Large Animal Review 6 - 2021

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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

06/21

Bimonthly, Year 27, Number 6, December 2021

LAR Large Animal Review

ISSN: 1124-4593

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

ORIGINAL ARTICLES BOVINE • Daily rhythm and seasonal variation of serum pancreatic alpha amilase in Holstein bovine maintained under natural environmental condition in Southern Hemisphere • Investigation of the efficacy of clinoptilolite in calves with cryptosporidiosis • Comparative efficacy of subconjunctival palpebral and bulbar injection of gentamicin-dexamethasone combination in dairy calves with epiphora • Effects of trimming on the thermographic pattern of claw sole in dairy cows • Assessment of animal and management based potential risk factor relation with claw health and lameness in dairy cows: A cross-sectional study • Frequency of White line disease and Sole ulcers and impact of hoof trimming in examined herds of Simmental cows • Effects of Thymus vulgaris L. Essential Oil and Compounds on Development and Quality of Bovine Preimplantation Embryos in vitro

OVINE • Investigation of the effect of Parapoxvirus ovis, Corynebacterium cutis lysate and vitamin c on immunosuppression caused by long-term transport Stress in Morkaraman sheep

CAPRINE • A novel approach for evaluating of goat milk quality: canonical correlation analysis between major milk composition parameters and fatty acid components in Damascus goats

PUBLIC HEALTH • In difesa delle razze autoctone

CASE REPORTS BOVINE • Extensive pleural abscess associated with congestive heart failure in a pregnant cow: an unusual presentation of hardware disease

EQUINE • Successful management of exuberant granulation tissue in two horses (Equus caballus) and a donkey (Equus asinus)

SOCIETÀ ITALIANA VETERINARI PER ANIMALI DA REDDITO ASSOCIAZIONE FEDERATA ANMVI

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R.D. Cerutti et al. Large Animal Review 2021; 27: 299-303

Daily rhythm and seasonal variation of serum pancreatic alpha amilase in Holstein bovine maintained under natural environmental condition in Southern Hemisphere

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RAÚL DELMAR CERUTTI1, MARÍA CRISTINA SCAGLIONE1, FRANCESCA ARFUSO2, CLAUDIA GIANNETTO2, ELISABETTA GIUDICE2, GIUSEPPE PICCIONE2* 1 2

Department of Veterinary Sciences, Universidad National de Litoral, Santa Fe, Argentina Department of Veterinary Sciences, University of Messina, Italy

SUMMARY In this study the effect of daily rhythm, season, gender and productive status on serum pancreatic alpha amylase was evaluated in clinically healthy Holstein cattle from Southern Hemisphere. A total of 18 animals were enrolled in the study and equally divided in three groups according to their gender and lactation class: Group A (dairy cows at first lactation class); Group B (dairy cows at third lactation class); Group C (bulls). Groups A and B were in the same productive throughout the investigation (dry period in winter, early lactation in spring, mild lactation in summer, end lactation in autumn). From each animal blood samples were collected every 3 hours over 24-hour period, every three months (winter, spring, summer, autumn) and on the serum obtained the concentration of serum alpha amylase has been assed. The application of two-way analysis of variance (ANOVA) showed a significant effect of season and group (gender and productive status) on alpha amylase. Cosinor rhythmometry showed a diurnal daily rhythmicity of alpha amylase in autumn, winter and spring in group A, in summer and autumn in group B and in all seasons in group C. This study suggests that seasonal and physiological conditions must be taken into consideration for the correct interpretation of serum chemistry in bovine in order to establish an accurate interpretation of laboratory data, critical in diagnosis, prognosis and treatment of diseases.

KEY WORDS Alpha amylase; bovine; daily rhythm; seasonal variation.

INTRODUCTION Different hematochemical parameters are affected by daily, circadian, and annual rhythm and, in particularly, when the daily rhythm has a large enough amplitude, it may modificate the clinical interpretation1,2. Animals have become adapted to the season of their environment by responding to the changes in the lengths of daylight and night in preparation for the climatic changes associated with the different seasons3. In particularly, in mammals, light signals detected from the photosensitive retinal ganglion cells serve entrain circadian rhythms to the daylight cycle that affect most physiological and biochemical processes4. As reported for other ungulates and mammals, bovine were expected to adjust their physiological responses on daily and seasonal basis, in accordance with the seasonal variations in the photoperiod and environmental conditions. Daily oscillation in the levels of physiological parameters has been described in a variety of animal species for a multiple of variables, including locomotor activity, body temperature, heart rate, blood pressure, hormonal secretion, and urinary excretion5-7.

Increasing the productivity or efficiency of farm animals necessarily involves changes in metabolism. In particular, several studies have been carried out in cows in order to evaluate the

Corresponding Author: Giuseppe Piccione (giuseppe.piccione@unime.it).

rhythmicity of the serum urea and ammonia concentration8, arterial blood gas9,10, blood electrolytes8, body temperature, peripheral concentrations of insulin, nitrogen11, acute phase proteins, redox states12,13, metabolic parameters including non-esterified fatty acids (NEFA) and total cholesterol14, as well as of the total locomotor activity15.

Alpha amylase catalyses the hydrolyses of alpha 1-4 glicosidic bonds present in starch, glycogen and other related carbohydrate. Serum alpha amylase concentration is normally low and fairly constant, and it is found to increase in different physiological and pathological conditions. It has been demonstrated that exocrine pancreas not only have 24-h variations of morphometric and functional parameters, but also it show a circadian modification of the pancreatic enzyme16. Nutrients play the most important role for regulation of secretory and motor activities at gastrointestinal level, whereas the influence of environmental factors such as circadian rhythm can be studied if the subjects are constantly fed or constantly fasting.

On the basis mentioned cases and considering that the environmental conditions are major physiological stressors which could affect the bovine’s biological system, the aim of this study was to evaluate and describe the daily and seasonal rhythm of the serum concentration of pancreatic alpha amylase in Holstein bovines under different environmental condition. Moreover, the possible difference in the daily and seasonal rhythmicity of this serum parameter between bulls and dairy cow was investigated.

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Daily rhythm and seasonal variation of serum pancreatic alpha amilase in Holstein bovine...

MATERIALS AND METHODS Animals Eighteen clinically healthy Holstein bovines were selected from a high production dairy farm in Villa Trinidad (Santa Fe, Argentina, longitude 26°13’ and latitude 58°17’). Their health status was evaluated based on rectal temperature, heart rate, respiratory profile, appetite, faecal consistency, and hematological profile. No subject showed any sign of diseases during the study. Animals were divided into three equal groups according to their gender and lactation classes: Group A (n=6) were dairy cows at first lactation class, aged 2 years old with a mean body weight of 500 ± 21 kg; Group B (n=6) were dairy cows at third lactation class, aged 4 years old with a mean body weight of 550 ± 20 kg; Group C (n=6) were bulls, aged 2 years old with a mean body weight of 520 ± 19 kg. The cows of the Groups A and B had the same gestational and productive period. Specifically, the cows were in dry period in winter, early lactation in spring, mild lactation in summer, end lactation in autumn. During lactation period dairy cows were milked twice a day at the same hour of the day (05:00 h and 15:00 h). All animals were raised under the uniform pasture conditions, and they were subject to natural variation in the light-dark cycle. Animals were under extensive management system allowing them to move in outdoors grazing area with pastures planted with alfalfa and water was available ad libitum. The protocol of this study was reviewed and approved in accordance with the standards recommended by the Guide for the Care and Use of Laboratory Animals and Directive 2010/63/EU.

Blood sampling From each animal blood samples were collected through jugular intravenous catheters (FEP G18*45mm) secured in place with suture (Vicryl, Ethicon, Somerville, NJ). Samples were collected every 3h over 24h period. Blood sampling was performed at the same hour of the day starting at 8:00 of day 1 and ending at 08:00 of day 2 in January (winter season), April (spring season), July (summer season) and October (autumn season).

The blood samples were centrifuged at 3.500 rpm for 10 min and the obtained sera stored at -20°C until analyzed. On serum samples the concentration of alpha amylase was evaluated by means of an automated analyzer (Metrolab 2100, Laboratory

Rodriguez Corswant SRL, Argentina) using commercially available kits.

At the same times of blood collection, thermal and hygrometric records were carried out for the whole study period by means of a data logger with a high reading accuracy and resolution (Model Tinytag Ultra 2, Gemini Data Logger, West Sussex, United Kingdom). Temperature-humidity index (THI), used as indicator of thermal comfort for cattle, was calculated using the U.S. Weather Bureau’s Temperature Humidity Index Formula for bovine specie17: THI[°C] = T° ambient + (0.36 * point of steam condensation) + 41.5.

Statistical analysis Two-way analysis of variance (ANOVA) was applied on the serum alpha amylase recorded at the same time of day (08:00) to assess significant effect of the experimental conditions (season, gender and productive status) on the tested parameter.

When significant differences were found Bonferroni’s post hoc comparison was applied. P values <0.05 were considered statistically significant.

Using cosinor rhythmometry18,19 four rhythmic parameters were determined: mesor (mean level), amplitude (half the range of oscillation), acrophase (time of peak) and robustness (strength of rhythmicity). Rhythm robustness (stationarity of a rhythm) was computed as the quotient of the variance associated with sinusoidal rhythmicity and the total variance of the time series6,19. Robustness greater than 60% is above noise level and indicates statistically significant rhythmicity. The cosinor procedure uses an F test to evaluate whether the amplitude of a cosine wave fitted to the data is significantly greater than zero6,19. Amplitude not significantly greater than zero implies absence of rhythmicity. All results were expressed as mean ± standard deviation (SD). P value <0.05 was considered statistically significant.

RESULTS The environmental temperature, relative humidity and THI recorded during the experimental period (winter, spring, summer and autumn) are shown in Figure 1.

Figure 1 - Means values of environmental temperature, relative humidity and temperature-humidity index (THI) recorded during the different seasons (winter, spring, summer and autumn).

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Figure 2 - Acrophase of alpha amylase daily rhythm observed in the three investigated groups (Group A - dairy cows at first lactation class; Group B - dairy cows at third lactation class; Group C - bulls) during the four seasons.

The application of two-way ANOVA showed a significant effect of season (F(3,60) = 54.25; p < 0.0001), gender and productive status (F(2,60) = 13.18; p < 0.0001) on alpha amylase. In particular, Bonferroni’s post hoc comparison showed statistically higher alpha amylase value during summer season than spring, winter and autumn in groups A and B. In group B the statistically lower alpha amylase value was observed in spring respect to summer, winter and autumn. In group C a statistically lower alpha amylase value was found in spring than winter. In all groups, the alpha amylase value was higher in winter than in autumn. During the summer season, group C showed a statistically lower alpha amylase value than the other two groups.

During spring season, group A showed a statistically higher alpha amylase value than the other two groups (Table 1).

A daily rhythmicity of alpha amylase was observed in autumn, winter and spring in group A, whereas the alpha amylase concentration obtained by cow belonging to group B showed a daily rhythmicity in summer and autumn. In group C daily rhythmicity of alpha amylase was observed in all seasons. The acrophase was always diurnal (Figure 2), and a percentage of robustness upper 60% was recorded (Figure 3).

DISCUSSION Our findings could be useful data for the evaluation of the animals’ adaptive mechanisms to environmental modifications

throughout the year. Ambient temperature between 5 and 25°C is considered the ideal ambient temperature for cow20. In the current study the ambient temperature and THI were within the critical zone during all seasons, except during the spring season, where these were upper that.

In physiological conditions, pancreatic exocrine secretion is closely correlated to intestinal motility; both are regulated by a complex interplay of hormonal and neural mediators to achieve optimal digestion and absorption of food and to maintain the physiological intraluminal milieu in the inter-digestive state. Moreover, morphological and functional parameters of the pancreas as well as intestinal motility are modulated by circadian rhythm and/or affected by the wake-sleep cycle16.

In our study, all groups were kept in pasture and were fed ad libitum. Alpha amylase showed a daily rhythmicity influenced by gender and productive conditions. In particular, bulls were the only group in which daily rhythmicity of alpha amylase was observed in all seasons. Moreover, in bulls the acrophase of daily rhythm of alpha amylase was recorded in the middle of the photophase during autumn, winter and spring.

Noteworthy, a nocturnal acrophase in summer season, precisely three hours after the sunset, was observed in bulls. This result needs a deeper investigation in order to clarify the reason of the mostly nocturnal activity found in bulls. Different daily fluctuations of alpha amylase were recorded in dairy cows, with an influence of age and stage of lactation. In particular, in dairy cows at the first lactation class a disruption of alpha amylase

Table 1 - Mean ± standard deviation of serum alpha amylase expressed in U/L recorded in the three experimental groups (Group A, dairy cows at first lactation class; Group B, dairy cows at third lactation class; Group C, bulls) at the same time of day (8:00) during the four seasons.

Autumn

Winter

Spring

Summer

Group A

33.58±7.00

75.43±11.38

63.65±8.88

148.24±57.55

Group B

41.09±4.73

77.46±19.91

7.70±1.72

142.33±33.53

Group C

38.58±5.37

74.56±18.37

27.01±4.78

51.64±13.91

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Daily rhythm and seasonal variation of serum pancreatic alpha amilase in Holstein bovine...

Figure 3 - Robustness of alpha amylase daily rhythm observed in the three investigated groups (Group A - dairy cows at first lactation class; Group B - dairy cows at third lactation class; Group C - bulls) during the four seasons. Vertical line indicates the noise level of 60% index of statistically significant rhythmicity.

daily rhythmicity was observed during summer season (mild lactation).

In the other three phase of lactation, acrophase was observed at five hours after sunrise.

In the dairy cows at the third lactation class, the disruption of alpha amylase daily rhythmicity was observed both during dry period and early lactation. Also, in this group, a nocturnal acrophase was recorded in the mild of lactation, five hours after sunset.

The only period in which the acrophase of alpha amylase daily rhythm was similar to the other groups was at the end of lactation in autumn.

Acknowledgments Not applicable.

Author Contributors Conceptualization, R.D.C., G.P., and M.C.S.; Methodology, C.G., F.A., and E.G.; Software, C.G.; Formal analysis, F.A., and C.G.; Investigation, G.P., and R.D.C.; Resources, M.C.S., and E.G.; Data curation, C.G., and F.A.; Writing original draft preparation, G.P.; Writing-review and editing, G.P., and C.G.; Visualization, R.D.C., M.C.S., and E.G.; Supervision, G.P.; Project administration, G.P. All authors have read and agreed to the published version of the manuscript.

Funding CONCLUSION According to the findings gathered in the present survey it can be concluded that various factors such as environmental conditions and productive levels influence the serum pancreatic alpha amylase concentration in Holstein cattle. This study provides new insights to plan properly the blood sampling schedule to assess the concentration of this metabolite according to the environmental conditions, sex and reproductive state of the animal. The knowledge of the temporal pattern of alpha amylase in relation to productive performance gives useful information about the time and the effect of administration of exogenous alpha amylase in cows. Also, this knowledge contributes to clarify the influence of the effect of some drugs on the alpha amylase secretion and activity.

Not applicable.

Ethical approval No approval from Ethics Committee was required. No invasive medical procedures were executed to perform the study.

The study was performed with the consent of the animals’ owner during the routinary clinical activity. Animal care and procedures are in accordance with the Guide for the Care and Use of Laboratory Animals and Directive 2010/63/EU for animal experiments (National law: D.L. 26/2014).

Data Availability Statement The data will be available by sending an email to the corresponding author.

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Conflict of Interest The authors declare no conflict of interest.

References 1.

Scaglione, M.C.; Cerutti R.D.; Arfuso F.; Rizzo M.; Pugliese M.; Piccione G. Daily rhythm of some haematological parameters in Holstein bovine maintained under natural conditions in southern hemisphere. Biol.

Rhythm Res. 2019, 50, 222-231.

2. Giannetto, C.; Piccione, G. Daily rhythms of 25 physiological variables in Bos taurus maintained under natural conditions. J. Appl. Biomed. 2009, 7, 55-61.

3. Koukkari, W.L.; Sothern, R.B. Introducing biological rhythm. New York: Springer Science Business Media Inc., 2006.

4. Mendez-Ferrer, S.; Chow, A.; Merad, M.; Frenette, P.S. Circadian rhythms influence hematopoietic stem cells. Curr. Opin. Hematol. 2009, 16, 235242.

5. Dunlap, J.C.; Loros, J.J.; De Coursey, P.J. Chronobiology: Biologcal Timekkeping. Sinauer, Sunderland, 2004.

6. Refinetti, R. Non stationary time series and the robustness of circadian rhythms. J. Theor. Biol. 2004, 571-581.

7. Piccione, G.; Caola, G.; Refinetti, R. Daily and estrous rhythmicity of body temperature in domestic cattle. BMC Physiol. 2003, 3, 7-14.

8. Piccione, G.; Grasso, F.; Fazio, F.; Assenza, A.; Caola, G. Influence of different schedules of feeding on daily rhythms of blood urea and ammonia concentrations in cows. Biol. Rhythm Res. 2007, 38, 133-139.

9. Piccione, G.; Caola, G.; Mortola, J.P. Day-night pattern of arterial blood gases in the cow. Resp. Physiol. Neurobiol. 2004, 140, 33-41.

10. Bajcsy, C.A.; Reiczigel, J.; Szenci, O. Circadian changes in blood ionized

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calcium, sodium, potassium and chloride concentrations and pH in cattle. Am. J. Vet. Res. 1999, 60, 945-948.

11. Lefcourt, A.M.; Huntngton, J.B.; Akers, R.M.; Wood, D.L.; Bitman, J. Circadian and ultradian rhythms of body temperature and peripheral concentrations of insulin and nitrogen in lactating dairy cow. Domest. Anim.

Endocrinol. 1999, 16, 41-55.

12. Giannetto, C.; Fazio, F.; Assenza, A.; Caola, G.; Pennisi, P.; Piccione, G. Circadian rhythms of redox states and total locomotor activity in dairy cattle. Czech J. Anim. Sci. 2010, 55, 183-189.

13. Giannetto, C.; Casella, S.; Giudice, E.; Marafioti, S.; Fazio, F.; Piccione, G. Daily rhythms of acute phase proteins in cattle under different natural environmental conditions. Livest. Sci. 2012, 149, 195-200.

14. Shehab-El-Deen, M.A.; Fadel, M.S.; Van Soom, A.; Saleh, S.Y.; Maes, D.; Leroy, J.L. Circadian rhythm of metabolic changes associated with summer heat stress in high-producing dairy cattle. Trop. Anim. Health Prod.

2010, 42(6), 1119-1125.

15. Piccione, G.; Giannetto, C.; Schembari, A.; Gianesella, M.; Morgante, M. A comparison of daily total locomotor activity between the lactation and the dry period in dairy cattle. Res. Vet. Sci. 2011, 91, 289-293.

16. Keller, J.; Layer, P. Circadian pancreatic enzyme pattern and relationship between secretory and motor activity in fasting humans. J. Appl. Physiol. 2002, 93, 592-600.

17. Monteverde, V.; Perillo, L.; Dara, S.; Vazzana, I.; Zumbo, A.; Piccione, G. Relationship between different environmental and house conditions on some acute phase markers in dairy cows. Large Anim. Rev. 2016, 22, 6569.

18. Nelson, W.; Tong, Y.L.; Lee, J.K.; Halberg, F. Methods for cosinor rhythmometry. Chronobiologia. 1979, 6, 305-323.

19. Refinetti, R. Circadian Physiology, 2nd ed. CRC Press, Boca Raton , 2005.

20. Roenfeldt, S. You can’t afford to ignore heat stress. Dairy Manag. 1998, 35(5), 6-12.

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P.F.P. Dinçer et al. Large Animal Review 2021; 27: 305-309

Investigation of the efficacy of clinoptilolite in calves with cryptosporidiosis

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PELIN FATOŞ POLAT DINÇER1*, ADEM ŞAHAN2, ISMAIL GÜNAL2, EGEMEN ERDEM ÖZTÜRK3 1 2 3

Departments of Internal Medicine, Faculty of Veterinary Medicine, Dokuz Eylül University, Izmir, Turkey Departments of Internal Medicine, Faculty of Veterinary Medicine, Harran University, Şanlıurfa, Turkey Departments of Biochemistry, Faculty of Veterinary Medicine, Harran University, Şanlıurfa, Turkey

SUMMARY This study aims to investigate the efficacy of clinoptilolite in calves infected with Cryptosporidium. For this purpose, 30 animals monoinfected with Cryptosporidium and 10 healthy calves as a control group were included in the study, and in total four groups were formed. Fluid therapy was administered to all three groups to correct dehydration and acidosis of the calves. Clinoptilolite was administered to the first group at an oral dose of 1 g/day and an oral dose of 1 g/day and standard treatment was applied to the second group. The third group was treated with only standard treatment. Blood gas (pH, HCO3-, Na+, Cl-, K+, Crea, BE) and blood biochemistry (AST, ALT, BUN, Glucose) measurements and feces scoring by inspection method (1=watery, 2=pudding consistency, 3=soft, 4=hard) were applied to the study groups. Statistical evaluation of the variables examined in the calves was made in SPSS 24.0 package program. In the second group, there was a significant increase in pH, HCO3- (p < 0.05) and BE (p < 0.01) compared to the first group, while a significant decrease in K+ (p < 0.05) value was detected. In the second group, increase in Na+, BE values (p < 0.05), and a significant decrease in K+ value (p < 0.05) compared to the third group were observed.

There was no significant difference among the groups in blood biochemistry results (p > 0.05). Feces scores of calves with diarrhea after treatment were statistically improved at p < 0.05 level in Groups 1 and 3, and at p < 0.01 level in Group 2. It was determined that there were 4 calves in the first group, 9 calves in the second group, and 7 in the third group which showed signs of improvement. As a result, it was concluded that, in addition to standard treatment, the inclusion of clinoptilolite at a daily dose of 1 g/kg orally for 5 days can contribute to the treatment of calves with Cryptosporidium, and that it is safe to be used on calves.

KEY WORDS Cryptosporidium, clinoptilolite, calf.

INTRODUCTION Zeolites have unique structures that can easily replace other molecules and cations with their negatively charged channels and positively charged alkali spaces. The most common zeolite mineral assemblages in nature are tuffs containing zeolite clinoptilolite and mordenite. The use of clinoptilolite-based products in veterinary and human medicine has increased in recent years due to its many positive medicinal properties such as ion exchange and adsorption capacity1.

Calf diarrhea is one of the leading neonatal period diseases, which is one of the important problems in calf breeding. The four most important factors reported in the world in calf diarrhea are rotavirus, coronavirus, Cryptosporidium parvum (C. parvum), and Escherichia coli (E. coli)2. Cryptosporidiosis is a zoonotic infection caused by protozoans of the genus Cryp-

Corresponding Author: Pelin Fatoş Polat Dinçer (pelinfatos.polat@deu.edu.tr).

tosporidium in mammals, reptiles, poultry, and fish. C. parvum is known to be the most disease-causing species in vertebrate animals and humans3. Although the life cycle of C. parvum is similar to cyst-forming microorganisms such as Toxoplasma and Eimeria, it is formed in Cryptosporidium in oocyst sporulation and thus is excreted with feces and shows infective characteristics4. Transmission occurs by oral ingestion of oocysts with food or water. It has been reported in aerosol transmission. The fact that oocysts can survive for an average of 6 months at 20 °C in the external environment and are resistant to disinfectants is an important factor in the spread of infection5. C. parvum is one of the most common pathogenic agents found in calves less than 3 weeks old. The disease, with symptoms such as severe diarrhea, dehydration, weight loss, and slow growth, causes death and serious economic losses6. Various anti-cryptosporidium (halofuginone lactate, spiramycin, lasalocid) effective substances have been used in its treatment, but with limited success5. In this context, new treatment options are needed for cryptosporidiosis.

Studies on the use of clinoptilolite in the treatment of enteric protozoa are limited in our country. It has been determined that

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Investigation of the efficacy of clinoptilolite in calves with cryptosporidiosis

different concentrations of nanoparticles containing clinoptilolite of Cryptosporidium, which causes diarrhea in humans and many animal species, have anti-cryptosporidium properties during in vitro infection7. Also, Ural et al. used oral clinoptilolite for Giardiasis control in goat kids in their study in 20178.

In this study, it was aimed to investigate the effectiveness of clinoptilolite in diarrhea caused by Cryptosporidium with high mortality and morbidity in calves.

MATERIALS AND METHODS The animal material of the study was composed of 30 calves with acute diarrhea and 10 healthy calves 7-30 days. They were of various breeds and sexes and were in public-owned enterprises in the Küçük Menderes region between September and December 2020. G. duodenalis, Coronavirus, Rotavirus, Cryptosporidium, and E. coli K99sp. antigens were screened through rapid test kits (Anigen Rapid Bovid-5 Ag Test Kit, Bionote Lab.) for the differential diagnosis of some infectious agents causing diarrhea. Thirty animals monoinfected with Cryptosporidium from a total of 116 animals suffering from acute diarrhea and 10 healthy calves as a control group were included in the study. Cryptosporidium positive calves were randomly divided into three groups with n=10 in each group. To correct the dehydration and acidosis of the calves in all three groups, fluid therapy was applied within the first 24 hours. Clinoptilolite treatment dose was determined as 1 g/kg9. Afterward, clinoptilolite was administered orally at a dose of 1 g/kg for 5 days to the 1st group, the 2nd group was given clinoptilolite at a dose of 1 g/kg orally and drug treatment (Aminosidin 70 mg/kg orally once a day for 5 days, Meloxicam® 0.5 mg/kg IV single dose), and the third group received drug treatment (Aminosidin 70 mg/kg orally once a day for 5 days, Meloxicam® 0.5 mg/kg IV single dose). The 4th group consisted of n = 10 healthy calves.

Feces scoring (1=watery, 2=pudding consistency, 3=soft, 4=hard) was performed on the 1st and 5th days of the study from all groups. For blood gas, blood was taken from V. Jugularis into 2 ml blood gas injector according to the procedure and without waiting, and pH, bicarbonate (HCO3-), sodium (Na++), chlorine (Cl-), potassium (K+), creatine (Crea), and Base Excess (BE) levels were measured in the blood gas device (Alere Epoc, Germany). For blood biochemistry, 5 ml of blood was taken from Jugular V. into tubes without anticoagulant and was centrifuged at 3000 rpm for 10 minutes. Serums were stored at -20° until measurements were made. Then AST (U/L), ALT (U/L), BUN (mmol/L) and glucose measurements were taken in the biochemistry device (Spotchem EZ SP-4430, Japan).This study was carried out with the approval of the Dokuz Eylül University Animal Experiments Local Ethics Committee, dated 12.08.2020, numbered 25/2020.

Statistical analysis The normality of distribution of continuous variables was tested by Shaphiro Wilk test. One-way ANOVA and LSD test (for normal data) and Kruskal Wallis and Dunn multiple comparison tests (for non-normal data) were used to compare numerical data across the three groups. Paired t test (for normal data) and Wilcoxon test (for non-normal data) were performed as before-after comparisons. Statistical analysis was performed with SPSS for Windows version 24.0 and a P< 0.05 was accepted as statistically significant.

RESULTS Study results are summarized in Tables 1-3. Four out of 10 animals in group 1 treated only with clinoptilolite, 9 out of 10 animals in group 2 treated with clinoptilolite and drug treatment together, 7 out of 10 animals in group 3 treated with medica-

Table 1 - Blood gas values of diarrheal calves before, after treatment and statistical comparison between groups.

Parameters

Group 1 B.T (n= 10)

Group 2 A.T (n= 4)

B.T (n= 10)

Group 3 A.T (n= 9)

B.T (n= 10)

Group 4

A.T (n= 7)

( n=10 )

pH

7.03 ± 0.08

7.22 ± 0.06

7.01 ± 0.1

7.28 ± 0.05

7.03 ± 0.08

7.3 ± 0.02

7.31 ± 0.03

HCO3-

12.48 ± 1.7

21.34 ± 5.48

13.36 ± 1.28

25.97 ± 3.29

13.41 ± 1.46

27.11 ± 2.2

25.68 ± 1.92

Na+ (mmol/L)

133.51 ± 2.22

139.28 ± 2.85

134.92 ± 2.78

140.44 ± 1.23

135.83 ± 1.62

138.12 ± 2.01

138.63 ± 3.81

Cl- (mmol/L)

95.83 ± 2.85

101.8 ± 1.17

97.5 ± 0.84

100.54 ± 1.62

97.14 ± 1.34

101.61 ± 1.27

102.29 ± 1.07

K (mmol/L)

6.22 ± 0.06

5.08 ± 0.09

6.01 ± 0.2

4.93 ± 0.02

6.18 ± 0.12

5.02 ± 0.1

4.21 ± 0.11

Crea (mg/dL)

1.66 ± 0.07

0.67 ± 0.04

1.68 ± 0.05

0.6 ± 0.06

1.68 ± 0.07

0.62 ± 0.05

0.71 ± 0.06

BE

-15.74 ± 3.8

3.72 ± 5.4

-15.36 ± 3.41

5.71 ± 0.23

-14.82 ± 3.99

5.64 ± 3.47

5.68 ± 1.08

+

Statistical Evaluation In-Groups

Between Groups

Group 1

Group 2

Group 3

Group 1/Group 2

Group 2/Group 3

pH

*

**

**

*

N.S

HCO3-

*

**

*

*

N.S

+

Na (mmol/L)

*

**

**

N.S

*

Cl- (mmol/L)

*

**

*

N.S

N.S

K+ (mmol/L)

*

**

*

*

*

Crea (mg/dL)

**

**

**

N.S

N.S

BE

*

**

*

**

*

N.S: Not Significant, * : P < 0.05, ** : P < 0,01, B.T: Before Treatment, A.T: After Treatment.

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307

Table 2 - Blood biochemistry values of diarrheal calves before, after treatment and statistical comparison between groups.

Parameters

Group 1 B.T (n= 10) A.T (n= 4)

Group 2 B.T (n= 10) A.T (n= 9)

Group 3 B.T (n= 10) A.T (n= 7)

Group 4 ( n=10 )

AST (U/L)

152.1 ± 40.2

42.2 ± 5.31

125.2 ± 27.7

43.11 ± 6.95

141.8 ± 36.58

45.14 ± 4.14

44.8 ± 2.49

ALT (U/L)

29 ± 6.93

12.2 ± 1.48

26.2 ± 4.61

12.89 ± 2.98

27 ± 5.46

11.71 ± 2.14

14.8 ± 2.35

BUN (mg/dL)

44.86 ± 4.5

5.78 ± 0.69

45.5 ± 3.65

5.61 ± 1.15

44.32 ± 4.39

6 ± 0.96

5.23 ± 1.1

Glucose (g/dL)

54.2 ± 10.75

74.6 ± 1.14

63 ± 6.57

73.89 ± 1.45

58.8 ± 9.89

74.71 ± 1.98

70 ± 6.48

Statistical Evaluation In-Groups

AST (U/L)

Between Groups

Group 1

Group 2

Group 3

Group 1/Group 2

Group 2/Group 3

*

**

*

N.S

N.S

ALT (U/L)

*

*

*

N.S

N.S

BUN (mg/dL)

**

**

**

N.S

N.S

Glucose (g/dL)

*

**

*

N.S

N.S

N.S: Not Significant, * : P < 0.05, ** : P < 0,01, B.T: Before Treatment, A.T: After Treatment.

tion alone showed improvement at the end of the study. Group results of diarrheal calves according to blood gas values before and after treatment are as follows: In Group 1 (clinoptilolite) calves, a significant increase (p < 0.05) was determined in blood pH, HCO3-, Na+, Cl-, BE values after treatment, while a significant decrease in K+ and Crea values (p < 0.05, p < 0.01) was detected. In group 2 (clinoptilolite+drug) calves showed a significant increase (p < 0.01) in blood pH, HCO3-, Na+, Cl-, BE values after treatment, and a significant decrease (p < 0.01) in K+ and Crea values. In group 3 (drug) calves, a significant increase was detected in blood pH, Na+ (p < 0.01) and HCO3-, Cl-, BE (p < 0.05) values after treatment, while a significant decrease in K+ and Crea values (p < 0.05, p < 0.01) was observed.

Post-treatment blood gas results among the groups are as follows: a significant increase in pH, HCO3- (p < 0.05) and BE (p < 0.01) was detected in Group 2 calves compared to Group 1, while a significant decrease in K+ value (p < 0.05) was detected. A significant increase in Na+, BE values (p < 0.05), and a significant decrease in K+ value (p < 0.05) were found in Group 2 calves compared to Group 3 (Table 1). In-group results of diarrheal calves according to blood biochemistry values before and after treatment are as follows: In group 1 calves, there was a significant decrease in AST, ALT (p < 0.05), and BUN (p < 0.01) values after treatment, and a significant increase in glucose value (p < 0.05). In group 2 calves, a significant decrease was detected in AST, BUN (p < 0.01) and ALT (p < 0.05) val-

Table 3 - Feces scores of calves with diarrhea before, after treatment and statistical comparison between groups.

Groups

B.T

A.T

Median [25%-75%]

Median [25%-75%]

1 [1 -2 ]

3 [3 -4 ]

*

Group 2

1 [1 -2 ]

4 [3 -4 ]

**

Group 3

1.5 [1 -2 ]

4 [4 -4 ]

*

Group 4

4 [4 -4 ]

4 [4 -4 ]

Group 1

Between group comparisons P

* : P < 0.05, ** : P < 0,01, B.T: Before Treatment, A.T: After Treatment 1=watery, 2=pudding consistency, 3=soft, 4=hard

ues, while a significant increase in glucose value (p < 0.01) was detected after treatment. After treatment in group 3 calves, a significant decrease in AST, ALT (p < 0.05) and BUN (p < 0.01) values and a significant increase in glucose value (p < 0.05) were detected in calves. No significant difference was found among the blood biochemistry results of the groups after treatment (Table 2). In the feces scores of calves with diarrhea after treatment, a statistically significant improvement at the level of p < 0.05 in Groups 1 and 3, and an improvement at the level of p < 0.01 in Group 2 were observed (Table 3).

DISCUSSION Cryptosporidium is particularly common in calves with insufficient immunity, who do not receive sufficient and high-quality colostrum and are exposed to stress such as cold and bad ventilation. Due to the lack of a complete and effective treatment, it is important to evaluate new treatment options. Clinoptilolite has been used by different researchers and its effectiveness in diarrhea cases has been investigated. Panousis et al. reported that clinoptilolite decreases intestinal transit rate, absorbs water, and tightens feces consistency10. Sadeghi and Shawrang reported in their study that clinoptilolite added to milk reduced the formation of diarrhea. It has been suggested that this effect is achieved by directly affecting the osmotic pressure in the gastrointestinal system and creating a change in metabolic acidosis9. In this study, the effectiveness of clinoptilolite in addition to the standard treatment protocol was investigated in cases of calf diarrhea caused by Cryptosporidium.

Kasari11 associated decreases in pH and HCO3- values with metabolic acidosis in 1999. In this study, in the comparisons within groups, it was determined that pH and HCO3- values increased significantly in all three groups after treatment. The findings obtained are consistent with the research12,13. When the post-treatment values in Groups 1, 2 and 3 were compared, there was no significant difference between Groups 2 and 3, while the difference between Groups 1 and 2 was significant. It was concluded that the clinoptilolite used for supportive medicine contributes to the healing.

In the comparisons within the group, it was observed that Na+

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Investigation of the efficacy of clinoptilolite in calves with cryptosporidiosis

and Cl- values increased after treatment. It has been reported that hyponatremia and hypochloremia occurring in diarrhea cases are associated with Na+ and Cl- lost by excessive feces and intestinal secretions14. However, the data we obtained were similar to the research12,15. Başer and Civelek reported13 in their study in 2013 that there was no significant difference in the amount of Na+ between healthy calves and calves with diarrhea. In the statistical evaluation after the treatment between the groups, it was found that the Na+ value of group 2 increased compared to group 3 where only the drug was used. BE values increased in all calves after treatment, and the findings are in parallel with the research16. In the statistical evaluation between all groups, a significant difference was found after the treatment.

