Substantial progress in research on sperm cryopreservation has occurred since the twentieth century, especially focusing on improving sperm freezing procedures and optimizing semen extenders. However, the cellular biological mechanisms of sperm freezing damage are still unclear, which greatly restricts the promotion and development of sperm cryopreservation. An essential component of sperm freezing damage is the occurrence of cell death. Considering the existence of multiple types of cell death pathways, this review discusses connections between characteristics of regulated cell death (e.g., apoptosis and ferroptosis), and accidental cell death (e.g., intracellular ice crystals) with sperm freezing damage and explores possible future research directions in this field.

The current study aimed to detect the relationship between the spermatozoa cryotolerance and the post-thawed sperm lipidome. Ejaculates from 20 goats, and performed a uniform frozen-thawed procedure in this study. According to the total motility of thawed sperm of goats, semen samples were classified into HF group (High Freezers, n = 8) with >60% total motility and LF group (Low Freezers, n = 8) with < 45% total motility. The lipidomic analysis based on UHPLC-MS/MS was utilized to investigate the relationship between sperm cryotolerance and their lipid metabolites expression. The results showed that the cryotolerance of sperm from different individual goats were in great variation. The total motility of post-thawed sperm in HF group (60.93 ± 2.43%) is significantly higher than that in LF group (34.04 ± 3.41%, P < 0.01). And the post-thawed sperm in HF group exhibited significantly higher plasma membrane (59.06 ± 2.34%) and acrosome integrity (62.93 ± 1.15%) than that in LF group (34.06 ± 4.85%, 44.92 ± 2.19% respectively, P < 0.01). The total of 29 lipid subclasses and 1,133 lipid molecules in the post-thawed goat sperm were identified by lipidomics analysis. The lipid content of thawed sperm in HF group was higher than that in LF group, the lipid profile in HF group was significantly separated from LF group, which indicated that the difference in lipid composition and lipid metabolism mode of sperm between the two groups was existed, especially the expression of phosphatidylcholine and triglyceride molecules. In conclusion, the cryotolerance of sperm from different individual goats were in great variation. Sperm with high cryotolerance may be able to uptake more lipids during cryopreservation. The increase in phosphatidylcholine and triglyceride content of thawed. Sperm may relate to more active lipid anabolic processes.

Sperm membrane stability is a key factor in determining sperm viability and fertilization capability, with broad implications ranging from basic reproductive biology to livestock breeding practices. This comprehensive review examines the structural and functional mechanisms underlying sperm membrane integrity, including defensive barrier functions, potentiometric ion channel regulation, and motility modulation that collectively optimize sperm survival, motility, and fertilization potential. Environmental factors such as temperature fluctuations, abnormal pH levels (outside the optimal 7.2–8.2 range), pathological conditions, and hormonal imbalances can compromise membrane stability by inducing oxidative stress and protein denaturation. Key regulatory proteins, notably NPC2 for cholesterol homeostasis, Flotillin proteins for lipid raft organization, and Annexin V for membrane repair mechanisms, demonstrate essential roles in maintaining structural integrity. In livestock reproduction, membrane stability research facilitates the optimization of cryoprotectant formulations and freezing protocols, resulting in 15–25% improvements in post-thaw sperm survival rates and enhanced artificial insemination success. These findings provide valuable insights for advancing assisted reproductive technologies and improving reproductive efficiency in animal husbandry.

Selenium-independent glutathione peroxidase (GPx5) is specifically expressed in the mammalian epididymis and plays an important role in protecting sperm from reactive oxygen species and lipid peroxidation damage. This study investigates GPx5 expression in the epididymis of Small Tail Han sheep. GPx5 expression was studied in three age groups: lamb (2 to 3 months), young (8 to 10 months), and adult (18 to 24 months). The epididymis of each age group divided into caput, corpus and cauda, respectively. Analysis the expression quantity of GPx5 in epididymis and testis by real-time fluorescent quantitative polymerase chain reaction and Western blot. Finally, GPx5 protein locating in the epididymis by immunohistochemical. The results demonstrate that in the lamb group, the GPx5 mRNA, but not protein, can be detected. GPx5 mRNA and expressed protein were detected in both the young and adult groups. Moreover, both the mRNA and protein levels of GPx5 were significantly higher in the young group than in other two groups. When the different segments of epididymis were investigated, GPx5 mRNA was expressed in each segment of epididymis regardless of age. Additionally, the mRNA level in the caput was significantly higher than that in corpus and cauda within same age group. The GPx5 protein was in the epithelial cells' cytoplasm. However, GPx5 mRNA and protein were not detected in the testis. These results suggest that GPx5 is mainly expressed in the epididymis of Small Tail Han sheep, and that the expression level of GPx5 is associated with age. Additionally, GPx5 was primarily expressed in the epithelial cells of the caput. Taken together, these studies indicate that GPx5 is expressed in the epididymis in all age grades.