Proteomic profiling of sperm with differential freezability in Wannan Black pig

Sperm freezability varies considerably among pigs owing to genetic, husbandry, and management differences. Boar sperm cryopreservation remains underutilized owing to its inconsistent freezability. Semen with good freezability (GF) increases sow litter size and reduces breeding costs. Previous studies identified freezability biomarkers in cattle, sheep, and pigs. To comprehensively investigate these biomarkers, we conducted proteomic profiling of sperm with poor freezability (PF) and GF in Wannan Black pigs. We detected 2,485 proteins, identifying 337 significantly differentially expressed proteins (DEPs). Gene Ontology annotation revealed the involvement of DEPs in crucial processes, such as protein homeostasis maintenance, zona pellucida binding, polyspermy blockade, and ATPase activation regulation. Its molecular functions include immune signaling and oxidative stress defense. KEGG pathway analysis highlighted DEPs enrichment in seminal adhesion complexes, nucleic acid helicase families, and mitochondrial membrane protein clusters. Subcellular localization showed predominant DEPs distribution in the cytoplasm, extracellular matrix, nucleus, mitochondria, and plasma membranes. Western blotting validation of differentially expressed proteins (HSP90AA1, ACE, ANXA2, DEFB1, and S100A8) demonstrated results consistent with the proteomic sequencing data. This study delineates the key proteomic differences underlying boar sperm freezability, providing insights into refining cryopreservation protocols and augmenting swine reproductive efficiency.