Blood K+ concentration increases as fluid and HCO3- loss increases in calves with diarrhea12,17. Despite the loss of K+ in diarrhea, the increased concentration in the blood was explained by the replacement of intracellular ion potassium with hydrogen26. The data by Awadalla reported18 that such an increase may be due to oliguria or anuria because the kidneys cannot eliminate excess potassium. It was observed that the intragroup K+ values decreased after the treatment and the results obtained were found to be compatible with the research11,12,13. When the values after treatment in Group 1, 2 and 3 were compared, a significant decrease was found in Group 2 and a statistically significant difference among the three groups.

Urea and creatinine levels increase in cases of infection, high fever, anorexia, and especially dehydration19. The reason for the increase in urea and creatinine in diarrhea calves has been explaıned as a decrease in renal perfusion along with dehydration12. In the study, it was found that serum creatinine concentration measured in dehydrated neonatal calves before treatment increased statistically after treatment. These findings are similar to the results of different studies12,20. When the post-treatment values were compared in groups 1, 2 and 3 no significant difference was found between creatinine levels. It has been reported that the addition of clinoptilolite at different rates to the diets of lambs21 and chickens22 did not make a significant difference in creatinine level. These results are in line with our study.

Increased AST activity in serum is a sensitive indicator of liver damage, and the damage is subclinical. In addition, AST enzyme level increases with anterior stomach tension, passive congestion, and muscle breakdown23. In this study, it is reported that the pre-treatment AST amount increased in all three groups in accordance with other studies13,20 in the in-group comparisons, and this may be related to liver damage caused by endotoxemia. When the post-treatment values in groups 1, 2 and 3 were compared, it was concluded that the difference between AST and ALT was not significant and that clinoptilolite had no effect on parenchymatous organs. The results obtained are similar to the results of different research groups24.

One of the most important complications of diarrhea in calves is hypoglycemia25. Since newborns are born with a limited energy reserve, any condition that affects energy intake will result in a disturbance in blood glucose balance26. In the study, it was determined that glucose increased statistically in the ingroup comparison after treatment. Santos et al. reported in their study in 2002 that Salmonella-infected calves with diarrhea were hypoglycemic27. Our study results are in line with the results of other research28. When the post-treatment values were compared in groups 1, 2 and 3 no significant difference was found among glucose levels. In their study conducted in 2018,

Uyarlar et al. reported that clinoptilolite did not affect blood glucose level in metabolic disease-free cows with similar milk yield up to the twenty-eighth day of lactation29. In addition, blood gas and blood biochemistry values obtained after treatment in the study were found to be between physiological values30.

Feces score was almost the same in the three groups before treatment (median 1-2), while a significant increase was seen after treatment (median 3-4). A statistically significant increase in feces score was found when Group 2 was compared with the other groups. With its ion exchange9 and toxic agents removal feature1, clinoptilolite can be viewed as an alternative in the treatment of acute diarrhea and as a supplement to homeostasis.

CONCLUSIONS As a result, in addition to standard treatment, oral clinoptilolite at a dose of 1 g/kg per day for 5 days can contribute to the treatment of calves with Cryptosporidium and its use in calves is safe. Considering the ineffectiveness of drugs used in the treatment of diarrhea caused by Cryptosporidium in calves, increasing calf deaths, and economic losses, the use of clinoptilolite can be considered as a strong alternative in increasing treatment success rate. In future studies, it is thought that the effectiveness of clinoptilolite can be demonstrated from different angles by including more infected animals with Cryptosporidium diarrhea, using different doses of clinoptilolite, and determining the number of Cryptosporidium oocysts in the feces.

References 1.

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2. Fuente R., Garcia A., Ruiz-Santa-Quiteria J.A., Luzón M., Cid D., Garcıa S., Orden J.A., Gómez-Bautista M. (1998). Proportional morbidity rates of enteropathogens among diarrheic dairy calves in central Spain.

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3. Özcel M.A., Özbel Y., Ak M. (2007). Özcel’in Tıbbi Parazit Hastalıkları.

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4. Abrahamsen M.S., Templeton T.J., Enomoto S., Abrahante J.E., Zhu G., Lancto C.A., Deng M., Liu C., Widmer G., Tzipori S., Buck G.A., Xu P., Bankier A.T., Dear P.H., Konfortov B.A., Spriggs H.F., Iyer L., Anantharaman V., Aravind L., Kapur V. (2004). Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Sci, 304: 441-445.

5. Sevinç F. (2004). Ruminatlarda cryptosporidiosis. Vet Bil Derg, 4: 7984.

6. Moore D.A., Zeman D.H. (1991). Cryptosporidiosis in neonatal calves: 277 cases (1986–1987). J. Am. Vet Med Assoc, 11: 1969-1971.

7. Karao lan G. (2017). Klinoptilolit içeren polimer nanopartiküllerin cryptosporidium parvum üzerine in vitro etkinli inin ara tırılması. Master’s Thesis. Adnan Menderes Üni Sa Bil Enst, Aydın.

8. Ural D.A., Erdo an H, Toplu S., Ayan A. (2017). Application of oral clinoptilolite intended for prevention of giardiasis in goat kids. Kocatepe Vet Derg, 10: 158-163.

9. Sadeghi A.A., Shawrang P. (2008). Effects of natural zeolite clinoptilolite on passive immunity and diarrhea in newborn Holstein calves.

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10. Pourliotis K., Karatzia M.A., Florou-Paneri P., Katsoulos P.D., Karatzias H. (2012). Effects of dietary inclusion of clinoptilolite in colostrum and milk of dairy calves on absorption of antibodies against Escherichia coli and the incidence of diarrhea. Anim Feed Sci Technol, 172: 136-140.

11. Kasari T.R. (1999). Metabolic acidosis in calves. Vet Clin North Am Food Anim Pract, 3: 473-486.

12. Sahal M., Kurtdede A., Börkü M.K., Ünsüren H., mren H.Y., Özlem M.B., Kalınbacak A. (1994). Yeni dogan ishalli buza ıların klinik bulguları ve asit-baz dengesi dikkate alınarak sodyum bikarbonat ve elek-

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13. Başer D.F., Cıvelek T. (2013). Akut shalli Neonatal Buza ılarda Venöz Asit-Baz Durumu ve Renal Fonksiyon Arası Korelasyon. Kocatepe Vet Derg, 1: 25-31.

14. Sobiech P., Rekawek W., Targonski M.A., Zarcznska K., Snarska A., Stopyra A. (2013). Changes in blood acid base balance parameters and coagulation profile during diarrhea in calves, Pol J Vet Sci,1: 453-459.

15. Ghanem M.M., Abd El-Raof Y.M., El-Nady H.A., El-khaiat HM. (2016). Clinical and haemato-biochemical changes of parasitic enteritis in buffalo with reference to acute phase protein. BJAS, 2: 75-83.

16. Trefz F.M., Lorch A., Feist M., Sauter-Louis C., Lorenz I. (2012). Construction and validation of a decision tree for treating metabolic acidosis in calves with neonatal diarrhea. BMC Vet Res, 8: 1-17.

17. Hall G.A., Jones P.W., Morgan J.H. (1992). Calf diarhoea. In: Bovine Medicine Disease and Husbandry of Cattle, Ed. Andrews A.H., Blowey R.W., Boyd H., Eddy R.G., 1st ed., Blackwell Science Ltd.,Oxford.

18. Awadalla S.A.M. (1996). Studies on enteritis in newly-born buffalo calves. PhD thesis, Fac of Vet Med, Zagazig Univ.

19. Gokce H.I., Woldehiwet Z. (1999). The effects of Ehrlichia (Cytoecetes) phagochytophila on the clinical chemistry of sheep and goats. J Vet Med, 46: 93-103.

20. Merhan O., Bozukluhan K., Gökçe G., Yılmaz Y. (2016). shalli buzaılarda haptoglobin, seruloplazmin ve bazı biyokimyasal parametre düzeylerinin ara tırılması. FÜ Sa Bil Vet Derg, 3: 195-198.

21. Toprak N.N., Yılmaz A., Öztürk E., Yigit O., Cedden F. (2016). Effect of micronized zeolite addition to lamb concentrate feeds on growth performance and some blood chemistry and metabolites. S Afr J Anim Sci, 3: 313-320.

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Zarcula S., Mircu C., Bonca G., Kirovski D., Otava G., Khoie-Ahmadi M., Tulcan C. (2014). Effects of clinoptilolite addition to colostrum on the concentration of serum proteins, minerals, enzyme activities in neonatal calves. IJAC, 2: 2290-2296.

Trefz F.M., Lorenz I., Lorch A., Constable P.D. (2017). Clinical signs, profound acidemia, hypoglycemia, and hypernatremia are predictive of mortality in 1,400 critically ill neonatal calves with diarrhea. PLoS ONE, 8e: 0182938.

Carlson G.P. (2009). Clinical chemistry tests. In: Large Animal Internal Medicine, Ed. Smith, B.P., 4th ed., 375-397, Mosby Elsevier, St. Louis.

Santos R.L., Tsolis R.M., Bäumler A.J., Adams L.G. (2002). Hematologic and serum biochemical changes in Salmonella ser Typhimurium-infected calves. Am J Vet Res, 8: 1145-1150.

Cleek J.L., Phillips R.W. (1981). Evaluation of a commercial preparation for oral therapy of diarrhea in neonatal calves: Administration by suckling versus intubation. JAVMA, 9: 977-981.

Uyarlar C., Akkaya A., Gültepe B., Eren E. (2018). Effects of yeast (Saccharomyces cerevisiae) and clinoptilolite administration on milk yield and some metabolic parameters in early lactation dairy cows. Vet Hek Der Derg, 1: 3-10.

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M. Fazili et al. Large Animal Review 2021; 27: 311-315

Comparative efficacy of subconjunctival palpebral and bulbar injection of gentamicin-dexamethasone combination in dairy calves with epiphora

311

N

MUJEEB FAZILIa*, AKEEL BEIGHa, ANEES SHAHa, MOHAMMAD ALTAF BHATb, AZMAT KHANc, RAMEEZ ALIa, AIJAZ DARa, SYED ANSARa, PARVAIZ RESHIa a

Mountain Livestock Research Institute (MLRI), Division of Veterinary Microbiology, Faculty of Veterinary Sciences & AH, c Associate Director, Directorate of Research, Shere Kashmir University of Agricultural Sciences & Technology of Kashmir, Manasbal, Safapora, District Ganderbal, Kashmir, India. Pin:193504 b

SUMMARY A prospective study was planned to compare the palpebral and bulbar subconjunctival injection of gentamicin-dexamethasone in dairy calves showing epiphora on exposure to dust pollution. Calves showing bilateral epiphora (n=12) and no epiphora (n=6) with mean age 6 months and weight 56.0 kg were included. The average periorbital matting of the facial hair extended to 4.8 cm.

The value was 4.92 ± 0.59 cm and 5.17 ± 0.90 cm in the eyes subjected subsequently to epibulbar or subpalpebral injection respectively. Schirmer Tear Test values (mean ± s.e) in epiphoric eyes (27.67 ± 3.30 mm/min) were non-significantly higher than non-epiphoric (21.00 ± 0.97 mm/min). Conjunctival swabs revealed Gram positive and -negative commensals. One eye was injected 0.5 mL of the combination epibulbar and the contralateral subpalpebral routes. Calves in the control group received 0.5mL normal saline. In six calves, the epiphora subsided in three days and in four more it took seven days. Compared to day 0 values, the reduction of 98.9 ± 7.1% and 86.1 ± 10.9% respectively in facial matting did not differ significantly (P > 0.05). Recurrence or additional cases were not noticed subsequently in the farm. Moderate rains nine days following treatment of calves settled dust and reduced air pollution. It is concluded that the eyes in the calves are more prone than adults to the dust pollution. Both subpalpebral and epibulbar injection of gentamicin-dexamethasone are similarly effective in epiphoric calves. The studies evaluating the deleterious effects of air pollution on the ophthalmic health in food animals deserve more attention in future.

KEY WORDS Calves; Epibulbar; Epiphora; Ophthalmology; Subconjunctival; Subpalpebral.

INTRODUCTION Epiphora is defined as an abnormal flow of tears down the face1.

Numerous etiologies that may lead to excessive tears include the following categories: appositional abnormalities of the eyelids, neurogenic lacrimal hypersecretory disorders, obstructive lacrimal drainage disorders, ocular surface disorders. The last two disorders are commonly encountered in bovine clinical practice. Unlike obstructive lacrimal drainage, the ocular surface disorders are predominantly encountered as outbreaks. Ocular surface dryness or irritation stimulates the reflex arc of the fifth and seventh cranial nerves, producing excessive tear secretion. When the lacrimal drainage system is unable to handle the increased tear volume, overflow occurs. Infectious Bovine Keratoconjunctivitis (IBK) or Pink Eye a severe transmissible ocular infectious disease caused by Moraxella bovis occurs during summer and autumn months when the fly populations are active2. The outbreaks of iritis and uveitis caused by Listeria monocytogens (silage eye) associated with

Corresponding Author: Mujeeb ur Rehman Fazili (fazili_mr@yahoo.co.in).

silage feeding are also accompanied by epiphora3. However, in most of the animals with these disease conditions, the alterations in cornea, conjunctiva and or eyelids are easily noticed. Environmental factors and causes including stress, dust, UV light and foreign bodies4, 5, 6. Air pollution is a serious human health issue causing complaints of eye redness, irritation, watering, foreign body sensation, and blurring of vision. According to the World Health Organization (WHO), the air pollution consists of different particulate matter (PM) size. PM10 with particle aerodynamic diameter < 10 micrometer are generated from construction and the road dust5.

Successful management of the tearing patient requires the clinician to determine the underlying cause of the epiphora. Being multifactorial in origin, the final diagnosis may always not be possible particularly under field clinical settings. Drugs may be delivered to the eye by topical application, subconjunctival injection, retrobulbar injection, intraocular injection and systemic administration. Systemic or topical use of antibiotics or a combination of both with or without corticosteroid are used in many infectious cases3. Subconjunctival administration of antimicrobials aims to reduce treatment costs and total dosages of drug while achieving higher ocular drug concentrations7.

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Comparative efficacy of subconjunctival palpebral and bulbar injection of gentamicin-dexamethasone combination

Figure 1 - Dusty path leading to the grazing area.

The objective of this investigation was to compare the therapeutic efficacy of gentamicin-dexamethasone combination injected subconjunctival via palpebral or bulbar routes in calves with epiphora.

MATERIALS AND METHODS The study was conducted in Mountain Livestock Research Institute (MLRI), SKUAST-Kashmir. Calves (n=34) were housed together in a room during the nights and in the paddock during the day time. They were fed concentrate feed 0.5 kg once and chapped fresh sorghum roughage 3.0 kg twice per day. Two sessions of grazing (2 hours each) in the nearby land were also allowed daily. The path leading to the grazing area and one located adjacent to the paddock was excessively dusty (Figure 1) due to prolonged dry spell.

The ailment (epiphora) developed naturally and suddenly in the calves included in this study. After obtaining consent from the head of the university farm, the treatment protocol was approved by the MLRI clinical board.

Fourteen14 of the 34 calves suddenly showed bilateral (n=12) or unilateral (n=2) epiphora (Figure 2) in the second week of November 2020. Majority of the affected calves were Jersey (n=11), the remaining (n=3) crossbred Holstein Frisian.

Females (n=10) outnumbered the males (n=4). The mean age and body weight of the calves was 6 months and 56.0 kgs respectively.

The animals with epiphora were watched in the paddock one by one up to 10 minutes to identify any vision and ophthalmic anatomical defect, blepharospasm, photophobia, soiling or scratching the ophthalmic areas.

For detailed clinical examination, they were restrained in standing position by two attendants, one holding the head and oth-

er preventing backward or side way movement. Starting from the dependent inner canthus of the eye, the extent to which the overflowing tears had matted the facial hair was assessed using an ordinary scale (Figure 2). Palpebral and corneal reflexes were tested before holding the eyelids apart for close ophthalmic examination.

The Schirmer Tear Test (STT) was conducted in six calves (one eye per calf) with unilateral or bilateral epiphora. Six more calves (one eye per calf) with no epiphora (negative control) were also subjected to this test. The calves were restrained in standing with minimal eye manipulations. A STT strip placed in the lower conjunctival fornix was removed after one minute and the value recoded in millimeters.

Taking aseptic measures, conjunctival swabs were obtained from a total of 12 calves (one eye per calf); six animals with and six

Figure 2 - Calf with epiphora: matted facial hair assessment.

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Figure 3 - Epibulbar injection in a calf with epiphora.

Figure 4 - Subpalpebral injection in a calf with epiphora.

more without epiphora. The swabs were inoculated on blood agar and maintained in aerobic incubation at 37 oC for 24 hours.

All the calves including those of the control group were instilled homatropine hydrobromide eye drops (Homide, 2%, Indoco Remidies, Ltd) bilaterally and shifted to the adjacent room with low light for a period of 15 minutes. The head of the calves was subsequently restrained in standing position by an attendant for fundus examination using direct opthaolomscope (Heine Mini3000 LED, Germany).

The calves with bilateral epiphora (n=12) and those (n=6) without epiphora were then physically restrained in lateral recumbency on a table in the paddock and given subconjunctival injection. Using an insulin syringe, 0.25 mL gentamicin sulphate (Inj. Gentalab-40 mg/ml, Laborate Pharma India Ltd.) mixed with 0.25 mL dexamethasone sodium (Inj. Dexona- 4 mg/ml, Zydus Health care Ltd.) was injected per eye in all the calves (n=12) included in the treatment group. Those (n=6) without epiphora (Control group) were injected 0.5 mL normal saline (NS).

A coin flip was utilized to randomly determine which eye was to be given the injection in the epibulbar area (Figure 3) and the contralateral was then included in the subpalpebral (Figure 4) group. Two calves with unilateral epiphora were excluded from the study and given treatment separately.

The detailed clinical examination and the vision tests were repeated on day 3 and day 7 in all the calves included in the study.

Student’s T test was applied to the Schirmer tear test and reduction percentage in the periocular matting values between the two routes of the drug administration. The significance was set at P < 0.05.

(serous) in all the calves. In the calves of the treatment group, the overall average distance traversed by tears in the periorbital skin was 4.8 cm (range 1.0 cm to 13.0 cm) on day 0. In the eyes given subsequently injection in the palpebral subconjunctiva, the tear over-flow was 4.92 ± 0.59 cm (mean ± s.e) and in those injected by bulbar route 5.17 ± 0.90 cm.

STT values (27.67 ± 3.30 mm/min) in epiphoric were higher than non-epiphoric (21.00 ± 0.97 mm/min) eyes but the difference was not statistically significant (P > 0.05).

All the conjunctival swab samples showed similar pattern of growth with mixed type of colonies. However, the samples collected from calves with epiphora showed more number of colonies in comparison to those with no epiphora. Upon Gram’s staining, both positive bacilli and cocci and negative bacilli (slender and stout) were observed. The bacterial growth seemed “commensals” only. Morexella bovis or Morexella like organisms were not noticed in any of the samples.

Direct bilateral ophthalmoscopy revealed no abnormality in any of the calves included in the study. The fundus reflection was clear with no shadow’s indicative of the alterations in the superficial structures. The surface of optic disc was flat, and its edge visible. Vasculature was normal and abnormalities like edema, hemorrhage, exudation or irregularities in the retinal pigment were not noticed.

The subconjunctival injections were performed easily in the recumbent animals. The use of the insulin syringe was suitable for low volume withdrawal of the drugs from the vials. Its short, narrow gauge, permanently attached needle made possible safe delivery of the drugs in the desired location. The bleb that developed while depositing the medicine in palpebral or bulbar conjunctiva subsided automatically within a day in all the calves.

In six calves, the epiphora subsided within three days. Their periorbital skin was dry. The average wet area had reduced to 2.3 cm on the right and 1.7 cm on the left side. The average matted area extended to 2.3 cm around eyes given palpebral subconjunctival injection and 1.4 cm in those injected via bulbar route. The reduction of facial matting values (mean ± s.e) was 65.5 ± 16.4% in eyes given epibulbar injection and 52.7 ± 22.4% in those administered the drugs via subpalpebral route. The P value was 0.648 and the values between the routes of drug administration showed no significant (P > 0.05) difference.

On day 7 post treatment, the epiphora had subsided completely in ten calves. In the remaining two animals, the matting was

RESULTS All the calves included in the study were free from systemic disease and were not on any medication.

The impaired vision was not noticed in any of the calves despite 41% (14/34) of them showing epiphora simultaneously. The superficial eye structures including eyelids, conjunctiva, cornea and sclera all appeared normal. Symptom’s indicative of pain or irritation in and around the affected eye/s was also not observed in any of the calves throughout the study period.

The overflowing tears matting the periorbital hair were colorless

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noticed up to 2.0 cm and 1.0 cm (subpalpebral group) and 2.0 cm and 3.0 cm (epibulbar group) respectively. As compared to the day 0 values, the reduction in facial matting values (mean ± s.e) was 98.9 ± 7.1% and 86.1 ± 10.9% in the epibulbar and the subpalpebral groups respectively. The P value was 0.616.

The values between the routes did not differ significantly (P > 0.05).

Calves in the control group neither showed epiphora nor any other abnormality subsequent to the subconjunctival injection.

Those given treatment also showed no recurrence or deleterious effects.

DISCUSSION Majority of the dairy cows maintained in this university farm belong to Jersey breed. Few Holstein Frisian cows are also reared.

Consequently, Jersey calves were therefore disproportionally represented more in this study.

The epiphora developed in calves only. The heifers, adult dry and milking cows housed in separate pens, although grazing in different locations but using same highly dusty paths within the farm showed no epiphora or any ophthalmic condition during the study period.

The overflowing tears were colorless (serous) in all the affected animals. Unlike commonly encountered microbial infections, the tears were neither purulent nor accompanied by ocular pain, corneal opacity and ulceration3, 8. The reflex tearing might have protected the calves from the adverse ocular surface effects of the dust5.

The severity of the epiphora assessed tentatively from the matted facial hair did not differ significantly between eyes and routes of subconjunctival injection; an important criterion for evaluation of the treatment protocol was thus fulfilled.

Schirmer tear test (STT) measures the quantity of tearing within a given time frame9. Recording for one minute gives satisfactory results in cattle1, 10. First described by Otto Schirmer in 1903, it is the fundamental diagnostic test used in veterinary ophthalmology11, 1. Tear production must be assessed before any agents have been instilled in the eye to prevent falsely elevated values1. The mean SST value obtained in our calves with no epiphora was within the normal range (> 20 mm/min) reported in this species10, 13. The values (27.67 ± 3.30 mm/min) obtained in calves with epiphora were non-significantly (P < 0.05) higher. Statistically significant difference is expected if larger number of calves can be included in such a study. The test was well tolerated by the calves and may be useful for evaluation of epiphora in calves.

The bacterial growth representing both gram positive and gram negative organisms with greater number of colonies developing from the swab samples obtained from our calves with epiphora seemed commensals only. It is difficult to ascertain the predominant organism with direct swab. Whether the composition or the immunological regulatory behavior of the microbiota residing on the ocular surface is affected by pollution is also not clear14.

Direct ophthalmoscopy revealed no retinal abnormality in any of the eyes in our calves. In cattle the fundus examination plays an important role in diagnosis of several diseases like hypovitaminosis A15. and consumption of moldy corn16.

Prompt treatment of the eye affections is essential. Drugs may be delivered to the eye in several ways: subconjunctival injection,

topical application and systemic administration. Due to the constant turnover of the precorneal tear film, the drugs applied topically are soon washed out of the eye. To be effective, their frequent application may not only be stressful to the animal but also laborious and time consuming for the personnel17.

The subconjunctival technique has the advantage of efficacy at a lower antibiotic dose18. It allows drugs to bypass the epithelium, one of the main barriers that limit drug entry17. Increase in drug absorption is also accompanied by prolonged contact time. Medications leak onto the cornea from the injection site and diffuse through the sclera into the globe19. Drugs with low solubility such as corticosteroids may provide a repository of drug lasting days to weeks20.

In order to deposit the solution at the desired subconjunctival site without backflow or spillage and to prevent inadvertent needle punctures to the vital eye structures, the calves included in this study were restrained in lateral recumbency. The ophthalmic examination of an adult cattle restrained in standing is a challenge and the attempts commonly made to give subconjunctival injections may be dangerous particularly in the fractious ones21.

In the calves of both the groups, similar volume (0.5 ml) and quantity of the drugs were administered subconjunctival. 0.5 mL per site is usually safe and effective in small animals20. Adult large animals are generally given up to 1.0 ml. Therapeutically effective tear concentrations are maintained for 24 hours or longer18. At all occasions, visible bleb developed at the site of injection indicating proper placement of the needle19.

Gentamicin - dexamethasone combination was used to manage epiphora in all the calves included in this trial. The dose of gentamicin and dexamethasone used in our calves was less than that recommended for adult cattle19. The corticosteroids in the form of ophthalmic ointments or subconjunctival injections are commonly used and are effective in acute inflammatory conditions; whether the etiology is allergic, traumatic, or infectious14, 22. Antimicrobial agents like chloramphenicol, gentamicin, neomycin and sulphonamides are the drugs of choice in ocular therapy14. Although the eyes in the affected calves included in this study showed no pathognomonic symptom (except epiphora) of any microbial infection of clinical significance, the Gentamicin was given to prevent flareup of infection under corticosteroid administration. Steroids reduce resistance to many types of such infections except simultaneously with an effective antibiotic or other antibacterial medications22.

The results of our study indicate that single dose subconjunctival injection of gentamicin - dexamethasone combination is effective in managing epiphora in calves. Subconjunctival injections allow drugs to bypass the epithelium; the barrier that limits their entry23. This route of administration is indicated for the treatment of lesions in the cornea, sclera, anterior uvea and vitreous. Animals given injection via subpalpebral or epibulbar conjunctiva showed no significant difference in their efficacy. In contrast to our findings, notable differences in efficacy of penicillin administered by these routes in calves with IBK was reported18, 24. However, in IBK appreciable involvement of several eye structures requires wide and deep spread of the drugs injected.

Calves in the control group did not develop any complications.

Adverse reactions were not reported in calves with IBK, given clindamycin or isotonic saline solution subconjunctival23.

No new cases or recurrence in calves included in the study may

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M. Fazili et al. Large Animal Review 2021; 27: 311-315

be attributed to the moderate rainfall nine days later and settling of the excessive dust.

CONCLUSION The results suggest that excessive dust in the environment may be associated with the development of serous epiphora in young calves. Subconjunctival administration of gentamicin - dexamethasone combination via the palpebral and bulbar routes are both effective for managing the ailment. However, more studies are needed to assess the deleterious ophthalmic effects of air pollution in the food animals so that various preventive or protective measures could be designed.

Acknowledgement The authors would like to thank Vice Chancellor, for permitting to use internal resources of the university to carry out the study.

Conflict of Interest The authors declare no conflict of interest related to the study.

References 1. Townsend W.M. (2010). Examination techniques and therapeutic regimens for the ruminant and camelid eye. Vet Clinics North America: Food Anim Pract, 26: 437-458.

2. Romano J.S., Mork T., Laaksonen S., Ågren E., Ågren E., Nymo I.H., Sunde M., Tryland M. (2018). Infectious keratoconjunctivitis in semidomesticated Eurasian tundra reindeer (Rangifer tarandus tarandus): microbiological study of clinically affected and unaffected animals with special reference to cervid herpesvirus 2. BMC Vet Res,14: 2-14.

3. Erdogan H.M. (2010). Listerial keratoconjunctivitis and uveitis (Silage eye). Vet Clinics North America: Food Animal Pract, 26 (3): 505-10.

4. Fazili M.R., Buchoo B.A., Bhattacharyya H.K. (2010). Successful management of a delayed case of a corneal foreign body in a cow. Turkish J Vet Animal Sci, 34 (3): 295-298.

5. Jung S.J., Mehta J.S., Tonglar L. (2018). Review Article: Effects of environment pollution on the ocular surface. Ocular Surface, 16 (2):198-205.

6. Hamba N., Gerbi A., Tesfaye S. (2021). Histopathological effects of ultraviolet radiation exposure on the ocular structures in animal studies - literature review. Trans Res Anatomy. 22, 100086.

7. George L.W. (1990). Antibiotic treatment of infectious bovine keratoconjunctivitis. Cornell Vet, 80 (3): 229-35.

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8. Sargison N.D., Hutner J.E., West D.M., Gwozdz M.J. (1996). Observations on the efficacy of mass treatment by subconjunctival penicillin injection for the control of an outbreak of infectious bovine keratoconjunctivitis. New Zealand Vet J, 44 (4):142-144.

9. Wieser B., Tichy A., Nell B. (2013.) Correlation between corneal sensitivity and quantity of reflex tearing in cows, horses, goats, sheep, dogs, cats, rabbits, and guinea pigs. Vet Ophthal, 16 (4): 251-262.

10. Tofflemire K.L., Whitley E.M., Gould S.A., Dewell R.D., Allbaugh R.A., Ben-Shlomo G., O’Connor A.M., Whitley R.D. (2015). Schirmer tear test I and rebound tonometry findings in healthy calves. Vet Ophthal, 18 (2): 147-151.

11. Yoon A., Liu C-C., Carter R.T., Lewin A.C. (2020). Environmental relative humidity affects Schirmer tear test results in normal dogs. Vet Ophthal, 23 (5): 923-926.

12. Featherstone H.J., Heinrich C.L. (2013). Ophthalmic examination and diagnostics Part 1: the eye examination and diagnostic procedures. In: Veterinary Ophthalmology, Ed. Gelatt K.N., 5th ed., 533-613. John Wiley & Sons, Inc., Ames, IA. 13. Moore C.P. (1990). Diseases of the eye. In: Large Animal Internal Medicine. Ed. Smith B., 1197-1203e., St Louis (MO): Mosby.

14. Chawla S.K., Panchbhai V.S., Gahlot T.K., Kumar P. (2020). The Special Sense Organs, Section A – Eye. In: Ruminant Surgery, Ed. Singh J., Singh S., Tyagi R.P.S., 2nd ed., 549-570., CBS Publishers and Distributors, New Delhi.

15. He X., Li Y., Li M., Jia G., Dong H., Zhang Y., He C., Wang C., Deng L., Yang Y. (2012.) Hypovitaminosis A coupled to secondary bacterial infection in beef cattle. BMC Vet Res, 8: 222.

16. Sandmeyer L.S., Vujanovic V., Petrie L., Campbell J.R., Bauer B.S., Allen A.L., Grahn B.H. (2015). Optic neuropathy in a herd of beef cattle in Alberta associated with consumption of moldy corn. Canadian Vet J, 56 (3): 249-56.

17. Stanley R.G. (2008). Ocular Clinical Pharmacology. In: Small Animal Clinical Pharmacology. Ed: Maddison J.E., Page W.S., Church D.B. 2nd ed. 557-573., Elsevier (USA).

18. McConnel C.S., Shum L., House J.K. (2007). Infectious bovine keratoconjunctivitis antimicrobial therapy. Australian Vet J, 85 (1-2): 65-9.

19. Ward D.A., Clark S.E. (1991). Ocular Pharmacology. Vet Clinics North America: Food Anim Pract, 7 (3): 779-791.

20. Whelan N. (2015). Routes of Administration for Ocular Medications.

MSD Manual Veterinary Manual. https://www.msdvetmanual.com Assessed on 05-09-2021.

21. Irby N.L. (2004). Surgical diseases of the eye in farm animals. In: Farm Animal Surgery, Ed. Fubini S.L., Ducharme N.G., 1st ed., 429. Saunders, Missouri.

22. Doug H. (1969). The use of corticosteroids in ocular disease of small animals. Lowa State Uni Vet 31(2): 3. https://lib.dr.iastate.edu/iowastate_veterinarian/vol31/iss2/3 (assessed 09-05-2021).

23. Senturk S., Cetin C., Temizel M., Ozel E. (2007). Evaluation of the clinical efficacy of subconjunctival injection of clindamycin in the treatment of naturally occurring infectious bovine keratoconjunctivitis. Vet Ophthal, 10 (3): 186-189.

24. Allen L.J., George L.W., Willits N.H. (1995). Effect of penicillin or penicillin and dexamethasone in cattle with infectious bovine keratoconjunctivitis. JAVMA, 206:1200-1203.

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Effects of trimming on the thermographic pattern of claw sole in dairy cows

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N

IBRAHIM1 AKIN*, YALCIN ALPER OZTURAN1 1

Faculty of Veterinary Medicine, Department of Surgery, Aydin Adnan Menderes University, Isikli, Aydin, TR, 09100

SUMMARY Lameness is one of the most significant health, economic and welfare issues in dairy farms. In the present study potential usefulness of infrared thermography (IRT) as a non-invasive instrument for rapidly screening the claw sole temperature alterations between baseline (before trimming) and after trimming was investigated. For this purpose, this study was aimed to reveal the effect of claw trimming on claw sole temperature by using infrared thermography (IRT). One hundred thirty IRT observations from the hind legs of non-lame, healthy cows were collected and examined from 65 cows. The maximum (Tmax), average (Tave), and minimum (Tmin) temperatures of the claw sole and the temperature difference (ΔT) between the lateral and medial claws were examined on the baseline and after trimming. Claw temperature values were evaluated for normality using the Shapiro Wilk test. Paired t-test was used for comparing results with normal distribution while Wilcoxon’s signed-rank test was also used for nonparametric comparisons. Average baseline and after trimming solar surface temperature values on lateral claws were determined as 19.91±2.67°C and 21.49±3.43°C, respectively. The solar surface temperature values on medial claws were baseline 19.96±2.52°C and after trimming 20.98±2.83°C. There were statistically significant differences between baseline with after trimming solar surface temperatures of lateral (Tmax: P=0.005; Tave: P<0.001; and Tmin: P=0.005), and medial claws (Tave: P<0.001).

After trimming, claw sole temperature values between the lateral and medial were statistically significant (Tmax: P=0.020; Tave: P=0.039; and Tmin: P=0.004). Medial-lateral claw temperature differences between baseline with after trimming values were also statistically significant (ΔTmax: P=0.050; ΔTave: P=0.001; and ΔTmin: P=0.019). In conclusion, claw trimming increased the solar surface temperature values of the claw sole in this study. In the present study, claw sole temperature values were relatively higher on lateral than medial claws both for the baseline and after trimming. In future studies, the reliability of the current method can be demonstrated by comparing thermography to other evaluation methods.

KEY WORDS Claw trimming, dairy cow, IRT, thermography.

INTRODUCTION Dairy cow lameness has a negative effect on welfare and dairy production, as well as decreasing reproductive capacity and increased culling rates in dairy farms1, 2. Claw disorders and lameness may cause considerable suffering for the cows because most cases of lameness are long-lasting and painful3. It is very important to identify lameness and claw lesions as early as possible to eliminate yield loss and reduce recovery time. The most common way of detecting lameness is to use locomotion scores4.

Locomotion scores may not be sensitive to detecting all related claw lesions5. Recent studies reported electronic lameness detection by recording dairy cattle locomotion parameters such

Corresponding Author: Ibrahim Akin (ibraak@adu.edu.tr).

as gait and weight distribution in walking or standing6,7,8. Routine claw trimming, from the view of the solar surface of claws, is a good management practice to detect and prevent claw diseases and treat lesions such as sole hemorrhages, sole ulcers, thin sole, white line separations/diseases, and heel erosion9.

However, over-trimming may predispose to claw injuries and disorders due to the thin sole10. Therefore, methods for assessing claw temperature measurements should be investigated as an alternative and assisting tool to trimmers with revealed temperature ranges of healthy claws.

Infrared thermography (IRT) is a non-invasive diagnostic technique that can measure the temperature of an object by emitted radiation and displays the information called a thermogram11,12,13. While the temperature of skin and extremities is determined by the underlying circulation and metabolism rate, some conditions such as inflammation may alter blood flow and surface temperature14. Thus, variation and superficial venous changes are related to inflammation can be measured and monitored by IRT15. Infrared thermography has been applied

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in equine medicine in the diagnosis of foot and leg problems and as an aid in the detection of lameness or inflammation in horses12. In dairy cows, IRT has been used to detect digital and interdigital dermatitis and inflammatory foot diseases15,16. Therefore, the objective of this study was to evaluate the potential role of using IRT to measure claw sole temperature differences between baseline and after claw trimming in healthy cows. In this study, the authors expected to observe temperature differences (minimum-average-maximum and temperature differences between claws) between lateral and medial claw sole temperatures baseline and after trimming.

