Large Animal Review 1 - 2022 by E.V. Soc. Cons. a r.l.

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Large Animal Review

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

ORIGINAL ARTICLES BOVINE • Unconventional effects of anti-microbial agents in bovine reproduction • Effects of herd size and bedding surfaces on milk yield and some health problems in dairy cow farms • Effects of dietary essential oil and live yeast supplementation on dairy performance, milk quality and fatty acid composition of dairy cows • Gross and histomorphometric differences in the caecum of domesticated cattle and water buffalo

OVINE • Analisi dei polimorfismi del gene della proteina prionica (PRNP) e suscettibilità alla Scrapie delle razze ovine autoctone del nordest d’Italia

CASE REPORTS BOVINE • Coccygeal osteosarcoma - a report in three cows

OVINE • Enzootic posthitis in post-weaning lambs a case series

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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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

01/22

Bimonthly, Year 28, Number 1, February 2022

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

INDEX

• Unconventional effects of anti-microbial agents in bovine reproduction • Effects of herd size and bedding surfaces on milk yield and some health problems in dairy cow farms • Effects of dietary essential oil and live yeast supplementation on dairy performance, milk quality and fatty acid composition of dairy cows • Gross and histomorphometric differences in the caecum of domesticated cattle and water buffalo

OVINE • Analisi dei polimorfismi del gene della proteina prionica (PRNP) e suscettibilità alla Scrapie delle razze ovine autoctone del nordest d’Italia

CASE REPORTS BOVINE • Coccygeal osteosarcoma - a report in three cows

ORIGINAL ARTICLES

OVINE • Enzootic posthitis in post-weaning lambs a case series

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

Anno 28, numero 1, Febbraio 2022 Rivista indicizzata su: CAB ABSTRACTS e GLOBAL HEALTH IMPACT FACTOR (2021): 0.417

BOVINE Unconventional effects of anti-microbial agents in bovine reproduction MARIAGRAZIA PICCINNO, EDOARDO LILLO, ANNALISA RIZZO, RAFFAELE LUIGI SCIORSCI

Editor in Chief: Enrico Fiore Editorial Board 2022-2024: Anna Rita Attili - Francesca Bonelli Marta Brscic - Enrico Fiore Giovanni Franzo - Matteo Gianesella Elisabetta Giudice - Giuseppe Stradaioli Annalisa Scollo - Marco Cullere Giorgio Marchesini - Rudi Cassini Sgoifo Rossi - Antonio Boccardo Cristina Sartori - Anastasia Lisuzzo

Effects of herd size and bedding surfaces on milk yield and some health problems in dairy cow farms KARSLIOGLU KARA NURCAN, GALIC ASKIN

Effects of dietary essential oil and live yeast supplementation on dairy performance, milk quality and fatty acid composition of dairy cows

Managing Editor: Matteo Gianesella

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Technical Editor: Anastasia Lisuzzo 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.

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Gross and histomorphometric differences in the caecum of domesticated cattle and water buffalo SHUKLA VINOD KUMAR, KUMAR ASHWANI, SINGH OPIDNER, SANGWAN VANDANA, PATHAK DEVENDRA

OVINE

Analisi dei polimorfismi del gene della proteina prionica (PRNP) e suscettibilità alla Scrapie delle razze ovine autoctone del nordest d’Italia FULVIO BORDIN, LAURA ZULIAN, ANNA GRANATO, MAURO CALDON, ROSA COLAMONICO, FRANCO MUTINELLI

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CASE REPORTS

BOVINE Coccygeal osteosarcoma - a report in three cows SANGWAN VANDANA, GILL KIRANDEEP, TANDIA NEELAM, KUMAR ASHWANI, GUPTA KULDIP

OVINE

Enzootic posthitis in post-weaning lambs a case series YİĞİT KAÇAR, MEHMET EMİN AKKAŞ, HAVVA KURNAZ, HASAN BATMAZ

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R.L. Sciorsci et al. Large Animal Review 2022; 28: 3-9

Unconventional effects of anti-microbial agents in bovine reproduction

MARIAGRAZIA PICCINNO, EDOARDO LILLO, ANNALISA RIZZO, RAFFAELE LUIGI SCIORSCI* Department of Veterinary Medicine, University of Bari Aldo Moro, S.P. per Casamassima km. 3 70010 Valenzano (BA), Italy

SUMMARY This review discusses the unconventional properties of some anti-microbial agents, with a particular focus on those administered in dairy cattle reproduction. Several antibiotics also possess inherent anti-inflammatory and immunomodulatory properties and could act synergistically with other anti-inflammatory drugs. For example, amoxicillin exerts a powerful anti-inflammatory action, modifying the transcriptional profile of specific cytokines, and it induces a relaxing effect on basal and oxytocin-induced myometrium contractility, in bovine. Macrolides act as anti-inflammatory agents by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and exert a relaxing effect on myometrium. Aminoglycosides are believed to increase the phagocytic activity of leukocytes and inhibit the induction of spontaneous contractions of the myometrium in non-pregnant and pregnant cows. Some fluoroquinolones modulate the leukocytes production and the synthesis of a wide spectrum of cytokines and chemokines with specific anti-inflammatory effects. Enrofloxacin was shown to increase basal uterine contractions in strips collected from bovine uterus in a concentration-dependent manner. Rifaximin exhibits anti-inflammatory and immunomodulatory properties and exerts a contractile effect during the follicular phase of cow. The association of Trimethoprim-sulfamethoxazole shows an anti-inflammatory effect but data regarding contractility are unavailable. The choice of antibiotics that may be used during pregnancy should also take into account the aforementioned unconventional properties and, therefore, favor the active principles that reduce uterine contractility and promote immune quiescence (such as amoxicillin, erythromycin, clarithromycin, gentamicin, rifaximin). Understanding these unconventional properties is mandatory to improve the use of antibiotics in a manner that is advantageous to both human and animal health and to enhance their beneficial effects. Therefore, a thorough understanding of the effects of antibiotics on the immune system and uterine smooth muscle cells may be useful in clinical practice, helping design efficient therapies, reduce the indiscriminate use of antibiotics, and prevent the antibiotic resistance. Moreover, this may help improve animal welfare and reduce the significant economic losses resulting from the impact of reproduction diseases on livestock.

KEY WORDS Antibiotics, anti-inflammatory, cow, immunomodulatory, muscle contractility.

INTRODUCTION The continuous discovery, development, and use of antibiotics in animal and human healthcare has immensely aided the fight against infectious diseases caused by bacteria and contributed to individual and social well-being. However, the persistent emergence of bacterial strains resistant to nearly all known antibiotics is a matter of serious concern for public health. The unwise and widespread use of antibiotics has contributed to the spread of these bacterial strains in the environment, thereby increasing the risk of antibiotic resistance 1. In addition to anti-microbial effects, several antibiotics show other properties, such as anti-inflammatory effects 2-5 and the ability to affect smooth muscle contractility in some organs,

Corresponding Author: Raffaele Luigi Sciorsci (raffaeleluigi.sciorsci@uniba.it).

such as the intestine 6,7 and the uterus 8,9. These unconventional properties are important to understand and standardise the use of antibiotics in a manner that is advantageous to both human and animal health. In this review, we discuss the unconventional properties of some anti-microbial agents, with a particular focus on those administered in dairy cattle reproduction (Table 1; Figure 1).

Classification of antibiotics Antibiotics are anti-microbial substances produced by bacteria and fungi. Apart of these, molecules of synthesis, such as sulphonamides and quinolones, are also included 10. Antibiotics can be classified according to different characteristics, such as on the type (bactericidal or bacteriostatic) 11 and spectrum of action (broad, medium, or narrow) 12, origin (natural, semisynthetic, or synthetic) 13, and resistance (access, watch, or reserve) 14,15. They can also be classified according to their mechanisms of action, including inhibition of cell wall synthesis, breakdown of cell membrane structure or function, prevention

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Unconventional effects of anti-microbial agents in bovine reproduction

Table 1 - Summary of the effects of antibiotics on inflammation. Mechanisms of action

Inhibition of cell wall synthesis

Antibiotic class

Molecules

Effects on inflammation

Penicillins

Amoxicillin

• Edits the transcriptional profile of IL-6, IL-10, TNF-α, TNF-β, and TNF-γ and increase production of IL-4 26,27 • Has pro-oxidant effect 29

Cephalosporins

Ceftiofur

Macrolides

Erythromycin

• Inhibits NFκB synthesis and gene expression of IL-2, IL-6, IL-8, TNF-α 55 • Decreases neuthrophil accumulation 56

Tetracyclines

Doxycyclin Tetracyclines in general

• Inhibits metalloproteinases activity 60,61 • Decrease neutrophil chemotaxis 62 • Scavenge ROS 63

Aminoglycosides

Gentamicin

• Induces production of IL-1b and IL-6 66

Enrofloxacin

• Modulates a wide spectrum of cytokines and chemokines, with specific anti-inflammatory effects 78,79 and induces an increase in ROS production 80 • Induce the release of PGF2α 88

Inhibition protein synthesis

Inhibition of the structure and function of nucleic acids

Blockage of key metabolic pathway

• Downregulates TNF-α, IL-1β, and IL-6 synthesis 35

Quinolones Norfloxacin and Enofloxacin

• Has anti-inflammatory and immunomodulatory properties that exert throught binding to Pregnane X receptor 103

Rifamycins

Rifampicin

Sulfonamides

trimethoprimsulfamethoxazole

• Decrease production of toxic metabolites by neutrophils 55 • Scavenge ROS 5

IL: interleukin; TNF: tumour necrosis factor; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; ROS: reactive oxygen species; PGF2α: prostaglandin F2α.

of bacterial protein synthesis by inhibition of ribosomal subunits (30S or 50S), disruption of the structure and function of nucleic acids, and blocking of key metabolic pathways 14. Here, the classification of antibiotics based on mechanism of action has been reviewed to discuss their unconventional properties.

Cell wall-formation inhibitors This class includes β-lactam antibiotics (penicillins, cephalosporins, cephamycins, β-lactamase inhibitors, monobactams, and carbapenems) 16, cycloserine, vancomycin, and bacitracin, all used in veterinary medicine 17.

post-partum diseases using amoxicillin (both alone and in association with oxytocin) is contraindicated 8: amoxicillin could affect uterine cleaning. While, during early pregnancy, amoxicillin could be useful for hindering embryonic resorption, for its relaxing effect and its mechanism of action 30. Csányi et al. 31 found that pre-treatment with amoxicillin enhanced uterine contractions on the final day of pregnancy in rats. The authors believed that this effect may have been achieved through the decrease in aquaporin 5 levels, a water channel protein, predominant before parturition, and not by a direct action of amoxicillin on uterine smooth muscle cells 31.

Penicillins

Cephalosporins

Amoxicillin is widely used in cattle medicine for the prevention and treatment of respiratory (e.g., pneumonia, shipping fever) 18, gastrointestinal (e.g., bacterial enteritis in calves), 19 and urinary 20 infections, as well as for the treatment of metritis and mastitis 21,22. In addition to the classical anti-microbial effect, amoxicillin also exerts a powerful anti-inflammatory action. It can modify the transcriptional profile of specific cytokines, including Interleukin (IL)-6, IL-10, Tumour Necrosis Factor (TNF)α, TNFβ, and TNFγ 23-26, and it can also influence the production of IL-4 by exerting inhibitory effects on interferon-γ expression 27. Furthermore, in contrast to other β-lactam antibiotics 28, this antibiotic seems to recognise a membrane receptor present on neutrophils capable of activating Nicotinamide Adenine Dinucleotide Phosphate (NADPH)-oxidase. The resulting pro-oxidant effect likely strengthens the bactericidal action of this drug 29. In cattle, amoxicillin was found to exert a relaxing effect on basal 8 (Fig. 1) and oxytocin-induced myometrium contractility during both follicular and luteal phases 30. Therefore, treatment of

Cephalosporins are grouped into 5 generations based on their target organism. While the use of first- and second-generation cephalosporins is approved primarily for the treatment of mastitis, third- and fourth-generation cephalosporins are used to treat respiratory infection, foot rot disease, metritis, and mastitis 32. Ceftiofur, a third-generation cephalosporin indicated for the treatment of bovine puerperal metritis caused by susceptible organisms 33, also exhibits immunomodulatory activities. It impairs pro-inflammatory cytokine secretion through the inhibition of NF-κB and mitogen-activated protein kinase activation. Ceftiofur inhibits p65-NF-κB translocation to the nucleus and down-regulates TNFα, IL-1β, and IL-6 34. Ci et al. 35 demonstrated that pre-treatment with ceftiofur reduced the mortality rate in mice that received a lethal dose of lipopolysaccharides (LPS) by modulating the production of these cytokines. Moreover, ceftiofur significantly decreased inflammation in a murine model of LPS-mediated acute lung injury (ALI) by reducing the levels of TNFα, IL-6, and IL-8 36.

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An in vitro study demonstrated that high doses of cephalosporins (500 µM, cumulative 1000 µM and 2000 µM) influenced uterine contractility by reducing the frequency, whereas they did not affect the amplitude and increased the area under the curve (AUC), that is an essential parameter for describing the effect of drugs as it reflects the exposure of tissue to the drug over time 37. This finding could have implications for its clinical use during pregnancy and in the post-partum period in cattle 37.

Agents that inhibit protein synthesis Drugs that inhibit bacterial protein synthesis can be divided into 50S inhibitors and 30S inhibitors 38,39 -.

Drugs that inhibit the 50S ribosomal subunit Antibacterial agents that inhibit the 50S ribosomal subunit include macrolides, lincosamides, phenicols, and linezolid 40,41. As the use of chloramphenicol is banned in food production

Figure 1 - Summary of the effects induced by antibiotics on in vitro contractility, in the bovine uterus under different hormonal conditions. Representative tracing of the drug has been performed by the authors. For each graph, the dotted line indicates the moment of the drug administration: the left side of the graphs represents the physiological contractility of the uterine strips; the right side shows the uterine strips behaviour after drug administration. Amoxicillin exerts a relaxing effect, whereas enrofloxacin promotes contractility in both phases of the cycle. Steroid hormones do not affect drug activity and for this reason their action has not been evaluated without steroid hormones. Rifaximin exerts a contractile effect only in the follicular phase 8 or in the absence of predominant steroid hormones 9. Tetracycline and streptomycin do not affect the contractility of the bovine uterus (unpublished data).

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Unconventional effects of anti-microbial agents in bovine reproduction

, tiamphemicol and florphenicol are the only two phenicols approved for use in cattle. These molecules are mostly used for the treatment of respiratory infections and infectious keratoconjunctivitis 43,44. Florphenicol also exhibits immunomodulatory activity. Zhang et al. 45 demonstrated that florphenicol inhibited TNF and IL-6 release in mice infected with LPS by suppressing the NF-κB pathway, which ultimately rescued them from LPS-induced death. Zhang et al. 46 also suggested the use of florphenicol as a potential treatment agent for ALI in LPSinfected mice based on a similar immunological effect on proinflammatory cytokines. Macrolides and lincosamides are widely used to treat common infections in cattle, such as respiratory and genital infections and foot lesions 47. Macrolides are indicated in the treatment of metritis caused by susceptible organisms; however, therapeutic regimens often emphasise the evacuation of uterine contents as the primary treatment, which has been confirmed by in vitro studies: Granovsky-Grisaru et al. 48 demonstrated that erythromycin inhibited oxytocin- and carbachol-induced uterine contractions in pregnant rats. A study reported that erythromycin inhibited PGF2α-induced uterus contractions in non-pregnant rats 49 and other study showed that clarithromycin inhibited contractions in the human uterus 50. Therefore, the use of antibiotics that exert a relaxing effect on myometrium could compromise the physiological self-cleaning phenomena and delay restitutio ad integrum in the treated subjects 51-54. Macrolides act as anti-inflammatory agents by inhibiting NF-κB, which further prevents the expression of other inflammatory mediators (IL-2, IL-6, IL-8, and TNF-α) 55. Macrolides also reduce neutrophil accumulation and encourage their apoptosis 56.

Agents that inhibit the 30S ribosomal subunit Some of antibacterial agents that inhibit the 30S ribosomal subunit are tetracycline, streptomycin, and spectinomycin 39,57. In cows, tetracycline can be used to treat respiratory, urinary, enteric, soft tissue, and skin infections 58. Intra-uterine tetracyclines are extensively used in association with systemic penicillin to treat metritis and retained fetal membrane (RFM) 22,59. In cases of RFM, tetracyclines can interfere with the normal placental detachment mechanisms owing to their inhibitory effects on metalloproteinase activity 60,61. Moreover, these antibiotics suppress neutrophil chemotaxis 62, act as scavengers of reactive oxygen species (ROS) 63, and are inhibitors of proinflammatory secretory phospholipase A2 64. Our in vitro study on uterine strips (unpublished data) indicates that tetracycline, at the concentrations of 10-6 M, 10-5 M, and 10-4 M, does not alter the contractility of the bovine uterus in pregnant animals (Figure 1). Csányi et al. 31 showed that doxycycline pre-treatment did not alter the AUC of oxytocin-induced contractions in pregnant rats. Therefore, it can be confidentially excluded that the risk of placental retention described by Kaitu’u et al. 60 and Beagley et al. 61 is related to the action of the antibiotic on uterine contractility.

Agents that react with the 30S ribosomal subunit and induce cell death Aminoglycosides are primarily used to treat septicaemia and infections of the digestive tract, respiratory tract, and urinary tract in adult cattle and calves.

Aminoglycosides are believed to increase the phagocytic activity of leukocytes 65. Additionally, in an in vitro study, Frieling et al. 66 demonstrated the potential of gentamicin to induce the expression of higher levels of IL-1β and IL-6 in whole blood samples upon stimulation with Escherichia coli, possibly due to the “remnants” of bacterial cell wall components broken down by gentamicin. Among aminoglycosides, gentamicin is particularly indicated for the treatment of infection caused by Pseudomonas aeruginosa and Mannheimia haemolytica in calves, as well as for those caused by multiple gram-positive bacteria, Mycoplasma spp., and Staphylococcus spp. 67. Some studies have shown that gentamicin sulphate inhibits the induction of spontaneous contractions of the myometrium in non-pregnant 53 and pregnant cows 54. In the isolated bovine uterus, it was observed to inhibit oxytocin-, PGF2α-, and KCl-induced contractions 53,54,68. Based on this, Yuksel et al. 54 claimed that gentamicin can be used as an antibacterial agent in cases of septic abortion, chorioamnionitis, pyelonephritis, and septic shock to prevent premature birth, miscarriage, and early contractions of the uterus. The relaxing effect was not exerted by streptomycine, as demonstrated in our in vitro study (unpublished data) (Figure 1). These results suggest that this drug may be used during pregnancy as well as in the treatment of post-partum pathologies, but more research is needed.

Nucleic acid synthesis inhibitors Nucleic acid synthesis inhibitors include fluoroquinolones and rifamycins 69,70.

Fluoroquinolones In buiatric practice, fluoroquinolones are mainly used for treating diarrhoea caused by E. coli 71 in calves, and respiratory infections caused by Pasteurella multocida, Pasteurella haemolytica, Haemophilus somnus, and Mycoplasma bovis in adult cattle 72,73. In combination with other antibiotics (such as rifampicin and streptomycin), fluoroquinolones are used to treat infections caused by Brucella spp., as they can reach moderately high intracellular concentrations in macrophages and neutrophils 74. Fluoroquinolones are also able to affect cytokine synthesis depending on the type of cell and cytokines 75-77. Some fluoroquinolones modulate the leukocytes production and the synthesis of a wide spectrum of cytokines and chemokines with specific anti-inflammatory effects 78,79. Three mechanisms may explain the various immunomodulatory effects: (1) action on intracellular cyclic adenosine-3’,5’-monophosphate and phosphodiesterases, (2) action on transcription factors, and (3) action on the eukaryotic equivalent of the bacterial SOS response (a global response to DNA damage), which is the bacterial response to DNA damage 78. On the other hand, enrofloxacin increases ROS production 80, which may be responsible for the typical side effects of the drug, such as cartilage damage and phototoxicity 81,82. The effects exerted by fluoroquinolones on smooth muscles in different organs and animal species have been highlighted in some studies. These drugs act as gamma aminobutyric acid (GABA) receptor antagonists 83 by blocking ATP-dependent potassium channels 84-87. In the intestine, they induce the release of prostaglandin F2α 88 which is responsible for cholinergic transmission in the myenteric plexus 89. In vitro studies on rat myometrium have also

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highlighted the effects of danofloxacin on oxytocin-induced uterine contractility. At lower concentrations (5-20 µmol), there is an increase in the frequency and amplitude of the peaks; however, at higher concentrations (40 and 80 µmol), this drug inhibits contractility as a consequence of hyperpolarisation 87. Enrofloxacin was shown to increase basal uterine contractions in strips collected from bovine uterus in a concentration-dependent manner; therefore, it was hypothesized to optimise uterine self-cleaning during post-partum complications, such as metritis and RFM 8 (Figure 1). Moreover, its association with ecbolic substances, such as oxytocin, could interfere with the formation of bacterial biofilms 90.

Rifamycins In veterinary medicine rifamycins are mostly used to treat foal infections caused by Rhodococcus equi or diseases caused by intracellular bacteria (e.g. paratuberculosis) 91,92. In cattle, rifaximin can be administered by the endo-uterine or intra-mammary route for the prevention and treatment endometritis and mastitis 93,94. The endo-uterine administration of rifaximin, in foam formulation, in acute metritis, induce the expansion of the uterine lumen, which allows the uniform distribution of the active principle on the entire endometrial surface. In vitro studies have shown that rifaximin exerts a contractile effect during the follicular phase under oestrogen control, and it has been speculated that it can be used alone or in association with oxytocin 8,90. Rifaximin can bind to the Pregnane X receptor (PXR), a phenomenon that can be affected by co-activation with steroids 95. Upon binding, there is a potentiation of the contracting/relaxing effect exerted by the steroid hormones predominantly expressed at that point 8 (Figure 1). The administration of rifaximin could represent an optimal therapy for the treatment of retained placenta and acute metritis in cattle 9. Additionally, rifaximin exhibits anti-inflammatory and immunomodulatory properties that involve its binding to PXR 96 . Activated PXR suppress the expression of NF-κB and cAMPresponse element (CREB) binding protein 97 and the release of pro-inflammatory cytokines 98, as indicated by the reduced levels of interferon-γ released by mononuclear cells (isolated from the gastrointestinal tract) after rifaximin treatment 99.

Agents that impair cellular metabolism Sulfonamides and sulphonamide potentiators Sulfonamides are commonly used owing to their efficacy in the treatment of several diseases of cattle and calf, including shipping fever complex, bacterial pneumonia, bacterial enteritis, peritonitis, metritis, mastitis, calf diphtheria, and foot rot disease 100. The association of sulfonamides with diaminopyrimidines, such as trimethoprim (TMP) (another competitive inhibitor that targets the bacterial folate production cascade), is commonly used with synergistic purpose 101. The use of TMP and sulfonamides (TMP-SU) expands the spectrum of bacteria and protozoa affected, thus the combination of drugs is used more often than sulfonamides alone. The intra-uterine administration of sulphonamides for the treatment of bovine endometritis is not recommended owing to the loss of efficacy in the presence of organic debris, pus, and necrotic tissues 102. Unfortunately, data regarding contractility induced

by sulphonamides or TMP-SU are currently unavailable. A nonanti-infective effect of trimethoprim-sulfamethoxazole (TMPSMZ) has been postulated. One study has suggested that the anti-inflammatory effects exerted by TMP-SMZ can be a consequence of the reduction in neutrophil production and interference with oxygen-derived free radicals owing to its action as a scavenger of reactive oxygen 5.

