Large Animal Review 1-2021 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

01/21

Bimonthly, Year 27, Number 1, February 2021

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 • Effects of teat end score on milk yield and quality in Holstein-Friesian cows • How effective is individual claw trimming in cattle? An ex-vivo study • Influence of niacin application on inflammatory parameters, non-esterified fatty acids and functional status of liver in cows during early lactation • Comparison of cytological, microbiological and histopathological findings of genital tracts in cows with different degree perineal conformation disorder OVINE • Development of sexual behaviour in ram lambs and its correlation to serum testosterone SWINE • The effect of dietary l-tryptophan on productive performance and behavior of weaned piglets POULTRY • Use of industrially produced litter material from waste paper sludge as litter in broiler houses REVIEWS BOVINE • Prevention of the main Clostridial diseases in cattle

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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INDEX

ORIGINAL ARTICLES

Anno 27, numero 1, Febbraio 2021 Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IF (2019): 0.299

KOÇ ATAKAN, AVCı MUSTAFA CAN, DOĞAN MEHMET ZEKI 3

How effective is individual claw trimming in cattle? An ex-vivo study

Editor in chief: Massimo Morgante 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

UYGUR CANATAN, HASAN KURT, MELIKE AKBALA, MELIKE ÇETIN, HILAL ACAR, VILDAN ASLAN, CANAN ALTıNCı SARıL, ELIF MEKIK TEMIZ, PELIN YIĞITGÖR, HAKAN SALCI

KOSTA PETROVIĆ, DRAGICA STOJANOVIĆ, MARKO R. CINCOVIĆ, BRANISLAVA BELIĆ, IVANA LAKIĆ, RADOJICA ĐOKOVIĆ

Technical Editor: Enrico Fiore 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.

Edizioni SCIVAC Palazzo Trecchi - 26100 Cremona Tel. 0372/460440 Iscrizione registro stampa del Tribunale di Cremona n. 299 del 25/9/1995 Direttore Responsabile Antonio Manfredi

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.

Comparison of cytological, microbiological and histopathological findings of genital tracts in cows with different degree perineal conformation disorder EMSAL SİNEM ÖZDEMİR SALCI, ÖZKAN YAVA, ÖZGE YILMAZ ARDIÇLI, GÜRSEL SÖNMEZ, SERPİL KAHYA DEMİRBİLEK, SENA ARDIÇLI, KAMIL SEYREK İNTAŞ

OVINE

Development of sexual behaviour in ram lambs and its correlation to serum testosterone NEVENA MAKSIMOVIĆ, SLAVČA HRISTOV, ALEKSANDAR MILOVANOVIĆ, TOMISLAV BARNA, IGOR STOJANOV, BOGDAN CEKI, IVANA MILOŠEVIĆ - STANKOVIĆ

SWINE

The effect of dietary l-tryptophan on productive performance and behavior of weaned piglets ŽIVKOVIĆ VLADIMIR, STANKOVIĆ BRANISLAV, HRISTOV SLAVČA, DELIĆ NIKOLA, NIKŠIĆ DRAGAN, SAMOLOVAC LJILJANA, PETRIČ EVIĆ MAJA

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

Influence of niacin application on inflammatory parameters, non-esterified fatty acids and functional status of liver in cows during early lactation

Managing Editor: Matteo Gianesella

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)

BOVINE Effects of teat end score on milk yield and quality in Holstein-Friesian cows

POULTRY

Use of industrially produced litter material from waste paper sludge as litter in broiler houses YETER BEYHAN

REVIEWS

BOVINE Prevention of the main Clostridial diseases in cattle RICCARDO COMPIANI, SILVIA GROSSI, LUIGI LUCINI, CARLO ANGELO SGOIFO ROSSI

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SAVE THE DATE Mercoledì 9 giugno Giovedì 10 giugno Venerdì 11 giugno Mercoledì 16 giugno Giovedì 17 giugno WEBINAR DALLE 15 ALLE 18 Per informazioni: Paola Orioli - Responsabile congressuale

info@sivarnet.it

0372 403539

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K. Atakan et al. Large Animal Review 2021; 27: 3-7

Effects of teat end score on milk yield and quality in Holstein-Friesian cows

KOÇ ATAKAN1*, AVCI MUSTAFA CAN1, DOĞAN MEHMET ZEKI1 1 Aydın Adnan Menderes University, Faculty of Agriculture, Department of Animal Science, 09100, Aydin/Turkey

SUMMARY Introduction - Hyperkeratosis develops in the teat end (TE) because of degeneration of the keratin layer. The functional errors and higher vacuum of the milking machine, increased milk yield, prolongation of milking, dirtiness of the animals and insufficient bedding are the reasons of the formation of hyperkeratosis on the TE. The lesions at the teats are risk for mastitis and there is a positive correlation between somatic cell count (SCC) and TE hyperkeratosis. Aim - The aim of this study was to determine hyperkeratosis level on the TE of Holstein-Friesian (HF) cows by using a scoring system and also the effect of TE score on the quality of milk. In addition, the effects of SCC on milk yield, fat content (FC, %) and non-fat dry matter content (NFDMC, %) and the loss of milk yield due to SCC were determined. Materials and methods - A HF herd in Aydın, Turkey, was visited monthly for nine months to score the TE for hyperkeratosis level, and to take milk samples for the determination of FC, NFDMC, and SCC. The level of hyperkeratosis on the TE were scored from 1 to 4. Correlation coefficients among the traits were also determined. Results and Discussion - The means of TE score, Log10SCC, morning (MMY) and evening (EMY) milk yield, FC and NFDMC were 2.45±0.069, 5.66±0.045, 10.88±0.289 kg, 10.88±0.283 kg, 3.96±0.070% and 10.38±0.043%, respectively. Among the parities, the lowest mean (2.02 ± 0.102) for TE score was determined in the first lactating cows, and the cows that calved in the summer (2.37 ± 0.090) had lower TE score than those that calved in winter (3.04 ± 0.109) (P <0.05). Log10SCC averages were higher in cows with higher TE score and higher parities (4th and 5th+). Log10SCC means of the cows whose TE score are 4 (6.26 ± 0.094) and 3 (5.86±0.093) are higher than those with 1 (5.48±0.118) and 2 (5.51±0.078) scores (P <0.05). High SCC in milk resulted in 1.73 kg (14.32%) decrease in MMY (P<0.05) and 3.24 kg (13.41%) in daily milk yield. Correlation coefficient between TE score and Log10SCC was positive and moderate (r=0.41). Conclusion - In conclusion, the TE hyperkeratosis caused by insufficient maintenance-management and barn and milking management conditions on the farm, leads to a significant increase in SCC and decrease in FC (24.4%). A significant milk yield loss were also determined in the daily milk yield due to high SCC. Scoring the TE of dairy cows gives an idea about the operating conditions on the dairy cattle farm and significant losses in milk yield and milk constituents could be prevented by decreasing SCC through improving TE score.

KEY WORDS Dairy cattle, Teat profile, Milk yield, Fat content, Somatic cell count, Correlation.

INTRODUCTION Milk quality is an important factor for the industrialists, the consumers as well as the producers. There is a direct relationship between the hygienic quality of milk and mastitis. Mastitis is an udder tissue inflammation commonly seen in dairy cattle, usually due to the pathogens, and is the most common and costly disease that causes significant economic losses in dairy cattle1. Due to higher relation with mastitis, the number of somatic cell count (SCC) in milk provides an idea about the health condition of the udder and the quality of milk produced.

Corresponding Author: Koç Atakan (acok@adu.edu.tr).

It has been stated that an infection of the mammary gland is the major factor affecting SCC2. In the studies on the prevalence of mastitis among enterprises and other factors affecting the frequency of mastitis according to the enterprises are barn conditions, hygiene and machine milking, season, feeding and milking techniques3. Breed, age or parity of cow and lactation period are the factors affecting SCC at cow level and in the first month of lactation SCC level is expected to be the highest4. One of the factor that causes an increase in mastitis prevalence and also SCC in milk is the formation of hyperkeratosis on the teat end (TE). Hyperkeratosis develops on the TE as a result of degeneration of the keratin layer, which is a physical barrier against the pathogens that cause infection in the udder5. The formation of hyperkeratosis on the TE may be resulted from

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Effects of teat end score on milk yield and quality in Holstein-Friesian cows

many factors such as higher vacuum and the functional errors of milking machines6, prolongation of milking7, increased milk yield, the dirtiness of the animals, insufficient bedding and also cow factors like teat shape, yield and genetics8. The reasons of the short, medium and long-term changes on the TE are reported by Mein et al.8 The changes in colour, firmness, thickness or swelling of teats, or degree of «openness» of the teat orifice are the short-term changes caused by the failure in milking machines or milking management. The medium- and long-term changes in the teat skin condition taking for a few days to 8 weeks are due to harsh weather or chemical irritation. Because of an adaptation in the teat, a ring with a diameter of 2 mm develops and it is stated that mastitis also increases with the increase of hyperkeratosis structures during lactation9. Hyperkeratosis is seen in cows with high milk yield especially in the fourth month of lactation10 and a positive correlation between TE hyperkeratosis and SCC in milk was reported11,12. A risk for mastitis due to the lesions at the teats was also mentioned13. Gleenson et al.11 reported increased risk of intramammary infection due to the TE hyperkeratosis. In an uninfected udder lobe, SCC in milk is generally below 200,000 cells / ml2, and it was stated that this value is expected to be below 100,000 cells / ml in cows in the first lactation, and the lowest SCC average was reported in the primaparous cows14. SCC in the udder lobes without infection may vary depending on environmental and physiological factors15. While milk yield increases with increasing parity, the sensitivity, deformations and contamination probability of the udder tissue with pathogenic microorganisms increase. A negative correlation between SCC and milk yield exists and, 0.92 kg of milk loss per day in primaparous cows and 1.52 kg of milk yield loss in multiparous cows with increased SCC were reported16. Some important changes in the composition of milk are also seen because of increasing SCC in raw milk17,18. Depending on the increase in SCC, the lactose content of milk decreases significantly while the amount of whey protein content increases18. The present study was conducted in a Holstein-Friesian (HF) herd in Aydın, Turkey, to examine the effects of TE score on the quality of milk as well as to determine the effects of SCC on milk yield, milk constituents and the loss of milk yield.

MATERIAL AND METHODS This research was carried out in a Holstein-Friesian (HF) herd at Aydın Adnan Menderes University (ADU), Faculty of Agriculture, Turkey. The herd was visited at morning milking once a month between June, 2019 and February, 2020. During the visit, besides scoring the TE of HF cows in terms of hyperkeratosis, a milk sample was taken from each cow to determine the milk fat content (FC, %), non-fat dry matter content (NFDMC, %) and SCC (cell/ml). The level of hyperkeratosis on the TE was scored and four different TE score were recorded from 1 to 4 (Figure 1). According to this scoring system; normal teat and no deformation (1 or N), a swollen ring at the TE (2 or S), a rough ring with keratin frons with 1-3 mm elongated from the nipple canal orifice (3 or R), and a fluffy and keratinized chapped and leafy ring (4 or VR) at the tip of the head8.

Figure 1 - Teat end (TE) hyperkeratosis scoring system (Mein et al., 2001).

A portable refractometer with the caliber 20% brix (Brand: ATC Refractometer 0-20% BRIX) was used to determine NFDMC (%) and, FC (%) was determined according to EAS19. In addition, the SCC level in milk was determined according to direct microscopic counting method20. A total of 203 milk samples were taken into the sampling containers of 50 cc per cow. The milk samples were kept in a cold chain and brought to ADU Faculty of Agriculture, Department of Animal Science, Animal Raising and Breeding Laboratory for the determination of FC, NFDMC and SCC. Milk yields produced by the cows in the morning and also evening milking were also recorded. The lactation period of the cows are divided into four lactation stages in 100-day interval, and two calving seasons are accepted as summer (May-October) and winter (November-April). In order to determine the effects of SCC on yield, loss and constituents of milk, four different SCC groups were accepted. According to this the cows with 0-200.000 cells/ml in milk included into the first group, cows with 200.001-400.000 cells/ml in milk included into group 2, cows with 400.001-1.000.000 cells/ml included into group 3 and the cows with SCC higher than 1.000.000 cells/ml included into group 4.

Statistical analysis The data obtained in the study were analyzed statistically with SAS12 package program, and the comparison of the subgroups was determined according to Tukey (P <0.05). Before the analysis, 10- base logarithmic transformation was applied to SCC data to provide a normality assumption22. The following statistical model was used in the analysis of TE score, Log10SCC, MMY, EMY, FC (%) and NFDMC (%) data. Yijklmno= µ+ ai + bj + ck + dl + fm+ gn + eijklmno Yijklmno: observation of the traits, µ: means of the traits, ai: parity effect (i=1, 2, 3, 4 and 5+), bj: calving season effect (j= winter and summer), ck: lactation period effect (k= 1, 2, 3 and 4), dl: observation month effect (l= April, May, … and February), fm: TE score effect (m= 1, 2, 3 and 4, this factor is used for the analysis of MMY, EMY, FC, NFDMC and Log10SCC), gn: SCC group effect (n= 1, 2, 3 and 4 and this factor was used only for the analysis of MMY, EMY, FC and NFDMC), eijklmno: random error. Phenotypic correlation coefficients among TE score, Log10SCC, MMY, EMY, FC and NFDMC were also calculated.

RESULTS The means and standard errors of TE score, Log10SCC, MMY, EMY, FC and NFDMC are given in Table 1. The overall averages of these traits are 2.45 ± 0.069, 5.66 ± 0.045 (457,088 cells / ml), 10.88 ± 0.289 kg, 10.88 ± 0.283 kg, 3.96 ± 0.070% and 10.38 ± 0.043%, respectively. The effects of parity, calving season, lactation stage and ob-

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K. Atakan et al. Large Animal Review 2021; 27: 3-7

Table 1 - LSMEANS and standard errors of teat end (TE) score, Log10SCC, fat (%), non-fat dry matter content (NFDMC, %) and morning and evening milk yield.

NS: not significant, *: P<0.05. **: P<0.01. A, B, C: Different letters show the significance level for P <0.01; a, b, c, d: Different letters show the significance level for P<0.05.

servation month on TE score were found to be statistically significant (P <0.05). TE score mean of the cows at the first lactation (2.02 ± 0.102) is the lowest and different from all other parities (P <0.05), and other differences among them are statistically insignificant (P> 0.05). As seen in Table 1, the cows that calved in winter (3.04±0.109) had higher TE score than those that calved in summer (2.37 ±0.090) (P<0.01). TE score was changed significantly (P<0.05) depending on the lactation stage and the lowest TE score mean was determined for the cows at the first stage of lactation and the highest for the third lactation stage. The difference between these two lactation stages was statistically significant (P<0.05), however other differences among the lactation stages were insignificant (P>0.05). For Log10SCC, the effects of parity, observation month and TE score were determined to be statistically important (P <0.05), however, calving season and lactation stage effects were insignificant (P> 0.05). As it can be seen from Table 1, the lowest Log10SCC mean was determined for the 2nd parity (5.57±0.081), and the highest was determined for the 4th (5.91±0.098) and 5th (5.91±0.135) parities. The difference between the 2nd and 4th parities are statistically significant (P<0.05), and other differences among the parities are insignificant (P>0.05). In parallel with the increase in TE score, the Log10SCC averages also increased gradually and the highest Log10SCC mean determined for TE score =4 (Table 1, Figure 2). The mean Log10SCC for TE score =4 is 6.26±0.094 (1,819,701 cells/ml) and this TE score is different from all other TE scores (P<0.05). In addition, TE score =3 (5.86±0.093; 724,436 cells/ml) was also

found to be statistically different from other TE scores (P<0.05). The effects of parity, lactation stage and month of observation on MMY and EMY were determined to be statistically significant (P <0.05), but calving season and TE score effects on these traits were insignificant (P>0.05). On the other hand, a statistically significant effect of SCC group on MMY was determined (P<0.05) but, SCC group effect on EMY was insignificant (P>0.05). For both MMY and EMY, the highest means were calculated for the first SCC groups, the means determined for the third and fourth SCC groups for both MMY and EMY are lower than those in the first and second SCC groups. The difference between the first and the fourth SCC groups for MMY

Figure 2 - Changes of SCC level depending on teat end (TE) score (A, B; significance for P<0.01, a, b, c: significance for P<0.05).

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Effects of teat end score on milk yield and quality in Holstein-Friesian cows Table 2 - Phenotypic correlation coefficients among TE score, Log10SCC, MMY, EMY, FC and NFDMC. TE score Log10SCC

Figure 3 - Daily milk yield loss depending on SCC group.

was found to be statistically different (P<0.05), but other differences among SCC groups were statistically insignificant (P>0.05) for MMY and EMY. As seen in Table 1, the cows in the fourth SCC group with the highest SCC mean had 1.73 kg (14.32%) and 1.15 kg (12.5%) less MMY and EMY than those with SCC less than 200,000 cells/ml at the first SCC group, respectively. In other words, daily milk loss of cows for the higher SCC groups can be calculated as 2,8 kg (11.59%) and 3.24 kg (13.41%), respectively for the third and fourth SCC groups (Figure 3). The milk loss per day for SCC group 2 is only 0.41 kg (1.7%). The losses per lactation can be calculated as 125 kg, 854 kg and 988.2 kg, respectively for the 2nd, 3rd and 4th SCC groups, compared to the first SCC group. As expected, MMY and EMY decreased gradually depending on the advance of lactation and revers to this, NFDMC (%) and FC (%) in milk increased. NFDMC (%) and FC (%) means for the first lactation stage were 9.98±0.10% and 3.77±0.18% and the means increased to 10.60±0.09% and 4.34±0.16% at the third stage of lactation, respectively. Among the factors, lactation stage and observation month had statistically significant effects (P<0.05) on NFDMC (%) and, for FC (%) the effects of parity, TE score and SCC group were determined to be statistically significant (P<0.05). As seen in Table 1, FC (%) decreased as TE score increased. The highest FC (%) was calculated as 4.55±0.21% for the first TE score and the mean decreased gradually to 3.44±0.18% for TE score =4. In other words, the cows had 9.23, 11.65 and 24.4% less fat in milk for 2nd, 3rd and 4th TE score than those the cows had TE score =1. Phenotypic correlation coefficients among TE score, Log10SCC, MMY, EMY, FC and NFDMC are given in Table 2. The phenotypic correlation between TE score and Log10SCC is positive and moderate (r=0.41). The phenotypic correlations of TE score with other traits were determined to be low. As expected the correlation between MMY and EMY is very high (r=0.90). Low to moderate negative phenotypic correlations of milk yield with FC and NFDMC were also calculated (Table 2).

DISCUSSION In the study, because the primiparous cows have both lower milk yield and less worn teats, TE score mean of them was also lower than multiparous cows. With the increase in the parity, the milk yield of the cows and the probability of deformed teats

Log10SCC

MMY

EMY

0.41**

MMY

0.12

-0.04

EMY

0.11

-0.05

-0.12

0.13*

-0.23**

-0.18**

NFDMC

0.02

-0.02

-0.31**

-0.38**

0.90**

0.14*

*: Significant for P<0.05. **: Significant for P<0.01. TE: Teat end, SCC: Somatic cell count, MMY: Morning milk yield, EMY: Evening milk yield, FC: Fat content, NFDMC: Non-fat dry matter content.

also increased. The higher TE score mean determined in winter calved cows are due mainly to wet and dirtiness conditions of the barns because of the insufficient usage of bedding of the barn depending on the higher rainfall and humidity in winter in the region. This situation can be seen much more clearly by the changes of TE score depending on the observing month. As the winter approached, the TE score mean increased especially in the months from November to February (Table 1). Because of less worn teats at the first stage of lactation the cows had the lowest TE score compared to other lactation stages. As emphasized before in some studies6,7, the causes of deformations occurring at the TE are vacuum differences due to the functional structure of the milking machine; continuing milking despite the cessation of milk flowing from the udder; wet and muddy conditions of the barn8 and insufficient bedding. Indeed, it was stated that in a study23, deformations at the TE are related to the quality of the management conditions of the farms. As a result of the cracking of the keratin layer, a suitable environment is formed at the TE to proliferate microorganisms, and these microorganisms pass through the TE into the udder tissue more easily, causing mastitis while increasing SCC in milk. The increase in SCC seen in this study depending on the increase of TE score agree with a study11. They reported an association of severe hyperkeratosis with clinical and subclinical mastitis and an increase in SCC in infected quarter. They also added that the risk of intermammary infection increased with the TE hyperkeratosis. The overall mean of back-transformed value of Log10SCC (457,088 cells / ml) found in this study is similar to the averages of a study14 for some herds in the same region. The estimated milk losses per day and per lactation found in this study due to high SCC agree with the results of other studies18,24,25. Similar to this study Jonas et al.25 estimated 3.4 kg loss milk yield per day at the first parity Hungarian Holstein cows. Hortet and Seegers24 determined the average loss per lactation is 300-400 kg (ie. 4-6%) and added that the loss could be increased to 950-1050 kg/lactation. Due to high SCC, in a study, a significant milk yield loss per milking (12.7%/milking) was reported18, besides 11.92% and 6.33% reduction in lactose content and NFDMC in Red-Holstein cows, respectively. A reverse situation to TE score was seen for the SCC group. As SCC in milk increased, FC decreased, gradually. The increase in the FC (%) as SCC increases could mainly be due to the decrease in milk yield. As milk yield decreases, FC (%) increas-

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K. Atakan et al. Large Animal Review 2021; 27: 3-7

es. In a study26, conducted in Jersey, Holstein and Guernsey dairy cattle, stated that while the level of yield increases according to the parity, protein and fat content decrease. As seen in Table 1, the milk yield is low from June to September for MMY and July to October in EMY. The low level of milk yield found in this study in these months is due to the high air temperature and humidity seen in these months in the region, and the increase in the yield in the November and December may be attributed to the suitable climatic conditions seen in the region. The effects of high air temperature and humidity also cause a decrease in the percentage of NFDMC in milk in July and August. It means that as the hyperkeratosis score increases, milk SCC and prevalence of mastitis increases as well. This finding agrees with the finding of Emre12 and showing that lesions at the TE pose a risk for mastitis13. Since the hyperkeratosis structures occurring at the TE negatively affect the anatomical defense system of the udder, an increase in SCC in milk is considered.

6. 7.

9. 10. 11.

12.

13.

CONCLUSIONS 14.

In this study, the changes of TE score depending on non-genetic factors and its effects on milk yield, constituents and SCC were determined in addition to the determination of the effects of SCC on milk yield, constituents and loss of milk yield. The cows with hyperkeratosis at the TE also had high TE score and SCC value in milk. TE hyperkeratosis caused by insufficient maintenance-management and barn and milking management conditions on the farm, besides treating the health of dairy cattle, leads to significant increase in SCC and decrease in FC (24.4%) in milk. A significant milk yield loss (3.24 kg or 13.41%) were also seen in the daily milk yield due to high SCC. As an indicator of the udder health, milking management, hygiene and barn conditions, scoring the TE of dairy cows gives an idea about the operating conditions on the dairy cattle farm and with this way significant losses in milk yield and milk constituents could be prevented by decreasing SCC through improving TE score.

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Atasever S., Erdem H. (2009). Estimation of milk yield and financial losses related to somatic cell count in Holstein cows raised in Turkey. Journal of Animal and Veterinary Advances, 8: 1491-1494. Dohoo I.R., Meek A. H. (1982). Somatic cell counts in bovine milk. Can. Vet. J. 23(4):119-125. Barkema H.W., Schukken Y.H., Lam T.J.G.M., Beiboer M.L., Wilmink H., Benedıctus G., Brand A. (1999). Management practices associated with the incidence rate of clinical mastitis. Journal of Dairy Science, 82: 1643-1654. Koç A. (2008). A study of somatic cell counts in the milk of HolsteinFriesian cows managed in Mediterranean climatic conditions. Turkish Journal of Veterinary and Animal Sciences. 32(1): 13-18. Blowey R., Edmondson P. (1995). Mastitis Control in Dairy Herds. United Kingdom: Farming Press.