Materials and methods Animals and housing The study was conducted on a commercial dairy farm in California with about 9.000 Holstein lactating cows. Non-lame, healthy cows were enrolled in the study as their claws were trimmed prior to dry-off. Sixty-five healthy and second parity Holstein cows (n=65) were selected in late lactation (228±8.23 days in milk). The present study included cows that exhibited no symptoms of systemic disease, claw lesions, or lameness. Cows were receiving preventive claw trimming once a year by a professional claw trimmer.

Cows were housed in a free-stall dairy barn. The flooring was grooved concrete and sand bedding. The alley next to the feed

bunk (3.50 m) was covered with a 1.5-cm thick flat rubber mat.

Alleys were cleaned 15 times a day with an automatic scraper.

Cows were fed a total mixed ration consisting of approximately 55% forage (corn silage, haylage, alfalfa hay, and wheat straw) and 45% concentrate (cornmeal, soybean meal, canola, and cottonseed). The feeding and water troughs were ad libitum. The cows were milked twice daily at 05:30 and 16:30 h in a double milking parlor.

Experimental design Infrared thermography images (thermograms) of enrolled cows’ hind legs were obtained daily before the afternoon milking. All cows walked into the trimming chute. Prior to the thermograms, claws were cleaned with a pressure washer to remove dirt. Following washing, the claws were dried using paper towels. The hind leg claws of the cows were trimmed by a professional claw trimmer by using a cutting disc. All thermograms were captured in the claw trimming area located next to the milking parlor, eliminating direct sunlight in the afternoon. All thermograms were accomplished in three days under the same condition and same time frame (13:00-18:00).

Clinical claw examination Claw lesions of the hind lateral and medial claws were examined during the routine herd trimming check. The diagnoses of claw lesions were performed before afternoon milking based on inspection, palpation, and evaluation of smells and pain re-

Figure 1 - Thermographic images (thermogram) of the claw’s solar surfaces (sole view area of the lateral and medial claw) baseline (b1: normal view, b2: hybrid view, and b3: thermographic view) and after trimming (a1: normal view, a2: hybrid view and a3: thermographic view); L = Lateral.

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actions with gooseneck claw tester as described by Espinasse et al. (1984)17. All visually lame and any disease-diagnosed animals were excluded from the study due to the study design.

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Other parameters with apparently non-normal distributions were compared with Wilcoxon’s signed-rank test. The significance level was set at P<0.05. The means and standard deviations are presented in Table 1, Table 2, and Table 3.

Thermal imaging Thermograms were obtained with TiR125 Thermal Imager (Fluke IR-Fusion Technology, Everett, WA, United States; emissivity value was set at 0.95 with a precision of ±0.01°C) to assess the maximum, average, and minimum surface temperatures of the claw sole. Thermograms were analyzed using the software SmartView 3.1.82.0 (Fluke IR-Fusion Technology, Everett WA, United States). The infrared camera was calibrated by the company and the calibration was tested every day by the thermal reading of an object of known temperature daily. Thermograms of all claws were scanned from solar aspects (Figure 1) while cows were standing on rubber mats placed on a hydraulic trimming chute. Legs were lifted with a hydraulic trimming chute, and all scans were performed at the same distance (0.5 m). The solar view area (Figure 1, the area inside the white line drawn by the first author (IA) for each claw via software program) of the lateral and medial claw in each image was measured with the help of the same software tool. All thermograms were captured in the claw trimming area, with no direct sunlight or detectable airflow, and they were taken immediately before and after trimming. Images were calibrated by relating each of the shades of gray on the digitized image to a specific temperature for all pixels. These values then were mapped to degrees centigrade, and the maximum (Tmax), average (Tave), and minimum (Tmin) temperatures of the solar area in each lateral and medial claw were determined before and after trimming. The software program [Smart View 3.1.82.0 (Fluke IR-Fusion Technology, Everett WA)] was adjusted to remove the effect of outlier maximum temperatures related to extraneous particles or too few (<5) pixels exhibiting the maximum temperature, the relative maximum temperature was calculated. The temperature differences (ΔT) between the sole of the lateral and the medial claws were also calculated via deducted from the «Medial claw sole temperature» to «Lateral claw sole temperature». Thus maximum (ΔTmax=Tmax medial Tmax lateral), average (ΔTave=Tave medial - Tave lateral), and minimum (ΔTmin=Tmin medial - Tmin lateral) temperatures were obtained with fewer variables.

Statistical analyses The statistical analyses were performed using SPSS 22 (IBM Corp. Armonk, NY, United States) statistical package program.

Descriptive statistics were performed to reveal the mean and standard deviation (SD) of every variable. Outcomes were checked for normality using the Shapiro Wilk test. For observing the effect of trimming on claw sole temperature, pre-trimming values of each parameter (Tmax-Tave-Tmin) and the temperature differences between the sole of the lateral and the medial claws (ΔTmax-ΔTave-ΔTmin) were determined as the predictor variables while after trimming values were determined as predicted variables. Similarly, medial claws were determined as the predictor variables to observe the temperature differences (ΔT) of lateral and medial claws before and after trimming, while lateral claws were determined as predicted variables. Before and after trimming average temperatures of lateral claws, ΔTmax, ΔTmin, and before and after trimming average temperatures of lateral claws were determined as normal distribution, and paired t-test was used for parametric comparison.

RESULTS Significant differences were found for Tmax (P=0.005), Tave (P<0.001), and Tmin (P=0.005) of lateral claws’ baseline and after trimming thermograms (Table 1). Only Tave of medial claws was found statistically significant (P<0.001) between baseline and after trimming thermograms. There was no statistical significance in baseline values (Tmax, Tave, and Tmin) between the lateral and medial claw sole thermograms (P>0.05).

However, after trimming, statistical differences were found on Tmax (P=0.020), Tave (P=0.039), and Tmin (P=0.004) values between lateral and medial claw thermograms (Table 2). The maximum (ΔTmax), average (ΔTave), and minimum (ΔTmin) temperature differences between medial and lateral claws (“medial claw solar surface temperature” - “lateral claw solar surface temperature”) showed a statistical significance (ΔTmax: P=0.050, ΔTave: P=0.001, and ΔTmin: P=0.019) between baseline and after trimming values (Table 3). The negative values were observed after trimming ΔTmax (-1.21±4.01°C), ΔTave (-0.51±2.43°C), and ΔTmin (-0.41±1.02°C) and baseline trimming ΔTmax (-0.29±2.67°C) since the lateral claw temperature values were higher than the medial claws (Table 3).

DISCUSSION The claw trimming leads to an increase in the surface temperature of the lateral and medial claw soles (Table 1). The sole temperatures of lateral and medial claws were statistically significant (P<0.05) after trimming compared to baseline values (Table 2). Similarly, there was a statistically significant increase (P≤0.05) in the temperature differences of medial and lateral claws (ΔT) between baseline and after trimming (Table 3). The authors investigated the thermal alterations on the sole surface of cow claws to detect a difference between baseline and after claw trimming. Our findings may demonstrate a new approach to preventive hoof trimming by focusing on the sole aspect (always faced and seen by the hoof trimmers) of the claws using IRT. IRT studies conducted on dairy cattle may be affected by cow level factors (inflammation, diseases, etc.) or ambient factors (sunlight, airflow, other heat sources, etc.) in dairy farms18. In the present study, we tried to establish the best possible study design for collecting data for avoiding or reducing that mentioned and preventable factors18.

Previously, lower sole horn thickness of the hind feet lateral claws9,19,20, uneven load distribution9, and length difference of paired digits21 were considered with the key practical claw trimming to reduce stress on the lateral hind claws and to redistribute some of the strain to the medial claw22,23. In the present study findings, ΔT values were more in favor of an increase in lateral claws. Lateral claw sole temperatures increased more than medial claws by hoof trimming and the temperature differences (maximum, average, and minimum) were statistically higher (ΔTMax: P=0.050, ΔTave:P=0.001, ΔTMin:P=0.019) after trimming compared to baseline trimming values. It may be interpreted

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Table 1 - Maximum (Tmax), average (Tave), and minimum temperature (Tmin) (°C; means and standard deviations) changes (means and standard deviations) between baseline and after trimming of lateral and medial claw solar area.

Lateral Claws (n=65)

Medial Claws (n=65)

Temperatures of claw sole area (°C)

BT (Mean ± SD)

AT (Mean ± SD)

P value

BT (Mean ± SD)

AT (Mean ± SD)

P value

Tmax

27.27±4.22

28.53±4.60

0.005

26.98±4.24

27.32±3.92

0.355

Tave

19.91±2.67

21.49±3.43

0.000

19.96±2.52

20.98±2.83

0.000

Tmin

15.93±2.10

16.41±2.50

0.005

15.95±2.19

16.00±2.27

0.958

BT: Baseline; AT: After trimming.

Table 2 - Comparison of medial and lateral claws solar surface maximum (Tmax), average (Tave), and minimum temperatures (Tmin) (°C; means and standard deviations) baseline and after trimming.

Baseline (n=65)

After Trimming (n= 65)

Temperatures of claw sole area (°C)

LC (Mean ± SD)

MC (Mean ± SD)

P value

LC (Mean ± SD)

MC (Mean ± SD)

P value

Tmax

27.27±4.22

26.98±4.24

0.291

28.53±4.60

27.32±3.92

0.020

Tave

19.91±2.67

19.96±2.52

0.506

21.49±3.43

20.98±2.83

0.039

Tmin

15.93±2.10

15.95±2.19

0.831

16.41±2.50

16.00±2.27

0.004

LC: Lateral claws; MC: Medial claws.

Table 3 - The temperature differences of claws (medial claw-lateral claw, means, and standard deviations) in maximum (ΔTmax), average (ΔTave), and minimum (ΔTmin) temperatures (°C; means and standard deviations).

Temperature Differences1 (°C)

BT (n=65, Mean ± SD)

AT (n=65, Mean ± SD)

P value

ΔTMax

-0.29±2.67

-1.21±4.01

0.050

ΔTAve

0.05±1.74

-0.51±2.43

0.001

ΔTMin

0.01±1.32

-0.41±1.02

0.019

1

Temperature differences (ΔT) of the claws (medial claw- lateral claw) between baseline and after trimming; a negative value indicates a lower medial claw surface temperature; BT: Baseline; AT: After trimming.

that these results may be indicating the possibility of encountering thin soles and/or over trimming. To this respect, ΔT values in this study can attribute to the difference, which has an important role in preventing thin sole and/or over trimming in both claws during hoof trimming. Moreover, compared to medial claws, Tmax, Tave, and Tmin temperatures of the lateral claw sole were significantly higher after trimming (P<0.05) in the present study. Nuss and Paulus (2006) reported a parallel finding that hind feet trimming could result in excessive lateral claw sole thinning10. Since the lateral claw bears more weight than the medial claw thus lateral claw wear is quicker24.

This is offset by increased growth of the claw horn25 and can be achieved by the proliferation of the corium cells, requiring increased blood and nutrient supply26. The higher temperature of lateral claws in this study may be triggered by these physiological conditions.

Thermographic identified hot spots occur at recognized weight-bearing sites, indicating injury or inflammation to the tissue15,16. In our data, especially considering after trimming temperatures, while maximum values may indicate local inflammation or any subclinical lesion of inner tissue of claw, minimum data may be indicated necrosis or low blood flow. Also, the higher average temperature may indicate a thin sole. That comment may be necessary for a call to more work to respond to a potential role of IRT on the diagnosis of subclinical or local inflammation or a claw disease. In our study, however, all animals were checked for lameness and claw lesions for achieving the healthy temperature range of claws by excluding all lame/sick cows. In this respect, this study may increase attention to achieve the normal temperature range of dairy cow

claws in future studies.

In the current study, practical claw trimming in the cows was carried out by a skilled claw trimmer. Claw trimming was correlated with an increase in claw sole temperature in the study.

This result is in line with the main principle of claw trimming.

It is supporting the argument of van der Tol et al. (2004), who found that claw trimming affected weight-bearing and pressure distribution on the hind claws22. They stated that the load of the lateral claws in the hind feet was reduced from 80 percent (pre-trimming) to 70 percent (2 wk post trimming). This argument is correlated with our finding’s fact that the after trimming lateral claws’ solar surface Tmax (28.53±4.60°C, P=0.020), Tave (21.49±3.43°C, P=0.039), and Tmin (16.41±2.50°C, P=0.004) were statistically higher than in the medial claws. Additionally, this temperature favor in lateral claws can be explained with the previous study van Amstel and Shearer (2006) reported that lateral claws were thinner than medial claws20.

In the present study, the temperature alterations between baseline and after trimming of claw sole have been observed and reported. Further analyses to predict the normal temperature range of healthy claws can be helpful in their decisions to trimmers and veterinarians for utilizing IRT as a non-invasive, automated diagnosis tool on trimming and claw health checks.

CONCLUSIONS We conclude that there was a significant temperature increase in claw sole temperatures between the paired hind claws by claw trimming. Lateral claw temperatures were relatively higher than

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Ibrahim Akin, Yalcin Alper Ozturan Large Animal Review 2021; 27: 317-321

medial claws baseline and after trimming. Lateral and medial claw temperatures can be evaluated and guided by the trimmers during and after trimming as they may be indicators of conditions such as weight-bearing and thin soles. Future studies may benefit from our current results in the hope of determining the temperature range of healthy claws on solar view.

Also, the reliability of the current method can be asserted by contrasting thermography with other evaluation methods in future studies to avoid thin soles and/or over-trimming.

Acknowledgments This study is produced from the project named “Hoof Trimmer Performance During Functional Hoof Trimming and Lame Cow Treatment in California Dairies - Implications on Lameness and Production” and all procedures were approved by the University of California Davis Institutional Animal Care and Use Committee (#17440, Davis, California, United States). The authors would like to thank the valuable contributions of Noelia Silva del Rio and Alfonso Lago. Also, we would like to thank Aykut Göktürk Üner for his help with the statistical analysis used in this study. Part of this study was presented in XV. National, I. International Turkey Veterinary Surgery Congress, 1114 May 2016, Erzurum, Turkey. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Assessment of animal and management based potential risk factor relation with claw health and lameness in dairy cows: A cross-sectional study

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KADRI KULUALP1*, YALCIN ALPER OZTURAN2, IBRAHIM AKIN2 1

*Faculty of Veterinary Medicine, Department of Surgery, Dokuz Eylul University, Kiraz, Izmir, TR, 35890 Faculty of Veterinary Medicine, Department of Surgery, Aydin Adnan Menderes University, Isikli, Aydin, TR, 09100 2

SUMMARY Lameness is one of the most common problems modern dairy industries and it may originate as infectious or noninfectious.

Lameness may also be related to housing and animal-based factors. In this regard, this study aimed to investigate the relationship between potential risk factors with lameness and claw lesions in dairy farms. Eleven dairy farms with 1685 cows were enrolled in this study. Relationships between lameness existence with hind limb conformation, claw conformation, days in milk (DIM), lactation number (LN), bedding type, claw trimming intervals, and relationships between infectious claw diseases (digital dermatitis-DD and heel erosion-HE) with LN, DIM, bedding type, footbath existence, footbath chemical, footbath changing frequency, and footbath solution volume per cow were investigated. A binary regression model was used to reveal relationships. There was no statistically significant difference in the relationship between cow breeds and lameness score, hind limb conformation, claw conformation, and claw diseases. A positive correlation between the existence of lameness with all risk factors was found. Cows with abnormal hind limb and claw conformation numbered 220 and were 2.3 times more prone to lameness (P<0.001), respectively. A positive correlation was found between infectious claw diseases with LN, DIM, bedding type, footbath chemical, and footbath changing frequency. However, a negative correlation was found between infectious claw diseases and footbath solution volume per cow. The relationship between the infectious claw disease existence with DIM and LN was found to be statistically significant (P<0.001). Also, an increase in LN and DIM were found to increase the risk of infectious claw lesion frequency by 3.3 and 2.2 times, respectively. Findings suggest that abnormal hind limb and claw conformations should be more closely monitored and investigated in terms of lameness. LN and DIM should also be monitored in terms of infectious claw diseases. The result of the study may help farmers to specify their potential animal and management-based risk factors related to lameness and infectious claw lesions in their farms.

KEY WORDS Dairy cow, footbath, claw conformation, hind limb conformation, claw diseases.

INTRODUCTION Lameness is one of the major causes of economic losses by reducing milk yield and reproductive performance in cows, increasing the culling rate and treatment costs in dairy farms1-3. Lameness also results in pain and welfare problems by adversely influencing the behavior of animals, including routine activities such as eating, drinking, and rest4,5. This situation is therefore considered in dairy farms as a herd health problem, not an individual one1,5.

Lameness is known as a complex and multifactorial problem3,6.

Lameness risk factors have been related to animal-based variables, and herd management practices have been reported to have an effect on their development2,7,8. Diet, genetic charac-

Corresponding Author: Kadri Kulualp (kadri.kulualp@deu.edu.tr).

teristics, breed, age, gender, days in milk (DIM) and lactation number (LN), limb and claw deformations may be considered as animal-based variables1. The alley dimensions, bedding used in paddocks and walking areas, and the presence of footbaths and, claw trimming interval, footbath chemicals, and its renewal frequency are factors that may be considered as herd management practices3,6,9. Claw lesions and lameness should be considered as a problem affected by potential risk factors3. Becker et al. (2014) argued that factors such as herd size, herd management practices, and housing systems may vary by location, so the relationship between claw lesions and lameness and risk factors should be separately disclosed in each geographical area7.

The objective of the present study was to investigate relationships between animal-based potential risk factors (breed, hind limb conformation, claw conformation, DIM, LN), and management-based risk factors (bedding types, claw trimming intervals, footbath existence, footbath chemical, footbath changing frequency, and footbath solution volume per cow) with lameness and infectious claw lesions (DD and HE).

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MATERIALS AND METHODS Farms and Animals This study was carried out in districts of Tire, Odemis, and Kiraz in the east of the Kucuk Menderes Basin of Izmir, Turkey between September 2020 and December 2020. Farms that had free-stall housing systems with no access to pasture and a herd size of ≥ 50 lactating cows were selected to ensure that the participating farms were representative of the majority of farms in the Kucuk Menderes Basin of Izmir. Eleven farms agreed to be included in the study. Alleys were cleaned 2 times a day with automatic scrapers. A TMR was fed ad libitum and supplied twice daily at 09:00 and 19:00. The cows were milked twice daily at 08:30 and 18:30. Cow claws were trimmed routinely at dryoff. No clinical systemic disease symptoms were observed in the enrolled cows. Breed, LN, DIM, and claw trimming interval data were recorded based on-farm records. Lameness scores, hind limb conformation, claw conformation, presence of DD and HE, bedding type, and information about footbaths were recorded by observations during the visits.

Lameness evaluation Lameness evaluation was carried out using the 4-point scale, (10) impaired mobility scale of the UK Agriculture and Horticulture Development Board (i.e., walks with shortened strides, an arched back, and uneven weight balance on all feet), where 0= good mobility (i.e., walks with long strides and an even weight balance on all feet), 1= imperfect mobility (i.e., walks with shortened strides or uneven steps without an immediately identifiable affected limb), 2= impaired mobility (i.e., walks with uneven weight bearing on a limb that is immediately identifiable or walks with obviously shortened strides), and 3= severely impaired mobility (i.e., walks with shortened strides, an arched back, and uneven weight balance on all feet) when cows were standing and walking. All cows were observed for lameness when they moved from the milking parlor to the alley.

Claw and hind limb evaluation Claw conformations and claw lesions were recorded during the milking time. Claw lesions were recorded as the presence or absence of specific claw lesions in each claw: DD, and HE. Briefly, cows’ feet were washed with water from a hose. The primary investigator (PI) examined claws for the existence of DD, and HE lesions. Simultaneously, claws were evaluated according to their conformations in terms of parameters such as normal, open claws, blunt claw, scissor claw, corkscrew claw, and big claws, and all data was transferred to an excel sheet. An external source of light was used in situations where the light source was inadequate during the examination. Assessment of hind limb conformations was performed by the PI who performed other examinations. The PI evaluated the hind limbs on feeding time in terms of criteria such as normal, open-limb, X, and bow-limbed, at a distance of about 5 meters, passing behind the cows without distracting them.

baths was obtained from the questionnaire. The footbath’s dimensions (length, width, and depth) were also measured to determine the volume of the footbath solution per cow (cm3) in each farm. Footbath dimensions were used to calculate the footbath solution volume (cm3) per cow by using the volume formula (lengthxwidthxdepth in cm).

Statistical analysis Explanatory variables such as animal-based information (breed, LN, DIM), claw and hind limb conformations, claw lesions, and management-based data (bedding type, claw trimming interval, footbath implementation, chemical, and changing frequency) were recorded in a spreadsheet program (Excel, Microsoft Corp., Redmond, WA, USA). For DIM data, cows were categorized into three groups based on the day in milk (DIM) according to the E-views equity test of means program: Group 1 (Early lactation): 0 ≤ DIM ≤ 45, group 2 (middle lactation): 46 ≤ DIM ≤75, and group 3 (late lactation): 76 ≤ DIM. Statistical analyses were carried out using SPSS 22 statistical package (IBM SPSS Statistics for Windows, Version 22.0, Armonk, NY, USA) program. Alpha values were set to <0.05 for statistical significance.

Relationships between breeds with the claw, hind limb conformations, lameness score, and claw diseases were investigated with the chi-square test. For achieving this, breeds (Simmental=0, Holstein=1), lameness (not exist=0, exist=1), claw and hind limb conformations (normal=0, abnormal=1), and claw lesions (healthy=0, lesion=1) were categorized to determine whether there is an association between categorical variables.

In order to investigate the influence of hind limb and claw conformations, categorized DIM, bedding type, claw trimming interval, and LN on the event of lameness, a statistical model was conducted with binary logistic regression. For this aim, binary dummy variables for outcomes from the recorded data were coded as follows: Claw conformation (normal=0, abnormal=1), hind limb conformations (normal=0, abnormal=1), DIM (≤45 days=0, >45 days=1), bedding type (concrete+sand=0, sand=1), claw trimming interval (less than one year=0, more than one year=1), LN (1, and 2=0; 3, 4, and 5=1), and lameness (not exist=0, exist=1). The Hosmer-Lemeshow test was used to determine the goodness of fit of the logistic regression model.

Similarly, a binary logistic regression model was performed to observe the influence of categorized DIM, bedding type, footbath implementation, footbath solution volume (cm3) per cow, footbath chemical, and footbath changing frequency on the event of claw diseases. For achieving this, dummy variables for covariates from the collected data were coded as follows: DIM (≤45 days=0, >45 days=1), LN (1, and 2=0; 3, 4, and 5=1), bedding type (concrete+sand=0, sand=1), footbath implementation (exist=0, not exist=1), footbath chemical (CuSO4+formalin=0, CuSO4=1), footbath changing frequency (everyday=0, every two days=1), footbath solution volume (cm3) per cow (more than 1 liter=0, less than 1 liter=1), and claw lesions (healthy=0, lesion=1). The Hosmer-Lemeshow goodness-of-fit test was used to evaluate the model fit.

Footbath evaluation A questionnaire was conducted at each farm. The questions were either open-ended (e.g.,» What is the active ingredient used as a footbath?) or closed-ended (e.g.,» Are you using a footbath? response scale: yes or no). Specific information on the frequency of its use (times/weeks) and the changing frequency of foot-

RESULTS Descriptive statistics of cows The average herd size was 187 lactating cows (range, 125 to 212).

The enrolled breeds, LN, and DIM of cows were described in

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Table 1 - Descriptive statistics of cows used in this study by their DIM and LN (n, %).

LN Breeds

DIM (day)

1 (n, %)

2 (n, %)

3 (n, %)

4 (n, %)

5 (n, %)

Total

Simmental

0-45 46-75 75<

58 (36.5) 32 (34.8) 41 (36.6)

46 (28.9) 28 (30.4) 24 (21.4)

34 (21.4) 13 (14.1) 25 (22.3)

17 (10.07) 17 (18.5) 16 (14.3)

4 (2.5) 2 (2.2) 6 (5.4)

159 (100) 92 (100) 112 (100)

Total

131 (36.09)

98 (27)

72 (19.83)

50 (13.77)

12 (3.31)

363 (21.54)

0-45 46-75 75<

188 (30.9) 78 (25.7) 111 (27)

209 (34.4) 113 (37.3) 154 (37.5)

103 (16.9) 68 (22.4) 73 (17.8)

88 (14.5) 35 (11.6) 63 (15.3)

20 (3.3) 9 (3.0) 10 (2.4)

608 (100) 303 (100) 411 (100)

Total

377 (28.52)

476 (36.01)

244 (18.46)

186 (14.07)

39 (2.95)

1322 (78.46)

Holstein

Table 1. Simmental breed cows (n=363, 21.54%) are less recorded than Holstein cows (n=1322, 78.46%), and no other breeds were encountered at farms used in this study. A total of 1685 dairy cows were enrolled in the study (Table 1). Second lactation (n=476, 36.01%) were the most common LN in Holstein breed cows and first lactation (n=131, 36.09%) was the most common LN in Simmental breed cows. The fifth lactation was the less encountered LN in both (n=39, 2.95% for Holstein and n=12, 3.31% for Simmental) breeds (Table 1). Early lactating (≤45 DIM) cows were the majority (608 Holstein and 159 Simmental) of the present study whereas mid-lactating cows (303 Holstein and 92 Simmental) were the less encountered cows in the study period (Table 1). There was no statistically significant difference between lactations within Simmental and Holstein breeds (P>0.05).

Comparison of recorded traits by breeds There was no statistically significant relationship between breeds (Holstein and Simmental) according to hind limb and claw conformation, lameness scores, and claw lesions (P>0.05, Table 2).

In terms of lameness, 89.7% (n=1186) of Holstein cows and 88.2% (n=320) of Simmental cows were scored «as 0» (Table 2). The most encountered lameness score was a mild (score 1)

in Holstein (n=71, 5.4%) and Simmental (n=24, 6.6%) cows (Table 2). Severe lameness (score 3) was less common in Holstein (n= 20, 1.5%) and Simmental (n=6, 1.7%) cows (Table 2). In terms of hind limb conformation, 1241 Holstein and 338 Simmental breed cows were recorded as normal. The highest and lowest number of abnormal hind limb conformation in both breeds were X and bow limbed, respectively (Table 2). On claw conformation evaluations, 1042 Holstein cows (78.8%) and 289 Simmental cows (79.6%) had normal claw shape.

Corkscrew claws (28 [7.7%] Holstein and 105 [7.9%] Simmental cows) were the most common abnormal claw conformation in both breeds. Big claw had the lowest recorded number in Holstein [10, (0.8%)] and Simmental breeds [4, (1.1%)] (Table 2).

On infectious claw disease evaluations, no lesions were found in 1178 (89.1%) Holstein and 320 (88.2%) Simmental cows.

The most common lesion was DD with 5.3% in Holstein and 6.3% in Simmental cows. The least prevalent one was HE with 0.5% in Holstein breeds, and white line disease with 1.1% in Simmental cows (Table 2).

Footbath Management Footbath specifications of participating farms are presented in (Table 3). Footbaths had a median length of 202 cm (range, 175 to 225), width 81 cm (range, 70 to 100), and a

Table 2 - The relationship between cow breeds and lameness score, hind limb conformation, claw conformation, and claw diseases (n, %).

Breeds Simmental (n, %)

Holstein (n, %)

P-value

Lameness score

0- none-lame 1- mild 2- moderate 3- severe

320 (88.1) 24 (6.6) 13 (3.6) 6 (1.7)

1186 (89.7) 71 (5.4) 45 (3.4) 20 (1.5)

0.821

Hind limb conformation

Normal Open limb Bow limbed X limbed

338 (93.1) 8 (2.2) 5 (1.4) 12 (3.3)

1241 (93.9) 29 (2.2) 11 (0.8) 41 (3.1)

0.814

Claw conformation

Normal Open claws Blunt claw Scissor claw Corkscrew claw Big claw

289 (79.6) 10 (2.8) 19 (5.2) 13 (3.6) 28 (7.7) 4 (1.1)

1042 (78.8) 29 (2.2) 72 (5.4) 64 (4.8) 105 (7.9) 10 (0.7)

0.869

Claw lesion

No-lesion White line DD HE Sole ulcer

320 (88.2) 4 (1.1) 23 (6.3) 6 (1.7) 10 (2.8)

1178 (89.1) 14 (1.1) 70 (5.3) 7 (0.5) 53 (4)

0.168

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Table 3 - Characteristics of footbath practices for lactating cows in enrolled farms.

Farm no

nLC (n)

Footbath chemical

Changing frequency

Dimensions (length x width x depth in cm)

Footbath solution volume per cow (cm3)

1

175

Formalin+CuSO4

Everyday

200x80x15

1371.42

2

146

CuSO4

Everyday

220x70x20

2109.58

3

145

Formalin+CuSO4

Everyday

200x75x25

2586.20

4

142

Formalin+CuSO4

Every two days

225x85x20

1346.83

5

203

CuSO4

Everyday

200x100x20

1970.76

6

130

CuSO4

Every two days

225x100x20

1730.76

7

212

-

-

-

-

8

136

Formalin+CuSO4

Everyday

180x70x15

1389.70

9

144

CuSO4

Everyday

200x80x20

2222.22

10

125

-

-

-

-

11

127

Formalin+CuSO4

Every two days

175x75x15

775.09

nLC: Number of lactating cows

mean depth of 18 cm (range, 15 to 25). Footbath design and footbath practices varied greatly among farms. Characteristics of footbath practices in enrolled cows are presented in (Table 3). There was no footbath implementation in the two farms. The combination of formalin+CuSO4 was used by 5 farms, whereas 4 farms only used CuSO4 as a footbath chemical. Six enrolled farms were changing their footbath solution every day, while 3 farms were renewing the footbath solution every two days (Table 3).

Estimated relative risk factors of lameness The data on the relationships between the hind limb and claw conformation, LN and DIM, bedding type, and claw trimming interval with the presence of lameness are presented in (Table 4). A positive correlation was found between all factors with the presence of lameness and this correlation was only statistically significant between the hind limb and claw conformations (P<0.001). Cows with abnormal hind limb and claw conformations were found to number 220 and were 2.3 times more prone to lameness, respectively (Table 4).

Estimated relative risk factors of DD and HE The data about the relationship between the presence of DD and HE with LN and DIM, bedding type, the footbath implementation, the footbath chemical, the footbath changing frequency, and the footbath solution volume per cow are presented in (Table 5). LN and DIM, the bedding type, the footbath chemical, and the footbath changing frequency were in positive correlation with DD and HE existence. However, footbath implementation and footbath solution volume per cow exhibited a negative correlation with DD and HE (Table 5). Only LN and DIM were found to have a statistically relevant relationship with the occurrence of DD and HE (P<0.001) and increasing LN and DIM raise the frequency of DD and HE by 3.39 and 2.20 times, respectively (Table 5).

DISCUSSION Lameness has a multifactorial, complex etiology and is characterized by pain, causes major economic losses by reducing

Table 4 - Estimated relative risk factors (odds ratio) and 95% confidence interval (CI) on lameness.

Variables

Hind limb conformation Claw conformation DIM Bedding type Claw trimming interval LN

Numerical value of variables

b

SE

P-value

Estimated odds ratio

95% CI for odds ratio

0=Normal, 1= Abnormal

5.394

0.395

0.000

220.033

101.525-476.873

0=Normal, 1= Abnormal

0.852

0.244

0.000

2.344

1.454-3.781

0=Until 45 days, 1= Over 45 days

0.252

0.229

0.272

1.286

0.821-2.014

0=Concrete + sand, 1= Sand

0.089

0.321

0.782

1.093

0.582-2.052

0=Less than one year, 1= More than one year

0.454

0.302

0.132

1.575

0.871-2.846

0=1 and 2, 1= 3, 4 and 5

0.139

0.231

0.548

1.149

0.731-1.806

b: Regression Coefficient, SE: Standard Error, CI: Confidence Interval

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Table 5 - Estimated relative risk (odds ratio) factors and 95% confidence interval (CI) on DD and HE. Variables

Numerical value of variables

b

SE

P-value

Estimated odds ratio

95% CI for odds ratio

LN

0=1 and 2, 1= 3, 4 and 5

1.221

0.160

0.000

3.392

2.476-4.645

DIM

0=Until 45 days, 1= Over 45 days

0.789

0.132

0.000

2.202

1.700-2.853

Bedding type

0=Concrete + sand, 1= Sand

0.215

0.164

0.190

1.240

0.899-1.710

Footbath application

0=Exist, 1= Non-exist

-0.24

0.210

0.253

0.786

0.521-1.187

Footbath solution volume 0=More than 1 liter, per cow (cm3) 1= Less than 1 liter

-0.269

0.261

0.302

0.764

0.458-1.274

Footbath chemical

0=CuSO4+ Formalin, 1= CuSO4

0.136

0.135

0.313

1.145

0.880-1.491

Footbath changing frequency

0=Everyday, 1= Every two days

0.120

0.164

0.462

1.128

0.818-1.555

b: Regression Coefficient, SE: Standard Error, CI: Confidence Interval

milk yield, lifespan, reproductive performance, and cow welfare2,3,5. When lameness is accompanied by infectious claw diseases, it becomes a herd issue, worsening existing losses; moreover, its prevalence and severity are often underestimated by farmers11.

Therefore, many studies have been carried out in recent years to examine the relationship between lameness and infectious claw diseases, with possible risk factors that may influence the development and prevalence of these issues1, 2, 5-8, 12.

Breed traits in dairy cows are considered as a potential risk factor for claw diseases, especially DD7,13. Becker et al. (2014) reported that Holstein cows were more prone to claw lesions than brown and red breeds7. Furthermore, the fact that Holstein cows’ horn claw structures are more vulnerable to diseases has been linked to their vulnerability to metabolic diseases13. Contrary to these views, Bielfeldt et al. (2005) reported that there is no significant difference between Simmental, Swiss brown, and Holstein breeds in terms of factors such as claw lesions and lameness (14). In the present study, there was no statistically significant difference between Simmental and Holstein breeds in terms of claw and hind limb conformation, claw lesions, or lameness (P>0.05).

Abnormalities in cows’ hind limb conformation and claw lesions may be related to claw diseases and lameness15,16. According to Toussaint Raven (1989), load variations on the paired claws can negatively affect hind limb conformation, potentially raising the risk of lameness17. Similarly, Olechnowicz et al. (2010) claimed that abnormal conformations may increase the risk of claw lesions and lameness16. It is also stated that abnormal claw conformations are a factor that increases the rate of both claw lesions and lameness18. Claw lesions were observed in 21%19 and 42%20 of cows with abnormal claw conformations, and these animals have a high risk for lameness. Abnormal claw conformation and claw lesions may have a reciprocal cause-and-effect relationship21,23. In the present study, cows with abnormal hind limb and claw conformations were found to be 220 and 2.3 times more likely to be lame than healthy cows, respectively (Table 4).

These results support the hypothesis16,21 that abnormal hind and

claw conformations both raise the risk of lameness.

Studies that have investigated the relationship between other risk factors and lameness have reported different results2-5, 7, 14, 24, 25.

The present study results showed that there was no statistically significant correlation between lameness and LN, DIM, bedding type, and claw trimming interval. Our findings are parallel to the results of the studies in which no relationship was found in terms of the factors mentioned5, 14, 25, and hence they contradict the results of other studies2,24. As reported in Becker et al. (2014) differences in geography and management may result in changes7. However, scientific reports from different locations may provide key points to prevent lameness for others.