CONCLUSION While antibiotics are commonly classified based on their action, they are never classified based on the alternative effects exerted, such as immunomodulation and uterine contractility. This review outlines the non-antimicrobial effects of antibiotics, and suggest its knowledge is of great value when choosing the most suitable drug in different cases. Drugs that stimulate uterine contractility (cephalosporin, enrofloxacin, and rifaximin) or at least do not inhibit it (tetracyclines and streptomycin) are a preferred choice in the treatment of uterine diseases. Antibiotics that impair physiological uterine cleaning (amoxicillin, erythromycin, clarithromycin, gentamicin) are suboptimal drugs. Substances that do not affect contractility can be used in pregnancy, and the relaxing properties of some agents (e.g., rifaximin) can be advantageously exploited to prevent premature birth, miscarriage, and early induction of uterine contraction together with more specific ones. Moreover, it could be interesting to select antibiotics based on their anti-inflammatory and/or immunomodulatory effect; e.g., ceftiofur and florphenicol may be particularly used to treat disease-associated endotoxemia, such as in the case of metritis, or to prevent septic abortion. Therefore, a thorough understanding of the effects of antibiotics on the immune system and uterine smooth muscle cells may be useful in clinical practice, helping design efficient therapies, reduce the indiscriminate use of antibiotics, and prevent the antibiotic resistance. Moreover, this may help improve animal welfare and reduce the significant economic losses resulting from the impact of reproduction diseases on livestock.

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Unconventional effects of anti-microbial agents in bovine reproduction

9. Sciorsci R.L., Piccinno M., Rizzo A. (2018). Contractile effect of rifaximin on bovine uterus in the presence of steroid hormone antagonists. Theriogenology, 110: 74-78. 10. Davies J. (2006). Are antibiotics naturally antibiotics? J. Ind Microbiol Biotechnol, 33: 496-499. 11. Nemeth J., Oesch G., Kuster S.P. (2015). Bacteriostatic versus bactericidal antibiotics for patients with serious bacterial infections: Systematic review and meta-analysis. J Antimicrob Chemother, 70: 382-395. 12. Acar J. (1997). Broad- and narrow-spectrum antibiotics: An unhelpful categorization Clin Microbiol Infect, 3: 395-396. 13. Peláez F. (2006). The historical delivery of antibiotics from microbial natural products - Can history repeat? Biochem. Pharmacol. 71:, 981-990. 14. Kapoor G., Saigal S., Elongavan A. (2017). Action and resistance mechanisms of antibiotics: A guide for clinicians. J Anaesthesiol Clin Pharmacol, 33: 300-305. 15. Hsia Y., Sharland M., Jackson C., Wong I.C.K., Magrini N., Bielicki J.A. (2019). Consumption of oral antibiotic formulations for young children according to the WHO Access, Watch, Reserve (AWaRe) antibiotic groups: an analysis of sales data from 70 middle-income and highincome countries. Lancet Infect Dis, 19: 67-75. 16. Tooke C.L., Hinchliffe P., Bragginton E.C., Colenso C.K., Hirvonen V.H.A., Takebayashi Y., Spencer J. (2019). β-Lactamases and β-Lactamase Inhibitors in the 21st Century. J Mol Biol, 431: 3472-3500. 17. Sarkar P., Yarlagadda V., Ghosh C., Haldar J. (2017). A review on cell wall synthesis inhibitors with an emphasis on glycopeptide antibiotics. Med Chem Comm 8: 516-533. 18. Apley M. (1997). Antimicrobial therapy of bovine respiratory disease. Vet Clin North Am Food Anim Pract, 13: 549-574. 19. Constable P.D. (2004). Antimicrobial Use in the Treatment of Calf Diarrhea Change in Small Intestinal Bacterial Flora in Calves with Diarrhea. J Vet Intern Med, 18: 8-17. 20. Alt D.P., Zuerner R.L., Bolin C.A. (2001). Evaluation of antibiotics for treatment of cattle infected with Leptospira borgpetersenii serovar hardjo. J Am Vet Med Assoc, 219: 636-639. 21. Roberson J.R., Warnick L.D., Moore G. (2004). Mild to moderate clinical mastitis: Efficacy of intramammary amoxicillin, frequent milk-out, a combined intramammary amoxicillin, and frequent milk-out treatment versus no treatment. J Dairy Sci, 87: 583-592. 22. Armengol R., Fraile L. (2015). Comparison of two treatment strategies for cows with metritis in high-risk lactating dairy cows. Theriogenology, 83: 1344-1351. 23. Melhus Å. (2001). Effects of amoxicillin on the expression of cytokines during experimental acute otitis media caused by non-typeable Haemophilus influenzae. J Antimicrob Chemother, 48: 397-402. 24. Demartini G., Esposti D., Marthyn P., Lapidari A., Fraschini F., Scaglione F. (2004). Effect of multiple doses of clarithromycin and amoxicillin on IL-6, IFNγ and IL-10 plasma levels in patients with community acquired pneumonia. J Chemother, 16: 82-85. 25. Khalil G., El-Sabban M., Al-Ghadban S., Azzi S., Shamra S., Khalifé S., Maroun R. (2008). Cytokine expression profile of sensitized human T lymphocytes following in vitro stimulation with amoxicillin. Eur Cytokine Netw, 19: 131-141. 26. Poll C., Lanieri L., Grubor B., Leonard V., Osborne C. (2012). Efficacy Of Amoxicillin In A Mouse Model Of Influenza And Streptococcus Pneumoniae Co-Infection. Page A3285-A3285 in: American Thoracic Society 2012 International Conference. 27. Wilson D.N. (2009). The A-Z of bacterial translation inhibitors. Crit Rev Biochem Mol Biol, 44: 393-433. 28. Hoeben D., Burvenich C., Heyneman R. (1998). Antibiotics Commonly Used to Treat Mastitis and Respiratory Burst of Bovine Polymorphonuclear Leukocytes. J Dairy Sci 81: 403-410. 29. Behra-Miellet J., Darchy A., Gressier B., Dine T., Luyckx M., Brunet C., Dubreuil L. (2007). Évaluation in Vitro De L’Activité De Deux Bêtalactamines Sur Le Métabolisme Oxydatif De Granulocytes Neutrophiles. Pathol Biol, 55: 390-397. 30. Piccinno M., Rizzo A., Cariello G., Staffieri F., Sciorsci R.L. (2016). Oxytocin plus antibiotics: A synergism of potentiation to enhance bovine uterine contractility. Theriogenology, 86: 1203-1211. 31. Csányi A., Hajagos-Tóth J., Kothencz A., Gaspar R., Ducza E. (2018). Effects of different antibiotics on the uterine contraction and the expression of aquaporin 5 in term pregnant rat. Reprod Toxicol, 81: 64-70. 32. Hornish R., Katarski S. (2002). Cephalosporins in Veterinary Medicine - Ceftiofur Use in Food Animals. Curr Top Med Chem, 2: 717-731. 33. Haimerl P., Arlt S., Borchardt S., Heuwieser W. (2017). Antibiotic treatment of metritis in dairy cows—A meta-analysis. J Dairy Sci, 100: 3783-3795. 34. Fornazari V.A.V., Szejnfeld D., Szejnfeld J., Bonduki C.E., Vayego S.A., Goldman S.M. (2019). Evaluation of Uterine Contractility by Magnetic Resonance Imaging in Women Undergoing Embolization of Uterine Fibroids. Cardiovasc Intervent Radiol, 42: 186-194.

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G. Askin et al. Large Animal Review 2022; 29: 11-14

Effects of herd size and bedding surfaces on milk yield and some health problems in dairy cow farms

KARSLIOGLU KARA NURCAN1, GALIC ASKIN2* 1 2

Department of Animal Science, Faculty of Agriculture, Bursa Uludag University, 16059 Bursa, Turkey Department of Animal Science, Faculty of Agriculture, Akdeniz University, 07059 Antalya, Turkey

SUMMARY Dairy farms around the world have undergone some changes over the years. One of these changes was the size of the herd, although its scale varied from country to country. Various factors such as cost, profitability and ease of solving possible problems are taken into consideration while deciding on the size of the herd in the planning phase of the investment. This study was carried out to determine the effect of herd size and use of different bedding materials on health and milk yield in Turkish dairy herds. A total of 44 dairy farms were used in the study, and all of them were free stall. Farms were grouped into three herd size categories; small (with less than 30 cows), medium (30 to 50 cows) and large (with more than 50 cows). 305-day milk yield data were collected at the end of lactation period from the database of Cattle Breeders’ Association of Bursa/Turkey. While health data (about dystocia, retained placenta, clinical mastitis and repeat breeding) were collected from the herd records, locomotion scoring was done by the researchers. The effects of herd size on repeat breeding, locomotion score, mastitis and milk yield were found significant. Bedding materials were examined in three different types (rubber, sand or without bedding-concrete surface) and their effects on repeat breeding, locomotion score, mastitis and 305-day milk yield were also found significant. The large size herds had higher milk yield (6993.24 ± 72.52 L) and better herd health than small and medium-size herds except for the repeat breeding and also, milk yield (7235.60 ± 110.94 L) and herd health were better in herds that used rubber bedding than concrete and sand except for the repeat breeding and dystocia. Consequently, herd health and milk yield were significantly affected by herd size and the bedding material that was used.

KEY WORDS Dairy herd, herd size, bedding type, milk yield, cow health.

INTRODUCTION The dairy industry in the developing world has undergone some changes over the recent decades like herd size. Herd sizes have increased, and large herds have started to adopt new technologies to improve both efficiency and profits. As a result of this, these herds have tended to increase production and reduce the cost per unit of milk1, but herd size has had and will probably continue to have some effects on the health of dairy cows2. To the best of our knowledge, there are just a few studies that have assessed the effects of herd size on animal health and milk yield3-6, and existing studies have provided conflicting results. While Wolf et al.6 reported that infectious disease incidence increased with increasing herd size, on the other hand, Chapinal et al.3 reported that larger dairy farms had a lower prevalence of lameness. Differently from the aforementioned researchers, Barkema et al.2 stated that herd size did not have a consistent, predictable association with health. Considering different studies about this topic and their different results, further research is needed to determine the effects of herd size on animal health.

Corresponding Author: Galic Askin (galic@akdeniz.edu.tr).

This is why the objective of this study was to investigate the effects of herd size on health and milk yield in dairy herds. Although there are no studies in the literature on this particular topic, dystocia, clinical mastitis, retained placenta, lameness and repeat breeding were evaluated as the health parameters in this study, because they are the most common health problems in the region where the study was conducted. Besides, the effects of bedding materials used for dairy herds on health and milk yield were also investigated as it was seen that the biggest difference among herds was bedding material in the course of the study. This study aims to guide breeders who want to improve their herds, to provide data for practice and to serve as a reference.

MATERIALS AND METHODS Selection of cows A total of 44 Holstein dairy herds located in west central Bursa/Turkey, including 1215 cows that had completed their first lactation period, were visited during the Spring season (March through May) of 2019. The records of 110 cows that had lactation lengths (LG) of 220<LG<550 days and 72 cows that had a 305-day milk yield (MY305) of 2,000<MY305<12,000 L were not included, because these records were not considered within normal managerial limits. 1033 cows that had completed their first lactation were included in the study.

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Effects of herd size and bedding surfaces on milk yield and some health problems in dairy cow farms

Selection of herds 44 different sized free stall herds were selected from among the herds of the members of the Cattle Breeder’s Association of Bursa. The herds were classified in 3 groups as small, medium and large according to the number of animals (NA) in each hear. Small referred to NA<30, medium referred to 30≤NA≤50, and large referred to NA>50.

Collection of disease and milk yield records For each cow, the following data were collected from the herd records: dystocia (yes, no), retained placenta (yes, no), clinical mastitis (yes, no), and repeat breeding (yes, no). Data on

lactation length (day) and 305-day milk yield (L) were collected at the end of the lactation period from the database of the Cattle Breeder’s Association of Bursa. Since there were no locomotion score records in the herds or in the Bursa Cattle Breeders’ Association records, the cows were evaluated for their locomotion score status by the researchers using a 4-point locomotion scoring method modified from the method reported by Sprecher et al.7, the rates of lameness with reference to the locomotion score were investigated in relation to 3 different bedding materials (sand, rubber or without bedding-concrete surface), and the assessments were made by the same trained professional.

Statistical analyses Table 1 - Effects of herd size and bedding material on milk yield. Herd Size

Mean±SE

Min

Max

Small

457

5990.86±47.95a

2621

9066

Medium

348

6484.85±72.98b

3569

11164

Large

228

6993.24±72.52c

4208

11464

715

6174.82±39.84a

Bedding Concrete surface Sand Rubber

abc

159

2926

9596

2621

11464

6437.44±106.81

159

7235.60±110.94

4208

11164

1033

6378.52±38.17

2621

11464

Means in a column with no common superscript differed significantly **(P<0.01).

The herds included in the study were divided into 3 groups according to their sizes (small: <30 cows, medium: 30-50 cows, large: >50 cows). Similarly, it was seen that 3 different bedding materials as rubber, sand and concrete were being used in the farms. SPSS8 was used for the statistical analyses. Chi-squared tests were performed to investigate the association between herd size and bedding materials with some herd health features (locomotion score, mastitis, dystocia, retained placenta and repeat breeding). Analysis of variance (ANOVA) was used to investigate the effects of herd size and bedding material on milk yield after checking the normal distribution of the data. The statistical model that was used in the study is given below: Yijk = µ + HSi + BMj + eijk where: Y = Milk yield; µ = overall mean; HS = Herd size; BM = Bedding material and e = Error

Table 2 - Effects of herd size and bedding material on herd health. Herd Size

Locomotion Score

0 1 2 3

Bedding

Small

Medium

Large

Concrete surface

Sand

Rubber

Total

226 175 45 11

165 131 40 12

161 52 10 5

343 275 75 22

101 46 9 3

108 37 11 3

552 358 95 28

** No Yes Mastitis

379 78

305 43

212 16

598 117

148 11

150 9

896 137

17.07

12.36

7.02

16.36

6.92

5.66

13.26

No Yes

419 38

323 25

213 15

654 61

154 5

147 12

955 78

Prevalence (%)

8.32

7.18

6.58

8.53

3.14

7.55

Prevalence (%)

Dystocia

Retained Placenta

No Yes

423 34

334 14

219 9

672 43

150 9

154 5

976 57

Prevalence (%)

7.44

4.02

3.95

6.01

5.66

3.14

5.52

Repeat Breeding

No Yes Prevalence (%)

174 283

132 216

63 165

258 457

70 89

41 118

369 664

61.93

62.07

72.37

63.92

55.97

74.21

64.28

* **,* denote statistical significance on the levels of P<0.01, P<0.05 respectively; NS, not significant.

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G. Askin et al. Large Animal Review 2022; 29: 11-14

RESULTS According to the study results, the effects of different herd sizes and bedding materials on milk yield were found significant (P<0.01). The results as shown in Table 1.When the results were assessed in terms of health problems, while effects of herd size on locomotion score, mastitis and repeat breeding was found significant (P<0.01, P<0.01 and P<0.05, respectively), its effects on dystocia and retained placenta was not. There were also found similar results in terms of the effects of bedding materials on health problems. While effects of bedding material on locomotion score, mastitis and repeat breeding was found significant (P<0.01), its effects on dystocia and retained placenta was not. The results are shown in Table 2.

DISCUSSION According to the herd size and bedding material effect on milk yield, the current study results were similar to Krpalkova et al.9 and Singh et al.10 for effects of herd size and similar to van Gastelen et al.11 and Astiz et al.12 for effects of bedding material. As seen in Table 1, milk yield increased when the herd size increased. There was a difference of approximately 1,000 liters between the mean milk yield in different herd size (small and large) and bedding (rubber and others) groups. The best explanation of these results may be that larger herds use more intensive management strategies than smaller herds, and a better understanding of how bedding management in these farms influences productivity and milk quality is needed as reported by Rowbotham and Ruegg13. When the effects of herd size and bedding material on health problems assessed, as seen in Table 2, the locomotion score was 0 in approximately half of the cows housed in the small and medium-sized herds (49.45% and 47.41%, respectively), whereas in the large herds, this rate was quite high (70.61%). In other words, the problem of lameness was encountered less frequently in the large herds as in the results reported by Chapinal et al.14. Also it was seen that the number of lame cows was higher in the herds that used no bedding material (concrete surface). Possible reasons for our result may be the time spent standing and its effects on the locomotion score. This is because lying time is shorter, and standing time is longer when dairy cows are forced to use hard surfaces, especially concrete15, and the prevalence of clinical lameness in cows kept in freestall barns using less soft bedding compared to sand or rubber bedding is higher and associated with more time spent standing16. While 78 of the 457 cows in the small herds had mastitis (17.07%), this rate was 12.36% for the medium-sized herds and only 7.02% for the large herds. The biggest possible reason for this result may be that the management practices - regular inspection of milking machine, use of milking parlor, central transport of milk, teat disinfection and dry cow treatment- performed for the herds became more reliable as the herd size increased. This finding was in a similar with the trend of better udder health and milk quality observed in larger herds in previous studies17; 18. Another reason could be the bedding material because sand or rubber bedding was used in most of the large herds (75%) within the scope of this study. According to the results of the comparison of different bedding materials, the herds with concrete bedding had disadvantages in terms of mastitis. In other words, mastitis cases were

encountered in 6-7% of the cows in the herds using sand or rubber as the bedding material, while in the herds that with concrete surface, it was seen that this ratio more than doubled. The effects of different bedding materials on mastitis have been proven in many other studies. Such comparative studies have reported a low mastitis incidence in herds that used sand or rubber bedding material19; 20. The problem of repeat breeding was observed in almost 2/3 of the cows included in this study. In the large herds, 72.37% of the cows had repeat breeding, whereas small ratios of approximately 10% were encountered for the small and medium-sized herds. When the results were assessed in terms of bedding material, the herds that used sand bedding were more advantaged in terms of conception. While the prevalence for repeat breeding was 55.97% in the herds that used sand bedding, this rate was 63.92% and 74.21% for the herds that used no bedding (concrete surface) and used rubber bedding, respectively. This result is noteworthy because a high prevalence for lameness and mastitis was seen in the herds for which concrete bedding was used, but the prevalence of repeat breeding in this group was low in comparison to the group of herds that used rubber bedding. One of the underlying reasons for this result may be differences in milk yield and changes in bedding options depending on the herd size. Accordingly, in this study, while most of the large herds (with higher milk yield and repeat breeding rates) were using rubber bedding, the rates of using this bedding material were low in most of the small and medium-sized herds (with lower milk yield and repeat breeding rates). The second reason may be the numbers of herds that were included in this study according to their size, because the number of small herds was higher than the others in this study. The third reason may be the selection of bedding materials for the cows because cows could prefer a softer area for lying or standing. Indeed there were observed all herds involved in this study have soil walkways. As a matter of fact, previous research has shown that cows tended to spend more time lying on softer surfaces, and adequate lying times led to an increase in cow health21.

CONCLUSIONS In conclusion, this study revealed that increased herd size or using rubber bedding resulted in increased milk production. This result suggested that there was paying more attention to maintenance and feeding had a big role in the health of the large herds. In terms of the health problems identified in the herds in this study, having large herds (except for repeat breeding) and rubber bedding provided an advantage. This result strengthened the interpretation given above. The most common health problem in the region is repeat breeding, which follows a contrary trend to the increase in the herd size and milk yield. Considering these issues together, it was concluded that it is necessary to be more careful about reproductive health and foot health in herd management especially in small and medium-sized herds, since these issues are in the shadow of milk yield in developing countries.

Acknowledgments All authors would like to thank the Cattle Breeder’s Association of Bursa and the breeders whose herds were involved in this long-term study.

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Effects of herd size and bedding surfaces on milk yield and some health problems in dairy cow farms

References 1. Wolf C.A. (2003). The economics of dairy production. Vet Clin North Am Food Anim Pract, 19: 271-293. 2. Barkema H.W., von Keyserlingk M.A.G., Kastelic J.P., Lam T.J.G.M., Luby C., Roy J.P., LeBlanc S.J., Keefe G.P., Kelton D.F. (2015). Changes in the dairy industry affecting dairy cattle health and welfare. J Dairy Sci, 98: 7426-7445. 3. Chapinal N., Barrientos A.K., von Keyserlingk M.A.G., Galo E., Weary D.M. (2013). Herd-level risk factors for lameness in free stall farms in the northeastern United States and California. J Dairy Sci, 96:318-328. 4. Shahid M.Q., Reneau J.K., Chester-Jones H., Chebel R.C., Endres M.I. (2015). Cow-and herd-level risk factors for on-farm mortality in Midwest US dairy herds. J Dairy Sci, 98: 4401-4413. 5. Sawa A., Bogucki M., Niewiadomski P. (2016). Cow longevity in herds of different milk production level and herd size. Ann Wars Univ Life Sci, 55: 261-266. 6. Wolf R., Barkema H.W., De Buck J., Slomp M., Flaig J., Haupstein D., Pickel C., Orsel K. (2014). High herd-level prevalence of Mycobacterium avium subspecies paratuberculosis in Western Canadian dairy farms, based on environmental sampling. J Dairy Sci, 97: 6250-6259. 7. Sprecher D.J., Hostetler D.E., Kaneene J.B. (1997). A lameness scoring system that uses posture and gait to predict dairy cattle reproductive performance. Theriogenology, 47: 1179-1187. 8. SPSS Inc. (2008). SPSS Statistics for Windows. (Version 17.0) [Computer software] SPSS for Windows Version 17.0, Chicago IL: SPSS Inc. 9. Krpalkova L., Cabrera V.E., Kvapilík J., Burdych J. (2016). Dairy farm profit according to the herd size, milk yield, and number of cows per worker. Agr Econ-Czech, 62: 225-234. 10. Singh P., Bhatti J.S., Hundal J.S., Kansal S.K. (2016). Effect of region and herd size on dairy herd performance parameters in Punjab. Indian J Ecol, 43: 373-374.

11. vanGastelen S., Westerlaan B., Houwers D.J., van Eerdenburg F.J.C.M. (2011). A study on cow comfort and risk for lameness and mastitis in relation to different types of bedding materials. J Dairy Sci, 94: 48784888. 12. Astiz S., Sebastian F., Fargas O. (2014). Enhanced udder health and milk yield of dairy cattle on compost bedding systems during the dry period: a comparative study. Livest Sci, 159: 161-164. 13. Rowbotham R.F., Ruegg P.L. (2015). Association of bedding types with management practices and indicators of milk quality on larger Wisconsin dairy farms. J Dairy Sci, 98: 7865-7885. 14. Chapinal N., Liang Y., Weary D.M., Wang Y., Von Keyserlingk M.A.G. (2014). Risk factors for lameness and hock injuries in Holstein herds in China. J Dairy Sci, 97: 4309-4316. 15. Haley D.B., de Passille A.M., Rushen J. (2001). Assessing cow comfort: effects of two floor types and two tie stall design on the behavior of lactating dairy cows. App Anim Behav Sci, 71: 105-117. 16. Cook N.B., Nordlund K.V., Oetzel G.R. (2004). Environmental influences on claw horn lesions associated with laminitis and subacute ruminal acidosis (SARA) in dairy cows. J Dairy Sci. 87: 36-46. 17. Plozza K., Lievaart J.J., Potts G., Barkema H.W. (2011). Subclinical mastitis and associated risk factors on dairy farms in New South Wales. Aust Vet J 89): 41-46. 18. Smith J.W., Ely L.O., Chapa A.M. (2000). Effect of region, herd size, and milk production on reasons cows leave the herd. J Dairy Sci, 83: 29802987. 19. Manninen E., De Passile A.M., Rushen J., Norring M., Saloniemi H. (2002). Preferences of dairy cows kept in unheated buildings for different kind of cubicle flooring. Appl Anim Behav Sci, 75: 281-292. 20. Weary D.M., Taszkun I. (2000). Hock lesions and free-stall design. J Dairy Sci, 83: 697-702. 21. Tucker C.B., Weary D.M. (2001). Cow comfort and stall design. J Adv Dairy Res, 13: 155-168.

