Heterologous Seminal Plasma Reduces the Intracellular Calcium and Sperm Viability of Cryopreserved Stallion Spermatozoa.

Despite the vital role of seminal plasma (SP) in maintaining sperm function and aiding gamete interaction in many species, SP is usually removed before cryopreservation of stallion sperm to improve cryosurvival of sperm. The present study assessed if the vital sperm functional parameters of genetically superior stallions producing poor quality semen can be enhanced by the supplementation of heterologous SP from the stallion producing high quality semen. Spermatozoa from poor quality semen producing stallions were divided into three aliquots: two aliquots were supplemented with SP obtained from good quality semen producing stallions at the rate of 20% and 30%, respectively, whereas the third aliquot remained as control (0% SP) and incubated at 37°C for 30 minutes. Sperm membrane integrity, mitochondrial membrane potential (MMP), mitochondrial superoxide (mtROS) generation, and intracellular calcium status were assessed at different time intervals during incubation by flow cytometry. It was observed that the dead sperm population increased (p < 0.01) during incubation in both the 20% and 30% SP-supplemented groups. However, no significant changes were observed in MMP in both the control and treatment groups at different time intervals. Interestingly, it was found that sperm mtROS production increased (p < 0.01) during incubation in the SP-supplemented groups compared with the control group. The proportion of live spermatozoa with high intracellular calcium was reduced (p < 0.01) during incubation in the SP-incubated groups. Collectively, heterologous SP addition could not repair the damages caused by the cryopreservation and further resulted in deterioration of semen quality as observed in our study by reducing viability, increasing reactive oxygen species (ROS) production possibly due to high proportion of dead cells, or some factors (yet to be identified) that are inducive of oxidative stress in stallion spermatozoa.

Sperm proteomic landscape is altered in breeding bulls with greater sperm DNA fragmentation index.

Although, it is well understood that sperm DNA damage is associated with infertility, the molecular details of how damaged sperm DNA affects fertility are not fully elucidated. Since sperm proteins play an important role in fertilization and post-fertilization events, the present study aimed to identify the sperm proteomic alterations in bulls with high sperm DNA Fragmentation Index (DFI%). Semen from Holstein-Friesian crossbred breeding bulls (n = 50) was subjected to Sperm Chromatin Structure Assay. Based on DFI%, bulls were classified into either high- (HDFI; n = 6), or low-DFI (LDFI; n = 6) and their spermatozoa were subjected to high throughput proteomic analysis. Liquid chromatography and mass spectrometry analysis identified 4567 proteins in bull spermatozoa. A total of 2660 proteins were found common to both the groups, while 1193 and 714 proteins were unique to HDFI and LDFI group, respectively. A total of 265 proteins were up regulated and 262 proteins were down regulated in HDFI group. It was found that proteins involved in capacitation [heparin binding (molecular function), ERK1 and ERK2 cascade (biological process), PI3K-Akt signalling (pathway), Jak-STAT signalling (pathway)], spermatogenesis [TLR signalling (pathway), gamete generation (biological process)] and DNA repair mechanism (biological process) were significantly altered in the bulls with high DFI%.

Establishment of a repertoire of fertility associated sperm proteins and their differential abundance in buffalo bulls (Bubalus bubalis) with contrasting fertility.

