Project description:The incidence of sub-fertility is higher in crossbred bulls compared to zebu bulls. In the present study, we analysed the metabolomic profile of seminal plasma from crossbred and zebu bulls and uncovered differentially expressed metabolites between these two breeds. Using a high-throughput LC-MS/MS-based approach, we identified 990 and 1,002 metabolites in crossbred and zebu bull seminal plasma respectively. After excluding the exogenous metabolites, we found that 50 and 68 putative metabolites were unique to crossbred and zebu bull seminal plasma, respectively, whilst 87 metabolites were common to both. After data normalisation, 63 metabolites were found to be dysregulated between crossbred and zebu bull seminal plasma. Observed pathways included Linoleic acid metabolism (observed metabolite was phosphatidylcholine) in crossbred bull seminal plasma whereas inositol phosphate metabolism (observed metabolites were phosphatidylinositol-3,4,5-trisphosphate/inositol 1,3,4,5,6-pentakisphosphate/myo-inositol hexakisphosphate) was observed in zebu bull seminal plasma. Abundance of Tetradecanoyl-CoA was significantly higher, whilst abundance of Taurine was significantly lower in crossbred bull seminal plasma. In conclusion, the present study established the seminal plasma metabolomic profile in crossbred and zebu bulls and suggest that increased lipid peroxidation coupled with low concentrations of antioxidants in seminal plasma might be associated with high incidence of sub-fertility in crossbred bulls.
Project description:To understand the etiology behind higher incidence of infertility in crossbred bulls, we performed transcriptomic analysis of testicular samples derived from crossbred males and compared with testicular transcriptomic profile of Zebu cattle
Project description:Abstract Buffalo bulls are the backbone of Indian dairy industry, and the quality of semen donating bulls determine the overall production efficiency of buffalo dairy farms. Seminal plasma (SP) of bulls harbor millions of lipid bilayer nanovesicles known as extracellular vesicles (EVs). These EVs carry a heterogenous cargo of essential biomolecules including fertility associated proteins which contribute to the fertilizing potential of spermatozoa. In this study, we explored various parameters of EVs such as size, concentration and the complete proteome profiles of SP EVs from two distinct fertility groups in order to unravel the differentially abundant proteins that potentially affect the overall fertility of bulls. Through Dynamic Light Scattering (DLS) it was found that the purified EVs were present in 7 to 14 size exclusion chromatographic (SEC) fractions with sizes ranging from 145 to 256 nm in the high fertile (HF) and low fertile (LF) bulls. Nanoparticle Tracking Analysis (NTA) confirmed the size of seminal EVs up to 200 nm, and concentrations varying from 2.84 to 6.82 × 1011 and 3.57 to 7.74 × 1011 particles per ml in HF and LF bulls, respectively across all the fractions. No significant difference was observed in the size and concentration of seminal EVs between the HF and LF groups. We identified a total of 1862 and 1807 proteins in seminal EVs of HF and LF bulls, respectively using high throughput LC-MS/MS approach. Out of these total proteins, 1754 proteins were common in both the groups and about 87 proteins were highly abundant in HF group while 1292 were less abundant as compared to LF bulls. Gene ontology (GO) analysis, revealed that the highly abundant proteins in HF group were mainly part of the nucleus and involved in nucleosome assembly along with DNA binding. In addition, highly abundant proteins in EVs of HF group were found to be involved in spermatogenesis, motility, acrosome reaction, capacitation, gamete fusion, and cryotolerance. Two highly abundant proteins of HF EVs, namely protein disulfide-isomerase A4 (PDIA4) and gelsolin (GSN), were successfully immunolocalized on spermatozoa, indicating that these proteins might be transferred to spermatozoa through EVs. The proteins; PDIA4 and GSN are intricately associated with sperm-oocyte fusion and acrosome reaction, respectively, thus they are vital for regulating the fertilizing capacity of sperm. Our evidences clearly support that the protein repertoires in EVs and subsequently their presence on sperm, are strongly associated with sperm functions. Altogether, the current investigation clearly indicates that SPEVs possess crucial protein repertoires which are essential for enhancing the sperm fertilizing capacity.