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Ö. Köknur et al. Large Animal Review 2021; 28: 15-20

Effects of dietary essential oil and live yeast supplementation on dairy performance, milk quality and fatty acid composition of dairy cows

ÖZLEM KÖKNUR1, SELMA BÜYÜKKıLıÇ BEYZI2, YUSUF KONCA2* 1 2

Saray Tarım ve Hayvancılık A.Ş, 38800 Develi, Kayseri, Turkey University of Erciyes, Faculty of Agriculture, Department of Animal Science, 38039 Kayseri, Turkey

SUMMARY This study aimed to determine the effects of live yeast (LY) and essential oil (EO) on dairy cattle diets on performance and milk composition traits. A total of 120 multiparous (in 2nd and 3rd lactations) Holstein dairy cows were used and 30 animals were allocated to each treatment group. Treatment groups were as follows: 1) control, (C, without any supplementation), 2) essential oil mixture addition (EO, 10 g/day/cattle) 3) live yeast, (LY, Saccharomyces cerevisiae, 10 g/day/cattle, 4×109 CFU/g) 4) EO+LY (10 g/day/cattle +10 g/day/cattle). Experiments were performed for 16 weeks. Body weight, milk yield, and feed conversion ratio were not influenced by the treatments. Milk fat increased with EO supplementation to the diet. Milk protein decreased in the LY+EO group. Somatic cell counts (SCC) decreased significantly with EO supplementation. Milk lactose, casein, and density were not significantly influenced by the treatments. Milk urea concentration increased in the LY group. Milk-free fatty acids significantly increased in the EO group. Milk citric acid increased in the LY group. The control (C) group had greater pentadecanoic acid (C15: 0) content than the other groups. The myristic acid ratio (C14: 0) of the C and EO groups was greater than the myristic acid ratio of the LY and LY+EO groups. Based on present findings, EO mixture supplementation to dairy cattle diets had positive effects on milk fat content and SCC.

KEY WORDS Milk fatty acids, somatic cell count, milk urea, natural feed additives.

INTRODUCTION Optimal ruminal fermentation and microbial digestion is a key part of the feeds to milk process in dairy animals. In this sense, feed additives are frequently used in animal feeding to ensure microbial growth, to control pathogenic microorganisms and to prevent negative effects of microorganisms on rumen fermentation 1,2. Use of some feed additives like antibiotics, hormones and hormone-like substances in ruminant feeding is restricted by laws since such substances have some negative effects on human health through cross-immunity against the pathogenic microorganisms. Therefore, various other additives with positive effects on animal metabolism and performance are widely used. These additives play a great role also in regulation of digestive system and protection of physiological balance and the other characteristics. These substances may also improve feed consumption, feed conversion ratio, meat and milk yields 3. Yeasts and plant essential oils are considered among such substances to be used in ruminant diets. Probiotics supplemented into ruminant diets may improve rumen conditions,

Corresponding Author: Yusuf Konca (yusufkonca@erciyes.edu.tr).

increase milk yield and quality. They do not allow harmful microorganisms to survive through producing substances in gastrointestinal system 4. Many plant extracts have been reported to have antibacterial, antiparasitic and antiviral properties5. Essential oils are known to have antimicrobial effects against a broad spectrum of microorganisms, including bacteria, protozoa and fungi 6,7. Essential oils may also increase digestion and absorption of nutrients through promotion of beneficial microbial population 6, anti-methanogenic affects8, thus improve feed conversion ratios and increase milk yield and quality 9,10. In a study, the leaf powder supplementation to diet were significantly reduced somatic cell count in the milk and were showed immunomodulatory effects with subclinical mastitis in dairy cows 11. Another study the effect of Origanum vulgare was given at 0.9 mL by intramammary infusion: S. aureus and E. coli were not detected in milk12. Also, the sage essential oil as intramammary infusion to ewes resulted in a significant decrease in somatic cell count. The infusion of aqueous extracts by intramammary (Fumariaindica, Nepatacataria and Adiantumcapillus, 750 mg/tube for 5 days) significantly improved sub-clinical mastitis ratio with all extracts in cows14. Those results summarize the essential oils in some plants which could be used as antimicrobials or as adjuvants, especially the

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Effects of dietary essential oil and live yeast supplementation on dairy performance...

control of mastitis. In a review study, essential oils related to animal nutrition, hygiene and protection; it is part of a sustainable, natural option to improve animal health and food derived from animal products and to reduce the use of antimicrobials in livestock has been reported 15. Essential oils and live yeast, as mentioned, with their strong antioxidant and antimicrobial properties, to support and improve animal performance and health; it is also a potential feed additive that can be used as a natural antioxidant in animal nutrition to improve the quality of products. In this context, the aim of this study was to evaluate the effects of in vivo dietary essential oils and live yeast (Saccharomycess cerevisia) supplementation on milk yield, composition and fatty acid profile of lactating dairy cows. In addition, it was aimed to evaluate whether essential oils and live yeast can be used as a natural feed additive that can improve product quality in milk.

MATERIAL AND METHODS Animal and feed material This study was carried out in Saray Agriculture and Livestock Inc. ranches, operating in Kayseri (38° 22 24.8376» and 35° 27 49.8384») province of Turkey. A total of 120 lactating and clinically healthy (parasitic drugs and other requirements for health were applied before the experiments) cows were used to determine the effects of essential oil mixture and live yeast supplementation to diet on their dairy performance and milk quality. All animals used in the study were cared for according to Erciyes University Ethics committee reports. The cows were selected among 1900 Holstein cows and allocated at age, lactation number, milk yield and live weight into 4 treatment groups of 30 cows each group in a paddock. The shelter barns were 120 m long and 28 m wide. They were free-stall barns with group-feeding and had free access to fresh water with automatic waterers. The barns have sufficient ventilation and suitable for animal breeding in all seasons and animal welfare. The treatment groups were as follows: 1: Control (no additives), 2: Essential oil mixture (EO, Bionat SB, Origanum (oregano), Cuminum (cumin), Cinnamomum (cinnamon), Allium (garlic) extract and Lignosulfonic acid as organic acid, similar doses to those reported by 16-18, 3: Live yeast (LY, contains 4x109 cfu/g yeast (Saccharomyces cerevisia NCYC R618) per gram, similar doses to those reported by 10 (Global Nutritech Co. Ltd., VA, USA, Branch of Turkey, KocaeliTurkey) and 4: EO + LY.

in the feeders every 7-day periods before feeding in the morning. The diets were formulated to meet nutrient requirements of dairy cows for lactation NRC 19 based on animal live weights and milk yields. The total mixture ration (TMR) composition is given in Table 1.

Determination of body weight Body weight (BW) of cattle was determined each month. However, the data are shown in only initial and final body weights in tables, because of the cows were not any significant differences in body weight of the treatment groups.

Determination of chemical composition of feed material Dry matter (DM), crude protein (CP), crude ash (CA), crude oil (CO) and crude cellulose (CS) analyses of silages were performed according to the methods specified in 20. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) analyses were performed according to 21 method.

Determination of chemical composition of milk samples The cows were machine milked twice daily with a commercial herd tracking system (Afimilk, version 4.5, Israel) with milkTable 1 - Ingredients and chemical composition of diets. Ingredients

Kg/d per cow

Corn

4.0

Barley

3.24

Soybean seed meal

2.75

Cotton seed meal

2.0

Sunflower seed meal

1.0

Wheat bran

1.0

Maize silage

21.0

Alfalfa hay

4.0

Vetch-wheat hay

0.5

Malt pulp

3.5

Salt

0.05

Marble powder

0.12

Vitamin-mineral premix1

0.10

By-pass oil

0.25

Chemical composition (g/kg DM) Dry matter

524.4

Experimental design

Crude protein

187.5

Following two weeks adaptation period, the experimental period was performed in 16 weeks. The experiment was performed in summer-autumn season, the average temperature was 20.1 ºC and the average humidity was 48.4% in the barn. During the experimental period, the health status and behavior of all cows was monitored on daily basis (data not shown). Alfalfa hay, corn silage and vetch-wheat grass were used as roughage. The feed additives used in the research were homogeneously mixed with milled grains and oilseed meal in 500 kg bunkers and transferred to the feed distribution wagons. The cows were fed at 4 times at day (08:00, 13:00, 17:00 and 21:00) and feed intake measured daily for each group. The amount of feed consumed to cows was monitoring for 7 days, and feed intake was calculated by collecting the remaining feeds

Ether extract

46.4

Organic matter

921.6

Acid detergent fiber

212.1

Neutral detergent fiber

394.2

Crude fiber

164.3

Non-fiber carbohydrates

293.5

Total digestibility

660.5

Metabolizable Energy2 Mcal/Kg 2

Net Energy Lactation Mcal/Kg

2.59 1.64

1 Each kg of premix provides; Vitamin A 15.000.000 IU, Vitamin D3 3.000.000 IU, Vitamin E 30.000 mg, Manganese 50.000 mg, Iron 50.000 mg, Zinc 50.000 mg, Copper 10.000 mg, Iodine 800 mg, Cobalt 150 mg and Selenium 150 mg. 2 Calculated according to NRC (19).

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Ö. Köknur et al. Large Animal Review 2021; 28: 15-20

ing machine. Milk yield, dry matter, fat, protein, and somatic cell counts was recorded at 14 days intervals from consecutive milkings. Milk samples were obtained from each cow from the animals every 14 days during 3 consecutive milking (all milking in a day), and pooled and kept refrigerated until chemical (4 °C) and fatty acid (FA) analysis (-20 °C). The milk density, acidity, lactose, casein, urea, free fatty acid, citric acid, and freezing point of the milk samples were analyzed in milk analyzer (Milko Scan FT 120, Foss, Padova, Italy).

Determination of fatty acid composition in milk The milk fatty acid composition were determined by gas chromatography (Shimadzu GC 2010 Plus) according to Fritsche and Steinhart22. The milk samples of each cattle were taken into numbered tubes and milk fat was separated and the fatty acid profile of those fat samples was analyzed).

Statistical analyses The data analyzed with SPSS statistical software 23. One-Way ANOVA procedure was used to determine whether the differences between the groups were significant. Duncan multiple comparison test was used to determine the differences between significant means. Significance level was considered as P<0.05.

fects on milk dry matter (P >0.05). Milk fat varied between 3.62 - 3.77 and milk fat increased significantly with EO supplementations. The greatest milk protein was observed in EO group, and this value was significantly greater than those of C and EO + LY groups. Present treatments did not have significant effects on milk density, freezing point, lactose, and casein. However, EO and LY treatments increased milk urea and the greatest value was observed in LY-supplemented group (P <0.01). The lowest acidity was observed in the control group, while the greatest value was observed in EO-supplemented group (P <0.05). The greatest free fatty acids were observed in LY group and the lowest value was observed in the control group (P <0.05). The greatest citric acid was observed in LY-supplemented group (P <0.05). EO supplementations into dairy cattle diets significantly reduced somatic cell counts.

Milk fatty acid composition Effects of EO, LY and EO+LY supplementation into cattle diets on milk fatty acid composition are provided in Table 4. Accordingly, milk fat C6:0, C16:0, C16:1, C18:0, C18:1 n9t, C18:2 n6c, C20:2, C22:0, C22:1 n9, C24:0, C22:6 n3 fatty acids were not influenced by the treatments (P>0.05). However, C14:0 fatty acid was found to be the greatest in control and EO-supplemented groups and the lowest in LY and EO+LY- supplemented groups (P <0.05). The control group also had the greatest C15:0 fatty acid content (P <0.05).

RESULTS DISCUSSION Performance Effects of EO, LY and EO+LY supplementations into dairy cattle diets on body weight (BW), feed intake, milk yield and FCR are provided in Table 2. Present treatments did not have significant effects on body weights (P >0.05). Dairy cattle were housed in a single compartment as a group during the experiment; therefore, the total feed intake was obtained as a group average (based on dry matter) for each dietary treatment. Therefore, the statistical analysis could not be done since there was no replicate for feed intake. EO, LY and EO+LY supplementation did not have significant effects on milk yield and FCR (P >0.05).

Milk composition Effects of EO, LY and EO+LY supplementations into dairy cattle diets on milk composition are given in Table 3. It was observed that present additives did not have any significant ef-

Performance Dietary EO, LY and EO+LY supplementations did not have significant effects on BW and feed conversion ratio. Such findings were supported by previous studies 16,24-31 indicating insignificant effects of live yeast additions to dairy cow diets on BW of cattle. However, in another studies, essential oil 32 and yeast additives increased body weight gain 33. On the other hand, some other researchers reported that such additives did not influence BWG of dairy cattle 34,35. Dairy cattle start to increase body condition after a negative energy balance (especially after the 2nd month of lactation) and weight gain may occur from the middle of lactation. In this study, there was not significant live weight losses or gains. At the same time, there were not any contributions of additives to that constant structure of cattle body.

Table 2 - Effects of essential oil, live yeast, and their combinations on dairy cow performance. Treatments C

EO+LY

SEM

Initial body weight, kg

617.97

620.07

620.67

619.03

7.98

0.636

Final body weight, kg

652.60

662.90

671.57

666.60

8.28

0.435

BW change, kg

34.63

42.83

50.9

47.57

1.28

0.454

Feed intake, DM, kg/day

22.73

22.80

23.07

Milk yield, L/day

24.65

25.26

25.16

25.80

0.74

0.752

Feed conversion ratio1

1.73

1.72

1.73

1.70

0.06

0.978

C: control; EO: essential oil addition 10 g/d per cow; LY: live yeast addition 40x109 cfu g/d per cow; EO+LY: essential oil+ live yeast; SEM: Standard error of means; P: probability; 1 The data calculated as: average daily dry matter intake / average daily milk yield.

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Effects of dietary essential oil and live yeast supplementation on dairy performance...

Table 3 - Effects of essential oil, live yeast, and their combinations on milk quality parameters. Treatments C Total solid, g/100 g

EO+LY

SEM

12.11

12.16

12.18

12.10

0.06

0.659

Fat, g/100 g

3.70

3.77

3.62

3.67

0.03

0.002

Protein, g/100 g

3.22b

3.28a

3.24ab

3.20b

0.02

0.013

Lactose, g/100 g

4.76

4.79

4.74

4.72

0.05

0.780

Casein, g/100 g

2.93

2.95

3.00

3.05

0.04

0.277

Urea, g/100 g

0.049

0.054

0.057

0.054

0.002

0.007

Acidity, SH

8.25b

9.67a

9.02ab

8.75b

0.19

0.001

Free fatty acid, Mol/L

7.86

12.08

13.71

11.27

0.87

0.001

Freezing point (-°C)

0.57

0.59

0.60

0.59

0.01

0.094

Citric Acid, g/kg

0.13c

0.15ab

0.16a

0.14bc

0.00

0.003

Density

1032.5

1032.4

1032.6

0.36

0.946

Somatic cell count

381627a

335831b

366631a

372419a

8319

0.001

C: control; EO: essential oil addition 10 g/d per cow; LY: live yeast addition 40x10 cfu g/d per cow; EO+LY: essential oil+ live yeast; SEM: Standard error of means; P: probability; a,b,c: Values with different superscript in a same line differ significantly between treatment groups.

The essential oil, live yeast and essential oil + live yeast supplementation to dairy cattle diets did not have significant effects on milk yields. 16-18,36 reported that addition of essential oil to dairy cattle rations did not affect milk yield. Contrarily, 16 reported that essential oil mixture containing thymol, eugenol vanillin, guaiacol and limonene increased milk yield as compared to the control group. Similarly, 37 reported that the addition of eucalyptus oil, menthol and peppermint oil to drinking water of lactating dairy cattle at a level of 16 mg/l improved milk yield however higher levels (32 and 48 mg/l) decreased milk yield. In addition, the addition of essential oil mix obtained from oregano, cinnamon and orange peels did not affect the milk yield of the cows 18. In a study, the addition of cinnamaldehyde and eugenol mixture with vitamins and minerals did not affect milk yield of dairy cattle36. Simi-

larly, some studies reported that addition of live yeast 38-40 did not affect milk yield. Another study indicated that 10 g/day yeast addition to the diet increased milk yield10. Although different results have been reported by the researchers, the effects of feed additives were more pronounced in animals that were housed and fed under unfavorable conditions 4,41.

Milk composition The essential oil, live yeast, and essential oil + live yeast supplementation to dairy cattle diets did not have any significant effects on milk dry matter. Milk protein, fat and mineral may affect the DM of milk. In this study, although milk fat and protein were affected, milk DM rate was not affected by the treatments. It was reported in previous study that essential oil addition to dairy cattle diets did not affect milk dry

Table 4 - Effects of essential oil, live yeast, and essential oil + live yeast addition to dairy cattle diets on milk fatty acid composition. Treatments C C6:0

3.77

7.50

4.52 b

EO+LY

SEM

4.86

1.22

0.303

C14:0

4.41

3.84

2.67

2.84

0.49

0.011

C15:0

1.39a

0.25b

0.46b

0.15b

0.27

0.028

C16:0

14.36

10.47

11.02

12.07

1.27

0.274

C16:1

6.44

6.94

5.84

5.57

0.43

0.241

C18:0

5.65

4.63

5.81

5.24

0.59

0.678

C18:1

7.31

7.47

10.52

8.99

0.76

0.092

C18:2

28.02

27.51

27.59

26.87

1.51

0.962

C20:2

9.47

11.19

10.94

10.87

0.70

0.375

C22:0

1.77

1.76

1.55

1.44

1.33

0.086

C22:1

2.21

3.52

2.71

2.52

0.59

0.976

C24:0

0.25

0.07

0.00

0.06

0.105

C22:6

0.08

0.59

0.00

0.16

0.303

C: control; EO: essential oil addition 10 g/d per cow; LY: live yeast addition 40x109 cfu g/d per cow; EO+LY: essential oil+ live yeast; SEM: Standard error of means; P: probability; a,b,c: Values with different superscript in a same line differ significantly between treatment groups.

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Ö. Köknur et al. Large Animal Review 2021; 28: 15-20

matter 17. Similarly, live yeast addition to the dairy cattle diet 31,40 did not affect dry matter of milk. However, in another study reported that live yeast addition to the diet increased the dry matter content of milk42. EO supplementations increased milk fat. Similarly, increasing milk fat and protein with essential oil mix including eugenol, geranyl acetate and coriander oil43. In another study determined that mixture of eucalyptus, menthol and peppermint oil with drinking water reduced milk fat yield37. However, some other studies reported that such supplementations did not have any significant effects on milk fat yield 16-18,36,44 . Similarly, addition of live yeast did not have any significant effects on milk fat 38-40,45,46. However, there are some other studies reporting increased milk fat contents with live yeast addition to dairy cattle rations 38,42,47,48. In another study reported that addition of 10 g/day live yeast per dairy cattle reduced milk fat10. Addition of herbal essential oil to dairy cattle diets increased milk protein. Milk protein can be influenced by a few factors and is generally more stable. In contrast, a study indicated that eugenol, geranyl acetate and coriander essential oil mixture increased milk protein content49. Contrarily, addition of live yeast to dairy cattle diets did not have any significant effects on milk protein 38,40,45,46. Previously reported that addition of live yeast to dairy cattle diets increased milk protein10,42. The supplementation of EO to dairy cow diets caused a significant decrease in milk somatic cell count (SCC). Somatic cell count in milk is an indicator of healthy udder and animal structure. It is well known that essential oils have a strong antimicrobial and antioxidant activity. The reduction in SCC obtained in this study can be attributed to the antimicrobial effect of the EO mixture. In another study reported that the addition of an EO mixture reduced the number of somatic cells in milk50. Similarly, it was found that yeast culture addition to dairy cow diets did not have significant effects on somatic cell counts 10,31,51,52. The addition of EO, LY and EO+LY to the diet did not have significant effects lactose, casein, and density of milk. There are not enough studies about the effects of live yeast and EO on chemical composition of milk. It was indicated in a previous study that addition of yeast culture to dairy cattle diet did not affect lactose of milk 35,46,51. However, a study stated that the addition of 10 g/animal of live yeast increased the lactose rate in milk, while the lactose rate in milk did not change when 14 g per day was used10. In another study reported that LY addition did not cause any changes in milk lactose52. Also reported that EO supplementation did not affect lactose of milk17. The addition of EO, LY and EO+LY to the diet influenced urea, acidity, free fatty acid, citric acid and freezing point of milk. It was determined that the amount of free fatty acid and citric acid in milk increased with yeast addition. Also, it was observed that the EO addition increased the acidity and freezing point of milk. However, there is not enough published experimental results on potential effects of such additives on milk quality traits. The greatest milk urea was observed in LY group and urea content of EO+LY mixtures was greater the control group. It was found that the addition of EO reduced the amount of urea in milk 50. It was reported in another study that the amount of urea in milk did not change by LY supplementation 51.

Milk fatty acid composition There was no significant effect of EO and LY and combinations on the fatty acid composition of milk. It was observed that only myristic acid (C14:0) ratio decreased in LY and EO+LY groups, C15:0 fatty acid also decreased in treatment groups. It was reported in a previous study that the essential oil additive did not have significant effects on fatty acid composition of milk 53 . It was reported in another study that flax oil increased n-3 fatty acid in milk, but the addition of yeast did not affect the fatty acid composition of milk 46. Also showed that live yeast additive had no effect on fatty acid composition of milk39.

CONCLUSION It was concluded based on present findings that essential oil mixture increased milk fat, protein, acidity, citric acid, and freezing point and decreased somatic cell counts. Despite some differences in milk urea and free fatty acids, live yeast and EO+LY groups generally had similar with the control group. Based on present findings, EO mixture can be recommended for dairy cattle diets just because of positive impacts on some milk quality parameters, but live yeast was not recommended for ruminant diets due to the addition of an extra feed cost. In addition, the income and outgoings obtained by adding EO mixture to the diet should be compared and it should be decided to use it as a feed additive.

ACKNOWLEDGEMENTS This paper presents the results were funded by Erciyes University Office of Scientific Research Projects (project no: FYL-20166467). Authors would like to thank Saray Farm Inc. for technical assistance in animal care and performance of thesis dissertation.

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Gross and histomorphometric differences in the caecum of domesticated cattle and water buffalo

VINOD KUMAR SHUKLA1, ASHWANI KUMAR1*, OPINDER SINGH2, VANDANA SANGWAN1, DEVENDRA PATHAK2 1

Department of Veterinary Surgery and Radiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141001, Punjab, India 2 Department of Veterinary Anatomy, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141001, Punjab, India

SUMMARY Domesticated cattle and water buffaloes are major dairy animals and considered alike morphologically. Recent studies highlighted differences in the behaviour, vital clinical parameters, the topography of abdominal organs and clinical manifestations of some disease conditions in cattle and buffaloes. This study investigated species differences in the gross and histomorphometry of the caecum and ileocaecal mesentery in domestic cattle and water buffalo. The study was conducted on 8 bovine cadavers (4 crossbred cattle and 4 Murrah water buffaloes that were euthanized due to causes other than gastrointestinal disorders) to evaluate the species-specific gross and histomorphometric differences in the caecum at the apex, and body including ileocaecal mesentery. Histomorphometry was done using H&E, Picrosirius Red and Verhoeff ’s Elastic stain. On gross examination, the mean length of the caecal apex (devoid of ileocaecal mesentery) and length of the ileocaecal mesentery were significantly less whereas the length and diameter of the caecal body were non-significantly less in buffaloes as compared to cattle. On histomorphometry, the thickness of the total caecal wall (at apex and body) along with its histological layers and the sub-epithelial connective tissue layer of the ileocaecal mesentery was also significantly less in buffaloes as compared to that in cattle. The collagen fibres were significantly less, quantitatively and qualitatively, in the caecal body and ileocaecal mesentery of buffaloes as compared to that in cattle. In conclusions, the caecum of domestic water buffalo and cattle show species specific gross and histomorphometric differences, which might have implications concerning the pathophysiology of caecal disorders or their sequel including surgical exploration.

KEY WORDS Bovine, caecum, gross anatomy, histology, large intestine, micrometry.