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Baştan A. (2010). Udder Health and Problems in Cows. Kardelen Press. İnönü Street. Ankara. Mundan D., Meral B, Demir, A, Doğaner M. (2015). Evaluation and economic of total bacteria and somatic cell count in dairy cattle farms. Harran University Journal of the Faculty of Veterinary Medicine, 4: 8489. Mein G.A., Neijenhuis F., Morgan W.F., Reinemann D.J., Hillerton J.E., Baines J.R., Ohnstad L., Rasmussen M.D., Timms L., Britt J.S., Farnsworth R., Cook N., Hemling R. (2001). Evaluation of bovine teat condition in commercial dairy herds: 1. Noninfectious factors. Pages 347-351 in Proc. 2nd Int. Symp. Mastitis and Milk Quality Vancouver, BC, Canada. Michel G., Seffner W., Schulz J. (1974). Hyperkeratosis of teat duct epithelium in cattle. MH Veterinary Medicine, 29: 570-574. Francis P.G. (1984). Teat skin lesions and mastitis. British Veterinary Journal, 5: 430-436. Gleenson D.E., Meaney W.J., O’Callaghan E.J., Rath M.V. (2004). Effect of teat hyperkeratosis on somatic cell counts of dairy cows. Intern J Appl Res Vet Med. 2(2): 115-122. Emre B. (2009). Distribution of lesions shaped in teat skin and hole in cows and effects of milk on the number of somatic cells. PhD Thesis. Ankara Univ. Health Science Institute, Ankara. Agger J.F., Willeberg P. (1986). Epidemiology of teat lesions in a dairy herd II: Associations with subclinical mastitis. Veterinarni Medicina, 38: 220-232. Koç A. (2006). Analysis of repeated milk somatic cell count of HolsteinFriesian cows raised in Mediterranean climatic conditions. J. of Biological Science. 6(6):1093-1097. Harmon R.J. 1994. Physiology of mastitis and factors affecting somatic cell counts. Journal of Dairy Science, 77: 2103-2112. Munro G.L., Grieve P.E., Kitchen B.J. (1984). Effects of mastitis on milk yield, milk composition, processing properties and yield and quality of milk products. The Australian Journal of Dairy Technology, March1984: 7-15. Atasever S., Erdem H. (2013). Relationships between somatic cell count and udder type scores in Holstein cows. Int. J. Agric. Biol., 15: 153-156. Koç A. (2015). Effects of somatic cell count and various environmental factors on milk yield and foremilk constituents of Red-Holstein cows. Journal of Agricultural Sciences. 21: 439-447. EAS. 2006. East African Standard. Determination of fat content (Routine method). Second Edition 2006. https://law.resource.org/ pub/eac/ibr/eas.164.2006.pdf Packard Jr., Tatini V.S., Fugua R., Heady J., Gilman C. (1992). Direct Microscopic Methods for Bacteria or Somatic Cells. In: Standard Methods for the Examination of Dairy Products, 16th edition, pp: 309325. Marshall, R.T. (ed.). American Public Health Association, Washington, DC, USA. SAS. 2002. SAS 9.4. Institute Inc., Cary, NC, USA. Shook G.E. (1982). Approaches to summarizing somatic cell count which improve interpretability. Annual Meeting of the National Mastitis Council, Proceedings 21st. Arlington. VA. National Mastitis Council. P: 150-166. Pirozok R.P., Mochire R.D., Helmboldt C.F. (1954). A method of reproducing teat topography (structure) for evaluation teat erosion. American Journal of Veterinary Research, 15: 140-142. Hortet P., Seegers H. (1998). Loss in milk yield and related composition changes resulting from clinical mastitis in dairy cows. Prev Vet Med. 37(1-4):1-20. Jonas E.M., Atasever A., Graft M., Erdem H. (2016). Influence of somatic cell count on daily milk yield production losses in primiparous Hungarian Holstein cows. Magyar Allatorvosok Lapja. 253-256. Schultz M.M., Hansen L.B., Steuernagel G.R., Reneau J.K., Kuck A.L. (1990). Genetic parameters for somatic cells, protein and fat in milk Holsteins. Journal of Dairy Science, 73: 494-502.

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CANATAN_imp_ok 20/02/21 11:36 Pagina 9

U. Canatan et al. Large Animal Review 2021; 27: 9-14

How effective is individual claw trimming in cattle? An ex-vivo study

UYGUR CANATAN, HASAN KURT, MELİKE AKBALA, MELİKE ÇETİN, HİLAL ACAR, VİLDAN ASLAN, CANAN ALTINCI SARIL, ELİF MEKİK TEMİZ, PELİN YİĞİTGÖR, HAKAN SALCI Bursa Uludag University, Faculty of Veterinary Medicine, Department of Surgery, Gorukle Campus, 16059, Bursa - Turkey

SUMMARY Claw trimming is an application to give a functional shape to the claws, to maintain foot weight distribution and to prevent foot diseases. Claw trimming should be done by experts. In this study, it was aimed to show the effectiveness of individual claw trimming applied in cattle claws by morphometric measurements. As material, 85 ex-vivo cattle feet of different races were used. The feet were divided into four groups as right front (n=17), left front (n=17), right hind (n=24) and left hind (n=27). In addition, the claws were divided laterally and medially. After the mechanical cleaning of the claws, functional nail cutting (Dutch method) was applied by different people and morphometric measurements of the claws (claw angle, dorsal wall length, claw height, diagonal length, heel height, inner heel height, sole length, sole width, abaxial white line width and axial white line width). The claw angle was 49° on the right hind lateral, 47° on the right hind medial, 49° on the left hind medial and within reference ranges on the other claws. Statistically, dorsal wall length of right anterior lateral (p=0.006) and left posterior medial (p=0.01) claws were significant. Significance was also detected left posterior lateral (p=0.01) and left posterior medial (p=0.01) claws in diagonal length. Inner heel height was significant on claws of left posterior lateral (p=0.01), and there was significant difference on right front lateral (p=0.02), left anterior medial (p=0.03) and left posterior medial (p=0.002) claws in terms of sole width. A positive correlation was found in the correlation analysis between all parameters except claw angle. As a conclusion, the claw trimming is important manipulation in large animal practice, regardless of the applied claw trimming technique, individual claw trimming has some difference on morphometric shape of the claws, and it should be performed by masters on this.

KEY WORDS Individual claw trimming, morphometry, claw, cattle.

INTRODUCTION Chiropody (claw trimming) is a forming process of the claw by cutting its length parts to make functional shape1. Chiropody also provides better body weight distribution in cattle1, 2, 3. Claw trimming is applied for the purpose of both diagnostic and prophylactic as well as therapeutically. In herds with high rates lameness, functional or prophylactic claw trimming should be performed4. Movement physiology of the extremities and foot biomechanics are only maintained with settled claw trimming in cattle. Improper claw trimming negatively affects the movement physiology5, and disrupts the physiological structures of the cattle. Irregular claw trimming is diagnosed if there is abnormal and asymmetrical appearance on heel horn and walls, axial and abax-

Corresponding Author: Hakan Salci (hsalci@uludag.edu.tr).

ial walls, toe, and soles4. Hoof care in cattle is important for herd management. The morphometric properties of the claw may differ genetically in cattle, therefore claw morphology should be taken into consideration in terms of preventing foot diseases and determining selection strategies6. By knowing the morphological features of the claw, structural differences that can affect the ability to absorb shock from the floor to the claw can be determined. In addition, the normal morphometric structure of the claws ensures optimum distribution of body weight on the base of the claw. Dorsal wall length, claw angle and heel height are generally expressed in claw morphometry. However, claw height, diagonal length, solear length and width are also important in terms of claw morphometry5. Weight distribution differs in cattle in front and hind legs. Body weight is more medial on the forelegs and lateral on the hind legs7. With the claw trimming, the broken weight distribution in the claw base is corrected, normal morphometric dimensions of the claw are provided, and better standing and walking of the cattle is ensured8.

CANATAN_imp_ok 20/02/21 11:36 Pagina 10

How effective is individual claw trimming in cattle? An ex-vivo study

Figure 1 - Claws’ morphometric parameters. α: claw angle, A: dorsal wall length, B: claw height, C: diagonal length, D: heel height, E: inner heel height, F: sole length, G: sole width, H: abaxial white line width and I: axial white line width.

The time for optimal claw trimming alters depending on the claw disorders and animal conformation. According to farming, housing system and individual features of animal qualities, chiropody time can be different. Thus, the periodically controls of claws is in a herd with higher lameness prevalence4. Generally, claw trimming is two times in a year for healthy herds8. The aim of this ex-vivo study is to show effectivity of the individual functional claw trimming by the morphometric measurements.

MATERIALS AND METHODS In total, 85 different breed cattle feet taken from the slaughterhouse were materials of the study. All feet were removed from carpal and tarsal joints, which were divided into four different groups [right front (n=17), left front (n=17), right hind (n=24) and left hind (n=27)]. In addition, medial and lateral claws were determined as right front lateral claw (RFL), right front medial claw (RFM), left front lateral claw (LFL), left front

medial claw (LFM), right hind lateral claw (RHL), right hind medial claw (RHM), left hind lateral claw (LHL) and left hind medial claw (LHM). Before trimming, the feet were mechanically cleaned and then all claws were trimmed according to Dutch method by different veterinary surgeons as described previously1. Trimming of the claws were performed using right-left edged knife, hoof clippers, file and electrical cutting tools. Calipers, ruler and a specific claw measuring device (ClawCheck®, Demotec, Germany) were utilized for morphometric measurements. Evaluated parameters pointed out in figure 1 were claw angle (α), dorsal wall length (A), claw height (B), diagonal length (C), heel height (D), inner heel height (E), sole length (F), sole width (G), abaxial white line width (H) and axial white line width (I) (Figure 1). Minimum, maximum, mean and standard deviations of the parameters in each group were calculated and statistical significance (p<0,05) between lateral and medial claws of fore and hindlimbs were analyzed using independent sample t-test for normal distribution and Man-Whitney U test for abnormal dis-

Table 1 - Morphometric measurements in right forelimb and P values (p<0.05). Parameter

Minimum L

Maximum M

Mean±Standart Deviations M

P values L

α (º)

47.05±4.16

46.82±4.57

0.722

0.56

A (cm)

4.3

5.1

8.9

9.1

7.51±1.03

7.75±0.98

0.006*

0.133

B (cm)

4.4

7.4

7.2

6.04±0.67

6.02±0.48

0.559

0.701

C (cm)

10.6

14.6

15.6

12.97±1.22

13.62±1.2

0.319

0.176

D (cm)

3.2

6.3

5.6

4.5±0.76

4.47±0.62

0.631

0.886

E (cm)

4.4

3.8

2.87±0.65

2.63±0.54

0.27

0.067

F (cm)

7.8

12.4

13.7

10.7±1.15

11.38±1.36

0.583

0.506

G (cm)

3.7

3.6

6.1

5.18±0.66

5.28±0.6

0.18

0.026*

H (cm)

0.1

0.6

0.8

0.46±0.13

0.52±0.17

0.001*

0.217

I (cm)

0.1

0.5

0.6

0.29±0.13

0.3±0.12

0.02*

0.132

α: Claw angle, A: Dorsal wall length, B: Claw height, C: Diagonal length, D: Heel height, E: Inner heel height, F: Sole length, G: Sole width, H: Abaxial white line width, I: Axial white line width, L: Lateral, M: Medial, (*): Significant difference.

CANATAN_imp_ok 20/02/21 11:36 Pagina 11

U. Canatan et al. Large Animal Review 2021; 27: 9-14

Table 2 - Morphometric measurements in left forelimb and P values (p<0.05). Parameter

Minimum

Maximum

Mean±Standart Deviations

P values

α (º)

46.76±4.84

47±3.87

0.07

0.429

A (cm)

6.3

6.6

10.4

10.3

8.18±1.02

8.3±1.02

0.315

0.774

B (cm)

5.2

5.6

7.4

6.25±0.56

6.2±0.38

0.459

0.303

C (cm)

12.7

12.5

16.3

16.8

13.91±0.95

14.3±1.07

0.079

0.633

D (cm)

4.5

4.2

6.3

5.37±0.51

5.14±0.58

0.899

0.062

E (cm)

2.5

2.2

4.2

4.1

3.31±0.52

3.2±0.55

0.46

0.604

F (cm)

9.5

13.1

14.2

11.03±1.16

11.65±1.27

0.884

0.418

G (cm)

4.5

6.1

5.8

5.35±0.5

5.18±0.48

0.265

0.033*

H (cm)

0.3

0.8

0.7

0.54±0.13

0.51±0.1

0.291

0.194

I (cm)

0.2

0.6

0.33±0.09

0.34±0.11

0.003*

0.065

Table 3 - Morphometric measurements in right hind limb and P values (p<0.05). Parameter

α (º)

Minimum

Maximum

Mean±Standart Deviations

P values

49.66±4.97

49.87±5

0.628

0.597

A (cm)

9.5

9.3

7.75±0.79

7.76±0.7

0.798

0.722

B (cm)

4.8

7.6

7.8

6.45±0.71

6.39±0.79

0.516

0.281

C (cm)

11.4

11.1

14.8

13.8

12.57±0.82

12.33±0.69

0.264

0.347

D (cm)

2.3

1.5

5.7

5.3

4.06±0.81

3.76±0.94

0.931

0.587

E (cm)

1.4

1.5

3.4

2.42±0.44

2.26±0.4

0.542

0.37

F (cm)

9.3

12.1

10.42±0.8

10.19±0.8

0.323

0.252

G (cm)

3.8

5.4

5±0.48

4.64±0.38

0.058

0.879

H (cm)

0.2

0.9

0.5±0.15

0.45±0.17

0.056

0.101

I (cm)

0.1

0.7

0.6

0.34±0.16

0.33±0.17

0.082

0.028*

Table 4 - Morphometric measurements in left hind limb and P values (p<0.05). Parameter

Minimum L

Maximum M

Mean±Standart Deviations

P values

α (º)

47.81±4.27

49.11±4.43

0.361

0.487

A (cm)

6.7

6.5

10.2

7.89±0.77

7.82±0.74

0.082

0.011*

B (cm)

5.1

4.9

8.2

7.6

6.41±0.74

6.29±0.67

0.957

0.839

C (cm)

10.7

10.5

15.1

14.6

12.61±0.86

12.44±0.8

0.012*

0.015*

D (cm)

2.8

5.2

5.4

4.13±0.75

4.08±0.76

0.137

0.724

E (cm)

1.2

1.7

4.4

2.6±0.64

2.46±0.51

0.013*

0.933

F (cm)

8.6

8.4

12.5

11.5

10.45±1.02

10±0.97

0.053

0.149

G (cm)

3.5

5.9

5.2

4.85±0.54

4.56±0.47

0.19

0.002*

H (cm)

0.2

0.3

0.8

0.9

0.5±0.13

0.49±0.13

0.035*

0.001*

I (cm)

0.1

0.7

0.32±0.15

0.35±0.15

0.035*

0.136

tribution. Pearson correlation analysis was performed to determine the correlation between the morphometric parameters of the lateral and medial claws on the front and hind legs. Statistical calculations were made on software (SPSS version 23, IBM®, USA).

RESULTS Minimum, maximum, mean, and standard deviations of the claw parameters were given in Table 1, 2, 3, and 4. Although the mean claw angles were found 49° in RHL and

CANATAN_imp_ok 20/02/21 11:36 Pagina 12

How effective is individual claw trimming in cattle? An ex-vivo study

Table 5 - Statistical findings between lateral and medial claws in fore-hind limbs (p<0.05). Parameter

α(º)

0.877

0.886

0.279

A (cm)

0.468

0.741

0.97

0.735

B (cm)

0.954

0.751

0.79

0.542

C (cm)

0.127

0.274

0.284

0.468

D (cm)

0.922

0.234

0.224

0.831

E (cm)

0.263

0.529

0.215

0.390

F (cm)

0.122

0.151

0.322

0.101

G (cm)

0.665

0.298

0.007*

0.05

H (cm)

0.17

0.492

0.348

0.632

I (cm)

0.737

0.811

0.546

0.476

RHM, 47° in LHL, 49° in LHM, angles of the other claws were normal. In terms of A parameters, there was a significant difference for RFL (p=0.006) and LHM (p=0.01). C parameters of the claws had significant difference LHL (p=0.01) and LHM (p=0.01). E parameters of LHL (p=0.01), G parameters of the RFL (p=0.02), LFM (p=0.03) and LHM (p=0.002) were also significant. The average value of H parameters was 0.46 cm in RFL, and lateral claw was thicker than medial claw at left hindlimb. The average value of I parameters were 0.29 cm in RFL, 0.33 cm in LFL, 0.33 cm in RHM and 0.32 cm in LHL. The fore and hind limb claws were grouped, statistical comparison results between lateral and medial claw parameters of these groups were given in Table 5. There were no significant difference between lateral and medial claws for right forelimb, left forelimb and left hindlimb (p<0.05). However, it was determined that the values of G parameter between lateral and

medial claws at right hind limb had significant difference (p=0.007). G parameter of the lateral claw is larger than medial claw. Correlation coefficient and p values between parameters are given in Tables 6, 7, 8 and 9. Of all parameters, only the claw angle had a negative correlation with the other parameters. In the correlation analysis of the claw angle with the other parameters, it was observed that there was a negative correlation between the lateral and medial claws of the forelimbs, as well as the A, C, F and G parameters of the hindlimb lateral claws, and A, C and F parameters of the hindlimbs medial claws. In addition, a positive correlation of hindlimbs medial claw angles with B and E parameters was determined. A positive correlation was found in the correlation analysis between all parameters except claw angle.

DISCUSSION Functional claw trimming helps to adjust weight distribution and balance on hooves in cows. Deformed or weakened claw horn is corrected with claw trimming. Routine claw trimming is an important manipulation for early diagnosis and treatment of the claw lesions as well as preventing the hoof diseases2,9. Morphometric measurements made before and after claw trimming provides information about the suitability of trimming10. Claw trimming rearranges claw angels and prevents the hoof lesion and disorders2,3,4. For this purpose, in this presented study, evaluation of the randomized individual functional hoof trimming were planned on cow claws and the morphometric measurements were taken on the claw to determinate whether how much individual claw trimming is functional or not. The purpose of the claw trimming is to prevent the claw lesions, and improve the locomotion by shaping the claws properly8. In the functional claw trimming, medial claw length should be 7.5 cm, the shape of the sole and dorsal length are symmetri-

Table 6 - Correlation coefficient and p values between parameters in lateral claws at forelimbs. Parameter

α (º)

A (cm)

B (cm)

C (cm)

D (cm)

E (cm)

F (cm)

G (cm)

A (cm)

-,450** ,008

B (cm)

-,071 ,690

,204 ,248

C (cm)

-,439** ,009

,866** ,000

,354* ,040

D (cm)

,143 ,420

,246 ,160

,283 ,105

,398* ,020

E (cm)

,017 ,923

,420* ,013

,273 ,118

,448** ,008

,691** ,000

F (cm)

-,528** ,001

,648** ,000

,200 ,256

,749** ,000

,006 ,974

,043 ,808

G (cm)

-,411* ,016

,509** ,002

,249 ,156

,682** ,000

,183 ,300

,241 ,170

,563** ,001

H (cm)

-,018 ,918

,288 ,099

,145 ,412

,298 ,087

,028 ,875

,055 ,756

,244 ,165

,287 ,100

I (cm)

,135 ,447

,065 ,714

-,006 ,972

,043 ,808

,242 ,168

,235 ,182

,030 ,866

-,028 ,873

H (cm)

,520** ,002

CANATAN_imp_ok 20/02/21 11:36 Pagina 13

U. Canatan et al. Large Animal Review 2021; 27: 9-14

Table 7 - Correlation coefficient and p values between parameters in medial claws at forelimbs. Parameter

α (º)

A (cm)

B (cm)

C (cm)

D (cm)

E (cm)

F (cm)

G (cm)

A (cm)

-,524** ,001

B (cm)

-,155 ,391

,322 ,064

C (cm)

-,653** ,000

,820** ,000

,410* ,016

D (cm)

,035 ,843

,449** ,008

,424* ,012

,461** ,006

E (cm)

-,144 ,417

,552** ,001

,432* ,011

,503** ,002

,826** ,000

F (cm)

-,531** ,001

,641** ,000

,325 ,061

,874** ,000

,334 ,053

,352* ,041

G (cm)

-,381** ,026

,554** ,001

,253 ,148

,674** ,000

,043 ,809

,151 ,393

,725** ,000

H (cm)

-,036 ,838

,307 ,077

,219 ,213

,233 ,185

,179 ,310

,219 ,214

,238 ,176

,312 ,072

I (cm)

,256 ,144

,046 ,795

,183 ,300

,096 ,588

,405* ,018

,352* ,041

,258 ,141

,276 ,114

H (cm)

,306 ,078

cally arranged. Solar surfaces in lateral and medial claw should be plain and axial wall should be concave9. In our study, although there was no significant difference between dorsal wall length of lateral and medial claws, there was a significant difference within the groups in terms of RFL (p=0.006) and LHM (p=0.01). A parameter of left forelimb was over 8 cm, and these results were close to normal value in the other limbs. No statistically difference was observed between D parameters. There are different claw trimming methods in cattle and all methods are performed to reconstitute the weight distribution in the claw. Aim of the claw trimming is to balance weight distribution between lateral and medial claws, and hoof trimmers

should also consider the individual hoof shape for re-constituting of the weight balance11. It has been informed that many claw trimming techniques (Dutch, white line method, Kansas and combine) are performed in the veterinary practice4. In the hoof trimming methods, Dutch method is the most commonly applied technique for functional claw trimming and this technique provides the best harmony among claws12. Considering the literature data, here, we applied to Dutch method as claw trimming technique and evaluated the functionality of the individual application of this technique. The reported morphometric measurements values of the claw parameters for about 500 kg cow are: claw angle (50° for

Table 8 - Correlation coefficient and p values between parameters in lateral claws at hindlimbs. Parameter

α (º)

A (cm)

B (cm)

C (cm)

D (cm)

E (cm)

F (cm)

G (cm)

H (cm)

A (cm)

-,353 ,011

B (cm)

,314* ,025

,213 ,134

C (cm)

-,286* ,042

,781** ,000

,319* ,022

D (cm)

,261 ,065

,151 ,290

,133 ,351

,289* ,040

E (cm)

,301* ,032

,155 ,277

-,056 ,696

,190 ,181

,610** ,000

F (cm)

-,423* ,002

,517** ,000

,155 ,276

,632** ,000

-,288* ,040

-,326* ,020

G (cm)

,259 ,067

,019 ,897

,390** ,005

,287* ,041

-,092 ,523

,030 ,837

,324* ,020

H (cm)

,209 ,141

-,019 ,895

,352* ,011

,050 ,725

,087 ,546

,017 ,907

,108 ,449

,429** ,002

I (cm)

,105 ,465

-,060 ,675

428** ,002

-,038 ,794

-,107 ,457

-,300* ,032

,257 ,069

,188 ,187

,476** ,000

CANATAN_imp_ok 20/02/21 11:36 Pagina 14

How effective is individual claw trimming in cattle? An ex-vivo study

Table 9 - Correlation coefficient and p values between parameters in medial claws at hindlimbs. Parameter

α (º)

A (cm)

B (cm)

C (cm)

D (cm)

E (cm)

F (cm)

G (cm)

A (cm)

-,328* ,019

B (cm)

,431** ,002

,023 ,871

C (cm)

-,142 ,319

,693** ,000

,109 ,447

D (cm)

,348* ,012

,101 ,480

,158 ,268

,339* ,015

E (cm)

,251 ,076

,060 ,673

,082 ,566

,112 ,433

,614** ,000

F (cm)

-,247 ,080

,419** ,002

,135 ,345

,554** ,000

-,313* ,025

-,401** ,004

G (cm)

,117 ,415

,228 ,107

,360** ,009

,294* ,036

,128 ,371

,020 ,888

,521** ,000

H (cm)

,056 ,696

-,070 ,628

,375** ,007

,064 ,654

-,001 ,992

-,111 ,440

,223 ,116

,374** ,007

I (cm)

,142 ,321

,042 ,771

,331* ,018

,087 ,543

,021 ,883

-,215 ,129

,272 ,054

,228 ,107

H (cm)

,460** ,001

front limb and 50-55° for hindlimb), the ratio of dorsal wall length and heel height (2/1), sole length (14 cm), sole width (5 cm), dorsal wall length (6-8 cm), diagonal length (10-14.5 cm), heel height (2.5-3 cm) and distance between sole and capsule (5 mm)5,13. However, it is informed that minimum dorsal wall length should be at least 9 cm for optimal claw weight distribution in cows12. In this study, the morphometric measurements values of the claw parameters were close to reported values, and except sole width of right hindlimb (p=0.007), there was no significant difference between the medial and lateral claws (Table 5). A positive correlation was found in the correlation analysis between all parameters except claw angle. On the other hand, positive and negative effects of the claw trimming are given: positively, routine claw trimming helps to detect unremarkable claw lesions and allow to early manipulations; negatively, if the cow in lactation period, trimming and similar manipulations lead to stress on cow11. Moreover, excessive shorting of the claws and inability to adjust the claw angle by trimming are the other negativities8,12,14. The most common trimming faults of this study were irregular adjustment of the claw angles and very short dorsal wall length, which resulted in decreasing sole thickness. The heel height was about 1.5 cm in some claws, which could be responsible to occurrence of the possible foot diseases, if it is practiced in routine.

