Proteomic analysis reveals the sperm protein difference among low, high quality and frozen semen of Sewa sheep in Tibet

The Tibetan Sewa sheep is a unique breed that inhabits the plateau area at an altitude of approximately 4,800 m. In this study, we conducted DIA quantitative proteomic analysis on high-quality semen (F), frozen semen (C), and low-quality semen (L) from Sewa sheep. A total of 33,073 peptide segments and 3,175 proteins were detected in the three groups of semen. Among them, 3,700 proteins were functionally annotated in the database. Comparison between the C and F groups revealed 163 differentially expressed proteins (DEPs) with 134 upregulated and 29 downregulated proteins. These DEPs were found to be associated with biological regulation and metabolic processes in Gene Ontology (GO) enrichment analysis; they also mapped to 97 pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Comparison between the L and F groups identified 459 DEPs with 319 upregulated and 140 downregulated proteins. These DEPs were found to be associated with metabolic processes and cellular component biogenesis in GO enrichment analysis; they also mapped to 107 pathways in KEGG enrichment analysis. Protein-protein interaction (PPI) analysis based on comparisons between C vs F group and L vs F group identified potential proteins that may affect sperm cryotolerance and sperm vitality; however, their specific functional mechanisms require further investigation. We have elucidated for the first time the differentially expressed proteins in various types of semen from Sewa sheep as well as their involvement in biological pathways, providing a theoretical basis for understanding sperm quality and freezing damage mechanisms in Sewa sheep.