INTRODUCTION The cattle and water buffaloes are major dairy animals that are considered alike as being belonging to the same subfamily (Bovidae). Besides, African buffalo, bison, yak and antelopes are also part of subfamily ‘Bovidae’. Studies have highlighted differences in the behaviour, vital clinical parameters, the topography of the reticulum and omasum, relative predisposition to long bone fractures, clinical manifestations of traumatic reticuloperitonitis, gross morphometry of caecum and pericarditis in cattle and buffaloes4-8. The caecum is a blind tube which extends backwards and upwards approximately at the level of 4th lumbar vertebra in the right flank region and its blind end commonly protrudes from the supraomental recess. The free end extends into pelvic cavity when fully distended. Caecal dilatation/impaction is commonly encountered in domestic cattle and water buffaloes. The condition of caecal volvulus is reported in cattle due to the free end of cecum being devoid of mesentery in cattle1. Right flank caecotomy is recommended in a standing position to decom-

Corresponding Author: Ashwani Kumar (drashwanikumar@rediffmail.com).

press the dilated caecum in cattle1. The cecum in domestic buffalo is mostly impacted along with the involvement of colon and decompression/evacuation through the right flank is difficult2, in clinical scenario due to the limitation in exteriorizing the cecum. This difficulty in exteriorizing the cecum in domestic buffalo in comparison to cattle is hypothesized to be due to the smaller apex of cecum (devoid of mesentery) and less elasticity of caecal tissue in domestic buffaloes in comparison to cattle. There is a paucity of literature on the gross and histomorphometry of caecum in the domestic water buffaloes and its comparison with the caecum of cattle. Therefore, this study was aimed to investigate the gross anatomy and histomorphometry of the caecum at the apex, body and ileocaecal mesentery in the domestic cattle and water buffaloes.

MATERIALS AND METHODS Animals: The study was carried out on 8 adult bovine cadavers [4 crossbred cattle (Bos taurus and Bos indicus) and 4 Murrah water buffaloes (Bubalus bubalis)] that were euthanized due to reason(s) unrelated to gastrointestinal ailments. The caecum along with the adjoining structures was identified, isolated and subjected to gross and microscopic study.

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Gross and histomorphometric differences in the caecum of domesticated cattle and water buffalo

Figure 1 - Photograph showing morphometric examination of a gross specimens of caecum in cattle (A) and buffalo (B). Apex (a), body (b).

Gross morphometric study: On isolated specimens of the caecum and adjoining structures in cattle and buffaloes (Fig. 1A and 1B), various measurements (cm) such as the length of caecal apex (devoid of the mesentery), length of the ileocaecal mesentery (with the qualitative status of fat content based on gross appearance), length (tip of the apex upto the entrance of ileum) and mid body diameter of the caecum (in cm) were obtained using inch tape (Table 1). Histomorphometric Study: The tissue samples collected from the caecal apex, body and ileocaecal mesentery were fixed in 10% neutral buffered formalin for 2 days. The samples were processed for paraffin block preparation by acetone benzene schedule and sections of 4-5µ thickness were obtained on glass slides using rotary microtome9. The cut sections were placed on the microscopic slides, de-paraffinized in xylene, rehydrated through descending grades of ethyl alcohol to running water. Sections were stained using Haematoxylin and Eosin (H&E) for histomorphometric study10, Verhoeff ’s stain for elastic fibres11 and Picrosirius Red for collagen fibres12. Microphotography and Histometry: Stained microscopic sections were examined and photographed using a light microscope (Nikon 80i) attached with a digital camera. Images were processed and measurements of the whole section from tunica mucosa to tunica serosa depicting total wall thickness and individually of each layer i.e. tunica mucosa, tunica sub-

mucosa, tunica muscularis and tunica serosa were obtained using Fiji (Image J) software13. Staining for collagen fibres: Collagen fibres were stained with Picrosirius red (PSR) according to published research14. Briefly, sections were de-paraffinized and hydrated to water. Weigert’s hematoxylin was applied for 7 minutes, and then washed in flowing tap water for 10 minutes before staining with 0.1% PSR in saturated picric acid solution for one hour. Stained slides were rinsed in acidified water and sections were dehydrated in alcohol, cleared in xylene and mounted with DPX. Quantification of collagen by staining intensity method: Counting the PSR stained area of stained samples was done using Fiji (ImageJ) software13 as described earlier15. A total of 10 representative photomicrographs (at 400 X magnification) were captured in from each tissue (one tissue/animal). “Image-typeRGB stack” was selected and the slider was placed on green colour channel, followed by selecting image, adjusting and thresholding. Then the setting of stack was adjusted by sliders until all the stained areas were selected. Thereafter, select “Analyse-Set Measurements” followed by “Area”, “Area fraction”, “Limit to threshold” and “Display label”. Finally, stained and unstained areas were analysed by selecting “Analyze-Measure” to get the results in tabular form. The results were saved individually for each animal. The data was pooled to each category of the treatment and was analysed statistically.

Table 1 - Various parameters recorded during gross morphometry of caecum. Parameter

Detailed description

Caecal apex (devoid of the mesentery)

From the point of caecal apex to the start point of the ileocaecal mesentery

Caecal body diameter

Width of caecum at the mid of body

Caecal body total length

From the point of caecal apex to the point of ileum entering into the caecum

Width of the ileocaecal mesentery

From point of attachment to the caecum to the point of attachment to the ileum

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Qualitative scoring of Collagen and Elastic Fibers: The qualitative analysis/grading of collagen and elastic fibres was done based on the microscopic presentation of collagen/elastic fibres in the microscopic slides i.e. their amount (nil to abundant) and on their arrangement; (loosely arranged or densely packed) as described in Table 2.

Statistical Analysis: The objective data on various gross and microscopic observations of the caecum and ileocaecal mesentery in cattle and buffaloes were processed for mean ± S.D. using Microsoft Excel and analysed for the level of significant differences concerning species and site (caecal apex and body) using t-test.

Table 2 - Microscopic qualitative grading of collagen and elastic fibres.

RESULTS

Amount of Collagen Fibers Nil

Qualitative Grade -

Very few

A significant number (but loosely packed)

Abundant (but loosely packed)

+++

Densely packed

Gross Morphometric Examination: The gross morphometric observations of the healthy caecum and adjoining structures in cattle and buffaloes are depicted in Table 3 and Fig. 2. The serosal surface of the caecum in cattle and buffaloes was smooth without any sacculations or bands. The mean length of caecal apex in buffaloes was 11.27 ± 1.32 cm, which was significantly (p=0.03) less than that in cattle (22.22 ± 5.63 cm)

Table 3 - Gross morphometric observations of the healthy caecum and adjoining structures in cattle and buffaloes. [statistical difference between cattle and buffaloes at p<0.01 (**) or p<0.05 (*)] S. No.

Parameters

Buffalo (Mean ± SD) (Range)

Cattle (Mean ± SD) (Range)

Age

4.88 ± 0.25 (4.5-5.0)

4.25 ± 1.19 (2.5-5.0)

Length of the caecal apex (cm)

11.27±1.32* (9.52-12.7)

22.22±5.63* (15.24-27.94)

Ileo-caecal mesentery length (cm)

4.57±1.21** (3.81-6.35)

12.7±2.92** (10.16-15.24)

Caecal body diameter (cm)

10.36±0.76 (9.65-11.43)

15.57±3.93 (10.16-19.2)

Total caecal body length (cm)

46.68±4.80 (41.91-50.8)

66.67±14.27 (48.26-76.2)

Presence of fat at the ileo-caecal mesentery Absent

3/4=75%

Fair

1/4=25%

Good

3/4=75%

Abundant

1/4=25%

Figure 2 - Bar diagram showing mean ± SD comparative gross morphometric parameters (cm) of healthy caecum and adjoining structures in buffalo and cattle.

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Gross and histomorphometric differences in the caecum of domesticated cattle and water buffalo

Figure 3 - Photograph showing measurement of length of caecal apex devoid of ileo-caecal mesentery in cattle (A) and buffalo (B).

(Fig. 3A and 3B). The mean length of the ileocaecal mesentery in buffaloes was 4.57 ± 1.21 cm, which was significantly (p=0.007) less than that in cattle (12.7 ± 2.92 cm). The mean length (p=0.075) and diameter (p=0.087) of the caecal body in buffaloes were 46.68 ± 4.80 and 10.36 ± 0.76 cm, respectively, were non-significantly less than that in the cattle (66.67 ± 14.27 and 15.57 ± 3.93 cm). The fat deposition was not observed in 3 out of 4 caecum specimens of buffaloes, whereas it was assessed as good in 3 out of 4 caecal specimens in cattle. The cattle and buffalo caecum were similar in topography extending from ventral end of last rib to right flank region, however there was limited mobility in buffalo caecum due to short ileocaecal mesentery.

Histomorphometric observations on caecal wall in Buffaloes The caecum at the apex and body in buffaloes was comprised of the four tunics namely, Tunica Mucosa, Tunica Submucosa, Tunica Muscularis and Tunica Serosa (Fig. 4A). The total wall thickness at the caecal apex was significantly (p=0.02) high than at the body in buffaloes. The comparative histomorphometric values of various layers of caecum at the apex and body in buffaloes are depicted in Table 4. Tunica Mucosa: The tunica mucosa of the buffalo caecum was composed of lamina epithelialis having simple columnar epithelium, lamina propria composed of loose connective tissue and lined by intestinal glands, and lamina muscularis mucosae composed of smooth muscles both at the apex (Fig. 4A) and

body of caecum. The tunica mucosa at the apex was significantly (p=0.04) thicker (258.51 ± 16.47µm) as compared to the body (203.36 ± 26.25µm). Tunica Submucosa: The tunica submucosa was predominantly composed of collagen fibres (Fig. 4B) and few elastic fibres (Fig. 4C) mainly in the tunica intima of blood vessels, lymphatics, neuronal elements, and connective tissue cells both at apex and body. The adipose tissue was scanty in the submucosa of buffalo both at the apex and body of caecum (Fig. 4D). The mean thickness of tunica submucosa at the apex was significantly (p=1.64E-03) thick as compared to that at the body of buffalo caecum. Tunica musularis: Tunica muscularis was composed of inner circular and outer longitudinal smooth muscle layers however randomly arranged smooth muscle bundles were frequently observed both at the apex and body of the cecum in buffalo (Fig. 4D, 5A). The muscle bundles in tunica muscularis were separated by welldeveloped connective tissue containing abundant collagen fibres (4D, 5B) and forming fascicles. There was non-significant (p=0.88) difference in the average thickness of tunica muscularis at apex and body of caecum in buffaloes. Tunica Serosa: The tunica serosa was a thick connective tissue layer both at the apex and body of the caecum in buffalo comprising of abundant collagen fibres, blood vessels, lymphatics and neuronal elements (Fig. 4A, 5A). Occasional adipose tissue was also observed in tunica serosa. The tunica serosa at the apex was significantly (p=0.04) thinner as compared to that at body of caecum in buffaloes.

Table 4 - Histometry (mean ± SD) of various layers of caecal apex and body in buffaloes (H&E stained sections). [Values with the same superscript differ significantly at p<0.05 (single superscript) and p<0.001 (triple superscript)] Mucosa (µm) (range)

Submucosa (µm) (range)

Muscularis (µm) (range)

Serosa (µm) (range)

Total wall (µm) (range)

Body

203.36 ± 26.25a (170.69-233.92)

112.30±4.43bbb (108.53-117.10)

833.05 ± 78.86 (720.70-895.69)

176.09 ±5.96c (170.10-183.33)

1386.65±73.50d (1298.20-1473.90)

Apex

258.51 ± 16.47a (235.19-273.98)

243.47 ± 20.88bbb (230.35-274.59)

823.45 ± 46.60 (764.69-892.75)

142.57 ±18.97c (123.29-166.37)

1464.36±46.05d (1406.78-1504.83)

Cecum

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V.K. Shukla et al. Large Animal Review 2022; 28: 21-32

Figure 4A - Section of apex of buffalo caecum showing tunica mucosa (TM), tunica submucosa (TS), tunica muscularis (ML) and tunic serosa (SE) (H&E X 4x).

Figure 4B - Section of apex of buffalo caecum showing lamina muscularis mucosae (lmm), tunica submucosa (TM), showing connective tissue fibers and blood vessels (bv) and inner circular (IC) and outer longitudinal (OL) muscle bundles separated by large interfascicular connective tissue (H&E X 10x).

Figure 4C - Section of apex of buffalo caecum showing elastic fibers in blood vessels (arrow) between muscle bundles (Verhoeff Vangieson X 40x).

Figure 4D - Section of apex of buffalo caecum showing abundant collagen fibers in tunica submucosa (TS) and muscle fascicles surrounded by collagen fibers (arrow) (Picrosirius Red X 10x).

Figure 5A - Section of body of buffalo caecum showing randomly arranged muscle bundles in tunica muscularis (TM) and thick tunica serosa (TS) (H&E X 10x).

Figure 5B - Section of body of buffalo caecum collagen fibers in tunica muscularis (TM) (Picrosirius Red X 10x).

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Gross and histomorphometric differences in the caecum of domesticated cattle and water buffalo

Histomorphometry of caecal wall in cattle The caecal wall at the apex and body in the cattle (Fig. 6A, 6B) was similar to that in buffaloes. The total wall thickness at the caecal body was significantly (p=4.47E-03) higher than at the apex in cattle. The comparative histometry of various layers of caecum at the apex and body in cattle are depicted in Table 5. Tunica mucosa: The tunica mucosa at the apex and body of caecum in cattle was similar to that in buffaloes. There was no significant (p=0.53) difference in the mean thickness of tunica mucosa at the body and apex of caecum in cattle.

Tunica submucosa: Tunica submucosa both at the apex and body of the cecum in cattle was similar to that in buffaloes, except for the distribution of adipose tissue. The distribution of adipose tissue varied in the apex and the body of the caecum. In the region of the apex, adipose tissue was present in the form of islands and enclosed by connective tissue fibres (Fig. 6B, 6C) which were mainly collagen fibres, however, some areas at apex were devoid of adipose tissue and mainly collagen fibres were present (Fig. 6D). In the region of the body of caecum a continuous layer of adipose tissue was observed in the submucosa (Fig. 7A, 7B) and it was enclosed by collagen fibres toward the

Table 5 - Micrometry (mean ± SD) of various layers of caecal apex and body in cattle (H&E stained sections). [Values with the same superscript shows significant difference in each column at p<0.001] Mucosa (µm) (range)

Submucosa (µm) (range)

Muscularis (µm) (range)

Serosa (µm) (range)

Total wall (µm) (range)

Body

632.58±55.92 (573.18-681.75)

1077.02±134.90aaa (888.46-1175.18)

2014.73±21.39bbb (1989.48-2039.37)

165.92±16.46 (146.44-185.47)

3890.70±137.56ccc (3719.64-4006.31)

Apex

666.76±52.10 (590.85-709.00)

567.10±51.62aaa (497.00-619.33)

1096.21±118.38bbb (969.35-1130.59)

184.01 ± 19.66 (158.34-199.61)

2434.08±299.18ccc (1994.12-2634.97)

Cecum

Figure 6A - Section of apex of caecum in cattle showing tunica mucosa (TM), tunica submucosa (TS), tunica muscularis (ML) and tunic serosa (SE) (H&E X 4x).

Figure 6B - Section of apex of caecum in cattle showing tunica mucosa comprising of intestinal glands (arrow) in lamina propria, thick lamina muscularis mucosae (MM) and adipose tissue (AT) in submucosa enclosed by connective tissue (H&E X 10x).

Figure 6C - Section of apex of caecum in cattle showing thick collagen bundles (arrow) surrounding adipose (AT) in submucosa. Tunica serosa (TS) and interfascicular connective tissue in tunica muscularis showing collagen fibers (Picrosirius Red X 4x).

Figure 6D - Section of apex caecum in cattle showing predominant collagen bundles in tunica submucosa (TS) and collagen fibers in interfascicular connective tissue (arrow) in tunica muscularis (Picrosirius Red X 10x).

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V.K. Shukla et al. Large Animal Review 2022; 28: 21-32

Figure 7A - Section of body of caecum in cattle showing the inner part of tunica submucosa entirely filled with adipose tissue (AT) and outer part having connective tissue fibers with blood vessels (bv) (Verhoeff Vangieson X 40x).

Figure 7B - Section of body of caecum in cattle showing adipose tissue (AT) with blood vessels (bv) extending into the tunica muscularis (TM) with randomly arranged muscle bundles (H&E X 4x).

Figure 8A - Section of body of caecum in cattle showing collagen bundles (arrow) close to lamina muscularis mucosae. Inner part of submucosa entirely filled by adipose tissue (AT). Tunica serosa (TS). Tunica muscularis (TM) showing randomly arranged muscle bundles (Picrosirius Red X 4x).

Figure 8B - Section of body of caecum in cattle showing abundant adipose tissue (AT) in submucosa and a few collagen fibers (Picrosirius Red X 10x).

Figure 8C - Section of buffalo mesentery showing thin subepithelial connective tissue (arrow) and abundant adipose tissue (H&E X 4x).

Figure 8D - Section of buffalo mesentery showing collagen fibrils (arrow) in subepithelial connective tissue and in between adipose tissue (Picrosirius Red X 4x).

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Gross and histomorphometric differences in the caecum of domesticated cattle and water buffalo

side of lamina muscularis mucosae (Fig. 8A, 8B). The mean thickness of submucosa at the apex of the caecum was significantly less (p=7.28E-03) than at the body in cattle; however, it was the thickest layer. Tunica muscularis: Similar to buffaloes, the tunica muscularis in the cattle was arranged in inner circular and outer longitudinal layers at the apex of the caecum. In the body of the caecum randomly arranged muscle fibres were frequently observed (Fig. 7A, 7B). The average thickness of tunica muscularis at the apex of the caecum in cattle was significantly less (p=4.62E04) as compared to that at the body. Tunica Serosa: The tunica serosa in cattle was similar to that in buffaloes. The tunica serosa in the cattle was composed of a connective tissue layer lined by mesothelium. The average thickness of tunica serosa at the apex and body of the caecum were non-significantly (p=0.38) different from each other.

In the caecal apex, all the layers; mucosa (p=2.29E-04), submucosa (p=3.33E-04), muscularis (p=0.01), serosa (p=0.02) and total wall thickness (p=6.69E-03) were significantly less in buffaloes as compared to that in cattle (Table 7). In comparison to cattle, there was a scanty amount of adipose tissue in buffaloes irrespective of the region of the caecum (apex or body). In cattle, islands of adipose tissue enclosed by thick bundles of collagen fibers were observed in the submucosa of apex region. On the other hand, the distribution of adipose tissue in the submucosa of the caecal body was in the form of a continuous layer which was lined by loosely arranged (compared to caecal apex) collagen fibres towards the side of lamina muscularis (Table 8). Tunica muscularis in both cattle and buffaloes were composed of both inner circular and outer longitudinal muscle layer, however a random arrangement of muscle fibres was also observed in both apex and body of buffaloes and only body in cattle.

Comparative histomorphometry of caecal wall in cattle and buffaloes

Ileo-caecal Mesentery in buffaloes: The mesentery in buffaloes was lined by mesothelium and was supported by thin sub-epithelial connective tissue (Fig. 8C) which had randomly arranged collagen fibrils (Fig. 8D). The mesentery was composed of adipose cells supported by collagen and few elastic fibres, connective tissue cells and blood vessels (Fig. 9A, 9B). The mean thickness of sub-epithelial connective tissue in buffaloes

The thickness of mucosa (p=1.04E-04), sub-mucosa (p=7.33E04), muscularis (p=3.14E-05) and total wall (p=1.42E-06) of the caecal body was significantly less in buffaloes as compared to that in cattle (Table 6). However, the serosa of the caecal body wall was non-significantly (p=0.31) thicker in buffaloes than in cattle.

Table 6 - Comparative histometry (mean ± SD) of various layers of caecal body wall between cattle and buffaloes (H&E stained). [Values with the same superscript differ significantly at p<0.001 level of significance] Mucosa (µm) (range)

Submucosa (µm) (range)

Muscularis (µm) (range)

Serosa (µm) (range)

Total wall (µm) (range)

Cattle

632.58±55.92aaa (573.18-681.75)

1077.02±134.90bbb (888.46-1175.18)

2014.73±21.39ccc (1989.48-2039.37)

165.92±16.46 (146.44-185.47)

3890.70±137.56ddd (3719.64-4006.31)

Buffaloes

203.36±26.25aaa (170.69-233.92)

112.30±4.43bbb (108.53-117.10)

833.05±78.86ccc (720.70-895.69)

176.09±5.96 (170.10-183.33)

1386.65±73.50ddd (1298.20-1473.90)

Species

Table 7 - Histometry (mean ± SD) of various layers of caecal apex wall between cattle and buffaloes (H&E stained). [Values with the same superscript differ significantly at p<0.05 (single superscript), p<0.01 (double superscript) and p<0.001 (triple superscript) level of significance] Mucosa (µm) (range)

Submucosa (µm) (range)

Muscularis (µm) (range)

Serosa (µm) (range)

Total wall (µm) (range)

Cattle

666.76 ± 52.10aaa (590.85-709.00)

567.10 ± 51.62bbb (497.00-619.33)

1096.21±118.38cc (969.35-1130.59)

184.01 ± 19.66d (158.34-199.61)

2434.08±299.18eee (1994.12-2634.97)

Buffaloes

258.51 ±16.47aaa (235.19-273.98)

243.47 ± 20.88bbb (230.35-274.59)

823.45 ± 46.60cc (764.69-892.75)

142.57 ±18.97d (123.29-166.37)

1464.36±46.05eee (1406.78-1504.83)

Species

Table 8 - Qualitative scoring of collagen fibres in cattle and buffaloes. Cattle Different layers

Buffaloes

Apex

Body

Apex

Body

Tunica mucosa

Tunica submucosa

+++*

++*

Tunica muscularis

++*

Tunica serosa

+++*

++*

* indicate densely packed collagen fibres; + indicate the amount of collagen fibres.

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V.K. Shukla et al. Large Animal Review 2022; 28: 21-32

was 147.96±9.03 µm. Randomly distributed connective tissue fibres particularly collagen fibres were abundant without any septa in the mesentery of buffaloes (Fig. 9A).

Ileo-caecal Mesentery in cattle: The ileocaecal mesentery in cattle was similar to that in buffaloes. The sub-epithelial connective tissue was thick and contained abundant collagen fibres (Fig.

10A, 10B). The major constituent of the mesentery was adipose cells, along with the connective tissue cells, collagen fibres and a few elastic fibres and blood vessels (Fig. 10A, 9C). The subepithelial connective tissue also sent connective tissue septa into the mesenteric folds (Fig. 9C). The mean thickness of sub-epithelial connective tissue in cattle was 339.55 ± 10.71 µm. The elastic fibres were abundant in the wall of blood cells (Fig. 9D).

Figure 9A - Section of buffalo mesentery showing thin subepithelial connective tissue with collagen fibers and collagen fibrils in the adipose tissue (Picrosirius Red X 4x).

Figure 9B - Section of mesentery of buffalo showing blood vessels (arrow) and collagen fibers in the adipose tissue (Verhoeff Vangieson X 4x).

Figure 9C - Section of mesentery of cattle showing collagen fibers (Ca) and collagen fibers (arrow) in adipose tissue (Picrosirius Red X 4x).

Figure 9D - Section of mesentery of cattle showing elastic fibers in the blood vessels (Verhoeff Vangieson X 40x).

Figure 10A - Section of mesentery of cattle showing thick subepithelial connective tissue (arrow) and abundant fat (H&E X 10x).

Figure 10B - Section of mesentery of cattle showing abundant collagen fibers (Ca) in subepithelial connective tissue and septa (arrow) extending into adipose tissue (Picrosirius Red X 10x).

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Gross and histomorphometric differences in the caecum of domesticated cattle and water buffalo

Comparison of Ileo-caecal Mesentery in cattle and buffaloes: The sub-epithelial connective layers of the ileocaecal mesentery in buffaloes (147.96 ± 9.03 µm; range 139.78 - 158.98 µm) was significantly (p=2.20E-07) thin as compared to that in cattle (339.55±10.71 µm; range 328.43 - 353.05 µm). The connective tissue septa that were seen in ileo-caecal mesentery of the cattle were found absent in the buffalo.

Histomorphological observation of elastic fibres in cattle and buffaloes Both cattle and buffalo samples showed very less amount elastic tissue fibres in both caecal apex and body. The elastic fibres were present in the tunica intima of blood vessels (Fig. 8C, 8D, 4C).

