Protective Effects of Seminal Plasma Supplemented in Boar Frozen Semen Thawing Media: Sperm Quality, Proteome, and Reproductive Performance

Boar semen cryopreservation enables elite genetic utilization, yet thawing-induced cryodamage impairs sperm quality. While seminal plasma shows promise as a natural cryoprotective additive, its effective concentration and molecular mechanisms remain unclear. This study aimed to screen for an effective seminal plasma concentration for thawing media and to elucidate its protective mechanisms in frozen–thawed boar sperm. Seminal plasma from freeze-tolerant (FT) and non-freeze-tolerant (NFT) boars was added to thawing media at gradient concentrations. Sperm quality was assessed post-thawing, followed by quantitative proteomics, functional enrichment, Western blot analysis, and artificial insemination (AI) fertility trials. Supplementation with 10–30% seminal plasma significantly improved post-thaw sperm motion parameters, and 20% showed the highest numerical values for most parameters among the tested concentrations. FT and NFT seminal plasma conferred similar protective effects, with no statistically significant difference detected between the two phenotypes. Proteomics identified 2965 sperm proteins, with only 11 differentially expressed proteins (DEPs) differing between FT and NFT groups, indicating substantial similarity in their sperm proteome profiles under the applied analysis. DEPs were significantly enriched in three core pathways: lysosome, complement and coagulation cascades, and sphingolipid metabolism. Western blot supported the proteomic expression trends for LGMN, PLS1, SERPINA1 and CLU. In field AI trials, frozen–thawed semen supplemented with 20% seminal plasma produced litter sizes and live piglet numbers that did not differ significantly from those of fresh semen; however, no contemporaneous conventional frozen-semen control group was included. Addition of 20% FT or NFT seminal plasma to thawing medium improves post-thaw sperm quality and fertilization capacity, possibly through the co-regulation of three core pathways. These findings provide a practical, operationally feasible protocol for commercial frozen boar semen production.