the current result, Carrasco et al.5 reported lower transversal measures, and decreased hindquarters compactness, chest rounded index, pelvic limb compactness and carcass compactness for Churra Tensina light lambs reared under grazing compared with drylot lambs with dams fed in confinement. Gallo et al.22 found higher carcass compactness index in feedlot lambs that fed a diet composed of 20% of roughage and 80% of concentrate than lambs raised on pasture. In that study, internal carcass length and leg length were also similar in pasture and feedlot lambs. Final weight group had no significant influence on subcutaneous fat colour parameters in both CON and PAS systems, except hue value in CON system. Ripoll et al.23 also reported no significant influence of carcass weight on subcutaneous fat lightness and chroma values. Fat of lamb carcasses in LOW group had lower hue value compared to that of MEDIUM and HIGH groups. Moreover, significant production system effect was only observed in LOW weight group for the yellowness of carcass fat. PAS lambs had more yellow fat colour with higher b* value compared with CON lambs. This may be attributed to the greater intake of herbage (and therefore pigments) in PAS lambs. Similarly, Carrasco et al.24 observed higher b* values in lambs from grazing groups than drylot lambs and explained this by more carotenoids deposition in grazed lambs due to pasture intake. In the other study, Dıaz et al.6 also found higher b* value for subcutaneous fat in lambs finished at pasture than that of sheepfold ones. The proportion of thoracic limb was higher in PAS lambs. With regard the pelvic limb proportion, PAS lambs in HIGH group had also higher mean values than that of

CON lambs. Ekiz et al.2 also observed lower proportions of thoracic and pelvic limbs in unweaned lambs finished with concentrates compared with lambs from grazing-based systems. Borton et al.14 noted that proportions of leg and foreshank joints, which are related to motor function, might be increased consistently in forage-finishing systems. Higher proportions of ribs and tail in HIGH weight group of CON lambs may be related to the higher fatness level of these lambs and/or lower proportions of the thoracic limb in these lambs compared to PAS lambs. Proportions of carcass joints found in the current study were comparable with the results of Aksoy et al.25 for Kivircik lambs finished with concentrate based system.

CONCLUSIONS In the conditions of the current study, CON lambs were significantly younger at slaughter. Moreover, CON lambs had higher dressing percentage, better carcass conformation with greater width measurements. Therefore, CON system can be preferred to produce a higher amount of lamb meat in a shorter time. Final weight did not influence on dressing percentage and subcutaneous fat colour. Therefore, among the groups investigated in the current study, final weight of 3536 kg might be preferred to increase lamb production, without any adverse effect on carcass quality for Kivircik lambs.

ACKNOWLEDGEMENTS The study was supported by Scientific Research Projects Coordination Unit of Istanbul University-Cerrahpasa (BEK2017-24916).

References 1. Ekiz B., Yilmaz A., Ozcan M., Kocak O. (2012). Effect of production systems on carcass measurements and meat quality of Kivircik lambs. Meat Sci, 90: 465-471. 2. Ekiz B., Yilmaz A., Demirel G., Yalcintan H., Kocak O., Ozcan M., Altinel A. (2013). Slaughter characteristics, carcass quality and fatty acid composition of lambs under four different production systems. Small Rum Res, 114: 26-34. 3. Boughalmi A., Araba A. (2016). Effect of feeding management from grass to concentrate feed on growth, carcass characteristics, meat quality and fatty acid profile of Timahdite lamb breed. Small Rum Res, 144, 158-163. 4. Cañeque V., Velasco S., Díaz M.T., de Huidobro F.R., Perez C., Lauzurica S. (2003). Use of whole barley with a protein supplement to fatten lambs under different management systems and its effect on meat and carcass quality. Anim Res, 52(3): 271-285. 5. Carrasco S., Ripoll G., Sanz A., Álvarez-Rodríguez J., Panea B., Revilla R., Joy M. (2009). Effect of feeding system on growth and carcass characteristics of Churra Tensina light lambs. Livest Sci, 121: 56-63. 6. Díaz M.T., Velasco S., Caneque V., Lauzurica S., Huidobro F., Perez C., Gonzales J., Manzanares C. (2002). Use of concentrate or pasture for fattening lambs and its effect on carcass and meat quality. Small Rum Res, 43: 257-268. 7. Abdullah A.Y., Qudsieh R.I. (2008). Carcass characteristics of Awassi ram lambs slaughtered at different weights. Livest Sci, 117: 165-175. 8. Díaz M.T., Velasco S., Pérez C., Lauzurica S., de Huidobro R.F., Cañeque V. (2003). Physico-chemical characteristics of carcass and meat Manchego-breed suckling lambs slaughtered at different weights. Meat Sci, 65(3): 1085-1093. 9. Lloyd W.R., Slyter A.L., Costello W.J. (1980). Effect of breed, sex and final weight on feedlot performance, carcass characteristics and meat palatability of lambs. J Anim Sci, 51: 316-320.

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10. Peña F., Cano T., Doménech V., Alcalde Ma J., Matos J., Garcia-Martinez A., Herrera M., Rodero E. (2005). Influence of sex, slaughter weight and carcass weight on “non-carcass” and carcass quality in Segureña lambs. Small Rum Res, 60: 247-254. 11. Anonymous (2009). Republic of Turkey, Ministry of Food, Agriculture and Livestock, General Directorate of Agricultural Research and Policies. Turkish Farm Animal Genetic Resources Catalogue. https://www.tarim.gov.tr/TAGEM/Belgeler/yayin/Katalog%20T%C3 %BCrk%C3%A7e.pdf 12. Ekiz B., Ozcan M., Yilmaz A., Tölü C., Sava T. (2010). Carcass measurements and meat quality characteristics of dairy suckling kids compared to an indigenous genotype. Meat Sci, 85: 245-249. 13. Colomer-Rocher F., Morand-Fehr P., Kirton A.H. (1987). Standard methods and procedures for goat carcass evaluation, jointing and tissue separation. Livest Prod Sci, 17: 149-159. 14. Borton R.J., Loerch S.C., McClure K.E., Wulf D.M. (2005). Comparison of characteristics of lambs fed concentrate or grazed on ryegrass to traditional or heavy slaughter weights. I. Production, carcass, and organoleptic characteristics. J Anim Sci, 83; 679-685. 15. Atti N., Mahouachi M. (2009). Effects of feeding system and nitrogen source on lamb growth, meat characteristics and fatty acid composition. Meat Sci., 81: 344-348. 16. Santos V.A.C., Silva S.R., Mena E.G., Azevedo J.M.T. (2007). Live weight and sex effects on carcass and meat quality of “Borrego terrinchoPDO” suckling lambs. Meat Sci, 77(4): 654-661. 17. Majdoub-Mathlouthi L., Saïd L., Say A., Kraiem K. (2013). Effect of concentrate level and slaughter body weight on growth performances, carcass traits and meat quality of Barbarine lambs fed oat hay based diet. Meat Sci, 93: 557-563.