Histomorphology of collagen fibres in Buffaloes

DISCUSSION

In an overall qualitative comparison of the caecal apex and body of buffaloes, the collagen fibres were densely packed in the caecal body. Tunica mucosa had a scanty to negligible amount of collagen fibres. The orientation of collagen fibres was wavy in appearance. The tunica submucosa, tunica muscularis and tunica serosa of the caecal body had more densely packed collagen fibres than that at the apex in buffalo (Fig. 4D, 5B).

Cattle anatomy is a reference for other bovine species16. The gross morphometry of the caecum has been described in cattle, deer17, giraffe18,19 and camel20. The caecum in cattle is a large mobile blind sac with a caudally directed apex and is the site where digestion of residual carbohydrates by passing fore-stomach takes place21. It is also involved in the absorption of volatile fatty acids and transportation of the chime into the colon. The caecum is located on the right side of the abdomen in the supraomental recess extending from the level of 4th lumbar at ileocolic junction and its blind free end protruding from the supraomental recess. Dorsally, the caecum is attached to the proximal loop of the ascending colon by caeco-colic fold, ventrally with ileum by ileo-caecal fold, cranially to the right side of the mesentery and caudally the apex is free which can be felt per-rectal at the pelvic inlet when distended22. Despite several studies, the aetiopathogenesis of the caecal dilatation and dislocation remained unclear but is considered similar to as that of abomasal displacement23 (Meylan 2008). Accumulation of gas in the blind sac of caecum causes its free end (caecal apex) to rotate in the clockwise or anticlock wise direction through the proximal colon that is relatively fixed structure24 (Smith 1987). Trans-rectal palpation was reported as sufficient to detect distended, displaced, or twisted cecum in about 95% of the sick cattle. Palpation of the caecal apex extending into the pelvic inlet indicates a simple dilatation; whereas, palpation of the caecal body suggest presence of retroflexion in which the caecal apex is directed cranially resulting in partial or complete cessation of defecation25. There is a paucity of literature on the gross and histomorphometry of caecum and adjoining structures in buffaloes. As per the author’s knowledge, this is the first report of its kind on the gross and micromorphometry of caecum in buffaloes. The study compared the gross and histometry of caecum between cattle and buffaloes. Scanty reports on caecal disorders in buffaloes in comparison to cattle and a difficult exteriorisation of caecal apex during right flank surgery prompted authors to plan comparative gross and histomorphometric studies on caecum in these species. The knowledge of gross and microscopic anatomy of various regions of the caecum and adjoining organs is important as these may get involved in many surgical disorders such as caecal dilatation with or without torsion or dislocation1. In cattle suffering from caecal dilatation, a long free end of caecum i.e. apex

Histomorphology of collagen fibres in cattle The collagen was found scanty in tunica mucosa of caecum in the cattle. It was present in the basement membrane of lamina epithelialis. Loosely arranged collagen fibres were seen in lamina propria. In the tunica sub-mucosa, thick bundles of collagen fibres were densely arranged especially towards lamina muscularis mucosae surrounded the island of adipose tissue. At locations where submucosa was devoid of adipose tissue, compactly arranged collagen fibers were present in caecal apex (Fig. 6C, 6D). In the submucosa of caecal body collagen fibers were loosely arranged especially toward lamina mucularis mucosae around a continuous layer of adipose tissue (Fig. 7A, 7B). Collagen fibers were also observed in the adipose tissue layer around adipose cells, both in caecal apex and body (Fig. 6C, 8A, 8B). The orientation of collagen fibers was wavy in appearance (Fig. 6C, 6D). In the tunica muscularis collagen fibres surrounded the muscle fascicles. A thin collagen layer was also observed inside the fascicles possibly representing the collagen in the perimysium (Fig 6C, 8A). In the tunica serosa layer collagen was densely packed (Fig. 6C) at the apex in comparison to body. Comparison of collagen fibres in cattle and buffaloes: The collagen fibres in the body (p=0.012) and mesentery (p=0.001) were significantly less in buffaloes as compared to cattle. However, no significant difference (p=0.34) was recorded in the collagen fibres of the apex of the cecum in the two species. The orientation of collagen fibres was similar in both the species but their arrangement varied (Table 8 and 9). Qualitatively, the amount of collagen fibres was more in cattle than in buffaloes. The amount of collagen fibres in the caecal apex were more and densely packed than at the caecal body in cattle which was contrary to that in buffaloes (Table 8 and 9).

Table 9 - Quantitative analysis of collagen fibres in cattle and buffaloes. [Significant difference between cattle and buffaloes at p<0.05 (single superscript) and p<0.01 (double superscript)] Species

Apex

Body

Mesentery

Cattle

24.33 ± 1.96

18.03 ± 1.63*

14.54 ± 1.93**

Buffaloes

22.22 ± 3.47

14.30 ± 1.28*

3.36 ± 0.35**

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and longer ileocaecal mesentery may a reason for the caecal apex to become dislocated or torsion. A paucity of literature on the similar sequel of caecal dilatation in buffaloes could be due to the smaller caecal apex devoid of the mesentery and short ileocaecal mesentery. The published literature lacks detailed description of caecal apex devoid of mesentery in various herbivores. During decompression surgery, dilated caecum is approached from the right flank and caecal apex in cattle is easily exteriorized by gently pushing the cecum from behind toward the body wall with the palm of the hand23 (Meylan 2008). The accumulated contents in the caecum and colon are drained via a small stab incision made at the apex. There are some reports on partial caecectomy (resection of the caecal apex) for cattle which have had a recurrence of caecal volvulus along with evidence of infarction. However, there is lack of literature concerning such complications in buffaloes suffering from caecal dilatation. So the longer caecal apex devoid of the mesentery in cattle might be facilitating the easy exteriorization of caecum during surgical intervention. Previous studies reveal that in buffaloes, it was difficult to exteriorise dilated caecum from the right flank incision so necessitating suturing of the caecum with skin margins before caecotomy2,3. The length and width of caecum in buffaloes were smaller than that in cattle which suggests species variation. Similarly in comparison to cattle, the buffaloes have been reported with shorter intestinal tract26. Grossly, the outer appearance of the caecum in cattle and buffaloes was smooth and without sacculations or bands and was similar to camel20, giraffe18,19 and deer17. Histologically, the caecal wall in cattle and buffaloes was similar to the previous findings in cattle21 and camel20, but the thickness in total and individual layers of caecal wall was significantly different in the two species. The fat content in the ileocaecal mesentery was more in cattle than buffaloes that corroborated with the previous findings27. In general, the various histological layers and total wall thickness in the caecum at the body and apex in buffaloes was significantly thinner than that of the cattle which might be due to species differences. Previous study also reported that tunica sub-mucosa of cattle to be thicker as compared to sheep and goat21. Another unique species-specific difference observed was that the tunica sub-mucosa of the caecal apex was significantly thicker in buffaloes and thinner in cattle in comparison to caecal body. The lesser collagen fibres of the caecal body and mesentery in buffaloes as compared to cattle indicated the caecum in cattle to be more elastic than that in buffaloes. More elasticity may be the reason for the higher incidence of caecal dilatation induced retroflexion or dislocation in cattle1 than in buffaloes2,3. In the current study, the mean length of the ileocaecal mesentery in buffalo was significantly less than cattle which might be the reason for comparative restricted mobility of the caecum in buffaloes. Another species-specific difference observed was that unlike cattle, ileo-caecal mesentry in buffalo was thin and lack the connective tissue septa. This study attempted to compare gross and histomorphometry of caecum between cattle and buffaloes; however, the smaller sample size was the limitation. Further investigations on a large sample size to compare other ruminant species are warranted. Based on the findings of the current study following conclusions were drawn:

• The caecum of buffalo differs grossly and in histometry from that of cattle. • The caecal apex (devoid of ileocaecal mesentery), body and ileocaecal mesentery were shorter in buffaloes as compared to that in cattle. • Grossly and histologically, the ileocaecal mesentery has markedly low-fat content in buffaloes as compared to that in cattle. Histologically submucosa of cattle caecum contained more adipose tissue in different parts as compared to buffaloes. • Histologically, the caecal wall at the body and apex and its various layers (particularly tunica submucosa and muscularis) are thinner in buffaloes as compared to cattle. • The buffalo caecum has lesser collagen content (qualitatively and quantitatively) as compared to cattle caecum.

Acknowledgements The authors are grateful to the Indian Council of Agricultural Research, New Delhi for providing financial support under the All India Network Program (ICAR-22) and Guru Angad Dev Veterinary and Animal Sciences University for proving the necessary facilities for conducting this study

References 1. Braun U., Beckmann C., Gerspach C., Hassig M., Muggli E., Schweizer G. K., Nuss, K. (2012). Clinical findings and treatment in cattle with caecal dilatation. BMC Vet Res, 8: 75. Doi: 10.1186/1746-6148-8-75. 2. Singh B. (2016). Clinical study on localization and surgical management of gastro-intestinal obstruction in bovine. MVSc thesis submitted to Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana. https://krishikosh.egranth.ac.in/displaybitstream?handle=1/5810064543. 3. Shukla V.K. (2020). Evaluation of ultrasonography as diagnostic and prognostic modality in cows and buffaloes suffering from caecal dilatation. MVSc thesis submitted to Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana. 4. Abdelal A.M., Floeck M., El Maghawry S., Baumgartner W. (2009). Clinical and ultrasonographic differences between cattle and buffaloes with various sequelae of traumatic reticuloperitonitis. Vet Med, 54(9): 399-406. 5. Kumar A., Saini N. S. (2011). Reliability of ultrasonography at the fifth intercostal space in the diagnosis of reticular diaphragmatic hernia. Vet Rec, 169(15): 391. DOI: 10.1136/vr.d4694. 6. Sangwan V., Mohindroo J., Kumar A., Randhawa C.S. (2018). Clinical, radiographic and ultrasonographic differences in the cows and buffaloes suffering from pericarditis. Intr J Livestock Res, 8(5): 255-263. 7. Sangwan V., Yadav G.P., Kumar A. (2020). Aluminium splint incorporated fiberglass cast preserves limb function in bovines with olecranon fracture. Vet Comp Orthop Traumatol, 33(6): 434-442. 8. Yadav G.P., Sangwan V., Kumar, A. (2020). Comparative occurrence pattern of fractures in cattle and buffaloes. Vet World, 12 (7): 11541159. 9. Pathak D., Bansal N., Singh O., Gupta K., Ghuman S.P.S. (2019). Immunolocalization of estrogen receptor (ER ) and progesterone receptor (PR) in uterus of buffalo during follicular and luteal phases of estrous cycle. J Anim Res, 9(1): 185-193. 10. Luna, L.G. (1968), Manual of histologic staining methods of the Armed Forces Institute of Pathology. 11. Sheehan C., Hrapchak B. (1973). Theory and Practice of Histotechnology. C. 8. Mosby Co., Saint Louis, p.86. 12. Junqueira L.C., Bignolas G., Brentani, R.R. (1979). Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J, 11(4): 447-455. 13. Schindelin J., Arganda-Carreras I., Frise E., Kaynig V., Longair M., Pietzsch T., Preibisch S., Rueden C., Saalfeld S., Schmid B., Tinevez J.Y., White D.J., Hartenstein V., Eliceiri K., Tomancak P., Cardona A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9: 676-682. 14. Vogel, B., Siebert, H., Hofmann, U., Frantz, S. (2015). Determination of collagen content within picrosirius red stained paraffin-embedded tissue sections using fluorescence microscopy. MethodsX, 2: 124-134.

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Gross and histomorphometric differences in the caecum of domesticated cattle and water buffalo

15. Choudhary RK, Choudhary S, Verma R., Pathak D. (2017). Mucin 1 Aberrantly Expresses in Goat Mammary Carcinoma. J Stem Cell Res Therap, 2(4): 00072. DOI: 10.15406/jsrt.2017.02.00072. 16. Pasquini C., Spurgeon T., Pasquini, S. (2003). Digestive system - large intestine. In: Anatomy of domestic animals - systemic and regional approach, 10th edition, Sudz Publishing, USA, pp- 280-283. 17. Perez W., Clauss M., Ungerfeld R. (2008). Observations on the macroscopic anatomy of the intestinal tract and its mesenteric folds in the Pampas deer (O. bezoarticus, Linnaeus, 1758). Anat Histol Embryol, 37: 317-321. 18. Perez W., Lima M., Clauss M. (2009). Gross Anatomy of intestine in the Giraffe (G. camelopardalis). Anat Histol Embryol, 38: 432-435. 19. Sauer C., Bertelsen M.F., Lund P., Weisbjerg M.R., Clauss M. (2016). Quantitative macroscopic anatomy of the giraffe (G. camelopardalis) digestive tract. Anat Histol Embryol, 45(5): 338-349. 20. Mohamed A.A., Kadhim K.H., Hussein, D.M. (2018). Morphological and histological study of the cecum and colon in adult local Camelus

dromedaries. Adv Anim Vet Sci, 6(7): 686-691. 21. Kadam S. D., Bhosle N. S., Kapadnis P. J. (2011). Comparative histological study of caecum in cattle, sheep and goat. Indian J Anim Res, 45(1): 67-69. 22. Fubini S. L. (1990). Surgery of the bovine large intestine. Vet Clin N Am Food Anim Prac, 6: 461-471. 23. Meylan, M. (2008). Surgery of the Bovine Large Intestine. Vet Clin Food Anim, 24: 479-496. 24. Smith D.F. (1987). Caecal dilatation and volvulus. Bovine Pract, 22: 165-167. 25. Braun, U., Steiner, A., Bearth, G. (1989). Therapy and clinical progress of cattle with dilatation and torsion of the caecum. Vet Rec, 125(17): 430-433. 26. Sengar, O. P. S., Singh, S. N. (1970). Studies on the digestive system of ruminants. 5. Structure of the intestine of buffalo-Bos bubalis L. Agra Univ J Res, 19(3): 13-31. 27. Smith D.F. (1984). Bovine Intestinal Surgery. Mod Vet Prac, 65(9): 705-710.

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F. Bordin et al. Large Animal Review 2022; 28: 33-40

Analisi dei polimorfismi del gene della proteina prionica (PRNP) e suscettibilità alla Scrapie delle razze ovine autoctone del nordest d’Italia

FULVIO BORDIN1§*, LAURA ZULIAN1§, ANNA GRANATO1, MAURO CALDON1, ROSA COLAMONICO1, FRANCO MUTINELLI1 1

Istituto Zooprofilattico Sperimentale delle Venezie, viale dell’Università 10, 35020, Legnaro (PD), Italy

Bordin Fulvio e Zulian Laura have contributed equally.

SUMMARY Prion protein (PRNP) gene polymorphisms are responsible for susceptibility to Scrapie, a transmissible spongiform encephalopathy affecting sheep and goats. As a result, different genotypes were defined based on the combination of polymorphic codons and their classification into risk groups of susceptibility (R1-R5) was proposed to plan the use of animals for reproductive purposes. Breeding programmes aimed at increasing the frequency of the ARR resistant allele in sheep population and to progressively eradicate PRNP susceptible alleles. In this study the relative frequency of PRNP haplotypes and genotypes were investigated in twelve local sheep breeds, some of them endangered, from three different Italian geographical areas: Alpagota, Brogna, Foza and Lamon from Veneto region, Carsolina and Plezzana from Friuli Venezia Giulia region, and Tiroler Steinschaf, Juraschaf, Tiroler Bergschaf, Schnalser Bergschaf, Schwarz-braunes Bergschaf and Villnoesser Brillenschaf from the Autonomous Province of Bolzano. All six known PRNP gene alleles were observed in the total sheep population analyzed and most of the animals carried genotypes with moderate to very high susceptibility to classical Scrapie. The predominant allele in all breeds was ARQ, with frequencies over 60% in some breeds (Foza, 63%, Alpagota, 63.9%, Tiroler Steinschaf, 68.4%). The ARR and VRQ alleles showed variable frequencies depending on the sheep breed with mean values of 27% and 6% respectively. The worst situation was observed in the Lamon breed with about 37.5% of the analysed animals carrying the VRQ risk allele, followed by Villnoesser Brillenschaf and Schnalser Bergschaf breeds with approximately 18% and 14.2% of the animals assigned to the high risk classes respectively. The different frequencies of the susceptible VRQ allele may depend both on the geographical isolation and the reproductive practices of the breeders. In addition, the presence of the ARK allele was demonstrated in eight breeds analysed, with a particularly significant frequency in the Foza and Tiroler Bergschaf breeds (6.8% and 5.7% respectively) compared to what is reported in literature. This study highlights the importance of PRNP gene genotyping of different sheep breeds in order to evaluate, guide and improve the organization of possible selection strategies that could lead to an improvement in resistance to Scrapie while preserving the genetic and cultural heritage of these autochthonous sheep populations.

KEY WORDS Alleles; autochthonous breed; genotyping; Scrapie; sheep.

INTRODUZIONE La Scrapie è una malattia infettiva degli ovini e dei caprini, appartenente al gruppo delle encefalopatie spongiformi trasmissibili, o malattie da prioni. La suscettibilità alla Scrapie è fortemente influenzata dai genotipi del gene codificante la proteina prionica (PRNP). Studi eseguiti su razze ovine presenti in Europa hanno permesso di rilevare la presenza di numerosi polimorfismi, ma solo quelli che interessano i codoni relativi agli aminoacidi in posizione 136, 154 e 171 hanno un chiaro effetto sulla suscettibilità alla Scrapie. Queste tre triplette nucleotidiche polimorfe codificano per sette alleli: A136R154Q171 (ARQ), ARR, AHQ, ARH, ARK, VRQ, TRQ. L’allele VRQ è, tra gli alleli, quello associato ad una maggiore suscettibilità alla Scra-

Corresponding Author: Fulvio Bordin (FBordin@izsvenezie.it).

pie, seguito da ARQ, mentre l’allele ARR conferisce resistenza alla malattia. L’allele AHQ è per lo più associato alla resistenza con dominanza incompleta, mentre il ruolo dell’allele ARH è ancora poco chiaro: in omozigosi è considerato a basso rischio, mentre in eterozigosi sembra essere “neutrale”, lasciando prevalere l’azione dell’allele a cui si trova associato. Per gli alleli ARK e TRQ non sono disponibili dati per stabilire il loro ruolo nella suscettibilità/resistenza alla Scrapie in quanto il primo è poco frequente mentre il secondo è molto raro1,2,3. L’estensione delle analisi genetiche alle popolazioni ovine in tutta Europa e il miglioramento delle capacità diagnostiche dimostrano che la classificazione di rischio dei genotipi e degli alleli può variare anche in relazione al ceppo di agente infettante, alla presenza di polimorfismi in codoni differenti da quelli considerati per la Scrapie classica e alla razza ovina considerata. In Italia il Piano Nazionale di Selezione Genetica (PNSG) degli ovini per la prevenzione della Scrapie classica è regolato dal Decreto del Ministero della Salute del 25 novembre 20154 e, come descritto nell’Allegato 1 - Parte A del suddetto DM, in-

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Analisi dei polimorfismi del gene della proteina prionica (PRNP) e suscettibilità alla Scrapie delle razze ovine autoctone

tende sollecitare l’utilizzo di riproduttori con genotipo resistente per ridurre la presenza, nelle popolazioni ovine italiane, degli alleli del gene PRNP responsabili della suscettibilità alla Scrapie, per contribuire alla creazione di “greggi a basso rischio” e per tutelare la salute umana e animale. Il piano si basa sulla genotipizzazione dei capi maschi da riproduzione con lo scopo di incrementare la frequenza dei caratteri di resistenza genetica alla Scrapie e quindi concorrere alla progressiva diminuzione della frequenza degli alleli di suscettibilità. Tuttavia, l’eventuale utilizzo di un numero limitato di capi nell’ambito dei programmi di selezione potrebbe avere un impatto negativo sulla variabilità genetica e sulle caratteristiche produttive delle razze ovine italiane, soprattutto per razze rare o a rischio di estinzione. Il PNSG nelle Regioni del Veneto, Friuli Venezia Giulia e nella Provincia Autonoma di Bolzano ha come finalità l’incremento della resistenza alla Scrapie classica degli ovini presenti con particolare attenzione alle razze autoctone e al loro patrimonio genetico. Nella Provincia Autonoma di Bolzano sono presenti sei razze ovine autoctone: Juraschaf, Tiroler Steinschaf, Schnalser Bergschaf, Schwarz-braunes Bergschaf, Villnoesser Brillenschaf e Tiroler Bergschaf. Queste razze sono storicamente legate alle comunità montane dell’Alto Adige e la loro conservazione contribuisce al mantenimento della biodiversità dell’ambiente montano locale e alla valorizzazione del territorio. La razza Juraschaf (pecora del Giura) è di origine svizzera, ma attualmente è allevata, oltre che in Svizzera, anche in Austria, Germania e Italia. Sono animali ben adattati all’ambiente montano, di taglia media, senza corna, di colore nero e marrone. Sono ovini caratterizzati da un’alta percentuale di nascite multiple, un ottimo istinto materno e un’elevata produzione di latte. La razza Tiroler Steinschaf (pecora tirolese delle rocce) è la razza ovina più antica del Tirolo e oggi è una delle razze locali a rischio di estinzione. Sono ovini fertili, robusti, di media taglia, con lana grigia o bianca, molto resistenti e perfetti per la vita in alta montagna. Attualmente la razza viene allevata per la produzione di carne e di lana. Le razze Schwarzbraunes Bergschaf (pecora di montagna nero-bruna), Tiroler Bergschaf (pecora tirolese di montagna) e Schnalser Bergschaf (pecora di montagna della Val Senales) derivano dall’incrocio della Tiroler Steinschaf con la Bergamasca. Sono razze ovine di taglia media molto diffuse nel Tirolo austriaco e italiano. La prima presenta un vello nero o castano ed è sprovvista di corna, mentre le altre due, hanno una lana completamente bianca e, come la prima, sono sprovviste di corna. Queste razze sono molto resistenti, in grado di adattarsi molto bene al clima rigido di alta montagna e sono perciò considerate razze polivalenti, allevate per la produzione di carne e lana e per la gestione della vegetazione dei pascoli alpini. La Villnoesser Brillenschaf (pecora della Val di Funes) è una razza in via di estinzione originaria della Slovenia, ma allevata anche in alcune zone di Austria, Germania e Italia. Si tratta di animali privi di corna, di taglia media, ben adattati ai pendii ripidi e ai pascoli di montagna. Sono principalmente di colore bianco con macchie nere intorno agli occhi che ricordano gli occhiali da vista, da cui il nome “Brillenschaf”, ovvero pecora con occhiali. Per migliorare la loro produttività sono stati fatti, nel tempo, incroci con le razze Bergamasca e Padovana, ottenendo animali di ottima qualità sia per la produzione di carne che per la produzione di lana5. Nella regione del Veneto sono quattro le razze ovine autoctone, diffuse principalmente nelle province di Vicenza, Verona

e Belluno: Foza (o Vicentina), Brogna, Lamon e Alpagota. Sono razze storicamente legate alle comunità montane del Veneto e da sempre utilizzate per la produzione di latticini o carne. La razza ovina Foza (o Vicentina) probabilmente originaria dell’altopiano di Asiago, in particolare nel comune di Foza, è costituita da animali di taglia medio-grande con carattere docile e buon istinto materno. È classificata come razza a duplice attitudine per carne e lana. Il tipo di allevamento è sia stanziale che transumante. L’attuale popolazione allevata è assai ridotta e a rischio estinzione e pertanto è importante provvedere alla salvaguardia dei suoi caratteri di rusticità e del patrimonio genetico. La razza Brogna è considerata autoctona della provincia di Verona ed è diffusa principalmente nell’altopiano della Lessinia e nelle valli adiacenti (Val d’Illasi, Val d’Alpone, Valle del Chiampo e Valpolicella). Sono ovini di taglia media, allevati in piccoli greggi stanziali. Tradizionalmente, era considerata una razza a triplice attitudine, ma ora la produzione principale è rappresentata dalla carne di agnello e secondariamente dal latte. La pecora di Lamon è autoctona dell’omonimo comune in provincia di Belluno e di altri comuni della Valbelluna. Utilizzata, in passato, nei greggi di pastorizia transumante, grazie alla sua elevata resistenza e adattabilità nei diversi ambienti montani, oggi, anche se considerata una razza a triplice attitudine, è allevata solo per la produzione di carne. La razza Lamon è in pericolo di estinzione a causa del progressivo abbandono dell’attività transumante come forma di allevamento, che ha portato ad una drastica riduzione del numero di capi e necessita, quindi, di interventi nella gestione della riproduzione e conservazione dei tratti morfologici della razza. La razza Alpagota, infine, è una popolazione ovina autoctona dell’altopiano Alpago-Cansiglio, nella parte sud-est della provincia di Belluno, allevata anche nei comuni limitrofi delle province di Belluno e Treviso. Classificata tra le pecore alpine, da cui si distingue per le sue caratteristiche di rusticità e frugalità, è di taglia medio-piccola e attualmente viene allevata principalmente per la produzione di carne. L’allevamento di questa razza è di tipo semi-stanziale con stabulazione invernale, pascolamento nei terreni vicini alle aziende in primavera e autunno e trasferimento in alpeggio, anche in pascoli ad alta quota, nel periodo estivo6. Nella regione Friuli-Venezia-Giulia sono due le principali razze ovine autoctone: Carsolina e Plezzana. La razza Carsolina (o Istriana) è originaria dell’area carsica nord adriatica ed è allevata nelle provincie di Gorizia, Pordenone, Udine e Trieste; è una razza di taglia media, l’allevamento è per lo più stanziale ed è considerata a duplice attitudine, ossia carne e latte. La valorizzazione delle produzioni locali lattiero casearie è favorita da Piani di Sviluppo Rurale che mirano a contribuire alla tutela e al recupero delle razze autoctone a rischio del territorio contrastando l’abbandono delle attività agricole montane. La razza Plezzana, internazionalmente conosciuta come Bovec o Bovska, è originaria dell’alta valle dell’Isonzo e deve il suo nome alla cittadina di Plezzo, situata in territorio sloveno. È una pecora di dimensioni ridotte, di colore chiaro o marrone, particolarmente adatta ai pascoli alpini grazie alla sua rusticità e frugalità. La Plezzana è allevata prevalentemente in piccoli nuclei stanziali, con alpeggio nella stagione estiva; è considerata una razza a duplice attitudine (latte e carne), ma viene prevalentemente utilizzata per la produzione di latte per la successiva trasformazione casearia in prodotti di alto valore aggiunto, contribuendo così a determinare una riqualificazione delle microeconomie locali6.

