Veterinary Research Forum

Abstract Infectious bovine rhinotracheitis, caused by bovine herpesvirus-1 (BHV-1) is a highly contagious viral disease affecting bovines, and clinically characterized by pyrexia, inappetence, respiratory distress, dyspnoea, conjunctivitis, nasal discharge, and sometimes abortions. In the present study, buffalo dairy farm having high mortality was investigated. The buffaloes were suffering from high rectal temperature, conjunctivitis, severe respiratory distress, and nasal discharge. Tissue samples from upper respiratory tract were collected aseptically following post-mortem examination of died buffaloes. Tracheal tissue samples were then processed for histopathological examination and DNA isolation. The presence of BHV-1 in the tissue samples was confirmed by nested polymerase chain reaction using glycoprotein B gene primers. The present study reported for the first time the clinical signs, post-mortem lesions, histopathological evidence, and detection of DNA of BHV-1 glycoprotein B gene through nested polymerase chain reaction assay during an active outbreak in buffaloes in India. The findings of this study are crucial for improving the diagnosis of BHV-1 and ultimately reducing financial losses within dairy industry.

Abstract Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, an oncogenic deltaretrovirus that has emerged as a potential zoonotic infection. The BLV naturally infects cattle and causes economic losses through a slow persistent infection with various clinical symtoms following preleukosis. The main objective of this study was to determine the seroprevalence of BLV antibodies in cattle and buffaloes in the border provinces of the Eastern Anatolia region, Türkiye, using the agar gel immunodiffusion (AGID) assay and  enzyme-linked immunosorbent assay (ELISA). For this purpose, a total of 1,033 serum samples were collected from 982 cattle and 51 buffaloes from the provinces of Ağrı (n = 178), Iğdır (n = 252), Kars (n = 317), Van (n = 221), and Hakkari (n = 65) during 2021 - 2022. In AGID and ELISA tests, seropositivity for BLV-specific antibodies was not detected in cattle and buffaloes from the mentioned provinces. This study revealed that BLV was not circulating in cattle and buffaloes in the easternmost border provinces of Türkiye during the sampling period and contributed to determine the status of BLV in the mentioned region. Due to the presence of virus in other regions of Türkiye and neighboring countries, Iran and Iraq, it is recommended to control animal movements, continue efforts to combat the transmission of the virus, and maintain control measures.

Abstract Obtaining information about the eye arterial supply in the buffalo can be a criterion for ocular diseases diagnosis and give a morphological basis for even more researches on the related clinical surgery of this animal. The aim of the present study was to describe the course and branches of the arteries supplying the eye in the adult river buffalo (Bubalus bubalis). In this study, the heads of eight buffaloes were prepared from the public slaughterhouse and dissected to study the eye arterial supply. This research results showed that the eye blood supply in the buffalo originates from the external ophthalmic, internal ophthalmic, superficial temporal and malar arteries. The external ophthalmic artery was given rise to branches to take part in the ophthalmic rete mirabile formation and also given off lacrimal, external ethmoidal, supraorbital and ciliary arteries as well as muscular branches to supply the eye ball and extra-ocular muscles. The internal ophthalmic artery was one of the sources of lateral posterior long ciliary artery. The superficial temporal artery was detached off some branches to supply the lateral angle of inferior and superior eyelids as well as lacrimal branch to supply lacrimal gland. The malar artery was originated from the infra-orbital artery and its branches were supplied the medial angle of inferior, superior and third eyelids. There were no obvious differences between buffalo and yak regarding arteries supplying the eyes. However, there were minor differences between buffalo and oxen, sheep and goat and there were obvious differences between buffalo and camel and giraffe.

Abstract The aim of this study was to evaluate the effects of adding sulfuric acid to sugarcane topssilage on rumen bacteria and whole rumen microorganisms (WRM) and compare the digestibility of sugarcane tops treated with different amount of urea, molasses and sulfuric acid between Holstein cow and Khouzestan buffalo. Regardless of the type of the treatment, potential of gas production (B) by cow WRM (130.670 mL) was more than buffalo (104.060 mL) (p < 0.05), but the rate of gas production (C) by buffalo WRM was greater than cow (0.021 and 0.014 mL per hr, respectively) (p < 0.05). The C in treatment containing only 2.40% sulfuric acid (0.033 mL per hr) was significantly highest (p < 0.05). Regardless of the type of the treatment, the B by cow rumen bacteria (75.040 mL) was more than buffalo (67.150 mL), (p < 0.05), while the C by rumen bacteria of buffalo (0.030 mL per hr) was more than cow (0.017 mL per hr), (p < 0.05). Regardless of the type of the animal, the B coefficient of rumen bacteria in treatment only containing 2.40% sulfuric acid was higher than control (p < 0.05). Therefore, the addition of sulfuric acid not only had no negative effect on microorganisms particularly bacteria, but also probably due to present of sulfur in acid, had positive effect on nutrients digestibility, and growth of microorganisms. The digestibility of sugarcane tops silage treated by cow rumen bacteria and whole microorganisms was higher than buffalo.