18. Oliveira F.G., Sousa W.H., Cartoxa F.Q., Cunha MG.G., Ramos J.P.F., Cezar M.F., Menezes L.M., Oliveira A.B. (2018). Carcass characteristics of Santa Ines sheep with different biotypes and slaughtering weights. Rev Bras Saúde Prod Anim Salvador, 19: 347-359. 19. McClure K.E., Solomon M.B., Loerch S.C. (2000). Body weight and tissue gain in lambs fed an all-concentrate diet and implanted with trenbolone acetate or grazed on alfalfa. J Anim Sci, 78: 1117-1124. 20. Kochewad S.A., Raghunandan T., Sarjan Rao K., Kondal Reddy K., Nalini Kumari N., Ramana D.B.V., Anil Kumar D., Kankarne Y, Kumar S., Meena L.R., Singh M. (2018). Productive performance, body condition score and carcass characteristics of Deccani lambs reared under different farming systems. Indian J Anim Res, 52: 444-448. 21. Ekiz B., Koçak O., Yalcintan H., Yilmaz A. (2016). Effects of suckling duration on growth, slaughtering and carcass quality characteristics of Kivircik lambs. Trop Anim Health Prod, 48: 395-401. 22. Gallo S.B., Arrigoni M.B., da Silva C. Lemos A.L., Haguiwara M.M.H., Bezerra H.V.A. (2019). Influence of lamb finishing system on animal performance and meat quality. Acta Scientiarum Anim Sci, v41i1.44742 (Doi: 10.4025/actascianimsci.v41i1.44742). 23. Ripoll G., Blanco M., Panea B., Joy M. (2019) The effect of carcass weight on fatness and muscle and fat colour of male Ojinegra de Teruel light lambs. Anim Prod Sci, 59: 1168-1175. 24. Carrasco S., Panea B., Ripoll G., Sanz A., Joy M. (2009). Influence of feeding systems on cortisol levels, fat colour and instrumental meat quality in light lambs. Meat Sci, 83: 50-56. 25. Aksoy Y., Uğurlu M., Önenç A., Şirin E., Şen U., Çiçek Ü., Ulutaş Z., Kuran M. (2018). Meat production characteristics of Turkish native breeds: I. Fattening, slaughter and carcass traits of lambs. S. Afr. J Anim Sci, 48: 665-672.

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C. Pesca et al. Large Animal Review 2020; 26: 73-76

Enzootic pneumonia in sheep: ewe and lamb immune response after Mannheimia haemolytica vaccine administration under field condition in Italy

CRISTINA PESCA1, KATIA FORTI1, ANDREA FELICI1, ELEONORA SCOCCIA1, CLAUDIO FORTE1, PIETRO ANTENUCCI2, SABINA MUNTONI2, LUCIA ANZALONE1, ANTONELLA DI PAOLO1, SILVIA CROTTI1 1

Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche “Togo Rosati”, Via G. Salvemini 1, 06126 Perugia, Italy 2 MSD Animal Health, Segrate, Milano, Italy

SUMMARY Mannheimia haemolytica related pneumonia is characterized by high morbidity and mortality in lambs and thus is a common cause of important economic losses in sheep industry due to reduction in lamb growing and decreased carcass value. Due to the frequency of the disease, vaccination is a very common practice in Italy but the extent and duration of colostral protection in Sarda lambs born from vaccinated dams is not known. In this study, the extent and length of colostral antibody protection in Sarda lambs born from M. haemolytica-vaccinated ewes was evaluated in field condition. The in-field trial took place in two different farms in Sardinia region (Italy). A total of forty-five adult healthy pregnant Sarda sheep were enrolled for the study (24 from the first flock and 21 from the second flock). Each flock in each farm was divided in two groups: unvaccinated control group (10 sheep from the first flock and 8 from the second flock) and vaccinated sheep (14 from the first flock and 13 from the second flock). A total of forty-five lambs born from both vaccinated (n=27) and unvaccinated (n=18) dams were included in the study. Colostrum was collected from all the dams within 6-12 hours after lambing. Blood samples were taken from all the lambs included in the study at 48 hours, 15, 30, 45, 60 days after parturition. Antibody responses following vaccinations were detected by ELISA test. In the trial, antibody titres both on serum of ewes and lambs and colostrum were comparatively examined and important differences were observed in vaccinated pregnant ewes where serum titres developed after vaccination resulted two times higher than in the unvaccinated group (p-value=0.0029); within the lamb groups, an increase of serum ELISA titres was found in lambs born from vaccinated ewes until 60 days after birth. Colostral titres didn’t show any significant difference. In conclusion, when the control group and the trial group were compared, vaccinating the lambs belonged to vaccinated dams showed significant differences concerning antibody responses against M. haemolytica.

KEY WORDS Vaccination; immunity, enzyme-linked immunosorbent assay; Sarda sheep.