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F. Bordin et al. Large Animal Review 2022; 28: 33-40

Scopo di questo studio è stato quello di caratterizzare dal punto di vista genetico la suscettibilità alla Scrapie classica di alcune razze ovine autoctone della Provincia Autonoma di Bolzano e delle regioni del Veneto e Friuli Venezia Giulia, alcune delle quali considerate a rischio di estinzione, ponendo le basi per la valutazione di possibili strategie di selezione genetica che possano portare ad un miglioramento della resistenza alla Scrapie conservando, al contempo, il patrimonio genetico delle diverse razze.

MATERIALI E METODI Nell’ambito del PNSG degli ovini, tra ottobre 2016 e febbraio 2021 sono state caratterizzate, dal punto di vista genetico, dodici razze ovine locali presenti in tre diverse aree geografiche: Provincia Autonoma di Bolzano, Veneto e Friuli Venezia Giulia, alcune delle quali a rischio di estinzione.

Animali Sono stati sottoposti ad analisi 6.164 capi di cui 4.208 arieti e 1.956 pecore di sei razze ovine della Provincia Autonoma di Bolzano; delle 1.956 femmine, 1.099 non rientravano nel PNSG. La popolazione ovina di ciascuna razza autoctona era così composta: - Juraschaf: 571 capi di cui 554 maschi e 17 femmine; - Tiroler Steinschaf: 258 capi di cui 224 maschi e 34 femmine; - Schnalser Bergschaf: 957 capi di cui 455 maschi e 502 femmine; - Schwarz-braunes Bergschaf: 706 capi di cui 573 maschi e 133 femmine; - Villnoesser Brillenschaf: 1.860 capi di cui 740 maschi e 1.120 femmine; - Tiroler Bergschaf: 1.812 capi di cui 1.662 maschi e 150 femmine. Sono stati sottoposti ad analisi 2.239 capi, di cui 640 arieti e 1.599 pecore, di quattro razze ovine autoctone venete (Foza, Brogna, Alpagota e Lamon) e 358 capi, di cui 170 maschi e 188 femmine, di due razze ovine autoctone friulane (Carsolina e Plezzana). Per queste sei razze l’analisi prevista dal PNSG ha riguardato 1.965 capi (605 maschi e 1.360 femmine), mentre i rimanenti 274 capi (35 maschi e 239 femmine) non rientravano nel PNSG. La popolazione ovina delle quattro razze autoctone della regione Veneto era così composta: - Foza: 96 capi di cui 51 maschi e 45 femmine; - Brogna: 1.322 capi di cui 323 maschi e 999 femmine; - Lamon: 283 capi di cui 143 maschi e 140 femmine; - Alpagota: 538 capi di cui 123 maschi e 415 femmine. Gli animali delle due razze ovine autoctone della regione Friuli Venezia Giulia erano così suddivisi: - Carsolina: 343 capi di cui 155 maschi e 188 femmine; - Plezzana: 15 capi maschi.

Estrazione del DNA Il sangue intero è stato raccolto in tubi contenenti K3-EDTA come anticoagulante e conservato a -20°C fino all’analisi. In funzione del numero di campioni da analizzare, l’estrazione del DNA è stata effettuata utilizzando o l’High Pure PCR Template Preparation Kit (Roche), seguendo le istruzioni fornite dal produttore, o il MagMAX CORE Nucleic Acid Purification Kit

(Thermo Fisher Scientific) per l’estrazione automatizzata dell’acido nucleico su strumento King Fisher™ 96 Flex Purification System (Thermo Fisher Scientific). I campioni di DNA sono stati conservati a -20°C fino all’uso.

Analisi dei polimorfismi ai condoni 136, 154 e 171 del gene PRNP ovino I polimorfismi del gene PRNP ai codoni 136, 154 e 171 sono stati identificati mediante real-time PCR su termociclatore LightCycler 2.0 (Roche), utilizzando il LightMix® PRNP Scrapie Susceptibility Mutation kit (TIB Molbiol) in combinazione con il kit LightCycler FastStart DNA Master Hybridization Probes (Roche), secondo le istruzioni e il profilo termico indicato dal produttore. Le diverse varianti alleliche dei polimorfismi sono state identificate mediante analisi delle temperature di melting. Qualora il risultato dell’analisi non fosse interpretabile o ci fossero dubbi sulla sua interpretazione si è proceduto all’amplificazione del DNA e al sequenziamento del prodotto di amplificazione. Per la reazione di amplificazione sono stati utilizzati i primer PRP-3S 5’-ATG AAG CAT GTG GCA GGA G-3’ e PRP-2A 5’CAG TTT CGG TGA AGT TCT CC-3’ in grado amplificare la regione del gene in cui sono presenti i tre codoni di interesse (136, 154 e 171). Il prodotto di amplificazione (280 bp), dopo corsa elettroforetica su gel di acrilamide al 7%, è stato visualizzato mediante colorazione con nitrato d’argento. Per il sequenziamento l’amplicone è stato purificato utilizzando il kit ExoSAP-IT Express PCR Product Cleanup (Applied Biosystems) e l’allestimento della reazione di sequenza è stata effettuata utilizzando il kit BrilliantDye™ Terminator Cycle Sequencing kit (Nimagen). Il prodotto della reazione di sequenza, purificato con il kit CENTRI-SEP 96 Well Plates (Princeton Separations), è stato caricato su sequenziatore ABI PRISM® 3130xl Genetic Analyzer (Applied Biosystems). La sequenza delle basi nucleotidiche presenti nei tre codoni di interesse (136, 154 e 171) e quindi il relativo amminoacido codificato è stata determinata mediante analisi dell’elettroferogramma ottenuto con il software SeqScape v3.0 (LifeTechnologies).

RISULTATI Razze ovine della Provincia Autonoma di Bolzano Dal 2016 ad oggi sono stati genotipizzati 6.164 ovini (4.208 arieti e 1.956 pecore) appartenenti a sei razze autoctone della Provincia Autonoma di Bolzano. Gli alleli e i genotipi rilevati per ciascuna razza e le relative frequenze sono riportati rispettivamente nelle tabelle 1, 2 e 3; inoltre, in base al genotipo, i capi sono stati suddivisi nelle cinque classi di rischio (R1→R5) di suscettibilità alla Scrapie classica7. Nella popolazione ovina totale l’allele maggiormente presente è ARQ, che varia dal 42,5% della razza Juraschaf al 68,4% della razza Tiroler Steinschaf. La predominanza dell’ARQ riguarda sia i maschi che le femmine di cinque razze su sei, eccetto la Juraschaf in cui l’allele predominante nelle femmine è ARR (50%), probabilmente dovuto al numero molto ridotto di pecore esaminate (n=17, Tabella 1). L’allele di resistenza ARR nella popolazione totale ha, invece, una frequenza che varia dal 18,4% della razza Tiroler Steinschaf al 31,4% della razza Juraschaf, mentre l’allele VRQ, che conferisce elevata suscettibilità alla Scrapie, varia

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Analisi dei polimorfismi del gene della proteina prionica (PRNP) e suscettibilità alla Scrapie delle razze ovine autoctone

Tabella 1 - Alleli del gene PRNP e relative frequenze espresse in percentuale rilevate nelle sei razze della Provincia Autonoma di Bolzano, nelle quattro razze della regione del Veneto e nelle due razze della regione Friuli Venezia Giulia. TIROLER STEINSCHAF

JURASCHAF

SCHNALSER BERGSCHAF

SCHWARZBRAUNES BERGSCHAF

VILLNOESSER BRILLENSCHAF

TIROLER BERGSCHAF

ALLELE

TOT (%)

M♂ (%)

F♀ (%)

TOT (%)

M♂ (%)

F♀ (%)

TOT (%)

M♂ (%)

F♀ (%)

TOT (%)

M♂ (%)

F♀ (%)

TOT (%)

M♂ (%)

F♀ (%)

TOT (%)

M♂ (%)

F♀ (%)

ARR

31,4

30,9

18,4

17,4

23,4

25,1

21,8

18,8

17,4

24,8

28,6

28,7

28,5

27,2

26,9

30,7

ARQ

42,5

42,6

38,2

68,4

71,4

48,5

54,9

53,6

53,9

55,1

48,9

50,5

49,6

51,1

55,2

55,4

AHQ

23,8

24,2

11,8

11,4

10,7

16,2

0,3

0,4

0,1

17,2

17,3

5,4

5,5

5,3

6,3

6,4

5,3

3,2

3,6

1,5

0,2

0,5

5,7

5,8

5,3

13,8

12,9

14,7

2,3

2,4

1,9

5,6

6,1

5,3

1,8

1,9

1,7

7,7

7,4

4,7

4,5

5,6

9,7

9,6

9,8

3,8

3,6

ARK

0,6

ARH

0,3

VRQ

1,4

0,2

1,6

ALPAGOTA

1,5

0,4

8,8

BROGNA

FOZA

LAMON

CARSOLINA

PLEZZANA

ALLELE

TOT (%)

M♂ (%)

F♀ (%)

TOT (%)

M♂ (%)

F♀ (%)

TOT (%)

M♂ (%)

F♀ (%)

TOT (%)

M♂ (%)

F♀ (%)

TOT (%)

M♂ (%)

F♀ (%)

TOT (%)

M♂ (%)

ARR

25,7

27,1

20,7

29,1

24,5

27,8

29,3

31,8

26,8

28,3

27,4

16,7

ARQ

63,9

67,1

59,4

59,8

66,7

46,3

44,4

48,2

47,2

50,6

44,4

AHQ

5,6

1,6

6,7

2,6

1,4

3,6

5,9

1,1

1,2

1,4

1,1

17,8

16,8

18,6

16,7

ARK

0,5

0,6

2,4

0,8

2,9

6,8

8,8

4,4

ARH

0,7

1,2

0,5

2,6

5,9

1,5

3,5

1,7

5,4

5,7

4,5

6,6

13,3

VRQ

3,6

4,1

3,5

6,3

13,2

4,1

19,6

20,6

18,6

0,6

1,3

3,3

0,5

dall’1,4% della razza Juraschaf al 9,7% della razza Villnoesser Brillenschaf. Da notare che nella razza Juraschaf tale allele è stato rilevato solo negli arieti e, come per l’allele ARR, anche in questo caso probabilmente è da attribuirsi al ridotto numero di pecore analizzate. Oltre agli altri alleli più comuni, l’allele ARK è presente in tutte le razze, ad eccezione della Schnalser Bergschaf, e la sua frequenza varia tra lo 0,2% delle razze Tiroler Steinschaf e Villnoesser Brillenschaf e il 5,7% della Tiroler Bergschaf (Tabella 1). Nella razza Tiroler Steinschaf non è stata rilevata la presenza dell’allele ARH. Nella razza Juraschaf, la popolazione totale (maschi e femmine) è composta per il 53,2% da capi appartenenti alle classi di rischio R1 e R2, scarsamente suscettibili alla Scrapie, mentre per il restante 46,8% da capi con un rischio medio alto di sviluppare la malattia. I genotipi predominanti nella razza Juraschaf sono ARR/ARQ (29,1%), ARQ/ARH (19,8%) e ARQ/ARQ (17%), mentre i capi resistenti omozigoti ARR/ARR costituiscono l’8,9% del totale (Tabella 2). Nella razza Tiroler Steinschaf i capi analizzati appartengono per il 67,1% alle classi di rischio R3→R5 e per il 32,9% alle classi R1 e R2. I genotipi predominanti nella razza sono ARQ/ARQ (47,3%), ARR/ARQ (24%) e ARQ/AHQ (15,1%), mentre i capi resistenti omozigoti ARR/ARR costituiscono il 3,9% del totale (Tabella 2). Sia nella razza Juraschaf che Tiroler Steinschaf non sono stati riscontrati capi omozigoti VRQ/VRQ. La razza Schnalser Bergschaf è composta da capi suscettibili alla Scrapie per il 61,7% (classi di rischio R3→R5) e per il 38,3% da capi scarsamente suscettibili o resistenti (classi di rischio R1 e R2). I genotipi predominanti nella razza sono ARQ/ARQ (29,7%), ARR/ARQ (26%) e ARQ/ARH (15,9%). I capi resistenti omozigoti ARR/ARR costituiscono il 5,3% del totale, mentre i capi omozigoti VRQ/VRQ rappresentano l’1,1% (Tabella 2).

F♀ (%)

Nella razza Schwarz-braunes Bergschaf i capi analizzati si suddividono nelle classi di rischio R3→R5 per il 69,6% della popolazione totale e nelle classi R1 e R2 per il restante 30,4%. I genotipi predominanti in questa razza sono ARQ/ARQ (30,6%), ARR/ARQ (18,4%) e ARQ/AHQ (18%). I capi omozigoti ARR/ARR resistenti costituiscono il 5,4% del totale, mentre non sono presenti ovini omozigoti VRQ/VRQ (Tabella 2). Le due razze autoctone con il maggior numero di capi analizzati nella Provincia Autonoma di Bolzano sono la Villnoesser Brillenschaf (1.860 ovini) e la Tiroler Bergschaf (1.812 ovini). Nella popolazione ovina Villnoesser Brillenschaf gli animali analizzati appartengono per il 57,7% alle classi di rischio R3→R5 e per il 42,3% alle classi R1 e R2. I genotipi predominanti sono ARQ/ARQ (28,4%) e ARR/ARQ (25,9%). I capi omozigoti ARR/ARR resistenti costituiscono il 9,2% del totale contro l’1,3% di quelli omozigoti VRQ/VRQ. La razza Tiroler Bergschaf invece si suddivide fra il 55,9% nelle classi di rischio R3→R5 e il 44,1% nelle classi R1 e R2. I genotipi predominanti sono ARQ/ARQ (30,8%) e ARR/ARQ (29,2%), mentre i capi omozigoti ARR/ARR resistenti costituiscono l’8,2% del totale. Il genotipo omozigote VRQ/VRQ è stato rilevato solo nei maschi e con una frequenza pari allo 0,5% (Tabella 2).

Razze ovine della regione del Veneto Nella regione del Veneto dal 2016 ad oggi, sono stati genotipizzati 2.239 capi (640 arieti e 1.599 pecore) appartenenti alle quattro razze autoctone Alpagota, Brogna, Foza e Lamon. Gli alleli e i genotipi rilevati per ciascuna razza e le relative frequenze sono riportati rispettivamente nelle tabelle 1 e 3. Nelle razze Alpagota e Brogna sono stati osservati tutti e sei gli alleli del gene PRNP, mentre nei capi appartenenti alle razze Foza e Lamon non è stata riscontrata la presenza, rispettivamente, dell’allele ARH e ARK. In tutte e quattro le razze ovine venete l’allele predominante, sia nei maschi che nelle femmine,

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F. Bordin et al. Large Animal Review 2022; 28: 33-40

Tabella 2 - Numero di capi con i relativi genotipi PRNP e frequenze (f) espresse in percentuale rilevate nelle sei razze della Provincia Autonoma di Bolzano. I genotipi sono stati suddivisi nelle cinque classi di rischio R1→R5 secondo il National Scrapie Plan for UK7: R1, ovini geneticamente resistenti alla Scrapie; R2, ovini geneticamente resistenti alla Scrapie, ma che necessitano di un'attenta selezione quando vengono utilizzati per la riproduzione; R3, ovini geneticamente poco resistenti alla Scrapie; R4, ovini geneticamente suscettibili alla Scrapie e non dovrebbero essere utilizzati per la riproduzione; R5, ovini altamente suscettibili alla Scrapie e non dovrebbero essere utilizzate per la riproduzione. JURASCHAF CLASSE DI RISCHIO

GENOTIPO

TOT

f (%)

ARR/ARR

8,9

8,5

ARR/ARQ

166

29,1

158

ARR/AHQ

14,7

0,5

♂

TIROLER STEINSCHAF

♀

f (%)

TOT

f (%)

23,5

3,9

28,5

47,1

5,9

f (%)

♂

f (%)

SCHNALSER BERGSCHAF F

♀

f (%)

TOT

f (%) 5,3

6,8

125

27,5

124

24,7

0,2

♂

f (%)

♀

f (%)

3,6

5,9

23,2

29,4

249

4,5

8,8

0,1

6,8

5,9

7,6

158

31,5

16,7

1,8

R2 ARR/ARH ARR/ARK ARQ/ARQ

17,1

11,8

122

47,3

116

51,8

17,6

284

29,7

126

27,7

ARQ/AHQ

113

19,8

112

20,2

5,9

15,1

15,2

14,7

0,1

0,2

ARQ/ARH

0,4

152

15,9

14,9

0,4

2,9

1,2

2,9

0,1

0,2

0,1

0,2

1,7

1,5

ARQ/ARK AHQ/AHQ

6,3

5,9

0,9

R3 AHQ/ARH AHQ/ARK

0,2

ARH/ARH

ARH/ARK

0,2

ARK/ARK

0,2

ARR/VRQ

0,7

ARQ/VRQ

1,8

2,7

AHQ/VRQ

0,4

0,9

14,7

2,9

ARH/VRQ

3,1

3,2

8,4

9,2

7,6

1,6

1,5

1,6

1,1

1,2

ARK/VRQ VRQ/VRQ TOTALE

571

554

258

SCHWARZ-BRAUNES BERGSCHAF CLASSE DI RISCHIO

GENOTIPO

TOT

f (%)

ARR/ARR

5,4

4,9

ARR/ARQ

130

18,4

ARR/AHQ

4,7

♂

f (%)

♀

224

957

455

VILLNOESSER BRILLENSCHAF M

♂

f (%)

502

TIROLER BERGSCHAF

♀

f (%)

TOT

f (%)

7,5

172

9,2

10,1

16,1

28,6

481

25,9

179

24,2

4,9

3,8

2,8

♂

f (%)

♀

f (%)

TOT

f (%)

8,7

148

8,2

132

7,9

302

530

29,2

485

29,2

2,8

3,4

2,7

4,1

10,7

R2 ARR/ARH

0,3

4,2

4,5

ARR/ARK

1,6

1,9

0,1

0,3

0,5

2,8

2,7

4,0

ARQ/ARQ

216

30,6

191

33,3

18,8

528

28,4

213

28,8

315

ARQ/AHQ

127

102

17,8

18,8

105

5,6

5,4

28,1

558

30,8

515

28,7

5,8

125

6,9

119

7,2

ARQ/ARH

2,8

4,2

5,0

3,7

ARQ/ARK

3,1

2,3

0,2

0,3

0,1

124

1,9

2,7

6,8

116

5,3

AHQ/AHQ

3,7

3,3

5,3

0,4

0,5

0,3

AHQ/ARH

1,2

0,4

0,3

0,4

0,6

0,3

0,7

0,5

0,4

1,3

0,2

0,5

0,3

AHQ/ARK

ARH/ARH

0,1

0,2

ARK/ARK

0,1

0,2

0,8

0,1

0,4

0,3

ARH/ARK

ARR/VRQ

1,8

1,7

2,3

104

5,6

5,3

5,8

2,1

3,3

ARQ/VRQ

4,4

3,7

7,5

155

8,3

6,8

105

9,4

3,8

3,7

4,7

AHQ/VRQ

2,4

2,8

0,8

1,1

0,4

0,3

ARH/VRQ

0,1

0,8

1,6

1,3

0,1

ARK/VRQ

0,6

0,1

0,3

1,3

1,9

0,4

0,5

0,7

VRQ/VRQ TOTALE

706

573

133

1860

740

1120

1812

1662

150

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Analisi dei polimorfismi del gene della proteina prionica (PRNP) e suscettibilità alla Scrapie delle razze ovine autoctone

Tabella 3 - Numero di capi con i relativi genotipi PRNP e frequenze (f) espresse in percentuale per le quattro razze della regione del Veneto e per le due razze della regione Friuli Venezia Giulia. I genotipi sono stati suddivisi nelle cinque classi di rischio R1→R5 secondo il National Scrapie Plan for UK7. ALPAGOTA CLASSE DI RISCHIO

GENOTIPO

TOT

f (%)

ARR/ARR

6,1

6,5

♂

f (%)

BROGNA F

♀

♂

f (%)

TOT

f (%)

109

8,2

f (%)

FOZA F

♀

f (%)

TOT

f (%)

♂

f (%)

♀

f (%)

6,3

4,2

2,1

19,8

ARR/ARQ

180

33,5

32,5

140

33,7

412

31,2

359

27,2

33,3

13,5

ARR/AHQ

1,6

3,4

1,8

0,2

1,6

ARR/ARH

0,4

0,8

0,2

0,7

0,2

0,5

ARR/ARK

0,6

0,7

1,1

4,2

2,1

ARQ/ARQ

221

41,1

162

470

35,6

123

9,3

347

26,2

38,5

18,8

19,8

ARQ/AHQ

6,1

0,8

7,7

0,4

2,6

6,3

5,2

ARQ/ARH

0,9

1,6

0,7

3,3

1,3

ARQ/ARK

0,4

0,5

2,7

0,2

2,5

9,4

7,3

2,1

AHQ/AHQ

0,7

AHQ/ARH

0,1

AHQ/ARK

0,2

0,1

0,2

ARH/ARH

0,2

ARH/ARK

0,2

ARK/ARK

0,2

0,2 1

ARR/VRQ

1,9

4,1

1,2

1,8

1,1

ARQ/VRQ

4,8

3,3

5,3

6,8

3,2

3,6

AHQ/VRQ

0,6

0,8

0,5

0,1

ARH/VRQ

0,6

0,5

0,2

ARK/VRQ

0,2

0,1

0,2

0,5

VRQ/VRQ TOTALE

538

123

415

1322

323

LAMON CLASSE DI RISCHIO R1

GENOTIPO

TOT

f (%)

♂

f (%)

999

CARSOLINA F

♀

f (%)

TOT

f (%)