References 1. 2.

3. 4. 5.

8. 9.

10.

11.

CONCLUSION In conclusion, morphometric parameters of the nail we use in this study is an auxiliary measure in determining the difference between the individual claw trimming. Complying to morphometric measurements, claw trimming in cattle also contributes to the appropriate distribution of body weight to the claws. Even though the claw trimming is important manipulation in large animal practice, regardless of the applied claw trimming technique, individual claw trimming has some difference on morphometric shape of the claws, and it should be performed by masters on this.

12.

13.

14.

Raven T. (1989): Cattle Footcare and Claw Trimming. 2nd ed, Farming Press, Ipswich, United Kingdom. Fjeldaas T., Sogstad A.M., Osteras O., 2006: Claw trimming routines in relation to claw lesions, claw shape and lameness in Norwegian dairy herds housed in tie stalls and free stalls. Prev Vet Med, 73: 255-271. Stoddard G.C., Cramer G. (2017) A review of the relationship between hoof trimming and dairy cattle welfare. Vet Clin Food Anim, 33(2): 365-375. Mahendran S., Bell N. (2015) Lameness in cattle 2. Managing claw health through appropriate trimming techniques. In Pract, 37: 231-242. Görgül O.S., Seyrek-Intas D., Salcı H., Gül N.Y. (2002) Süt sığirlarında tırnak uzamasının morfometrik değerlendirilmesi ve tırnak biyomekaniğine etkisi. Veteriner Cerrahi Dergisi, 8(3-4): 57-62. Kasarda R., Vlcek M., Candrak J., Moravcikova N. (2018) Estimation of heritability for claw traits in Holstein cattle using Bayesian and REML approaches. J Cent Eur Agric, 19(4): 784-790. Raulkar R.V., Thorat M.G., Kuralkar S.V., Chepte S.D., Waghmare S.P., Kharwadkar M.D. (2016) Morphometric evaluation of hooves in different affections of hoof in cattle. Int J Agric Sci, 8(54): 2858-2861. Manske T., Hultgren J., Bergsten C. (2002) The effect of claw trimming on the hoof health of Swedish dairy cattle. Prev Vet Med, 54: 113-129. Van der Tol P.P.J., Van der Beek S.S., Metz J.H.M., Noordhuizen-Stassen E.N., Back W., Braam C.R., Weijs W.A. (2004) The effect of preventive trimming on weight bearing and force balance on the claws of dairy cattle. J Dairy Sci, 87: 1732-1738. Nuss K., Paulus N. (2006) Measurements of claw dimensions in cows before and after functional trimming: A post-mortem study. Vet J, 172: 284292. Van Hertem T., Parmet Y., Steensels M., Maltz E., Antler A., Schlageter-Tello A.A., Lokhorst C., Romanini C.E., Viazzi S., Bahr C., Berckmans D., Halachmi I. (2014) The effect or routine hoof trimming on locomotion score, ruminating time, activity, and milk yield of dairy cows. J Dairy Sci, 97: 48524863. Archer S.C., Newsome R., Dibble H., Sturrock C.J., Chagunda M.G.G., Mason C.S., Huxley N. (2015) Claw length recommendations for dairy cow foot trimming. Vet Rec, 177(9): 222. Radisic B., Waticic D., Vnuk D., Lipar M., Balic I.M., Ditko B., Smolec O., Orak A., Capak H., Kos J. (2012) Measurements of healthy and pathologically altered hooves, their interrelation and correlation with body mass in Simmental breeding bulls. Vet Arh, 82(6): 531-544. Olechnowicz J., Jaśkowski J.M. (2010) Hoof measurements related to locomotion scores and claw disorders in dairy primiparous cows. B Vet I Pulawy, 54: 87-92.

CANATAN_imp_ok 20/02/21 11:36 Pagina 15

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PETROVIC_imp_ok 20/02/21 11:38 Pagina 17

K. Petrović et al. Large Animal Review 2020; 27: 17-21

Influence of niacin application on inflammatory parameters, non-esterified fatty acids and functional status of liver in cows during early lactation

KOSTA PETROVIĆ1, DRAGICA STOJANOVIĆ1, MARKO R. CINCOVIĆ1, BRANISLAVA BELIĆ1, IVANA LAKIĆ1, RADOJICA ĐOKOVIĆ2 1

Department of Veterinary medicine, Faculty of Agriculture, University of Novi Sad, Sq. Dositej Obradovic 8, 21000 Novi Sad, Serbia 2 Faculty of Agronomy, University of Kragujevac, Cara Dušana str. 34, 32000 Čačak, Serbia

SUMMARY Metabolic stress in periparturient period in cows is characterized by reduced food intake, negative energy balance, increased lipolysis, ketogenesis, insulin resistance and inflammation. Central metabolic organ in early lactation is liver. Lipid peroxidation and ketogenesis can trigger inflammatory response. Niacin has anti-lipolytic action and potential anti-inflammatory effect. The aim of this study was to determine influence of niacin application on inflammatory response and functional status of liver (expressed with liver functional index, LFI) in cows in early lactation. 30 Holstein-Friesian cows were included in the experiment. Niacin was applied to 15 cows two weeks before and two weeks after calving and 15 cows were included in negative control group. Blood samples were taken by venipuncture of v. coccigea before morning feeding, in the moment of calving and then at first and second week after calving. Reduction of TNF-α, haptoglobin, total bilirubin and NEFA concentration, but increase of albumin and cholesterol parameters was caused by niacin application. Fibrinogen concentration was unchanged. Increased values of albumin, cholesterol and reduced value of bilirubin followed by increase of LFI was noted after niacin application. That indicates improved liver hepatocytes function. Positive correlations between TNF-α and fibrinogen, NEFA, haptoglobin and negative with albumin were noted. That proves significance of albumin as a negative protein of acut phase and role of lipolysis in inflammation development in cows in early lactation. Negative correlation between LFI and TNF-α, haptoglobin and NEFA was noted. Correlation between LFI and NEFA and correlation of LFI and haptoglobin was controlled by TNF-α. This indicates that niacin can have dominant anti-inflammatory effect in liver protection. Besides anti-lipolytic effect, niacin has showed anti-inflammatory action that can be significant in hepoatocyte protection in early lactation in dairy cows. Significant influence of niacin on TNF-α was noted. This cytokine controls correlations of LFI, lipolysis and inflammatory response.

KEY WORDS Cows, inflammation, liver, niacin, tumor necrosis factor alpha.

INTRODUCTION Metabolic stress in periparturient period in cows is characterized by reduced food intake, negative energy balance, increased lipolysis, ketogenesis and insulin resistance development1,2. Increased lipolysis in cows can cause discharge of proinflammatory cytokines (adipokines) from fat tissue. The greatest role of them is given to the tumor necrosis factor alpha (TNF-α)3. Central metabolic organ in early lactation is liver. Liver processes increased lipid component. Lipid peroxidation and ketogenesis can trigger inflammatory response. Ohtsuka et al.4 showed increased activity of tumor necrosis factor alpha in cows with mild and severe fatty liver syndrome. Haptoglobin and serum amyloid A concentrations are increased in blood plasma of cows that developed fatty liver5. Bertoni et al.6 confirmed

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

that cows classified as cows with high inflammatory index express higher values of bilirubin, AST, GGT and reduced levels of albumin and cholesterol. That indicates fatty liver biochemical profile. Trevisi et al.7 confirmed that inflammatory mediators can directly cause metabolic changes. Niacin expresses anti-lipolytic effect, reduces insulin resistance in early lactation and has a positive influence at functional status of hepatocytes8,9. Application of anti-inflammatory drugs in peripartal period causes reduction in lipolysis and improves hepatic functions10. Anti-lipolytic effect of nicotinic acid is mediated by nicotine receptor GPR109A. Nicotine-amid reduces bonding ability to GPR109A11. Activation of this receptor causes inhibition of adenylate-cyclase. After that proteinkinase A is inactivated and reduced phosphorilation of hormone sensitive lipase occurs, and consequently lipolysis is reduced. Considering that lipolysis triggers inflammatory response and niacin acts anti-lipolytic there is a possibility of anti-inflammatory action of niacin. It has been showed that nicotine receptor GPR109A can inhibit cytokine production12 that indicates anti-inflammatory effect.

PETROVIC_imp_ok 20/02/21 11:38 Pagina 18

Influence of niacin application on inflammatory parameters, non-esterified fatty acids and...

The aim of this study was to determine niacin influence at inflammatory response and functional status of liver in cows in early lactation.

MATERIAL AND METHODS Animals and management - 30 cows of Holstein- Friesian breed in second and third lactation were included in experiment. No health disorders were noted and milk production was 7000±500 L. Cows were kept in free system at deep rug. In transitional period cows were kept bounded in a maternity ward. Cows were fed with limited meals except in maternity yard were food was given ad libitum. Mixed meals that satisfied all individual needs were given to animals. Water was given ad libitum. Meal for cows in early lactation should contain proteins (17.5-19.5% crude proteins, 30-33% degradable proteins, 35-40% non-degradable proteins), carbohydrates (ADF min. 17-21%, NDF min. 28-31%, NDF from forage min. 18-23%, non-structure carbohydrates 35-42%, level of dry matter from forage min 40-45%), energy (NEL 7-7.4 MJ/kg meal dry matter) fats 5-7%. Niacin application- Niacin was given mixed with foods per os.

Fine granular powder of nicotinic acid (Rovimix®Niacin) was used at dosage that allows 6-12 g available in gastrointestinal tract. Dosage was applied daily (60-120 g/day in total mixed ration meal). In recent studies that dosage was suggested as optimal biologic concentration. Niacin was applied during two week period before and after calving. Blood sampling- Blood was taken by venipunction of v. coccigea in cows before morning meal in order to avoid postprandial effect at metabolite values. Further blood was sampled at the moment of calving, then one and two weeks after calving. Samples for biochemical analyzes were taken in test tubes that contained gel separator (BD Vacutainer® SST II Advance, BD Plymoth, UK). Gel contained silicone that activates coagulation cascade and gel that represents barrier between coagulum and serum after centrifugation. Sampled blood was processed in laboratory in the shortest possible time. Determination of blood parameters- Biochemical analyzes were taken by automatic spectrophotometer Chemray (Rayto, PRC). NEFA, albumin, cholesterol and bilirubin concentrations was determined by standard colorimetric kits (Biosystem, Spain). Tumor necrosis factor alpha (TNF-α), haptoglobin and fibrinogen were determined by standard kits of

Table 1 - Influence of niacin application at determined parameter values. Week TNF-α (ng/mL)

Niacin

Control

SEM

Niacin influence

0.004

p<0.01

0.012

p<0.01

0.015

0.42

p<0.01

0.21

p<0.01

0.024

p<0.01

0.011

p<0.01

0.047

p<0.01

0.24

0.39

0.28

0.52

0.27

0.61

Haptoglobin (mg/dL)

0.39

0.76

0.44

0.82

0.47

0.89

Fibrinogen (g/L)

0.92

0.96

0.94

0.98

0.93

0.96

Albumin (g/L)

39.1

32.2

37.82

31.5

38.9

33.6

T.Bilirubin ( mol/L)

6.8

8.1

7.2

9.5

8.1

10.2

Cholesterol (mmol/L)

2.05

1.82

2.19

1.93

2.41

2.08

NEFA (mmol/L)

LFI

0.36

0.48

0.35

0.77

0.35

0.45

22.1

10.2

24.2

11.2

25.9

12.7

PETROVIC_imp_ok 20/02/21 11:38 Pagina 19

K. Petrović et al. Large Animal Review 2020; 27: 17-21

Table 2 - Correlation matrix of parameters in experiment. TNF-α TNF-α

Haptoglobin

Fibrinogen

NEFA

ALB

LFI

Haptoglobin

0.858**

Fibrinogen

0.115

0.077

NEFA

0.685

0.608

-0.001

ALB

-0.531**

-0.452**

-0.046

-0.512**

-0.091

LFI

-0.464

-0.398

Cloud-Clone Corp (PRC) and ELISA reader (Rayto, PRC). Liver function index (LFI)- This index was determined based on formula LFI = (ALB - 17.71) / 1.08 + (CHOL - 2.57) / 0.43 - (TBIL - 4.01) / 1.21 (Trevisi and Minuti, 2018)13. Statistical analyses- Influence of niacin application, week of sampling and their interaction at parameter values were determined by GLM model. Correlations between TNF-α, haptoglobin, fibrinogen, albumin, NEFA and LFI were determined by Pearsons correlation coefficient. By partial correlation was determined if TNF-α controls relation of LFI and other parameters with which showed significant correlation. Partial correlation was visualized by Graph of linear regression between non-standardized values obtained by analyzes of linear regression between determined parameters.

RESULTS Reduction of TNF-α, haptoglobin, total bilirubin and NEFA concentration, but increase of albumin and cholesterol parameters was caused by niacin application. Fibrinogen concentration was unchanged. Increased values of albumin, cholesterol and reduced value of bilirubin followed by increase of LFI was noted after niacin application. That indicates improved liver hepatocytes function. Results are presented on Table 1. Positive correlations between TNF-α and fibrinogen, NEFA, haptoglobin and negative with albumin were noted (Table 2). That proves significance of albumin as a negative protein of acut phase and role of lipolysis in inflammation development in cows in early lactation. Negative correlation between LFI and TNFα, haptoglobin and NEFA was noted (Table 3). Correlation between LFI and NEFA and correlation of LFI and haptoglobin was controlled by TNF-α (Figure 1-4). This indicates that niacin can have dominant anti-inflammatory effect in liver protection.

-0.396

1 0.963**

DISCUSSION Lipolisis in dairy cows is in relation with ketogenesis and change in liver function14. Reduced lipolysis and inflammatory response in cows was showed in the results. Anti-lipolytic effect of niacin is well known and these results are showed in review study of Niehoff et al.15. Niacin expresses anti-inflammatory actions in fatty tissue. Wanders et al.16 showed that in high fat meal fed mice niacin causes reduction in inflammatory response with increased expression of anti-inflammatory cytokines and reduction in expression of pro-inflammatory cytokines. Research of anti-inflammatory effect of niacin at human monocytes showed that niacin reduces secretion of TNF-α and other inflammatory cytokines17. Significant change in fibrinogen value wasn’t noted in our research after niacin application, but haptoglobin value was reduced. Morey et al.18 have not found statistically significant difference between haptoglobin value but tendency of niacin to reduce haptoglobin concentration in cows in early lactation was noted (p<0.1). At non-alcoholic fatty liver disease model was showed that niacin application directly affects lipid metabolism and indirectly affects inflammatory cascade showing protective role19. Liver showed immunologic and anti-inflammatory role by regulation of acute phase response20. During acute phase liver produces proteins of acute phase. They are great opsonins against bacteria and their toxins and they trigger complement reaction and other protective reactions21. For listed reasons, the main role of liver in albumin production is decreased due to competition between precursors. Albumins, many enzymes and apolipoproteins are known as negative proteins of acute phase. Presence of negative proteins of acute phase is crucial for maintaining metabolic integrity of the whole organism so their deficit is rapidly noticeable in cows that express health problems22. Aggregate index that combines, analyzes and

Table 3 - Summary of regression analysis for TNF-α, NEFA and haptoglobin variables predict LFI before (left) and after TNF-α exclusion (right part-partial) as a control parameter. Coefficient Model 1

Correlations

Beta

(Constant)

25.91

1.45

Haptoglobin

0.06

4.1

0.003 -0.467

Sig.

Zero-order

Partial

Part

17.93

<0.01

0.015

-0.398

0.002

0.001

-2.52

<0.05

-0.464

-0.261

-0.240

19.22

<0.01

TNF-α

-18

7.14

(Constant)

26.4

1.37

NEFA

-3.61

3.17

-0.147

-1.14

-0.396

-0.121

-0.107

TNF-α

-14.01

4.99

-0.363

-2.81

<0.01

-0.464

-0.288

-0.265

PETROVIC_imp_ok 20/02/21 11:38 Pagina 20

Influence of niacin application on inflammatory parameters, non-esterified fatty acids and...

Graphs 1-2 - Correlation of haptoglobin and LFI before (left) and after exclusion of TNF-α as a control factor (right).

Graphs 3-4 Correlation of NEFA and LFI before (left) and after exclusion of TNF-α as a control factor (right).

compares negative proteins of acute phase and similar parameters was developed23,24. The main goal of this index is to reveal and determine cows with inflammatory processes and liver damage. Aggregate indexes calculate index of functional status of the liver (LFI) and index of liver activity (LAI). Using these indices allows early identification of cows with inflammatory processes in periparturient period even in subclinical states25. Generally poor LFI and LAI indicate reduced food intake and reduced digestive activity26, greater lipid mobilization and reduced milk production27. Increased concentration of albumins and cholesterol and reduced levels of bilirubin followed by increased LFI after niacin application was noted in this research. That indicates improvement of liver hepatocytes function. It is not unusual that cows do not show clinical manifestations of metabolic disturbances and inflammatory processes until the calving and lactation. Calving triggers metabolic changes and their expression but some parameter in blood can be great indicators and signalize early development of metabolic disturbances and inflammation. The most commonly used are values of pro-inflammatory cytokines, pos-

itive proteins of acute phase and negative proteins of acute phase28. According to Trevisi et al.29 the most significant biomarkers that indicate inflammatory processes and disturbances in cows 3-4 weeks before calving are positive proteins of acute phase. Positive proteins of acute phase are positively correlated with low LFI index and parameters included in calculation of LFI30.

CONCLUSION Negative correlation of LFI and TNF-α was noted in our research because niacin cause increase of LFI index while decreases TNFα, NEFA and haptoglobin concentrations. Anti-lipolytic and antiinflammatory action of niacin was noted. That is significant mechanism in liver hepatocytes protection in early lactation. Significant influence of niacin on TNF-α concentration was noted and that is very important because this cytokine controls correlations between indices of functional status of liver hepatocytes, lipolysis parameters and inflammatory response.

PETROVIC_imp_ok 20/02/21 11:38 Pagina 21

K. Petrović et al. Large Animal Review 2020; 27: 17-21

DISCLOSURE STATEMENT No conflict of interest was reported by the authors.

13. 14.

ACKNOWLEDGMENT This research is supported by project “Influence and clinical evaluation of blood serum tumor necrosis factor alpha (TNFα) in inflammatory response of ruminants and dogs”, Province secretariat of higher education and science, Vojvodina, Serbia.

15. 16.

17.

References Cincović M. R., Ðoković R., Belić B., Lakić I., Stojanac N., Stevančević O., Staničkov N. (2018). Insulin resistance in cows during the periparturient period. Acta Agric Serb, 23(46): 233-245. 2. McFadden J.W. (2020). Lipid biology in the periparturient dairy cow: contemporary perspectives. Animal, 14(S1): s165-s175. 3. Sordillo L.M., Raphael W. (2013). Significance of Metabolic Stress, Lipid Mobilization, and Inflammation on Transition Cow Disorders. Vet Clin Food Anim, 29: 267-278. 4. Ohtsuka H., Koiwa M., Hatsugaya A., Kudo K., Hoshi F., Itoh N., Yokota H., Okada H. and Kawamura S. (2001). Relationship between serum TNF activity and insulin resistance in dairy cows affected with naturally occurring fatty liver. J Vet Med Sci, 63:1021-1025. 5. Ametaj B.N., Bradford B.J., Bobe G., Nafikov R.A., Young J.W., Beitz D.C. (2005). Strong relationships between mediators of the acute phase response and fatty liver in dairy cows. Can J Anim Sci, 85(2): 165-175. 6. Bertoni G., Trevisi E., Han X., Bionaz M. (2008). Effects of inflammatory conditions on liver activity in puerperium period and consequences for performance in dairy cows. J Dairy Sci. 91(9): 3300-3310. 7. Trevisi E., M. Amadori A. M. Bakudila, G. Bertoni (2009). Metabolic changes in dairy cows induced by oral, low-dose interferon-alpha treatment. J. Anim Sci, 87(9): 3020-3029. 8. Cincović M., Hristovska T., Belić B. (2018a). Niacin, Metabolic Stress and Insulin Resistance in Dairy Cows. pp107-126. In: Niacin, Metabolic Stress and Insulin Resistance in Dairy Cows, B Group Vitamins - Current Uses and Perspectives, Jean Guy LeBlanc and Graciela Savoy de Giori, IntechOpen, London. 9. Petrović K., Stojanović D. (2020). Effects of anti-inflammatory effect of niacin on liver enzymes and oxidative stress of cows in early lactation. Veterinarski pregled, 1, 1:105-111.[in Serbian] 10. Kovačević Z., Stojanović D., Cincović M., Belić B., Davidov I., Stojanac N., Galfi A. (2019). Effect of postpartum administration of ketoprofen on proinflammatory cytokine concentration and their correlation with lipogenesis and ketogenesis in Holstein dairy cows. Pol J Vet Sci, 22(3): 609-615. 11. Gille A., Bodor E.T., Ahmed K., Offermanns S. (2008). Nicotic acid: Pharmacological effects and mechanisms of action. Ann Rev Pharmacol Toxicol 48:79-106. 12. Li Z., Li X., Lin S., Chen Y., Ma S., Fu Y., Wei C., Xu W. (2017). Nicotinic Acid Receptor GPR109A Exerts Anti-Inflammatory Effects Through In-

18.

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20. 21. 22.

23.

24.

25.

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27.

28.

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30.

hibiting the Akt/mTOR Signaling Pathway in MIN6 Pancreatic β cells. Ann Clin Lab Sci, 47(6): 729-737. Trevisi E., Minuti A. (2018). Assessment of the innate immune response in the periparturient cow. Res Vet Sci, 116: 47-54. Fiore E., Piccione G., Perillo L., Barberio A., Manuali E., Morgante M., Gianesella M. (2017). Hepatic lipidosis in high-yielding dairy cows during the transition period: haematochemical and histopathological findings. Anim Prod Sci, 57(1): 74-80. Niehoff I.D., Hüther L., Lebzien, P. (2008). Niacin for dairy cattle: a review. Br J Nutr, 101(1): 5-19. Wanders D., Graff E.C., White B.D., Judd R.L. (2013). Niacin increases adiponectin and decreases adipose tissue inflammation in high fat dietfed mice. Plos one, 8(8): e71285. Digby J.E., Martinez F., Jefferson A., Ruparelia N., Chai J., Wamil M., Greaves D.R., Choudhury R.P. (2012). Anti-inflammatory effects of nicotinic acid in human monocytes are mediated by GPR109A dependent mechanisms. Arterioscler Thromb Vasc Biol, 32(3): 669-676. Morey S.D., Mamedova L.K., Anderson D.E., Armendariz C.K., Titgemeyer E.C., Bradford, B.J. (2011). Effects of encapsulated niacin on metabolism and production of periparturient dairy cows. J Dairy Sci, 94(10): 50905104. Kashyap M.L., Ganji S., Nakra N.K., Kamanna V.S. (2019). Niacin for treatment of nonalcoholic fatty liver disease (NAFLD): novel use for an old drug? J Clin Lipidol, 13: 873-879. Ceciliani F., Ceron J.J., Eckersall P.D., Sauerwein H. (2012). Acute phase proteins in ruminants. J Proteome 75: 4207-4231. Bertoni G., Minuti A., Trevisi E. (2015). Immune system, inflammation and nutrition. Anim Prod Sci, 55(6): 354-360. Epelman S., Liu P.P., Mann D.L. (2015). Role of innate and adaptive immune mechanisms in cardiac injury and repair. Nat Rev Immunol 15(2): 117-129. Bionaz M., Trevisi E., Calamari L., Librandi L., Ferrari A., Bertoni G. (2007). Plasma paraoxonase, inflammatory conditions, liver functionality and health problems in transition dairy cows. J Dairy Sci 90: 1740-1750. Bertoni G., Trevisi E. (2013). Use of the liver activity index and other metabolic variables in the assessment of metabolic health in dairy herds. Vet Clin Food Anim, 29: 413-431. Trevisi E., Amadori M., Archetti I., Lacetera N., Bertoni G. (2011). Inflammatory response and acute phase proteins in the transition period of high-yielding dairy cows. pp. 355-380, In: Veas, F. (Ed.), Acute Phase Protein/Book 2. InTech, Rijeka (Croatia). Zhou Z., Loor J.J., Piccioli-Cappelli F., Librandi F., Lobley G.E., Trevisi E. (2016). Circulating amino acids in blood plasma during the peripartal period in dairy cows with different liver functionality index. J Dairy Sci, 99(3): 2257-2267. Lacetera N., Scalia D., Bernabucci U., Ronchi B., Pirazzi D., Nardone A. (2005). Lymphocyte functions in overconditioned cows around parturition. J Dairy Sci. 88: 2010-2016. Loor J.J., Bertoni G., Hosseini A., Roche J.R., Trevisi E. (2013). Functional welfare - using biochemical and molecular technologies to understand better the welfare state of peripartal dairy cattle. Anim Prod Sci, 53(9): 931-953. Trevisi E., Amadori M., Cogrossi S., Razzuoli E., Bertoni G. (2012). Metabolic stress and inflammatory response in high-yielding, periparturient dairy cows. Res Vet Sci, 93: 695-704. Amadori M., Fusi F., Bilato D., Archetti I.L., Lorenzi V., Bertocchi L. (2015). Disease risk assessment by clinical immunology analyses in periparturient dairy cows. Res Vet Sci 102: 25-26.

