Influence of season and climatic variables on testicular cytology, semen quality and melatonin concentrations in crossbred bucks reared under subtropical climate.

Seasonality in reproduction and effects of climatic variables on testicular cytology and semen quality in bucks reared under subtropical climatic conditions were not well understood. In the present study, using testicular cytology, semen evaluation and melatonin concentrations assessed over a period of 1 year, we report that bucks reared under subtropical climatic conditions did not show seasonality in reproduction. Climatic variables including temperature, relative humidity, temperature-humidity index (THI), sunshine hours and day length were recorded daily during the whole period of experimentation (one complete year). Ejaculates were collected from crossbred (Alpine X Beetal) males (n = 6) biweekly using artificial vagina, and semen quality (volume, mass activity, sperm concentration, motility, viability, membrane integrity and protamine deficiency) was assessed. To understand the seasonal influence at testicular level, using fine needle aspiration biopsy method, testicular cells were aspirated and different types of cells and testicular cytology indices were quantified. Blood was collected biweekly for estimation of melatonin concentrations. Mass activity was higher (P < 0.05) during rainy season while individual sperm motility and sperm concentration were higher (P < 0.05) during rainy and autumn seasons as compared to other seasons. Sperm functional parameters did not show any differences during different seasons. Sertoli cell count, spermatogenic cell count and testicular indices did not differ among the seasons. Melatonin concentrations also did not differ significantly among the four seasons studied. Among the climatic parameters, THI had significant (P < 0.05) influence on sperm quality. The proportion of Sertoli cell in the testicular cytology had a significant and positive relationship with RH, THI and day length. It was concluded that seasonal variations are less evident in terms of spermatogenesis and semen quality in Alpine X Beetal crossbred bucks reared under subtropical climatic conditions.

Identification of suitable combinations of in vitro sperm-function test for the prediction of fertility in buffalo bull.

The present study assessed sperm functional characteristics in the frozen-thawed semen of buffalo bulls and estimated their relationship with field fertility. Frozen semen samples from three different freezing operations each from nine Murrah buffalo bulls were used for the assessment of different sperm functions related to fertilizing potential. Bulls were classified into high (n = 2), medium (n = 5), and low (n = 2) fertile based on adjusted field fertility. The sperm functions estimated included membrane integrity using carboxyfluorescein diacetate-propidium iodide, acrosome reaction status using fluorescein isothiocyanate peanut agglutinine, status of apoptosis using Annexin-V, protamine deficiency using Chromomycin A3, membrane stability using Merocyanine 540 and lipid peroxidation status using 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene. The relationship between the proportion of live acrosome-intact spermatozoa and fertility was positive and significant (r = 0.59; P = 0.001). The proportion of moribund spermatozoa showed a significantly negative correlation with fertility (r = -0.50; P = 0.008). Similarly, the relationship of spermatozoa with unstable membrane (r = -0.51; P = 0.007), necrotic (r = - 0.42; P = 0.028), early necrotic (r = -0.42; P = 0.031), and apoptotic spermatozoa (r = -0.39; P = 0.046) with bull fertility was negative and significant. The correlation between the protamine-deficient spermatozoa and fertility was negative, but not significant. Among different combinations of tests, live acrosome-intact spermatozoa and lipid peroxidation status of spermatozoa revealed high positive correlation with buffalo bull fertility (adjusted R2 = 0.73, C[p] = 0.80). These preliminary findings may help in developing tools for assessing fertility of buffalo bulls, once validated in more animals.

Morphometric evaluation of seminiferous tubule and proportionate numerical analysis of Sertoli and spermatogenic cells indicate differences between crossbred and purebred bulls.

AIM: The present study compared the testicular cytology and histology between crossbred (Holstein-Friesian [HF] × Tharparkar) and purebred (HF and Tharparkar) bulls to find out differences if any. MATERIALS AND METHODS: Four peripubertal bulls from each breed were utilized for the study. Through percutaneous needle aspiration biopsy, Sertoli and spermatogenic cells were extracted, and morphometry was studied. For histological studies, testicular tissues obtained through unilateral castration were utilized. Sertoli cells specific GATA4 antibody was used to study the population of Sertoli cells in the seminiferous tubule through immunofluorescence. RESULTS: The testicular weight, volume, and scrotal circumference differed significantly among the breeds. The diameter and area of the seminiferous tubule was high in HF, followed by Karan Fries (KF), and Tharparkar bulls. However, the degree of compactness, based on qualitative evaluation, was high in Tharparkar followed by KF and HF bulls. The intensity of Leydig cells was higher in Tharparkar bulls followed by KF and HF. The proportion of Sertoli cells was higher (p<0.05) in HF and Tharparkar bulls compared to KF bulls. CONCLUSION: It may be concluded that variations exist in testicular components of the breeds studied and the proportion of Sertoli cells in relation to spermatogenic cells was significantly lower in crossbred bulls compared to purebred bulls.

Differential proteomic profile of spermatogenic and Sertoli cells from peri-pubertal testes of three different bovine breeds.

Sub-fertility is one of the most common problems observed in crossbred males, but the etiology remains unknown in most of the cases. Although proteomic differences in the spermatozoa and seminal plasma between breeds have been investigated, the possible differences at the sperm precursor cells and supporting/nourishing cells have not been studied. The present study reports the differential proteomic profile of spermatogenic and Sertoli cells in crossbred and purebred bulls. Testis was removed by unilateral castration of 12 peri-pubertal bulls (10 months age), four each from crossbred (Holstein Friesian × Tharparkar), exotic purebred [Holstein Friesian (HF)] and indigenous purebred [Tharparkar (TP)] bulls. Spermatogenic and Sertoli cells were isolated and subjected to proteomic analysis. Protein extracts from the Sertoli and spermatogenic cells of each breed were analyzed with 2-dimensional difference gel electrophoresis (2D-DIGE) and analyzed with Decyder™ software. Compared to HF, 26 protein spots were over expressed and 14 protein spots were under expressed in spermatogenic cells of crossbred bulls. Similarly, 7 protein spots were over expressed and 15 protein spots were under expressed in the spermatogenic cells of TP bulls compared to that of crossbred bulls. Out of 12 selected protein spots identified through mass spectrometry, Phosphatidyl ethanolamine binding protein was found to be over expressed in the spermatogenic cells of crossbred bulls compared to TP bulls. The protein, gamma actin was found to be over expressed in the Sertoli cells of HF bulls, whereas Speedy Protein-A was found to be over expressed in Sertoli cells of crossbred bulls. It may be concluded that certain proteomic level differences exist in sperm precursor cells and nourishing cells between breeds, which might be associated with differences in the fertility among these breeds.