Abstract Leptin, the 16-kDa product of the obese (ob) gene, primarily secreted from adipose tissue, has been implicated to play an important role in the regulation of food intake and energy expenditure. This study investigated protective effect of leptin on trichostatin A-induced apoptotic on in vitro maturation ratio of buffalo oocytes. Ovaries were collected from abattoir and were transported immediately to the laboratory by a thermos flask containing sterile normal saline with antibiotics. Oocytes were aspirated from 2 to 8 mm visible follicles. Oocytes were placed in a culture plate and then incubated at 38.5 ˚C with 5% CO₂ in air for 24 hr. The maturation of oocytes was evaluated under a stereomicroscope. The FITC-Annexin V and propidium iodide staining method was used to detect oocyte apoptosis. In leptin treated groups with 0, 10, 50 and 100 ng mL^-1 and groups that apoptosis was induced, the percentage of oocytes maturation was 77.03, 86.12, 85.08, and 79.89% and 59.96, 56.93 and 51.98, respectively, while the percentage of apoptosis was 8.83, 7.90, 8.58, and 9.39%, and 10.37, 11.57 and 12.03, respectively. Our findings showed that addition of 10 and 50 ng mL^-1 leptin to IVM medium of buffalo oocytes could increase oocyte nuclear maturation, and could decrease oocyte apoptosis when trichostatin A added for inducing apoptosis.

Abstract To investigate uterine fluid and serum copper (Cu) and zinc (Zn) variations during the estrous cycle in water buffaloes, 71 genital tracts and blood samples were collected from the abattoir in Urmia, Iran. The phase of the estrous cycle was determined by examining ovarian structures; 18, 15, 16 and 22 were pro-estrous, estrous, met-estrous and diestrous, respectively. The uterine fluid was collected by gentle scraping of the uterine mucosa with a curette. Blood serum and uterine fluid samples of 71 pre-pubertal buffalo calves were also collected and treated in similar manners. The mean (± SEM) total serum (77.10 ± 1.50 µg dL^-1) and uterine fluid (296.40 ± 9.40 μg dL^-1) Cu in cyclic cows was higher than the values of 54.00 ± 1.10 μg dL^-1 and 133.40 ± 5.70 μg dL^-1 in pre-pubertal calves, respectively. Blood serum (114.60 ± 3.20 μg dL^-1) and the uterine fluid (349.90 ± 8.90 μg dL^-1) Zn content in cyclic cows were also higher than those (98.80 ± 1.50 μg dL^-1 and 246.6 ± 4.50 μg dL^-1 respectively) in pre-pubertal calves. Serum Cu in pro-estrus and estrus were lower than those in other stages and also lower than those in the uterine fluid. The lowest serum Zn content was recorded in pro- and met-estrus, while in the uterine fluid it was observed in estrus. In all stages of estrous cycle the uterine fluid Zn content was significantly higher than those of the serum. These results suggested that during the estrous cycle in the buffalo cows, Cu and Zn were actively secreted in uterine lumen and were not dependent on blood serum. The values also increased after puberty.

Abstract To investigate uterine fluid and serum calcium (Ca) and Magnesium (Mg) variations during the estrus cycle in water buffaloes, 71 genital tracts and blood samples were collected from the abattoir in Urmia. The phase of the estrous cycle was determined by examining ovarian structures. 18 animals were pro-estrous, 15 estrous, 16 met-estrous and 22 diestrous. The uterine fluid was collected by gentle scraping of the uterine mucosa with a curette. Blood serum and uterine fluid samples of 71 pre-pubertal buffalo calves were also collected and treated in similar manners. The mean ± SEM total serum and uterine fluid Ca in cyclic buffaloes were 8.68 ± 0.28 mg dL^-1 and 8.10 ± 0.2 mg dL^-1 vs. 6.76 ± 0.65 mg dL^-1 and 7.90 ± 0.15 mg dL^-1 in pre-pubertal calves, respectively. Blood serum Mg was not different in cyclic and pre-pubertal animals but the uterine fluid Mg in cyclic cows was higher than those in pre-pubertal calves. Serum Ca in pro-estrus and estrus were higher than those in other stages and also higher than those in the uterine fluid. The lowest Mg content of serum was recorded in diestrus, while in the uterine fluid it was observed in estrus. In all stages of estrous cycle except for estrus the uterine fluid Mg content was significantly higher than those of the serum. These results suggested that during the estrous cycle in the buffalo cows, Ca was passively secreted in uterine lumen and mostly dependent on blood serum Ca concentrations but Mg was secreted independently. The values (except for serum total Mg) also increased after puberty.

