Identification of Biomarkers Affecting Cryopreservation Recovery Ratio in Ram Spermatozoa Using Tandem Mass Tags (TMT)-Based Quantitative Proteomics Approach

SIMPLE SUMMARY: Sperm cryopreservation is one of the key procedures in artificial insemination technology. With the continuous development of cryopreservation technology, the survival rate of frozen sperm has been greatly improved, and it is widely used in artificial insemination technology. However, the molecular regulation of sperm cryopreservation recovery ratio in rams remains poorly understood. The main objective of this study was to gain a better understanding of the biomarkers that play a role in sperm freezing tolerance in the proteome levels of the rams. Our results confirm that some proteins and pathways associated with high and low cryopreservation recovery ratios were identified. These findings related to sperm freezing tolerance improve our understanding of molecular mechanisms of sperm resistance to low-temperature environments, and are helpful for livestock breeding. ABSTRACT: Sperm proteins play vital roles in improving sperm freezing resilience in domestic animals. However, it remains poorly defined which proteins regulate the freezing resilience of spermatozoa in rams (Ovis aries). Here, we compared the proteome of ram sperm with a high cryopreservation recovery ratio (HCR) with that of ram sperm with a low cryopreservation recovery ratio (LCR) using a tandem mass tag-based quantitative proteomics approach. Bioinformatic analysis was performed to evaluate differentially expressed proteins (DEPs). A total of 2464 proteins were identified, and 184 DEPs were screened. Seventy-two proteins were higher in the LCR group. One hundred and twelve proteins were more abundant in the HCR group, and they were mainly involved in the regulation of oxidative phosphorylation and thermogenesis pathways. Proteins in high abundance in the HCR group included the S100A family, such as S100A8, S100A9, S100A14, and S100A16, effectively controlling for CA(2+) and maintaining flagella structure; HYOU1 and PRDX1, which participate in antioxidant protection and anti-apoptosis to prevent cell death; and HSP90B1, which maintains cell activity and immune response. Our results could help illuminate the molecular mechanisms underlying cryopreservation of ram semen and expand the potential direction of cryopreservation of high-quality semen.