INTRODUCTION Sheep farming is a significant industry in Italy. According to 2018 data reported by the National Data Bank (BDN), more than a half of the total amount of sheep reared belongs to Sarda breed. Pneumonia is responsible for important economic losses in sheep farming worldwide1. Mannheimia haemolytica is a Gram-negative, facultative anaerobic, non-motile, opportunistic pathogen bacteria and is one of the most important respiratory pathogens in the family Pasteurellaceae able to cause serious outbreaks of acute pneumonia in sheep of all ages including in neonatal, weaned and growing lambs. M. haemolytica and the related pneumonia is common and is distributed worldwide although the prevalence of serotypes may vary by region and flock; it is a commensal of the upper respiratory tract of healthy sheep that can cause pneumonia

Corresponding Author: Cristina Pesca (c.pesca@izsum.it).

either alone or in conjunction with other organisms. The interaction of the host with respiratory pathogens such as respiratory syncytial virus, adenovirus, parainfluenza virus type 3, and Mycoplasma spp. in particular, under the influence of environmental factors and stressors are thought to break down the mucosal barrier integrity of the lower respiratory tract, allowing to M. haemolytica to colonize, proliferate and induce significant tissue damage2,3,4,5,6. The disease is also called “shipping fever” because it appears to occur most often in animals that have undergone recent stress such as weaning, transportation, changes in diet or cohabitation with new animals from different farms. Clinical signs include an acute onset of hyperthermia (>40,5°C), dyspnea, anorexia, depression, inappetance, lethargy. In lambs pneumonia causes mortality, decreased growth at considerable cost to the industry, along with animal welfare implications. Death losses are high in severely affected animals within 12 hours of the onset of any clinical signs7. Gross lesions typically show a fibrinous, necrotic pneumonia. The introduction of an effective antibiotic therapy is necessary when the disease outbreaks into a flock. Treatment shall begin at the rise of the pathology be-

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Enzootic pneumonia in sheep: ewe and lamb immune response after Mannheimia haemolytica vaccine administration

cause of the rapid progression of lung damage and endotoxin release. Administering prophylactic antibiotics to at-risk lambs may be beneficial even if this would bring towards the development of antimicrobial resistance. Management measures to reduce known stressors such as overcrowding, poor ventilation, high and cold temperatures should also be considered. In severely affected flocks response to therapy is often disappointing because of the widely use of antimicrobials and the related resistance. For these reasons, for the economic costs of treatment and reduction of lamb weight in survivors, application of prophylactic measures would be desirable. Due to the frequency of the disease, vaccination is a very common practice in Italy but the extent and duration of colostral protection in Sarda lambs born from vaccinated dams is not known. Due to the lack of studies on immunities of M. haemolytica in Sarda sheep lambs, a key question is how long lambs born from M. haemolytica-vaccinated ewes under field condition are protected. The aim of this study was to measure the extent and length of colostral antibody protection in Sarda lambs born from M. haemolytica-vaccinated ewes. The immune response of both ewes and lambs with until 60 days of life was evaluated by measuring specific serum and colostral antibody titres produced against the bacterium by ELISA test.

were taken from all the lambs included in the study at 48 hours, 15, 30, 45, 60 days after parturition. A total of 5 lambs came to death during the study period (Table 1). Colostrum samples and serum samples from both dams and lambs were tested for M. haemolytica antibodies by ELISA home made test.

Vaccination The sheep were vaccinated subcutaneously with 2 ml per injection on the site around the neck. The vaccine was obtained from formalin killed cells of the epidemiologically most important serotypes of M. haemolytica (AI, A2, A6, A7 and A9), grown under iron restricted conditions: 5x108 cells per strain in buffered physiological saline adsorbed onto aluminum hydroxide. Thiomersal was included as a preservative.

ELISA assay Blood samples were taken lambs from the jugular vein using vacutainer tubes six times and sera were stored at −20°C until serological tests were performed. The serum of a newborn lamb born from an unvaccinated ewe was used as negative control of the test. ELISA test was performed for the determination of antibody levels in lambs born from vaccinated and unvaccinated dams as follows: the vaccine was diluted in carbonate/bicarbonate buffer (0.05 M, pH 9.6) to a final concentration of 2.5x106 cells/well and used to coat 96-well plate with 100 µl/well (MaxiSorp). To normalize the effect of the adjuvant two lane were coated with adjuvant diluted in carbonate/bicarbonate buffer to 1.25%. After incubation over night at +4°C, the plate was washed three times in PBS containing 0.05% Tween 20 (PBST) and blocked by adding 200 µl/well of buffer containing 1% of casein in PBST (saturating buffer) and incubated at room temperature under stirring (300 rpm) for 1 h. After decanting the saturating buffer, 2fold serial diluted (1:100-1:12.800) in saturating buffer, of vaccinated and unvaccinated sera were added in duplicate to the plate at 100 µl/well. In each plate a negative control (serum taken from a lamb immediately after birth) was added. After 1 h of incubation at room temperature under stirring (300 rpm), the plate was washed with PBST and an antibody rabbit anti-sheep peroxidase-conjugated (Santa Cruz Biotechnology, Inc) was added at the dilution of 1: 5000 in saturating buffer for 1 h. The plate was washed with PBST and incubated in the dark, with a peroxidase substrate solution (1-step Ultra TMB-Elisa-Thermo Scientific) for 20 minutes at room temperature, and finally the reaction was blocked with a 0.5% sulfuric acid solution and the optical density was measured at 450 nm by spectrophotometer microplate reader (Sunrise™ Basic Absorbance Reader -Tecan Trading AG). The averages of the duplicated OD values subtracted from the adjuvant value were recorded.

