Deep Metabolomic Profiling Reveals Alterations in Fatty Acid Synthesis and Ketone Body Degradations in Spermatozoa and Seminal Plasma of Astheno-Oligozoospermic Bulls.

Male fertility is extremely important in dairy animals because semen from a single bull is used to inseminate several thousand females. Asthenozoospermia (reduced sperm motility) and oligozoospermia (reduced sperm concentration) are the two important reasons cited for idiopathic infertility in crossbred bulls; however, the etiology remains elusive. In this study, using a non-targeted liquid chromatography with tandem mass spectrometry-based approach, we carried out a deep metabolomic analysis of spermatozoa and seminal plasma derived from normozoospermic and astheno-oligozoospermic bulls. Using bioinformatics tools, alterations in metabolites and metabolic pathways between normozoospermia and astheno-oligozoospermia were elucidated. A total of 299 and 167 metabolites in spermatozoa and 183 and 147 metabolites in seminal plasma were detected in astheno-oligozoospermic and normozoospermic bulls, respectively. Among the mapped metabolites, 75 sperm metabolites were common to both the groups, whereas 166 and 50 sperm metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Similarly, 86 metabolites were common to both the groups, whereas 45 and 37 seminal plasma metabolites were unique to astheno-oligozoospermic and normozoospermic bulls, respectively. Among the differentially expressed metabolites, 62 sperm metabolites and 56 seminal plasma metabolites were significantly dysregulated in astheno-oligozoospermic bulls. In spermatozoa, selenocysteine, deoxyuridine triphosphate, and nitroprusside showed significant enrichment in astheno-oligozoospermic bulls. In seminal plasma, malonic acid, 5-diphosphoinositol pentakisphosphate, D-cysteine, and nicotinamide adenine dinucleotide phosphate were significantly upregulated, whereas tetradecanoyl-CoA was significantly downregulated in the astheno-oligozoospermia. Spermatozoa from astheno-oligozoospermic bulls showed alterations in the metabolism of fatty acid and fatty acid elongation in mitochondria pathways, whereas seminal plasma from astheno-oligozoospermic bulls showed alterations in synthesis and degradation of ketone bodies, pyruvate metabolism, and inositol phosphate metabolism pathways. The present study revealed vital information related to semen metabolomic differences between astheno-oligozoospermic and normospermic crossbred breeding bulls. It is inferred that fatty acid synthesis and ketone body degradations are altered in the spermatozoa and seminal plasma of astheno-oligozoospermic crossbred bulls. These results open up new avenues for further research, and current findings can be applied for the modulation of identified pathways to restore sperm motility and concentration in astheno-oligozoospermic 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 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.