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E.S.Ö. Salci et al. Large Animal Review 2021; 27: 23-29

Comparison of cytological, microbiological and histopathological findings of genital tracts in cows with different degree perineal conformation disorder

EMSAL SİNEM ÖZDEMİR SALCI1, ÖZKAN YAVAŞ2, ÖZGE YILMAZ ARDIÇLI3, GÜRSEL SÖNMEZ2, SERPİL KAHYA DEMİRBİLEK3, SENA ARDIÇLI4, KAMİL SEYREK İNTAŞ5 1

Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Bursa Uludag University, 16059-Bursa / Turkey Department of Pathology, Faculty of Veterinary Medicine, Bursa Uludag University, 16059-Bursa / Turkey 3 Department of Microbiology, Faculty of Veterinary Medicine, Bursa Uludag University, 16059-Bursa / Turkey 4 Department of Genetics, Faculty of Veterinary Medicine, Bursa Uludag University, 16059-Bursa / Turkey 5 Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Near East University, Nicosia / Cyprus 2

SUMMARY The aim of this study is to investigate cytological, microbiological and histopathological findings of the genital tracts in cows with different degree perineal conformation (PC) disorder. Totally, 28 cows brought the slaughterhouse were materials of the study. Information about to age, body weight, reproductive status, number of parturitions, days in milk (DIM) and the reason for slaughtering of the cows was obtained in the anamnesis learned from the owners. Before slaughtering, cows were evaluated in terms of PC and then they were divided into four groups with equal numbers of cows according to PC disorders: group I (GRI) normal; group II (GRII) mild; group III (GRIII) moderate and group IV (GRIV) severe. PC disorders of the cows were graded on a scale in terms of vulvar angle, vulvar length over ischial arch, depth of anus and perineal length. A body condition score (BCS) was also evaluated in the cows. The perineal region and vagina were inspected for vaginal discharge and vaginal mucosal appearance, respectively. Pneumovagina was classified as negative, suspicious and positive. Sterile swab samples were taken from vagina, cervix and uterus for bacteriological culture and identification. For cytological examination, smears were taken from vagina, cervix and uterus. Tissue samples were taken from vagina, cervix and uterus for histopathological examination. Statistically, Pearson’s correlation, Fisher’s exact tests and regression analysis were performed for all data. Clinically, characters of the vaginal discharge (serous, foamy, mucous, purulent and urine-mixed) and vaginal hyperemia increased in GRIII and GRIV. Microbiologically, as parallel to the PC disorder, E. coli in Enterobacteriaceae family was the most common bacterium in Group III and Group IV. As PC disorder in the groups increased, cytological examination findings were found to be significant, similar to clinical and microbiological examination. However, histopathological examination gave more meaningful results in groups. Statistic results pointed out that difference between the groups in terms of vaginal mucosa color, cervical and uterine microbiology was significant. In conclusion, PC disorder in cows constitutes predisposition for many genital canal diseases that may be the cause of infertility. The presence of genital canal diseases can be suspected in cattle using the PC scale. Therefore, it is appropriate to evaluate the health status of the genital canal with other diagnostic methods (microbiology, cytology and histopathology), especially in cows with PC disorder.

KEY WORDS Cow, cytology, histopathology, microbiology, scoring of perineal conformation disorder.

INTRODUCTION Anatomo-morphological changes in the perineal region and the form of the vulva affect the reproductive performance in large animals1,2. Maintaining the anatomical structures of the genital canal is important for the continuity of reproductive performance, and this can be implied clinically by the presence of ideal perineal conformation (PC)2,3,4. For an ideal PC in cows, anal sphincter and vulvar lips should be on the same line, and 80% of vulvar lips should form an angle of at least 80° below the baseline of the pelvic and horizontal plane3. If the vulvar lips angle is greater than 45° from the vertical plane, this vulva is called horizontal vulva; however, the other types are called vertical vulva5,6.

Corresponding Author: Emsal Sinem Özdemir Salci (ssalci@uludag.edu.tr).

Because of different factors such as increasing number of parturitions, perineal lacerations etc., if the vulvar lips and hymeneal sphincter is not sufficiently closed and the air fills into the vagina, this condition is called pneumovagina3,6,7. Urovagina is the collection of urine in the cranial part of the vaginal canal due to vesico-vaginal reflux5,8. Although urovagina is considered as a different pathology, it is also presented in severe pneumovagina cases6. In cows, pneumovagina is formed, if the angle of the upper vulvar commissure with the ischial arch is greater than 20 degrees; thus, the vulvar angle measurements are important both diagnosis and evaluation of the pneumovagina cases3,6. Particularly, in cows with pneumovagina, this angle is inclined to 40.2%, and it is reported that only 7.3% of healthy cows have this inclination. In cows with severe pneumovagina, there is a marked change in the PC. In these cows, the anus is inclined towards cranially, and the vulva is over-angled, and perineal atrophy is seen with a rate of 6.7% due to connective tissue weakness. In addition, the vulva is longer longitudinally in cows with pneumovagina6.

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Comparison of cytological, microbiological and histopathological findings of genital tracts in cows

The air in the vagina leads to vaginal irritation in cows with pneumovagina. Depending on the straining induced by this irritation, either during the passive movements of the cow or when abdominal relaxation occurs, air enters to vagina and exits from the vulvar lips spontaneously7. This condition causes drying in vaginal mucosa, which makes the vaginal mucosa susceptible to the infections including vaginitis, cervicitis and endometritis1,3. According to our best of knowledge, in the literature, there was no study in which PC was scored and investigated cytological, microbiological and histopathological changes of the genital tracts using this scale in cows. Therefore, this presented study aimed to investigate cytological, microbiological and histopathological findings of the genital tracts in cows with different degree PC disorder.

and character of the fluid in the vagina, cervix and uterus were recorded.

Microbiological examinations Sterile swaps were used to collect samples from vagina, cervix and uterus. Vaginal samples were taken before the clinic vaginal examinations. The cervix and uterus samples were obtained immediately following to slaughter of the cows after opening of the cervix and uterus lumens with a sterile scalpel. All samples were taken to the microbiology laboratory and processed within 2 hours. The samples were cultured for bacteria and yeasts with 3 pair 5% defibrinated sheep blood and incubated at 37°C. After incubation for 24-48 h, plates were examined for growth. Colonies are stained with Gram stain. BBLTM CrystalTM biochemical tests were used for identification of colonies according to instructions of manufacturers.

MATERIALS AND METHODS

Cytological examinations

This study was approved by Bursa Uludag University Animal Experiments Local Ethics Committee (Decision no: 2017-07 / 05).

Smear preparations were prepared from the samples of vagina, cervix and uterus and then they were fixed in methanol after air-drying. Following fixation, the preparations were stained with Diff-Quick. All preparations were evaluated under a light microscope (Olympus®, CX41).

Cows A total of 28 cows (different age and breed) were materials of the study, which were brought to a slaughterhouse for slaughtering. Perineal region of these cows were evaluated under four groups according to PC scale as given in Table 1 [group I (GRI) (n=7) normal; group II (GRII) (n=7) mild; group III (GRIII) (n=7) moderate and group IV (GRIV) (n=7) severe]. A body condition score (BCS) was determined in cows based on the 1-5 scale as reported previously9.

Histopathological examinations Tissue samples taken from the vagina, cervix and uterus were fixed in 10% formaldehyde solution. Paraffin blocks were prepared by going through routine tissue follow-up procedures. Sections taken from paraffin blocks with a thickness of 5 µm were stained with hematoxylin-eosin (H&E) and examined under a light microscope (Olympus®, CX41) for presence of pathological changes.

History of the cows and PC scoring

Statistical analysis

Information about to age, body weight, reproductive status, number of parturitions, days in milk (DIM) and the reason for slaughtering of the cows was obtained from the owners. A routine clinical examination was performed, and the measurements of the perineal region including vulvar angle, vulvar length over ischial arch, depth of anus and perineal length was obtained with caliper, miter and ruler before slaughtering for scoring the PC. Following the measurements, the cows were included in their group according to the PC scale.

The means and standard deviations of the obtained metric results of the cows in groups were calculated. In order to investigate any association between the selected traits and reproductive parameters in cows, multiple linear regression analysis was applied. In this context, the evaluation was performed in three steps. Initially, the best subsets regression analysis was performed to compare all possible models with respect to a specified set of predictors and to identify the bestfitting models related to the present study. The results provides the information to choose among candidate models with strongest fit for each number of predictors and the model giving the largest adjusted explained variance (R2 adjusted)10. The two-way interactions between the parameters were added to the model and tested for significance. The assessment was maintained until achieving the R2 value, which do not increase. As a final step, ultimate analysis with the selected variables determined by subsets regression was performed, and the results were expressed as regression coefficients and the corresponding statistical significance. Correlation coefficients were estimated using the Pearson’s correlation test. Correlations were classified into three groups according to levels of Pearson’s correlation coefficients (PCC) generated from the analysis: low correlation (PCC<0.30), intermediate correlation (PCC=0.30-0.70), and high correlation (PCC>0.70). The differences between the groups generated by the evaluation of parameters including degree of vulvar angle, vulvar length over ischial arch, depth of anus, and perineal length (as shown in Table 1) were determined by the two-sided Fish-

Clinical examinations The perineal region was inspected in terms of vaginal discharge, anatomical structures of the vulva and any pathological changes around vulvar region. In the examination of the vagina with the speculum, mucosal appearance, presence and character of the vaginal discharge (serous, seromucous, mucous, foamy, purulent and urine mixed) were noted. The diagnosis of the pneumovagina was made according to the presence of vaginal air absorption and/or outflow by per-rectal vaginal pressure. Pneumovagina was classified as negative, suspicious and positive. While the presence of vaginal air was uncertain in suspicious cases, severe vaginal air was present in positive cases.

Macroscopic examinations After taking sterile swap samples for cytological and microbiological examination, the presence of any macroscopic pathology related to vagina, cervix and uterus was evaluated, and any existing lesions were noted. In addition, the presence

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E.S.Ö. Salci et al. Large Animal Review 2021; 27: 23-29

Table 1 - Criteria of the perineal conformation, and grouping of the study. Normal (group I)

Mild (group II)

Moderate (group III)

Severe (group IV)

Degree of vulvar angle

0-10

10-30

30-55

> 55

Vulvar length over ischial arch (cm)

0-1

1-2.5

2.5-4

Depth of anus (cm)

4-6

6-8

> 8 cm

Perineal length (cm)

Parameters

er’s exact probability test. The statistical analyses were carried out with Minitab version 19.1.1 (Minitab, Inc., State College, PA, USA). For all statistical comparisons a probability level of p<0.05 was accepted as statistically significant.

RESULTS Findings of the cows classified as PC are given in Table 2. The cows in the study were Holstein (n=26) and Simental (n=2) breeds. Age, body weight, number of parturitions, DIM, the reason for slaughtering and BCS of the cows are given in Table 3.

Reproductive status of the cows In GRI, all cows were open and their DIMs were >100 days. Only 6th and 7th cases in this group were repeat breeder cows. All cows in the GRII were open and the other cows’ DIMs were >100, except for case 1 (DIM between 60-100 days). The 5th and 7th cases in this group were repeat breeder cows. The cows in the GRIII were open and the DIMs of the cows, except for the 6th case (DIM 60-100 days), were >100 days. The 1st case in this group was repeat breeder. The cows in GRIV were open and their DIMs were >100 days. The 6th and 7th cases in this group were repeat breeder cows (Table 3).

Clinical findings In the inspection of the perineal region, no pathology was observed on the anatomical structures of the genital organs of the cows in groups. In the mucosal examination of the vagina, 1st and 2nd cases of GRI; 1st, 3rd and 7th cases of GRII; all cases of GRIII and GRIV had hyperemic appearance. Seromucous and serous vaginal discharge were detected in 1st and 2nd cases of GRI, respectively. Foamy mucous vaginal discharge was present in 3th case of GRII. In GRIII, foamy seromucous in the 2nd case and mucous discharge in the 1st, 6th and 7th cases were observed. Vaginal discharge characters encountered in GRIV were purulent-urine mixed in the 1st case, seromucous-urine mixed in 2nd case, and mucous in 4th case. Pneumovagina was only observed in the cases of GRII, GRIII and GRIV; however, pneumovagina was suspected 2nd, 4th, 6th and 7th cases of GRII, because presence of the vaginal air was not clearly determined in these cases. In addition, 1st and 2nd cases of the GRIV were complicated with urovagina.

Macroscopic findings Macroscopically, no pathological findings were found in the vagina and cervix of the cases in the groups. However, in GR I, purulent and serous discharge was observed only in the uterus of 1st and 2nd cases, respectively. In GRII, there was a mucous

Table 2 - Means and standard deviations of the parameters in the groups Normal (group I)

Mild (group II)

Moderate (group III)

Severe (group IV)

Degree of vulvar angle

1.85±3.28

19.42±5.28

38.14±5.33

67.28±9.35

Vulvar length over ischial arch (cm)

0.07±0.18

1.68±0.36

3.01±0.42

5.08±1.14

Depth of anus (cm)

2.05±1.57

4.92±0.76

6.58±0.49

9.85±0.95

Perineal length (cm)

5.12±0.76

4.07±0.52

3.27±0.76

2.64±0.82

Parameters

Table 3 - Means and standard deviations of the parameters and the reasons for slaughtering of the cows in the groups. Normal (group I)

Mild (group II)

Moderate (group III)

Severe (group IV)

6.01±1.52

5.07±1.78

5.64±1.10

5.85±2.11

614.28±37.79

521.42±95.11

492.85±67.25

504.28±58.83

3±1

2.28±1.38

2.57±1.27

3.42±1.61

Days-in-milk

345.85±134.09

312±195.64

212.28±80.9

232.85±123.03

Reason for slaughtering

Infertility, udder problem, lameness

Infertility, udder problem

Infertility, lameness

3.46±0.65

2.5±0.82

2.46±0.63

2.61±1.05

Parameters Age Body weight Number of parturitions

Body condition score

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Comparison of cytological, microbiological and histopathological findings of genital tracts in cows

fluid in the uterus of the 3rd case. The uterus contents of the cases in GRIII were mucous in 3rd and 6th cases. The contents encountered in the uterus in GRIV were mucous in 2nd and 4th cases.

case), Aeromonas (6th case) and Proteus mirabilis (7th case) from vagina; Klebsiella spp. (2nd case), E. coli (3rd, 5th and 6th cases) and Enterococcus spp. (7th case) from cervix; E. coli (in all cases) from uterus.

Microbiological findings

Cytological findings

According to the culture results of the groups, the microorganisms obtained from the vagina, cervix and uterus are listed below. In GR I, microbiological isolations were Escherichia coli (1st, 2nd, 5th and 6th cases) from vagina; E. coli (1st case), Enterococcus spp. (1st case), Gardnerella vaginalis (1st case), Proteus mirabilis (5th case) and Aeromonas spp. (6th case) from cervix; E. coli (1st case), Gardnerella vaginalis (6th case), Proteus mirabilis (1st case) and Aeromonas spp. (3rd case) from uterus. In GR II, microbiological isolations were E. coli (1st, 2nd, 3rd, 5th, 6th and 7th cases), Enterococcus spp. (6th case) and Gardnerella vaginalis (7th case) from vagina; E. coli (1st, 2nd, 3rd, 4th, 6th and 7th cases) from cervix; E. coli (2nd, 4th and 7th cases), Klebsiella spp. (1st case) and Enterobacter spp. (3rd and 4th cases) from uterus. In GR III, microbiological isolations were E. coli (1st and 5th cases), Klebsiella spp. (1st case), Candida spp. (2nd case) and Staphylococcus spp. (7th case) from vagina; E. coli (1st, 2nd, 3rd and 4th cases), Enterococcus spp. (2nd case), Proteus mirabilis (5th case) and Klebsiella spp. (6th and 7th case) from cervix; E. coli (1st, 2nd, 3rd, 4th, 6th and 7th cases), Aeromonas spp. (4th case) and Klebsiella spp. (5th case) from uterus. In GR IV, microbiological isolations were Streptococcus bovis (1st case), E. coli (1st, 2nd, 3rd, 4th and 5th cases), Enterobacter (3rd

In GRI, only 1st case was cytologically positive, and plenty of polymorph nuclear cells (neutrophils) were seen in the examination of the samples taken from the vagina, cervix and uterus. No sign of inflammation was determined in the examinations of the samples of vagina, cervix and uterus taken from all cases in GRII. Cytological examinations in GRIII revealed numerous epithelial cells in uterine samples of 4th case, severe polymorph nuclear cell infiltration in vaginal samples of 6th case, and severe inflammatory cells in vagina (Figure 1) and uterine (Figure 2) samples of 7th case. In GRIV, a small number of neutrophils and lymphocytes in the vaginal samples of the 1st case. And in 4th case, only a few lymphocytes were found in the samples taken from uterus.

Figure 1 - This cytological view of case 7 in GRIII points out polymorph nuclear cells (arrow heads) and epithelial cells (arrows), which is obtained from vagina, (Diff-Quick, X 200).

Figure 2 - Arrows demonstrate polymorph nuclear cells on cytological view of uterus in case 7 of GRIII, (Diff-Quick, X 200).

Histopathological findings In GRI, epithelial desquamation in vaginal mucosa, and diffuse, moderate mononuclear cell infiltration in lamina propria were observed in 1st case, which was indicating to signs of vaginitis. Diffuse, moderate mononuclear cell infiltrations were detected in the lamina propria of cervix (cervicitis). In the uterus, desquamation of mucosal epithelial cells, and diffuse, severe mononuclear and polymorph nuclear cell infiltrations in lamina propria were noted, which was pointing out signs of endometritis (Figure 3). In 2nd case, only in the cervix, mild mononuclear cell infiltration in the lamina propria was present. In GRII, focal, mild mononuclear cell infiltration was observed in the vaginal mucosa of 2nd and 7th cases, which was compatible with vaginitis. In 1st, 2nd, 3rd, 4th and 7th cases, desquamation in the mucosal epithelium, focal, multifocal or diffuse, mild to moderate mononuclear cell infiltrations were noted in the lamina propria of the uterus (endometritis). In GRIII, chronic, granulomatous vaginitis findings (central caseification necrosis and calcification, macrophages and Langhans type multinucleated giant cells surrounded by connective tissue) were observed in 2nd case (Figure 4). In 3rd, multifocal, mild to moderate mononuclear cell infiltrations in the lamina propria of the cervix and uterus were noted. These findings pointed out the cervicitis and endometritis in this case. In 4th case, degeneration and desquamation in the glandular ep-

Figure 3 - This microscopic view shows the endometritis in case 1 of GRI: mononuclear and polymorph nuclear cell infiltrations (arrows) in the lamina propria of uterus, (H&E, X 100).

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E.S.Ö. Salci et al. Large Animal Review 2021; 27: 23-29

In GRIV, in 1st case, multifocal, severe mononuclear cell infiltration was noted in the lamina propria and submucosa of the vagina (vaginitis). In 2nd case, mild mononuclear cell infiltration was observed in the lamina propria of the vagina and cervix, and there were also multifocal to diffuse mononuclear cell infiltrations in the periglandular areas and lamina propria of the uterus. Diffuse moderate mononuclear cell infiltration was present in the uterus of 4th case. Focal, mild mononuclear cell infiltrations were observed in the vagina and cervix of case 7.

Statistical analysis findings Regression coefficients and statistical significance of parameters (number of parturition, BCS, body weight, DIM and age on vaginal mucosa color, vaginal discharge, histopathology, cytology, microbiology and macroscopic discharge in the uterus) were given in Table 4.

Figure 4 - Chronic granulomatous vaginitis finding of the case 2 in GRIII. Arrows points out Langhans type multinucleated giant cells, (H&Eosin, X 200).

Correlations:

ithelium, diffuse, severe mononuclear cell infiltrations and periglandular fibrosis were observed in the lamina propria of the uterus. In 6th case, a large number of free erythrocytes were found in the lamina propria and submucosa in the uterus. In addition, abundant iron-laden macrophages (hemosiderinophage) were noteworthy in these areas. In 7th case, hyperplasia in the epithelial layer of the vaginal mucosa, and diffuse, severe and mostly mononuclear cell infiltrations in the lamina propria were observed.

There is a positive correlation between vaginal discharge and vaginal cytology (R=0.548, p=0.003), cervical histopathology (R=0.431, p=0.022) and the presence of macroscopic discharge in the uterus (R=0.775, p=0,000). A positive correlation was observed between vaginal cytology and vaginal histopathology (R=0.420, p=0.026), cervical cytology (R=0.471, p=0.011) and uterine cytology (R=0.533, p=0.003).

Table 4 - Regression coefficients and statistical significance (p) of the parameters. Parameter Vaginal Mucosal Color Vaginal Discharge Vaginal Histopathology Vaginal Cytology Vaginal Microbiology Cervical Histopathology Cervical Cytology Cervical Microbiology Uterine Histopathology Uterine Cytology Uterine Microbiology Macroscopic Uterine Discharge

BCS

+0.298

+0.28

0.17

0.277

Age

-0.001569 *0.005

BCS × DIM

*0.032

-0.001620

-0.132

0.626

0.885

*0.043

0.3

-0.00151

+0.00170

+0.054

+0.067

NP × BCS -0.135

0.00016

+0.054

-0.000642

0.673

0.853

0.472

0.941

0.751

0.556

0.369

+0.001

+0.088

-0.00074

+0.00027

-0.097

+0.0449

-0.000355

0.383

0.489

0.628

0.308

0.442

0.503

0.494

+0.251

+0.025

+0.00151

+0.00008

-0.086

-0.096

-0.000168

0.938

0.177

0.436

0.588

0.661

0.262

0.796

-0.004

+0.539

-0.00097

+0.00197

+0.115

-0.0251

-0.001032

0.642

0.324

0.566

0.253

0.408

0.732

0.080

-0.025

-0.037

+0.000489

-0.000181

+0.0143

+0.0008

-0.000004

0.788

0.723

0.592

0.637

0.847

0.983

0.989

+0.134

+0.066

-0.00137

+0.00040

+0.056

-0.0652

+0.000093

0.801

0.67

0.489

0.459

0.73

0.452

0.889

-0.003

+0.000522

+0.0476 p

DIM

+0.077

-0.231 p

*0.021

0.465

-0.141

0.5 -0.31

+0.00039

-0.00086

-0.142

+0.0901

+0.000127

0.538

0.921

0.833

0.545

0.359

0.276

0.840

+0.0439

-0.0029

+0.045 0.797

0.541

*0.036

-0.0022

+0.031

-0.001441

0.973

0.762

*0.023

NP: Number of parturitions, BCS: Body condition score, BW: Body weight, DIM: Days in milk, *: Statistically significant effect observed in the regression model. Models were selected according to the best subsets regression analysis.

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Comparison of cytological, microbiological and histopathological findings of genital tracts in cows

There is a positive correlation between cervical histopathology and cervical cytology (R=0.413, p=0.029) and the presence of macroscopic discharge in the uterus (R=0.592, p=0.001), cervical microbiology and uterine microbiology (R=0.503, p=0.006). There is a positive correlation between the presence of macroscopic discharge in the uterus and uterine histopathology (R=0.0549, p=0.002). There is a negative correlation between body weight and uterine histopathology (R=-0.430, p=0.022) and uterine microbiology (R=-0.496, p=0.0007).

Comparisons between groups: There was a statistically significant difference between the groups in terms of vaginal mucosa color (p=0.004), cervical microbiology (p=0.032) and uterine microbiology (p=0.006). As PC disorder became more severe, the contamination of the cervix (p=0.032) and the uterus (p=0.006) with bacteria of fecal origin increased. Moreover, the colors of the vaginal mucosa were more hyperemic (p=0.004).