MATERIALS AND METHODS Study design, sample size and animals The in-field trial took place in two different farms in Sardinia region (Italy). A total of forty-five adult healthy pregnant Sarda sheep were enrolled for the study (24 from the first flock and 21 from the second flock). Sheep selected were similar for body condition score, age (between 2 and 3 years) and at the same month of pregnancy. Each flock in each farm was divided in two groups: unvaccinated control group (10 sheep from the first flock and 8 from the second flock) and vaccinated sheep (14 from the first flock and 13 from the second flock). A total of eighteen unvaccinated sheep from the two flocks and the relative lambs were used as unvaccinated controls in the study. In total eighteen lambs were born from these ewes. Twenty-seven ewes from the two flocks were vaccinated for the first time six weeks prior to lambing. Vaccinated ewes were reinoculated at 2 week intervals. Serum was collected 2 weeks after the second vaccination (two weeks before lambing). In total twenty-seven lambs were born from these ewes. A total of forty-five lambs born from both vaccinated (n=27) and unvaccinated (n=18) dams were included in the study. The farmer checked carefully that all the lambs received colostrum. Colostrum was thus collected from all the dams within 6-12 hours after lambing. Serum samples

Table 1 - Number of lambs sampled from 48 hours until 60 days after birth in both farms. Farm

Group

48 hours

15 days

30 days

45 days

60 days

Unvaccinated dams

Vaccinated dams

Unvaccinated dams

Vaccinated dams

Total

Statistical analysis The Pearson χ2 test was used to evaluate the difference between vaccinated and unvaccinated sheep and the presence of antibodies in dams, colostrum and lambs. The non parametric Mann-Whitney test was used to assess at each time the quantitative difference between groups (vacci-

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C. Pesca et al. Large Animal Review 2020; 26: 73-76

nated vs. unvaccinated). A p-value ≤ 0.05 was considered statistically significant. Calculations were carried out in Stata 11.2 and Miscrosoft Excel 2013.

RESULTS Serum and colostrum antibodies titres on vaccinated and unvaccinated dams To determine ELISA titres we considered that if the vaccine was effective in inducing specific antibody, the vaccinated animals would have higher specific antibody titres than control animals. In order of that, in this ELISA test, the cut off values were calculated similar to the methods described by Mehmet et al.8 and on the unvaccinated control groups (sheep serum, colostrum, lambs at 48 hours, lambs at 15, 30, 60 days). The cut off values were calculated to the average values of the OD 450 nm obtained at the sample serum dilution 1:1600 adding 3 standard deviations. OD results were evaluated at the median serum diluition of 1:800 and each sample was considered “negative” when the value was below the cut-off and “positive” when was above. Of the twenty-seven vaccinated ewes, 77.8% developed antibody titres >1: 800 detectable on serum by ELISA while of the eighteen unvaccinated ewes, 66.7% developed antibody titres ≤1: 800 (Table 2); a significant difference was found between these groups (p-value=0.0029). Of the twenty-seven colostrum samples belonging to the vaccinated dams, only 18.5% developed antibody titres > 1:800 detectable by ELISA (Table 3). Colostrum samples did not showed any difference between the two groups (p-value=0.506).

Table 2 - Number of serum samples below and above the cut off value evaluated at the median serum diluition of 1:800 in vaccinated and no vaccinated ewes against M. haemolytica (% calculated on the total column). Groups

Serum samples ≤ 1: 800

> 1: 800

Total

Vaccinated ewes

6 (33.3%)

21 (77.8%)

27 (60%)

Unvaccinated ewes

12 (66.7%)

6 (22.2%)

18 (40%)

Total

18 (100%)

27 (100%)

45 (100%)

Table 3 - Number of colostrum samples below and above the cut off value evaluated at the median serum diluition of 1:800 in vaccinated and no vaccinated dams against M. haemolytica (% calculated on the total column). Groups

Colostrum ≤ 1: 800

> 1: 800

Total

Vaccinated dams

22 (57.9%)

5 (71.4%)

27 (60%)

Unvaccinated dams

16 (42.1%)

2 (28.6%)

18 (40%)

Total

38 (100%)

7 (100%)

45 (100%)

from the unvaccinated dams (Figure 1). In particular, significant differences were demonstrated at 15 days (p-value=0.04) after birth, whereas no significant differences were found at 2 days (p-value=0.501), 30 days (p-value=0.43), 45 days (p-value=0.74) and 60 days (p-value=0.246).

Serum antibodies titres on lambs born from vaccinated and unvaccinated dams

DISCUSSION

The results on the total of the serum samples (n=118) of the lambs born from vaccinated dams showed higher anti-M. haemolytica titres time by time until sixty days after birth in comparison to the samples from the lambs (n=90) born

Infectious respiratory diseases of sheep and goats contribute to 5.6 percent of the total diseases of small ruminants9. M. haemolytica constitutes one of the most significant agents of Figure 1 Mean antibody titres in serum against M. haemolytica in lambs from vaccinated and no vaccinated ewes at each sampling time.

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Enzootic pneumonia in sheep: ewe and lamb immune response after Mannheimia haemolytica vaccine administration

respiratory infection, which is a major problem for sheep farmers. In a large Italian survey, 43.5% of the lambs between 25 and 35 days of life tested positive for M. haemolytica by PCR3. This result was in agreement with other reports in literature, which assess that lambs are most susceptible to the infection by this pathogen during the first months of life7,10. Until now, there are no published data on Sarda sheep supporting the knowledge about the extent and length of colostral antibody protection against M. haemolytica in lambs born from vaccinated ewes under field condition. This could lead to ensure that farmers can vaccinate their ewes at the optimal time prior to lambing to achieve maximum levels of colostral antibody transfer, maximizing the duration of colostral immunity, while also indicating when lambs should then be newly immunized without interfering with colostral immunity. Prevention of M. haemolytica infection by using a good vaccination program both on ewes and lambs would be expected to decrease pneumonia in lambs by developing good maternal and passive colostral immunity. In this study, the length and efficacy of immunization with a vaccine against M. haemolytica infection in Sarda sheep were investigated. Serological determination of antibody titres in serum and colostrum in a group of vaccinated dams and relative lambs and that one of unvaccinated dams and relative lambs was tested. Antibodies were detected among the pregnant ewes and their lambs bled at 48 hours, 15, 30, 45, 60 days after birth by ELISA test. To the authors knowledge, the serum ELISA titres obtained in this study could not be compared because no similar studies were performed in Italy on Sarda sheep; on the contrary, for the determination of antibody titres obtained after M. haemolytica vaccinations, many foreign studies have been performed and different antigens and techniques have been used11,12. Amongst these techniques, ELISA is widely used13. Mosier et al.14, showed a positive correlation between the ELISA titres and resistance to experimental pasteurellosis in cattle and reported that ELISA was a more feasible and faster technique considering the number of sera that could be tested readily. In this study, significant increase of serum ELISA titres following vaccination in pregnant ewes and its efficacy on immunity on lambs is shown. In vaccinated pregnant ewes, serum titres developed after vaccination resulted two times higher than in the unvaccinated group (Table 2), this difference was statistically significant (p-value=0.0029). On the contrary, colostral titres didn’t show any significant difference between the two groups examined (Table 3). This result suggested that in many cases colostrum couldn’t be sampled within the 6-12 hours after birth and it must be considered that this kind of matrix is highly degradable15. Within the lamb groups, an increase of serum ELISA titres was found in lambs born from vaccinated ewes until 60 days after birth (Figure 1). After this period, lamb passive immunity seems dangerously to decrease and this can lead to the development of pneumonia. The result is in agreement with the reports which assess that the major incidence of respiratory diseases (over 30%) is registered in sheep between 3 and 6 months of life7,10. The major differences regarding the concentration of antibody titres against M. haemolytica were found at 15 days (p-value=0.04). According to the results obtained in this investigation, M. haemolytica vaccination on pregnant ewes between six and four weeks prior to lambing seems to provide a good passive immunity to lambs until 60 days after birth.

