Proteomic signatures of sperm and extracellular vesicles associated with sperm freezability in Holstein bulls

Semen cryopreservation and artificial insemination have crucial and beneficial effects on cattle breeding. The freezability of sperm, as primarily reflected by post-thaw sperm motility (PTM), is essential for evaluating semen quality. Some studies have shown notable differences in sperm freezability among various bulls. Here, we compared protein profiles of sperm cells and seminal plasma extracellular vesicles (SPEVs) in the high sperm freezability group and the low sperm freezability group of Holstein bulls to identify the important proteins and their mechanisms that affect sperm freezability. As a result, 432 and 394 differentially expressed proteins (DEPs) were identified in sperm and SPEVs between high and low freezability groups. The results of weighted correlation network analysis (WGCNA) showed that the blue module was significantly ( r = 0.89, P = 9 × 10 − 6 ) associated with sperm freezability. In addition, the pathway analysis revealed that “Metabolic pathways” and “Oxidative phosphorylation” were the predominant biological processes represented. Furthermore, 17 DEPs were found located in the previously identified QTLs related to post-thaw sperm motility, indicating possible variation in their genes. Interestingly, the expression of 142 protein pairs in sperm were significantly (|r| >0.9, P < 0.05) correlated with their expression in SPEVs. Finally, we detected genetic variations in six important candidate genes ( STK38 , HSPA1A , HSP90B1 , LPO , DNASE2 and CUTA ), and found that a missense mutation (Chr23g. 23:27522566 A > G) in HSPA1A may affect sperm freezability by decreasing the expression of HSPA1A. Our study highlighted the different protein characteristics of sperm and SPEVs in samples with distinct sperm freezability. These proteins, together with relevant SNPs might be useful markers for selecting bulls with high sperm freezability.