DISCUSSION Reproductive problems and infertility cause economic losses in dairy cows7,11. Reproductive problems in cows are multi-factorial, and although endometritis and metritis are the most important pathological conditions of cows’ reproductive tracts, vaginitis and cervicitis should be evaluated to determinate the cause of the infertility6,12,13. Bacterial contaminations of the reproductive tracts lead to vaginitis and cervicitis, which are resulted from PC disorder, pneumovagina, urovagina and traumatic recto-vulvar injuries, and then this bacterial contamination result in ascending infective uterine pathologies that responsible to early embryonic death, placentitis, abortion and premature newborns6,8,14. PC disorder can be congenital or acquired, and it results from perineal atrophy and rectovaginal lacerations in various degrees, which predispose the cows to endometritis and other genital canal diseases6. According to our literature review, there is no study where PC has been classified in cows; thus, in this study, PC has been classified in the cows with different parameters (degree of vulvar angle, vulvar length over ischial arch, depth of anus and perineal length). The results obtained by clinical examinations (body condition, pneumovagina, vaginal inspections, etc.) and the cytological, microbiological and histopathological findings of the vagina, cervix and uterus in cows to be slaughtered were grouped according to this classification. As mentioned above, PC disorder may occur due to some general reasons including age, number of parturitions and low body condition score6,8. Abnormalities of the pelvic angle and cranioventral displacement of the vulva following parturition are the other causes of the PC disorder4,6,8. PC disorder is a reason of the infertility; thus, considering to the vulvar angle (VA) and vulvar length, surgical correction could be planned on the perineal region to prevent diseases (pneumovagina, urovagina and etc.) in horses2,15,16 and in cow3,8. As stated above, in this study, a scale was determined using perinal region conformation such as vulvar angle, vulvar length over ischial arch, depth of anus, and perineum length in the evaluation of pneumovagina, urovagina and the other infectious diseases, which cause infertility in cows.

Different diagnostic methods including vaginal examination, cytology and biopsy have been used to evaluate healthy status of the genital tracts13,17,18. In cases of pneumovagina, air suction to the vagina and fecal contamination of the genital tracts is possible11. The diagnosis of pneumovagina is made by the detection of the airflow into the vagina (or outflow from the vagina) and the presence of foamy vaginal discharge or mucosal hyperemia in the examination of the vagina with the speculum1,6. Evaluation of the vaginal secretions is not always a reliable diagnostic method for all parts of genital canal infections, because vaginal, cervical and uterine disease can be seen together or alone13. If there is a microbial contamination, purulent vaginal discharge can be seen in clinical examination7. On the other hand, in a slaughter study, it is reported that mucous, bloody mucous, mucopurulent and hemorrhagic character vaginal fluids do not always pointed out the uterine infections13. In this presented study, while serous and seromucous vaginal discharges were observed in cows with normal PC, the presence of foamy and mucous character discharge was determined in GRIII and IV due to the presence of pneumovagina, because PC disorder was more severe in these groups. Particularly, in cases had severe PC disorder (pneumovagina and urovagina), there were purulent and mucous discharges mixed with urine. But, statistically no difference was found in groups. Hyperemic appearance in the vaginal mucosa was also more common in cases with severe degree PC disorder. Depending on the urine accumulated in the vagina, inflammation in the cervix and vagina, and endometritis may be formed by contact of the cervical urine accumulated4,5,8. If vesicovaginal reflux is permanent, endometritis may result in periglandular fibrosis5. Urovagina accompanied by pneumovagina, causes endometritis, leading to infertility problems in cows4,5,8. Macroscopically, as in the presented study (case 1 in GRI), it is possible to encounter genital canal pathologies in cows with normal PC. Because PC disorder became moderate and severe in GRIII and GRIV, macroscopically different contents (purulent, serous, mucous, etc.) were encountered in the uterus. Moreover, macroscopic findings in the uterus were compatible with microbiological and histopathological findings. The genital system microflora in cattle shows a very dynamic structure consisting of aerobic, facultative anaerobic and anaerobic bacteria19,20. Vagina also forms a natural habitat for many saprophytic microorganisms that can be opportunistic pathogens21. Microbiologically, different species has been reported in the genital canal of cows. Particularly, reported bacteria are in vagina; Bacillus spp., E. coli, Enterococcus, Pediococcus, Lactobacillus20, in cervix; Enterococcus faecalis13 and in uterus; E. coli, Streprococcus bovis, Streprococcus uberis, Enterococcus spp., Proteus mirabilis, Aerococcus spp., Trueperella spp. Klebsiella spp., Moraxella spp., Staphylococcus spp.6,11,22. Additionally, Enterobacteriaceae are the dominant microorganisms in the vaginal flora20. In the presented study, only E. coli was seen in the vagina of cows with normal PC (GRI). In the cervix and uterus, E.coli, Enterobacter spp, Proteus spp, Aeromonas spp. and Gardinella vaginalis were encountered. Ascending bacterial infections of the reproductive tracts are most important causes for infertility in cows, and uterine contaminant bacteria are nonspecific and belong to a great number of bacterial species11,13. In cases where pneumovagina is complicated with urovagina, the genital canal becomes susceptible

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E.S.Ö. Salci et al. Large Animal Review 2021; 27: 23-29

to secondary infections4,5,8. Moreover, if there is a failure of physical barriers such as pneumovagina and urovagina, pathogen bacteria introduce the uterus11. It is reported that the number of fecal origin bacteria is higher in the uterus of pneumovagina cows6,11. E. coli, Streptococcus bovis, Streptococcus equinus and other different bacteria have been identified in the bacteriological examination of samples taken from the uterus in cows with pneumovagina11. Enterococcus spp., Staphylococcus spp. and E. coli are the other detected genital canal microorganisms in slaughtered cows13. In this study, as the degree of PC disorder increased in cows, microorganism flora difference was observed in the cervix and uterus. Most of these microorganisms were of fecal origin. In cows with moderate and severe PC disorder (GRIII and GRIV), the frequency of microorganism in the genital canal was higher than the cows with normal PC due to the effect of possible pneumovagina. The dominant microorganism seen in cows with PC disorder was E. coli from Enterobacteriaceae family. Bacteriological examination of the genital canal cannot provide meaningful and reliable information unless it is evaluated together with cytological and histological examination23. Cytological examination is an effective, easy-to-perform and reliable method in terms of clinical practice18,23. However, cytological examination of the genital tracts is not commonly used method in the diagnosis of the reproductive disorders of the cows12,18. Cell types encountered in cytological examination are; epithelial cells, granulocytes, neutrophils, lymphocytes, monocytes, eosinophils and basophils12,13,17,18. In a post-mortem study, it has been reported that vaginitis, cervicitis and endometritis are detected according to cytological and histopathological findings13. In the presented study, polymorph nuclear cell infiltration and inflammation cells were evaluated histopathologically. Since cytological findings are not sufficient in the evaluation for PC disorder, cytological findings should be evaluated together with microbiological and histopathological findings. In the cytological and histopathological examination of the uterus, findings such as mononuclear cell infiltration are important for the diagnosis of endometritis17,18. However, for the definitive diagnosis of endometritis, endometrial biopsy, which is not applicable in clinical conditions, should be preferred17,18,23. Since the presented study was carried out on the post-mortem material, the examination of the genital canal can be done easily by histopathological examination. When the cases in the groups were evaluated, vaginitis and endometritis were more prominent in the cows with PC disorder. Therefore, it was obvious that histopathological examination gave more meaningful results.

CONCLUSIONS In conclusion, PC disorder in cows is predisposing factor for many genital canal diseases that may cause of infertility. As planned in this study, the presence of genital canal diseases can be suspected in cattle using the PC scale. Therefore, it is appropriate to evaluate the health status of the genital canal with other diagnostic methods (microbiology, cytology and histopathology), especially in cows with PC disorder.

ACKNOWLEDGEMENT This study was performed by supporting of a scientific research project in Bursa Uludag University, Turkey (Project no: KUAP(V) - 2017/10).

References 1. England. G.C.W. (2005). Fertility and obstetrics in the horse. 3rd ed., 117120, Blackwell Publishing, UK. 2. Robinson N.E., Sprayberry K. (2009). Current therapy in equine medicine. 6th ed., 764-765, Saunders, USA. 3. Dehghani S.N., Yavari M., Kafi M. (2011). Treatment of pneumovaginitis in dairy cattle by caslick operation. Res Opin Anim Vet Sci, 1(6): 349-351. 4. Youngquist R.S., Braun W.F.Jr. (1993). Abnormalities of the tubular genital organs. Vet Clin North Am Food Anim Pract, 9(2): 309-322. 5. Gautam G., Nakao T. (2009). Prevelance of urovagina and its effect on reproductive performance in Holstein cows. Theriogenology, 71:1451-1461. 6. Goncagul G., Seyrek Intas K., Kumru I.H., Seyrek Intas D. (2012). Prevalence and accompanying signs of pneumovagina and urovagina in dairy cows in the Southern Marmara region. Tierärzt Prax Ausg G Grosstiere Nutztiere, 40(6): 359-366. 7. Demet Ö. (2010). İneklerde pnömovagina olgusu; önemli bir döl tutmama sorunu. Dicle Üniv Vet Fak Derg 1(1): 26-28. 8. Ay S.S., Fındık M. (2011). İneklerde ürovaginanın nedenleri ve sağaltım seçenekleri. Vet Hekim Der Derg 82(1): 63-68. 9. Ferguson J.D., Galligan D.T., Thomsen N. (1994). Principal descriptors of body condition score in dairy cattle. J Dairy Sci, 77: 2695-2703. 10. Chatterjee S., Simonoff J.S. (2013). Handbook of regression analysis. 3034, John Wiley & Sons, USA. 11. Goncagul G., Seyrek-Intas K., Kumru I.H., Ozakin C., Ozdemir Salci E.S., Weiss R., Prenger-Berninghoff E. (2012). Bacterial infertility and ascending uterine infections with respect to pneumovagina and urovagina in cows. Res Opin Anim Vet Sci, 2(12), 583-586. 12. Brodzki P., Brodzki A., Kurek L., Marczuk J., Tatara M.R. (2015). Endometrial cytology at luteal and follicular phases of the ovarian cycle in cows. Ann Anim Sci, 15(1): 107-117. 13. Casarin J.B.S., Martini A.P., Trentin J.M., Fiorenza M.F., Pessoa G.A., Barros S.S., Rubin M.I.B. (2018). Bacteriological, cytological and histopathological evaluation of the reproductive tract of slaughtered cows. Pesq Vet Bras, 38(1): 53-58. 14. Pouret E.J.M. (1982). Surgical technique for the correction of pneumoand urovagina. Equine Vet J, 14(3): 249-250. 15. Caslick E.A. (1937). The vulva and the vulvo-vaginal orifice and its relation to genital health of the thoroughbred mare. Cornell Vet, 27(2): 178-187. 16. Pascoe R.R. (1979). Observations on the length and angle of declination of the vulva and its relation to fertility in the mare. J Reprod Fertil Suppl, 27: 299-305. 17. Kasimanickam R., Duffield T.F., Foster R.A., Gartley C.J., Leslie K.E., Walton J.S., Johnson W.H. (2004). Endometrial cytology and ultrasonography for the detection of subclinical endometritis in postpartum dairy cows. Theriogenology, 62(1-2):9-23. 18. Prieto M., Barrio M. Quintela L.A. Perez-Marin C.C., Becerra J.J., Vigo M., Diaz C., Cainzos J., Prieto A., Fernandez F.I., Martinez D., Herradon P.G. (2012). Validation of a simple method for the interpretation of uterine cytology in cows. Vet Med-Czech, 57(7): 360-363. 19. Otero C., Saavedra L., Silva de Ruiz C., Wilde O., Holgado A.R., NaderMacias M.E. (2000). Vaginal bacterial microflora modifications during the growth of healthy cows. Lett Appl Microbiol, 31(3): 251-254. 20. Wang Y., Ametaj B.N., Ambrose D.J., Ganzle M.G. (2013). Characterisation of the bacterial microbiota of the vagina of dairy cows and isolation of pediocin-producing Pediococcus acidilactici. BMC Microbiol, 13:19. 21. Panangala V.S., Fish N.A., Barnum D.A. (1978). Microflora of the cervico-vaginal mucus of repeat breeder cows. Can Vet J, 19(4): 83-89. 22. Fuentes B.M., Arias L.A.Q., Gonzales J.J.B., del Sol L., Feijoo J.E.M., Punal J.L.G., Lopez M.B., Lago A.P., Cao J.M.D., Rodriguez G.F., Herradon P.J.G., Martinez A.I.P. (2017). Agreement between postmortem endometrial cytology, biopsy and bacteriology in culled dairy cows. Anim Reprod, 14(4): 1024-1033. 23. Hemberg E., Lundeheim N., Einarsson S. (2005). Retrospective study on vulvar conformation in relation to endometrial cytology and fertility in Thoroughbreed mares. J Vet Med A Physiol Pathol Clin Med, 52(9): 474477.

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N. Maksimović et al. Large Animal Review 2021; 27: 31-34

Development of sexual behaviour in ram lambs and its correlation to serum testosterone

NEVENA MAKSIMOVIĆ1*, SLAVČA HRISTOV2, ALEKSANDAR MILOVANOVIĆ3, TOMISLAV BARNA3, IGOR STOJANOV3, BOGDAN CEKIĆ1, IVANA MILOŠEVIĆ-STANKOVIĆ4 1

Institute for Animal Husbandry, Autoput 16, 11080 Belgarde, Serbia University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia 3 Scientific Veterinary Institute, “Novi Sad”, Rumenački put 20, 21000 Novi Sad, Serbia 4 PhD student, University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia 2

SUMMARY The drive to mate females is variable among the rams and can have a major impact on sheep production. Androgen testosterone may be a key mediator within the expression of various morphological and behavioural traits in mammals, but the factors underlying individual variation in circulating testosterone levels are poorly understood. The aim of this study was to investigate the development of sexual behaviour patterns in ram lambs as well as the role of testosterone in the expression of their libido. Research was carried out on the sheep farm of the Institute for Animal Husbandry, Belgrade, Serbia. The study included 20 crossbred ram lambs (autochthonous breed Pirot Pramenka x Merinolandschaf x Ile de France). All ram lambs used in the study were of same age and rearing conditions. They were weaned at 2 months of age and kept indoors from birth throughout the whole study. Animals were introduced in the study at the age of 3 months and the trial was completed at the rams’ age of 17 months. Blood samples for testosterone levels were taken bimonthly (at the age of 3, 5, 7, 9, 11, 13, 15 and 17 months), as well as were male-female and male-male interactions recorded. Male-female interactions included the following elements of behaviour: nosing (or anogenital sniffing), pawing, flehmen response, attempted mounts. Also, duration of all male activities directed towards ewe was recorded (male-female interactions in total). Male-male interactions involved the frequency of male-male mounts. Performed investigations showed that sexual behaviour of rams was age dependent, but poorly correlated to serum testosterone. The average serum testosterone levels ranged from 1.83-13.28 ng/mL, and were age dependant (P<0.05). Male-female oriented behaviour developed linearly with age, while male-male specific behaviour was characterized by high intensity in young age and then pronounced variability in later test periods. None of the studied behavioural interactions were highly correlated to serum testosterone. These findings support standpoint of more than one factor influencing development of sexual behaviour of ram lambs.

KEY WORDS Rams, sexual behaviour, testosterone, correlative relationships.

INTRODUCTION The desire to mate is variable among the rams and can have a major impact on sheep production, especially when only one ram is used for reproduction in the flock. Libido or sexual desire refers to sexual motivation and is manifested through certain forms of behaviour such as: searching for sheep, detection of sheep in the oestrus, courting and mating. Rams exhibit a wide range of different libido levels, from none to extremely aggressive, which is focused solely on the search and mating of female animals, while sacrificing all other needs, such as food, water and rest1. There are several stereotyped forms of behaviour that the ram can exhibit just before the first mount, which are defined as courtship behaviour. These include anogenital sniffing, ewe’s flunk nudging, impatient foot

Corresponding Author: Nevena Maksimović (nevena_maksimovic@yahoo.com).

stomping, lifting the head and neck while simultaneously raising the upper lip as a reaction to the smell of the ovine urine, called flehmen response, as well as emitting low-pitched ‘gargling’ vocalizations2, 3. Mating behaviour and libido of rams can be estimated through so called serving capacity tests in which rams are exposed to ewes confined to a pen of limited size4. These tests are also known as pen tests and usually imply using females in heat and are repeated in order for rams to be assigned with a serving capacity score, which is the average number of ejaculations each ram achieves over repeated 30 minutes testing periods. Sexual behaviour can be very variable among rams and while some become immediately attracted by present female and begin courtship even if female is out of heat, others never approach ewes or take a long time before they do. These strong individual variations in sexual behaviour of rams are not completely understood. Testosterone as main androgen was often considered as a predominant for expressing and maintaining libido in rams as reported in previous studies of various authors3, 5, 6. However, many

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Development of sexual behaviour in ram lambs and its correlation to serum testosterone

other studies showed no significant correlation between androgen levels and sexual behaviour in rams7, 8. Testosterone may be a key mediator in the expression of various morphological and behavioural traits in mammals, but the factors underlying individual variation in circulating testosterone levels are not completely understood9. Understanding the scheme of sexual behaviour of rams is not only important from the aspect of selection of quality males, but also in order to determine the best male to female rations for mating. The aim of this study was to investigate the development of sexual behaviour patterns in ram lambs as well as the role of testosterone in the expression of their libido.

MATERIAL AND METHODS Investigations were carried out on an experimental sheep farm of the Institute for Animal Husbandry, Belgrade. The study included 20 crossbred rams (autochthonous breed Pirot Pramenka x Merinolandschaf x Ile de France). All ram lambs used in the study were of same age and conditions. Prior to weaning, male lambs were kept with their mothers and, starting from the second week of age, additionally fed with alfalfa hay and concentrate (160 g protein/kg plus vitamins and minerals) ad libitum. Lambs were weaned at the age of 2 months and were kept in the barn system in one group until the end of the study. The post weaning diet was based on the use of alfalfa hay and concentrates. Animals were introduced in the study at the age of 3 months and the trial was completed at the rams’ age of 17 months. Blood samples for testosterone level were collected every two months (at the age of 3, 5, 7, 9, 11, 13, 15 and 17 months, respectively) from the jugular vein in the morning period from 8 to 9 am. After one hour, blood samples were centrifuged, and the blood serum was extracted, which was then used for the analysis. Analysis was performed by radioimmunoassay (RAİ) method using a commercial kit (Immulite® total testosterone, Siemens healthcare diagnostic inc., UK) for animal use. The aspects of sexual behaviour of rams were studied through male-female and male-male interactions every two months, i.e. at the age of rams of 3, 5, 7, 9, 11, 13 and 15 months. Male-female interactions were monitored individually by introducing a ram into a pen with non-oestrous restrained ewe for 20 minutes10. These interactions included the following elements of behaviour: 1) the incidence of direct contact by sniffing anogenital region of ewe (nosing), 2) the frequency of impatient foot

stomping of the ram (pawing), 3) the frequency of the manifestation of the flehmen response (upper lip response), 4) the frequency of attempted mounts, 5) duration of all activities directed towards ewe (male-female interactions in total). All interactions were tracked by directly observing and recording all the relevant activities, as well as by video recording. Male-male interactions were monitored in a group box for 8 hours per day by video recording, and the frequency of malemale mounts was recorded. Statistical analysis of the experimental data was performed using the statistical package IBM SPSS Statistics 21. Statistical significance of differences of all examined parameters were determined by means of the one way ANOVA, followed by the Tukey HSD test. The relative dependence of the traits was determined by Pearson’s correlation coefficient, whose significance was tested. Analyses were performed for the significance levels of 5% and 1%, and the results are presented as mean ± SEM. Animal experimentation was conducted within standard ethical norms.

RESULTS Testosterone concentration changed with rams’ age and also showed high variability throughout the study period (Figure 1), which was previously investigated by Maksimović et al.11. The average values of the testosterone level in the blood serum of the rams ranged from 1.83 ng/ml at three months of age to 13.28 ng/ml at the end of the test period (when rams were at the age of 17 months), which was significant at P<0.05. Table 1 shows mean values, variability and statistical difference of analysed sexual activity patterns expressed through malefemale interactions in different ages of rams. Incidence of all analysed interactions between rams and ewe was elevated with age of rams, and this was statistically significant (P<0.05). Frequency of these interactions rose linearly from third to ninth month of age, after which it showed variability in terms of declining and rising throughout rest of study period. The most frequent male-female interactions in forms of nosing, pawing and mounts were observed in rams at the age of 13 months, as well as the longest duration of all activities directed towards ewe. Male-male interactions analysed through frequency of attempted mounts in different age of rams are given in Table 2. From the data presented it can be seen that a total of 175 malemale mounts were registered for the whole study period with

Table 1 - Mean ± SEM of analysed male-female interactions. Age of rams (months)

Nosing (frequency)*

Pawing (frequency)*

Flehmen (frequency)*

Mounts (frequency)*

Total male-female interactions (minutes)*

3.05d ± 0.56

0.10d ± 0.10

0.15d ± 0.10

0.00e ± 0.00

0.51d ± 0.15

3.35d ± 0.52

0.25d ± 0.25

0.25cd ± 0.09

0.10de ± 0.10

0.81d ± 0.20

8.20c ± 1.44

4.35bc ± 1.85

1.05abc ± 0.32

6.00bc ± 3.31

4.48c ± 1.12

* P<0.05;

13.30 ± 1.61

5.30 ± 1.87

2.40 ± 0.69

9.55 ± 4.22

8.14b ± 1.27

9.20bc ± 1.07

2.20cd ± 1.39

1.80ab ± 0.61

2.00cd ± 1.56

4.38c ± 0.60

15.60a ± 1.65

20.10a ± 3.82

1.50ab ± 0.38

15.45a ± 3.18

12.66a ± 1.02

9.20bc ± 1.21

12.35b ± 3.80

0.75bcd ± 0.35

11.15ab ± 3.28

6.62bc ± 1.01

a,b,c,d

- column means with different letters differ significantly.

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N. Maksimović et al. Large Animal Review 2021; 27: 31-34

Age of rams (months)

No of mounts/ram

Mounts (sum)

0.1c ± 0.1

3.2 ± 1.29

1.75a ± 0.88

0.25bc ± 0.20

abc

0.9

± 0.69

1.55 ± 0.75

0.9abc ± 0.61

Testosterone ng/ml

Table 2 - Mean ± SEM of analysed male-male interactions.

Age (months)

* P<0.05; a,b,c - column means with different letters differ significantly.

Figure 1 - Serum testosterone levels in rams depending on the age.

Table 3 - Correlation coefficients between serum testosterone and sexual behaviour activities.

Parameters

Nosing

Pawing

Flehmen

Male-female mounts

Male-female interactions in total (duration)

Male-male mounts

Testosterone

0.35**

0.19*

0.05

0.12

0.29**

0.18*

* P<0.05; ** P<0.01.

average number of mounts per ram ranging from 0.1 to 3.2. The highest activity was recorded at the age of 5 months. The determined frequencies of male-male mounts were significantly dependent on the age (P<0.05), and they showed significant variability throughout the test period. Table 3 shows correlation coefficients determined between testosterone concentration and sexual activity patterns of rams. The obtained results of the analysed correlations between testosterone concentration and the aspects of sexual behaviour of rams showed a weak to moderate relationship. Weakest link was between testosterone and flehmen response and the strongest one was with nosing behaviour, which was also statistically significant (P<0.01).