CONCLUSIONS Nowadays, the Italian sheep industry is facing many difficulties because of the minimal public funds given to the farmers and the lowest profits in the agricultural industry. Given the significant economic losses due to M. haemolytica infection and the increasing antibiotic resistance, it is necessary to adopt useful preventive tools for the benefits of the whole sheep industry. The results obtained from this investigation showed that immunization of ewes and lambs with a vaccine containing M. haemolytica AI, A2, A6, A7 and A9 bacterins, under field condition and between the sixth and the fourth week prior to lambing significantly increases serum antibody titres and provides good protection to lambs.

References 1. Brodgen K.A., Lehmkuhl H.D., Cutlip R.C. (1998) Pasteurella haemolytica complicated respiratory infections in sheep and goats. Vet Res, 29: 233-254. 2. Ackermann M.R. (2014) Lab model of respiratory syncytial virus-associated lung disease: insights to pathogenesis and novel treatments. ILAR J, 55: 4-15. doi: 10.1093/ilar/ilu003. 3. Bottinelli M., Schnee C., Lepri E., Stefanetti V., Filippini G., Gobbi M., Sebastianelli M., Antenucci P., Rampacci E., Coletti M., Passamonti F. (2017) Investigation on mycoplasma populations in pneumonic dairy lamb lungs using a DNA microarray assay. Small Ruminant Res, 147: 96-100. doi: 10.1016/j.smallrumres.2016.12.038. 4. Lin Y.C., Miles R.J., Nicholas R.A.J., Kelly D.P., Wood A.P. (2008) Isolation and Immunological detection of and Immunological detection of Mycoplasma ovipneumoniae in sheep with atypical pneumonia, and lack of a role for Mycoplasma arginini. Res Vet Sci, 84: 367-373. doi: 10.1016/j.rvsc.2007.06.004. 5. Nicholas R., Ayling R., McAuliffe L. (2008) Respiratory Disease of Small Ruminants. In: Mycoplasma Diseases of Ruminants, Ed. Nicholas R., Ayling R., McAuliffe L., 1st ed., 169-198, CAB International, Oxforshire, UK. 6. Rodger J.L. (1989) Parainfluenza 3 vaccination of sheep Vet. Rec., 125: 453-456. 7. Alley M.R. (1991) Pneumonia in sheep. Veterinary Annual, 31: 51-58. 8. Mehmet A., Tanar O., Baris S., Rifki H., Osman Y., Zafer C. (2006). Vaccination studies of lambs against experimental Mannheimia (Pasteurella) haemolytica infection. Small Rumin Res 65, 44-50. doi: 10.1016/j.smallrumres.2005.05.020 9. Amit K., Suresh K. Tikoo, Praveen Malik and Aruna T. Kumar (2014) Respiratory Diseases of Small Ruminants. Vet Med Int, 2014: 2. doi: 10.1155/2014/373642. 10. Radostits O.M., Clive C.G., Hinchcliff K.W., Constable P.D. (2007) In: Veterinary Medicine: A textbook of the diseases of cattle, horses, sheep, pigs and goats, Ed. 10th, 946-949, Elsevier, Saunders, Edinburgh. 11. Srinand S., Hsuan S.L., Yoo H.S., Maheswaran S.K., Ames T.R., Werdin R.E. (1996) Comparative evaluation of antibodies induced by commercial Pasteurella haemolytica vaccines using solid phase immunoassays. Vet Microbiol, 49: 181-195. doi.org/10.1016/0378-1135(95)00187-5. 12. Tao S. (2009) Evaluation of a vaccine against Mannheimia haemolytica and Pasteurella multocida in sheep. Honors Thesis Presented to the College of Agriculture and Life Sciences, Department of Animal Science of Cornell University in Partial Fulfillment of the Requirements for the Research Honors Program. 13. Akan M., Öncel T., Sareyyüpoğlu B., Hazırŏglu R., Yaşar Tel O., Cantekin Z. (2006) Vaccination studies of lambs against experimental Mannheimia (Pasteurella) haemolytica infection. Small Ruminant Res, 65: 44-50. doi: 10.1016/j.smallrumres.2005.05.020. 14. Mosier D.A., Confer A.W., Hall S.M., Gentry M.J., Panciera R.J. (1986) Enzyme-linked immunosorbent assay for detection of serum antibodies to Pasteurella haemolytica cytotoxin (leukotoxin) in cattle. J Clin Microbiol, 24: 218-222. 15. Gautier J. M., Corbière F. (2017) Better knowledge for sheep colostrum quality and passive immune transfer, Ninth International Sheep Veterinary Congress, Harrogate, UK, 22-26.

