TMT-based quantitative proteomics analysis reveals the differential proteins between fresh and frozen-thawed sperm of yak (Bos grunniens)

Sperm cryopreservation is one of the most effective methods for the conservation of germplasm re-sources and used of superior sires widely. However, the motility of yak (Bos grunniens) sperm was low after thawing and the proteomics changes in sperm cryopreservation remain unknown. Therefore, the aim of this study was to explore the differences between fresh sperm and frozen sperm of yak through the proteomic analysis and thus improve the understanding of sperm cryodamage. The Tandem Mass Tags (TMT) technology was used to screen differentially expressed proteins (DEPs) before and after freezing. Then, GO and KEGG analysis were conducted to analyze the DEPs enriched signaling pathways. Finally, the DEPs, including superoxide dismutase 1 (SOD1) and NADH ubiquinone oxidoreductase core subunit S8 (NDUFS8) were verified by the immunofluorescence technique. The results showed that there were 229 DEPs between fresh and frozen-thawed yak sperm. Compared with the fresh sperm, 120 proteins were up-regulated and 109 proteins were down-regulated in frozen-thawed sperm. The GO annotation showed that the up-regulated proteins enriched in metabolic and cytoskeleton-related processes, including lipoprotein metabolic process, lipid transport, extracellular region and intermedi-ate filament cytoskeleton organization. In contrast, the down-regulated proteins enriched in biological processes including single fertilization, sperm capacitation and response to unfolded protein. KEGG pathway analysis indicated that freezing and thawing affected the oxidative phosphorylation pathway, the fructose and mannose metabolic pathway and the glycerolipid metabolic pathway of yak sperm. Immunofluorescence results showed that the protein expression level of SOD1 protein in the frozen group was significantly lower than that in the fresh group (P < 0.01), and the protein expression level of NDUFS8 protein was significantly higher in frozen group (P < 0.01). This study revealed the DEPs be-tween fresh and frozen-thawed sperm and provides a theoretical basis to further explore the exertion of normal biological functions of yak sperm after freezing and thawing. (c) 2023 Elsevier Inc. All rights reserved.