Sperm harbours a wide range of proteins regulating their functions and fertility. In the present study, we made an effort to characterize and quantify the proteome of buffalo bull spermatozoa, and to identify fertility associated sperm proteins through comparative proteomics. Using high-throughput mass spectrometry platform, we identified 1305 proteins from buffalo spermatozoa and found that these proteins were mostly enriched in glycolytic process, mitochondrial respiratory chain, tricarboxylic acid cycle, protein folding, spermatogenesis, sperm motility and sperm binding to zona pellucida (p < 7.74E-08) besides metabolic (p = 4.42E-31) and reactive oxygen species (p = 1.81E-30) pathways. Differential proteomic analysis revealed that 844 proteins were commonly expressed in spermatozoa from both the groups while 77 and 52 proteins were exclusively expressed in high- and low-fertile bulls, respectively. In low-fertile bulls, 75 proteins were significantly (p < 0.05) upregulated and 176 proteins were significantly (p < 0.05) downregulated; these proteins were highly enriched in mitochondrial respiratory chain complex I assembly (p = 2.63E-07) and flagellated sperm motility (p = 7.02E-05) processes besides oxidative phosphorylation pathway (p = 6.61E-15). The down regulated proteins in low-fertile bulls were involved in sperm motility, metabolism, sperm-egg recognition and fertilization. These variations in the sperm proteome could be used as potential markers for the selection of buffalo bulls for fertility.

High-throughput proteomic characterization of seminal plasma from bulls with contrasting semen quality.

Seminal plasma proteins are the major extrinsic factors that can modulate the sperm quality and functions. The present study was carried out to compare the proteomic profiles of seminal plasma from breeding bulls producing good and poor quality semen in an effort to understand the possible proteins associated with semen quality. A total of 910 and 715 proteins were detected in the seminal plasma of poor and good quality semen producing bulls, respectively. A total of 705 proteins were common to both the groups, in which 380 proteins were upregulated and 89 proteins were downregulated in the seminal plasma of poor quality semen, while 236 proteins were co-expressed. The proteins negatively influencing sperm functions such as CCL2, UQCRC2, and SAA1 were among the top ten upregulated proteins in the seminal plasma of poor quality semen. Proteins having a positive role in sperm functions (NGF, EEF1A2, COL1A2, IZUMO4, PRSS1, COL1A1, WFDC2) were among the top ten downregulated proteins in the seminal plasma of poor quality semen. The upregulation of oxidation-reduction process-related proteins, histone proteins (HIST3H2A, H2AFJ, H2AFZ, H2AFX, HIST2H2AB, H2AFV, HIST1H2AC, HIST2H2AC, LOC104975684, LOC524236, LOC614970, LOC529277), and ubiquinol-cytochrome-c reductase proteins (UQCRB, UQCRFS1, UQCRQ, UQCRC1, UQCRC2) indicate deranged oxidation-reduction equilibrium, chromatin condensation and spermatogenesis in poor quality semen producing bulls. The expression of proteins essential for motile cilium (CCDC114, CFAP206, TEKT4), chromatin integrity (PRM2), gamete fusion (IZUMO4, EQTN), hyperactivation, tyrosine phosphorylation, and capacitation [PI3K-Akt signalling pathway-related proteins (COL1A1, COL2A1, COL1A2, SPP1, PDGFA, NGF)] were down regulated in poor quality semen producing bulls. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03474-6.

Single nucleotide polymorphisms cumulating to genetic variation for fertility in crossbred (Bos taurus × Bos indicus) bull spermatozoa.

Spermatozoa from high-fertile (HF) and low-fertile (LF) breeding bulls were subjected to high-throughput next-generation sequencing to identify important Single nucleotide polymorphisms (SNPs) and novel variants associated with fertility. A total of 77,038 genome-wide SNPs were identified, among which, 10,788 were novel variants. A total of 42,290 and 34,748 variants were recorded with 6115 and 4673 novel variants in in HF and LF bulls, respectively. Higher number of SNPs were identified in HF compared to LF bulls. GO analysis of filtered genes with significant variations in HF bulls indicated their involvement in oxidative phosphorylation and metabolic pathways. GO analysis of filtered genes with significant variation in LF bulls revealed their involvement in Ca2++ ion binding, structural constituent of ribosome, and biological processes like translation and ribosomal small subunit assembly. The study identified SNPs in candidate genes including TPT1, BOLA-DRA, CD74, RPS17, RPS28, RPS29, RPL14, RPL13, and RPS27A, which are linked to sperm functionality, survival, oxidative stress, and bull fertility. The identified SNPs could be used in selection of bulls for high fertility and the variation in these genes could be established as an explanation for the fertility differences in bulls upon validation in large number of bulls.