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Infezione da Ovine herpesvirus tipo 2 in allevamenti ovini in Umbria

ORIANA RAFFAELE1, EMILIA DEL ROSSI1, LORENZO CASTELLI2, STEFANO PIGNANI2, STEFANO PETRINI3, MARIA TERESA MANDARA1, MARIA LUISA MARENZONI1 1

Dipartimento di Medicina Veterinaria, Università degli Studi di Perugia Associazione Regionale Allevatori Umbria 3 Istituto Zooprofilattico Sperimentale Umbria e Marche “Togo Rosati” 2

RIASSUNTO Ovine gammaherpesvirus 2 (OvHV-2) è un gammaherpesvirus che causa un’infezione asintomatica nella pecora e una malattia sistemica molto grave in altre specie di ruminanti e nel maiale, denominata febbre catarrale maligna (FCM). Il controllo dell’infezione nella pecora risulta essere un fattore chiave nel contenimento della FCM. Inoltre non può essere escluso un ruolo di questo virus nella patologia ovina. Scopo del presente studio è stato quello di indagare la presenza di OvHV-2 negli allevamenti ovini in Umbria e di comprendere la relazione di alcuni fattori di rischio dell’infezione individuati negli allevamenti controllati. Sono stati analizzati, tramite nested PCR, 50 tamponi nasali prelevati rispettivamente da 39 pecore e da 11 capre, provenienti da 9 diverse aziende, per ricercare la presenza del DNA di OvHV-2. La specie, l’età, l’attitudine produttiva, la promiscuità con specie diverse (bovini e/o capre) e l’effetto del singolo allevamento sono stati valutati come possibili fattori di rischio dell’infezione mediante analisi statistica univariabile e multivariabile. Ventinove su 39 (74,4%) pecore e 3 su 11 (27,3%) capre sono risultate positive. I fattori di rischio presi in considerazione si sono dimostrati significativi nell’analisi univariabile, ma solo l’effetto dell’allevamento è rimasto statisticamente significativo nell’analisi multivariabile. Probabilmente il singolo allevamento contiene al suo interno altre variabili in grado di spiegare una modificazione nella prevalenza dell’infezione da OvHV-2. In futuro dovranno essere esaminati altri fattori tipici di ciascun allevamento al fine di comprendere meglio l’epidemiologia dell’infezione.

PAROLE CHIAVE Ovine gammaherpesvirus 2, Regione Umbria, pecora, PCR, allevamento ovino.

INTRODUZIONE Ovine gammaherpesvirus 2 (OvHV-2) è un gammaherpesvirus che causa un’infezione pressoché asintomatica nelle pecore, mentre è associato a forme cliniche, anche molto gravi, in specie affini ma non completamente permissive. In quest’ultimo caso la malattia è conosciuta come febbre catarrale maligna (FCM). Nonostante siano stati fatti passi avanti nelle conoscenze della patogenesi, ancora si è lontani dal capire il reale meccanismo che è alla base sia dell’infezione nella pecora sia della FCM nelle specie affini suscettibili. Si pensa che la trasmissione di OvHV-2 avvenga prevalentemente tramite la via inalatoria1,2, ma anche la via venerea è stata ritenuta possibile, in particolare da parte dell’ariete, condizione che potrebbe determinare una stagionalità dell’infezione3. Il sito di replicazione primaria del virus negli ovini è rappresentato dal polmone4, mentre la successiva fase di latenza è localizzata nei linfociti1,5. Per quanto riguarda la malattia nelle specie secondarie, sembra che l’evento chiave nel determinismo della FCM sia una disregolazione delle cellule T, determinata dal virus6,7.

Corresponding Author: Maria Luisa Marenzoni (marialuisa.marenzoni@unipg.it).

La FCM ha una manifestazione clinica abbastanza variabile. Esiste una forma classica, acuta, costituita da una sindrome caratterizzata da linfoproliferazione sistemica, vasculite e lesioni infiammatorie distribuite in molti tessuti, e che presenta un tasso di letalità del 50-70%8. Esistono inoltre forme croniche, che possono esitare in guarigione parziale o completa, e anche infezioni asintomatiche, che probabilmente non vengono diagnosticate9,10. Questa stessa malattia, in realtà, può essere causata da diversi gammaherpesvirus del genere Macavirus: oltre a OvHV-2, anche alcelaphine herpesvirus-1 (AlHV-1), caprine herpesvirus-2 (CpHV-2), ibex malignant catarrhal fever virus, alcelaphine herpes virus-2like e un virus di origine ancora non definita, che ha causato la malattia nel cervo dalla coda bianca, sono stati associati a forme cliniche di FCM in diverse specie animali. Tutti questi virus, oltre a infettare in maniera asintomatica il rispettivo ospite primario (pecore, gnu, capre, ibex, alce, etc.), sono in grado di infettare specie affini, ma secondarie, dove causano appunto la FCM. Tra le specie più colpite da FCM vi sono i bovini, i bufali, il bisonte americano, varie specie di cervidi, nonché altri ruminanti selvatici, ma anche i suini11,12. Alcune specie di animali sono considerate altamente suscettibili11,12. La FCM si manifesta con depressione, anoressia, febbre alta, linfoadenopatia, congiuntivite, opacità corneale, infiammazione, ulcerazione ed essudazione del tratto respiratorio e digestivo superiore, diarrea e deficit neurologici, fino ad arri-

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Infezione da Ovine herpesvirus tipo 2 in allevamenti ovini in Umbria