Infectious claw diseases, especially DD and HE, have been the most common infectious causes of lameness, have a multifactorial etiology12, 26, 27, and must be handled without causing major economic losses or herd welfare issues3,14,28. DD and HE have been linked to LN14, DIM29, bedding material30, footbath implementation12, chemical properties of the footbaths27, and footbath change frequency28. Gomez et al. (2015)29 and Bielfeldt et al. (2005)14 reported that the 60-120 DIM cows and LN and DIM period increase the risk of HE, respectively. In agreement with other studies, we found a statistically significant positive relationship between the presence of DD and HE with LN and DIM, with changes in LN and DIM increasing the incidence of DD and HE by 3.39 and 2.20 times, respectively (Table 5). In extensive dairy farms, efficient and proper use of footbaths has become crucial in preventing infectious claw diseases12,28,30. A strong relationship between HE3 and DD12 with footbath usage has been stated. The association between footbath usage with the existence of DD and HE lesions was found to be statistically insignificant (P>0.05) in our findings.

The chemicals, concentrations, and dimensions of footbaths used in the farms were evaluated as consistent with the literature12, 26, 27, 28, 30. The similarity of the management practices of footbath usage in study farms and the existence of only 2 of 11 farms (farm number: 7 and 10, 337/1685 cows) which have not been using footbaths may be thought to be the reason for this insignificant relationship.

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328

Assessment of animal and management based potential risk factor relation with claw health and lameness...

CONCLUSION The present association between the infectious claw disease existence with DIM and LN and lameness with the hind limb conformation and claw conformation may be considered in dairy farms as a management practice of prevention of lameness and infectious claw diseases. The result of the study may help farmers to specify their potential animal and management-based risk factors related to lameness and infectious claw lesions in their farms. Dairy farmers may be able to prevent future losses by closely observing mid and late-lactating cows for lameness and infectious claw diseases.

Statement of conflict of interest Authors have declared no conflict of interest.

References 1.

Ristevski, M., Toholj, B., Cincovi , M., Trojacanec, P., Staric, J., Smolec, O. (2017). Milk production, body condition score and metabolic parameters at the peak of lactation as risk factors for chronic lameness in dairy cows. Kafkas Univ Vet Fak Derg, 23 (5): 721-727.

2. Jewell, M. T., Cameron, M., Spears, J., McKenna, S. L., Cockram, M. S., Sanchez, J., Keefe, G. P. (2019). Prevalence of lameness and associated risk factors on dairy farms in the Maritime Provinces of Canada. J Dairy Sci, 102(4): 3392-3405.

3. Moreira, T. F., Nicolino, R. R., Meneses, R. M., Fonseca, G. V., Rodrigues, L. M., Facury Filho, E. J., Carvalho, A. U. (2019). Risk factors associated with lameness and hoof lesions in pasture-based dairy cattle systems in southeast Brazil. J Dairy Sci, 102(11): 10369-10378.

4. Weigele, H.C., Gygax, L., Steiner, A., Wechsler, B., Burla, J. B., (2018).

Moderate lameness leads to marked behavioral changes in dairy cows.

J Dairy Sci, 101(3): 2370-2382.

5. Oehm, A. W., Jensen, K. C., Tautenhahn, A., Mueller, K. E., Feist, M., Merle, R. (2020). Factors Associated with Lameness in Tie Stall Housed Dairy Cows in South Germany. Front Vet Sci, 7: 1086.

6. Novotna, I., Langova, L., Havlicek, Z. (2019). Risk factors and detection of lameness using infrared thermography in dairy cows-A review. Ann Anim Sci, 19(3): 563-578.

7. Becker, J., Steiner, A., Kohler, S., Koller-Bähler, A., Wüthrich, M., Reist, M. (2014). Lameness and foot lesions in Swiss dairy cows: II. Risk factors. Schweiz Arch Tierheilkd, 156(2): 79-89.

8. Davis-Unger, J., Schwartzkopf-Genswein, K. S., Pajor, E. A., Hendrick, S., Marti, S., Dorin, C., Orsel, K. (2019). Prevalence and lameness-associated risk factors in Alberta feedlot cattle. Transl Anim Sci, 3(2): 595606.

9. Solano, L., H. W. Barkema, E. A. Pajor, S. Mason, S. J. LeBlanc, and K. Orsel. (2016). Prevalence and distribution of foot lesions in dairy cattle in Alberta, Canada. J Dairy Sci, 99: 6828-6841.

10. Internet, https: / / dairy. ahdb .org .uk/ resources -library/ technical information/ health -welfare/ mobility -score -instructions/ #.

W3SDV7pOlFF; [Accession Date: December 16, 2021].

11. Westrik, J.D., Keulen, K.A.S., Schukken, Y., Brand, A. (1994). The effects of lameness on reproductive performance, milk production and culling

in Dutch dairy farms. Prev Vet Med, 20(4): 249-259.

12. Solano, L., Barkema, H. W., Pickel, C., Orsel, K. (2017). Effectiveness of a standardized footbath protocol for prevention of digital dermatitis. J Dairy Sci, 100(2): 1295-1307.

13. Grohn Y. T., Rajala-Schultz P. J., Allore H. G., DeLorenzo M. A., Hertl J. A., Galligan D. T. (2003). Optimizing replacement of dairy cows: modeling the effects of diseases. Prev Vet Med, 61: 27-43.

14. Bielfeldt, J. C., Badertscher, R., Tölle, K. H., Krieter, J. (2005). Risk factors influencing lameness and claw disorders in dairy cows. Livest Prod Sci, 95(3): 265-271.

15. Vermunt, J. J., Greenough, P. R. (1996). Claw conformation of dairy heifers in two management systems. Br Vet J, 152(3): 321-331.

16. Olechnowicz, J., Jaskowski, J. M., Nowak, W. (2010). Effect of hind limb conformation on claw disorders in dairy primiparous cows. Bull Vet Inst Pulawy, 54: 437-439.

17. Toussaint Raven E. (1989). Structure and functions. In: Cattle foot care and claw trimming. Edited by E. Toussaint Raven, Farming Press, Ipswich, UK, pp.27-34.

18. Ward, W. R. (1994). Recent studies on the epidemiology of lameness. In Proceedings of the VIIIth International symposium on Disorders of the Ruminant Digit and International Conference on Bovine Lameness, pp. 197-203. Banff, Canada.

19. Eddy, R. G., Scott, C. P. (1980). Some observations on the incidence of lameness in dairy cattle in Somerset. Vet Rec, 106(7): 140-144.

20. Russell, A. M., Rowlands, G. J., Shaw, S. R., Weaver, A. D. (1982). Survey of lameness in British dairy cattle. Vet Rec, 111(8): 155-160.

21. Telezhenko, E., Bergsten, C., Magnusson, M., Nilsson, C. (2009). Effect of different flooring systems on claw conformation of dairy cows. J Dairy Sci, 92(6): 2625-2633.

22. Toussaint Raven, E. (1973). Lameness in cattle and foot care. Netherlands Journal of Veterinary Science 5: 105-11.

23. Peterse, D. J. (1986). Claw measurements as parameters for claw quality in dairy cattle. In Proceedings of the 5th International Symposium on Disorders of the Ruminant Digit, pp. 87-91. Dublin, Republic of Ireland.

24. Westin, R., Vaughan, A., De Passillé, A. M., DeVries, T. J., Pajor, E. A., Pellerin, D., Rushen, J. (2016). Cow-and farm-level risk factors for lameness on dairy farms with automated milking systems. J Dairy Sci, 99(5): 3732-3743.

25. Somers, J. R., Huxley, J. N., Doherty, M. L., O’Grady, L. E. (2019). Routine herd health data as cow-based risk factors associated with lameness in pasture-based, spring calving irish dairy cows. Animals, 9(5): 204.

26. Cook, N. B., Rieman, J., Gomez, A., Burgi, K. (2012). Observations on the design and use of footbaths for the control of infectious hoof disease in dairy cattle. Vet J, 193(3): 669-673.

27. Jacobs, C., Beninger, C., Hazlewood, G. S., Orsel, K., Barkema, H. W. (2019). Effect of footbath protocols for prevention and treatment of digital dermatitis in dairy cattle: A systematic review and network meta-analysis. Prev Vet Med, 164: 56-71.

28. Cook, N. B. (2017). A review of the design and management of footbaths for dairy cattle. Veterinary Clinics: Food Anim Pract, 33(2): 195225.

29. Gomez, A., Cook, N., Socha, M., Döpfer, D. (2015). First-lactation performance in cows affected by digital dermatitis during the rearing period. J Dairy Sci, 98: 4487-4498.

30. Solano, L., Barkema, H. W., Pajor, E. A., Mason, S., LeBlanc, S. J., Heyerhoff, J. Z., Orsel, K. (2015). Prevalence of lameness and associated risk factors in Canadian Holstein-Friesian cows housed in freestall barns. J Dairy Sci, 98(10): 6978-6991.

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M. Ninković et al. Large Animal Review 2021; 27: 329-332

Frequency of White line disease and Sole ulcers and impact of hoof trimming in examined herds of Simmental cows

329

N

MILAN NINKOVIĆ1*, SVETA ARSIĆ2, JADRANKA ŽUTIĆ1, NEMANJA ZDRAVKOVIĆ1, DIMITRIJE GLIŠIĆ1, ZORANA ZUROVAC SAPUNDŽIĆ1, JOVAN BOJKOVSKI2 1 2

Department for Healthcare, Institute of Veterinary Medicine of Serbia, Belgrade, Serbia Department of Ruminants and Swine diseases, University of Belgrade, Faculty of Veterinary Medicine, University of Belgrade, Belgrade, Serbia

SUMMARY Lameness as a consequence of hoof disorders is gaining significance in animal health considerations with a particular impact in tie-hold systems. Preventive hoof trimming becomes an important zootechnic measure and procedure for the health and welfare improvement of cows. The objective of our study was to demonstrate findings of sole ulcers and white line diseases of cattle on smallholder farms. Another aspect was to display our observations on hoof trimming within the perspective of sole ulcers and white line disease in the examined herds. A total of 108 Simmental cows from 14 small noncommercial farms located in the Mačva district, Western Serbia, from March to October in 2020 were observed. The trimming procedure was performed by the Dutch method with cows fixed in a mobile chute for hoof trimming. Also, medical treatment of the hoof was provided as needed, using orthopedic foot blocks (wooden and rubberized blocks). Before treatment the white line disease and sole ulcers were present in 12 herds (85.7%). After treatment, there was a decrease in the frequency of sole ulcers positive herds (from 85.7% to 78.5%), while the frequency of white line disease in herds remained unchanged. Regarding cows, a frequency of white line disease of 25.0% was reduced to 18.5%, showing a significant difference (p<0.05) before and after treatment. The frequency of sole ulcers in cows of 23.1% was reduced to 15.7% on re-examination six months after trimming though the difference was not significant, respectively (P<0.05). White line disease and sole ulcers are widespread diseases of hooves on small noncommercial farms in Serbia. This study also provides insights that show the health status of cows’ hooves and demonstrated the importance of regular hoof trimming. Our findings suggest that regular hoof trimming (twice a year) and adequate housing conditions are crucial for improving the health of hooves.

KEY WORDS Hoof trimming; Lameness; Simmental cows; Sole ulcers; White line disease.

INTRODUCTION Lameness of dairy cows is one of the most common reasons for their exclusion from production1. Factors that lead to lameness are constant holding in deep litter straw beds, insufficient wear of the horn, irregular trimming of the hooves, poor horn quality, mechanical lesions, and wet conditions in stalls as well as the breed and nutrition2. The most usual noninfective lesions which occur in dairy cattle are sole ulcers and white line disease3, which together lead to the greatest economic losses among all foot diseases in dairy cattle4. They directly decrease the milk yield, lead to weight loss5,6 and affect the occurrence of reproductive disorders7. High milk yield, age, and body condition of cows can all be risk factors for the development of white line disease and sole ulcers of cows8. In Hungary economic losses due to lameness were € 61.60 per cow or € 320.10 per case9. The main cause of white line disease and sole ulcers is laminitis10. The sole ulcer is specific lesions of the sole, and has a multifactorial etiology11. As a consequence of laminitis, there are circulatory changes resulting from rotation of the phalangeal bone, these statistical-mechanical changes lead to pressure on the corium of the soles and development of haemorrhagic-necrotic changes and

Corresponding Author: Milan Ninković (milan.ninkovic1992@gmail.com).

predisposition to the appearance of sole ulcers12, 13. Vascular changes at the site ulcer include edema, hemmorhages, necrosis and an increased number of arterio-venous anastomoses14.

The pathological process in sole ulcers is the most prevalently located on the hind legs, on the outer claw of the hoof because it carries more weight than the inner claw15. Neglecting of hoof trimming procedure leads to horn overgrowth followed by the increased incidence of sole ulcers16. The risk of developing sole ulcers is higher if cows stand longer on a solid surface11. White line disease involves hemorrhage, fissure, and abscess between the sole and the wall of the hoof and is predominantly localized to the outer claw of the hind hoof17,18. Laminitis, which disrupts blood flow to the corium, leads to poor production and quality of keratinocytes that result in cracks in between keratinocytes along the white line and the development of while line disease10.

Due to changes along the white line, a separation wall occurs between the hoof wall and the sole of hoof18. Loss of continuity between the sole and the wall of the hoof allows the colonization of pathogenic bacteria aided by environmental factors moisture and dirty surroundings, and penetration to the corium can result in the appearance of lameness19.

Cows with lameness are 25% less likely to remain pregnant, which is in line with the fact that the highest prevalence of sole ulcers is around the peak of lactation8. Studies have shown that the impact of sole ulcers and white lines can lead to a reduction of milk production by 574 kg and 369 kg per lactation, respectively20.

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Frequency of White line disease and Sole ulcers and impact of hoof trimming in examined herds of Simmental cows

Cows affected with white line disease and sole ulcers in the first third of lactation had milk production reduced by 324 kg compared with later lactation stages21. Hoof trimming aims to improve the balance of the foot, provide relief from the overgrown horn, improve the health of hooves, and to prevent the onset of hoof diseases, and usage of therapeutics22,23. We have estimated the frequencies of white line disease and sole ulcers on small farms in Serbia because of the lack of detailed data on the frequencies of these diseases in Serbia. The subject is also of particular interest due to the choice of breed of cows, and their housing. Thus, the aim of this report is to deliver the first information on white line and sole ulcers in smallholders’ farms in the Ma va district in the Republic of Serbia and to study the effect of hoof treatment on the frequency of hoof diseases.

exclusively found on hind feet, with no alterations exhibited on front feet hoofs. The history of previous hoof trimming was not disclosed by all farmers. The trimming procedure was performed using the Dutch method, with cows fixed in a mobile chute for hoof trimming. In additional, medical treatment of the hoof was provided as needed, using orthopedic foot blocks (wooden and rubberized block).

Statistical analysis Descriptive statistics were prepared using the office software package (Microsoft® Excel® MSO, 2016), while the significance of differences before and after treatment was tested using a paired-sample t-test in GraphPad® Prism®6 software. Farms with fewer than 7 cows were excluded from the statistical comparison of the observed groups. P-values ≤0.05 were considered significant.

MATERIAL AND METHODS Statement of ethical compliance Study population This study was conducted on a total of 108 Simmental dairy cows from 14 smallholders’ farms located in four municipalities (Vladimirci, Koceljeva, Šabac, and Loznica) in the Ma va district, Western Serbia. Ages of cows were from 3 to 9 years.

All cows enrolled in this study were kept in tie-stall farms, from the first to seventh lactation with an average yearly milk yield of 5700 kg. The diet, although not uniform in all farms, was based on corn silage, alfalfa hay, and concentrate with about 16% protein in the meal. All herds had a concrete stall base with deep litter straw beds. The study took place from March to October 2020. All cows had their hooves trimmed between 60 and 180 days of lactation.

Data collected Information regarding milk yield and feeding was obtained by interview with farm owners. The clinical examination focused on all possible visible alterations on the hooves. Changes were

The experiment was done in compliance with Serbian Law on Animal Welfare (Official Gazette of the Republic of Serbia No 41/09) and Ordinance on the conditions for registration for experimental animals and the keeping of such a register, training programs on welfare on experimental animals, request forms for approval of conducting experiments on animals, standing, treatment and killing experimental animals and reproduction, circulation, or implementation experiments on animals (Official Gazette of the Republic of Serbia No 39/10).

RESULTS The average size of the herds in this study was 7.71 with standard deviation of 4.18 cows. In this study, both lesions concomitantly, white line disease and sole ulcers, were present in 10 of 14 herds (71.43%). Data of the frequency of white line disease and sole ulcers on smallholders’ farms are presented in Table 1.

Table 1 - Occurrence of sole ulcer and white line diseases in cows on smallholders’ farms.

Before treatment

After treatment

Number of cows in herd

White line disease

Sole ulcers

White line disease

Sole ulcers

1

15

4

2

4

0

2

3

0

1

0

1

3

8

2

2

1

2

4

12

3

3

1

2

5

8

0

2

1

2

6

3

1

0

1

0

7

6

1

2

2

0

8

14

3

3

2

1

9

11

2

5

1

4

10

1

1

1

0

1

11

7

3

0

2

1

12

6

2

1

1

1

13

5

3

1

3

1

14

9

2

2

1

1

Total

108

27

25

20

17

Frequency %

25

23.1

18.5

15.7

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M. Ninković et al. Large Animal Review 2021; 27: 329-332

White line disease Before treatment the frequency of white line disease was present in 12 out of 14 herds (85.71%). Amongst the 108 examined cows, the frequency of white line disease was 27 (25.0%). After treatment, the frequency of herds with white line disease remained unchanged, but the frequency of white line disease on the 108 cows was reduced to 20 (18.5%); a significant difference in the occurrence of white line disease in cows before and after hoof trimming of P<0.05 (P=0.0479).

331

Using deep litter straw beds for beddings was found to reduce the prevalence of white line disease and sole ulcers30, and our general observation supports these findings for decreased frequencies of white line disease and sole ulcers. On the other hand, there are reports that hoof trimming may not lead to reduced prevalence of sole ulcers31 and our findings haven’t shown a statistically significant reduction of sole ulcer frequency on the observed farms (P>0.05). The appearance of white line disease and sole ulcers in Simmental cows has been rarely investigated concerning lameness.

Sole ulcer Prior to treatment, sole ulcer frequency was esssentially the same as the white line disease i.e. present in 12 of the 14 herds (85.71%), and found in 25 (23.1%) cows. Subsequent to treatment, the number of herds affected dropped to 11 (78.6%), and the number of findings dropped to 17 (15.7%) cows. Decrease of sole ulcers showed no significant difference before and after treatment (P>0.05, P=0.0796).

CONCLUSION White line disease and sole ulcers are widespread diseases of hooves on small noncommercial farms in Serbia. This study also provided insights into the health status of cows’ hooves and demonstrated the importance of regular hoof trimming.

Regular hoof trimming twice a year with adequate ambient conditions can maintain the good health of hooves.

DISCUSSION Competing interests Regular hoof trimming enables insight into the health condition of the hoof and detection of subclinical claw lesions before the appearance of visible lameness. The importance of determining the frequency of hoof disorders in the herd is considerable, because farmers notice when cows become lame.

White line disease and sole ulcers are the most common claw lesions in dairy cows12. On the territory of Serbia, previous authors have estimated the frequency of white line disease and sole ulcers in cows to be 6.18% and 3.35% on mini-farms in mountainous Serbian areas24. Contrary to this, Bojkovski et al.13 estimated the frequency of sole ulcers 21.83% cows on seven farms with tie stall housing. The variable frequencies of white line disease and sole ulcer may be due to the fact that some cows develop pathological lesions without observable lameness25. The prevalences of white line disease and sole ulcers were 12.1% and 9.4%, respectively, in free stalls with deep straw bedding in Slovakia26. Four main risk factors may have caused the findings in our study: irregular hoof trimming, non-adequate conditions, wet, dirty surroundings, the negative effects of concrete flooring with deep litter straw beds, so the probabilities for the occurrence of white line disease and sole ulcers were high in the presence of these predisposing factors27. Body condition score (BCS) and digital cushion thickness are factors that may affect our results. Cows with BCS <3.5 have higher probabilities of foot disease, and composition digital cushion, therefore ratio of connective and adipose tissue have plays in the onset of hoof disease28. Due to insufficient walking surfaces on the farms, the tied way of keeping the cows does not lead to even wear of the horn and is itself an introduction to the appearance of hoof disorders. However, a significant factor influencing the overall health of cows is hoof trimming29, which had a positive impact, leading to a decrease in the frequency of white line disease and sole ulcers in our study.

A prevalence of white line disease of 8.8% was found in Sweden12. Sole ulcers were reported in 85% of herds in the Netherlands with a 1-5% prevalence among cows11. A factor that may have affected the results in our study was the lack of previous or irregular trimming practice of cows amongst the farms we studied.

The authors declare that they have no conflict of interest.

Aknowledgements Thank you very much for reading manuscript Steve Quarrie.

This paper is founded by Ministry of Education, Sciences and Technological development of Republic of Serbia under contact No.451-03-68/2020-14/200030 on TR 31079 project.

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22. Bryan M., Tacoma H., Hoekstra F. (2012). The effect of hind claw height differential and subsequent trimming on lameness in large dairy cattle herds in Canterbury, New Zealand. N Z Vet J, 60 (6): 349-355.

23. Canatan U., Hasan K., Akbala M., Cetin M., Acar H., Aslan V., Altinci Saril C., Mekik Temiz E., Yigitgor P., Salci H. (2021). How effective is individual claw trimming in cattle? An ex-vivo study. Large Anim. Rev. 27: 9-14.

24. Milosavljević P., Savić-Stevanović V. (2013). Frequency of some acropodium diseases in dairy cows in Serbia. Acta Vet-Belgrade, 63 (2-3); 247-254.

25. Solano L., Barkema H.W., Mason S., Pajor E.A., LeBlanc S.J., Orsel K. (2016). Prevalence and distribution of foot lesions in dairy cattle in Alberta, Canada. Journal of Dairy Science 2016; 99 (8): 6828-6841.

26. Mudroň P. (2016). Effects of manure bedding on the rate of claw diseases in dairy cows. Folia Vet, 60 (4): 14-19.

27. Barker Z.E., Amory J.R., Wright J.L., Blowey R.W., Green L.E. (2007).

Management factors associated with impaired locomotion in dairy cows in England and Wales. J Dairy Sci, 90 (7): 3270-3277.

28. Fabbri G, Gianesella M, Morgante M, Armato L, Bonato O, Fiore E. (2020). Ultrasonographic alterations of bovine claws sole soft tissues associated with claw horn disruption lesions, body condition score and locomotion score in Holstein dairy cows. Res in Vet Sci, 131: 146-152 29. Gudaj R, Brydle E, and Lehoczky J. (2013). Different management methods on prevalence of lameness in 25 holstein-friesian herds in Hungary. Acta Vet-Belgrade, 63 (4): 405-420.

30. Livesey C., Harrington T., Johnston A., May S., Metcalf J. (1998) The effect of diet and housing on the development of sole haemorrhages white line haemorrhages and heel erosions in Holstein heifers. Anim Sci, 67(1): 9-16.

31. Van der Tol P.P.J., Van der Beek S.S., Metz J.H.M., Noordhuizen-Stassen E.N., Back W., Weijs W.A. (2004). The effect of preventive trimming on weight bearing and force balance on the claws of dairy cattle. J Dairy Sci, 87 (6): 1732-1738.

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Extensive pleural abscess associated with congestive heart failure in a pregnant cow: an unusual presentation of hardware disease

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DOMENICO CAIVANO1*, LORENZO PISELLO1, PIERO BONI2, RODOLFO GIALLETTI1, LAKAMY SYLLA1, ILARIA PORCELLATO1, CHIARA BRACHELENTE1, FABRIZIO RUECA1 1 2

Department of Veterinary Medicine, University of Perugia, Via San Costanzo 4, 06126 Perugia, Italy Veterinary Practicioner, Via Martiri di Modena 2, 06033 Cannara, Perugia, Italy

SUMMARY A four-year-old pregnant Friesian cow was referred to the Veterinary Teaching Hospital of Perugia University for right-sided congestive heart failure signs. Thoracic auscultation revealed decreased lung sounds and absence of heart sounds on the left side.

On right site, increased breathing sounds and normal heart sounds were auscultated. Thoracic ultrasonographic examination allowed to visualize an extensive intrathoracic abscess displacing the heart against the thoracic wall with compression of the right atrium and ventricle. Congestive heart failure signs resolved after abscess drainage and the cow delivered a healthy bull-calf. Radiographic examination performed after delivery, showed a thoracic radiopaque foreign body cranial to the diaphragm. Due to worsening of the clinical health condition with subsequent poor prognosis, euthanasia was elected by the cow’s owner. At the postmortem examination, a diffuse fibrinous pleuritis with a large abscess extending among thoracic wall, heart, and lung in the left thoracic cavity and containing a metal wire were observed. Multiple adhesions were present in both thoracic and abdominal cavities. This report describes clinical, radiographic and ultrasonographic, and postmortem findings in a cow affected by a traumatic pleural abscess and clinical signs of right-sided congestive heart failure. Pleural abscesses in cows, if particularly extensive, can cause cardiac displacement against the thoracic wall with right heart dysfunction leading to congestive heart failure.

KEY WORDS Bovine; congestive heart failure; foreign body; ultrasonography.

INTRODUCTION Traumatic reticoloperitonitis (TRP), or hardware disease, is a complex disease that occurs when foreign bodies, such as wires or nails, perforate the reticular wall and penetrate into the peritoneal cavity1-3. Thoracic cavity can also be involved in the inflammatory process if the foreign body perforates the diaphragm4-5. When foreign bodies penetrate the pericardial sac, pericardial effusion and cardiac tamponade can occur; this condition is commonly called traumatic pericarditis (TP).

Tachycardia, distension/pulsation of the jugular veins, subcutaneous edema, and muffled heart sounds have been previously reported as common clinical signs of TP6-8. Severity of these clinical signs depends on the degree of cardiac tamponade6-8.

Conversely, when foreign bodies cause pleuritis or thoracic abscesses, respiratory signs such as coughing, dyspnea, and abnormal lung sounds have been commonly reported4-5.

In the present case report we describe an unusual presentation

Corresponding Author: Domenico Caivano (domenico.caivano@unipg.it).

of TRP in a cow with a diffuse fibrinous pleuritis and a large pleural abscess associated with clinical signs of right-sided congestive heart failure (CHF).

CASE PRESENTATION A four-year-old Friesian cow in 9th month of gestation was referred to the Veterinary Teaching Hospital of Perugia University with severe edema of the submandibular region and brisket.

Clinical examination showed poor body condition score (graded as 2/5), fasciculations of anconeus muscles, abduction of elbows and bilateral engorgement of the jugular veins (Figure 1).

The rectal temperature was 39.3° C and respiratory rate was 40 breaths/minute. The heart rate was 100 beats/minute with normal rhythm. Thoracic auscultation revealed decreased lung sounds over the middle third of the pulmonary fields and absence of heart sounds on the left side. Conversely, increased breathing sounds and normal heart sounds could be auscultated on right site. Hematological parameters revealed leukocytosis (21.8 × 103/µL, reference range 6.0-17.0 × 103/µL) de-

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Extensive pleural abscess associated with congestive heart failure in a pregnant cow

Figure 1 - Edema of the submandibular region and brisket with distension of the jugular vein.

creased hemoglobin (9.7 g/dL, reference range 12.0-18.0 g/dL) and hematocrit (29.7 %, reference range 35.0-55.0 %).

Serum chemistry profile showed increased hepatic enzymes (AST 340 U/L, reference range 50-150 U/L; ALKP 443 U/L, reference range 28-233 U/L) and decreased albumin (1.94 g/dL; reference range 2.5-3.5 g/dL). Ultrasonographic examination of the right thoracic cavity showed cardiac displacement against the right thoracic wall with compression of the right atrium and ventricle (Figure 2). Cardiac displacement was due to a well-defined, encapsulated mass occupying the left sided thoracic cavity. Ultrasonographic examination of the left thoracic cavity allowed to visualize this mass with hypoechoic fluid collection and multiple bright spots (Figure 2). The mass was 35 cm in diameter with a capsule of 3 cm thick. Being an extensive intrathoracic mass, its precise origin was unclear. Pleural effusion was also visualized. The ultrasonographic findings

were typical of an intrathoracic abscess and clinical signs of right-sided CHF was considered secondary to compression of right atrium and ventricle against the thoracic wall.

A thoracic drainage tube was placed, and 25 liters of purulent exudate were drained. Echocardiographic examination was repeated after abscess drainage and showed normal dimension and function of the right atrium and ventricle. However, extensive pulmonary fields on the left-sided hemithorax were characterized by consolidation areas. Based on the antimicrobial susceptibility test from purulent exudate sample (Trueperella pyogenes was isolated), sulfadimethoxine and trimethoprim (Sulfaprim®, 10ml/100kg once a day EV) were administered. Although signs of CHF disappeared in 7 days, respiratory effort continued to be present and, after 14 days from admission, the cow delivered a healthy bull-calf weighing 28.5 kg. Three days following delivery, radiographic examination was performed and a thoracic radiopaque long, thin foreign body cranial to the diaphragm was identified (Figure 3).

Due to worsening of the clinical condition and poor prognosis, euthanasia was elected by the cow’s owner. Seven days following delivery, the cow was euthanized in our hospital (after sedation, a euthanasia solution was intravenously administered) and submitted to necroscopy. A complete postmortem examination was performed. In the thoracic cavity, a diffuse bilateral fibrinopurulent pleuritis with moderate pleural effusion was evident. A large pleural abscess (about 20 cm in diameter, with 8 cm thick fibrous capsule) containing a metal wire of 7 cm was found in the left thoracic cavity (Figure 3). The abscess compressed the pulmonary parenchyma, without directly involving the lungs and showed multiple fibrous adherences with the surrounding tissues (lung and pericardium). Multiple adhesions were also present between pericardium and epicardium.

Consolidation of the left lung with interlobular emphysema and tracheobronchial/mediastinal lymphadenomegaly were also present. Within the abdominal cavity, hepatomegaly and fibrinous adhesions between the diaphragm, liver, and spleen were present.

After the euthanasia of the dam, the calf was bottle-fed with milk replacer to an amount of approximately 10% of his body weight per day in 4 times during the first 3 weeks, then twice

Figure 2 - Ultrasonographic images of the thoracic cavity from right (A) and left (B) parasternal view. (A) Only left ventricle (LV) can be visualized as the right ventricle is compressed against the thoracic wall by a mass (abscess). (B) This well-defined, encapsulated mass (abscess) is characterized by a hypoechoic fluid collection with multiple bright spots, extending for up to 35 cm from the chest wall into the left thoracic cavity. Pleural effusion was also visualized (arrow).

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Figure 3 - Radiographic (A) and postmortem (B) images. (A) Laterolateral radiograph taken at the level of the diaphragm showing a thoracic radiopaque long, thin foreign body (white arrow) cranial to the diaphragm. (B) A large pleural abscess containing a metal wire of 7 cm long (black arrow) in the left-sided thoracic cavity.

a day until weaning. He was encouraged to eat concentrates as early as possible. Upon the 3 weeks, starter concentrate intake increased and the calf started growing significantly.

DISCUSSION Clinical signs of right-sided CHF in cows are commonly due to TP, valvular endocarditis, idiopathic pericarditis, or cardiac tumors6-11. Traumatic pericarditis secondary to foreign body penetration through the reticulum is considered the main cause of right-sided CHF in cattle6-8,12-14. Perforation of the pericardium by a foreign body can cause pericarditis with fluid accumulation in the pericardial sac: if too much fluid builds up, it increases the intrapericardial pressure with subsequent cardiac compression and hemodynamic compromise6-8,12-14.

Clinical signs depend on the degree of cardiac tamponade6-8,1214 . Tachycardia, distension/pulsation of the jugular veins, subcutaneous edema, muffled heart sounds, have been commonly reported in cows affected by TP6-8,12-14. In the cow described in this report, these clinical signs were present as a consequence of an extensive pleural abscess secondary to a foreign body migration, compressing the heart. The large abscess displaced the heart against the thoracic wall with compression of the right atrium and ventricle. This dramatically decreased right ventricular and atrial diastolic volume, increasing hydrostatic pressure in the cranial and caudal vena cava and causing distension/pulsation of the jugular veins, subcutaneous edema, and abdominal effusion.

Migration of the metallic foreign body from the reticular wall can be promoted by the pressure of the fetus in advanced gestation14-16. As in the cow of this report, the pregnant uterus can apply a physical pressure to the rumen and reticulum collab-

orating for penetration by an existing foreign body in the reticulum, mainly in the final third of the pregnancy, when the weight and size of the uterus are remarkably increased17.

Echocardiography is a safe and noninvasive imaging technique that can be useful to confirm or rule out the presence of cardiac disease in cattle18-20. Moreover, this diagnostic tool is readily available and can be performed in veterinary hospitals as well as in farms. Echocardiography in animals with TRP has proven a good diagnostic procedure for distinguishing traumatic pericarditis from thoracic abscesses7. In the present case, echocardiography allowed to obtain the diagnosis of an intrathoracic abscess compressing right atrium and ventricle against the thoracic wall. Additionally, echocardiography showed the resolution of right atrial and ventricular compression with normal dimensions and function of the rightsided cardiac chambers after pleural abscess drainage, elicited by the disappearance of right-sided CHF clinical signs within 7 days.

Radiographic examination can be useful to visualize radiodense foreign bodies such as wire or nails. Laterolateral radiographic views of the caudoventral thorax and reticulum are commonly carried out with the cow in standing1,7,21,22. A dorsoventral radiographic view would also be required for accurate localization of the foreign body, however this is not possible in adult cattle due to the high dorsoventral depth of the thorax7. Frequently, the size of the cow cannot allow to obtain a conclusive radiograph with clear thoracic details. If a foreign body is not seen at the radiographic examination, its presence cannot be ruled out because thick radiodense adhesions can obscure the foreign body. Furthermore, the foreign body could be migrated back into the reticulum. Thoracic radiographic findings can be suggestive, but not conclusive for TP or pleural effusion/abscess, due to indistinguishable diaphragmatic outline

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and cardiopulmonary silhouette. In the cow of this report, laterolateral radiograph performed after the drainage of the intrathoracic abscess, showed a thoracic radiopaque long, thin foreign body cranial to the diaphragm into the caudal mediastinum.

The prognosis in cows affected by TRP depends on the severity and chronicity of the disease: medical or surgical management in cases that are treated early shows a good to guarded prognosis8,12,23; TRP with diffuse peritonitis or chronic conditions have a poor prognosis8,12,24. In our case report, humane euthanasia was elected by the cow’s owner because of severity and chronicity of the disease.

In conclusion, we have reported clinical, radiographic and ultrasonographic, and postmortem findings in a cow with a traumatic pleural abscess associated with clinical signs of right-sided CHF. Pleural abscesses in cows, if particularly extensive, can cause cardiac displacement against the thoracic wall with right heart dysfunction leading to CHF.

6.

7.

8.

9.

10.

11.

12.

13.

Acknowledgments No third-party funding or support was received in connection with this study or the writing or publication of the manuscript.

14.

15.

Conflict of Interest

16.

The authors declare that there were no conflicts of interest.

17.

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Effects of Thymus vulgaris L. essential oil and compounds on development and quality of bovine preimplantation embryos in vitro

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YUSUF ZIYA GUZEY Hatay Mustafa Kemal University, Faculty of Agriculture, Tayfur Sokmen Campus, 31060, Antakya, Hatay / Turkey

SUMMARY Thyme oil is already being used in humans and animals for its various activities; however, little is known about its influence on preimplantation embryos. We aimed to investigate the effects of thyme oil on the growth, development and quality of embryos produced in vitro. A total of 266 blastocysts were harvested from 936 oocytes in 11 replicates. The addition of thyme oil significantly increased cleavage rate (P<0.05) morulae (P<0.01) and blastocyst (P<0.001) yield. Embryo quality parameters were found to be enhanced by antioxidant supplementation at atmospheric oxygen level (P<0.001). High oxygen tension during in vitro culture of bovine embryos is detrimental probably due to the accumulation of reactive oxygen species. Reduction of oxidative stress during in vitro production of bovine embryos may support subsequent development and improve the quality of blastocysts (P<0.001).

Thyme oil can safely be used to decrease these adverse effects of oxidative stress in this regard. Moreover, in vitro culture of bovine embryos can be performed using carbon dioxide incubators instead of expensive ones.

KEY WORDS Embryo; in vitro culture; antioxidant; oxidative stress.

INTRODUCTION The oxygen tension in the lumen of the female reproductive tract is about one-third of the in vitro conditions. Suppression of oxygen tension to 5% positively affects the developmental competence of in vitro fertilized embryos, reduces the production of free radicals and ensures arrest of fewer embryos at 8-16 cell block. Moreover, high oxygen tension results extended 4-cell cycle in most embryos1. Embryos, cultured in vitro, are exposed to OS as a result of insufficient defence mechanisms. Nevertheless, surrounding cumulus cells isolate the oocyte from the extracellular media and thus the oocytes are not exposed to high oxygen. Therefore, extracellular antioxidant supplementation during in vitro maturation has no beneficial effect on cumulus-oocyte complexes (COC)2. COC can tolerate low levels of hydrogen peroxide (H2O2) exposure3.

Moreover, antioxidant supplementation to the media of fertilization may reduce the number of embryos harvested due to the increased amount of H2O2. There are conflicting reports on whether antioxidant supplementation is necessary, especially at low oxygen concentrations, during embryonic development.

Supplementation of various extracellular antioxidants to glucose-free media during the developmental period does not have a significant effect under low oxygen levels. The effect of oxygen tension on the development of in vitro cultured embryos varies depending on the culture media and the culture system.

High oxygen tension may be more detrimental due to excessive free radical production as a result of increasing OS, par-

Corresponding Author: Yusuf Ziya Guzey (yzguzey@gmail.com).

ticularly in defined and semi-defined culture systems1.

Antioxidant supplementation to the culture media is considered the best strategy to reduce oxidation. Antioxidants reduce these harmful effects by preventing the formation of free radicals and reduce apoptosis by preventing the peroxidation of lipids and lipoproteins4. The most effective antioxidants are those which break free radical chain reaction. These antioxidants consist of aromatic and phenolic ring exchange H ion to free radicals5. Since phenolic compounds are considered potential antioxidants, their use in the prevention of oxidative stress-related diseases has been subjected to many researches6. Both synthetic and natural antioxidants counteract the detrimental effects of oxidative stress on reproduction. Synthetic antioxidants may have possible side effects in addition to their toxicity7. Thyme oil and compounds are promising in studies of replacing synthetic antioxidants with natural ones8. Thymus vulgaris L. is one of the strongest species that exhibit antioxidant activities among herbs and is now widely cultivated as tea, spice and herbal medicine9. Two main active compounds of thyme oil are thymol and carvacrol. Many reports are available regarding the antioxidant activities of thymol and carvacrol6. The phenolic content of thyme oil generates this effect10. Phenolic phytochemicals protect cell contents against free radical-induced damage by contributing to optimal health with their antioxidant and free radical scavenging abilities6. The in vitro antioxidant activities of plant extracts are due to their ability to scavenge free radicals or suppress singlet oxygen formation11.

Oregano oil components provide protection against peroxide and mutagen-induced DNA damage6. Flavonoids and also phenolic phytochemicals such as carvacrol and thymol protect the cell against free radicals and oxidative stress but phenolic compounds can have both antioxidant and pro-oxidant effects dosedependently12.

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Currently, more than three-quarters of all IVF cycles in the world are performed under atmospheric oxygen conditions, albeit partially13. It is mostly hard to stabilize oxygen concentration in old generation incubators due to recurrent opening/closing of the solid frame and replacement of these incubators may slow over time because of high costs. To maintain continuity of in vitro processes and due to economic purposes, we also prefer to keep an old generation incubator at least for in vitro maturation and fertilization.

Preventing oxidative stress, which is a major problem particularly in embryo production from slaughtered cattle, and increasing the success in embryo production, as well as reducing the cost of incubators and developing natural tools, will lead to significant conveniences and developments in in vitro embryo production. From this point of view, the objective of this study was to investigate the effects of thyme oil and compounds on the development and quality of bovine embryos produced in vitro. Moreover, the potential of performing the whole process of in vitro embryo culture within carbon dioxide incubators was also investigated.

MATERIAL AND METHODS Media for in vitro production of embryos were obtained as follows. Hepes–Tyrode’s Lactate (TL), in vitro fertilization (IVF)TL, sperm preparation (SP)-TL and potassium simplex optimized medium including amino acids (KSOM+AA) were purchased from Caisson (Sugar City, ID, USA) to prepare HEPES– Tyrode’s albumin lactate pyruvate (TALP), IVF-TALP, SP-TALP and KSOM – bovine embryo (KSOM-BE) as previously described

by Parrish et al.14 and Loureiro et al.15. Thyme oil (W306509), carvacrol (W224511) and thymol (T0501) were purchased from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany).

Oocyte collection media (OCM) was tissue culture medium 199 (TCM-199) with Hanks’ salts, L-glutamine, hepes and indicator of phenol red (Thermo Fisher Scientific) supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 5% v/v fetal bovine serum (FBS). Oocyte maturation media (OMM) was TCM-199 with GlutamaxTM and phenol red without hepes (Thermo Fisher Scientific) supplemented with 2.2 mg/mL sodium bicarbonate, 10% v/v FBS, 5 µg/mL gentamycin, 0.22 mg/mL sodium pyruvate, oestradiol and gonadotropins.

COCs were rinsed in Hepes-TALP and transferred into untreated 4-well dishes containing OMM (30-40 oocytes/well) for maturation (Day -1). In vitro maturation took place in a humidified atmosphere of 5% CO2 in the air and lasted for 1822 h. COCs were then rinsed in Hepes-TALP and transferred into IVF-TALP media. Frozen semen from a single bull was thawed and the swim-up method was used to separate the motile fraction of thawed semen14. Briefly, frozen-thawed semen was gently transferred to the bottom of a sterile centrifuge tube containing SP-TALP medium and incubated for 30-45 min under a humidified atmosphere of 5% CO2 in the air. After that, the supernatant retarding the pellet was transferred into another sterile centrifuge tube and centrifuged at 400×G for 10 min. The supernatant was removed gently without disrupting the pellet containing live sperm cells (~100 µL). Sperm concentration was determined using a haemocytometer. The fertilization procedure was completed after the addition of diluted sperm, heparin and PHE cocktail (20 µM penicillamine, 10 µM hypotaurine, 1 µM epinephrine in final concentration) solution into fertilization media containing oocytes. Sperm and oocytes were cultured together for about 8-12 h under conditions of humidified atmosphere and 5% CO2 in the air. Putative zygotes were denuded of cumulus cells by gentle vortexing in HEPES-TALP at the highest speed for 3 min post-insemination and transferred randomly into KSOM-BE media supplemented with fatty acid free-bovine serum albumin (FAFBSA) and gentamicin.

In vitro culture of embryos was performed under a humidified atmosphere of 5% CO2 and 5% O2 with the balance of N2.

Cleavage rates were assessed on Day 3 (Day 0 regarded as the day of in vitro fertilization). Blastocyst formation, morphology and quality parameters were assessed on days 7 and 8. Embryos were stained for 10 min in 1 µg/mL Hoechst 33342 according to the method of Moreira et al.19 and the number of cells for each embryo was counted using fluorescent images of Hoechst-stained nuclei.

In vitro production of embryos Ovaries were derived from a local abattoir in Hatay/Turkey (35° 52 – 37° 4 N, 35° 40 – 36° 35 E) and transported in saline (0.9% w/v NaCl) supplemented with penicillin/streptomycin, at 30-35ºC within 2 h following the collection of the first ovary.

In vitro production of embryos was performed as previously described16) but in brief, the follicular fluid including cumulus-oocyte complexes (COCs) from follicles (2-8 mm diameter) was aspirated using a 21-gauge needle attached to a disposable syringe and pooled into a sterile centrifuge tube. After allowing COCs to gravitate, supernatant follicular fluid was removed carefully by not disrupting the residue. Debris, including COCs, was then transferred into petri dishes containing OCM. Classification of COCs performed under a stereomicroscope (×10~40) according to the method of Boni et al.17 and Tasripoo et al.18. COCs with at least 3 layers of compact cumulus cells surrounding a homogenous evenly granulated cytoplasm were classified as immature. Following classification,

Figure 1 - Embryo quality assessment. Photomicrographs of (A) bovine oocyte, (B) Cleavage after 36-48 h, (C) d 8 blastocyst (scale bar = 100 µm) and (D) stained cells (scale bar = 100 µm) after Hoechst 33342 staining.

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Y. Z. Guzey; Large Animal Review 2021; 27: 337-342

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Experimental design Solution of thymol at a concentration of 0.05 µM was prepared in ethanol. Designated treatment groups were [1] Control at low oxygen (5%) tension (CLO), [2] Control at high oxygen (20%) tension (CHO), [3] KSOM-BE supplemented with 0.05 µM carvacrol at low oxygen tension (CrvLO), [4] KSOM-BE supplemented with 0.05 µM carvacrol at high oxygen tension (CrvHO), [5] KSOM-BE supplemented with 0.05 µM thymol at low oxygen tension (TLO), [6] KSOM-BE supplemented with 0.05 µM thymol at high oxygen (THO), [7] KSOM-BE supplemented with 0.025% thyme oil at low oxygen tension (ThyLO) and [8] KSOM-BE supplemented with 0.025% thyme oil at high oxygen tension (ThyHO). Embryos were randomly assigned to the treatment groups and compared for embryo development and quality.

Figure 2 - Embryo yields under a controlled oxygen concentration.

Statistical analyses Data were collected in 11 replicates over days for the experiment. Data were arcsine transformed and analysed using a General Linear Model (GLM) for evaluation of differences between developmental stages of IVP embryos. Embryo diameters and blastomere counts were analysed using GLM. The model included as main effects oxygen tension (5 and 20 per cent) and the antioxidant resource (control, carvacrol, thymol and thyme oil). The interaction between the two tested effects was also considered in the following model;

here µ is general mean, αi is the effect of oxygen tension, ßj is the effect of antioxidant, (αß)ij is the interaction between oxygen tension and antioxidant, and eijk is random error. Embryo quality scores were evaluated utilizing chi-square. All analysis was performed using SPSS software (v23.0, SPSS Inc., Chicago, IL). Data were presented as mean±standard error (SE). The p-value used to determine significance in all tests was 0.05.

Ethics As stated by Animal Protection Act article 2c of the directive 2011/28914, embryos are not included in the list of organisms that require specific authorization, so no approval was requested.

RESULTS The effect of oxygen tension on cleavage was significant in favour of low oxygen tension (p<0.01), in the present experiment. The

Figure 3 - Embryo yields under an atmospheric oxygen concentration.

number of cleaved embryos under low oxygen tension was 456 from 486 oocytes in all replicates (Figure 2). However, the effect of antioxidant supplementation to the culture media was not significant. Cleavage rates obtained for low oxygen tension was similar according to multiple comparisons of Tukey HSD (Table 1). Antioxidant supplementation to the culture media significantly enhanced the cleavage rate of embryos exposed to a gas phase of atmospheric oxygen (p<0.05). The number of cleaved embryos for high oxygen tension was 399 from 450 oocytes (Figure 3). Effect of antioxidant addition to the cul-

Table 1 - Embryo counts during in vitro development.

Control

Carvacrol

Thymol

Thyme Oil

SEM

P AO

OT

AO × OT

Cleavage (%)

LO HO

93.13 78.98a

92.91 92.18b

95.44 92.58b

93.65 90.83b

0.96 1.47

0.01 9

0.00 2

0.023

Morulae (%)

LO HO

59.72a,b 50.37a

56.92a 55.99b

60.41b 61.32c

59.60a,b 61.49c

0.76 1.32

0.00 0

NS

0.003

Blastocyst (%)

LO HO

32.03 16.52a

32.19 31.24b

33.80 34.23b

33.28 32.30b

0.44 1.43

0.00 0

0.00 0

0.000

LO: low oxygen tension; HO: high oxygen tension; AO: antioxidant; OT: oxygen tension; NS: non-significant; B: blastocyst; SEM: Standard error of the mean, different letters of superscript in a row represent different groups according to Tukey HSD.

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ture media of IVP embryos under controlled and high oxygen tension found to be significant (p<0.05).

Antioxidant addition to the culture media did not result in any significant development rate under the condition of controlled oxygen tension. Nevertheless, a very slight increase was confirmed by thymol supplementation while a slight decrease was observed for those supplemented with carvacrol. The number of embryos developed to morulae was 269 under low oxygen tension (Figure 2), whereas it was 229 for atmospheric oxygen concentration (Figure 3). Development rates of embryos to morulae significantly increased by antioxidant addition under atmospheric levels of oxygen (p=0.000). Table 1 indicates an enhancement of antioxidant supplementation depending on oxygen tension (p<0.01).

An apparent decrease in blastocyst yield is noticeable from Table 1 at atmospheric oxygen concentration. The addition of antioxidants to the culture media significantly improved the blastocyst yield of IVP bovine embryos (p=0.000) at the atmospheric level of oxygen. Blastocyst yield for low oxygen tension did not enhance by antioxidant supplementation and was also similar in all groups. Blastocyst yield was 149 for low oxygen tension (Figure 2) and 117 at atmospheric oxygen concentration (Figure 3). According to these results, we can claim that the addition of thyme oil and/or compounds to the culture media of IVP bovine embryos significantly improved blastocyst yield depending on oxygen concentration (p=0.000).

Another remarkable parameter widely accepted other than blastocyst yield is embryo quality in in vitro embryo production.

Embryo diameter, blastomere count and embryo quality scores were recorded during the experiment as qualitative and quantitative parameters in this regard (Table 2). The effect of oxygen tension on embryo diameter was significant and, the addition of antioxidants to the culture media improved embryo diameter under both low and high oxygen conditions (p=0.000). Best results were obtained by the addition of carvacrol and thymol independently of oxygen concentration. A dramatic decrease in embryo diameter was also recorded at atmospheric oxygen level.

Similar results were obtained for blastomere count, the addition of antioxidants to the culture media significantly improved blastomere numbers (p=0.000).

Maximum blastomere number recorded for thyme oil group, independently of oxygen concentration. Embryo quality score was significantly improved by the addition of antioxidants to the culture media (p=0.000).

DISCUSSION The developmental ability of oocytes, derived from slaughtered cows is relatively low than those in vivo. The major challenge for scientists is to mimic in vivo conditions for in vitro embryo production. Disturbances in the redox state of the cell may cause toxic effects via the production of peroxides and free radicals and thus may damage protein, lipid and even DNA structure.

Antioxidant supplementation to the media may prevent mitochondria and DNA damages4.

In the present study, a significant decrease in cleavage rates was observed under the condition of atmospheric oxygen concentration. Oocytes rely on the energy provided by the mitochondria. Oxidative phosphorylation accounts for about onethird of ATP synthesis during the process reach up to cleavage20.

Reactive oxygen species can initiate mitochondrial dysfunction21.

ROS emerging as a by-product of mitochondrial metabolism can trigger oxidative damage and impair the ability to synthesize ATP. Reduced mitochondrial activity at this stage may result in the arrest of the oocyte, disrupted fertilization and impaired embryo development4, 20, 22, 23. Even though we disregarded individual oxygen consumption and ROS measurement, putative zygotes in the CHO group presumably suffered from oxidative stress.

Supplementation of culture media with thyme oil, carvacrol and thymol significantly improved the cleavage, morulae and blastocyst yields in vitro, independently of the oxygen tension (Table 1). Hydrogen peroxide does not interact directly with DNA. Crossing the biological membrane, H2O2 penetrates the cell nucleus and interacts with iron and copper ions to form OH radicals, which cause lesions and breaks in DNA. This protective effect of carvacrol and thymol against DNA lesions is due to the potent oxidant H2O224. However, excessive production of hydrogen peroxide and superoxide anion can damage mitochondria. Chromatin condensation and fragmentation, apoptosis, disturbed cell division and embryonic arrest may occur as a result of protease and caspase activation, subsequently25.

Reactive oxygen species can initiate detrimental effects and are able to pass through the cell membrane. This may result in the cell block of embryos only in IVF. Unfavourable conditions, such as OS, may challenge in vitro development of bovine embryos from one cell to blastocyst and result 8-16 cell block. This may have been perceived as a reason for the low morulae yield observed in this experiment. Metal chelators have a beneficial effect on embryo development21. Thyme oil and compounds seem

Table 2 - Blastomere number, diameter and quality score of blastocysts.

Control

Carvacrol

Thymol

Thyme Oil

SEM

P AO

OT

AO × OT

Diameter (µm)

LO HO

171.14a 155.91a

231.14c 228.82c

232.64c 227.59c

207.73b 201.09b

4.48 5.22

0.000

0.000

0.000

Embryo quality

LO HO

1.8 2.0

1.8 1.8

1.73 1.77

1.80 2.41

0.34

0.000

NS

NS

Cell count

LO HO

80.45a 67.09a

93.18c 93.18c

89.18b 88.55b

95.00c 93.55c

1.12 1.96

0.000

0.000

0.000

LO: low oxygen tension; HO: high oxygen tension; AO: antioxidant; OT: oxygen tension; NS: non-significant; B: blastocyst; SEM: Standard error of the mean, different letters of superscript in a row represent different groups according to Tukey HSD.

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to enhance morulae yield by reducing ROS formation due to its strong radical scavenger characteristic. Such antioxidants prevent lipid oxidation stimulated by Fe2+/ascorbate and Fe2+/H2O2 and thus aid to overcome this cell block5.

Oxygen tension at the atmospheric gas phase resulted in a decrease in embryo diameter and blastomere count. However, antioxidant supplementation to the culture media under high oxygen tension enhanced diameter, blastomere count and also embryo quality (Table 2). A mechanism involving a cascade of metabolic reactions may cause the production of free oxygen radicals and such suboptimal conditions may result in arrested cell division and can influence programmed cell death. Higher oxygen tension triggers an upsurge of ROS in the cytoplasm of the embryo and thus a decrease in in vitro development26.

The beneficial effect of thyme oil may partly occur due to the detoxification of highly diffusible reactive species. Antioxidant supplementation to the media permits embryo development, probably by limiting the peroxidation process. Germ cells are much more sensitive to ROS and OS than somatic cells. High ROS in culture media causes a decrease in embryo development and blastocyst formation2. Antioxidants serve to prevent the formation or circulation of free radicals and thus delay autoxidation. Most research exhibited that antioxidant supplementation is essential for better quality embryo production, increased longevity and blastulation5.

Embryos may have different sensitivities to ROS at different developmental stages because of various defence mechanism thresholds. Both spermatozoa and embryos are sources of ROS, which may lead to oxidative damage and thus impaired embryo development in vitro. Blastocysts are more sensitive to free radicals21, 27. Today it is widely accepted that low oxygen tension is present in the mammalian genital tract but embryos are exposed to 20 per cent atmospheric oxygen during in vitro fertilization.

Reduced oxygen tension in in vitro embryo culture is related to better blastocyst formation and blastomere number in many species. Embryos expose decreasing amount of oxygen tension along their journey from the oviduct to the uterus, and finally lowest tension at blastocyst formation following compaction28. Gene expression, proteome, metabolism and genome are adversely affected in embryos under atmospheric conditions23. We can subjectively claim that OS is one of the environmental factors responsible for this critical drop in blastocyst yield by half. In corroborate with the results that report higher developmental rates of embryos cultured in vitro22, we obtained high blastocyst yield under low oxygen tension and high oxygen tension by supplementation of thyme oil and compounds to the culture media.

It has been reported that the compounds of thyme oil can modulate DNA damage in a dose-dependent manner through peroxide and some genotoxins6. Hydrogen peroxide mainly causes DNA breaks through the formation of hydroxyl radicals by the Haber-Weiss reaction catalysed by ferric ions that can diffuse in cells and tissues. ROS pass through the cell membrane and cause changes in macromolecules in the cell, mitochondrial alterations, meiotic arrest of the oocyte, embryonic block and even apoptosis29. Phenolic compounds have the ability to chelate metal ions. The protective effect of flavonoids against H2O2-induced DNA damage is provided by the intracellular binding of iron11. In particular, thymol and carvacrol are effective in scavenging peroxyl radicals by reducing the peroxidation of phospholipid liposomes and the presence of iron

341

and ascorbate8.

Although optimum conditions have not been set down yet, controlled oxygen tension (~5%) stands for an accepted scientific fact for in vitro embryo production. Cumulus-cell matrix that surrounds oocyte during in vivo embryo development assist protection from intracellular pH alterations since such switch may cause impaired development. Our hypothesis here is that the beneficial effects of low oxygen tension are not essential when oocytes are enclosed in their cumulus cells. It has been reported that genes expressing proteins with antioxidant function are at a lower level at the 2-4 cell stage at 20% oxygen concentration. The effect here is due to the use of maternal storage antioxidants under OS conditions prior to genome activation in the embryo. However, it has been reported that the amount of antioxidants before the 8-cell stage is not induced by OS30.

Herein, the addition of thyme oil and its compounds seem like an effective alternative to disburden the detrimental effect of oxidative stress at a gas phase of atmospheric oxygen. Regarding the complex composition of essential oils, we aimed to reveal the possible mechanism of action of thyme oil and its compounds on the reduction of OS. In conclusion, carvacrol and thymol can be metabolized by cells during in vitro culture of bovine embryos in glucose-free semidefined media, and can safely be used in terms of cyto- and genotoxicity to obtain improved developmental rate. Moreover, it was concluded that in vitro embryo culture with supplementation of thyme oil can also be performed in conventional carbon dioxide incubators.

In the present experiment, thymol and thyme oil increased the rate of bovine embryos developed into morulae and blastocyst in vitro. These results, in the first place, showed that herbal antioxidants can safely be used in terms of cytotoxicity dose-dependently and can enhance the environmental conditions of bovine embryos produced in vitro. Reduction of oxidative stress during in vitro production of bovine embryos may support subsequent development and improve the quality of blastocysts as a consequence. Further research on quantitative analysis of embryonic oxidative status would be crucial to acquire a precise definition of the anti-oxidative effects of thyme oil and compounds. Moreover, the effects of thyme oil and its compounds on reactive nitrogen species as well as reactive oxygen species should be the subject of further studies.

ACKNOWLEDGEMENT The financial support of the Scientific Research Projects Coordination Unit of Hatay Mustafa Kemal University (Grant No. 15113) is gratefully acknowledged.

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2. Abedelahi, A., Salehnia, M., Allameh, A. A., Davoodi, D. (2010). Sodium selenite improves the in vitro follicular development by reducing the reactive oxygen species level and increasing the total antioxidant capacity and glutathione peroxide activity. Hum Reprod, 25: 977-985.

3. Loren, P., Sánchez, R., Arias, M.-E., Felmer, R., Risopatrón, J., Cheuquemán, C. (2017). Melatonin Scavenger Properties against Oxidative and Nitrosative Stress: Impact on Gamete Handling and In Vitro Embryo Production in Humans and Other Mammals. Int J Mol Sci, 18: 1119.

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Brewer, M. S. (2011). Natural Antioxidants: Sources, Compounds, Mechanisms of Action, and Potential Applications. Compr Rev Food Sci F, 10: 221-247. Ündeğer, Ü., Başaran, A., Degen, G. H., Başaran, N. (2009). Antioxidant activities of major thyme ingredients and lack of (oxidative) DNA damage in V79 Chinese hamster lung fibroblast cells at low levels of carvacrol and thymol. Food Chem Toxicol, 47: 2037-2043.

Onal, A. G., Guzey, Y. Z. (2019). Effect of Ferula communis L. on reproductive parameters in Awassi ewes. J Hellenic Vet Med Soc, 70: 1625-1630.

Aydın, S., Başaran, A. A., Başaran, N. (2005). Modulating Effects of Thyme and Its Major Ingredients on Oxidative DNA Damage in Human Lymphocytes. J Agric Food Chem, 53: 1299-1305.

Domaracký, M., Rehák, P., Juhás, S., Koppel, J. (2007). Effects of selected plant essential oils on the growth and development of mouse preimplantation embryos in vivo. Physiol Res, 56: 97-104.

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Kozics, K., Klusová, V., Srančíková, A., Mučaji, P., Slameňová, D., Hunáková, L., Kusznierewicz, B., Horváthová, E. (2013). Effects of Salvia officinalis and Thymus vulgaris on oxidant-induced DNA damage and antioxidant status in HepG2 cells. Food Chem, 141: 2198-2206.

Ismail, A. A., Abdel-Khalek, A.-K. E., Khalil, W. A., Yousif, A. I., Saadeldin, I. M., Abomughaid, M. M., El-Harairy, M. A. (2020). Effects of mint, thyme, and curcumin extract nanoformulations on the sperm quality, apoptosis, chromatin decondensation, enzyme activity, and oxidative status of cryopreserved goat semen. Cryobiology, 97: 144-152.

Sovernigo, T., Adona, P., Monzani, P., Guemra, S., Barros, F., Lopes, F., Leal, C. (2017). Effects of supplementation of medium with different antioxidants during in vitro maturation of bovine oocytes on subsequent embryo production. Reprod Domest Anim, 52: 561-569.

Parrish, J. J. (2014). Bovine in vitro fertilization: in vitro oocyte maturation and sperm capacitation with heparin. Theriogenology, 81: 67-73.

Loureiro, B., Brad, A. M., Hansen, P. J. (2007). Heat shock and tumor necrosis factor-alpha induce apoptosis in bovine preimplantation embryos through a caspase-9-dependent mechanism. Reproduction, 133: 1129-1137.

Soto, P., Natzke, R. P., Hansen, P. J. (2003). Actions of tumor necrosis factor-alpha on oocyte maturation and embryonic development in cattle. Am J Reprod Immunol, 50: 380-388.

Boni, R., Cuomo, A., Tosti, E. (2002). Developmental Potential in Bovine Oocytes Is Related to Cumulus-Oocyte Complex Grade, Calcium

Current Activity, and Calcium Stores. Biol Reprod, 66: 836-842.

18. Tasripoo, K., Srisakwattana, K., Nualchuen, W., Sophon, S. (2012). Effects of various activators on bovine embryonic development following intracytoplasmic sperm injection. Iran J Appl Anim Sci, 2: 167-173.

19. Moreira, F., Paula-Lopes, F. F., Hernández-Cerón, J., Moore, K., Hansen, P. J. (2001). Protocol to Count Cell Number of Preimplantation Embryos using Nuclear Staining with Hoechst 33342 or DAPI, Available at: https://animal.ifas.ufl.edu/hansen/lab_protocol_docs/protocol_count_cell_number_preimplantation_embryos_nuclear_staining_hoechst_33342_dapi.pdf, Access date: 11.05.2017.

20. Lopes, A. S., Lane, M., Thompson, J. G. (2010). Oxygen consumption and ROS production are increased at the time of fertilization and cell cleavage in bovine zygotes. Hum Reprod, 25: 2762-2773.

21. Guérin, P., El Mouatassim, S., Ménézo, Y. (2001). Oxidative stress and protection against reactive oxygen species in the pre-implantation embryo and its surroundings. Hum Reprod Update, 7: 175-189.

22. Takahashi, T., Sasaki, K., Somfai, T., Nagai, T., Manabe, N., Edashige, K. (2016). N, N-Dimethylglycine decreases oxidative stress and improves in vitro development of bovine embryos. J Reprod Dev, 62: 209-212.

23. Truong, T., Gardner, D. K. (2017). Antioxidants improve IVF outcome and subsequent embryo development in the mouse. Hum Reprod, 32: 2404-2413.

24. Horvathova, E., Turcaniova, V., Slamenova, D. (2007). Comparative study of DNA-damaging and DNA-protective effects of selected components of essential plant oils in human leukemic cells K562. Neoplasma, 54: 478-483.

25. Cheuquemán, C., Arias, M. E., Risopatrón, J., Felmer, R., Álvarez, J., Mogas, T., Sánchez, R. (2015). Supplementation of IVF medium with melatonin: effect on sperm functionality and in vitro produced bovine embryos. Andrologia, 47: 604-615.

26. Kitagawa, Y., Suzuki, K., Yoneda, A., Watanabe, T. (2004). Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology, 62: 1186-1197.

27. de Assis, P. M., Castro, L. S., Siqueira, A. F., Delgado Jde, C., Hamilton, T. R., Goissis, M. D., Mendes, C. M., Nichi, M., Visintin, J. A., Assumpção, M. E. (2015). System for evaluation of oxidative stress on invitro-produced bovine embryos. Reprod Biomed Online, 31: 577-580.

28. Harvey, A. J. (2007). The role of oxygen in ruminant preimplantation embryo development and metabolism. Anim Reprod Sci, 98: 113-128.

29. Khazaei, M., Aghaz, F. (2017). Reactive Oxygen Species Generation and Use of Antioxidants during In Vitro Maturation of Oocytes. International J Fertil Steril, 11: 63-70.

30. Amin, A., Gad, A., Salilew-Wondim, D., Prastowo, S., Held, E., Hoelker, M., Rings, F., Tholen, E., Neuhoff, C., Looft, C., Schellander, K., Tesfaye, D. (2014). Bovine embryo survival under oxidative-stress conditions is associated with activity of the NRF2-mediated oxidative-stress-response pathway. Mol Reprod Dev, 81: 497-513.

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K.E. Yanar, M.S. Aktas et al. Large Animal Review 2021; 27: 343-350

Investigation of the effect of Parapoxvirus ovis, Corynebacterium cutis lysate and vitamin c on immunosuppression caused by long-term transport stress in Morkaraman sheep*

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KERIM EMRE YANAR*, MUSTAFA SINAN AKTAS Department of Internal Medicine, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey

SUMMARY The purpose of the study is to investigate the effectiveness of the inactive Parapoxvirus ovis (iPPVO), Corynebacterium cutis Lysate (CCL) as well as Vitamin C (Vit-C) on immunosuppression induced by long-term transport stress in Morkaraman ewes. The study consisted of 4 groups: 1 control, 3 trial groups (iPPVO, CCL and Vit-C), and 8 ewes were allocated into each treatment group (n=32). While levels of cortisol and adrenaline significantly (P<0.05) increased at post-transport (PT), the lowest increase was obtained from the Vit-C group. Lymphocyte counts in iPPVO group significantly (P<0.05) increased at PT. Additionally, ADA activities in the iPPVO, CCL and Vit-C groups significantly (P<0.05) increased. However, activities of ADA in the control group decreased (P<0.05). A significant (P<0.05) positive correlation (r=0.24) was also obtained between ADA activities and lymphocyte counts. Hp levels of the control group at 7th days after transport (PT7) approached levels at Pre-transport (PrT).

However, the Hp levels at PT, PT1 and PT7 were similar to that of the iPPVO group at PrT. In the CCL group, the Hp level at first day after transport (PT1) attained a similar level at PrT. In conclusion, immediately before the transport, administration of Vit-C reduced transport stress more than others. Additionally, it could be suggested that the administration of iPPVO before transport was more effective toward immunosuppression occurring after transport, when lymphocyte counts, ADA activities and Hp levels were taken into consideration.

KEY WORDS Transport, stress, Parapoxvirus ovis, Corynebacterium cutis lysate, vitamin C, sheep.

INTRODUCTION Animal transport, which is an important element for comprehensive production systems, is very common especially for sheep1. Various reasons, for example the use of pasture-based farming systems, breeding of animals, fattening, slaughter, grazing opportunities, or sales lead to the need for transport2. Road transport is the most economically important form of transportation in the European Union countries. Twenty-five million cattle, 7 million calves, 171 million pigs, 75 million sheep and lambs and 9 million goats are transported annually, and 90% of the animal transportation is carried out by road3. If the journey distance of the animals is shorter than 50 km, the transportation is considered as short distance journey4. On the other hand, it was reported that the duration of the transportation was longer than 8 hours (including loading and unloading time) as well as distance of the journey was greater than 400 km in the long route transport of animals5,6.

* This study was supported by a grant from the Administration of Scientific Research Projects of Ataturk University, Turkey, (Project number: TDK2019/7206).

The manuscript is result of the Doctorate Thesis of the first author.

Corresponding Author: Kerim Emre Yanar (emre.yanar@atauni.edu.tr).

In the road transport of ruminants, many factors, for instance, loading and unloading moments, duration of the journey, improper vehicle use, bad road conditions, vibration and vehicle movements, very hot or cold climate, insufficient ventilation, high stocking densities, water and feed insufficiency result in stress, and adversely affect animal welfare, animal health, animal performance as well as the quality of the products7. The hypothalamo-pituitary-adrenocortical axis (HPA) and sympatho-adrenomedullary axis activation are held responsible for the stress8. Researchers have reported increases in cortisol and catecholamines levels at different stages of the transport due to the HPA activation9,10. Increases in the activity of the Adrenal cortex negatively affect animal welfare and may lead to immunosuppression11.

Behavioral changes, hormonal changes, oxidative stress markers, immunological indicators, genomic and proteomic indicators are used as biomarkers for the determination of stress in animals12,13. The immunological responses of ruminants to HPA activation that occurred during transport are leukocytosis and lymphopenia14. Results of the studies revealed that especially the T lymphocytes counts decreased during transport.

On the other hand, there was no significant change in the B lymphocytes counts15,16.

ADA is an enzyme widely found in tissues and body fluids. However, the most important biologic activity is associated with lymphoid tissue, since ADA is required for the proliferation and differentiation of T lymphocytes17. A positive significant correlation between ADA activity and lymphocyte count was also determined by Rao et al.18 and Abdi et al.19. It was also reported

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that the decrease in ADA activity was associated with suppression of lymphocyte count, lymphocyte proliferation and differentiation, which led to a lack of immune response20,22. Additionally, Lomborg et al.23 suggested the usage of Hp as a physiological indicator of stress in cattle during road transportation. It was also noted that increased Hp levels were responsible for immunosuppression after transport, and the HP fraction (33 k+20 K Dalton) obtained from serums led to dose-dependent suppression in lymphocyte blastogenesis24.

Purposes of the immunomodulation in animals are as follows: activating a strong and long-term immune response to diseases caused by microorganisms; accelerating the maturation of specific and non-specific immunity in the neonatal period and young animals; developing local protective immune reactions in sensitive areas such as the mammary gland in dairy cattle or the gastrointestinal tract in neonatal ruminants; reducing the immunosuppressive effects of stress and environmental pollution; increasing immunity time after vaccination25. Neuroendocrine hormones, thymus products, inactive Parapoxvirus ovis, Corynebacterium cutis lysate, Probiotics, Levamisole, Vit-E and Vit-C are some of the immunomodulators26,29.

Vit-C has been shown to enhance differentiation and proliferation of B- and T-cells as well as maturation of immature T cells30,31. Parapoxvirus ovis and Corynebacterium cutis lysate are non-selective (innate) immunmodulators which often resulted in the activation and modulation of T lymphocyte function32.

Therefore, they could have potential positive effect on reducing immunosuppression caused by long route transport stress.

To the best of our knowledge, no single study exists which compares the immunomodulatory efficacy of iPPVO, CCL and Vit-C on the ADA activity and Hp in long-term road transport of sheep. Therefore, it was aimed to compare the effects of some immunostimulants, such as iPPVO, CCL and Vit-C on immunosuppression that occurred as a result of stress-induced by long-term road transportation of Morkaraman ewes in this study.

MATERIALS AND METHODS The study has been approved by the ethics committee for animal experiments in Ataturk University (Decision No: 2018/78).

A total of 32 Morkaraman ewes at 1-2 years old which were subjected to the same care and feeding conditions were used in the current study. After the feces of the animals were examined regarding diarrhea, blood and constipation, and then healthy ewes were included in the research. Prior to one week from the beginning of the study, all animals were administered 1 ml/50 kg single dose of the antiparasitic drug (Ivomec®, Boehringer Ingelheim, France) containing 10 mg/ml ivermectin as well as 1ml/20 kg single dose of antibiotic (Reptopen®, Ceva, France) containing 200.000 IU/ml Benzilpenicilinprokain and 200 mg/ml Dihidrostreptomicin sülfate.