DISCUSSION The concept of sexual behaviour of male individuals is quite complex, both in its basis, in terms of differentiation, as well as in terms of the way and the strength of expression. As it was pointed out, individual differences in libido and sexual preferences that exist among rams are not completely understood. When exposed to ewe, most rams will exhibit courtship behaviour usually consisted of anogenital sniffing, ewe’s flunk nudging, impatient foot stomping and flehmen response, followed by completed mounts. In contrast, a small percentage of rams develop a same-sex preference for other rams even when raised with females12, 13. In current study, all of the examined aspects of sexual behaviour showed almost an identical trend, with the increase in value during the period from 3 to 9 months of rams’ age, followed by subsequent periods of lower and higher activities, with a very significant jump of activity at the age of 13 months, when the highest values of these parameters were determined. This is in agreement with the research by Ungerfeld and Gonzalez-Pensado10, who monitored the development

and manifestation of sexual behaviour of rams from 6 weeks to 9 months of age, in which it is emphasized that the frequency of expressing sexual behaviour toward non-oestrous ewe progressively increased with age. Observations of sexual activity patterns of individual rams showed that not all of them exhibited all forms of courting and mating behaviour, but all of them engaged in some form of sexual interaction at some point even though the female used were out of heat. Male-male interactions that are expressed in the context of sexual behaviour are quite common among domestic animals. Rams begin to develop and manifest this kind of sexual behaviour early, even within the first 10 weeks of life10, which is characterized by mounting attempts and simulation of copular behaviour that is directed at other males in the group. A total of 175 male-male mounts were registered for the whole study, with most frequent interactions being recorded in 5 months old lambs. The determined values were statistically significant (P<0.05) depending on the age, but showed significant variability during the entire test period. The very number of identified interactions itself is difficult to compare with the number determined in other studies, due to the difference in the number of animals, the length of the tests and the calculation methodology, but also because of very few such studies. However, this study is in agreement with previous findings of Ungerfeld et al.14 who determined intensive male-male interactions in ram lambs age 5 to 6 months. This is also consistent with the findings of Grubb15, who reported intense courting behaviour among the rams when they were about six months old. Nonetheless, although this study showed a significant effect of the age, it cannot be concluded that there was a linear trend in increasing these activities with age, which is contrary to the findings of Ungerfeld and Gonzalez-Pensado10. Grubb16 reported that among wild Soay male lambs, male-male mounts were more frequently observed than male-female

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Development of sexual behaviour in ram lambs and its correlation to serum testosterone

mounts. Some authors proposed that this kind of behaviour is related to establishment of social hierarchy15, 17. Some consider that rams mount opportunistically, e.g., if one ram is eating at the feeder or drinking, the other ram will try to mount him, or when rams are clogged in the crowd while moving, they will mount the individuals in front of them3,18. However, there is little information about this form of behaviour and its physiological significance. The results obtained in this study showed that sexual behaviour of rams wasn’t directly related to serum testosterone. Tested correlations between testosterone concentrations and aspects of sexual behaviour were generally weak to moderate. Even though some of these correlations were statistically significant, the strength of established correlative relationships is not convincing enough to support the distinct testosterone-dependent nature of rams’ sexual behaviour. This is in agreement with other work, which reported that there is no correlation between plasma testosterone and libido19, 20. It is likely that in the basis of male reproductive behaviour, testosterone has a significant place, but that it is not solely responsible for its expression, i.e. that there are other systems and mechanisms involved whose role cannot be ignored or excluded. As proposed by some authors, sexual behaviour displayed by adult rams requires minimum concentrations of circulating testosterone21, 22, so when this minimum requirement is met sexual performance won’t be directly related to testosterone concentrations. Roselli et al.,23 consider that hormones themselves are important but not directly responsible for the regulation of sexual behaviour, but that a significant role in this process has a nervous system, more specifically a region that includes a medial preoptic area/anterior hypothalamus (MPOA/AH). The MPOA/AH comprises a steroid-sensitive brain region that contains high concentrations of androgen and oestrogen receptors24. Roselli et al.,25 explain the hypothesis that a critical period exists in early perinatal life during which circulating testosterone produced from the foetal testis masculinises and defeminises both the neuroendocrine and behavioural potential of the brain.

CONCLUSIONS Performed investigations concluded that sexual behaviour of rams was age dependent, but poorly correlated to serum testosterone. Male-female oriented behaviour developed linearly with age, while male-male specific behaviour was characterized by high intensity in young age and then pronounced variability in later test periods. None of the studied behavioural interactions were highly correlated to serum testosterone. These findings support standpoint of more than one factor influencing development of sexual behaviour of ram lambs.

DISCLOSURE STATEMENT No conflict of interest was reported by the authors.

ACKNOWLEDGMENTS This research was supported and financed by the Ministry of Education, Science and Technological Development of the Republic of Serbia No. 451-03-68/2020-14.

References 1. Roselli C.E., Stormshak F., Stellflug J.N., Resko J.A. (2002). Relationship of serum testosterone concentrations to mate preferences in rams. Biol Reprod, 67: 263-268. 2. Bernon E.D., Shrestha B.N.J. (1984). Sexual activity patterns in rams. Can J Comp Med, 48: 42-46. 3. Perkins A., Roselli E.C. (2007). The ram as a model for behavioral neuroendocrinology. Horm Behav, 52: 70-77. 4. Mattner P.E., Braden A.W.H., George J.M. (1971). The relation of libido tests to subsequent service activity of young rams. Aust J Exp Agric, 11: 473. 5. Parott R.F., Baldwin B.A. (1984). Sexual and aggressive behaviour of castrated male sheep after injection of gonadal steroids and implantation of androgens in the hypothalamus: a preliminary study. Theriogenology, 21: 533-542. 6. D'Occhio M.J., Galil K.A.A., Brooks D.E., Setchell B.P. (1985). Differential effects of gonadectomy on sensitivity to testosterone of brain centres associated with gonadotrophin negative feedback and with mating behaviour in rams. J Endocrinol, 104: 69-75. 7. Perkins A., Fitzgerald J.A. Price E.O. (1992). Luteinizing hormone and testosterone response of sexually active and inactive rams. J Anim Sci, 70: 2086-2093. 8. Stellflug J.N. (2006). Comparison of cortisol, luteinizing hormone and testosterone responses to a defined stressor in sexually inactive rams and sexually active female-oriented and male-oriented rams. J Anim Sci, 84: 463-468. 9. Preston T.B., Stevenson R.I., Lincoln A.G., Monfort L.S., Pilkington G.J., Wilson K. (2011). Testes size, testosterone production and reproductive behaviour in a natural mammalian mating system. J Anim Ecol, 81, 1, 296-305. 10. Ungerfeld R., Gonzalez-Pensado P.S. (2008). Social rank affects reproductive development in male lambs. Anim Reprod Sci, 109: 161-171. 11. Maksimovic N., Hristov S., Stankovic B., Petrovic M.P., Mekic C., Ruzic-Muslic D., Caro-Petrovic V. (2016). Investigation of serum testosterone level, scrotal circumference, body mass, semen characteristics, and their correlations in developing MIS lambs. Turk J Vet Anim Sci, 40, 1: 53-59. 12. Katz S.L., Price O.E., Wallach R.J.S., Zenchak J.J. (1988). Sexual performance of rams reared with and without females after weaning. J Anim Sci, 33: 1166-1171. 13. Price E.O., Borgwardt R., Blackshaw J.K., Blackshaw A., Dally M.R., Erhard H. (1994). Effect of early experience on the sexual performance of yearling rams. Appl Anim Behav Sci, 42: 41-48. 14. Ungerfeld R., Ramos A.M., Bielli A. (2007). Relationship between malemale and male-female sexual behaviour in 5-6-month-old male lambs. Anim Reprod Sci, 100: 385-390. 15. Grubb P. (1974). The rut and behaviour of Soay rams. In:, Island Survivors: The Ecology of the Soay Sheep of St. Kilda, Eds. Jewell P., Milner C., Morton Boyd J., 195-223, The Athlone Press, University of London, London. 16. Grubb, P. (1974). Social organization of Soay sheep and the behaviour of ewes and lambs. In:, Island Survivors: The Ecology of the Soay Sheep of St. Kilda, Eds. Jewell P., Milner C., Morton Boyd J.,131-159, The Athlone Press, University of London, London. 17. Orgeur P., Mimouni P., Signoret J.P. (1990). The influence of rearing conditions on the social relationships of young male goats (Capra hircus). Appl Anim Behav Sci, 27: 105-113. 18. Price E.O., Katz L.S., Wallach S.J.R., Zenchak J.J., (1988). The relationship of male-male mounting to the sexual preferences of young rams. Appl Anim Behav Sci, 21: 347-352. 19. Howles C.M., Webster G.M., Haynes N.B. (1980). The effect of rearing under a long or short photoperiod on testis growth, plasma testosterone and prolactin concentrations, and the development of sexual behavior in rams. Reproduction, 60, 4: 437-447. 20. Moghaddam H.G., Pourseif M.M., Rafat A.S. (2012). Seasonal variation in semen quantity and quality traits if Iranian crossbred rams. Slovak J Anim Sci, 45, 3: 67-75. 21. Schanbacher B.D., Lunstra D.D. (1976). Seasonal changes in sexual activity and serum levels of LH and testosterone in Finnish landrace and Suffolk rams. Publications from USDA-ARS/UNL Faculty. Paper 753. 22. Holmes R.J. (1986). Sexual behavior of sheep. In: Current Therapy in Theriogenology, Diagnosis, Treatment and Prevention of Reproduction Diseases in Small and Large Animals, Ed. Morrow D.A., 2nd ed., 870-873, WB Saunders. 23. Roselli E.C., Larkin K., Resko J.A., Stellflug J.N., Stormshak F. (2004). The volume of a sexually dimorphic nucleus in the ovine medial preoptic area/anterior hypothalamus varies with sexual partner preference. Endocrinology, 145: 478-483. 24. Simerly R.B. (1995). Hormonal regulation of limbic and hypothalamic pathways. In: Neurobiological effects of sex steroid hormones, Eds. Micevych P.E., Hammer R.P.J., 85-114, Cambridge University Press, Cambridge, UK. 25. Roselli E.C., Reddy C.R., Kaufman R.K. (2011). The development of maleoriented behaviour in rams. Front Neuroendocrinol, 32: 164-169.

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V. Živković et al. Large Animal Review 2021; 27: 37-41

The effect of dietary l-tryptophan on productive performance and behavior of weaned piglets

ŽIVKOVIĆ VLADIMIR1*, STANKOVIĆ BRANISLAV2, HRISTOV SLAVČA2, DELIĆ NIKOLA1, NIKŠIĆ DRAGAN1, SAMOLOVAC LJILJANA1, PETRIČEVIĆ MAJA1 1 2

Institute for Animal Husbandry, Autoput 16, 11080 Belgarde, Serbia University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia

SUMMARY This study was carried to determine if dietary tryptophan can be beneficial for piglets in period of weaning. Trial was conducted on 84 individuals (Landrace×Yorkshire) female and castrated male piglets, at 30 days of age, and of 9.78±0.42 kg. Test subjects were penned into four groups and allocated to four different diets. This was done opposite to standard farm procedure; all piglets penned in same group came from different litters. Animals were fed ad libitum for six days. Piglets in trial groups were fed with basically same mixture with different levels of digestible L-tryptophan (0.1; 0.2; 0.3%). Productive performance (FCR; ADG; ADFI) was calculated and behavior characteristics (postures, mounting, abnormal and aggressive behavior) were observed using CCTV cameras. For the purpose of production performance analysis one way ANOVA was used while the Tukey test served to determine the statistical significance of the differences between individual means values. Considering that there is no normal distribution for behavioral parameters, we used non parametric Kruskal-Wallis test with multiple comparisons of mean rank between groups. Productive results showed that control group had significantly better results for average daily gain compared to all trial groups (p<0.05), other productive parameters didn’t show any significant difference. On the other hand, statistically significant results occurred for two behavioral characteristics. Fighting differed significantly (p<0.05), during first day of weaning between control and trial groups, intensive ear biting occurred at day two and three after weaning, and different significantly on day three (p<0.05). According to our results tryptophan had no positive effects on productive performance (feed intake, daily weight gain and feed conversion), but had some positive effects on reducing aggressive behavior. Conclusion could be that implementing small doses of tryptophan on weaning can be beneficial to reducing stress and behavioral anomalies of piglets. Further more extensive studies should be carried to verify these results.

KEY WORDS Rearing, stress, nutrition, aggressive behavior.

INTRODUCTION In commercial swine production, weaning is probably one of the most stressful periods in the life of a pig. Intensive swine production has led to decreasment of weaning age. For better utilization of housing facilities piglets were usually reared at an early stage of life1, 2. Today, most piglets on commercial farms are separated from their mothers at the age of 3 to 4 weeks3. Piglets experience certain change at a time when usually they are not supposed to face them. Those changes include: separation from the sow, change of environment, mixing with piglets from different litters and change from a liquid based diets to a solid ones. Weaning exposes piglets to different conditions that affect their welfare, which in turn causes major stress. Tryptophan is known to perform several physiological functions. When tryptophan is supplemented in higher amounts than the requirements, it can be used as a therapeutic supplement. Tryptophan has been shown to affect brain and nervous system function through interference with serotonergic neurotransmission4. Tryptophan is widely regarded as fourth or fifth limiting amino acid (AA) in maize based diets. In some stud-

ies tryptophan has been associated with the control of stress, immune response and health maintenance5. Tryptophan serves as precursor for serotonin synthesis, and tryptophan-induced serotonergic activity in the brain has been associated with the regulation of some behavioral and physiological processes such as change of mood, control of aggression, sensitivity to stress, sleep patterns, and feed intake6, 7. Behavioral observations had already been made in numerous studies, mostly dealing with the dominance in the relationships, which are often connected with other behavioral and physiological characteristics of the animals8. Rearing environment can influence the development of some behavior, which can also influence behavioral response to stressful situations in later stages of life2. We hypothesized that an increase in tryptophan concentration in the diet above the assumed normal requirement value would reduce behavioral and physiological stress at weaning and that it will positively affect productive performance of piglets.

MATERIALS AND METHODS Diet and housing

Corresponding Author: Živković Vladimir (vladimirzivkovic_87@yahoo.com).

Trial was conducted on experimental farm of Institute for animal husbandry, Belgrade, Serbia. Crossbred (Landrace×Yorkshire) female and castrated male piglets, at 30 days of age and

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The effect of dietary l-tryptophan on productive performance and behavior of weaned piglets

bodyweight of 9.78±0.42 kg, were used in trial. Piglets from seven litters of ten pigs each were penned in four groups of seven (4:3; male to female ratio) and allocated to four different diets. This was done opposite to standard farm procedure; all piglets penned in same group came from different litters. Trial comprised 3 replications with total of 84 piglets. Pen was rectangle shape, three square meters in size, with seven separate feeding places. Every pen is equipped with one nipple drinker. Pens are cage based with combined floors (concrete and plastic). Ventilation, temperature and relative humidity were constant during the trial. Animals were fed ad libitum for six days. All animals were fed morning before weaning with trial mixtures, so that tryptophan can take function before weaning. The feed was pre-weighed at start of trial in daily portions for each pen according to the expected feed consumption. The pigs had access to the feed at all times through manual feeders placed in the front of each pen. Average daily feed consumption (ADFI) was calculated by subtracting unconsumed feed at the end of trial from the pre-

weighed amount and splited by the days. Body mass were weighted at start and at the end of trial. Piglets were weighed at the beginning and at the end of the experiment and the Average daily gain (ADG) were calculated with the following equation: Final weight - Initial weight ADG =

Duration of the experiment (days)

Feed conversion (FCR) was also calculated: Daily feed intake FCR =

Daily weight gain

A basal mixture (Table 1) was formulated to contain sufficient amounts of all essential amino acids. The piglets in trial groups were fed with basically same mixture with different levels of digestible L-tryptophan (0.1; 0.2; 0.3%, respectively).

Table 1 - Composition of diets for weaned piglets in the trial. Treatment

Maize

578.00

577.90

577.80

577.70

Soybean cake

240.10

Soybean meal

100.00

Calcium carbonate

15.00

Ingredients, g/kg

Sodium chloride

3.70

3.75

Monocalcium phosphate

10.10

L-Lysine

1.10

1.00

2.00

3.00

L-Tryptophan Minazel*

2.00

Mineral-vitamin premix**

50.00

* Natural mycotoxin adsorbent. ** Added per kg diet: 15,000 IU Vitamin A, 1500 IU Vitamin D3, 40 IU Vitamin E, 1.0 mg Vitamin K3, 2.0 mg Vitamin B1, 4 mg Vitamin B2, 10 mg d-Pantothenic acid, 18 mg Niacin, 70 mg Biotin, 18 mg Vitamin C, 0.03 mg Vitamin B12, 4 mg Vitamin B6, 170 mg Fe: Fe(II) sulphate, 4 mg Cu: Cu(II) sulphate, 16 mg Zn: Zn(II) oxide, 50 mg Mn: Mn(II) oxide, 0.304mg KI, 0.3 mg Se: Se-selenite.

Table 2 - Ethogram of behaviors observed during continuous observations of all pigs in the experiment after mixing10. Behavior

Description

Posture Lying Sitting Standing

Pig lying with eyes open or with eyes closed and without movement. Dog-sitting position. Pig standing on all four feet.

Aggressive Fights Headknocks

Mutual pushing, ramming or pushing the opponent with the head, with or without biting. Lifting the opponent by pushing the head under its body11. Knocking heads between two pigs. Three or more knocks is considered a fight12.

Mounting Mounts

Placing feet on the back of another pig with or without pelvic movement13.

Abnormal Tail directed Ear directed Flank directed

Tail positioned in the mouth of another pig. Ranges from tail being gently manipulated to tail being chewed or bitten14. Ear positioned in the mouth of another pig: ranges from ear being gently manipulated to being chewed or bitten14. Biting directed towards the flank of another pig.

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Table 3 - Statistical indicators (mean ± SE) for production performance of piglets. Treatments

319.68±0.06

ADFI, g/d

324.29±0.05

ADG, g/d

154.60±0.02

132.85±0.03

132.22±0.03

115.47±0.01b

FCR, g/g

2.09±0.15

2.40±0.22

2.29±0.06

2.54±0.17

303.97±0.07 b

SEM, Standard error of the means; ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion rate; a different superscript differ significantly (p<0.05).

Behavior Observation of test subject was done by using four CCTV cameras which recorded from 6:00h to 20:00h for 6 days post weaning. Cameras were placed on the ceiling at three meters height, so it can provide us top view of the pen. All cameras equipped with motion detection sensor and night recording. Color images were captured with a frame rate of 24 frames per second and a resolution of 1280×720 pixels. All observed behaviors are classified in table 2. For the analysis of the collected date we used Behavioral Observation Research Interactive Software9.

Statistical analyses All statistical analyses were performed using R-project software15. For the purpose of production performance analysis one way ANOVA was used while the Tukey test served to determine the statistical significance of the differences between individual means values. Considering that there is no normal distribution for behavioral parameters, we used non parametric KruskalWallis test with multiple comparisons of mean rank between groups.

a, b

294.21±0.09

, in a row, the least squares means with

RESULTS Production performance Results (Table 3) have shown that control group had better production results for both ADG and FCR compared to the trial groups. Only statistical significance occurred in ADG between control group and the rest.

Behavior Postures. Treatments had no significant effect on the postures of pigs in all groups (P>0.05). Aggressive behavior. There were significantly more aggressive behaviors in control group compared to trial (Figure 1) during first 24h post weaning (P<0.05). Fighting time in pens differed from 1 to 256 seconds. Mounting behavior. All mounts occurred in the first three days after mixing. Mounts lasted from 3 to 12 seconds. There were no statistical significance between treatments (P>0.05). Abnormal behavior. On the third day there were statistical difference between control and trial groups (P<0.05), and also there were very big difference between days. First 24h post weaning

Figure 1 - Number of fights for the whole trial period, Different letters indicate significant differences (P<0.05).

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The effect of dietary l-tryptophan on productive performance and behavior of weaned piglets

Figure 2 - Number of cases of abnormal behaviors for the whole trial period; Different letters indicate significant differences (P < 0.05).

there were only few cases of abnormal behavior. Than in next couple of day’s number of cases multiplied several times (Figure 2). On the fourth day again number of cases dropped.

DISCUSSION Pigs are very adaptable to their environment, but nutritional status may affect how they respond to environmental stimulus. Apart from being a one of the major amino acids for protein synthesis, brain serotonin content is increased as the result of increased dietary tryptophan16, and also plays a key role in appetite regulation. Our result has shown that dietary l-tryptophan had no positive effect on growth performance of the piglets. Control group had best results for ADG and FCR. Some researchers come to same conclusion as us, that dietary tryptophan had no effect on daily gain17, 18; while others concluded tryptophan in small amounts could improve ADG and FCR19, 20. Martínez-Trejo et al.21 also reported that tryptophan supplementation of piglets positively affected some categories related with their behavior, however, it had no effect on their productive performance (feed intake, daily weight gain and feed conversion), which is pretty similar to our results. This suggests that when giving tryptophan above the requirements, feed intake does not decrease. Those results also agrees with the one reported by Li et al.7. Janczak et al.22, observed a tendency to decrease aggressive behavior among animals when supplementing tryptophan in the drinking water of mice. Meunier-Salaün et al.16 reported that dietary tryptophan levels induced minor changes in behavioral responses. Some researchers concluded that adding dietary tryptophan induces lethargic behavior23. Peeters et al.24 reported that

pigs provided with tryptophan in their drinking water spent more time lying during simulated transport than control pigs, but no other differences were observed. When tryptophan is supplemented, some nutritional factors can influence the passage of tryptophan across the blood-brain barrier to convert it into serotonin. Level of the Large Neutral Amino Acids (LNAA; leucine, isoleucine, methionine, valine, phenylalanine and tyrosine) in diets, are competing with tryptophan for carrier proteins across the cell membrane; and also level of carbohydrates in the diet where high glycemic index can increase insulin which results removal of selectively the LNAA from plasma with less effect on tryptophan25 and also the concentration of fatty acids, which compete with tryptophan for binding to albumin26.

CONCLUSION According to our results tryptophan had no positive effects on productive performance (feed intake, daily weight gain and feed conversion), but had some positive effects on reducing aggressive behavior. Conclusion could be that implementing small doses of tryptophan on weaning can be beneficial to reducing stress and behavioral anomalies of piglets. Further detailed studies must be conducted to verify these results.

ACKNOWLEDGEMENTS Research was financed by the Ministry of Education, Science and Technological Development of Republic of Serbia No. 45103-68/2020-14.

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References 1. Benson G.J., Rollin B.E. (2004). The well-being of farm animals: challenges and solutions. Blackwell Publishing, Ames, IA: USA. 2. Li Y., Wang L. (2011). Effects of previous housing system on agonistic behaviors of growing pigs at mixing. Appl. Anim. Behav. Sci., 132: 20-26. 3. Fels M., Hoy S., Hartung J. (2012). Influence of origin litter on social rank, agonistic behaviour and growth performance of piglets after weaning. Appl. Anim. Behav. Sci., 139: 225-232. 4. Huether G., Kochen W., Simat T.J., Steinhart H. (1999). Tryptophan, serotonin, and melatonin: Basic aspects and applications. Kluwer Academic/Plenum Publ., New York. 5. Le Floc’h N., Sève B. (2007). Biological roles of tryptophan and its metabolism: potential implications for pig feeding. Livest, 112: 23-32. 6. Markus C.R., Olivier B., Panhuysen G.E.M., van der Gugten J., Alles M.S., Tuiten A., Westenberg H.G., Fekkes D., Koppeschaar H.F., de Haan E.E. (2000). The bovine protein alpha-lactalbumin increases the plasma ratio of tryptophan to the other large neutral amino acids, and in vulnerable subjects raises brain serotonin activity, reduces cortisol concentration, and improves mood under stress. Am. J. Clin. Nutr., 71: 1536- 1544. 7. Li Y.Z., Kerr B.J., Kidd M.T., Gonyou H.W. (2006). Use of supplementary tryptophan to modify the behavior of pigs. J. Anim. Sci., 84: 212-220. 8. Langbein J., Puppe B. (2004). Analysing dominance relationships by sociometric methods - a plea for amore standardised and precise approach in farm animals. Appl. Anim. Behav. Sci., 87: 293-315. 9. Friard O.P., Gamba M. (2016). Behavioral Observation Research Interactive Software (BORIS). Università di Torino. 10. van Staaveren N., Lemos Teixeira D., Hanlon A., Ann Boyle L. (2015). The Effect of Mixing Entire Male Pigs Prior to Transport to Slaughter on Behaviour, Welfare and Carcass Lesions. PLoS One., 10(4). 11. Stewart C.L., O’Connell N.E., Boyle L. (2008). Influence of access to straw provided in racks on the welfare of sows in large dynamic groups. Appl. Anim. Behav. Sci., 112: 235-247. 12. Keeling L.J., Gonyou H.W. (2001). Social Behavior in Farm Animals. CAB International, Wallingford, UK, 147-176. 13. Fàbrega E., Puigvert X., Soler J., Tibau J., Dalmau A. (2013). Effect of on farm mixing and slaughter strategy on behaviour, welfare and productivity in Duroc finished entire male pigs. Appl. Anim. Behav. Sci., 143(1): 31-39. 14. O’Connell N.E., Beattie V.E., Watt D. (2005). Influence of regrouping strategy on performance, behaviour and carcass parameters in pigs. Livestock Production Science, 97: 107-115. 15. R Core Team. (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria.