Integrated multi-omics analyses reveals molecules governing sperm metabolism potentially influence bull fertility.

Bull fertility is of paramount importance in bovine industry because semen from a single bull is used to breed several thousands of cows; however, so far, no reliable test is available for bull fertility prediction. In the present study, spermatozoa from high- and low-fertility bulls were subjected to high-throughput transcriptomic, proteomic and metabolomic analysis. Using an integrated multi-omics approach the molecular differences between high- and low-fertility bulls were identified. We identified a total of 18,068 transcripts, 5041 proteins and 3704 metabolites in bull spermatozoa, of which the expression of 4766 transcripts, 785 proteins and 33 metabolites were dysregulated between high- and low-fertility bulls. At transcript level, several genes involved in oxidative phosphorylation pathway were found to be downregulated, while at protein level genes involved in metabolic pathways were significantly downregulated in low-fertility bulls. We found that metabolites involved in Taurine and hypotaurine metabolism were significantly downregulated in low-fertility bulls. Integrated multi-omics analysis revealed the interaction of dysregulated transcripts, proteins and metabolites in major metabolic pathways, including Butanoate metabolism, Glycolysis and gluconeogenesis, Methionine and cysteine metabolism, Phosphatidyl inositol phosphate, pyrimidine metabolism and saturated fatty acid beta oxidation. These findings collectively indicate that molecules governing sperm metabolism potentially influence bull fertility.

High throughput deep proteomic analysis of seminal plasma from stallions with contrasting semen quality.

Seminal plasma proteins and pathways associated with sperm motility have not been elucidated in stallions. Therefore, in the current study, using the high throughput LC/MS-MS approach, we profiled stallion seminal plasma proteins and identified the proteins and pathways associated with sperm motility. Seminal plasma from six stallions producing semen with contrasting sperm motility (n = 3 each high-and low-motile group) was utilized for proteomic analysis. We identified a total of 1687 proteins in stallion seminal plasma, of which 1627 and 1496 proteins were expressed in high- (HM) and low- motile (LM) sperm of stallions, respectively. A total number of 1436 proteins were co-expressed in both the groups; 191 (11%) and 60 (3.5%) proteins were exclusively detected in HM and LM groups, respectively. A total of 220 proteins were upregulated (>1-fold change) and 386 proteins were downregulated in SP from LM group stallions as compared to HM group stallions, while 830 proteins were neutrally expressed in both the groups. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed dysregulation of the important proteins related to mitochondrial function, acrosome, and sperm cytoskeleton in the seminal plasma of stallions producing ejaculates with low sperm motility. High abundance of peroxiredoxins and low abundance of seminal Chaperonin Containing TCP1 Complex (CCT) complex and Annexins indicate dysregulated oxidative metabolism, which might be the underlying etiology for poor sperm motility in LM group stallions. In conclusion, the current study identified the seminal plasma proteomic alterations associated with poor sperm motility in stallions; the results indicate that poor sperm motility in stallions could be associated with altered expression of seminal plasma proteins involved in oxidative metabolism.

Deciphering metabolomic alterations in seminal plasma of crossbred (Bos taurus X Bos indicus) bulls through comparative deep metabolomic analysis.

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.

Epididymosomes: A potential male fertility influencer.

During transit and storage in epididymis, spermatozoa undergo final maturation, acquire motility, functional competence and the ability to fertilise an oocyte. Epididymal secretions contain a complex biochemical milieu of diverse inorganic ions, proteins, metabolites and other molecules. Since it is believed that spermatozoa are translationally silent, proteins appearing in them are thought to be synthesised elsewhere, including epididymis, and then incorporated to the cells. One of the important mechanisms suggested to be involved in transfer of epididymal secretions to spermatozoa is through exosomes called epididymosomes. Epididymosomes released from the epididymal epithelium contain proteins, noncoding RNAs and distinct set of lipids that are transferred to spermatozoa while they pass through the different epididymal regions. Owing to the importance of these molecules for sperm maturation and fertilising ability, research on epididymosomes has gained increasing attention during the last decade. This review is focused on epididymosomes, with emphasis on recent advances in the understanding of mechanisms of epididymosomal cargo transfer to spermatozoa and potential roles of epididymosomes in sperm function and beyond. Possibilities of utilising the molecular signatures of epididymosomes as a tool for male fertility assessment are also discussed.