♂

f (%)

PLEZZANA F

♀

f (%)

TOT

f (%)

♂

f (%)

ARR/ARR

9,5

7,4

2,1

10,5

11,6

9,6

6,7

ARR/ARQ

21,6

9,2

12,4

23,3

25,8

21,3

ARR/AHQ

1,1

0,7

0,4

7,9

5,2

10,1

ARR/ARH

2,8

0,7

2,1

3,2

5,9

23,9

26,5

21,8

26,7

18,4

16,8

19,7

6,7

4,1

5,2

3,2

13,3

3,2

4,5

2,1

6,7

2,9

2,6

3,2

13,3

0,6

0,5

6,7

♀

ARR/ARK ARQ/ARQ ARQ/AHQ ARQ/ARH

3,2

0,7

2,5

ARQ/ARK AHQ/AHQ R3 AHQ/ARH

0,4

AHQ/ARK ARH/ARH ARH/ARK ARK/ARK R4

ARR/VRQ

14,1

6,7

7,4

1,2

0,6

1,6

ARQ/VRQ

19,8

8,8

0,9

0,6

1,1

AHQ/VRQ

1,1

0,7

0,4

ARH/VRQ

0,7

0,4

1,8

1,1

0,7

ARK/VRQ VRQ/VRQ TOTALE

283

143

140

343

155

188

f (%)

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F. Bordin et al. Large Animal Review 2022; 28: 33-40

è ARQ, con una frequenza che varia dal 46,3% della razza Lamon al 63,9% dell’Alpagota. L’allele di resistenza ARR mostra una frequenza compresa tra il 25,7% della razza Alpagota e il 29,3% della Lamon, mentre quella dell’allele VRQ varia dallo 0,5% della razza Foza al 19,6% della Lamon. La frequenza dell’allele ARK, in generale poco comune nelle popolazioni ovine, è compresa tra lo 0,5% della razza Alpagota e il 6,8% della Foza (Tabella 1). Nella razza Alpagota la popolazione totale (maschi e femmine) è composta per il 56,5% da capi appartenenti alle classi di rischio R3→R5, mentre il restante 43,5% appartiene alle classi R1 e R2, scarsamente suscettibili alla Scrapie. I genotipi predominanti nella razza sono ARQ/ARQ (41,1%) e ARR/ARQ (33,5%). I capi resistenti omozigoti ARR/ARR rappresentano il 6,1% del totale, mentre non sono stati rilevati omozigoti VRQ/VRQ (Tabella 3). Nella razza Brogna i capi analizzati appartengono alle classi di rischio R3→R5 per il 57% e per il restante 43% alle classi R1 e R2. I genotipi predominanti nella razza sono ARQ/ARQ (35,6%) e ARR/ARQ (31,2%), mentre i capi omozigoti ARR/ARR resistenti costituiscono l’8,2% del totale. Solo l’1% della popolazione è costituito da capi omozigoti VRQ/VRQ (Tabella 3). La razza Foza è composta da capi suscettibili alla Scrapie per il 55,2% (classi di rischio R3→R5) e per il 44,8% da capi scarsamente suscettibili o resistenti (classi R1 e R2). I genotipi predominanti nella razza sono ARQ/ARQ (38,5%), ARR/ARQ (33,3%) e ARQ/ARK (9,4%). I capi omozigoti ARR/ARR resistenti costituiscono il 6,3% del totale, mentre non sono stati riscontrati omozigoti VRQ/VRQ (Tabella 3). La razza Lamon è suddivisa per il 65% nelle classi di rischio R3→R5 e per il 35% nelle classi R1 e R2. I genotipi predominanti nella razza sono ARQ/ARQ (24%), ARR/ARQ (21,6%) e ARQ/VRQ (19,8%). I capi omozigoti ARR/ARR costituiscono il 9,5% del totale mentre quelli omozigoti VRQ/VRQ l’1,8% (Tabella 3).

Razze ovine della regione Friuli Venezia Giulia Delle razze autoctone Carsolina e Plezzana della regione Friuli Venezia Giulia sono stati genotipizzati 358 capi (170 arieti e 188 pecore). Gli alleli e i genotipi rilevati per ciascuna razza e le relative frequenze sono riportati rispettivamente nelle tabelle 1 e 3. Nelle due razze sono stati osservati tutti gli alleli del gene PRNP ad esclusione dell’allele ARK. ARQ è l’allele predominante, con una frequenza del 47,2% nella Carsolina e del 50% nella Plezzana, mentre l’allele ARR, che conferisce resistenza alla Scrapie, è presente nel 28,3% di capi della razza Carsolina e nel 16,7% di quelli della razza Plezzana. La frequenza dell’allele VRQ è dell’1% e del 3,3% rispettivamente nella razza Carsolina e nella Plezzana (Tabella 1). Nella razza Carsolina la popolazione totale (maschi e femmine) è composta per il 55,1% da capi appartenenti alle classi di rischio R3→R5, mentre per il restante 44,9% appartenenti alle classi R1 e R2. I genotipi predominanti nella razza sono ARQ/ARQ (23,9%), ARR/ARQ (23,3%), e ARQ/AHQ (18,4%). I capi resistenti omozigoti ARR/ARR costituiscono il 10,5% del totale, mentre non sono stati riscontrati capi omozigoti VRQ/VRQ (Tabella 3). Per la razza Plezzana sono stati genotipizzati solo 15 arieti. Nonostante il numero ridotto di campioni è stato tuttavia possi-

bile osservare otto diversi genotipi che hanno portato alla suddivisione dei capi nelle classi di rischio R3→R5 per 73,3% e per il restante 26,7% nelle classi R1 e R2. I genotipi predominanti nella razza sono ARQ/ARQ (26,7%), ARR/ARQ (20,0%), ARQ/ARH (13,3%) e AHQ/ARH (13,3%). È stato identificato un solo individuo resistente omozigote ARR/ARR, mentre non sono stati rilevati capi omozigoti VRQ/VRQ (Tabella 3).

DISCUSSIONE In questo studio sono state analizzate le frequenze alleliche di alcune razze ovine autoctone presenti nella Provincia Autonoma di Bolzano, nella regione del Veneto e nella regione Friuli Venezia Giulia. Nella popolazione ovina oggetto di studio sono stati osservati tutti e sei gli alleli noti del gene PRNP e la maggior parte degli animali rientra nelle classi di rischio R3→R5 con suscettibilità alla Scrapie classica da moderata a molto alta. L’allele predominante in tutte le razze è ARQ, con frequenze superiori al 60% nelle razze Foza (63%), Alpagota (63,9%) e Tiroler Steinschaf (68,4%). In quest’ultima, l’elevata frequenza dell’allele ARQ conferma quanto già rilevato da Sipos et al.10 nello studio del genotipo del gene PRNP di alcune razze ovine austriache. Gli alleli ARR e VRQ mostrano frequenze variabili a seconda della razza ovina, con un valore medio sul totale della popolazione del 25% e del 5,3% rispettivamente. Le frequenze relativamente elevate dell’allele ARR (> 20%) in quasi tutte le razze oggetto dello studio confermano ciò che è stato descritto, in particolare per le razze della Provincia Autonoma di Bolzano, da Sipos et al.10 nelle razze ovine austriache e da Cubric Curik et al.11 nelle pecore istriane. Questo dato è piuttosto rilevante perché, in relazione al PNSG in atto, la presenza dell’allele ARR fornisce l’opportunità di aumentare la resistenza alla Scrapie classica in queste razze. Nonostante la significativa presenza di genotipi resistenti e semi-resistenti (ARR/-) vi sono numerosi capi che presentano alleli di suscettibilità alla Scrapie da moderata ad alta. Tra tutte le razze, la Lamon presenta la situazione peggiore in questo senso, in quanto la frequenza dell’allele VRQ è elevata (19,8%) e circa il 37,5% degli animali appartiene alle classi di rischio R4 e R5. Ciò significa che questi capi non dovrebbero essere utilizzati come riproduttori secondo il Regolamento della Commissione (EU) No 630/2013 del 28 giugno 201312. A tal proposito, nonostante nella medesima razza la frequenza dell’allele VRQ si sia ridotta rispetto a quanto riportato da Granato et al.9 nel 2013 e il piano di selezione per la resistenza alla Scrapie sia in vigore da almeno cinque anni, in questi ovini permangono condizioni di elevato rischio di erosione genetica in relazione al limitato numero di capi allevati, allevamenti ed arieti utilizzati. La frequenza dell’allele VRQ osservata nelle razza Lamon è paragonabile a quanto rilevato da Lühken et al.13 nella razza ovina inglese Exmoor Horn e greca Anogeiano (frequenza VRQ prossima al 18% e 16% rispettivamente), e da Townsend et al.14 in alcune rare razze ovine inglesi (Galway e White Face Dartmoor) tra cui la razza ovina Boreray, in cui la frequenza dell’allele VRQ è del 28,4%. Tuttavia, mentre gli autori di quest’ultimo studio hanno suggerito che questo allele si sarebbe involontariamente mantenuto come sottoprodotto degli schemi di selezione utilizzati dagli allevatori, per la razza Lamon è più probabile che l’alta frequenza dell’allele VRQ sia il risultato

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Analisi dei polimorfismi del gene della proteina prionica (PRNP) e suscettibilità alla Scrapie delle razze ovine autoctone

della drastica riduzione dei capi negli ultimi anni o di un effetto fondatore in seguito ad un prolungato isolamento9. Anche le razze altoatesine Villnoesser Brillenschaf e Schnalser Bergschaf presentano circa il 18% e il 14,2% rispettivamente dei capi assegnati alle classi di rischio R4 e R5, ma in questo caso le diverse frequenze dell’allele VRQ rilevato nelle razze presenti nella stessa area possono dipendere sia dall’isolamento geografico, che ne ha diminuito lo scambio genetico, sia dalle pratiche riproduttive attuate dagli allevatori8. Interessante è stato inoltre rilevare la presenza dell’allele ARK in otto delle razze oggetto di questo studio, con una frequenza particolarmente significativa nella Foza e nella Tiroler Bergschaf (6,8% e 5,7% rispettivamente). Lühken et al.13 avevano già ipotizzato che l’allele ARK potesse essere un tratto distintivo di alcune razze alpine, come nel caso delle pecore Bergamasca e Biellese, anche se è stato trovato in razze non prettamente alpine, ma in tutti i casi con una frequenza inferiore all’1%2, 15, 16, 17, 18, 19, 20, 21. Anche in questo caso, si può supporre che la frequenza relativamente elevata dell’allele ARK sia dovuta alle dimensioni ridotte della popolazione ovina analizzata (ad esempio la razza Foza) o all’isolamento geografico degli allevamenti di montagna (ad esempio la razza Tiroler Bergschaf).

CONCLUSIONI Il presente studio ha evidenziato l’importanza della genotipizzazione del gene PRNP di alcune razze ovine autoctone dell’Alto Adige, del Veneto e del Friuli Venezia Giulia, al fine di definire il grado di suscettibilità alla Scrapie classica. Un’attenzione particolare è rivolta alla conservazione di queste popolazioni ovine autoctone ed è dovuta al fatto che, essendo già a rischio di estinzione, la salvaguardia del patrimonio zootecnico e culturale potrebbe essere messa in pericolo se entrassero in contatto con l’agente della Scrapie. I risultati ottenuti dalle analisi hanno rivelato alcune caratteristiche che possono aiutare ad indirizzare e migliorare l’organizzazione del piano di conservazione e programmare quindi gli accoppiamenti degli animali con la realizzazione di gruppi di monta. A tal proposito infatti, oltre a genotipizzare gli arieti riproduttori, si è provveduto a determinare il grado di suscettibilità alla Scrapie anche di numerose pecore di alcune delle razze considerate. Inoltre, l’analisi del genotipo ha evidenziato caratteristiche genetiche distintive che confermano o aggiornano le informazioni già presenti in letteratura, sottolineando l’importanza di preservare le risorse genetiche degli animali allevati non solo per ragioni culturali o etiche, ma anche scientifiche. In conclusione, possiamo affermare che i programmi di allevamento per controllare la suscettibilità alla Scrapie classica devono basarsi su un’adeguata strategia di selezione oltre che su un’attenta gestione degli accoppiamenti per le diverse razze in esame, al fine di aumentare la frequenza dell’allele ARR riducendo quella degli altri alleli sensibili, in primis VRQ. Per la conservazione della razza e soprattutto nel caso di una razza rara o in via di estinzione è quindi importante trovare un giusto compromesso tra l’utilizzo di soli arieti delle classi di rischio R1 e R2 (resistenti), con conseguente drastica riduzione del numero di arieti disponibili e un prolungato utilizzo degli stessi, e l’eliminazione degli arieti più suscettibili in quanto questo comporterebbe una grave perdita di variabilità genetica e una riduzione delle dimensioni delle popolazioni ovine, mettendone così a rischio la sopravvivenza futura.

Bibliografia 1. Acin C., Martin-Burriel I., Goldmann W., Lyahyai J., Monzon, M., Bolea R., Zaragoza P. (2004). Prion protein gene polymorphisms in healthy and scrapie-affected Spanish sheep. J Gen Virol, 85: 2103-2110. doi: 10.1099/vir.0.80047-0 2. Billinis C., Psychas V., Leontides L., Spyrou V., Argyroudis S., Vlemmas I., Papadopoulos O. (2004). Prion protein gene polymorphisms in healthy and scrapie-affected sheep in Greece. J Gen Virol, 85: 547-554. doi: 10.1099/vir.0.19520-0 3. Detwiler L. A., Baylis M. (2003). The epidemiology of scrapie. Rev Sci Tech Off Int Epiz, 22: 121-143. 4. Italian Ministry of Health (2015). Misure di prevenzione su base genetica per l'eradicazione della scrapie ovina classica, finalizzate all'incremento dell'allele di resistenza della proteina prionica (ARR) nell'intero patrimonio ovino nazionale. Retrieved from https://www.gazzettaufficiale.it/atto/serie_generale/caricaDettaglioAtto/originario?atto.dataPubblicazioneGazzetta=2016-01-27&atto.codiceRedazionale= 16A00566&elenco30giorni=false.+ 5. Bigi D., Zanon A. (2020). Atlante delle razze autoctone. Bovini, Equini, Ovicaprini, Suini allevati in Italia (2nd ed., pp. 483, Bologna: Edagricole-New Business Media. 6. Registro Genealogico giovane bestiame. Registro Anagrafico delle popolazioni Ovi-Caprine autoctone a limitata diffusione. Associazione nazionale pastorizia. Retrieved from www.assonapa.com 7. Dawson M., Del Rio Vilas V. (2008). Control of classical scrapie in Great Britain. In Practice, 30: 330-333. https://doi.org/10.1136/inpract.30.6.330 8. Bordin F., Dalvit C., Caldon M., Zulian L., Colamonico R., Trincanato S., Mock B., Reale S., Mutinelli F., Granato A. (2020). Genetic variability following selection for scrapie resistance in six autochthonous sheep breeds in the province of Bolzano (northern Italy). J Anim Breed Genet, 137: 395-406. doi: 10.1111/jbg.12478 9. Granato A., Dalvit C., Caldon M., Colamonico R., Barberio A., Mutinelli F. (2013). PRNP gene polymorphism in native Italian sheep breeds undergoing in situ conservation. Small Ruminant Res, 113: 323-328. https://doi.org/10.1016/j.smallrumres.2013.03.009 10. Sipos W., Kraus M., Schmoll F., Achmann R., Baumgartner W. (2002). PrP genotyping of Austrian sheep breeds. J Vet Med Sci, 49: 415-418. doi: 10.1046/j.1439-0442.2002.00472.x. 11. Cubric Curik V., Feligini M., Ferencakovic M., Dzidic A., Salajpal K., Ambriovic-Ristov A., Curik I. (2010). Sequence polymorphism of PrP exon 3 gene in Istrian and Crossbred sheep. Ital J Anim Sci, 8: 86-88. 12. Commission Regulation (EU) No 630/2013 of 28 June 2013 amending the Annexes to Regulation (EC) No 999/2001 of the European Parliament and of the Council laying down rules for the prevention, control and eradication of certain transmissible spongiform encephalopathies. OJ, L 179: 60-83. Retrieved from https://eur-lex.europa.eu/legal-content/GA/TXT/?uri=CELEX:32013R0630 13. Lühken G., Lipsky S., Peter C., Erhardt G. (2008). Prion protein polymorphisms in autochthonous European sheep breeds in respect to scrapie eradication in affected flocks. Small Ruminant Res, 75: 43-47. https://doi.org/10.1016/j.smallrumres.2007.07.010 14. Townsend S.J., Warner R., Dawson M. (2005). PrP genotypes of rare breeds of sheep in Great Britain. Vet Rec, 156: 131-134. doi: 10.1136/vr.156.5.131. 15. Gombojav A., Ishiguro N., Horiuchi M., Serjmyadag D., Byambaa B., Shinagawa M. (2003). Amino acid polymorphisms of PrP gene in Mongolian sheep. J Vet Sci, 65: 75-81. 16. Lan Z., Wang Z.L., Liu Y., Zhang X. (2006). Prion protein gene (PRNP) polymorphisms in Xinjiang local sheep breeds in China. Arch Virol, 151: 2095-2101. https://doi.org/10.1292/jvms.65.75 17. Ekateriniadou L.V., Panagiotidis C.H., Terzis A., Ploumi K., Triantafyllidis A., Deligiannidis P., Triantaphyllidis K., Sklaviadis T. (2007). Sheep genotyping for PrP gene polymorphisms in rare Greek breeds. Vet Rec, 160: 194-195. https://doi.org/10.1136/vr.160.6.194 18. Gootwine E., Abdulkhaliq A., Jawasreh K., Valle Zárate A. (2008). Screening for allele frequency at the PrP locus in (PRNP) Awassi and Assaf populations in Israel, the Palestinian Authority and Jordan. Small Ruminant Res, 77: 83-88. https://doi.org/10.1016/j.smallrumres.2008.02.008 19. Pongolini S., Bergamini F., Iori A., Migliore S., Corradi A., Bassi, S. (2009). Prion protein genotypes of Italian sheep breeds with lysine171 and phenylalanine-141 detection. Vet Microbiol, 137: 18-23. https://doi.org/10.1016/j.vetmic.2008.12.012 20. Alvarez L., Gutierrez-Gil B., Uzun M., San Primitivo F., Arranz J.J. (2011). Genetic variability in the prion protein gene in five indigenous Turkish sheep breeds. Small Ruminant Res, 99: 93-98. https://doi.org/ 10.1016/j.smallrumres.2011.03.043 21. Oner Y., Yesilbag K., Tuncel E., Elmaci C. (2011). Prion protein gene (PrP) polymorphisms in healthy sheep in Turkey. Animals, 11: 17281733. https://doi.org/10.1017/S1751731111000942

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V. Sangwan et al. Large Animal Review 2021; 28: 41-45

Coccygeal osteosarcoma A report in three cows

VANDANA SANGWAN*1, KIRANDEEP GILL2, NEELAM TANDIA3, ASHWANI KUMAR4, KULDIP GUPTA5 1

Associate Professor, Department of Veterinary Surgery and Radiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India Research Fellow, Department of Veterinary Surgery and Radiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India Assistant Professor, Department of Veterinary Surgery and Radiology, College of Veterinary Science, Nanaji Deshmukh Veterinary Science University, Jabalpur, Madhya Pradesh, India Professor, Department of Veterinary Surgery and Radiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India Professor, Department of Veterinary Pathology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana-141004, Punjab, India

SUMMARY The report describes a rare occurrence of coccygeal osteosarcoma in three adult cows (2 cross-bred and one local Zebu cow). All cows had a hard ulcerated swelling at the base of tail from 4 to 6 months making it heavy and stiff. The swelling was pressing the anus and the cows had difficulty in defecation, lifting and moving the tail. Lateral radiographs revealed the lysis of the initial coccygeal vertebra (varying from C1 to C6 vertebrae) with sunburst appearance, indicating advanced stage of bone tumour. The tails were amputated, as cranial as possible, under general anaesthesia (midazolam, ketamine and isoflurane), in lateral recumbency. Histology showed bony spicules along with proliferation of the osteoblasts with pleomorphic and spindle shaped plump oval nuclei, confirming coccygeal osteosarcoma in all the cows. The cows are lived a quality life for 3 to 6 months after surgical excision of tail and died within 4 to 12 months. In conclusion, coccygeal osteosarcoma bears a poor prognosis due to delay in presentation, extensive involvement and consequent re-occurrence.

KEY WORDS Amputation; bovine; coccygeal; osteosarcoma; surgery; tail tumour.

INTRODUCTION Osteosarcoma is one of the most common bone tumour encountered in dogs, cats and humans1. A few cases of osteosarcoma have been reported in cattle, predominantly in the bones of head2; maxilla3, 4, mandible5, nasal cavity6, 7 and ischium8. Osteosarcoma originating from the appendicular skeleton like scapula9 and metacarpal bone10 is also reported. As per author’s knowledge, osteosarcoma originating from the coccygeal vertebra has not been reported in cattle. This case report describes the clinical presentation, radiographic, surgical and histological findings of coccygeal osteosarcoma in three adult cows.

Corresponding Author: Sangwan Vandana (drvandanasangwan@rediffmail.com).

HISTORY AND CLINICAL OBSERVATIONS (Table 1) The three non-gravid cows were presented to the University Veterinary Hospital at different time in an interval of total 20 months. Two of the cows were crossbred of exotic breeds while one was local Zebu humped cow. All the cows had hard swelling/mass with a few ulcerated spots near the base of the tail, which was gradually increasing in size (Figure 1). The tail was heavy and stiff and the cows were unable to lift or move it freely, the severity of which increased with the size of the mass. The mass was pressing the anus and it was impossible to do per rectal examination in cow 1 (Figure 1a, b, c). All the three cows had nearly normal appetite. The first 2 cows were moderately anaemic and the tail involvement was also extensive. Lateral radiographs of the coccygeal regions revealed lytic changes in the proximal coccygeal vertebrae with sunburst appearance (Figure 1c, f, i) suggestive of bone tumour. The involvement of number of coccygeal ver-

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Coccygeal osteosarcoma - A report in three cows

tebra varied in cows. The clinical and radiographic findings indicated advanced progression of the disease condition, whereas, prognosis was poor for surgical correction. However, the owners did not agree for euthanasia, and tail amputation was decided under general anaesthesia. The technical challenges associated with the amputation from the base of tail in cow 1 and 2 was the broad base, incomplete excision of the tumorous mass and inadequate skin flap for the closure of surgical wound. The cows were called off feed and off water for 24-36 hours on the day of surgery. Intravenous fluids (dextrose normal saline solution 5%) were administered during this period.

SURGICAL MANAGEMENT The cows were restrained in lateral recumbency for surgery. Premedication was done using injection midazolam (Neon Laboratories Ltd., Mumbai, India) (0.2 mg/kg), intravenously, followed by induction with injection ketamine hydrochloride (Neon Laboratories Ltd., Maharashtra, India) (4mg/kg) intravenously, till effect. Endotracheal intubation was done with 20mm (ID) tube and the anaesthesia was maintained using 2-3% Isoflurane (Raman and Weil Pvt. Ltd, Daman, India) inhalant anaesthesia mixed with 100% oxygen using partial re-

Figure 1 - Photograph showing a tumorous mass at the base of the tail in cows (caudal and lateral view) and their respective radiographs in lateral view (Cow 1: a, b, c; Cow 2: d, e, f; Cow 3: g, h, i).

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V. Sangwan et al. Large Animal Review 2021; 28: 41-45

Table 1 - Detailed description of cows with coccygeal osteosarcoma. Cow 1

Cow 2

Cow 3

Breed

Holstein crossbred

Zebu humped

Jersey crossbred

Age

6 year

9 year

Weight

360 Kg

345 Kg

450 Kg

Period of illness

6 months

4 months

6 months

Size of mass

30 X 25 cm

18 X 14 cm

20 X 15 cm

Vertebrae involvement on radiograph (Figure 1c, f, i)

C1-C2-C3-C4-C5 (C1 and C5 showing partial lysis, C2,C3 and C4 showing complete lysis with sunburst appearance)

C3-C4-C5 (C3 partial lysis, C4-C5 showing extensive lysis and sunburst appearance)

C5-C6 (C5 showing complete lysis with sunburst appearance, C6 partial lysis)

Hb (g/dl)

8.5

7.0

11.0

TLC / mL

12650

9200

11000

N (%)

L (%)

PCV (%)

25.2

21.0

33.0

Platelets (/µL)

340000

292000

325000

Re-occurrence at 3 months and the cow died within next one month with hematoma and swelling in sacral and pubis region.