16. Meunier-Salaün M.C., Monnier M., Colléaux Y., Seve B., Henry Y. (1991). Impact of dietary tryptophan and behavioral type on behavior, plasma cortisol and brain metabolites of young pigs. J. Anim. Sci., 69: 36893698. 17. Susenbeth A., Lucanus U. (2005). The effect of tryptophan supplementation of diets of restricted and unrestricted-fed young pigs. J. Anim. Physiol. Anim. Nutr. (Berl.), 89 (9-10): 331-336. 18. Gomes L.M., ade Mello Miassi G., dos Santos L.S., Dib Saleh M.A., Sartori J.R., Panhoza Tse M.L., Berto D.A. (2018). Impact of two light programs and two levels of dietary tryptophan for weanling piglets. Livestock Science, 216: 191-196. 19. Trevisi P, Melchior D, Mazzoni M, Casini L, De Filippi S., Minieri L., Lalatta-Costerbosa G., Bosi P. (2009). A tryptophan enriched diet improves feed intake and growth performance of susceptible weanling pigs orally challenged with E.coli K88. J. Anim. Sci., 87: 148-156. 20. Capozzalo M.M., Kim J.C., Htoo J.K., de Lange C.F.M., Mullan B.P., Resink J., Hansen C., Stumbles P., Hampson D., Ferguson N., Pluske J. (2020) Estimating the standardized ileal digestible tryptophan requirement of pigs kept under commercial conditions in the immediate post-weaning period. Animal Feed Science and Technology, 259: 114342. 21. Martinez-Trejo G., Ortega-Cerrilla M.E., Rodarte-Covarrubias L.F., Herrera-Haro J.G., Figueroa-Velasco J.L. Galindo-Maldonado F., SanchezMartinez O., Lara-Bueno A. (2009) Aggressiveness and Productive Performance of Piglets Supplemented with Tryptophan. Journal of Animal and Veterinary Advances, 8: 608-611. 22. Janczak A.M., Bakken M., Braastad B.O. (2001). A cautionary note regarding the use of nutritional L-tryptophan to alter aversion-related behavior in mice. Applied Anim. Behav. Sci., 72: 365-373. 23. Koopmans S.J., Guzik A.C., van der Meulen J., Dekker R., Kogut J., Kerr B.J., Southern L.L. (2006). Effects of supplemental L-tryptophan on serotonin, cortisol, intestinal integrity, and behavior in weanling piglets. J. Anim. Sci., 84: 963-971. 24. Peeters E., Driessen B., Steegmans R., Henot D., Geers R. (2004). Effect of supplemental tryptophan, vitamin E, and a herbal product on responses by pigs to vibration. J. Anim. Sci., 82: 2410-2420. 25. Clark J., Mills D.S. (1997). Design Considerations for the Evaluation of Tryptophan Supplementation in the Modification of Equine Behavior. In: Mills, D.S., S.E. Heath and L.J. Harrington (Eds.). Page 164-173 in Proc. of the First International Conference on Veterinary Behavioural Medicine. Universities Federation for Animal Welfare, Potters Bar, April 1-2, Birmingham, UK. 26. Grimmett A., Sillence M.N. (2005). Calmatives for the excitable horse: A review of L-tryptophan. Vet. J., 170: 24-32.

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Yeter B., Large Animal Review 2021; 27: 43-49

Use of industrially produced litter material from waste paper sludge as litter in broiler houses

BEYHAN YETER Department of Animal Science, Faculty of Agriculture, University of Kahramanmaras Sutcu Imam, Kahramanmaras, Turkey

SUMMARY The aim of the current study was to evaluate the “Gaia” as a bedding material and find out the possible effects on the broiler performance, foot pad dermatitis (FPD), breast feather loss (BFL) and litter characteristics. The study was conducted with 3492 mixed-sex Ross 308 chicks reared under standard commercial-broiler growing conditions in two environmentally similar closed poultry houses and using either pine shavings (PS, 4.5 kg/m2) or paper waste (PW, 3.5 kg/m2) as litter. Three replicates were used for each litter type. The results showed litter material had no effect on poultry performance in terms of live weight, feed efficiency, or viability; however, in comparison to the PS group, the PW group had significantly higher mean foot pad dermatitis (FPD) scores (0.07 vs 0.10; P<0.05) and lower breast feather loss (BFL) scores (1.83 vs. 62; P<0.05). There is no significant difference among treatments in terms of litter pH, ammonia concentrations, and moisture levels. Moreover, due to the high drying temperature of the PW, it has no microbial load. As a result, it was concluded that because PW has no adverse effect on poultry performance characteristics, the material can be used as poultry litter.

KEY WORDS Breast feather score, Broiler performance, Foot pad dermatitis, Litter, Moisture, pH.

INTRODUCTION Litter is used in broiler production to prevent chicks from coming into direct contact with the ground. Choice of litter material is based upon numerous factors, including availability, usability, price, insulating capacity, water holding capacity, animal comfort, lack of harmful effects to animals, humans and the environment, and opportunities for subsequent use as fertilizer or fuel1,2. Litter is an important material in ensuring that animal health and welfare, food safety and environmental requirements are maintained at an optimum level and that production performance and efficiency is maintained2,3,4. According to Toghyani et al.5, the humidity, pH, ammonia, dust and microbiological properties of litter are as important as inhouse conditions in poultry production. Litter material has an effect on broiler physiological and behavioral characteristics1, growth characteristics, and animal welfare and immune systems6. Specifically, negative litter characteristics not only may lead to the development of breast, foot joint (hock), foot-pad and finger lesions but also adversely affect carcass characteristics, which may results in economic losses1. It is more likely that during the production, litter materials are mixed with manure, feathers, feed and water containing a variety of microorganisms, salts, nutrients and other wastes7. Ammonia level8,9 and digestive tract microbiota10,11 have a crucial effect on growth and other characteristics of broilers.

Corresponding Author: Yeter Beyhan (byeter@gmail.com).

In many countries, wood shavings, which have a particle size of about 1-3 cm and are obtained from the lumber and furniture industries, are preferred as litter material for intensive broiler production4. Recently the demand of wood shavings has increased not only in broiler production capacity but also increase in the use of fuel and hardboard production. Increase in the price of wood shavings has resulted in the increased cost of broiler production13,14,15. Therefore considerable attempts has been diverted in finding alternative litter material such as paper wastes16, gypsum17, hardwood bark18, kenaf19, hazelnut husks20, rice and wheat straw21, rice hulls22, rice hull ash23 and sawdust24, sand, zeolite, vermiculite, and sepiolite8,27,29, tree leaves30,31,32, composted municipal garbage33 and cellulose-based industrial wastewater byproducts14. A number of studies have also examined the possibilities of reuse litter13,34,35. Recently waste sludge has been converted into a commercial product called “Gaia” in Turkey (IKMAK Plastic Industry Trade Limited, 2016, Duzce, Turkey) for bedding material. The commercial product is the waste sludge produced during the recycling of paper and cardboard production and may be an alternative to the commonly used wood shavings. Ritz et al.14 also suggested that cellulose-based material mixed with feces at the end of broiler production can be utilized as a fertilizer for plant production. Although the sludge has been used alone or in combination with rice hulls as bedding materials in broiler house36,50, so far, this commercial product called “Gaia” has not been tested as a bedding material in broiler production. Therefore the aim of the current project was to evaluate the Gaia as a bedding material and find out the possible effects on the broiler performance, foot pad dermatitis (FPD), breast feather loss (BFL) and litter characteristics.

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Use of industrially produced litter material from waste paper sludge as litter in broiler houses

Table 1 - Material characteristics of the PW litter used in the study. Analysis paremeters pH (24°C)

Unit

Analysis results*

Max. criteria

5.6

Organic matter

77.6

Humidity (70°C)

13.8

Organic Carbon (C)

55.5

C/N

16.8

Total humic+fulvic acid

36.04

Total Nitrogen (N)

3.3

Total P2O5

1.4

Soluble in water K2O

2.6

Heavy metals accepted upper limits Cadmium (Cd)

mg/kg

0.83

Copper (Cu)

mg/kg

76.97

450

Nickel (Ni)

mg/kg

8.34

120

Lead (Pb)

mg/kg

2.47

150

Zinc (Zn)

mg/kg

315.3

1100

Mercury (Hg)

mg/kg

<0.01

Chromium (Cr)

mg/kg

4.37

350

Tin (Sn)

mg/kg

<0.01

* Analysis was performed at the LABEN Agricultural Analysis Laboratory, a Turkish-accredited institution in Antalya, Turkey on April 20, 2016.

Figure 1 - Pine shavings (PS) and material obtained from wastepaper processing sludge (PW).

MATERIAL AND METHODS The study was conducted at the Animal Production and Research Center of the Sutcu Imam University Faculty of Agricultural in Kahramanmaras, Turkey. The study complied with ethical guidelines and was conducted with the approval of the Local Ethics Committee for Animal Experiments (Project No. 2016/06).

Litter materials The material used in this experiment is a cellulose slurry comprised of cellulose fibers and sediment sludge that is produced as a byproduct of recycled paper and cardboard processing and has an average moisture level of 75-80%. Prior to packaging, the material is dried at 120°C in a rotary-drum oven for 3540 minutes, cooled, and passed through a 5-mm sieve. PW material characteristics are listed in Table 1. Dust- and resin-free pine shavings (PS) were used as litter in the control group. Figure 1 shows examples of both the PW and PS litter. Litter was spread on the floor of poultry houses (PW: 3.5 kg/m²; PS: 4.5 kg/m²). Water-holding capacity was calculated according to Tüzüner37 using the formula WHC = (wet net weight - dried net weight) / dried net weight x 100. WHC values for the litter materials used were found to be 268.3% (PS) and 140.6% (PW).

Production environment, animal material and rearing conditions The study was carried out in two environmentally controlled poultry houses constructed at the research center during the same time period. Each house is 7 m x 19 m, with 3-m high walls and an interior divided into 3 equal 44.27 m2 sections (2.33 m x 19 m), with 60-cm wire fences. The experiment was conducted with 3.492 mixed-sex Ross-308 broiler chicks. Chicks were randomly divided into 2 groups (PW: Experimental group; PS: Control group) of three replications each.

Table 2 - Nutritional values of the feeds used in the trial. Starter 1-10. days

Grower 1 11-20. days

Grower 2 21-33. days

Finisher 34-42. days

Dry matter (%)

88.0

Crude protein (%)

23.0

22.0

Crude cellulose (%)

6.0

Crude ash (%)

8.0

2805

2850

3050

Nutrient contents

Metabolic Energy Kcal/kg Metabolic Energy MJ/kg

11.744

11.932

12.770

Ca (%)

1.0-1.5

0.9-1.5

0.8-1.2

0.70

0.65

0.60

0.15-0.30

P (%) Na (%) NaCl (%)

0.35

Lysine (%)

1.30

1.20

1.10

0.90

Methionine (%)

0.60

0.50

0.35

Cystine (%)

0.32

0.40

0.30

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Yeter B., Large Animal Review 2021; 27: 43-49

Table 3 - Temperature and humidity values (at animal level) applied in the house during the experiment. Temperature (0C)

Relative Humidity (%)

Age (Week) PS, Min, Max

PW, Min, Max

PS, Min, Max

PW, Min, Max

32.0 (30.0-33.0)

60.0 (55.0-65.0)

60.0 (56.0-65.0)

28.0 (27.0-29.0)

28.0 (27.0-29.1)

60.0 (57.0-67.0)

60.0 (58.0-67.0)

27.1 (24.1-31.1)

27.4 (24.9-31.4)

60.8 (36.5-70.7)

59.2 (63.4-65.9)

26.3 (24.0-29.7)

26.9 (23.6-30.7)

55.7 (28.3-70.8)

56.9 (31.3-71.0)

25.1 (21.7-27.0)

25.1 (21.6-27.0)

53.9 (31.1-69.6)

55.7 (32.6-68.5)

24.0 (18.6-26.9)

24.3 (19.1-26.2)

52.0 (39.5-75.9)

62.0 (38.1-76.3)

Chicks in the PW group were raised in 1 house, and chicks in the PS group were raised in the other house. A total of 582 chicks placed in each compartment (13.1 chick/m2). Chicks were fed with commercial broiler rations obtained from a commercial feed factory. Nutrient contents of the feed used during the experiment are given in Table 2. Feed was provided from spiral feeders (22 feeders per pen), and water was provided by watering lines with nipple drinkers (68 per pen). Both feed and water were given ad-libitum throughout the experiment. Chicks were vaccinated against Newcastle, Gumboro and Infectious Bronchitis via drinking water during the growing period. A light/dark regime of 23/1 hours was applied during the experiment. Illumination was provided by white bulbs. A light intensity of 18 lux was maintained at the level of feeders and monitored by a light monitor (Lutron LX-101). Houses were heated by thermostat-controlled infra-red electric heaters, with 3 heaters per house. Temperature and humidity values of both houses were kept similar during the trial period. In-house temperature and humidity values were automatically recorded every 15 minutes by HOBO U12 External Data Loggers and used to calculate daily and weekly average temperature and humidity. The average, maximum and minimum values are given in Table 3.

Litter traits The ammonia concentration of the air just above the litter were measured at 35 and 42 days using ammonia analyzer (Drager Safety, Inc., 101 Technology Dr., Pittsburg, PA) placed at litter level in 3 separate zones in each compartment of the house,

Score 0

Figure 2 - Foot pad dermatitis scoring.

Score 1

and mean, maximum, and minimum values were calculated. pH and moisture content of litter were measured at the end of the experiment in triplicate.

Broiler performance traits Mortality was recorded daily, and weekly mortality rates were calculated cumulatively for each replication. Animals were weighed together at 7, 14, 28 and 35 days of age to determine total live weights per replication and weighed individually at 21 and 42 days of age to determine individual live weights. Feed consumption and feed efficiency were calculated for each replication at days 7, 14, 21, 28, 35 and 42. All animals were slaughtered at day 42. Foot pad dermatitis (FPD) was evaluated at the end of the trial (42 days) from the left foot of each chicken. FPD scores were recorded as follows: 0: No lesions; 1: small point lesions, 2: lesions covering more than 25% of the foot; 3: lesions covering more than 50% of the foot; 4: lesions covering the entire sole of the foot (12,38,49, Figure 2). Feather loss was evaluated on day 42 from the breast of each chicken and scored as follows: 0: no feather loss; 1: up to 25% feather loss; 2: up to 50% feather loss; 3: up to 75% feather loss; 4: complete loss of feathers (Figure 312,21).

Statistical analysis Statistical analysis was performed using the statistical software package SPSS (Version 21). Body weights, feed efficiency, mortality, and litter ammonia and fertilizer levels of the groups were compared using t tests, and FPD scores and BFL scores were compared using Mann-Whitney U tests. Statistical significance was set at a level of 0.05.

Score 2

Score 3

Score 4

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Use of industrially produced litter material from waste paper sludge as litter in broiler houses

Score 1

Score 2

Score 3

Score 4

Figure 3 - Feather loss scoring.

RESULTS AND DISCUSSION Broiler performance traits No health problems were observed during the experiment, and no additional medication was used. Live weights and feed efficiency (7 to 42 days of age) and mortality rates (0 to 42 days of age) for both litter groups are presented in Table 4, and changes in live weights, feed efficiency and mortality rates are shown in Figures 4, 5, and 6, respectively. Broiler chickens in both groups reached live weights of more than 2 kg in 35 days. Statistical analysis showed live weights, feed efficiency and mortality rates were not significantly affected by litter material. Feed

efficiency at 42 days of age were also similar for both groups as were mortality rates. While the majority of studies examining commonly used litter materials as well as litter materials particular to certain regions have reported no differences in broiler live weights, feed consumption, feed efficiency, or viability in connection with litter type14,23,29,40, some studies have reported litter materials to have significant effects on various performance traits4,20,27. The PW litter material used in this study is similar to the litter material used by Ritz et al.14 and Villagra et al.50 they stated that the material can be used without affecting broiler performance.

Table 4 - Mean body weight (g), feed efficiency (g:g), and mortality (%) of Ross-308 broilers commercially reared with 2 different litter systems (PS vs PW). _ _ _ Mortality (%) X±Sx_ Body weight (g) X±Sx_ Feed efficiency (g/g) X±Sx_ Treatment d42 d7 d21 d42 d7 d21 d42 PS

1.89±0.062

165.9±0.15

931.2±6.78

2820.6±10.11

0.94±0.01

1.21±0.01

1.59±0.01

2.34±0.057

165.3±1.08

922.7±2.93

2829.3±14.71

0.94±0.02

1.23±0.01

1.59±0.01

Mean

2.12

165.6

926.9

2824.9

0.94

1.23

1.59

p-value

0.164

0.612

0.316

0.823

0.864

0.162

0.793

Significance NS: Non significant differences.

Figure 4 - Broiler live weights (g) by litter system (PS and PW) and age (1-42 days).

Figure 5 - Broiler feed conversion rates (kg feed: kg CA) by litter system (PS and PW) and age (7-42 days).

Figure 6 - Broiler mortality (%) by litter system (PS and PW) and age (7-42 days).

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Yeter B., Large Animal Review 2021; 27: 43-49

Broiler welfare traits While the present study found no differences in broiler performance characteristics, FPD levels and BFL scores at 42 days of age were significantly affected by litter material (P < 0.05; Table 5), with mean FPD scores significantly higher (P < 0.05) in the PS group (0.07) as compared to the PW group (0.10). However, FPD scores of both litter groups were acceptable, with 96.40% of the PS group and 91.20% of the PW group scoring ‘0’. Previous studies have noted that foot and breast defects as well as some welfare parameters may be affected by litter material2,4,45,46. For example, Yildiz et al.28 reported statistically significant differences (p < 0.05) in FPD scores for sawdust (1.98), sawdust+vermiculite (1.02), and vermiculite (1.06) litter, and Bintas at al.29 similarly found that adding either soil or zeolite to sawdust litter lowered FPD levels. Tercic et al.12 also reported significant differences in FPD scores for wood shavings (1.00), shredded paper (1.15), and chopped wheat-straw pad litter (1.48). In contrast to these findings, Ritz et al.14 found that while adding a cellulose waste-water by-product to pine-shaving litter significantly improved FPD scores during the early stages of broiler growth, the improvement was no longer significant at the end of the growth period, and Bilgili et al.26 reported similar rates of FPD for sand and pine-shaving litters, with rates ranging between 10.1% and 28.0% over 3 different trials. Zikic et al.46 found litter type and litter treatment with enzymaticbacterial production incidence to have a significant effect on the severity of FPD in broilers, with scores ranging between 1.94 (Un-chopped) and 1.47 (Chopped). El-Wahab et al.40 found the addition of Biotin and ZnO to feed of broilers raised on wood shavings resulted in significant differences in FPD scores, with scores ranging between 0.6-1.4 on a 7-point scale. Yamak et al.35 reported that reuse of sawdust litter resulted in significant increases (P < 0.05) in FPD scores from 2.39 for fresh litter to 2.64 for reused litter, with the authors attributing these increases to increases in litter moisture and ammonia levels. El-Wahab et al.40 also reported a significant relationship has between FPD levels and litter moisture and pH, with a 30% moisture level reported to be the critical cut-off point. Lien et al.16

determined the breast-blisters scores in pine shavings and recycled paper chip litters as 13.2% and 10.7%, and foot abnormalities as 8.2% and 6.9% whereas FPD scores in the present study were significantly higher for the PW group, feather loss scores were significantly higher in the PS group (PS: 1.62; PW:1.83; p < 0.05). Tercic et al.12 reported BFL to vary significantly (P < 0.05) according to litter type, with scores of 1.62, 1.81 and 2.71, respectively, for wood shavings, shredded paper and chopped wheat straw litter. Yildiz et al.28 also found significant differences in breast feather scores between wood shavings (2.92), vermiculite (2.93), and wood shavings+vermiculite (2.89). Lima et al.41 found that adding soil, sand and similar materials to litter as well as increasing litter height reduced breast feather loss. In terms of breast blisters, different studies have reported different results for different litter material. Although Willis et al.30 reported similar scores of 1.4, 1.3, and 1.4, respectively, for wood shavings, 50% wood shavings + leaves, and leaves used as a litter, Bilgili et al.26 reported breast-blister scores for different litters to range between 0-1.7, with scores lower for sand as compared to pine shavings, and Sarica and Cam20 reported scores for various plant-product litter to range between 1.3 - 2.6, with the highest breast-blister scores occurring with wheat stalk litter.

Litter traits Litter traits (ammonia, pH, moisture levels) did not vary significantly between the PS and PW groups (Table 6). Ammonia concentrations measured at the litter surface on days 35 and 42 were similar and acceptable for both groups (day 35: PS, 10.1 ppm; PW, 11.8 ppm, day 42: PS: 11.8 ppm; PW: 13.3 ppm). Sarica and Demir25 reported ammonia at litter levels to be 23.40 ppm for sawdust and 16.50 ppm for sawdust + zeolite. Chablee and Yeatman23 evaluated ammonia concentrations in 3 different litter treatments (pine shavings, rice hull ash, 50% pine shavings + 50% rice hull ash) and reported levels of 22, 24 and 21 ppm, respectively. Bintas et al.29 reported differences in sawdust litter particle size had no significant effect on ammonia levels, which ranged between 31.6 ppm-35.2 ppm. Miles

Table 5 - FPD and BFL scores of broilers at 42 days of age. FPD Scores Treatment

_ X±Sx_

BFL Scores

_ X±Sx_

0.07±0.01b (0:0-4)

96.40

3.00

0.23

0.06

1.83±0.03a (2:1-4)

49.10

28.10

14.10

8.70

0.10±0.01a (0:0-4)

91.20

8.10

0.23

0.41

0.12

1.62±0.02b (1:1-4)

60.40

22.90

11.50

5.20

a, b

: scores indicated by different letters are different according to Mann-Whitney U test (P < 0.05).

Table 6 - Mean moisture, pH and ammonia concentrations (%) of litter materials. Moisture content (%)

Ammonia concentration (ppm)

Treatments Initial

42 d

35 d

42 d

35 d 1

42 d

12.2

25.3

24.2

7.12

7.62

10.1 (8 -14 )

11.8 (101-162)

12.8

26.4

25.6

7.43

7.79

11.8 (71-162)

13.3 (121-172)

Mean

12.5

25.9

24.9

7.28

7.71

10.95

12.55

p-value

0.089

0.078

0.092

0.350

0.430

0.091

0.098

Significance 1

35 d

: Min, : Max.

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Use of industrially produced litter material from waste paper sludge as litter in broiler houses

et al.48 found ammonia levels measured at house walls, drinkers, and feeders to be 5.68 ppm, 12.3 ppm and 4.52 ppm, respectively. Ritz et al.14 found ammonia levels to vary according to litter material, with levels ranging from 42.4 ppm to 75.4 ppm. These levels are high when compared to other studies. Apart from substrate, ventilation, litter moisture and settlement frequency also have a significant effect on litter ammonia levels34. In the present study, pH levels for the PS and PW litter groups were 7.12 and 7.62, respectively, on day 35 and 7.43 and 7.79, respectively, on day 42. The differences between litter groups were not statistically significant. The pH levels found in our study are similar to those reported by Onbasilar et al.47 for wood shavings (7.5) and rice hulls (7.4). A study by El-Deek et al.44 determined pH levels as 6.67 to 7.24 on different litter, and in the second research, they determined between 6.51 and 6.93. Studies by El-Wahab et al.40 determined as higher pH value compared to reported research results (Experiment 1 between 8.48 and 8.61; Experiment 2 between 8.11 and 8.18). Similar high pH value (8.24 - 8.26) is determined by Lima et al.41 and the findings from both studies are higher than the pH results from this study. Moisture content of litter is the most important factor affecting ammonia levels, litter pH, and house humidity. The present study found initial moisture levels to be slightly higher for the PS group (PS: 12.2%; PW: 12.8%), whereas values were slightly higher for the PW group on day 35 (PS: 25.3%; PW: 26.4%) and day 42 (24.2% and 25.6%); however, the differences between groups were not statistically significant (Table 6). In general, when compared to the present study, previous studies reported higher litter moisture levels18,20,29,39. A number of studies have noted that when the moisture content in poultry house litter drops to 30% or below, both ammonia levels and FPD levels decrease as well12,14,34,40. As a result, based on the results obtained for broiler performance characteristics, FPD and BFL scores, and litter characteristics, commercial product called “Gaia” could be used as litter material in broiler production without compromising the broiler performance. However further investigation is required to test the feasibility of this commercial product in poultry production.