Abstract High ambient temperature is the major constraint on Buffalo productivity. The aim of this study was to evaluate the reproductive performance and thyroid gland function in winter and summer seasons in Khuzestan buffalo bulls. Six male indigenous buffaloes of Khuzestan with nearly the same age (2-3 years old) and weight were used. Semen and blood samples through jugular vein were collected, every two weeks throughout the summer and winter seasons. The thyroid hormones and thyrotropin stimulating hormone (TSH) concentration in blood serum were measured by radioimmunoassay method. Semen quality was determined, using computer assisted sperm analyzer (CASA) and routine methods. The concentration of thyroxin (T4) was lower in winter than summer (p ≤ 0.05). The level of T3 uptake was higher in cold season than that of in hot season (p ≤ 0.05). The differences of tri-iodotyronine (T3) and TSH concentrations, as well as free thyroxin index were not significant between seasons. The semen volume and spermatozoa parameters including concentration, progressive motility, linear velocity, mean velocity, beat cross frequency, linear coefficient and straightness coefficient were higher in winter than summer (p ≤ 0.05). Semen pH and amplitude of lateral head displacement of spermatozoa were higher in summer than winter (p ≤ 0.05). In winter, there was positive correlation between spermatozoa concentration and T3 value of blood serum (p ≤ 0.05). There were positive correlations between values of semen volume and T4, progressive spermatozoa motility percent and TSH, as well as, total motility of spermatozoa and TSH in summer (p ≤ 0.05). In general, thyroid function and semen quality of Khuzestan buffaloes may be affected by seasons.

Abstract The purpose of this study was to evaluate the probable effects of leptin addition in different levels to the semen extender on sperm quality (motility and motility parameters, viability, sperm membrane integrity, and DNA damage). Semen specimens were evaluated immediately after leptin addition, equilibration time and after thawing the frozen semen. Five healthy buffalo bulls (5 ejaculates from each bull) were used. Each ejaculate was diluted at 37 ˚C with tris-based extender containing 0 (control), 10, 20, 50, 100, and 200 ng mL^-1 leptin. The diluted semen was kept 4 hr in refrigerator to reach to the equilibration time and then packed in 0.5 mL French straws and frozen in liquid nitrogen. Our results showed that, in the fresh semen, no significant difference was observed in all sperm quality parameters evaluated among all of the examined leptin concentrations. Addition of 10 ng mL^-1 leptin into semen extender significantly preserved sperm motility, all of the motility parameters, and viability in equilibrated semen compared to that of control group. However, in vitro addition of 200 ng mL^-1 leptin, significantly decreased theses parameters. In the frozen thawed semen, all leptin concentrations decreased sperm motility and viability, but significant decrease was observed in concentrations of 100 and 200 ng mL^-1. Adding leptin to semen extender did not have any significant influence on sperm DNA damage and sperm membrane integrity in all examined groups. These findings suggest that in vitro addition of 10 ng mL^-1 leptin could preserve sperm motility and viability in cooled semen of buffaloes.

Abstract This study was carried out to investigate effects of copper sulphate (CuSO₄) additive to semen extenders on sperm parameters: progressive motility, viability, membrane integrity and DNA damage, after semen dilution and cryopreservation. Semen samples of 5 buffalo bulls of 3-5 years old were collected at 5 different occasions during the autumn 2011. A total number of 25 samples were used in each examination. Sperm progressive motility and viability were measured at 0 (T₀), 60 (T₁) and 120 (T₂) min after diluting semen in tris-citric acid extender containing 0 (control), 0.004, 0.008, 0.016, 0.032 and 0.064 mg L^-1 CuSO₄. Later, semen was diluted in a tris-citric acid-egg yolk-glycerol extender containing the same amounts of CuSO₄, cooled to 4 ˚C and kept refrigerated for 4 hr to equilibrate, sperm progressive motility, viability, membrane integrity and DNA damage were estimated. Then, semen was packed in 0.5 mL French straws and frozen in liquid nitrogen. Later, the frozen semen was thawed in 37 ˚C water bath for 30 sec, and the same parameters as well as total antioxidant capacity (TAC) of the frozen-thawed semen were estimated. The results showed that copper additive at the rate of 0.032 mg L^-1 gives a better protection of sperms through the process of dilution, equilibration and freeze-thawing than that in control and other Cu concentrations, while 0.064 mg L^-1 CuSO₄ had deleterious effect on the sperm.