Experimental design The study consisted of 4 groups as 1 control and 3 experimental groups (iPPVO, CCL and Vit-C). A total of 32 ewes was randomly allotted to 4 groups containing 8 ewes in each one. In Group I, 2 ml of a commercial product (Zylexis®, Zoetis, USA) containing a minimum of 230 IFN inactive strain of Parapoxvirus ovis (iPPVO) D 1701 in per 1 ml was administered intramuscularly to ewes at two times with 3-days interval33. An-

imals in Group II were injected subcutaneously with 2 ml of Ultra-Corn® (Virbac, France) containing 20 mg of Corynebacterium cutis lysate (CCL) per 1 ml, at 2 times with an interval of 8 days34. Last injections of ewes in Group I (iPPVO) and Group II (CCL) were performed 24 h before transport. In Group III, another commercial drug (Maxivit-C®, Bavet, Turkey) containing 200 mg of ascorbic acid per 1 ml was administered intramuscularly at a single dose of 200 mg/kg to ewes just before transport35. Animals in the control group (Group IV) were received 10 ml of 0.9 % saline intramuscularly (placebo) 24 h before transport.

Transport procedure The animals from different treatment groups were mixed and loaded on the same truck by using a loading ramp. The sizes of the trailer of the truck utilized for transportation were 8 m in length and 2.2 m in width. Therefore, stocking density was about 0,4 m2/ewe. The distance traveled was 510 km, and the journey took 8 hours including the time needed for loading and unloading.

Obtaining clinical findings Temperature (°C), pulsation (beat/min) and respiration rate (per min), cough, runny nose/tear, diarrhea and pathological lung sounds of animals in all groups were determined and recorded at pre-transport (PrT), post-transport (PT) as well as 1st (PT1) and 7th (PT7) days after transport.

Blood sampling Blood samples from Vena Jugularis of all ewes were taken in 10 ml serum tubes (Vacutainer tube with clot activator, Becton Dickinson Co. USA) and sterile test tubes with 0.14% anticoagulant (EDTA K3, Pty Ltd., Adelaide, SA, Australia) in the sampling days at PrT, PT, PT1 and PT7. In group III, PrT measurements were taken before administering the treatment. Serum samples were obtained after centrifugation at 3000 g for 10 min, and were put into Eppendorf tubes. They were stored in a freezer at -80 °C until they were analyzed. For hematological analysis, Abacus® Junior Vet 5 brand hemogram device was used.

Adrenaline and cortisol levels at PrT and PT as well as ADA activity and Hp levels at PrT, PT, PT1 and PT7 were determined by using EL SA specific test kits for sheep (YL Biont® Sheep Epinephrine/Adrenaline, Cortisol, ADA and Haptoglobin ELISA Kit, Shanghai YL Biotech Co., Ltd.).

Statistical analysis According to the results of the normality test, it was found out that data obtained from this study had a normal distribution.

Therefore, the data were statistically analyzed by the One-Way ANOVA procedure of the SPSS program (version 22.0)36. Different immunomodulators (iPPVO, CCL, Vit-C and control groups) and sampling days (PrT, PT, PT1 and PT7) were considered as main effects in the statistical analysis and the main effects were analyzed separately. The comparisons were first made among immunomodulator groups, and then within the same group among the data obtained at PrT, PT, PT1 and PT7 sampling days.

The Duncan’s Multiple Comparison Test was applied for comparison of subclass means when F-test for the main effect was significant. Pearson correlation coefficients were also calculated to analyze the covariation between lymphocyte and ADA variables by the SPSS computer software program36.

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RESULTS Clinical findings Temperature, pulsation and respiration rate obtained at PrT, PT, PT1 and PT7 are presented in Table 1. At PT, temperature, pulsation and respiration rate were major clinical findings exhibited by the animals. The temperature in all groups except for the Vit-C group increased in PT compared to PrT. On the other hand, the value in PT1 decreased in comparison with PT. However, pulsation and respiration rate significantly increased in all groups immediately after transport (PT) comparing to their baselines (Table 1).

In this study, cough, nasal discharge, tear discharge, diarrhea, and pathological sounds in the lungs were not observed in any animal at PrT, PT and PT7. However, 2 animals in the iPPVO

group, 3 animals in the CCL group, 4 animals in Vit-C group as well as 7 animals in the control group had a slight serous and mucopurulent nasal discharge at PT1. Pathological lung sounds were also detected in 2 animals in the iPPVO group and 1 animal in the CCL group at PT1. Additionally, medium pitched vesicular sounds were determined in the lungs of 3 animals in the Vit-C Group in addition to 7 animals in the control group. None of the animals had diarrhea.

Hematological findings Hematological results of ewes in iPPVO, CCL, Vit-C and control groups are presented in Table 2. Statistically significant (P<0.05) increases concerning leukocyte counts at PT and PT1 comparing to PrT were detected in the iPPVO group and VitC groups respectively. Lymphocyte counts in iPPVO group at

Table 1 - Means and standard errors for clinical findings at PrT, PT, PT1 and PT7 days.

Parameters

Temperature °C

Pulsation (Beat/min)

Respiration (Rate/min)

iPPVO (Group I) X̄±SE

CCL (Group II) X̄±SE

Vit-C (Group III) X̄±SE

Control (Group IV) X̄±SE

PrT PT PT1 PT7

39.41±0.07AB 39.96±0.09C 38.96±0.24A 39.58±0.15B P<0.05

39.52±0.11A 39.97±0.12B 39.42±0.15A 39.50±0.16A P<0.05

39.55±0.12AB 39.82±0.16B 39.33±0.09A 39.42±0.16AB P<0.05

39.61±0.1A 40.11±0.06B 39.47±0.17A 39.73±0.1AB P<0.05

P>0.05 P>0.05 P>0.05 P>0.05

PrT PT PT1 PT7

94.12±3.89Aab 119.5±2.44B 100.5±4.04A 99.75±4.41A P<0.05

94.12±3.19Aa 117.50±3.01B 96±2.59A 96.75±4.27A P<0.05

107.12±4.24Ab 125.50±4.06B 106.25±3.53A 112.75±7.2AB P<0.05

99.5±4.08Aab 117.75±3.0B 97.25±4.86A 110±7.86AB P<0.05

P<0.05 P>0.05 P>0.05 P>0.05

PrT PT PT1 PT7

44.12±2.42A 65.50±3.14B 48.75±2.78AB 53±3.29B P<0.05

51.62±2.92AB 65.50±5.44C 48.25±1.75A 60±2.26BC P<0.05

44.5±2.57A 55.5±3.81B 51.75±3.57AB 53.75±2.65AB P<0.05

45±3.27A 57.75±3.53B 52.25±4.58AB 59.25±4.86B P<0.05

P>0.05 P>0.05 P>0.05 P>0.05

Sampling Days

A, B, C a, b

The means shown in different capital letters within the group (in the column) are statistically significant.

The means shown in different lowercase letters between the groups (on the line) are statistically significant.

Table 2 - Means and standard errors for hematological findings at PrT, PT, PT1 and PT7 days.

Parameters

Leukocyte (x103/µL)

Lymphocyte (x103/µL)

Neutrophil (x103/µL)

A, B a, b

iPPVO (Group I) X̄±SE

CCL (Group II) X̄±SE

Vit-C (Group III) X̄±SE

Control (Group IV) X̄±SE

PrT PT PT1 PT7

9.27±0.39Aa 11.23±0.52B 10.48±0.24AB 10.41±0.41AB P<0.05

10.09±0.70ab 12.48±2.49 10.60±1.88 11.25±2.82 P>0.05

10.11±0.37Ab 11.31±0.86AB 12.34±0.59B 10.84±0.61AB P<0.05

11.82±0.81b 12.08±0.77 12.27±0.96 10.86±0.54 P>0.05

P<0.05 P>0.05 P>0.05 P>0.05

PrT PT PT1 PT7

6.24±0.19Aa 7.46±0.29B 6.96±0.18ABa 7.48±0.94B P<0.05

6.26±0.52a 7.31±0.54 6.70±0.46a 7.63±0.79 P>0.05

6.59±0.38a 6.93±0.49 7.79±0.59ab 7.55±0.56 P>0.05

8.79±0.50b 7.61±0.48 8.61±0.62b 8.49±0.38 P>0.05

P<0.05 P>0.05 P<0.05 P>0.05

PrT PT PT1 PT7

2.98±0.25AB 3.71±0.27Ba 3.47±0.19AB 2.88±0.30Aab P<0.05

3.78±0.28A 5.10±0.52Bb 3.84±0.32A 3.56±0.28Ab P<0.05

3.46±0.47 4.33±0.73ab 4.48±0.44 3.22±0.50ab P>0.05

3.32±0.59AB 4.12±0.57Bab 3.81±0.62AB 2.32±0.27Aa P<0.05

P>0.05 P<0.05 P>0.05 P<0.05

Sampling Days

345

The means shown in different capital letters within the group (in the column) are statistically significant.

The means shown in different lowercase letters between the groups (on the line) are statistically significant.

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PT and PT1 sampling days were significantly higher (P<0.05) than those at PrT. iPPVO and CCL groups at PT1 had significantly (P<0.05) lower lymphocyte counts than Vit-C and control groups.

Biochemical findings Means for adrenaline, cortisol, ADA activity and Hp levels at PrT and AT sampling days are presented in Table 3. Although adrenaline and cortisol levels in all groups increased significantly (P<0.05) after transport, the values in the Vit-C group were lower (P<0.05) than those in iPPVO, CCL and control groups at PT. ADA activity in the control group decreased significantly

(P<0.05) at PT compared to PrT. However, activities of ADA in iPPVO, CCL and Vit-C groups increased significantly (P<0.05) on the contrary of the control group (Table 3). ADA activity in the control group at PT1 approached the ADA level at PrT (Figure 1). On the other hand, ADA activity in other groups at PT1 remained significantly higher (P<0.05) than ADA levels at PrT. Hp levels increased (P<0.05) in CCL, Vit-C and control groups at PT, PT1 and PT7 days, while those in the iPPVO group did not significantly change during the period between PrT and PT (Table 3). In the control group, Hp level (P<0.05) returned to the initial level at PT7, while the Hp level in the CCL group

Figure 1 - ADA activities at PrT, PT, PT1 and PT7 sampling days.

Table 3 - Means and standard errors for biochemical Findings at PrT and PT as well as ADA and Hp levels at PrT, PT, PT1 and PT7 days.

Parameters

Cortisol (pg/ml) Adrenaline (pg/ml)

ADA Activities (pg/ml)

Hp (pg/ml)

A, B, C a, b, c

iPPVO (Group I) X̄±SE

CCL (Group II) X̄±SE

Vit-C (Group III) X̄±SE

Control (Group IV) X̄±SE

PrT PT

1.58±0.08A 2.84±0.18Bb P<0.05

1.61±0.13A 2.69±0.12Bb P<0.05

1.65±0.05A 1.96±0.04Ba P<0.05

1.67±0.03A 3.00±0.10Bb P<0.05

P>0.05 P<0.05

PrT PT

6.09±0.32A 12.37±0.79Bb P<0.05

6.43±0.29A 11.45±0.36Bb P<0.05

6.63±0.24A 8.44±0.24Ba P<0.05

6.30±0.17A 12.23±0.40Bb P<0.05

P>0.05 P<0.05

PrT PT PT1 PT7

2.55±1.12Aa 16.72±1.94Cb 8.2±1.03Ba 4.54±1.27ABa P<0.05

6.55±1.10Ab 13.65±0.78Bb 13.28±0.23Bc 12.53±0.40Bc P<0.05

3.75±0.97Aa 8.31±0.56Ba 9.25±0.61Bab 8.56±0.70Bb P<0.05

10.80±0.37Bc 9.45±0.28Aa 10.35±0.60ABb 10.66±0.26ABbc P<0.05

P<0.05 P<0.05 P<0.05 P<0.05

PrT PT PT1 PT7

24.13±1.03ABb 22.28±1.22Aa 26.89±1.22Ba 26.63±1.40Bb P<0.05

21.31±0.84Aab 28.66±1.21Bb 22.75±0.80Ab 21.94±0.38Aa P<0.05

20.69±1.22Aa 28.92±1.26Cb 26.30±0.68BCa 24.94±0.73Bb P<0.05

22.26±0.80Aab 31.01±1.14Bb 27.10±0.65Ba 18.27±2.68Aa P<0.05

P<0.05 P<0.05 P<0.05 P<0.05

Sampling Days

The means shown in different capital letters within the group (in the column) are statistically significant.

The means shown in different lowercase letters between the groups (on the line) are statistically significant.

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Figure 2 - Hp levels at PrT, PT, PT1 and PT7 sampling days.

was measured at a level similar to PrT in PT1 (Figure 2). On the other hand, the Hp levels of the Vit-C group in PT, PT1 and PT7 were statistically higher (P<0.05) than these at PrT (Table 3).

DISCUSSION Cortisol is an important marker for determining of the stress in animals37. Aktas et al.38 found out that there was an increase in adrenaline level in all dairy cattle groups after 22 hours of transport. Similarly, studies carried out on sheep, goats, pigs and horses have reported increases in adrenaline and cortisol levels at post-transport39,42. The results were in accordance with findings of the current study which revealed significantly higher levels of adrenaline and cortisol in all groups at PT in comparison with PrT. These findings also indicated that 8 hours (including loading and unloading) and approximately 510 km of road transportation resulted in stress on Morkaraman ewes in iPPVO, CCL, Vit-C and control groups.

In the present study, the least increases of the adrenaline and cortisol levels were obtained from ewes in the Vit-C group, although all of the animals were exposed to the same loadingtransport-unloading processes. Vit-C is known as an anti-stress agent43. Kassab and Mohammed44 found out that the increase in the cortisol level of the Vit-C group was lower than that of the control group following 225 km road journey of sheep. They also pointed out that the result was due to the cortisol inhibitory role of Vit-C during the transport. Similarly, studies on different animal species also revealed that Vit-C can alleviate the negative effects of transport45,47. For this reason, in the current study, the lower adrenaline and cortisol levels of the Vit-C group at PT compared to the PrT could be attributed to the stress preventing/reducing effect of the Vit-C as stated in the literature.

The increasing body temperature during the transport reflects the animal’s physical reaction to maintain balance as the vehicle moves48. In other studies, the temperature increased in animals at the beginning of transport, but decreased in the fol-

lowing hours49,50. It has been reported that the temperature of cattle after 14 hours of transport started to decrease only at rest51.

In the current study, the temperature in the iPPVO, CCL and control groups increased significantly (P<0.05) at PT compared to PrT. The result could be due to the adrenal cortex response during long-term road transport.

Increases in pulsation take place from the beginning of transport, and this condition continues as long as the transportation52,53. It was stated that the pulsation in cattle during loading and unloading was higher than these during the transport54.

Respiration rate is one of the most important physiological parameters for evaluating the health status of animals and the body against stress47. Kassab and Mohammed44 noted that the respiration rate of ewes significantly higher at the end of journey (225 km) compared to prior to transport. The result was attributed to the road transport stress. Similar to the findings of the other studies, in the current study, respiration rate and pulsation significantly increased in PT comparing to PrT in all groups. The increased pulsation and respiration rate could be ascribed to the release of cortisol and catecholamines into the bloodstream during stress as reported by Getabalew et al.55 Additionally, in the current study, increases of the cortisol and adrenaline levels at PT could also support their suggestion.

Stress causes a decrease in the number of lymphocytes and an increase in the number of neutrophils56. In a study conducted on sheep, while the percentage of leukocytes, neutrophils and monocytes increased after transport, a decrease was observed in the percentage of lymphocytes57. In another study carried out on sheep, the percentage of leukocytes, neutrophils and monocytes increased, while the percentage of lymphocytes decreased after transport compared to the control group44. In the present study, an increase in lymphocyte and ADA activity was observed in the iPPVO group after transport, as a result of the immunomodulatory effect of iPPVO on Morkaraman ewes.

There are not limited number of studies concerning the effect of iPPVO on immunosuppression induced by long-term transport in livestock. In a study, Winnicka et al.58 reported that the CD4+/CD8+ showing the ratio of helper cytotoxic T lym-

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phocytes in goats injected with iPPVO against the immunosuppression resulting from glucocorticoid administration was higher than the reference value. In the present study, statistically insignificant an increase in lymphocyte levels at PT1 comparing to PRT was determined in the iPPVO group. There was also a slight level of serous runny nose and a slightly noticeable increase in pathological lung sounds during clinical examination in sheep at PT1. However, lymphocyte counts of ewes in the iPPVO group at PT and PT7 were significantly higher (P<0.05) than those at PrT, and no clinical abnormalities such as cough, nasal discharge, tear discharge, diarrhea and pathological sounds in the lungs were observed. Ziebell et al.59 investigated the clinical findings of the horses for 2 weeks after transport. They reported that the clinical score of the iPPVO group was better than the control group and that iPPVO administration was a successful tool to prevent serious clinical consequences caused by road stress. In the current study, it was suggested that the increase in lymphocyte levels in the iPPVO group at PT, PT1 and PT7 might be related to the administration of iPPVO. The possible cause of this result could be the increase in the number of T lymphocytes as already reported by Winnicka et al.58. In the current study, higher ADA activities of the iPPVO group after transport may also support this thought.

Similarly, an increase in ADA activity was observed in CCL as well as in Vit-C groups after transport and the elevation continued until AT7 days. In the literature, there is no study investigating the effects of CCL application on hematological parameters in acute or chronic stress situations. Dik et al.60 reported that the IL-12 and IL-6 levels were higher in sheep after 1 day of CCL administration with PPR vaccine compared to the group in which PPR vaccine was administered alone.

Trinchieri61 indicated that IL-12 stimulates cytotoxic T lymphocytes and plays a specific role in defense against viruses, and also shows synergistic effects with IFN, IL-4 and IL-6 in the early cellular immune response. In the present study, the increase in ADA activity after transport in the CCL group could be ascribed to the hypothesis that CCL enhances IL-12 level and IL12 stimulates the production of T lymphocytes, consequently, an increase in lymphocyte count has been formed.

In addition to cortisol inhibition, Vit-C has been reported to be a chain-breaking antioxidant, which plays a role in preventing and restricting free radical chain formation. Thus, it protects blood cells including neutrophils and lymphocytes from oxidative damage34. On the other hand, in vitro studies demonstrate that Vit-C is required for the development of T lymphocytes. Additionally, Vit-C increases the proliferation of T lymphocytes and may affect their function. There are limited and controversial results regarding the effects of Vit-C on B lymphocytes that mediate humoral immunity62. However, Vit-C also increases the proliferation of NK cells, a group of cytotoxic natural lymphocytes. In summary, Vit-C positively affects lymphocyte development and function. In the present study, it was suggested that the increase in ADA activity in the Vit-C group could be due to the positive enhancement of lymphocyte development and function as well as the anti-stress properties of Vit-C. Hp levels were determined in various animal species that were transported, and different results were obtained. Kołacz et al.63 noted that the increased Hp level of pigs after transport did not return to a normal level with rest. On the contrary, Hp concentration was 86.2% higher than that measured at the 22nd hours after transport. Fazio et al.64 reported that the Hp level in

sheep and cattle reached a peak at the 48th hour after transport.

Pascual Alonso et al.57 stated that the Hp level, which increased after 4 hours of transport, continued for 24 hours in sheep. A significant positive correlation between dose-dependent Hp concentration and serum lymphocyte suppression was also determined by Murata and Miyamoto24. In the present study, Hp levels in PT and PT1 in the control group were found to be higher than PrT. The induction of the acute phase protein (APP) response in animals under stress is based on the hypothesis that “non-inflammatory and psychological stresses activate the sympatho-adrenal and HPA axis via the afferent nervous system”65,66. The sympatho-adrenal axis releases catecholamines, which stimulate immune-related cells and cause them to produce pro-inflammatory cytokines in response to stress65. HPA pathway controls glucocorticoid release from the adrenal cortex66. Both glucocorticoids and proinflammatory cytokines such as IL-1, IL-6, and TNF- are indicated as potential direct stimuli of the acute phase response67. In the current study, it is highly possible that the results obtained from high Hp levels in the control group emerged as the effect of catecholamines and glucocorticoids as already stated by Johnson et al.65 and Leonard66.

Additionally, high adrenaline and cortisol levels obtained from the control group also support this information. Therefore, transport as a stress factor is seen to induce two main stress pathways (acute and chronic) and stimulates the production of APPs such as Hp. It could be suggested that the high level of Hp resulted in negative effects on the immune system and the result was in accordance with clinical findings of the study.

Hp level increased after transport in the CCL and Vit-C groups.

Hp level in the CCL group at PT1 was similar to PrT. It was suggested that pre-transport CCL administration partially stopped the increase of Hp, one of the immune response markers of the body due to stress, and may have a positive effect on the ewes.

Clinical findings of the CCL group partially coincide with this result. On the other hand, Vit-C administration at PrT did not have a reducing effect on serum Hp concentration.

CONCLUSION In conclusion, it was demonstrated that iPPVO administration could be more effective against immunosuppression induced by long-term road transport of Morkaraman ewes, taking into consideration lymphocyte count, ADA activity and Hp levels.

A positive correlation between ADA activity and lymphocyte count was also determined in this study. Additionally, since VitC administration before transportation reduced road transport stress more efficiently than others, it could be suggested as a stress inhibitor for ewes.

Acknowledgements This study was supported by a grant from the Administration of Scientific Research Projects of Atatürk University, (Project #: TDK-2019/7206) and produced from the Doctorate Thesis of the first author that was entitled “Investigation of the Effect of Parapoxvirus ovis, Corynebacterium cutis Lysate and Vitamin C on Immune Suppression Caused by Long-Term Transport Stress in Morkaraman Sheep”.

Conflicts of interest The authors declare no conflicts of interest with respect to their authorship and/or the publication of this manuscript.

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A novel approach for evaluating of goat milk quality: canonical correlation analysis between major milk composition parameters and fatty acid components in Damascus goats

351

j

UFUK KAYA1*, HÜSEYIN ÖZKAN2, BARAN ÇAMDEVIREN3, İREM KARAASLAN4, YAKAN AKIN2,4 1

Department of Biostatistics, Faculty of Veterinary Medicine, Hatay Mustafa Kemal University, 31060 Hatay, Turkey Department of Genetics, Faculty of Veterinary Medicine, Hatay Mustafa Kemal University, 31060 Hatay, Turkey 3 Department of Molecular Biochemistry and Genetics, Institute of Health Sciences, Hatay Mustafa Kemal University, 31060 Hatay, Turkey 4 Technology and Research and Development Center (MARGEM), Hatay Mustafa Kemal University, 31060 Hatay, Turkey 2

SUMMARY The aim of this study was to investigate the effects of major milk composition parameters (lactose, protein and fat) and somatic cell score (SCS) on the variation of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA), unsaturated fatty acids (UFA), omega 6 (ω6), omega 3 (ω3), odour index (OI) and nutritive value (NV) of goat milk in the first month of the lactation. For this aim, 120 milk samples were collected in different times post-partum (0, 4, 7, 14 and 28 days) from 24 Damascus goats. Composition parameters and fatty acid components of milk samples were determined. Pearson correlation coefficient and canonical correlation analysis (CCA) were used to evaluate the relationships between major milk parameters and fatty acids components. In addition to positive correlations between major milk parameters, significant and variable correlations were found between major milk composition parameters and fatty acid components according to the Pearson Correlation coefficient. Most of the fatty acids components were also correlated with each other. However, only the first of four calculated pairs of canonical variables was found to be significant, strongly (rc= 0.857, P<0.001).

According to the calculations of canonical loading and cross-loading results, protein and lactose from the set of independent variables and OI and NV from the set of dependent variables were found to be the most important variables. On the other hand, redundancy indexes results showed that the fatty acid contents may be explained by the milk components in goat milk. CCA results has revealed that multidimensional evaluation of measurements in dependent and independent variable sets can provide to researchers with important and innovative information for assessment of milk quality. Considering the multiple relationships with a multivariate approach, strong and significant relations were found for the first time in terms of major milk quality parameters in goats.

KEY WORDS Canonical correlation; Damascus goat; Goat milk; Milk composition; Milk fatty acid profile.

INTRODUCTION Animals’ products are the main components of human diets.

Goat, one of the oldest domesticated animal, is an important source of animal food, particularly for milk and dairy products1. Furthermore, goat milk is commonly chosen by people with digestive system problems or allergies. It is greater in carbohydrate, fat and protein percentages compared to cow’s milk.

Moreover, it is richer in short and medium-length chain fatty acids2. Composition and fatty acid content of milk influence significantly the nutritional value and quality of milk3. Furthermore, they are important for monitoring the mammary gland’s status and the health of goat kids. For these reasons, they are important features in animal management systems2. In addition, relationships between composition and fatty acids com-

Corresponding Author: Ufuk Kaya (u.kaya@mku.edu.tr).

ponents of milk have critical importance for livestock. In this way, it has been aimed to determine the relationship between the fatty acid components of milk, which is more difficult to determine due to application difficulties and expensiveness, and the composition parameters that are easily determined4. The simple correlation coefficient is generally used to determine these relationships5,6. Although correlation analysis evaluates the strength and direction of the relationships between measurements, it remains insufficient to detect latent or indirect relations7. The multivariate statistical methods of canonical correlation analysis (CCA) can be used to successfully explain these relationships. CCA explains the relationship by calculating linear combinations between two data sets. In addition, CCA can produce structural and spatial inferences by handling two data sets simultaneously8.

The aim of this study was to investigate the effects of major milk composition parameters (somatic cell count, lactose, protein and fat) on the variation in milk fatty acids profile in the first month of the lactation stage (early lactation stage, ELS) with CCA in goat milk.

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MATERIALS AND METHODS Study design, animals and sample collection All procedures and practices complied with The Regulation on the Studying Procedures and Principles of Animal Experiments of Ethics Committees of the Ministry of Agriculture and Forestry (2014, Turkey). The study was conducted in a farm located at 36° 21′ 52.6′′ N and 36° 15′ 14.6′′ E at an altitude of 82 m above sea level in Hatay, Turkey. The enrolled goats aged 3-4 years were in their 2nd-3rd lactation periods. In addition, all enrolled goats presented a singleton birth. The average of lactation period was maintained approximately 215 days. For this study, milk samples were collected from 24 healthy Damascus goats randomly selected from 200 goats for five times after parturition (0th, 4th, 7th, 14th, and 28th days). The health status was evaluated by veterinarians. Animals were fed with 1.2 kg/goat concentrate feed (88.90% dry matter, 16.50% crude protein, and 2649 kcal/kg total ME) and 1.0 kg/goat wheat straw. 50 ml of milk for each animal were collected to sterile falcon tubes at morning milk and brought to laboratory in 30 min in the cold box (4 °C) at all experimental times. The samples were homogenized by manual mixing and divided into two aliquots.

A total of 120 milk samples were collected. Before sampling, goats’ udders were washed with alcohol-based disinfectant and cleaned with water and sterile cotton gauze swabs.

Determination of milk composition All measurements were performed in the laboratory of genetics department (Faculty of Veterinary Medicine, Hatay Mustafa Kemal University). The compositional parameters of milk (fat, protein and lactose) samples were measured with milk analyzer (Milkotester Master Classic LM2-P1, BULGARIA) while somatic cell count (SCC) was determined with somatic cell counter (Lactoscan SCC 6010, BULGARIA). Obtained SCC data were normalized and transformed into Somatic Cell Score (SCS=log2(SCC/100.000) + 3)9. Somatic cell count and composition of milk were determined in validated devices.

Cream layer collection and fatty acids evaluation All measurements for fatty acids were performed in an accredited laboratory (Technology and Research and Development Center, Hatay Mustafa Kemal University). Milk samples were centrifuged (NF800R, NUVE, TURKEY) at +4 °C at 1800 xg for 15 min for cream layer collection. Thereafter, the samples were kept at -20 °C for 15 min and the cream layers were collected into 1.5 mL volume sterile tubes. After, about 500 µL cream layers was added 2 mL 2N methanolic and waited for 4 min at room temperature. Following the incubation, 4 ml nHeptane was added and samples were incubated at room temperature for 2 min. The samples were then centrifuged for 5 min at 200 xg and the aqua phases containing methyl esters were transferred to 1.5 mL vials.

Individual fatty acids of milk samples were identified using a gas chromatograph (GC-2025, Shimadzu, JAPAN) equipped with flame ionization detector. Separation was performed with a Restek Rt-2560 column (100 m length, 0.25 mm internal diameter x 0. 20 µm film thickness). After injection at 100 °C (held for 2 min), the oven temperature was raised to 250 °C at a rate 4 °C/min and finally held constant for 15 min. The flame ionization and injector temperatures were set at 250 °C with the

Table 1 - Fatty acid related parameters.

Parameters SFA MUFA ω6 ω3 PUFA UFA

Formulas of parameters* C4:0+C6:0+C8:0+C10:0+C12:0+C14:0+C15:0+ C16:0+C17:0+C18:0+C20:0+C22:0 C14:1 ω5+C15:1 ω7+C16:1 ω7+C17:1 ω7+C18:1 ω9+C20:1 ω9+C24:1 ω9 C18:2 trans+C18:2 cis+C18:3 ω6+C20:2 ω6+C20:4 ω6+C22:2 ω6 C18:3 ω3+C20:3 ω3+C20:5 ω3+C22:6 ω3 ω6+ω3 MUFA+PUFA

OI

C4:0+C6:0+C8:0+C10:0

NV

(C18:0+C18:1 ω9)/C16:0

SFA: Saturated fatty acids; MUFA: Monounsaturated fatty acids; PUFA: Polyunsaturated fatty acids; UFA: Unsaturated fatty acids; ω6: Omega 6; ω3: Omega 3; OI: Odour index; NV: Nutritive value; *: 2, 28.

following gas flow: carrier gas hydrogen: 40 mL/min, instrument air: 400 mL/min. The hydrogen flow rate was 1.2 mL/min, the split ratio was 1:50 and injection volume was set to 1 L. FAME peaks were identified by comparing their retention times with those of Restek FAME Mix Standard. While ω6 and ω3 percentages were calculated using the fatty acids in the formulas as C18:2 trans+C18:2 cis+C18:3 ω6+C20:2 ω6+C20:4 ω6+C22:2 ω6 and C18:3 ω3+C20:3 ω3+C20:5 ω3+C22:6 ω3, respectively, OI percentage and NV were calculated using the fatty acids in the formulas as C4:0+C6:0+C8:0+C10:0 and (C18:0+C18:1 ω9)/C16:0, respectively (Table 1).

Statistical analysis Descriptive statistics, Pearson correlation coefficient and CCA were performed using the IBM SPSS Statistics software Version 23.0. Descriptive statistics of variables were calculated before statistical analysis. The variables included in the study were checked in terms of normality assumption by KolmogorovSmirnov test and in terms of the homogeneity of variance assumption by Levene test. The relationship between major milk composition parameters (SCS, fat, protein and lactose) and fatty acid components (SFA, MUFA, PUFA, UFA, ω6, ω3, OI, NV) was determined using the Pearson correlation coefficient. Before canonical correlation analysis, variables were evaluated in terms of multicollinearity problem. Accordingly, MUFA and UFA were excluded from the study due to their high Variance Inflation Factor (VIF) values (VIF>10). Then, the CCA was used to evaluate the relationships between the independent set (X) of the milk composition parameters (SCS, milk fat, protein and lactose) and the dependent set (Y) of the fatty acids’ components (SFA, PUFA, ω6, ω3, OI and NV). With this analysis, the relations between variables (milk composition parameters) in a set and variables (fatty acid components) in another set can be examined.

The F values of the canonical correlation coefficients were examined using Wilk’s Lambda values from the significance tests.

According to CCA, canonical variables were created with linear combinations of these variables. Canonical variable pairs (U and V) were calculated. How much of the variance in one data set is explained by the other data set was calculated with the redundancy index. Significance level was accepted as P<0.05.

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Figure 1 - The major composition parameters (a) and fatty acid components (b) (Mean ± SEM).

Figure 1a: major composition parameters; Figure 1b: fatty acid components; SFA: Saturated Fatty Acids, MUFA: Monounsaturated fatty acids, PUFA: Polyunsaturated fatty acids, UFA: Unsaturated fatty acids.

RESULTS The major milk composition parameters and fatty acids components were summarized in Figure 1. SCS average was 11.83 ± 0.26 (SCC: 1873 x 103/mL). The sum of n6 and n3 values (%) were 6.66 and 1.56, respectively. The OI (%) and NV were also determined as 9.52 ± 0.44 and 1.66 ± 0.03, respectively.

Relations of milk composition parameters and fatty acid components were determined by Pearson correlation coefficient. Positive correlations were found between SCS and fat, protein and lactose. In addition, lactose and protein were positively correlated. While SFA and OI had a positive relation, a negative relation was found with PUFA, ω6, ω3 and NV. Positive correlations were determined between PUFA and ω6, ω3 and NV variables and between ω6 and ω3 and NV variables.

A negative correlation was determined between OI and NV. PUFA, ω6, ω3 and OI in the dependent set Y were negatively correlated with milk fat. On the other hand, positive correlations were found between MUFA and protein and lactose. Finally, negative correlations were found between OI and SCS, fat, protein and lactose parameters (Figure 2).

According to CCA results, four different pairs of canonical variables and four different canonical correlation coefficients were obtained from the set X and set Y (Table 2). It was determined that only the first canonical correlation coefficient (0.857) calculated from the first canonical variable pair was significant (P<0.001). This result showed that there were significant relationships between linear combinations of major milk com-

Figure 2 - Heatmap showing Pearson correlations between major composition parameters and fatty acids components.

SCS: Somatic cell score, SFA: Saturated fatty acids, MUFA: Monounsaturated fatty acids, PUFA: Polyunsaturated fatty acids, UFA: Unsaturated fatty acids, ω6: Omega 6, ω3: Omega 3, OI: Odour Index, NV: Nutritive Value; *P < 0.05; **P < 0.01; ***P < 0.001.

Table 2 - Results of canonical correlation analysis.

Canonical variables

Canonical correlation

Canonical R2

Eigenvalue

F test

Wilks’ Lambda

P

1

0.857

0.735

2.770

6.74

0.220

< 0.001

2

0.352

0.124

0.142

1.11

0.830

0.353

3

0.165

0.027

0.028

0.587

0.948

0.787

4

0.159

0.025

0.026

0.763

0.975

0.518

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position parameters and fatty acids components. Canonical correlation coefficients calculated with other canonical variable pairs were found to be non-significant.

The linear components of the first canonical variable (U and V) were presented in Table 3. According to the results, the model was obtained through with standardized canonical coefficients as U1 = - 0.052 (SCS) - 0.061 (Fat) + 1.638 (Lactose) - 2.567 (Protein) and V1 = 0.449 (SFA) + 2.407 (PUFA) - 1.639 (ω6) - 0.726 (ω3) + 0.851 (OI) + 0.695 (NV).

Table 3 - Standardized Coefficients of the First Canonical Variables for set X and set Y. Independent variables (X)

U1

Dependent variables (Y)

V1

SCS

-0.052

SFA

0.449

Fat

-0.061

PUFA

2.407

Lactose

1.638

ω6

-1.639

Protein

-2.567

ω3

-0.726

OI

0.851

NV

0.695

SCS: Somatic cell score, SFA: Saturated fatty acids, PUFA: Polyunsaturated fatty acids, ω6: Omega 6, ω3: Omega 3, OI: Odour Index, NV: Nutritive Value.

Table 4 - Canonical loading and cross-loading for the first canonical variables.

Canonical loadings

Canonical cross-loadings

SCS

-0.531

-0.455

Fat

-0.493

-0.422

Lactose

-0.969

-0.831

Protein

-0.985

-0.845

SFA

0.119

0.102

PUFA

0.151

0.129

ω6

0.069

0.059

ω3

0.226

0.194

OI

0.815

0.698

NV

0.242

0.207

Independent variables (X)

Dependent variables (Y)

SCS: Somatic cell score, SFA: Saturated fatty acids, PUFA: Polyunsaturated fatty acids, ω6: Omega 6, ω3: Omega 3, OI: Odour Index, NV: Nutritive Value.

Table 5 - Result of redundancy analysis.

Canonical variables

Variable Set

Explained by canonical variables

Explained variation

1

X

U

60.9

1

X

V

44.8

1

Y

U

10.0

1

Y

V

13.6

Canonical loadings and canonical cross-loadings between the canonical variables and the original variables within their own sets were presented in Table 4. It was determined that the largest contribution to the canonical variable U1 was provided by protein with the largest canonical loading (-0.985), followed by lactose (-0.969), SCS (-0.531) and fat (-0.493), respectively. In addition, OI (0.815) made the heaviest contribution to the canonical variable V1. In canonical cross-loadings, protein and OI also displayed higher weights than other variables.