Project description:The aim of this study was to profile the transcriptome and perform histological analysis of the bovine uterus in response to sperm from high fertility (HF) and low fertility (LF) bulls
Project description:Crossbred bulls produced by crossing Bos taurus and Bos indicus suffer with high incidence of infertility/subfertility problems; however, the etiology remains poorly understood. The uncertain predictability and the inability of semen evaluation techniques to maintain constant correlation with fertility demand for alternate methods for bull fertility prediction. Therefore, in this study, the global differential gene expression between high- and low-fertile crossbred bull sperm was assessed using a high-throughput RNA sequencing technique with the aim to identify transcripts associated with crossbred bull fertility. Crossbred bull sperm contained transcripts for 13,563 genes, in which 2,093 were unique to high-fertile and 5,454 were unique to low-fertile bulls. After normalization of data, a total of 776 transcripts were detected, in which 84 and 168 transcripts were unique to high-fertile and low-fertile bulls, respectively. A total of 176 transcripts were upregulated (fold change > 1) and 209 were downregulated (<1) in low-fertile bulls. Gene ontology analysis identified that the sperm transcripts involved in the oxidative phosphorylation pathway and biological process such as multicellular organism development, spermatogenesis, and in utero embryonic development were downregulated in low-fertile crossbred bull sperm. Sperm transcripts upregulated and unique to low-fertile bulls were majorly involved in translation (biological process) and ribosomal pathway. With the use of RT-qPCR, selected sperm transcripts (n = 12) were validated in crossbred bulls (n = 12) with different fertility ratings and found that the transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes was significantly (p < 0.05) lower in low-fertile bulls than high-fertile bulls and was positively (p < 0.05) correlated with conception rate. It is inferred that impaired oxidative phosphorylation could be the predominant reason for low fertility in crossbred bulls and that transcriptional abundance of ZNF706, CRISP2, TNP2, and TNP1 genes could serve as potential biomarkers for fertility in crossbred bulls.
Project description:Abstract The water buffalo (Bubalus bubalis) is an indispensable part of the Indian dairy sector and in several instances, the farmers incur economic losses due to failed pregnancy after artificial insemination (AI). One of the key factors for the failure of conception is the use of semen from the bulls of low fertilizing potential and hence, it becomes important to predict the fertility status before performing AI. In this study, the global proteomic profile of high fertile (HF) and low fertile (LF) buffalo bull spermatozoa was established using a high-throughput LC-MS/MS technique. A total of 1385 proteins (≥ 1 high-quality PSM/s, ≥ 1 unique peptides, P < 0.05, FDR < 0.01) were identified out of which, 1002 were common between both the HF and LF groups while 288 and 95 proteins were unique to HF and LF groups respectively. We observed 211 and 342 significantly upregulated (log Fc ≥2) and downregulated in HF (log Fc ≤0.5) spermatozoa (p <0.05). Gene ontology analysis revealed that the fertility associated upregulated proteins were involved in spermatogenesis, sperm motility, acrosome integrity, zona pellucida binding and other associated sperm functions. Besides this, the downregulated proteins were involved in glycolysis, fatty acid degradation and inflammation. Furthermore, fertility related differentially abundant proteins (DAPs) on sperm viz., AKAP3, Sp17 and DLD were validated through Western blotting and immunocytochemistry which was in coherence with the LC-MS/MS data. The DAPs identified in this study may be used as potential protein candidates for predicting fertility in buffaloes. Our findings provide an opportunity in mitigating the economic losses that farmers incur due to male infertility.
Project description:The objective of the study was to identify the fertility-associated metabolites in bovine spermatozoa using liquid chromatography-mass spectrometry (LC-MS). Six Holstein Friesian crossbred bulls (three high-fertile and three low-fertile bulls) were the experimental animals. Sperm proteins were isolated and protein-normalized samples were processed for metabolite extraction and subjected to LC-MS/MS analysis. Mass spectrometry data were processed using iMETQ software and metabolites were identified using Human Metabolome DataBase while, Metaboanalyst 4.0 tool was used for statistical and pathway analysis. A total of 3,704 metabolites belonging to various chemical classes were identified in bull spermatozoa. After sorting out exogenous metabolites, 56 metabolites were observed common to both the groups while 44 and 35 metabolites were found unique to high- and low-fertile spermatozoa, respectively. Among the common metabolites, concentrations of 19 metabolites were higher in high-fertile compared to low-fertile spermatozoa (fold change > 1.00). Spermatozoa metabolites with variable importance in projections score of more than 1.5 included hypotaurine, d-cysteine, selenocystine. In addition, metabolites such as spermine and l-cysteine were identified exclusively in high-fertile spermatozoa. Collectively, the present study established the metabolic profile of bovine spermatozoa and identified the metabolomic differences between spermatozoa from high- and low-fertile bulls. Among the sperm metabolites, hypotaurine, selenocysteine, l-malic acid, d-cysteine, and chondroitin 4-sulfate hold the potential to be recognized as fertility-associated metabolites.