Abstract The aim of the present study was to investigate the effect of in vitro supplementation of selenium on fresh and frozen spermatozoa quality of buffalo (Bubalus bubalis) bulls. Five healthy buffalo bulls (5 ejaculates from each bull) were used. Each ejaculate was diluted at 37 ˚C with tris-based extender containing 0 (control), 0.5, 1, 2, 4 and 8 μg mL-1 sodium selenite and the sperm motility and viability were evaluated at 0 (T0) (immediately after dilution), 60 (T1) and 120 (T2) min after diluting semen. In the second step, semen samples were diluted with tris-egg yolk-glycerol extender containing the same amounts of sodium selenite, cooled to 4 ˚C, equilibrated and semen parameters (motility, viability, membrane integrity and DNA damage) were estimated. Then, the semen was packed in 0.5 mL French straws and frozen in liquid nitrogen. Later, the semen was thawed and analyzed for the same parameters, as well as total antioxidant capacity. Results showed that addition of 1 and 2 μgmL-1 selenium to the semen extender significantly increased the sperm motility of fresh and equilibrated semen compared to the control without affecting other parameters. However, in frozen-thawed semen, extenders containing 1 and 2 μg mL-1 selenium significantly improved sperm motility, viability, membrane integrity and semen total antioxidant capacity and also resulted in lower DNA damaged sperms. In this study selenium supplementation of semen extender of 4 and 8 μg mL-1 had deleterious effects on sperm parameters as early as the samples were prepared for freezing.

Abstract The aim of this study was to evaluate the defense cells changes of cervical mucous during follicular and luteal phases of estrus cycle in river buffalo. Reproductive organs of the adult and apparently healthy female buffaloes were collected from the slaughterhouse. By visual investigation of both the ovaries for presence of corpus luteum and growing follicles, the luteal and follicular phase of each buffalo was specified. Cervical discharge samples were collected by sterile swabs and then spread over the glass slides, dried and fixed with methanol. The specimens were undergone Giemsa staining. The percentage of lymphocytes, neutrophils, monocytes (macrophages), eosinophils and basophils in each case (for both the follicular and luteal phases) were obtained at 20 microscopic fields. The percentage of lymphocytes, neutrophils and basophils in luteal phase were higher than the follicular phase. The percentage of eosinophils in follicular phase was higher than the luteal phase. The percentage of monocytes (macrophages) in luteal and follicular phases was nearly equal. The statistical analysis showed that the differences of all cells between follicular and luteal phase were not significant (P > 0.05). The most defense cells in discharges of external os of cervix (both follicular and luteal phases) were neutrophils and lymphocytes.

Abstract In order to determine calcium (Ca), magnesium (Mg) content and total antioxidant capacity (TAC) of seminal plasma in buffalo and to study their associations with the semen characteristics, 54 semen samples were collected from 10 buffalo bulls; semen quality was evaluated, seminal plasma was then harvested by centrifugation and its Ca and Mg content were estimated and its TAC determined. The Ca and Mg content of the seminal plasma (Mean ± SEM) were recorded as 22.36 ± 0.52 mg dl-1 and 11.94 ± 0.36 mg dl-1 respectively, while, its mean TAC value was 1.50 ± 0.02 mmol L-1. The mean Ca value was highly associated with sperm progressive motility, gross motility, viability (P = 0.000 for all), negatively with semen volume (P = 0.01), and with Mg and TAC values (P = 0.000 for both). The mean Mg values was highly associated with sperm progressive motility, gross motility and viability and seminal plasma Ca and TAC (P = 0.000 for all) and negatively associated with semen volume (P = 0.014). The mean TAC values was highly associated with sperm progressive motility, gross motility and viability and seminal plasma Ca and Mg (P = 0.000 for all). For further clarification of these associations, the data was categorized in three groups of excellent (Ex, >90% motile, n = 33), good (Go, 80-89% motile, n = 15) and moderate (Mo, Keywords: