M.K. Muhammad Aslam

Identification of putative fertility markers in seminal plasma of crossbred bulls through differential proteomics

Sub-fertility is a major problem in crossbred bulls leading to disintegration of breeding systems and huge economic loss. Identification of some potential biomarkers to determine the latent fertility of bulls accurately has long been the interest of researchers. In this study, we analyzed the proteome of seminal plasma (SP) from bulls with varying fertility to identify the fertility-associated proteins. The proteomic profile of high- and low-fertile bulls was compared by two-dimensional difference gel electrophoresis and differentially expressed proteins were identified through matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. Out of the 18 differentially expressed proteins (P < 0.05), 9 were overexpressed in SP of high-fertile bulls and 9 were overexpressed in SP of low-fertile bulls. The differential expressions ranged from 1.5- to 5.5-fold between the two groups, where protection of telomeres-1 protein (POT1) was highly overexpressed (2.9-fold) in high-fertile group and prostaglandin E2 receptor EP3 (PTGER3) was highly abundant (5.5-fold) in low-fertile group. The protein interaction network was elucidated using STRING software tool, and the functional bioinformatics analysis was done using Blast2Go software. Most of the differentially expressed proteins were found to be involved in cellular processes and biological regulation with binding and catalytic function. It is inferred that the expression of certain proteins in the SP varied with bull fertility, and concurrent appraisal of their expression along with other fertility assays may help in determining bull fertility.

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.

Testicular Cell Indices and Peripheral Blood Testosterone Concentrations in Relation to Age and Semen Quality in Crossbred (Holstein Friesian×Tharparkar) Bulls

Present study analyzed the changes in peripheral blood testosterone concentrations and testicular cytogram in relation to age and semen quality in crossbred males. Three different age groups of crossbred males viz. bull calves (6 months, n = 5), young bulls (15 months, n = 5) and adult bulls (4 to 6 years, n = 8) were utilized for the study. Testicular fine needle aspiration cytology technique was used to quantify testicular cytology and their indices. Peripheral blood testosterone concentrations were measured using enzyme-linked immunosorbent assay method. Semen samples collected from adult bulls were microscopically evaluated for quality parameters. Mean peripheral blood testosterone concentrations in bull calves, young bulls and adult bulls were 2.28±0.09 ng/mL, 1.42±0.22 ng/mL and 5.66±1.08 ng/mL respectively, and that in adult bulls were significantly different (p<0.01) from young bulls and bull calves. There was no significant difference between the proportion of different testicular cells in bull calves and young bulls. Between young and adult bulls, significant differences (p<0.01) were observed in the proportion of spermatocytes, spermatozoa, and sperm: Sertoli cell ratio. The proportions of Sertoli cells showed a significant difference (p<0.01) between the three age groups. The number of primary spermatocytes had a positive correlation with peripheral blood testosterone concentrations in bull calves (r = 0.719, p<0.01). Number of Sertoli cells per 100 germ cells was negatively correlated with blood testosterone concentration in young bulls (r = −0.713, p<0.01). Among different semen parameters in adult bulls, ejaculate volume (r = 0.790, p<0.05) had positive relationship, and sperm motility had significant negative correlation (r = −0.711, p<0.05) with testosterone concentrations. The number of Sertoli cells and Sertoli cell index had a positive correlation with various semen quality parameters (p<0.001). Results of the present study conclude that number of Sertoli cells and Sertoli cell index are good indicators of semen quality, but peripheral blood testosterone concentrations may not have a direct relationship with various seminal attributes in crossbred bulls.

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.

Identification of biomarker candidates for fertility in spermatozoa of crossbred bulls through comparative proteomics

Associations between expression of some proteins in spermatozoa and fertility have been sought in recent years to identify the male fertility markers. Since the incidence of sub-fertility is high in crossbred bulls, the present investigation was carried out on high- and low-fertile crossbred bulls to identify fertility markers in spermatozoa through proteomics approach. Sperm proteome of high-fertile bulls were compared with low-fertile bulls using 2D-DIGE and MALDI-TOF-MS techniques and the results were validated with immuno-blotting. The proteins MDH2, ENO1, RIBC1, CAPN7, ATP5D, LacA like protein-2 like, NCAPD3, DECR1, GCNT2, GDI2, TOP and USP12 were over expressed in high-fertile spermatozoa, whereas DST like isoform 1, TMEM43 and BSP1 were over expressed in low-fertile spermatozoa (P < 0.05). The differential expression ranged from 1.57 (GDI2) to 5.1 (BSP1) fold between the two groups. Based on the GO annotation, majority of them were involved in cellular and metabolic processes, with catalytic and binding activities, and localized in cell and organelles. Among these proteins, ENO1 and BSP1 were selected based on the degree of differential expression and reliability in identification, for further validation. Immuno-blotting studies indicated that ENO1expression was positively correlated (P < 0.05) while the expression of BSP1 was negatively (P < 0.01) correlated with bull fertility. The proportion of capacitated spermatozoa in frozen thawed spermatozoa of low-fertile bulls was higher (P < 0.05) as compared to high-fertile bulls. Collectively, the study identified some potential molecules in spermatozoa of bulls, which may act as a panel of biomarkers for fertility.

Effect of functional traits on subsequent reproduction performance of Murrah buffaloes in India

ABSTRACT A study was conducted on calving records of Murrah buffaloes to study the influence of various functional traits on subsequent reproductive performance. The effects of calving abnormalities, uterine health problems, female fertility problems and udder health problem on calving to first service (CFS), service period (SP), dry period (DP) and calving interval (CI) were studied by the least-squares method using the SAS package. The increase in CFS, SP, DP and CI was 27.07%, 18.71%, 96.74% and 6.70%, respectively. Subsequently, the average milk yield per day of calving interval was found to be reduced (from 4.91 to 2.10 kg/day) in these animals in comparison with the normal calvers. A highly significant increase in average CFS, SP, DP and CI was found among animals susceptible to metritis (16.93%, 73.88%, 53.92% and 20.04%, respectively). The decrease in average milk yield per day of calving interval (MY/CI) was 18.55% in comparison with the animals not affected by metritis. The average MY/CI was observed to be 16.47% lower in the anoestrus-affected animals in comparison with normal animals. The increase in average SP, DP and CI associated with repeat breeding cases was 162.63%, 87.58% and 50.15%, respectively. The increase in average DP and CI due to incidence of mastitis was 10.48% and 4.26%, respectively. The increase in average dry period associated with the incidence of mastitis during early stage of lactation (1–60 days) was 13.63%. The incidence of mastitis during early stage of lactation significantly reduced the average MY/CI by 5.50 and 14.20%, respectively.