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Yeter B., Large Animal Review 2021; 27: 43-49 31. Chakma S., Miah M.Y., Ara A., Kawsar M.H. (2012). Feasibility of using fallen tea leaves as litter in broiler rearing. Bangladesh J. Anim. Sci., 41: 52-54. https://doi.org/10.3329/bjas.v41i1.11978 32. Sharma G., Khan A., Singh S., Kumar Anand A. (2015). Efficacy of pine leaves as an alternative bedding material for broiler chicks during summer season. Vet. World, 8: 1219-1224. https://doi.org/10.14202/vetworld.2015.1219-1224 33. Malone G.W., Chaloupka G.W., Eckroade R.J. (1983a). Composted municipal garbage for broiler litter. Poult. Sci., 62: 414-418. https://doi.org/10.3382/ps.0620414 34. Bolan N.S., Szogi A.A., Chuasavathi T., Seshadri B., Rotrock M.J., Panneerselvam P. (2010). Uses and management of poultry litter. World’s Poult. Sci. J., 66: 673-698. https://doi.org/10.1017/S0043933910000656 35. Yamak U.S., Sarica M., Boz M.A. (2016). Ucar A. Effect of reusing litter on broiler performance, foot-pad dermatitis and litter quality in chickens with different growth rates. Kafkas Univ. Vet. Fak. Derg., 22: 85-91. 36. Ozlu S., Shiranjang R., Elibol O., Karaca A., Turkoglu M. (2017). Effect of paper waste products as a litter material on broiler performance. Tavukçuluk Ara . Derg., 14: 12-17. 37. Tuzuner A. (1990). Soil and water analysis laboratories handbook. Ministry of Agriculture, Forestry and Rural Affairs. General Directorate of Rural Services. Ankara/Turkey 38. Mayne R.K. (2005). A review of the aetiology and possible causative factors of foot paddermatitis in growing turkeys and broilers. World’s Poult. Sci. J., 61: 256-267. https://doi.org/10.1079/WPS200458 39. Atapattu N.S.B.M., Wickramasinghe K.P. (2007). The use of refused tea as litter material for broiler chickens. Poult. Sci., 86: 968-972. https://doi.org/10.1093/ps/86.5.968 40. El-Wahab A.A., Radko D., Kamphues J. (2013). High dietary levels of biotin and zinc to improve health of foot pads in broilers exposed experimentally to litter with critical moisture content. Poult. Sci., 92: 1774-1782. https://doi.org/10.3382/ps.2013-03054 41. Lima R.C., Freitas E.R., Gomes H.M., Cruz C.E.B., Fernandes D.E. (2018). Performance of broiler chickens reared at two stocking densities and coir

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litter with different height. Revista Ciencia Agronomica, 49: 1-14. https://doi.org/10.5935/1806-6690.20180059 Malone G.W., Chaloupka G.W., Saylor W.W. (1983b). Influence of litter type and size on broiler performance. 1. Factors affecting litter consumption. Poult. Sci., 62: 1741-1746. https://doi.org/10.3382/ ps.0621741 Sarica M., Bicer A. (2004). Effect of using hazelnut husks and wood shawings as broiler litter materials at different deepness’s on performance and litter properties in broiler production. 4th National Zootechni Congress Book 1 Isparta, Turkey, p. 102-111. El-Deek A.A., Al-Harthi M.A., Khalifah M.M., Elbanoby M.M., Alharby T. (2011). Impact of newspaper as bedding material in arid land on broiler performance. Egyptian Poult. Sci., 31: 715-725. Sigroha R., Bidhan D.S., Yadav D.C., Sihag S.S., Malik A.K. (2017). Effect of different litter materials on the performance of broiler chicken. J. Anim. Research, 7: 665. https://doi.org/10.5958/2277-940X.2017.00102.4 Zikic D., Djukic-Stojcic M., Bjedov S., Peric L., Stojanovic S., Uscebrka G. (2017). Effect of litter on development and severity of foot-pad dermatitis and behavior of broiler chickens. Bre. J. Poult. Sci., 19: 247-254. https://doi.org/10.1590/1806-9061-2016-0396 Onbasilar E., Erdem E., Unal N., Kocakaya A., Torlak E. (2013). Effect of yucca schidigera spraying in different litter materials on some litter traits and breast burn of broilers at the fifth week of production. Kafkas Univ. Vet. Fak. Derg., 19: 749-753. https://doi.org/10.9775/kvfd.2013.8627 Miles D.M., Brooks J.P., McLaughlin M.R., Rowe D.E. (2013). Broiler litter ammonia emissions near sidewalls, feeders, and waterers. Poult. Sci., 92: 1693-1698. https://doi.org/10.3382/ps.2012-02809 Hocking, P.M., Mayne, R.K. Else, R.W. French, N.A. & Gatchlife, J. (2008). Standard European footpad dermatitis scoring system for use in turkey processing plants. World’s Poult. Sci. J. 64, 323-328. https://doi.org/10.1017/ S0043933908000068 Villagra A., Olivas I., Benitez V., Lainez, M. (2011). Evaluation of sludge from paper recycling as bedding material for broilers. Poult. Sci., 90: 953957. https://doi.org/10.3382/ps.2010-00935

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LAR

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Large Animal Review is a bimonthly magazine published by SIVAR (Italian Society of Farm Animals Veterinary Practitioners) for scientific updating of veterinarians who deal with animals in livestock production and the supply chain control in the production of food industry. The topics of main interest for the journal are those of internal medicine, surgery, obstetrics, animal nutrition, zootechnics, infectious and parasitic diseases, food safety and security, animal welfare, prevention and management.

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R. Compiani et al. Large Animal Review 2021; 27: 51-56

Prevention of the main Clostridial diseases in cattle

R. COMPIANI1, S. GROSSI1, L. LUCINI2, C.A. SGOIFO ROSSI1* 1

Dipartimento di Scienze Veterinarie per la Salute, la Produzione Animale e la Sicurezza Alimentare (VESPA) - Università degli Studi di Milano 2 Dipartimento di Scienze e Tecnologie Alimentari per una filiera agro-alimentare sostenibile (DiSTAS) Università Cattolica del Sacro Cuore

SUMMARY Clostridial diseases of cattle are an economic and welfare issue worldwide. Clostridia are obligate anaerobic spore-forming grampositive bacteria able to cause a wide range of pathologies in humans and animals. Pathogenicity is expressed by sporulation in favourable environmental condition with release of toxins. Toxins produced and tissues damaged are generally characteristic for each clostridial. The incidence of clostridiosis is relatively low however the outcome is generally very poor despite the bacteria being sensitive to the most common antibiotic therapies. The generally rapid course of the disease prevents any intervention. Despite a continually developing classification, clostridium that affect cattle can be classified based on their target tissue and pathogenic expression, as neurotoxic, histotoxic and enterotoxic. Scientific knowledge about different clostridial toxins, their aetiopathological mechanisms, risk factors and pathologies involved are generally limited due to the large number of bacteria strains and types involved. Alongside the more studied neurotoxic C. tetani and C. botulinum for their implications in human medicine, there are lots less known pathogenic strains capable of causing extremely severe clinical patterns in veterinary medicine. In particular regarding enterotoxic clostridia, the incidence of necro-haemorrhagic enteritis and enterotoxaemia is probably wrongly estimated because complete post-mortem investigation is rarely performed and several other reasons can lead to sudden death. The aim of this review is to describe the main clostridial diseases that can affect cattle and some of the possible prevention strategies as controlling major known risk factors and the use of vaccination.

KEY WORDS Clostridia, clostridiosis, cattle.

INTRODUCTION Clostridia are obligate anaerobic spore-forming gram-positive bacteria. The genus Clostridium consists of dozens of strains characterized by different pathogenicity, many of which are able to cause illness in humans and other animals. Pathogenicity is expressed not by the presence of clostridial bacteria but by replication; in favourable environmental conditions, sporulation with release of toxins takes place. Clostridial toxins are biologically active proteins that are antigenic in nature. Toxins produced and tissues damaged are generally characteristic for each clostridial1. In beef and dairy rearing systems, clostridium-associated diseases are both a welfare and an economic issue. The incidence of clostridiosis is relatively low however the outcome is generally very poor despite the bacteria being sensitive to the most common antibiotic therapies. The rapid course of the disease, in most cases, prevents any intervention. The aim of this review is to describe the main clostridial diseases that can affect cattle and the prevention strategies that field veterinarians can implement to support the farming system.

Corresponding Author: Carlo Angelo Sgoifo Rossi (carlo.sgoifo@unimi.it).

Bovine clostridiosis The classification of clostridia is a continually developing topic for researchers and is mainly based on the types of toxins produced. From a practical point of view, clostridium that affect cattle can be classified based on their target tissue and pathogenic expression, as neurotoxic, histotoxic and enterotoxic (Table 1).

Neurotoxic clostridia The main neurotoxic clostridia which affect cattle are C. tetani and C. botulinum. Tetanus is an acute, often fatal disease of almost all domestic animal species caused by the neurotoxins produced by Clostridium tetani in anaerobic conditions. This usually develops after contamination of deep and penetrating wounds. The neurotoxin produced causes the characteristic rigidity and muscle spasms2-4. C. tetani is present in soil and faeces and there can be several means of entry into the animal. Published literature reports infections of the umbilical cord, infection of wounds caused by barbed wire or pitchfork injuries, injection sites, hoof and interdigital space lesions, oral mucosal wounds caused by coarse forages, vaginal laceration incurred during dystocic calving, uterine prolapse or placental extraction, bedsores or surgical site lesions, dehorning and castration activities. Penicillin or tetracycline treatment to reduce bacteria proliferation in addition to anti-tetanus homologous

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Prevention of the main Clostridial diseases in cattle

Table 1 - Bovine clostridiosis classification. Type Neurotoxic clostridia

Histotoxic clostridia

Strain

Pathology

C. tetani

Tetanus

C. botulinum

Botulism

C. septicum C. chauvoei C. novy C. sordellii

Gangrene or tissue infections (muscles or muscles fascia, subcutaneous tissue, liver, abomasum, kidney, etc.)

C. perfringens C. haemolyticum Enterotoxic clostridia

C. sordellii

Enteritis and Enterotoxaemia

C. perfringens C. septicum C. difficile

serum and careful wound/entry site disinfection can improve prognosis. Considering the aetiology, prevention should be based on improving the hygiene of the housing environment and farming/vet tools5. Botulism is a neuro-paralytic disease of humans and animals, caused by the neurotoxins produced by Clostridium botulinum. C. botulinum is a ubiquitous soil-borne pathogen that finds an excellent growing environment in decaying organic matter6. In cattle, the signs are generally associated with the ingestion of feed or water contaminated with the remains of carcasses. There are even reported outbreaks linked to contact with poultry litter. Furthermore, C. botulinum can directly proliferate in forages without carcasses, in cases of inaccurate collecting and storage procedures (presence of organic refuse, excess soil contamination, high humidity and temperature)5,7-8. Botulism usually results in fatality since the neuronal flaccid paralysis cannot be reversed by available therapeutic options except for administration of antitoxin9. Its prevention is simply based on good management practice during harvesting and storing feed.

Histotoxic clostridia Among the different pathologies caused by histotoxic clostridial strains, the two main diseases affecting cattle are ‘Blackleg’ and ‘Malignant oedema’. The differences between these two diseases include the clostridial strains implicated and the entry point into the animal. Malignant oedema is considered to be an ‘‘exogenous’’ disease because different clostridia, such as C. septicum, C. chauvoei, C. novyi, C. sordellii, and C. perfringens, from the environment gain access into the tissues after skin or mucosal wounds and development of an anaerobic environment. Main types of trauma that may lead to malignant oedema include, but are not restricted to, intramuscular injections, parturition, shearing, castration, surgery and tail docking10-11. Clinically, the involved tissues rapidly develop oedema, characterised by a variable presence of gas, high fever and inappetance. In case of a nonhyperacute form, resulting in sudden death, treatment with penicillin or tetracycline may be effective only if started very early in the disease process and preferably in addition to polyvalent serum administration and surgical curettage of the wound. Despite rapid intervention the prognosis is poor and death typically occurs within 2-5 days after trauma5. Blackleg, in contrast, is caused by C. chauvoei alone and is con-

sidered to be an ‘‘endogenous’’ disease1,12-14. Even without a consensus from the scientific world about blackleg pathogenesis, the infection is acquired by the ingestion of C. chauvoei spores that are transported from the intestine to the muscles and tissues by macrophages across Peyer’s patches. Other hypotheses include entry by oral mucosal wounds due to teething, forestomach traumatic injures by foreign bodies and lesions of the enteric mucosa15-17. The spores remain dormant in the target tissues until a traumatic injury induces the anaerobic conditions ideal for their germination, multiplication and toxin production17-18. The predisposing traumatic events reported include bumps, blows, mounting behaviour, competition at the feed bunk or at water points, constricted passing in narrow places, goading and transport in general5. This infectious disease is acute and globally spread among ruminants, causing significant loss in livestock production19 as it is generally fatal, being included among the causative agents of sudden death. The disease is typically observed during the warm season, and young cattle aged between 6 to 24 months are mostly affected18. C. chauvoei is one of the clostridia supporting a fatal hyperacute form counted among the causative agents of sudden death. Moreover, the classical forms are reported with swelling and crepitus of affected skeletal muscles due to a neutrophilic necrotizing myositis20. Rarely diagnosed forms include fibrinous pleuritis, pericarditis, epicarditis, meningoencephalitis, severe acute necrotizing enteritis or myositis of sublingual muscles and diaphragm21-25. Avoiding soil-contaminated pasture, forages and litter is the most effective preventive strategy especially in those regions characterized by very high annual rainfall that can expose and activate latent spores, after soil excavation or areas with a history of flooding25-26.

Enterotoxic clostridia Among enterotoxic clostridia, Clostridium perfringens is the major cause of necrotic and haemorrhagic enteritis and enterotoxaemia both in humans and other animal species. In accordance with the other diseases caused by clostridia, although morbidity is rather low, therapy is largely ineffective and mortality is close to 100%, making it an economically important disease27. Moreover, it is one of the most widespread bacteria, ubiquitously present in the environment, in soil, food, manure and the normal intestinal microbiota of both humans and animals28. C. perfringens is classified into five toxinotypes (A, B, C, D and

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R. Compiani et al. Large Animal Review 2021; 27: 51-56

E), based on their ability to produce different toxins and extracellular enzymes. In addition to producing a combination of the four major toxins (alpha, beta, epsilon and iota toxin), C. perfringens strains can produce additional but not less harmful toxins such as enterotoxin and necrotic enteritis B-like toxin1,29-31. Despite an ongoing debate among researchers, C. perfringens type A strains are the suspected agent in cases of bovine alimentary tract disorders such as clostridial abomasitis and necro-haemorrhagic enteritis. Type D, however, seems to be the aetiological agent of proper “enterotoxaemia”32. Indeed, the term “enterotoxaemia” is wrongly applied to generic diseases caused by C. perfringens, but should be used only in cases where major signs are caused by systemic actions of the toxins without the presence of important intestinal lesions1,30-31. From a practical point of view, since the predisposing factors and outcome of the clinical condition are basically the same, and the correct differential diagnosis does not lead to more valid therapeutic options, the debate is only formal and detailed to the laboratory. Enterotoxic clostridial disease is in fact characterized by a high case fatality rate, sudden deaths, more or less evident lesions of necrotic and haemorrhagic enteritis of the small intestine and, most often, an absence of other clinical signs28,33-34. When premonitory signs are noticed by the farmer, cattle death is expected within 5 hours due to necro-haemorrhagic enteritis35-36. Those signs are typically lateral recumbency and cold extremities, as consequence of cardiovascular shock. Other signs detected less frequently are colic, respiratory distress, nervous signs, distended abdomen and diarrhoea25,35-36. Considering the huge presence of C. perfringens in the environment, the development of the disease is linked to the type of bacteria present and the relative pattern of toxins produced, the amount of spores within the enteric tract and the host susceptibility. Several nutritional predisposing factors are in fact described to predispose the enteric environment to the germination and multiplication of C. perfringens. Since C. perfringens proliferate using the amount of digestible carbohydrates that exceed the digestive and absorptive capacity of the intestinal mucosa, high protein and energy-rich diets predispose to the disease27-37. Other dietary issues such as sudden change of feed composition, change of pasture, moving from pasture to burns and high protein concentration in association with low amounts of fibre may alter the microbiota composition promoting clostridial overgrowth36. C. perfringens also affects preweaned calves. Indeed, necro-haemorrhagic enteritis is more frequently observed in veal calves and suckler calves. The whey present in cow’s milk or milk replacer contains high quality, readily available amino acids, potentially predisposing to clostridial overgrowth27,38-40. Increasing host susceptibility is not only a matter of nutrition but also stressful environmental conditions, such as regrouping, transport, handling and medical treatments are risk factors for necro-haemorrhagic enteritis27. In cases of stress, the consequent intestinal microbiota modification can lead to a less efficient digestive process with more nutrients available for bacterial growth41-44. The enteritis slows down intestinal motility and the consequent intestinal stasis diminishes the flushing of bacteria and toxins contributing to further bacterial overgrowth36,45.

Prevention strategies Management risk factors In reviewing the main bovine clostridial diseases, it is evident that prevention of risk factors is fundamental because bacte-

ria are ubiquitous in the environment and that therapy is often in vain due to the very rapid course of disease. The preventive strategies are based on applying good farm management practices in order to avoid animals coming into contact with the pathogens, and limiting their susceptibility to infection through vaccination. Good management practices and high hygiene levels of tools and structures for handling the animals can avoid the risk of tetanus and malignant oedema since wounds or injuries are pre-requisites for the development of disease. Ergonomic structures and effective management could minimize the traumatic events resulting in the onset of blackleg even if complete prevention can only happen by avoiding the presence of Clostridia from the digestive tract. Good management practices in harvesting, storing and feeding the animals can prevent not only blackleg, but even botulism and diseases associated with enterotoxic clostridia. In Table 2 are reported those dietary features able to destabilize the intestinal environment, altering the pH, the transit speed and the balance among the commensal microorganism populations, and thus potentially promoting the ideal conditions for clostridial germination and replication. To the authors knowledge, no evidence is reported of a direct correlation between other diseases and toxicoses or clostridiosis in cattle. Based on the hypothesis that clostridial enteric colonization is promoted by those factors able to reduce the effectiveness of the digestive process and modulate the intestinal microbiota, it is not possible to exclude that other diseases may promote clostridial germination and proliferation in the gut, for example enteric parasitosis or general diarrhea-based diseases. Furthermore, ruminal acidosis is a condition able to alter intestinal pH and increase tissue permeability in the proximal and distal colon46. Moreover, there is clear evidence that mycotoxins initiating intestinal damage are able to promote C. perfringens proliferation and the development of necrotic enteritis in poultry47.

Vaccination Considering that clostridia-associated diseases are often quickly fatal, other than managing all the risk factors, vaccination is usually the only possible intervention. The available clostridial vaccines are a combination of active compounds against several clostridial strains, often including toxoids of several toxinotypes of C. perfringens, mainly type C and D. In addition, toxoids from several other clostridial species are usually present in the same commercial products, such as: C. chauvoei, C. novyi, C. sordellii, C. septicum and C. tetani27. It is an incorrect, common opinion that toxoid-vaccines are not able to stimulate self-antibody production. Regarding blackleg, the study of Araujo et al., (2010)48 showed that booster shots significantly increased beef calves’ serological response at 30 days post-immunization. The higher serum IgG levels against C. chauvoei were found in those calves vaccinated at four months of age, followed by a booster dose one month later, and then annually repeated48. Furthermore, the interaction between toxins and body defences can explain why calves in veal production systems seems to be more susceptible to necro-haemorrhagic enteritis27,35-36,40,49. Calves receiving exclusively milk replacer do not develop an active immunity towards C. perfringens alpha toxin, when maternal immunity declines, unlike calves raised for beef production, in which a fluid transition from passive maternal to active immunity is observed40,50. Also based on this evidence, the actual scientific opinion gives more impor-

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Table 2 - Dietary and feed management issues affecting digestive effectiveness. Changes in the diet

- Sudden changes in energy and protein dietary content

Lack of fibre

- Forages and concentrates separated - Short cut forages - Diet with low peNDF - Inaccurate TMR charging and/or mixing

Excess fermentation

- Excess starch from barley/wheat/high moisture corn/flaked cereals - Reduced particle size

Protein imbalance

- Imbalance between protein and energy - High soluble protein - Excess of non-protein nitrogen - Not well fermented or stoked silages - High nitrates in water or feed - Inaccurate TMR charging and/or mixing

Feed bunk fermentation -

Unstable silages Water added to the feed Warm season and too humid diet Feed bunk with poor hygiene level

Intake variations

- Lack of feed in the feed bunk for a too long period - Competition - Illness - Water availability and its temperature - Season and burns microclimate

Diet inhomogeneity

- Inaccurate TMR charging and/or mixing - Too short or too long TMR mixing time - Too long cut forages - Absence of binder or appetizer in dry diet

tance to C. perfringens alpha toxin in the pathogenesis of necrohaemorrhagic enteritis51. Alpha toxin toxoid was not always included in commercial vaccines, or more accurately, not enough focus was put on its preservation and titration. Furthermore, the purification method can affect its presence in vaccines since it is well known that the protective antigenicity of alpha toxin is easily destroyed by formaldehyde inactivation during the vaccines production52-56. Clostridial components in vaccines are produced in a reasonably standardized manner by successive passaging of bacteri-

al culture in a medium for growth, in order to obtain the necessary volume of bacteria required for manufacture of the vaccine. At the end of the growth phase the bacteria are inactivated. Bacterial cells and the culture medium are then separated by centrifugation. The resulting supernatant is concentrated and the associated toxins are detoxified to finally constitute the active ingredients of the vaccine. Despite the standard approach to manufacture, the bacterial cells and their toxins are very sensitive to their environment. Small variations (e.g. pH and temperature) within and between manufacturing processes may impact each clostridial component regarding the type of toxins produced and their quantity57-59. Implementing the vaccines productive systems and the titration assay can improve the vaccine quality. For example, applying a consolidated shotgun proteomic approach, it was possible to find and quantify the toxoid of alpha toxin in a commercial vaccine (Miloxan - Boehringer-Ingelheim) that had no such characteristic registered. In the Lab of the University of Sacro Cuore of Piacenza, Italy, the following procedure was applied. Total proteins were extracted in phenol, precipitated and then re-suspended in urea/thiourea and protein amount determined by the Bradford colorimetric approach. The same amount of proteins (50 ug) from each sample, was reduced and alkylated, then digested in trypsin overnight (again, the very classical bottom-up approach). Peptides were finally analysed by nano LC-CHIP QTOF tandem mass spectrometry, using a data-dependent approach and label-free quantitation. Peptides were validated at 1% false discovery rate, and protein inference was then done against the proteome downloaded from UniProt (selecting all those proteins having “Clostridium” in organism taxonomy). Briefly, protein inference is based on the matching between the peptide list from our experiments and the peptide list gained from in-silico digestion of the proteome downloaded from UniProt. Single peptide identification was allowed for unique peptides only. Results are reported in Table 3.

CONCLUSIONS Clostridial diseases of cattle are an economic and welfare issue worldwide. Clostridiosis are non-contagious diseases characterized by low incidence and rapid clinical course that renders therapeutic intervention ineffective. Our knowledge

Table 3 - Quantification of toxoid in a commercial vaccine. Toxin

Vaccine tested* Media Total Intensity

% toxin/total intensity

Q46149

Alpha-toxin

5.39E+05

B1R9V5

Beta-toxin

7.25E+06

27%

Q46342

Cytotoxin L

1.57E+07

59%

E7D8R1

Epsilon-toxin (Fragment)

5.23E+05

P04958

Tetanus toxin

1.92E+06

U3YLU7

Toxin B

8.43E+05

Total intensity (toxins)

2.67E+07

100%

*Miloxan - Boehringer-Ingelheim

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R. Compiani et al. Large Animal Review 2021; 27: 51-56

about different clostridial toxins, their aetiopathological mechanisms, risk factors and pathologies involved are generally limited due to the large number of bacteria strains and types involved. Furthermore, and in particular regarding enterotoxic clostridia, the incidence of necro-haemorrhagic enteritis and enterotoxaemia is probably wrongly estimated because complete post-mortem investigation is rarely performed and several other reasons can lead to sudden death. Controlling predisposing risk factors and vaccinating, especially young cattle, could minimize this issue. However, it must always be taken into account that vaccine failure can still occur because clostridia are ubiquitous in the environment, some strains are extremely low-dose pathogens, and that, even in a production system where vaccination is regularly implemented, never are all possible antigens and toxoids present in any one commercial product.

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