The surgical wound did not heal by primary intention. After 6 months, swelling appeared in the sacral region. The cow became recumbent and died within 12 months.

Swelling appeared at the surgical site after 4 months nd spread to sacrum and apubis. The cow had difficulty in passing faeces and died at 7 months.

CBC

Follow up

CBC: Complete Blood Count, Hb: Haemoglobin, TLC: Total Leucocyte Count, N: Neutrophils, L: lymphocytes, PCV: Packed Cell Volume

breathing circuit. Incisions were made on the either sides of the tumorous mass. Subcutaneous dissection was done to make a skin flap for the closure of tail stump after amputation. In cow 1, the tumorous mass had multiple bone pieces and was firmly adhered to the rectal wall. During dissection, an almost 6 cm linear tear occurred in the rectal wall, which was later repaired using polyglactin 910 (no. 3-0) in a simple continuous manner. The tumorous mass was resected upto the sacrum (Figure 2). The remaining lytic bone pieces and affected tissues were also removed.

Figure 2 - Photograph showing cows after tail amputation.

All major blood vessels encountered were ligated and the surgical wound was flushed with normal saline solution. The subcutaneous sutures were applied using polyglactin 910 (no.2-0) and the skin closure using silk no. 2 in horizontal/cross mattress pattern. The anaesthetic recovery showed slight shortage of skin on ventral aspect leading to widening of the anus in cow 1. In cow 2 and 3, the surgery was comparatively simple, with no rectal involvement and the amputation was done after C1 and C2 vertebrae, respectively. Though, in cow 2, there were pus pockets in the tissue leading to contamination of the surgical wound.

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Coccygeal osteosarcoma - A report in three cows

Figure 3 - Photomicrograph showing bony spicules (black star) along with proliferation of osteoblasts (black arrow) suggestive of osteosarcoma (H&E, 20X).

POSTOPERATIVE CARE AND COMPLICATIONS Post-operative care included injection ampicillin-cloxacillin (10 mg/kg), twice daily for 5 days, injection gentamicin sulphate (4mg/kg) divided in 2 doses daily for 3 days and injection meloxicam (0.2mg/kg) once daily for 3 days, intramuscularly. The surgical wound healed uneventfully in cow 1 and 3; however, cow 2 had surgical wound dehiscence. After surgery, cow 1, 2 and 3were survived for 3, 6 and 4 months, respectively and reported re-occurrence of tumour involving sacral and pelvic regions. Subsequently, all the cows died in a time period of 4, 12 and 7 months, respectively.

HISTOLOGY The biopsy samples were stained with haematoxylin and eosin (H&E) stain as per the standard procedure. Microscopically, the slides showed bony spicules along with proliferation of the osteoblasts with pleomorphic and spindle shaped plump oval nuclei (Figure 3), suggestive of osteosarcoma.

DISCUSSION Tail tumours are rare in bovine. There are a few reports of mixed apocrine adenocarcinoma in a cow11 and a buffalo calf12. Adult buffaloes were not reported for tail tumours; although buffaloes have common occurrence of tail varicosity13, tail gangrene and dislocations14. Such lesions, particularly, when present on the base of the tail make difficulty during defecation and calving13. In the present report also, cow 1 had difficulty in passing faeces as the anal region was compressed by the tumour mass. Radiography of bone with lysis and sunburst appearance is typical of osteosarcoma15 which was also confirmed on histopathology in the present study. An osteosarcoma or osteogenic sarcoma is a cancerous tumour of a bone. It is an aggressive malignant neoplasm that arises from the primitive transformed cells of mesenchymal origin and it

exhibits osteoblastic differentiation. Histologically, it is characterized by the production of osteoid and/or immature bone tissue by neoplastic osteoblasts2. Osteosarcoma revealed tumorous cells with pleomorphic and spindle shaped plump oval nuclei. These cells are usually accompanied by strands of osteoid which become distinct islands4. Tail amputation for coccygeal osteosarcoma, in the present study, was a high risk surgery as there were chances of uncontrolled and diffused haemorrhages from the aggressive tumorous lesion. Tail amputation for gangrene or varicosity is usually recommended in standing position under epidural anaesthesia13. But, in the present cows, the surgery was done under general anaesthesia so to avoid unwanted movement of the cow, intraoperatively, and to facilitate adequate resection and ligation of the vessels. Besides, the tumorous mass was extensive and caudal epidural space was not available for injecting local anaesthesia, particularly in cow 1 and 2.

CONCLUSION Coccygeal osteosarcoma is a rare clinical entity in cows and bears poor prognosis due to delay in presentation, extensive involvement and consequent re-occurrence. Extensive cases of coccygeal osteosarcomas can be operated guardedly by tail amputation, under general anaesthesia, considering the short term welfare of the cow.

Conflicts of interest The authors have no conflicts of interests with anyone.

Authors contribution Author 1, 2, 3 are the surgeons, Author 4 anaesthesiologist and Author 5 pathologist. All authors have scrutinized and approved the manuscript.

Acknowledgements The authors acknowledge the Indian Council of Agricultural Research, New Delhi, India and Guru Angad Dev Veterinary and Animal Sciences University, Ludhiana, India for providing necessary facilities to treat the cows.

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V. Sangwan et al. Large Animal Review 2021; 28: 41-45

References 8. 1.

3. 4. 5.

Szewczyk M., Lechowski R., Zabielska K. (2015). What do we know about canine osteosarcoma treatment? - Review. Vet Res Commun, 39(1): 6167. ThompsonK.G., Pool R.R. (2002). Tumors of Bones. In: Tumors in Domestic Animals, Ed. Meuten D. J.,4th ed., 266-283, Iowa State Press, Ames, IA. Plumlee K.H., Haynes J.S., Kersting K.W., Thompson J. R. (1993). Osteosarcoma in a cow. J Am Vet Med Assoc, 202: 95-96. Prins D.G., Wittek T., Barrett D.C. (2012). Maxillary osteosarcoma in a beef suckler cow. Irish Vet J, 65 (1): 1-4. Pérez-Martínez C., Escudero-Diez A., García-Iglesias M.J., Ferreras-Estrada M.C., García-Fernández R. A., Espinosa-Alvarez, J. (1999). Fibroblastic osteosarcoma in a chamois (Rupicaprapyrenaicaparva). Vet Rec, doi: 10.1136/vr.144.6.154. Pospischil A., Weiland F., von Sandersleben J., Hänichen T., Schäffler H. (1982). Endemic ethmoidal tumours in cattle: sarcomas and carcinosarcomas. A light and electron microscopic study. Zentralbl Veterinarmed A, doi: 10.1111/j.1439-0442.1982.tb01822.x, Yoshimoto K., Komagata M., Chiba S., Hiro M., Kobayashi Y., Matsumoto K., Inokuma H. (2011).A case of nasal osteosarcoma in a Holstein cow. J

9. 10. 11.

12.

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Jpn Vet Med Assoc, 64: 457-460. Nagamine E., Matsuda K., Ishii C., Koiwa M., Taniyama H. (2014). Primary ischial osteosarcoma occupying the pelvic cavity in a Japanese black cow. J Vet Med Sci, 76(6): 891-894. Sastry G.A., Twiehaus M.J. (1964). Multiple neoplasia in a cow. Indian J Pathol Bacteriol, 158: 199-201. Heimann W. (1975).Post-traumatic osteosarcoma in cattle (short communication). Dtsch Tierarztl Wochenschr, 82: 16-17. Tessele B., Rissi D.R., Langohr I.M., Vielmo A., Barros C.S.L. (2015). Mixed apocrine adenocarcinoma of the tail in a cow. Brazilian J Vet Pathol, 8(2), 72 - 75. Phaneendra M.S.S.V., Veena V., Saibaba M., Prasad W.L.N.V. (2016). Apocrine Gland Adenocarcinoma of tail in a Buffalo Calf (Bubalus bubalis)A case report. J Livestock Sci, 7: 165-167. Sangwan V., Mahajan S.K., Kumar A., Singh T., Saini N.S. (2015). Surgical management of massive coccygeal varicosity in a Murrah Buffalo. Buffalo Bull, 34 (2): 149-152. Satyanarayana G., Sreenu M., Prasad V.D., Naidu G.V. (2014). Management of Tail Affections in Buffaloes. Int J Agric Sci & Vet Med, 27-33. Resnick D. (2002). Tumors and tumor-like lesions of bone: Radiographic principles. In: Diagnosis of bone and joint disorders, Ed. Resnick D., 4th ed., 3745-4128, Saunders Philadelphia, USA.

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IL PORTALE DEL VETERINARIO

DI FIDUCIA

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

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CODICE EGO

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

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ISCRIVITI ALLA SIVAR PER IL 2020 E RICHIEDI L’ABILITAZIONE AI SERVIZI DEL PORTALE VETERINARIO DI FIDUCIA: AVRAI ACCESSO A DES, DDD E FORUM INVIO RICHIESTA A info@sivarnet.it o vetdifiducia@anmvi.it

KACAR_404_ok 27/02/22 12:12 Pagina 47

Y. Kaçar et al. Large Animal Review 2022; 28: 47-49

Enzootic posthitis in post-weaning lambs: a case series

YİĞİT KAÇAR1*, MEHMET EMİN AKKAŞ1, HAVVA KURNAZ2, HASAN BATMAZ1 1 2

Department of Internal Medicine, Faculty of Veterinary Medicine, Uludag University, Gorukle, Bursa, Turkey Department of Microbiology, Faculty of Veterinary Medicine, Uludag University, Gorukle, Bursa, Turkey

SUMMARY Enzootic posthitis is a disease that causes ulcerative lesions in the external genitalia of lambs, leading to negative effects on animal welfare, animal health, and profitability. The disease can be observed anywhere in the world where sheep breeding is common. Enzootic posthitis has a multifactorial aetiology but is primarily caused by rations with high protein concentration. Older lambs and adult rams during the mating term are more commonly affected, whereas the occurrence in younger lambs is a rare finding. This report describes the disease detected in 6 Merino male lambs 75-90 days old and compares their clinical and laboratory findings with six healthy lambs. Approximately 100 male lambs in a flock of 300 reportedly had varying degrees of difficulty in urinating, preputial lesions, and bloody urine. Clinical examination of the affected lambs revealed an inflamed prepuce (hot, painful, and swollen) and lesions with slight hyperemia to ulcerative changes. Preputial lesions generally consisted of circular ulcerative lesions and scabs, and the tissue was prone to bleeding when the scabs were removed. Blood and urine samples, as well as preputial swabs, were taken for laboratory analyses. Corynebacterium renale, Staphylococcus intermedius, Streptococcus uberis, and Truperella pyogenes were isolated during the microbiological examination of the preputial samples. A diagnosis of enzootic posthitis was made based upon the clinical and laboratory findings of the affected lambs. For comparison, six healthy lambs were selected and evaluated. Clinical examination and some laboratory analyses performed for all lambs revealed no statistical differences in body temperature (39.78 ± 0.13 and 39.43 ± 0.13 ºC), total leukocyte count (7773.33 ± 649.13 and 6916.66 ± 802.25/mm3), and hematocrit (PCV%) (47.00% ± 3.54 and 39.33% ± 1.45) between the diseased and healthy lambs; however, the mean urine pH values were 8.41 and 7.66, respectively (p <0.05). After the diagnosis, ration was rearranged with addition of ammonium chloride and reduction of protein concentration. Additionally, amoxicillin (15 mg/kg, two doses 48 hours apart, IM), metamizole sodium (20 mg/kg daily for two days, IM), and vitamin C (20 mg/kg for three days, IM) were administered together with local treatment of preputial lesions by using pomade rivanol for three days. With this treatment and management practices, lesions in many animals were healed on the 7th day of treatment and completely healed on the 10th day. Here, for the first time to the best of our knowledge, we document the presence of severe and widespread enzootic posthitis in male lambs aged 75-90 days to emphasize the importance of correct ration planning during the rearing period.

KEY WORDS Enzootic posthitis, ulcerative posthitis, lambs.

INTRODUCTION ‘Enzootic balanoposthitis’, also called ‘enzootic posthitis’, ‘ulcerative posthitis’, ‘pizzle rot’, ‘sheath rot’, and ‘peestersiekte’, is a multifactorial disease characterized by ulcerative changes in the external genital organs 1-6. Although enzootic posthitis has been reported mainly in Australia, New Zealand, Scotland, England, Spain, South Africa, and South America, the disease can be seen in all regions where sheep breeding occurs 2,7,8,9,10. While the disease has been identified in different sheep breeds, Merino sheep reportedly have a higher incidence rate because of the long hairs surrounding the prepuce, which can cause contamination with urine and the causative agent 2,7,8,11.

Corresponding Author: Yiǧit Kaçar (yigitkacar@uludag.edu.tr).

Alkaline urine output, associated with a high protein ration (> 16%, especially 18%), is the most important predisposing factor for disease formation 2,12,13. Pellet feeds with high protein concentration, legume-rich rations, and even grazing on pastures treated with nitrogen fertilizers can play a predisposing role in this disease 2,9,13. While enzootic posthitis has generally been described in adults or older lambs 6,8,9,13, we aimed to evaluate weaning young lambs for the disease based on their clinical and laboratory findings and compare those findings with that of healthy lambs.

CASE PRESENTATION The study animals consisted of six male lambs from a Merino flock with preputial lesions exhibiting varying degrees of difficulty urinating, along with six healthy lambs as a control group. In the flock anamnesis, it was stated that, in the last 20 days, about 100 out of 300 male lambs, 75-90 days old, had dif-

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Enzootic posthitis in post-weaning lambs: a case series

Figure 1 - Ulcerative lesions in the prepuce.

ficulty urinating, bloody urination, or intermittent urination (stranguria). It was reported that three diseased lambs died, and four were slaughtered during this period due to these problems. Based on the flock feeding and management regimen, all lambs were weaned and consumed lamb starter feed (17% protein, 5.15% cellulose, 8.34% crude ash) and roughage (hay and alfalfa mix) ad libitum. It was also reported that one spoon of ammonium chloride (about 10 g) was added to the drinking water (50 liters) once a week for the flock. Examination of the flock revealed the prepuce of sick lambs to be usually hot and painful on palpation, and preputial lesions from mild hyperemia to severe ulcerative changes were observed. Some lambs had necrosis and scabs on the prepuce, and the tissue was prone to bleeding when the scabs were removed. In addition, the prepuce of severely affected lambs was edematous, with circular ulcerative lesions at the external orifice of the urethra, narrowing the preputial entrance (Figure 1). The temperatures (ºC) of healthy and diseased lambs were 39.43 ± 0.13 and 39.78 ± 0.13, respectively. White blood cell (WBC) (/mm3) values in healthy and diseased lambs were 6916.66 ± 802.25 and 7773.33 ± 649.13, respectively. Similarly packet cell volumes (PCV) were 39.33% ± 1.45 and 47.00% ± 3.54 and urine pH levels were 7.66 ± 0.10 and 8.41 ± 0.15 (p <0.05) (Table 1). Corynebacterium renale, Staphylococcus intermedius, Streptococcus uberis, and Truperella pyogenes were isolated following microbiological cultivation of preputial swab samples from two diseased lambs. In addition, it was observed that one affected lamb had marked crystalluria (magnesium ammonium phosphate = struvite) in the urine sediment (Figure 2).

Table 1 - Clinical and laboratory findings with mean and standard error vaules of diseased and healthy lambs.

Parameters

Diseased group (n=6)

Healthy group (n=6)

Significance

Temperature (°C)

39.78 ± 0.13

39.43 ± 0.13

p = 0.092

Urine pH

8.41 ± 0.15

7.66 ± 0.10

p = 0.002

WBC (/mm )

7773.33 ± 649.13

6916.66 ± 802.25

p = 0.426

PCV %

47.00 ± 3.54

39.33 ± 1.45

p = 0.073

Figure 2 - Struvite (magnesium ammonium phosphate) crystals in urine sediment. Struvite crystals are refractile, colorless, tabular, threedimensional, wedge-like, prism-like crystals.

Based upon the clinical and laboratory examinations, a diagnosis of enzootic posthitis was made. For treatment, amoxicillin (Moksidif LA®, Ceva; 15 mg/kg, two doses 48 hours apart, IM), metamizole sodium (Calor®, Ekomed; 20 mg/kg daily for two days, IM), and vitamin C (Tekno-C®, Teknovet; 20 mg/kg for three days, IM) were provided and pomade rivanol (Rivanol®, Ülkem İlaç) was applied to the prepuce for three days. In addition, the feed ration was changed, reducing the lamb starter feed to half the amount and replacing it with corn-barley. Ammonium chloride was added to the drinking water three times a week. With this treatment and management practices, lesions in many animals were healed on the 7th day of treatment and completely healed on the 10th day.

DISCUSSION It has been reported that ulcerative posthitis can be seen in lambs of almost all ages, but it is more common in rams 1-4 years of age 6,9, especially in castrated rams older than three years 2. In many flocks, the disease has been described in older rams, especially during and after the mating period 6,8,13, but rarely reported in young lambs 4-5 months of age 11. It has been suggested that young lambs are more resistant to this disease because they use more protein for growth and development, and since adult lambs have a lower rate of growth, they can not tolerate excess protein intake 9,11. Also, the larger and wider preputial surface of older rams increases the incidence of this disease 9,11. Based on these claims, some suggest that young lambs are more resistant to this disease; however, here, we present severe and widespread enzootic posthitis in 75 to 90-day-old male lambs for the first time. Isolation of Corynebacterium renale and Truperella pyogenes in microbiological cultivation of preputial swabs from sick lambs support the literature 5,7,8. The destruction of epithelial cells by ammonia, which occurs with Corynebacterium renale, is of great importance in the pathogenesis of the disease 2,9,14 and Truperella pyogenes contributes to the pathogenesis of the disease by causing cell damage with its virulence factors 7,15.

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Y. Kaçar et al. Large Animal Review 2022; 28: 47-49

Apart from these findings, other bacteria isolated, such as Staphylococcus intermedius and Streptococcus uberis, were interpreted as contaminant bacteria. In this case report, similar to another study 9, body temperature was similar in sick and healthy lambs. The absence of a significant difference in total leukocyte counts between healthy and affected lambs has been interpreted as a result of the disease’s local course. Although there was no statistical difference in PCV% values between the sick and healthy lambs, the PCV% value was higher in the sick lambs (47.00 ± 3.54 vs. 39.33 ± 1.45). This situation is thought to be caused by the decrease in water consumption due to pain in the sick lambs and hemoconcentration due to anuria-dysuria. In this study, urine pH values increased in diseased lambs (p <0.05). In a study conducted by Loste et al.9 on diseased rams, the mean urine pH was 8.6, similar to our study. Although the urine pH levels were increased in both studies, differences between pH values in the two reports may be related to differences in animal ages (2.5-3 months vs. 1-4 years) and rations. It is known that as the amount of protein in the ration increases, the urea concentration in the urine, the urine pH, and the severity of lesions increase in parallel 9,11,12. Likewise, Loste et al.9 demonstrated the relationship between rations with high protein (16.8%), alkaline rumen (pH: 7.7), alkaline urine (pH: 8.6), and posthitis which led them to emphasize the importance of ration protein content. Similarly, in our study, lambs were fed lamb starter feed containing 17% protein, and sick lamb urine pH values were higher than healthy lambs. Struvite crystals, which are refractile, colorless, tabular, three-dimensional, wedge-like, prism-like, or coffin lid in appearance, can be seen in alkaline urine conditions 16. Thus, the detection of struvite crystals in the urine of one of the diseased lambs supports the clinical diagnosis of enzootic posthitis due to high protein ration consumption and alkaline urine pH.

CONCLUSION In conclusion, although ulcerative posthitis is known to affect male lambs of different ages, especially older lambs, for the first time in this case series, clinical and laboratory findings of the disease are described in young lambs aged 75-90 days after wean-

ing. Based on these findings, we emphasize that care should be taken in selecting lamb grower feeds and ration planning as prophylaxis for this disease.

References 1. Batmaz H. (2019). Enzootik Balanoposthitis. In: Koyun ve Keçilerin İç Hastalıkları-Semptomdan Tanıya Tanıdan Sağaltıma. 2nd ed., 154, Nobel Tıp Kitabevi, Ankara, Turkey. 2. Constable P.D., Hinchcliff K.W., Done S.H., Grünberg W. (2016).Enzootic Posthitis (Pizzle Rot, Sheath Rot, Balanoposthitis) and Vulvovaginitis (Scabby Ulcer). In: Veterinary Medicine 11th ed., 1552-1554, Elsevier, St. Louis Missouri. 3. Greig A. (2007). Ulcerative balanitis and vulvitis. In: Diseases of Sheep, Ed. Aitken I.D., 4th ed., 143-145, Blackwell publishing, USA. 4. Jones M., Miesner M.D., Baird A.N., Pugh D.G. (2012). Diseases of the Urinary System. In: Sheep and Goat Medicine, Eds. Pugh D.G., Baird A.N., 2nd ed., 357-359, Elsevier, Maryland Heights, Missouri. 5. Van Metre D.C, Rao S., Kimberling C.V., Morley P.S. (2002). Factors associated with failure in breeding soundness examination of Western USA rams. Prev Vet Med, 105(1-2): 118-126. 6. Watt B., Wait P., Slattery S. (2016). Ulcerative Balanitis in Rams-An Enigmatic Disease of Unknown Aetiology. Page 9-11 in Australian Sheep Veterinarian’s Conference, Dubbo. 7. Kidanemariam A., Gouws J., van Vuuren M., Gummow B. (2005). Ulcerative balanitis and vulvitis of Dorper sheep in South Africa: a study on its aetiology and clinical features. J S Afr Vet Assoc, 76(4): 197-203. 8. Pritchard G.C., Scholes S.F., Foster A.P., Mitchell E.S., Lawes J., Ibata G., Banks M. (2008). Ulcerative vulvitis and balanitis in sheep flocks. Vet Rec, 163(3): 86-89. 9. Loste A., Ramos J.J, García L., Ferrer L.M., Verde M.T. (2005). High prevalence of ulcerative posthitis in Rasa Aragonesa rams associated with a legume-rich diet. J Vet Med, A Physiol Pathol Clin Med, 52(4): 176-179. 10. Doherty M.L. (1985). Outbreak of posthitis in grazing wethers in Scotland. Vet Rec, 116(14): 372-373. 11. Kimberling C.V., Arnold K.S. (1983). Diseases of the urinary system of sheep and goats. Vet Clin North Am Large Anim Pract, 5(3): 637-655. 12. Van Metre D.C., Dawson Soto D.R. (2014). Diseases of the Renal System. In: Large Animal Internal Medicine, Eds. Smith B.P., Smith B.P., 5th ed., 895-897, Elsevier, St. Louis, Missouri. 13. Lapham-Simpson C. (2018). Unusual outbreak of penile lesions in rams. Page 145-148 in Proceedings of the Society of Sheep and Beef Cattle Veterinarians of the NZVA, New Zealand. 14. Scott P.R. (2015). Sheep Medicine, 2nd ed., 283-284, Boca Raton London New York, CRC Press. 15. Billington S.J, Songer J.G., Jost B.H. (2001). Molecular characterization of the pore-forming toxin, pyolysin, a major virulence determinant of Arcanobacterium pyogenes. Vet Microbiol, 82(3): 261-274. 16. Manjusha K.M., Sharun K., Kalaiselvan E., Kumar R., Saxena A.C., Kinjavdekar P., Pawde, A.M. (2021). Management of post-urethral urinary obstruction due to struvite uroliths in a female buffalo calf (Bubalus bubalis). Large Anim Rev, 27(3), 175-177.

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