The total variances explained by canonical variables were determined by redundancy analysis (Table 5). 60.9% of the total variation in the X variable set was explained by U1 and 44.8%

by V1. Otherwise, 10.0% of the total variation in the Y variable set was explained by U1 and 13.6% by V1.

DISCUSSION The lactation stage in goats varies depending on many factors such as breeds, season, and management. However, it is possible to separate lactation into three stage: early, mid and late lactation1, 10. SCS strongly reflects mammary physiology and health of animal. According to obtained results, it has been found to be high as expected. Because, colostrum and transition milk have quietly high SCC2. Different studies reported a higher SCS value in goat milk during ELS, with different levels according to breed2, 6, 11, 12. Particularly, Damascus goats showed variable SCS in ELS between 31 to 9089 x 103 / ml13, 14 . Since SCC in goats is variable and related with mammary infections, Koop et al. (2010) reported that bacteriological examination or milk yield measurements were necessary for the detection of healthy goats that were not subclinical mastitis.

Even if the yield or bacterial status of milk are valuable markers for subclinical mastitis, milk and colostrum were presented to kids ad libitum for nutrition in ELS and therefore milk yield could not be measured in this study.

In addition, although the animals were determined to be physiologically healthy, it was thought that the lack of bacterial examination in the milk was a factor to be taken into account.

According to Pearson correlation coefficient, SCS has positively correlated with major milk composition parameters (fat, protein and lactose). Studies have been showed that there are controversial relationships between SCS and major milk composition parameters depending on factors such as birth season and stage of lactation15, 16.

In our study, the major composition parameters of goat milk during ELS showed differences compared to animals reported in different studies12, 17. However, significant interactions were detected between major composition parameters of milk (fat, protein and lactose) as reported5, 18. While El-Tarabany et al. (2019) found that lactose presented a negative relationship with protein, and no relationship with fat, positive correlations were found between lactose, protein, and fat in our study. Although these differences are mainly due to the breed type, they are significantly influenced by environmental factors such as season and ration6.

Fatty acids components of milk are remarkable for milk quality as well as major components that are the basic elements of milk composition19. Recently, the increased concern of food quality led to focus on fatty acids components of milk20. In this study, significant and remarkable correlations were found between fatty acids components as other studies6, 21. These cor-

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relations showed that the quality of milk should be assessed with SFA, MUFA and UFA contents of milk. NV and OI indices (Table 1) used to evaluate fatty acids are very important due to their nutritional value and composition of desirable fatty acids (such as C4:0, C18:0, and C18:1 ω9) because of human health22, 23. Different studies in goat milk showed short chain fatty acids and C16:0 are lower, and C18:0 and C18:1 are higher in ELS compared to other stages10, 22. Moreover, C16:0 which is one of the fatty acids used in calculation of NV increase in metabolic disorders such as subclinical ketosis. This condition adversely affects milk quality by decreasing in NV23. It has been clearly understood that the negative relations of NV and OI has potent effect on the milk quality in ELS, although the fatty acids (shorter and longer fatty acids) used in the calculation of these parameters have been different.

Variable interactions were found between major composition parameters and fatty acids. PUFA, ω6, and ω3, were negatively correlated with milk fat. It was reported in ruminants that the relationships between milk fat and these fatty acid parameters were similar24. In another study, PUFA, ω6 and ω3 were found to be high in animals with low milk fat25. This situation confirms that milk fat presented negative relationships with PUFA, ω6 and ω3 in our study. OI showed a negative correlation with all major component parameters and SCS as expected. High SCS in ELS decreases the synthesis of short chain fatty acids by affecting secretory activity in the udder2, 26.

On the other hand, MUFA was positively correlated with protein and lactose. Complex molecular mechanisms play a role in the formation of quantitative characters such as milk yield and milk quality in ruminants. It is thought that these relationships in ELS may be regulated by complex and interrelated mechanisms2, 6.

However, a univariate statistical approach is insufficient to consider multiple relationships between these variables. On the other hand, CCA allows examining the effects between different variables’ groups while preserving the relationships between the original variables in each group27. Considering the multiple relationships with a multivariate approach used in this study, strong and significant relationships were found between major composition parameters and fatty acids components in terms of quality parameters in goat milk during ELS. Lactose, protein and OI had the heaviest weight in the analysis of data sets through CCA. Lactose and protein are necessary for the feeding the newborns, because the energy source can be digested faster than fat28. It was reported that OI tended to decrease from the beginning to mid lactation stage, and to increase in the last lactation stage1, 10. The short-chain fatty acids are related to the nutrition and growing of the offspring due to the immaturity of digestive systems and immunity2. The NV was constituted by the ratio between C18:0 and C18:1 with C16:0 fatty acids. The C18:0 and C18:1 concentrations increased in milk during ELS, while C16:0 concentration decreased respectively. Despite NV did not show an important weight, it is one of the main factors for the quality of milk2, 29. According to CCA, protein, lactose, OI, and NV of milk might be said to be the most influential variables in terms of both dependent (fatty acids components) and independent (major composition parameters) variables. However, canonical loadings should be considered as a set, since they have been obtained through linear combinations of variables.

As known, the quality parameters of goat milk in ELS can be explained by the effects of many dependent and independent

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variables. Therefore, both Pearson correlation analysis that examines cases in one dimension and CCA, that examines cases in multidimensional were preferred in this study. The relationships between major composition parameters and fatty acids components in goat milk in ELS were evaluated separately with Pearson correlation coefficient, while the effects of all parameters in independent and dependent variable sets were examined with CCA. In addition, the parameters that contributed the most to the correlation between data sets and how much the dependent variables could be explained by the independent variables were determined by CCA in this study.

In general, it has been shown with explained variance ratios calculated with the redundancy index (Table 5) in our study that the change in the fatty acid profile in ELS may be explained by some of the quality parameters of milk. In addition, researchers have stated that the explained variation in parameters are important7, 18.

Although there is no acceptable general guideline for the redundancy index that is used in the calculation of the explained variance ratios, it could provide important information to the researcher even if the variations are low30.

CONCLUSION CCA might be considered as a convenient method to evaluate the relationships between data sets and to identify influencing factors. Negative relationships were obtained between major milk composition parameters (independent variable set) and milk fatty acids components (dependent variable set) in goat’s milk during ELS. Goat milk that is a quality food due to its macronutrients (protein, carbohydrate and fat) and essential fatty acids in human nutrition is important in terms of nutritional value and health. Although the first 28-days milk quality parameters of Damascus goats seem to be acceptable in terms of human nutrition, it has deduced that milk is actually more necessary for the goat kids because of the health and development.

In conclusion, information about independent data set may estimate milk fatty acids, considering that the analysis of the dependent data set is more difficult and expensive. To the best of our knowledge, this is the first study that attempts to reveal the relationship between major milk composition parameters and fatty acid components with CCA in ELS milk of Damascus goats. The outputs of this study clearly indicate that more studies are needed to examine the relationships between milk composition and fatty acids components in different breeds of goats and even different livestock animals.

DECLARATION OF CONFLICTING INTEREST The authors declare no conflict of interest.

ACKNOWLEDGMENTS This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) with the 119O716 project number.

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2. Yakan A., Özkan H., Çamdeviren B., Kaya U., Karaaslan İ., Dalkıran S. (2021). Expression patterns of major genes in fatty acid synthesis, inflammation, oxidative stress pathways from colostrum to milk in Damascus goats. Sci Rep, 11: 1-10.

3. García V., Rovira S., Boutoial K., López M.B. (2014). Improvements in goat milk quality: A review. Small Rumin Res, 121: 51-57.

4. Amores G., Virto M. (2019). Total and free fatty acids analysis in milk and dairy fat. Separations, 6: 14.

5. El-Tarabany M.S., Abdel-Hamid T.M., Ahmed-Farid O.A., Al-Marakby K.M. (2019). Characterization of progesterone profile, physiological responses, milk composition and blood biochemical and hematological indices at the early stage of lactation in goats. Biol Rhythm Res, 50: 647657.

6. Özkan H., Yakan A. (2020). The Relationship Between Milk Fatty Acid Profile and Expression Levels of SCD, FASN and SREBPF1 Genes in Damascus Dairy Goats. Kocatepe Vet Derg, 13: 1.

7. Özen, D., Kocakaya A., Ozbeyaz C. (2021). Estimating relationship between live body weight and type traits at weaning and six months of age in Bafra lambs using canonical correlation analysis. JAPS J Anim Plant Sci, 31(2): 386-393.

8. Hotelling, H. (1992). Relations between two sets of variates. In: Breakthroughs in statistics, Eds., Kotz S., Johnson N.L., 1th ed., Springer, New York, USA. 9. Ali A.K.A., Shook G.E. (1980). An optimum transformation for somatic cell concentration in milk. J Dairy Sci, 63: 487-490.

10. Yakan, A., Özkan H., Eraslan Şakar A., Ateş C.T., Ünal N., Koçak Ö., Doğruer G., Özbeyaz C. (2019). Milk yield and quality traits in different lactation stages of Damascus goats: Concentrate and pasture based feeding systems. Ankara Üni Vet Fak Derg, 66: 117-129.

11. Barrón-Bravo O.G., Gutiérrez-Chávez A.J., Ángel-Sahagún C.A. (2013).

Losses in milk yield, fat and protein contents according to different levels of somatic cell count in dairy goats. Small Rumin Res, 113: 421-31.

12. Sánchez-Macías D., Moreno-Indias I., Castro N., Morales-Delanuez A., Argüello A. (2014). From goat colostrum to milk: Physical, chemical, and immune evolution from partum to 90 days postpartum. J Dairy Sci, 97: 10-16.

13. Doaa F.T., Hafasa F.H.Y., El-Baz A.M., El-Sherbieny M.A. (2014). Relationship between somatic cell count and udder health in Damascus goats.

Egypt J Sheep Goat Sci 9: 31-42.

14. Gocmen H., Darbaz I., Ergene O., Esendal O.M., Aslan S. (2019). The relationships between somatic cell count, total bacterial count and intramammary infection in milk samples of Damascus goats during postpartum days. Small Rumin Res, 180: 1-5.

15. Margatho G., Rodríguez-Estévez V., Medeiros L. (2018). Seasonal variation of Serrana goat milk contents in mountain grazing system for cheese manufacture. Rev Med Vet, 169: 157-65.

16. Sandrucci A., Bava L., Tamburini A. (2019). Management practices and milk quality in dairy goat farms in Northern Italy. Ital J Anim Sci, 18: 1-12.

17. Kuchtík J., Králíčková Š., Zapletal D., Węglarzy K., Šustová K., Skrzyżala I. (2015). Changes in physico-chemical characteristics, somatic cell count and fatty acid profile of Brown Short-haired goat milk during lactation. Anim Sci Pap Reports, 33: 71-83.

18. Stürmer M., Busanello M., Velho J.P., Heck V.I., Haygert-Velho I.M.P. (2018). Relationship between climatic variables and the variation in bulk tank milk composition using canonical correlation analysis. Int J Biometeorol, 62: 1663-1674.

19. Markiewicz-Kęszycka M., Czyżak-Runowska G., Lipińska P., Wójtowski J. (2013). Fatty acid profile of milk-a review. Bull Vet Inst Pulawy, 57: 135-139.

20. Lopez A., Vasconi M., Moretti V.M., Bellagamba F. (2019). Fatty acid profile in goat milk from high-and low-input conventional and organic systems. Animals, 9: 452.

21. Strzałkowska N., Jóźwik A., Bagnicka E., Krzyźewski J., Horbańczuk K., Pyzel B., Horbańczuk J.O. (2009). Chemical composition, physical traits and fatty acid profile of goat milk as related to the stage of lactation. Anim Sci Pap Reports, 27: 311-320.

22. Alizadehasl M., Ünal N. (2021). The investigation of milk yield, composition, quality, and fatty acids in Angora goats based on rangeland feeding conditions. Large Anim Rev, 27: 83-90.

23. Fiore E., Lisuzzo A., Tessari R., Spissu N., Moscati L., Morgante M., Gianesella M., Badon T., Mazzotta E., Berlanda M., Contiero B., Fiore F. (2021). Milk fatty acids composition changes according to β-hydroxybutyrate concentrations in ewes during early lactation. Animals, 11: 1371.

24. Kirchnerová K., Foltys V., Špička J. (2021). Impact of lactation stage on milk fat fatty acids profile in grazing dairy cows. J. Microbiol Biotechnol Food Sci, 2021: 1164-74.

25. Conte G., Dimauro C., Serra A. (2018). A canonical discriminant analysis to study the association between milk fatty acids of ruminal origin and milk fat depression in dairy cows. J Dairy Sci, 101: 6497-6510.

26. Sramek Á., Bodnár Á., Póti P. (2018). The effect of udder health on mineral concentrations and fatty acid composition of alpine goat milk. Anim Sci Pap Reports, 36: 4.

27. Gittins R. (2012). Canonical analysis: a review with applications in ecology. Ed. Gittins R., 1st ed., Springer-Verlag, New York, USA. 28. Moreno-Indias I., Sánchez-Macías D., Castro N., Morales-delaNuez A., Hernández-Castellano L.E., Capote J., Argüello A. (2012). Chemical composition and immune status of dairy goat colostrum fractions during the first 10 h after partum. Small Rumin Res, 103: 220-224.

29. Virto M., Chávarri F., Bustamante M.A., Barron L.J.R., Aramburu M., Vicente M.S., Pérez-Elortondo F.J., Albisu M., De Renobales M. (2003). Lamb rennet paste in ovine cheese manufacture. Lipolysis and flavour. Int Dairy J, 13: 391-399.

30. Hair, J.F. 2009. Multivariate data analysis. 7th ed., Kennesaw State University Press, Georgia, USA. 31. Koop G., Van Werven T., Schuiling H.J., Nielen M. (2010). The effect of subclinical mastitis on milk yield in dairy goats. J Dairy Sci, 93: 5809-5817.

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GIUSEPPE PALLANTE* DMV, specialista Clinica Bovina; specialista Diritto e Legislazione Veterinaria.

Libero Professionista, Veterinario Aziendale

SOMMARIO Fermare l’erosione genetica e gestire in modo sostenibile le razze minori di bestiame sono tra le prossime sfide della professione veterinaria. La difesa delle razze autoctone rappresenta una voce centrale che la Commissione Europea ha individuato per la salvaguardia del clima. La veterinaria, in una visione di Global Health, è chiamata a contribuire a questa strategia indirizzando gli allevatori e fornendo informazioni utili ai consumatori che chiedono alimenti sani in un’ottica di corresponsabilità decisionale. Nel presente articolo la questione viene declinata attraverso tre punti nodali: la figura del veterinario aziendale, la necessità di una formazione intellettuale che coniughi al sapere tecnico scientifico una visione ecologica ed etica, e, per finire, i risultati raggiunti attraverso la ricerca nell’ambito accademico.

PAROLE CHIAVE Veterinario aziendale, cambiamenti climatici, razze autoctone, zoo etica, Commissione Europea.

Ci hanno detto che, come specie, gli esseri umani sono programmati per non guardare lontano (E se smettessimo di fingere? Franzen J., 2020)

PREMESSA La UE entro il 2050 vuole rendere l’Europa il primo continente climaticamente neutro in termini di contenimento di CO2 atmosferica. Per centrare questo obiettivo la Commissione Europea ha individuato una nuova strategia di crescita sostenibile e inclusiva per rilanciare l’economia, migliorare la salute e favorire la qualità della vita dei propri cittadini.

A questo proposito il 20 maggio del 2020 la Commissione UE inviava una “comunicazione al Parlamento Europeo, al Consiglio, al Comitato Economico e Sociale Europeo e al Comitato delle Regioni” con il sottotitolo “Una strategia dalla fattoria alla tavola per un sistema alimentare, equo e rispettoso dell’ambiente”.1 Tutte le aree tematiche definite al suo interno richiamano in qualche modo il ruolo e le responsabilità della professione veterinaria.

In particolar modo è significativo come con questo documento la UE affronti, per la prima volta, la pandemia del COVID-19 dal punto di vista alimentare riconoscendo “i legami inestricabili tra persone sane, società sane e pianeta sano” e come il nostro sistema alimentare sia minacciato dalla “crescente ri-

Corresponding Author: Giuseppe Pallante (g.pallante@csiz.eu).

corrente siccità, inondazioni, incendi boschivi e nuovi parassiti” e che pertanto deve diventare più sostenibile e resiliente.

Si potrebbe obiettare, per quanti leggono oggi questo articolo, che la strategia proposta e i tempi di riferimento, 2050, prevedono sicuramente un salto generazionale e quindi non necessariamente potremmo sentirci coinvolti. Ma è da credere che solo accompagnando le nuove leve di colleghi veterinari e formando sempre più allevatori consapevoli che si potrà arrivare a raggiungere, step by step, gli obiettivi preposti dalla Commissione. Pertanto è necessario iniziare ad aprire una riflessione come categoria responsabile, favorendo contributi e iniziative in merito.

IL RUOLO DEL VETERINARIO AZIENDALE La pandemia COVID-19 ha sottolineato l’importanza di un sistema alimentare solido e “ci ha resi profondamente consapevoli delle interrelazioni tra la nostra salute, gli ecosistemi, le catene di approvvigionamento, i modelli di consumo e i confini planetari”.2 Questa dichiarazione, e le conseguenze in campo, hanno permesso di riconoscere l’alta formazione della categoria del veterinario e rilanciato una figura, quella del buiatra, spesso appiattita, nell’immaginario sociale, solo alle richieste dell’allevatore e di conseguenza all’economia del mercato. Il salto di definizione concettuale, e non solo in campo applicativo, raggiunto con la qualifica di “veterinario aziendale”, offre oggi al professionista quelle opportunità di formazione e di indirizzo consulenziale all’allevatore, che fino a ieri risultava esposto

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alla esclusiva mercé del primo imbonitore.

Non solo, la One Health, tradotta in termini applicativi in One Medicine, richiama la professione veterinaria non solo ad un indirizzo di zoo-tecnica ma in ultima analisi di zoo-etica, ovvero una visione complessiva di cui, come professionisti, in linea con il nuovo Codice Deontologico Veterinario (box 1), non possiamo esimerci: Nel contesto deontologico questo dovere implica una obbligatorietà morale che il professionista “sente” e che lo fa agire non per adempiere ad un obbligo di legge o eseguire un ordine esterno o per evitare, disattendendo quella disposizione o quell’ordine, una sanzione; ma agisce invece riconoscendo il dovere di agire conformemente ad un modello di condotta che ha alla base una valenza morale dell’agire professionale caratterizzato da un ampio spazio di autonomia e libertà.3 Bisogna riconoscere come negli ultimi 50 anni, a fronte di un impetuoso sviluppo della tecnologia, il buiatra ha avuto pochi margini di trattativa nel far valere il proprio parere professionale, e ancor meno di possedere quella adeguata formazione etica in grado di riflettere su un progresso tecnico scientifico appiattito all’esclusivo valore economico.4 Certo sarà necessario per tutti fare un salto, magari non sanitario, ma etico e culturale, per far emergere non solo le nostre indiscusse doti professionali ma quelle umane, che da sempre accompagnano il nostro vissuto quotidiano. Non sarà facile ricreare una qualche cornice d’ordine, ma un cambio di paradigma oggi più che mai è necessario proprio come conseguenza delle aree tematiche trattate dalla “comunicazione” della Commissione UE, e che non richiedono solo adesioni passive della categoria agli indirizzi istituzionali, ma sostanziali contributi riflessivi in merito (Figura 1).

Figura 1 - Intergovernmental Panel on Climate Change (IPCC, ONU) https://www.ipcc.ch/

Box 1 - CODICE DEONTOLOGICO VETERINARIO Art. 1 Il Medico Veterinario svolge la propria attività professionale al servizio della collettività e a tutela della salute degli animali e dell’uomo. In particolare dedica la sua opera: • alla protezione dell’uomo e degli animali dai pericoli e danni derivanti dall’ambiente, dalle malattie degli animali e dal consumo delle derrate o altri prodotti di origine animale; • alla prevenzione, alla diagnosi e alla cura delle malattie degli animali e alla tutela del loro benessere; • alla conservazione e allo sviluppo funzionale del patrimonio zootecnico; • alla conservazione e alla salvaguardia dell’ambiente e del patrimonio faunistico ispirate ai principi di tutela della diversità biologica e della coesistenza compatibile con l’uomo; • alle attività legate alla vita degli animali d’affezione, da competizione sportiva ed esotici; • alla promozione del rispetto degli animali e del loro benessere in quanto esseri senzienti; • alla promozione di campagne di prevenzione igienico-sanitaria ed educazione per uncorretto rapporto uomo-animali-ambiente;alle attività collegate alle produzioni alimentari, alla loro corretta gestione e alla valutazione dei rischi connessi alla gestione della sicurezza alimentare

Abbiamo da tempo dimenticato, tranne che in sporadiche occasioni, che siamo una professione intellettuale e che, prima di qualunque altra categoria sanitaria, siamo coinvolti in campo applicativo. Basta andare con la mente alla fecondazione assistita, la tradizionale FA quotidiana, praticata da oltre 40 anni con successo e che solo da poco più di vent’anni ha iniziato ad essere esercitata in campo umano. O alla gestione del fine ultimo, l’eutanasia, argomento tabù ancora per molti e con cui noi ci confrontiamo da decenni, per concludere con il mondo della ricerca. A dicembre del 2020 è apparsa in prima pagina, su tutti i principali quotidiani e non solo nazionali, la foto della prima bimba “proveniente dal freddo”, ossia da un embrione congelato. Un evento quanto mai quotidiano per il buiatra che applica l’embryo transfer da almeno 30 anni, ma capace ancora di far notizia quando investe il campo umano.

Questi eventi, centrali per molti versi per lo sviluppo e la coscienza dell’umanità, sono il nostro patrimonio che ci definisce e che richiedono riflessioni mature e condivise, prima che altri esprimano giudizi privi della pur minima esperienza in campo. E tutto ciò non perché il veterinario abbia acquisito un qualche ammaestramento specialistico, ma per quell’acume speciale, e forse anche più vero, che nasce dall’osservazione amorevole e partecipata del vissuto quotidiano.

Nello specifico della dichiarazione della Commissione UE si fa rilevare come oggi “i cittadini vogliono alimenti sani provenienti da un pianeta sano, e meritano informazioni chiare che consentano loro di compiere scelte informate”. Questa affermazione pone al centro un quesito squisitamente etico: come vogliamo vivere e quali scelte di conseguenza si devono compiere?

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Noi non possiamo tirarci indietro, o fingerci estranei a queste domande, ma possiamo fornire il nostro parere motivato e il nostro contributo in campo. L’allevatore non è solo il nostro datore di lavoro ma il nostro partner, noi dipendiamo da lui, come lui dipende da noi. Come tale sempre più dovrà essere reso partecipe su scelte, e formato su obiettivi trasversali, che se non condivisi gli cadranno sulla testa come tegole ovvero danni diretti, e che inevitabilmente non potranno non avere ricadute collaterali anche sul professionista rimasto indifferente.

La strategia Farm to Fork proposta dalla Commissione Europea è al centro del Green Deal Europeo. Essa si propone di affrontare una crescita sostenibile, migliorare la salute e la qualità della vita e “affronta in modo completo le sfide dei sistemi alimentari sostenibili e riconosce i legami inestricabili tra persone sane, società sane e un pianeta sano”.5 Si teme un rischio in agguato in ogni piatto, e nel contempo si inventano soluzioni fantasiose: secondo alcuni a causa dell’effetto serra nel 2050 sarà difficile pensare a un pranzo con una bistecca alla fiorentina, un bicchiere di Chianti o di champagne e delle ciliegie. Dovremo accontentarci di antipasti fatti di scarabei fritti, di hamburger di carne sintetica ottenuta in laboratorio, patate e tiramisù realizzati con una stampante in 3D.6 Le imprese si stanno unendo per guidare il cambiamento climatico e, attraverso la ricerca, investono e annunciano nuovi alimenti del tutto artificiali modificando e diminuendo il consumo della carne.7 Un allarme è espresso anche dalla più autorevole rivista di cultura alimentare, l’Accademia Italiana della Cucina, che preoccupata afferma come: “modificare il nostro sistema alimentare diminuendo il consumo della nostra amata carne è un po’ come recidere le radici del nostro passato, con la nostra storia”.8 Di fatto si oscilla tra stucchevoli proposte di ritorno ad una natura e ad un sistema puro e incontaminato con allevamenti da copertina per riviste patinate e, contemporaneamente, si procede alla sistematica distruzione dell’ambiente.

Cos’altro aspettiamo a dire la nostra? Che ci chiamino per nome?

Chiaramente i punti affrontati sono tali e tanti che ogni collega troverà la sezione a lui più confacente da coniugare, ed eventualmente da rinforzare con lo studio, così da fornire pareri motivati, ma nessuno può chiamarsi estraneo, pena l’inevitabile caduta non solo di immagine, ma di credibilità di tutta la professione.

Siamo chiamati ad un nuovo paradigma etico professionale: non solo “far bene”, ma far bene per qualcosa di buono, di bello e in ultima analisi, di giusto. Nuova linfa, ossia nuove idee da mettere in campo, e nuova vita; questa la sfida per la categoria del futuro.

LE RAZZE AUTOCTONE La strategia dell’UE per la biodiversità per il 2030 è un piano completo, ambizioso e a lungo termine, per proteggere la natura e invertire il degrado degli ecosistemi.

Ci sarà una migliore tracciabilità per garantire che almeno il 30% dell’importo totale del bilancio dell’Unione Europea e delle spese dell’UE per la ripresa sostenga gli obiettivi di protezione del clima, e che il 7,5% della spesa annuale sia dedicata agli obiettivi di biodiversità, a partire dal 2024, e il 10% dal 2026 in poi.9 Secondo un rapporto FAO del 2016 circa il 17%, ossia 1458, delle specie animali domestiche sono a rischio estinzione (Box

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2). Mentre sullo stato di rischio di molte altre specie, il 58%, semplicemente non si sa molto, a causa della mancanza di dati sulle dimensioni e sulla struttura delle loro popolazioni: di fatto tra il 2000 e il 2014 si sono estinte quasi 100 razze di bestiame (Figura 2).

Per la Commissione Europea, e per nostro centrale interesse professionale, va evidenziato come il benessere animale si traduce non solo nel miglioramento della salute degli animali, ma deve contribuire anche a preservare la biodiversità. Viene così evidenziato come, per lo sviluppo di una zootecnia sostenibile, sarà vincolante la sua valutazione in termini di impatto ambientale e climatico: “la PAC accompagnerà la transizione verso sistemi di allevamento sostenibile supportando soluzioni innovative e pratiche di produzione sostenibile”.10 Al primo punto della dichiarazione della CE infatti risulta elen-

Box 2, CLASSIFICAZIONE DELLE RAZZE A RISCHIO DI ESTINZIONE (FAO, 2003) Le Secondary guidelines for development on National farm animal genetic resources management plans della FAO (2003), suddivide le razze in 7 categorie di rischio. La classificazione si basa sulla dimensione complessiva della popolazione, sul numero di femmine riproduttive e sul trend della popolazione (in aumento, stabile o in decremento): • Estinta: razza per la quale non è più possibile ricreare la popolazione; l’estinzione è inevitabile, perché non esistono riproduttori maschi (seme) o femmine (oociti) né embrioni.

• Critica: razza con meno di 100 femmine riproduttive o con meno (o al massimo) 5 maschi riproduttori, ovvero razza con popolazione complessiva di circa 100 animali, ma in diminuzione, e percentuale di femmine allevate in purezza inferiore all’80%.

• Critica conservata: condizioni identiche a quelle di una razza in situazione critica, ma per la quale sono attivi programmi di conservazione, ovvero le popolazioni sono mantenute da compagnie commerciali o da istituti di ricerca.

• Minacciata: razza con un numero totale di femmine compreso tra 100 e 1000, o con un numero di maschi inferiore o pari a 20 ma maggiore di 5; ovvero razza con popolazione complessiva di circa 100 animali ma in aumento, e percentuale di femmine allevate in purezza maggiore dell’80%; ovvero razza con popolazione complessiva di circa 1000 animali, ma in decremento, e percentuale di femmine allevate in purezza inferiore all’80%.

• Minacciata conservata: condizioni identiche a quelle di una razza minacciata, ma per la quale sono attivi programmi di conservazione, ovvero le popolazioni sono mantenute da compagnie commerciali o da istituti di ricerca.

• Non a rischio: razza con numero totale di femmine e maschi riproduttori maggiore rispettivamente di 1000 e 20; ovvero razza con popolazione complessiva di circa 1000 animali, in aumento e con una percentuale di femmine allevate in purezza pari a circa il 100%.

• Stato sconosciuto: la consistenza della popolazione non è nota e necessita di indagini conoscitive.

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Figura 2 - Stato delle razze d’allevamento. Tratto dal sito ufficiale della FAO. http://www.fao.org/fileadmin/user_upload/newsroom/images/status-worlds-domestic-animal-breeds-fao.jpg

cata, tra le azioni di protezione, la necessità di invertire la curva di perdita della biodiversità: “è urgente ridurre la dipendenza da pesticidi e antimicrobici, ridurre la fertilizzazione in eccesso, aumentare l’agricoltura biologica, migliorare il benessere animale e invertire la perdita della biodiversità”.11 Il legame che sostiene la difesa delle razze autoctone nasce dalla loro funzione di favorire un pascolo altrimenti non recuperabile e a basso impatto ambientale perché “non trattato”, oltre che mantenere quella biodiversità indispensabile per lo stesso futuro delle attuali razze che vanno per la maggiore.

Secondo gli studi effettuati dal barometro di Eurostat, l’agricoltura è responsabile del 10,3% delle emissioni di gas serra, e di questi quasi il 70% proviene dal settore animale. Inoltre allo stato attuale il 68% della superficie agricola totale è utilizzato per la produzione di alimenti per animali.12 Le razze autoctone possono aiutare a ridurre l’impatto ambientale e climatico delle produzioni alimentari e così sostenere la transizione verso un allevamento più sostenibile.

Il concetto di razza anche se in campo umano è abbondantemente superato non solo dal punto di vista culturale ma scientifico, resta invece ancora tenacemente presente nel campo degli animali domestici.

Il Borgioli definisce le razze come “un complesso di individui della stessa specie, che si distinguono per caratteristiche somatiche e funzionali”. 13 La varietà di razze all’interno di una specie rappresenta la garanzia maggiore per tutelare la stessa specie: più razze, più opportunità per l’uomo di garantirsi sempre nuove variabili capaci di soddisfare i suoi obiettivi.

Questo spiega il grande salto, e la sua fortuna, ad esempio della specie canina che da poco più di un centinaio di razze cen-

site alla fine del XIX secolo ha prodotto le oltre 400 razze attuali. Cani da lavoro, cani da caccia con tutte le sue varianti, ma anche cani da soccorso, cani da compagnia, cani da gregge, cani da corsa e da oggi finanche cani terapeutici. Il cane continua a segnare le infinite variabili dell’evoluzione dei bisogni della specie sapiens, che non ha dovuto di volta in volta servirsi di nuove specie, ma sfruttare l’enorme ricchezza del patrimonio genetico di una singola specie.

Accanto alla continua attenzione degli allevatori di cani alle richieste della società umana, in grado non solo di prevederne i loro bisogni ma di indirizzarne le scelte, gli allevatori di allevamenti per alimenti si stanno sempre più imbottigliando in un cul de sac di cui sembra non esserci altra via d’uscita.

Certo si potrebbe obiettare che mentre nel primo caso si parla di specie domestiche allevate per venire incontro alle esigenze “sociali” dell’uomo, nel secondo si parla di animali da reddito destinati alle esclusive produzioni alimentari. Ma è una dicotomia solo apparente in quanto, sia coloro che allevano cani che altre specie domestiche, hanno tutti come obiettivo finale il reddito con cui sostenersi a prescindere dal loro indirizzo produttivo.

Fino a 40 anni fa i testi di Zoognostica per molte specie parlavano, in termini di apprezzamento, di animali a duplice o triplice attitudine, a dimostrazione che la specificità di un attributo non era affatto un parametro da privilegiare, ma bensì rappresentasse un potenziale svantaggio per quella razza.

A conferma di ciò Teleosforo Bonadonna, più di 50 anni fa, già metteva in evidenza come gli indirizzi selettivi di una razza verso un unico scopo, se avessero garantito il suo successo, enfatizzando alcune caratteristiche, sarebbero stati anche la loro tomba nell’ipotesi di un cambio di indirizzo negli usi e costumi del-

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le popolazioni umane.14 Le finalità di ogni allevatore non cambiano con il cambiare delle specie, piuttosto a cambiare sono le sensibilità e l’attenzione che costoro dimostrano nel leggere non solo il presente, ma prevedere per quanto possibile i trend futuri. E magari, quando il mercato è stagnante, incentivare il potenziale acquirente favorendo nuove razze in grado di promuovere nuove sensibilità.

Ad animare le nuove aziende agricole e i nuovi allevatori ci sono vocazioni professionali, nuove tendenze e sensibilità sociali, e la soddisfazione che il lavoro materiale, quando è una scelta e non una condanna, porti ad una gratificazione interiore.

Ricostruire questo percorso risulta indispensabile e allo stesso tempo a basso costo, in quanto il profilo del prodotto delle razze autoctone è già fonte di una serie di attributi impossibili da ricreare nella catena industriale e quindi non concorrenziale. L’origine della specie, la qualità della razza, la tipizzazione del territorio, la valorizzazione dell’ambiente e il preservare la biodiversità rappresentano in sé un unicum insostituibile e che non possiede processi di competizione nell’anonima catena di distribuzione di massa, e quindi della concorrenza al ribasso.

Ciò che oggi colpisce nelle scelte del consumatore è il totale appiattimento delle proprie necessità al processo industriale. Non valgono più distinguo di razza, ma solo un generico indirizzo produttivo, latte/carne, la fa da discriminante: La carne affettata e cellofanata (dove si deve dedurre che chi l’ha confezionata ha già stabilito per noi dosi di consumo e modalità d’impiego) esposta nelle catene dei supermercati è l’icona che meglio sintetizza il pensiero del suo futuro acquirente.15 Se da un lato, per la specie cane, l’uomo risulta provvisto di innumerevoli sfaccettature emotive e di bisogni, tanto da ottenere una ricchezza di varietà di razze profondamente diverse l’una dall’altra, dall’altro, per quanto riguarda le specie ad indirizzo produttivo, le variabili risultano estremamente limitate allo stretto necessario e tutto viene omologato a poche offerte.

Com’è possibile che per una specie, il cane, diversifichiamo e riconosciamo oltre 400 razze e per altre, come nei bovini, oltre il 40% mondiale degli esemplari è oggi rappresentato da una sola razza, la Frisona?

Quando appare una contraddizione “significa che l’analisi non è stata approfondita abbastanza e che taluni caratteri distintivi sono passati inosservati”.16 Una valutazione di massa che siano litri di latte, o indice di conversione in carne, del resto importa poco o nulla. Lo stesso concetto di qualità si presta ad essere tirato per qualsiasi uso: Infatti da parte dell’industria, e di riflesso del consumatore, vanno sempre più definendosi due atteggiamenti diversi di affrontare il prodotto di qualità: alcuni elaborano un prodotto di qualità partendo da una visione antica, tradizionale, che immagina alimenti che scandiscono le occasioni e la quotidianità, o comunque seguono una traccia di altri tempi; mentre una frangia, più ristretta alle grandi industrie, adotta una scansione che vincola la qualità alle sole garanzie tecnologiche.17 Com’è possibile che lo stesso uomo che ha modellato campioni di razza per ogni prestazione, adattando una stessa specie, il cane, a infinite attitudini, abbia rinunciato a comprendere il valore insito in ogni potenziale razza presente su di un preciso territorio per ciò che riguarda il suo cibo? Possibile che il cibo, e tutto ciò che esso rappresenta di simbolico e culturale possa omologarsi ad un anonimo prodotto privo di storia e ancor meno

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di memoria sociale?

Se una cosa diventa buona da mangiare quando è anche buona da pensare,18 come è possibile che nel giro di una sola generazione si sia perso il valore culturale e rituale del cibo?