Sperm phenotypic characteristics and oviduct binding ability are altered in breeding bulls with high sperm DNA fragmentation index.

In the present study, we standardized an in vitro oviduct explants model for cattle and assessed the oviduct explants binding ability and phenotypic characteristics of spermatozoa obtained from breeding bulls with high- and low-sperm DNA fragmentation index (DFI%). Cryopreserved spermatozoa from Holstein Friesian crossbred breeding bulls (n = 45) with known field fertility were assessed for DFI% and were classified into either high DFI% or low DFI% category. Flow cytometry was used to assess sperm membrane integrity, acrosome reaction status, mitochondrial membrane potential and intracellular calcium concentrations. It was found that spermatozoa from bulls with low DFI% had significantly higher (P < 0.05) membrane integrity, acrosome intactness, and mitochondrial membrane potential. To assess the sperm oviduct binding ability, oviduct explants were prepared by incubating the oviduct cells overnight in TCM-199 medium at 38.5 °C under 5% CO2. Different sperm concentrations and times of incubation were evaluated and found that 2 million spermatozoa and 1-h incubation yielded high binding index (BI). The BI was also significantly (P < 0.01) higher (>2 times) in the bulls with low-DFI% as compared to high DFI% bulls. The correlation between binding index and DFI% was negative and significant (r = -0.528; P < 0.05). Further, the binding index was positively correlated with conception rate (r = 0.703), intact sperm membrane (r = 0.631) and mitochondrial membrane potential (r = 0.609). It is inferred that sperm phenotypic characteristics and oviduct binding ability are impaired in breeding bulls with high sperm DFI%, which might be associated with low conception rates in these bulls.

Transcriptomic Profiling of Buffalo Spermatozoa Reveals Dysregulation of Functionally Relevant mRNAs in Low-Fertile Bulls.

Although, it is known that spermatozoa harbor a variety of RNAs that may influence embryonic development, little is understood about sperm transcriptomic differences in relation to fertility, especially in buffaloes. In the present study, we compared the differences in sperm functional attributes and transcriptomic profile between high- and low-fertile buffalo bulls. Sperm membrane and acrosomal integrity were lower (P < 0.05), while protamine deficiency and lipid peroxidation were higher (P < 0.05) in low- compared to high-fertile bulls. Transcriptomic analysis using mRNA microarray technology detected a total of 51,282 transcripts in buffalo spermatozoa, of which 4,050 transcripts were differentially expressed, and 709 transcripts were found to be significantly dysregulated (P < 0.05 and fold change >1) between high- and low-fertile bulls. Majority of the dysregulated transcripts were related to binding activity, transcription, translation, and metabolic processes with primary localization in the cell nucleus, nucleoplasm, and in cytosol. Pathways related to MAPK signaling, ribosome pathway, and oxidative phosphorylation were dysregulated in low-fertile bull spermatozoa. Using bioinformatics analysis, we observed that several genes related to sperm functional attributes were significantly downregulated in low-fertile bull spermatozoa. Validation of the results of microarray analysis was carried out using real-time qPCR expression analysis of selected genes (YBX1, ORAI3, and TFAP2C). The relative expression of these genes followed the same trend in both the techniques. Collectively, this is the first study to report the transcriptomic profile of buffalo spermatozoa and to demonstrate the dysregulation of functionally relevant transcripts in low-fertile bull spermatozoa. The results of the present study open up new avenues for understanding the etiology for poor fertility in buffalo bulls and to identify fertility biomarkers.