vare alla morte dell’animale colpito11,12. Le possibilità terapeutiche sono pressoché nulle, per cui l’infezione grave è quasi invariabilmente letale. Il virus è difficilmente isolabile13 e questo ha contribuito all’incapacità di sviluppare un vaccino, ragion per cui non è possibile effettuare neanche una profilassi indiretta. Al contempo le diagnosi differenziali che occorre considerare in questi casi includono malattie quali l’afta epizootica, la diarrea virale bovina, la rinotracheite infettiva bovina, la cheratocongiuntivite infettiva da herpesvirus, la cheratocongiuntivite infettiva da Moraxella bovis, la stomatite papulosa e la blue tongue, che prevedono anche l’adozione di misure sanitarie di prevenzione restrittive, quali sequestro dell’allevamento infetto, blocco delle movimentazioni, abbattimenti, etc. In Italia, a parte i casi registrati nei giardini zoologici14,15, dove la possibilità che la specie di virus coinvolto sia più ampia, i virus che maggiormente vengono associati alla FCM sono OvHV-2 e CpHV-2. Gli episodi di FCM segnalati nel nostro territorio sono relativamente rari, occasionalmente con andamento epidemico, ma in generale probabilmente sottodiagnosticati. Generalmente questi casi sono riconducibili a situazioni di allevamenti promiscui di bovini/ovini o bovini/ovini/caprini16,17,18 o bufali/bovini/ovini19. In uno studio effettuato in Italia si è arrivati alla conclusione che la FCM, nella sua forma associata alle pecore, ossia da OvHV-2, è da considerare una malattia endemica20. Tuttavia, non esistono dati che indichino quale sia la reale prevalenza di infezione da OvHV-2 in Italia e anche a livello internazionale sono poche le indicazioni a tal proposito. La Tabella 1 riassume le prevalenze riportate per OvHV-2 nel mondo. Tra i fattori di rischio indicati per l’infezione da OvHV-2 compaiono l’età delle pecore (gli agnelli di età compresa tra i 6 e i 9 mesi diffondono il virus molto più frequentemente e più intensamente degli adulti), gli allevamenti promiscui (bovini/ovini, bovini/ovini/caprini e bufali/bovini/ovini), la densità degli animali in allevamento (maggiore è la densità, mag-

giore è il rischio di contagio per gli animali suscettibili), la pratica della transumanza (maggior probabilità di contatto diretto tra animali infetti e suscettibili), i fattori climatici, la presenza di vettori e il livello di stress dovuto alle variazioni climatiche o alle pratiche di management10,12,16,17,18,19,21,22,23,24. Non rientrerebbero invece tra i possibili fattori di rischio la razza e il sesso, né l’attitudine dell’allevamento (latte, carne o misto)22,25. Per tale motivo è importante conoscere se e quanto questi virus circolano negli allevamenti ovini e caprini del territorio e i fattori che possono influenzare la loro diffusione, che costituiscono il punto chiave per contenere l’infezione. Scopo del presente studio è stato quello di valutare la presenza di OvHV-2, potenziale trigger di FCM, in allevamenti ovini del territorio umbro e verificare se alcuni fattori di rischio sono presenti nei nostri allevamenti al fine di aumentare le conoscenze sull’epidemiologia di questa infezione nel territorio.

MATERIALI E METODI Nel periodo ottobre 2016 - febbraio 2017 sono stati prelevati, previo consenso informato degli allevatori, dei tamponi nasali (TN) da pecore in quanto considerati un campione indicativo della possibilità di trasmissione del virus, che avviene principalmente per via inalatoria, a seguito di contatto ravvicinato. Gli allevamenti testati sono stati selezionati in quanto iscritti all’Associazione Regionale Allevatori (ARA) dell’Umbria e considerati rappresentativi della realtà produttiva ovina e caprina locale. Gli elementi che sono stati registrati al momento del prelievo erano il codice identificativo del soggetto campionato; la razza; l’età; il sesso; l’attitudine produttiva; la consistenza numerica dell’allevamento; la tipologia di allevamento (intensivo, semi-intensivo, estensivo); il numero ap-

Tabella 1 - Prevalenza dell’infezione da OvHV-2 riportata in precedenti studi nel mondo. Area geografica

Test applicato

Prevalenza OvHV-2

Bibliografia

INDIA (Regione del Kashmir)

PCR da sangue

pecore: 84,4% (28/33) agnelli: 95,2% (20/21) capre: 61,5% (16/26)

Wani et al., 20068

INDIA (Regione del Karnataka)

PCR da sangue

pecore: 24,44% (87/356)

Premkrishnan et al., 201521

KENYA

PCR da sangue

pecore: 90,4% (161/178)

Mirangi et al., 199730

PORTOGALLO

PCR da sangue

pecore (allevamento monospecifico): 81,3% (65/80) allevamento solo bovini (allevamento monospecifico): 0% (0/18) pecore (allevamento misto bovini-pecore): 70,5% (31/44) bovini (allevamento misto bovini-pecore): 7,4% (2/27)

Cortez et al., 200810

USA

PCR da sangue CI-ELISA§

pecore: 99% (143/144) pecore (non associate a casi di FCM nel bovino): 53% (282/531) pecore (associate a casi clinici di FCM nel bovino): 59% (88/149) capre: 61% (177/291)

Li et al., 199625

SUD AFRICA

PCR da sangue

pecore: 75,3% (130/170)

Bremer et al., 201022

TURCHIA

PCR da sangue

pecore: 62,7% (37/59)

Kalayci et al., 201531

GERMANIA

PCR da sangue CI-ELISA§

pecore: 100% (20/20) pecore: 72% (36/50)

Frolich et al., 199840

GIAPPONE

SN*

pecore: 37,6% (56/154) capre: 40% (2/5)

Giangaspero et al., 201341

CI-ELISA: competitive inibition ELISA; * SN: sieroneutralizzazione.

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O. Raffaele et al. Large Animal Review 2020; 26: 79-85

prossimativo di movimentazioni in entrata o in uscita dall’allevamento; l’ubicazione dell’allevamento; la densità degli animali nell’allevamento; l’effettuazione della pratica della transumanza; la gestione delle rimonte e l’età di separazione degli agnelli dalle madri; infine, se l’allevamento era promiscuo con caprini, bovini o altri ruminanti. Da ogni allevamento sono stati campionati almeno 5 animali fino a un massimo di 10. I TN sono stati effettuati utilizzando tamponi di cotone che sono stati strofinati all’interno delle narici delle pecore e successivamente posti in 500 µl di tampone fosfato salino (PBS).

Estrazione del DNA Il DNA è stato estratto partendo da 200 µl del liquido di sospensione del tampone nasale, utilizzando un kit commerciale, secondo le indicazioni del produttore (QIAamp DNA Mini kit, Qiagen).