Cosa perdiamo quando buttiamo via il passato? La formula perfetta che inchioda tutti: “que sais-je?”, che cosa so io? 19

IL CONTRIBUTO DELLA RICERCA Quando si prendono in considerazione le preoccupazioni relative alla perdita di biodiversità delle specie, la risposta del mondo scientifico, su eventuali modelli da proporre in campo applicativo, evidenzia come non esiste una strategia di selezione universale che possa essere utilizzata per ottimizzare il numero effettivo in una piccola popolazione che si vuole proteggere.

In un recente studio sulla “gazzella di Cuvier” l’equipe scientifica si è concentrata sui sistemi di accoppiamento per ridurre al minimo la scomparsa della variabilità genetica. Il pericolo dietro l’angolo nei programmi di accoppiamento è che non si può considerare il semplice aumento di popolazione come un parametro di successo. Nello specifico il passaggio da un valore di popolazione basso ad un valore medio infatti non aveva massimizzato l’obiettivo di ampliare la biodiversità genetica di specie. È necessario quindi avvalersi di simulazioni con modelli matematici sviluppati al computer che identificano parametri utili per più scenari: a breve, medio e lungo termine.20 Così, a seconda delle circostanze, a volte è consigliabile limitare i piani di accoppiamento anche in contesti dove sarebbe possibile beneficiare di tutte le femmine e/o maschi disponibili ad avere una prole. Pertanto, ogni particolare popolazione può avere le sue strategie di accoppiamento ottimali e ciò dovrebbe essere studiato attentamente prima di incorporarlo in un piano di allevamento.

Altri studi in campo applicativo hanno riguardato i guadagni genetici nei programmi di allevamento attraverso la selezione ottimale di materiale genomico utilizzando tecniche di ovulazione multipla e trasferimento degli embrioni. Anche in questo caso sono stati simulati stocasticamente vari scenari di programmi di allevamento valutando i potenziali benefici del potenziale genetico e ponendo una penalità sul tasso di consanguineità.21-22 Implementare lo studio di razze autoctone non esaurisce la sua missione con la valorizzazione del patrimonio genetico in sé, ma presenta ricadute “trasversali” nell’ecosistema.

In linea su questo piano di ricerca da diversi anni sta lavorando il professore Sabbioni della Facoltà di Veterinaria dell’Università di Parma. Un’operazione che non ha precedenti in ambito nazionale e che ha visto la facoltà come referente scientifico di un progetto che si è andato maturando in considerazione che: un allevamento di poche razze altamente selezionate comporta automaticamente la perdita di varietà di geni potenzialmente utili per la produzione, la resistenza alle malattie e l’adattamento a condizioni climatiche estreme e profondamente diverse. La perdita di variabilità genetica rappresenta quindi un grave pericolo per noi e per le generazioni future.23 Una visione quella della valorizzazione delle razze autoctone, di scarso impatto economico, e che anche in ambito della ricerca universitaria, appiattita alle sole produzioni intensive, era vista come un’operazione di retroguardia. Nulla di più sbagliato:

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Le razze locali hanno assunto un ruolo nella produzione zootecnica legata alle produzioni di qualità, in virtù del loro legame con il territorio. Sono quindi un segnale che ci riporta al concetto di produzioni sostenibili e ad una zootecnia rispettosa dell’ambiente, sulle basi delle attuali conoscenze scientifiche.24 Ad essere coinvolti nel tempo sono stati il suino “nero” di Parma, e la pecora Cornigliese dell’Appennino Parmense, ma anche polli Modenesi e Romagnoli, tacchino di Parma e Piacenza, il cavallo Bardigiano e i bovini locali, la rossa Reggiana e la bianca Modenese, conosciuta anche come bianca della Val Padana.

Le ricerche partite dalla caratterizzazione fenotipica sono poi passate alla ricerca delle caratteristiche chimiche-bromatologiche delle loro produzioni per concludere poi sulla variabilità genetica finalizzate alla genotipizzazione. In tal senso vanno ampliate e coordinate le collaborazioni regionali attraverso il nuovo “Network Europeo di Banche Genetiche” (EUGENA) per gestire e migliorare le razze nel loro habitat naturale.

Recenti studi hanno dimostrato come un elemento basilare per la conservazione di questi ambienti è proprio la presenza di bestiame al pascolo.25 Gli ambienti d’alta quota infatti, oltre a essere tra gli elementi più interessanti e caratteristici del paesaggio nazionale costituiscono un insieme di habitat di specie ad elevata diversità e tra i più minacciati tra quelli presenti sul territorio europeo.

Venendo meno la presenza delle specie autoctone in grado di pascolare anche in aree marginali e non accessibili all’uomo, come il territorio alpino e la dorsale appenninica, si è subìto il ben noto e visibile ritorno della vegetazione arboreo arbustiva, con perdita di habitat vitali per molte specie. Ciò che risulta dalle Linee Guida è come la difesa delle razze autoctone non solo prevede un valore diretto del proprio patrimonio genetico, ma presenta un enorme valore indiretto per svariate altre specie che premono sullo stesso territorio.

Un esempio concreto si è rilevato con la riduzione della presenza della coturnice (Alectoris graeca) dal suo areale. Nonostante negli ultimi anni la caccia sia stata interdetta, questa specie infatti è scomparsa da molti settori montani in seguito alla riforestazione naturale dei versanti aperti e dei pascoli e all’abbandono delle coltivazioni cerealicole in quota, ideali habitat di svernamento. Nulla di più delle significative conclusioni del report del MUSE (Museo di Scienze, Trento): La condizione ottimale è pertanto rappresentata da uno sfruttamento regolare, non eccessivo e adeguatamente pianificato, in grado di riprodurre un “disturbo ecologico” intermedio, necessario per mantenere gli habitat cui sono associate le specie degli ambienti aperti d’alta quota. Il pascolo bovino non intensivo è idoneo, in quanto favorisce la presenza di aree con erba bassa o rada all’interno dell’ambiente prativo, importanti per molte specie per la cattura delle proprie prede; inoltre, favorisce la presenza di una ricca entomofauna e di un mosaico vegetazionale apprezzato da molte specie.26 Proprio attraverso la loro presenza in situ è possibile riconoscere anche il valore culturale di queste razze. I punti elencati sono talmente centrali ed evidenti che ci sarebbe da chiedere piuttosto come mai si fa fatica a penetrare nel mercato.

La questione così come ora esposta stravolge il percorso finora giustificativo del valore delle razze autoctone, la cui sopravvivenza oggi è vincolata solo ai finanziamenti a fondo perduto di piatte politiche agrarie. È necessario piuttosto ribaltare il paradigma del “più e meglio”, e inchiodare alle loro respon-

sabilità quanti hanno privilegiato la monocultura alimentare e la selezione unidirezionale di specie e di razza. Sono costoro che devono giustificare le loro scelte, e non chi ha costruito il proprio percorso nella diversificazione rispondendo a principi economici, etici e scientifici coerenti.

Il finanziamento per le razze autoctone non può essere assegnato come se fosse l’integrazione giustificativa al mancato guadagno che si sarebbe potuto ottenere facendo scelte più aggressive. Questo è quanto di più sbagliato come indirizzo politico e culturale perché fa credere che seguendo altri percorsi lo stesso allevatore avrebbe ottenuto maggiore reddito e che la comunità sociale, sentendosi in colpa, bilancia attraverso una sovvenzione per mancato guadagno. È un messaggio profondamente contradditorio perché, falsamente, si fa credere alle grandi opportunità a cui rinuncia chi fa queste scelte, senza valorizzare il senso profondo delle scelte alternative. La questione non va posta come scelta alternativa ma come responsabilizzazione verso la società e valorizzazione del proprio ruolo e compiti.

ASPETTI ETICI E SOCIOCULTURALI È comprensibile come il bestiame allevato sia al centro di molteplici questioni sul tema della sostenibilità, sia che si consideri la sicurezza alimentare che la lotta alla povertà o ai cambiamenti climatici. I singoli allevatori di tutto il mondo stanno abbandonando molte razze che si sono adattate localmente nel corso di migliaia di anni a favore di nuove razze cosmopolite ma più produttive. In parte questa transizione si può spiegare con l’economia, poiché l’uniformità alimentare della dieta verso alimenti cereali più economici, e gli strumenti genetici moderni, hanno reso più redditizia l’agricoltura intensiva.27 Anche le politiche governative mal progettate potrebbero aver contribuito al presente declino. Resta quindi da interrogarsi su quale ruolo dovrebbero continuare ad avere le razze autoctone nelle economie locali. E quali approcci si possono proporre nel far coesistere i valori sociali e la memoria storica identitaria di una popolazione proteggendo le razze economicamente svantaggiate e nel contempo salvaguardare la biodiversità genetica, garantendo comunque un reddito all’allevatore?

Assolutamente centrale rilevare come la FAO ha attualizzato la stessa definizione di razza come: ciascun sottogruppo specifico di animali di interesse zootecnico, con caratteristiche esteriori definibili e identificabili, che può essere separato dagli altri gruppi definiti in modo simile all’interno della stessa specie mediante stima visiva. O un gruppo per il quale la separazione geografica o culturale da gruppi fenotipicamente differenti ha indotto ad accettare la propria identità separata.28 Una definizione assolutamente innovativa, che contiene in un termine unico due rami portanti: elementi fenotipici e aspetti identitari.

Si è visto come le razze autoctone possono contribuire a modellare l’identità culturale umana a causa del coinvolgimento di queste in molti riti e tradizioni locali, rappresentando un importante ponte di collegamento per gli esseri umani con le loro culture e ambienti.29 L’uso della transumanza è stato inserito nel 2019 dall’UNESCO nella Lista del Patrimonio Culturale Immateriale, che ha riconosciuto il valore della sua pratica sulla base di una candi-

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datura transnazionale presentata da Italia, Austria e Grecia.

Marsoner et al., in un articolo scientifico del 2018, hanno riconosciuto le razze autoctone come indicatori dell’offerta di servizi Eco Sistemici Culturali (CES) in precise aree territoriali (ad es. Arco Alpino). In particolare lo studio ha rilevato il potenziale non sfruttato per iniziative economiche di integrazione al reddito. 30 Nella conclusione dello studio si individua come strumento identificativo la creazione di marchi per prodotti limitati al luogo d’origine (Denominazione di Origine Protetta, DOP), contestualizzando anche gli aspetti sociali e culturali. Tale iniziativa potrebbero contribuire alla conservazione a lungo termine delle razze in via di estinzione, e di conseguenza, costituire una strategia di conservazione promettente.

Appare legittimo quindi interrogarsi sul legame tra risorse genetiche utilizzate e servizi forniti.

Per rispondere a questa esigenza il MIPAAF, in coordinamento con il Comitato Permanente per le Risorse Genetiche, ha promosso la redazione di Linee Guida per la conservazione e la caratterizzazione della biodiversità vegetale, animale e microbica di interesse per l’agricoltura, a cui ha contribuito un nutrito gruppo di esperti predisponendo tre distinti manuali dedicati rispettivamente alla biodiversità vegetale, animale e microbica.31 La versatilità dei valori cui si presta la razza autoctona è così esplicitata: Per il settore zootecnico, il documento propone un approccio innovativo nella definizione delle strategie di conservazione sinora adottate in Italia: la scelta delle razze da conservare prioritariamente va basata non solo sullo stato di rischio delle razze, ma anche, e soprattutto, sulla valutazione della loro importanza attuale e futura in vari ambiti (economico-produttivo, sociale, storico, culturale, ecologico, paesaggistico, etc.). Con questo approccio si intende puntare non solo all’obiettivo di diminuire o azzerare il rischio di estinzione di tutte le razze allevate ma, contemporaneamente, anche a quello di migliorare o massimizzare l’utilità derivante dal loro impiego (intendendo per “utilità” una combinazione ponderata di caratteri, valori e caratteristiche di ciascuna razza), fino a giungere all’auto-sostentamento economico-produttivo della razza.32 Non va dimenticato inoltre che la razza autoctona è facilmente riconoscibile dal consumatore perché parte integrante, non solo del territorio di appartenenza in cui vive, ma della memoria e degli usi alimentari della tradizione. Una garanzia ulteriore perché ricostruisce quell’anello mancante tra cibo-tradizionerito, che negli ultimi anni è venuto a mancare e che resta essenziale per dar senso a ciò che mangiamo. La centralità della “civiltà della tavola”, che ha rappresentato da sempre il senso del buono da pensare, e non solo del buono da mangiare, elaborando una serie di interrogativi etici.33 Harris sviluppa la sua riflessione dalla considerazione che “Il cibo ha ben poco a che fare con il nutrimento”.34 Questa riflessione antropologica è stata del tutto annullata dalla grande distribuzione rendendo il consumatore stesso merce tra le merci. La cura e l’amore che giustificavano un consumo attento della carne rappresentava il picco delle delizie da servire in tavola. Da allora non vi è ricerca antropologica che non preveda una sezione, anche solo una riflessione a margine, del “valore” cibo.

Il cibo resta una componente centrale del patrimonio dell’umanità, così da essere inserito nel paradigma delle “culturali universali”35 dell’umanità e che hanno caratterizzato le due gran-

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di rivoluzioni dell’uomo: la prima, la rivoluzione agricola, che ha portato la società umana da cacciatori/raccoglitori a quella di agricoltori/allevatori, in cui centrale resta tutt’ora presente, a macchie di leopardo, il ruolo della pastorizia, e la seconda, la rivoluzione urbana, con il passaggio di masse ingenti di popolazione trasferita dalla campagna alla città.

Il passaggio epocale tra questi due sistemi ha prodotto diverse eccezioni al concetto di cultura, che è passato da fenomeno identitario esclusivo interno di precise popolazioni, una visione rigida e statica, ad uno scambio di scenari sempre più complessi costituendo nuovi habitat di significati in parte ancora da sistematizzare. Una “ragnatela di significati”36, così come attualmente viene riconosciuta, che sposta l’attenzione dalle rappresentazioni e dalle strutture culturali che la compongono alle sue modalità interpretative, al suo significato individuale che rispondono alla domanda: “che senso ha?”. O ancora meglio: “che senso do a ciò che mangio?”.

A questo proposito resta fondamentale il contributo analitico di Geertz in questo campo, che seppe affrancarsi dal paradigma universalista per porgere attenzione alle più “umili” manifestazioni della vita quotidiana di un popolo. I cambiamenti dei sistemi alimentari sono tra le tendenze che maggiormente devono essere monitorate per essere poi in grado di prevedere il loro impatto sulla domanda, coinvolgendo inevitabilmente specie e razze. Un’antropologia interpretativa basata sulla sostanziale simbolicità dell’esperienza umana che si manifesta in ciascuna cultura e che si evidenzia in modo implicito nel comportamento quotidiano, a volte anche in forma inconscia, degli individui. Un’attività di decodificazione del significato delle azioni, delle pratiche e dei riti negli individui, offrendosi all’interpretazione suggerita “tra l’atto di mangiare e l’atto di copulare che si riscontra in tutte le lingue del mondo”.37 È evidente come in quest’ottica il cibo, con i suoi usi e divieti, rituali e tradizioni, la fa da padrone. Capace di esprimere significati e linguaggi simbolici, il cibo, e il cibo carne in assoluto, ha ben poco a che fare, concordando con Harris, con il nutrimento. Proprio questa sostanziale valenza di significati si riflette poi non solo sulle specie alimentari, ma spesso proprio sulla tipologia di razza che identifica le successive modalità di elaborazione alimentare.

CONCLUSIONI Le razze autoctone rappresentano una necessità almeno per 3 motivi: 1) Garantiscono la biodiversità in quanto valore in sé non negoziabile.

2) Contribuiscono agli obiettivi di una zootecnia sostenibile e ad una maggiore attenzione del consumatore alle offerte del mercato.

3) Recuperano la memoria storica di un territorio attraverso prodotti e tradizioni, privilegiando valori socioculturali condivisi.

Se da sempre la valorizzazione delle razze autoctone ha rappresentato una battaglia di retroguardia per lo scarso impatto economico e con prospettive scientifiche altrettanto scarse, oggi le razze locali assumono un ruolo centrale nelle produzioni zootecniche di qualità, in virtù del loro legame con il territorio. Non solo, oggi la presenza di più razze autoctone è in-

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In difesa delle razze autoctone

dispensabile perché, evitando l’erosione genetica, assicura la vita alle stesse razze cosmopolite più rappresentate, che senza innesti di sangue perderebbero qualunque sfida del futuro. Valori scientifici importanti quindi, con ricadute per gli allevatori attraverso il recupero dei pascoli marginali e la difesa del paesaggio, ma anche per il loro specifico contesto culturale, che le razze autoctone mantengono attraverso le produzioni tipiche di un territorio. Aspetti che se ben declinati toccano tre macroaree fondamentali per il nostro vivere: a) l’economia di base, attraverso un’accorta gestione delle risorse alimentari; b) la cultura, non solo attraverso la difesa del territorio e la valorizzazione del paesaggio, ma favorendo un tessuto di obblighi reciproci tra produttore e consumatore che accomuna entrambi attraverso un sapere condiviso; c) la ricerca scientifica, attraverso la difesa della biodiversità che, come la FAO riconosce: “la diversità genetica del patrimonio zootecnico può aiutare a nutrire un mondo più caldo e in condizioni ambientali meno favorevoli”.38 1

Commissione Europea, COM/2020/381; visionabile sul sito: https://eur-lex.europa.eu/legal-content/EN/TXT/?qid=1590404602495 &uri=CELEX:52020DC0381 2 idem 3 AAVV; Gruppo di Lavoro FNOVI “Animali, uomo ambiente”, La responsabilità del medico veterinario verso l’ambiente e l’uomo; Mondo Sanitario; Anno XXVI, n.10, Novembre 2019; p.14.

4 Pallante G., Il veterinario e l’allevatore: è possibile fare della bioetica in zootecnia?; in La questione animale a cura di Castiglione S., Lombardi Vallauri L.; TRATTATO DI BIODIRITTO, Rodotà S., Zatti P.; Giuffrè ed., Milano, 2012; p.233243.

5 Commissione Europea, sito cit.; 6 Teti V., Fine pasto. Il cibo che verrà; Einaudi, Torino; 2015, p.15.

7 Word Economic Forum (WEF), rapporto visionabile sul sito: http://www3.weforum.org/docs/WEF_Two_Degrees_of_Transformation.pdf 8 Debiaggi M, Il futuro della carne e la carne del futuro; in Civiltà della tavola. Accademia Italiana della cucina; anno I, n.5/2020; p. 4.

9 Bilancio UE 2021-2027, visionabile sul sito: https://www.europarl.europa.eu/news/it/headlines/priorities/qfp/20201211IPR936 21/il-parlamento-approva-definitivamente-il-bilancio-ue-2021-2027 10 Commissione Europea, sito citato.

11 Idem.

12 Eurostat ha certificato 39,1 milioni di ettari di cereali e semi oleosi e 70,7 milioni di ettari di prati su 161 milioni di ettari di terreno agricolo nell’UE a 27 Stati. Fonte Eurostat Rapporto 2019. Visionabile su Sito ufficiale: https://ec.europa.eu/eurostat 13 Borgioli E.; Genetica e miglioramento degli animali domestici; Edagricole; Bologna, 1985.

14 Bonadonna T., Etnologia Zootecnica; UTET, Torino, 1976.

15 Pallante G., Uomo cibo animale. Per una Zooantropologia della complessità, in L’animale relazionale. Simbologia Etica Cibo; a cura di Pallante G.; La Finestra ed.; Lavis (TN) 2006.

16 Levi-Strauss C., Il crudo e il cotto; Il Saggiatore, Milano 2004; p. 216 17 Pallante G., Obiettivo latte: ridiscutere la qualità; Large Animals Review, Anno VIII, n.4, Agosto 2002.

18 Harris M. , Buono da mangiare. Enigmi del gusto e consuetudini alimentari; Einaudi; Torino 1985 19 De Montaigne M., Saggi; Bompiani, Firenze, 2017 20 Gicquel E., Boettcher P., Besbes B., Furre S., Fernández J., Danchin-Burge C., Berger B., Baumung R., Feijóo J.R.J., Leroy G., Impact of conservation measures on demography and genetic variability of livestock breeds, Animal,14, Cambridge, 2020; p. 670-680 21 Granleese, T.; Clark, SA; Cigno, AA; Van der Werf, JH; Increased genetic gains in sheep, beef and dairy breeding programs from using female reproductive technologies combined with optimal contribution selection and genomic breeding values. Genetic Selection Evolution (GSE), 2015; p.47-70.

22 Shadma F, Bhong C.D., Rank D.N., Joshi C.G.; Genetic variability and bottleneck studies in Zalawadi, Gohilwadi and Surti goat breeds of Gujarat (India) using microsatellites. Small Ruminant Research 77 (2008) p. 58-64.

23 Sabbioni A. , La tutela della biodiversità e delle razze locali in zootecnia; UNIPR Ricerca, Visionabile anche sul sito:

https://www.unipr.it/sites/default/files/allegatiparagrafo/16-12-2020/unipr_ricerca_dicembre_2020.pdf 24 idem.

25 Pedrini P., Brambilla M.; Linee guida per la conservazione di specie focali di interesse comunitario. Specie Ornitiche degli ambienti d’alta quota; MUSE, Trento. http://www.lifeten.tn.it/binary/pat_lifeten/azioni_preparatorie/LifeTEN_A8_LineeGuida_ornitiche_alta_quota.1453911696.pdf 26 Idem.

27 Mendelson R., The challenge of conserving indigenous domesticated animals, Ecological Economics, 45 (2003), pp. 501-510, 28 Sito FAO: http://www.fao.org/news/story/it/item/381412/icode/ 29 Hoffmann, I., From, T., Boerma, D., 2014. Ecosystem services provided by livestock species and breeds, with special consideration to the contributions of smallscale livestock keepers and pastoralists.; The Contribution of Livestock Species and Breeds to Ecosystem Service; FAO, Rome, 2014 Sito: http://www.fao.org/3/i6482e/i6482e.pdf 30 Marsoner T., Vigl L., Manck F., Jaritz G., Tappainer U., Tasser E., Indigenous livestock breeds as indicators for Cultural Ecosystem Service (CES): a spatial analysis within the Alpine Space; Ecological Indicators; vol.94; 2018 ; p. 55-63.

Visionabile sul sito di Science Direct: https://www.sciencedirect.com/science/article/pii/S1470160X17303886 31 Governo Italiano, MIPAAF; Linee Guida per la conservazione e la caratterizzazione della biodiversità animale di interesse per l’agricoltura.

32 idem 33 Pallante G., Pallante V., Quanta sofferenza sei disposto a tollerare per il tuo cibo?

Come e perché i vitelli ingrassano; in Emotività animali. Ricerche e discipline a confronto; Andreozzi M., Castignone S., Massaro A., (a cura di). IRENE, Interdisciplinary Researches on Ethics and Natural Environment, Edizioni Universitarie di Lettere Economia e Diritto; Milano, 2013 34 Harris M., op. cit.¸ p. 5 35 Amselle J.L., Connessioni. Antropologia dell’universalità delle culture; Bollati Boringhieri; Milano, 2001 36 Geetz C., Interpretazioni di culture; il Mulino; Bologna, 1998 37 Lévi Strauss C., Siamo tutti cannibali, il Mulino; Bologna, 2015; p. 124 38 Sito FAO: http://www.fao.org/news/story/it/item/381412/icode/#:~: text=Le%20razze%20sono%20popolazioni%20distinte,solo%201.056%20sono% 20razze%20transfrontaliere.

Bibliografia AAVV; Gruppo di Lavoro FNOVI: “Animali, uomo ambiente”, La responsabilità del medico veterinario verso l’ambiente e l’uomo; in Mondo Sanitario; Anno XXVI, n.10, Novembre 2019; Amselle J.L., Connessioni. Antropologia dell’universalità delle culture; Bollati Boringhieri; Milano, 2001 Bonadonna, T.; Etnologia Zootecnica; in Trattato di Scienza e Tecnologia delle Produzioni Animali UTET, Torino, 1976.

Borgioli E.; Genetica e miglioramento degli animali domestici; Edagricole; Bologna, 1985.

De Montaigne M., Saggi; Bompiani, trad. Garavini F., Firenze, 2017.

Harris M. , Buono da mangiare. Enigmi del gusto e consuetudini alimentari; trad.

Arlorio P., Einaudi; Torino, 1985.

Franzen J., E se smettessimo di fingere?; trad. Pareschi S., Einaudi, Torino 2020 Geetz C., Interpretazioni di culture; il Mulino; Bologna, 1998 Gicquel E., Boettcher P., Besbes B., Furre S., Fernández J., Danchin-Burge C., Berger B., Baumung R., Feijóo J.R.J., Leroy G., Impact of conservation measures on demography and genetic variability of livestock breeds, Animal,14, Cambridge, 2020; Granleese, T.; Clark, SA; Cigno, AA; Van der Werf, JH; Increased genetic gains in sheep, beef and dairy breeding programs from using female reproductive technologies combined with optimal contribution selection and genomic breeding values. Genetic Selection Evolution (GSE), 2015; p.47-70.

Governo Italiano, MIPAAF; Linee Guida per la conservazione e la caratterizzazione della biodiversità animale di interesse per l’agricoltura Hoffmann, I., From, T., Boerma, D., 2014. Ecosystem services provided by livestock species and breeds, with special consideration to the contributions of smallscale livestock keepers and pastoralists.; The Contribution of Livestock Species and Breeds to Ecosystem Service; FAO, Rome, 2014 Levi-Strauss C., Il crudo e il cotto; trad. Bonomi A., Il Saggiatore, Milano, 2004.

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G. Pallante Large Animal Review 2021; 27: 357-365

Riferimenti in rete

Levi Strauss C., Siamo tutti cannibali, trad. Ferrara R., il Mulino; Bologna, 2015.

Marsoner T., Vigl L., Manck F., Jaritz G., Tappainer U., Tasser E., Indigenous livestock breeds as indicators for Cultural Ecosystem Service (CES): a spatial analysis within the Alpine Space; Ecological Indicators; vol.94; 2018.

Mendelson R., The challenge of conserving indigenous domesticated animals, Ecological Economics, 45 (2003).

Pallante G., Il veterinario e l’allevatore: è possibile fare della bioetica in zootecnia?; in La questione animale di Castiglione S., Lombardi Vallauri L. (a cura di); Trattato di Biodiritto, Rodotà S., Zatti P.; Giuffrè ed., Milano, 2012; p.233-243.

Pallante G., Uomo cibo animale. Per una Zooantropologia della complessità, in L’animale relazionale. Simbologia Etica Cibo, Pallante G., (a cura di); La Finestra ed.; Lavis (TN), 2006.

Pallante G., Obiettivo latte: ridiscutere la qualità; Large Animals Review, Anno VIII, n.4, Agosto 2002.

Pallante G., Pallante V., Quanta sofferenza sei disposto a tollerare per il tuo cibo?

Come e perché i vitelli ingrassano; in Emotività animali. Ricerche e discipline a confronto; Andreozzi M., Castignone S., Massaro A., (a cura di).

IRENE, Interdisciplinary Researches on Ethics and Natural Environment, Edizioni Universitarie di Lettere Economia e Diritto; Milano, 2013 Pedrini P., Brambilla M.; Linee guida per la conservazione di specie focali di interesse comunitario. Specie Ornitiche degli ambienti d’alta quota; MUSE, Trento.

Shadma F, Bhong C.D., Rank D.N., Joshi C.G.; Genetic variability and bottleneck studies in Zalawadi, Gohilwadi and Surti goat breeds of Gujarat (India) using microsatellites. Small Ruminant Research; n.77, 2008.

Teti V., Fine pasto, il cibo che verrà, Einaudi; 2015.

365

BILANCIO UE 2021-2027: https://www.europarl.europa.eu/news/it/headlines/priorities/qfp/20201211IPR9 3621/il-parlamento-approva-definitivamente-il-bilancio-ue-2021-2027 COMMISSIONE EUROPEA: https://eur-lex.europa.eu/legal-content/EN/TXT/?qid=1590404602495&uri= CELEX:52020DC0381 EUROSTAT: https://ec.europa.eu/eurostat FAO: http://www.fao.org/news/story/it/item/381412/icode/ http://www.fao.org/news/story/it/item/381412/icode/#:~:text=Le%20razze%20 sono%20popolazioni%20distinte,solo%201.056%20sono%20razze%20 transfrontaliere MUSE: http://www.lifeten.tn.it/binary/pat_lifeten/azioni_preparatorie/LifeTEN_A8_ LineeGuida_ornitiche_alta_quota.1453911696.pdf UNIVERSITÀ DI PARMA: https://www.unipr.it/sites/default/files/allegatiparagrafo/16-12-2020/unipr_ ricerca_dicembre_2020.pdf WEF (Word Economic Forum): http://www3.weforum.org/docs/WEF_Two_Degrees_of_Transformation.pdf

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S. Shivaraju et al. Large Animal Review 2021; 27: 367-369

Successful management of exuberant granulation tissue in two horses (Equus caballus) and a donkey (Equus asinus)

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z

S. SHIVARAJU1*, KUMAR MAITI SWAPAN2, E. KALAISELVAN1, G.V. PRAKASH1, MOHAN DIVYA5, GOPINATHAN ASWATHY4, S.P. SUNIL KUMAR6 1

PhD Scholar, Division of Veterinary surgery, ICAR-IVRI, Izatnagar, Bareilly, Uttar Pradesh, India -243 122.

Principal scientist, Division of Veterinary surgery, ICAR-IVRI, Izatnagar, Bareilly, Uttar Pradesh, India -243 122.

3 MVSc Scholar, Division of Veterinary surgery, ICAR-IVRI, Izatnagar, Bareilly, Uttar Pradesh, India -243 122.

4 Scientist, Division of Veterinary surgery, ICAR-IVRI, Izatnagar, Bareilly, Uttar Pradesh, India -243 122.

5 Veterinary surgeon, Cheriyamundam, Govt. of Kerala, India.

6 PhD Scholar, Division of Extension Education, ICAR-IVRI, Izatnagar, Bareilly, Uttar Pradesh, India -243 122.

2

SUMMARY Proud flesh is a complication of wound healing. In this study, two horses and one donkey were presented with proud flesh on the pastern joint of left hind limb (n=2) and cranial aspect of right hock region, respectively. Surgical excision of proud flesh was done in all three equids under general anaesthesia and were treated post operatively, with copper sulphate (CuSO4), potassium permanganate (KMNO4) and glycerine paste in the ratio of 1:1:5. The present report accounts for the successful management of proud flesh in 3 equines by the combination of surgical excision and chemical methods for wound management.

KEY WORDS Donkey, granulation, horse, proud flesh, surgical excision.

INTRODUCTION

HISTORY AND CLINICAL FINDINGS

Exuberant granulation tissue (EGT) is commonly known as proud flesh/ fibroplasia. Granulation tissue reduces the wound size and dead space by contraction, and acts as a barrier for external contaminants1. Whereas, EGT is a complication of wound healing in equines and is composed of dark red fleshy exuberant granulation tissue with sero-sanguinous fluid discharge and protrudes out from the wound and prevents normal wound healing2, 3. The pathophysiology of proud flesh in horse is not yet clear but several contributing factors such as anatomy and function of the distal limb, high motion areas, low oxygen tension and blood flow, chronic contamination, a difference in concentration of growth factors (TGF-β1), the disparity between the synthesis and degradation of collagen and a prolonged low grade of inflammation favours the genesis of proud flesh in equines4-7. Histological examination of a biopsy specimen of the proud flesh can confirm the condition8. The surgical resection of the exuberant granulation tissue9-11 is considered the best treatment modality. This paper discusses the successful management of EGT in the distal extremities of two horses (Equus caballus) and a donkey (Equus asinus) using a combination of surgery and medicine.

Case 1 and 2: Two male horses weighing 300kgs each were presented to this referral institute with the history of trauma leading to laceration and later growth in the pastern joint of the left hind limb for two months (Figure 1a and 1b). The equines were unsuccessfully treated at field level.

Case 3: A female donkey aged 5 years and weighing 220 kgs was presented with an ulcerative growth on the cranial aspect of the right hock region. The equine had a history of barbed wire injury 45 days back (Figure 1c) On physical examination of the equines, a firm granular tissue was observed in the respective wound areas. The physiological parameters of all the equines were within the normal range. Biopsy samples were taken prior to surgery to rule out sarcoids or tumour. Histological examination of the biopsy samples revealed a chaotic, unorganized, cellular appearance (Figure 2). After the confirmation of the sample as an exuberant granulation tissue, surgical excision was decided as the first line of treatment followed by wound management.

Corresponding Author: S. Shivaraju (id:shivaraju558@gmail.com).

SURGICAL PROCEDURE All the equines were fasted 12 hrs and were administered with inj. Tetanus toxoid (1500 IU) and prophylactic antibiotic (ceftriaxone) prior to surgery. The equines were pre-medicated with inj. xylazine (1.1 mg/kg body weight) and inj. butorphanol (0.03

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Successful management of exuberant granulation tissue in two horses (Equus caballus) and a donkey (Equus asinus)

Figure 1 - Photographs showing exuberant granulation tissue at the left hind pastern joint (a and b) and cranial to hock joint (c).

mg/kg body weight), intravenously. Anaesthesia was induced and maintained with inj. thiopentone sodium solution (5%)12.

The proud flesh were excised aseptically and the bleeding vessels were ligated using absorbable suture material or were arrested by local application of potassium permanganate crystals (KMNO4). Relaxation sutures were applied on the skin of both the horses. The growth on hock was removed and the skin was sutured with horizontal mattress sutures (Case 3). The weights of the excised granulation tissue masses were 300g, 1.2kg and 400grams in equines1, 2 and 3, respectively.

POSTOPERATIVE CARE The surgical wound was daily flushed with potassium permanganate (KMNO4) solution and pressure bandage was applied with the paste made of 10 parts of copper sulphate (CuSO4), 10 parts potassium permanganate (KMNO4) and 50 parts of glycerine for until (almost 7 days) the wound surface went 2mm below the skin16. Postoperative antibiotic (Amoxicillin-Sulbactam, 3gm, IM) and anti-inflammatory (Flunixin meglumine 1.1 mg/kg, IV) were given BID for five days. The

skin sutures were removed on 15th, 18th, 20th post-operative day in equine 1,2 and 3, respectively. The complete cicatrix formation in wounds took more than 30 days in all the equine without the evidence of re-occurrence.

DISCUSSION The incidence of traumatic wounds is higher in equine than in any other species and healing is usually complicated13 attributing to differences in the inflammatory response and wound contraction property14. The exuberant granulation tissue is commonly seen in the limbs of the horses due to multiple reasons (less skin, more mobility near joints and improper wound management) that causes oxygen gradient (increase) between tissue and wound surface and create a warm and moist environment, and in turn favours the granulation tissue formation10. Any treatment that arrests wound contraction or epithelization promotes excessive granulation tissue formation.

In the current paper all the equines had granulation tissue on joints (2 on pastern, 1 in hock region) and had a history of being treated with caustic agents topically. Though there are reports on the successful treatment of proud flesh using caustic agents16, but the present equines treated in field, could not make any successful remarks, which may be due to use of improper concentration and application.

Many treatment modalities for the management of proud flesh have been debated in the literature such as chemical cautery, thermo cautery cryosurgery, bandaging, electromagnetic stimulation, topical antibiotics, gentian violet, tetra-chloro-decaoxide and boric ointment impregnated gauge, steroids application or surgical excision16,17 followed by skin grafting2,16.

Neovascularization with the proliferation of endothelial cells along with infiltration of different mono-nuclear cells and fibroblasts on H&E staining of the biopsy tissue sample is considered as confirmation for proud flesh15.

CONCLUSION The present report accounts for the successful management of proud flesh in 3 equines by the combination of surgical excision and chemical methods for wound management.

Figure 2 - Micro-photograph showing neovascularization (blue marks: small arterioles) with infiltration of different cells and fibrosis (40x) on H&E stain.

Conflict of interest The authors do not have any conflict of interest.

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S. Shivaraju et al. Large Animal Review 2021; 27: 367-369

Authors Contribution All the authors have contributed in terms of giving their technical knowledge to frame the article.

8.

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