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.

The cryopreservation process induces alterations in proteins associated with bull sperm quality: The equilibration process could be a probable critical control point.

The present study quantitatively characterized the proteomic changes in bull spermatozoa induced by the cryopreservation process. We performed high-throughput comparative global proteomic profiling of freshly ejaculated (before cryopreservation), equilibrated (refrigerated storage; during cryopreservation), and frozen (ultralow temperature; after cryopreservation) bull spermatozoa. Using the liquid chromatography-mass spectrometry (LC-MS/MS) technique, a total of 1,692, 1,415, and 1,286 proteins were identified in fresh, equilibrated, and cryopreserved spermatozoa, respectively. When the proteome of fresh spermatozoa was compared with equilibrated spermatozoa, we found that 166 proteins were differentially expressed. When equilibrated spermatozoa were compared with cryopreserved spermatozoa, we found that 147 proteins were differentially expressed between them. Similarly, we found that 156 proteins were differentially expressed between fresh and cryopreserved spermatozoa. Among these proteins, the abundance of 105 proteins was lowered during the equilibration process itself, while the abundance of 43 proteins was lowered during ultralow temperature preservation. Remarkably, the equilibration process lowered the abundance of sperm proteins involved in energy metabolism, structural integrity, and DNA repair and increased the abundance of proteins associated with proteolysis and protein degradation. The abundance of sperm proteins associated with metabolism, cGMP-PKG (cyclic guanosine 3',5'-monophosphate-dependent protein kinase G) signaling, and regulation of the actin cytoskeleton was also altered during the equilibration process. Collectively, the present study showed that the equilibration step in the bull sperm cryopreservation process was the critical point for sperm proteome, during which a majority of proteomic alterations in sperm occurred. These findings are valuable for developing efficient protocols to minimize protein damage and to improve the quality and fertility of cryopreserved bull spermatozoa.

Genom-wide analysis identifies single nucleotide polymorphism variations and altered pathways associated with poor semen quality in breeding bulls.

The reason for poor semen quality among the breeding bulls is not well understood. In the present study, we performed high-throughput RNAseq analysis of spermatozoa to identify the SNPs present in good and poor-quality semen-producing Holstein Friesian breeding bulls. A total of 21,360 and 44,650 SNPs were identified in good and poor-quality semen with a minimum read depth of 20, among which 4780 and 8710 novel variants were observed in good and poor-quality semen, respectively. Greater SNPs and indels variations were observed in poor compared to good-quality semen. In poor-quality semen, SNP variations were observed in ZNF280B, SLC26A2, DMXL1, OR52A1, MACROD2 and REV1 genes, which are associated with regulation of spermatogenesis, post-testicular maturation, Cl- channel activity, V-ATPase-mediated intracellular vesicle acidification, a mono-ADP-ribosyl hydrolase and ATR-Chk1 checkpoint activation. GO analysis of filtered genes with significant variations between good and poor-quality semen showed enrichment in important pathways related to semen quality such as MAPK signalling pathway, Akt signalling pathway, focal adhesion, cAMP signalling pathway, and Rap1 signalling pathway. Network analysis of filtered genes in poor-quality semen showed variations in pathways of purine metabolism, pyrimidine metabolism, prolactin signalling pathway and RNA cap-binding complex. It is inferred that SNP in genes involved in maintaining sperm functions could be the reason for poor-quality semen production in bulls, and the identified SNPs hold potential to be used as biomarkers for semen quality in bulls.

Sperm Transcripts Associated With Odorant Binding and Olfactory Transduction Pathways Are Altered in Breeding Bulls Producing Poor-Quality Semen.

Spermatozoa carries a reservoir of mRNAs regulating sperm functions and fertilizing potential. Although it is well recognized that a considerable proportion of high genetic merit breeding bulls produce poor-quality semen, the transcriptomic alterations in spermatozoa from such bulls are not understood. In the present study, comparative high-throughput transcriptomic profiling of spermatozoa from good and poor-quality semen-producing bulls was carried out to identify the transcripts associated with semen quality. Using next-generation sequencing (NGS), we identified 11,632 transcripts in Holstein Friesian bull spermatozoa; after total hit normalization, a total of 544 transcripts were detected, of which 185 transcripts were common to both good and poor-quality semen, while 181 sperm transcripts were unique to good quality semen, and 178 transcripts were unique to poor-quality semen. Among the co-expressed transcripts, 31 were upregulated, while 108 were downregulated, and 46 were neutrally expressed in poor-quality semen. Bioinformatics analysis revealed that the dysregulated transcripts were predominantly involved in molecular function, such as olfactory receptor activity and odor binding, and in biological process, such as detection of chemical stimulus involved in sensory perception, sensory perception of smell, signal transduction, and signal synaptic transmission. Since a majority of the dysregulated transcripts were involved in the olfactory pathway (85% of enriched dysregulated genes were involved in this pathway), the expression of selected five transcripts associated with this pathway (OR2T11, OR10S1, ORIL3, OR5M11, and PRRX1) were validated using real-time qPCR, and it was found that their transcriptional abundance followed the same trend as observed in NGS; the sperm transcriptional abundance of OR2T11 and OR10S1 differed significantly (p < 0.05) between good and poor-quality semen. It is concluded that poor-quality semen showed altered expression of transcripts associated with olfactory receptors and pathways indicating the relationship between olfactory pathway and semen quality in bulls.

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.

The proportion of tyrosine phosphorylated spermatozoa in cryopreserved semen is negatively related to crossbred bull fertility.

Sub-fertility is a major problem in crossbred bulls. Identification of subtle differences in the quality of cryopreserved spermatozoa among bulls belonging to different fertility rankings would help determine the latent fertility of semen before their use at field conditions. In the present study, we assessed the status of tyrosine phosphorylation, membrane integrity and acrosome reaction of cryopreserved spermatozoa in crossbred bulls (n = 22) with different levels of field fertility and assessed their relationship with fertility. Bulls were categorized into above-average (n = 4), average (n = 14) and below-average (n = 4) based on their different field fertility rates. The progressive sperm motility was significantly (P < 0.05) higher in above-average fertile bulls compared to either average or below-average fertile bulls whereas sperm membrane integrity and acrosomal reaction status did not differ among the three groups. The proportion of live tyrosine-phosphorylated spermatozoa were significantly (P < 0.05) higher in below-average and average fertile bulls compared to above-average bulls. Immunolocalization of protein tyrosine phosphorylation in spermatozoa revealed that the proportion of spermatozoa showing tyrosine phosphorylation at acrosome and post-acrosomal area (APA) and at acrosome, post-acrosome and tail (APAT) were significantly (P < 0.05) higher in below-average fertile bulls than other groups. The APA pattern (r = -0.605; P < 0.01) and APAT (r = 0.507; P < 0.05) pattern were significantly and negatively correlated with bull fertility. It was concluded that the proportion of live tyrosine-phosphorylated spermatozoa in cryopreserved semen was negatively related to bull fertility.