Protocolli di PCR Al fine di identificare OvHV-2 sono stati utilizzati inizialmente tre protocolli di PCR. Il primo era un protocollo di PCR in grado identificare tutti i membri della famiglia Herpesviridae, avendo come gene target un frammento della DNA polimerasi comune a tutti gli herpesvirus26. In parallelo, nelle prime fasi, sono stati utilizzati due ulteriori protocolli di PCR OvHV-2-specifici, secondo quanto indicato dalla World Organisation for Animal Health (OIE)27,28,29. I protocolli di PCR sono stati sottoposti a un settaggio iniziale, tramite PCR a gradiente, che ha valutato temperature comprese tra 53 e 66°C per verificare se il profilo termico indicato nei protocolli pubblicati fosse quello ottimale o se la temperatura di annealing dei primer andasse modificata per ottenere un protocollo più sensibile. Le temperature di annealing sono state verificate tramite diluizioni seriali del controllo positivo (DNA di OvHV-2 sequenziato). I tre diversi protocolli di PCR sono stati poi comparati tramite diluizioni seriali del controllo positivo per valutare la diversa sensibilità. Ogni set di reazione allestito aveva un controllo positivo (DNA di OvHV-2 precedentemente sequenziato) e un controllo negativo (senza DNA di OvHV-2).

Analisi statistica La stima della prevalenza dell’infezione da OvHV-2 è stata definita come proporzione di animali positivi alla PCR da TN sul totale degli animali testati. Le variabili indipendenti registrate al momento del prelievo e considerate potenziali fattori di rischio per l’infezione (specie, età, allevamento, attitudine produttiva, promiscuità tra specie) sono state analizzate separatamente per accertare la possibile relazione tra potenziale fattore di rischio e infezione da OvHV-2. L’età è stata analizzata categorizzandola in due gruppi: soggetti di età > di 1 anno oppure > di 2 anni. Il test F di Fisher o il test χ2 sono stati utilizzati secondo il metodo più appropriato ai dati in studio. Variabili che sono risultate con una probabilità (P) ≤0,20 nel modello univariabile sono state incluse nel modello multivariabile, inserendo manualmente le variabili ed eliminandole con la modalità backward. La stima dell’Odds Ratio (OR) e dei corrispondenti intervalli di confidenza al 95% (95% CI) è stata ottenuta tramite regressione logistica. I dati sono stati analizzati tramite il software R, version 2.8.1 (R, Development Core Team 2007). Un valore di P<0.05 è stato considerato significativo.

RISULTATI Scelta del protocollo di PCR Dai risultati ottenuti con la PCR a gradiente, le temperature indicate nei protocolli pubblicati da Baxter et al. (1993)28 e Flach et al. (2002)29 non rappresentavano le migliori e quindi sono state utilizzate nuove temperature di annealing, come indicato nella Tabella 2. Il protocollo più sensibile è risultato quello descritto da Flach et al., 200229, che è stato utilizzato per eseguire le analisi su tutti i soggetti.

Prevalenza e analisi dei fattori di rischio In totale sono stati campionati 50 animali, di cui 39 pecore e 11 capre. Sette animali erano sotto l’anno di età, 23 di età compresa tra 1 e 2 anni e 20 di età superiore ai 2 anni. Nel periodo dell’anno in cui è stato effettuato il campionamento non erano presenti soggetti di 6-9 mesi di età, che rappresentano la fascia di età più suscettibile all’infezione. Gli allevamenti avevano caratteristiche uniformi per quanto riguarda la densità degli animali (conforme alle indicazioni del Decreto Legislativo 26 marzo 2001, n. 146, recante l’Attuazione della direttiva 98/58/CE sulla protezione degli animali negli allevamenti, GU n. 95 del 24-04-2001), la transumanza (che non era praticata da nessun allevamento), la separazione degli agnelli dalle madri (che avveniva a circa 30 giorni di vita) e l’introduzione delle rimonte nel gruppo degli adulti (che avveniva dopo il primo parto, a circa 15 mesi di età). Gli allevamenti erano tutti di tipo semiestensivo, ad eccezione di uno intensivo e uno estensivo. L’attitudine produttiva era da latte per sei aziende e da carne per tre. La consistenza numerica degli allevamenti variava da 70 a 1200 capi, con un valore medio di 471 animali e una mediana di 460. Solo un’azienda aveva una consistenza inferiore ai 100 capi. Il numero di movimentazioni era basso per tutte le aziende, da considerarsi occasionale, sebbene in alcuni casi una singola movimentazione poteva consistere anche nell’acquisizione di circa 100 nuovi soggetti. Tutti gli allevamenti erano ubicati vicino ad altri allevamenti, riserve naturali o zone di ripopolamento, in cui erano comunque presenti altri ruminanti selvatici o domestici. Quattro aziende allevavano esclusivamente ovini, due avevano sia pecore che capre, due anche capre e bovini e una era promiscua con bovini. In genere erano associate pecore da carne e bovini da ingrasso. Un allevamento prima promiscuo con pecore e capre ha inserito i bovini da ingrasso nel periodo successivo allo studio. La prevalenza totale dell’infezione riscontrata da TN era del 64% (32/50). La Tabella 3 riporta le prevalenze ottenute, considerando le singole variabili esaminate nell’analisi statistica. All’analisi univariabile sono risultati significativi tutti i fattori indagati, ossia la specie (pecora vs capra), l’età superiore a 1 anno o superiore a 2 anni, l’allevamento, l’attitudine (latte vs carne), la promiscuità tra specie. L’unico fattore che invece è risultato significativo anche nell’analisi multivariabile è l’allevamento, che ha ottenuto un OR=0.55, CI 95%: 0.38-0.79, P=0.02. Pertanto, l’allevamento risulta un fattore più determinante rispetto agli altri, perché probabilmente assorbe in un unico parametro tutte le altre variazioni considerate.

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Infezione da Ovine herpesvirus tipo 2 in allevamenti ovini in Umbria

Tabella 2 - Primer utilizzati nello studio.

Nome primer

Gene target

DFA (F)

DNA polimerasi degli herpesvirus

ILK (F)

DNA polimerasi degli herpesvirus

KG1 (R)

DNA polimerasi degli herpesvirus

TGV (FN)

DNA polimerasi degli herpesvirus

IYG (RN)

DNA polimerasi degli herpesvirus

556 (F; FN)