Proteomic Profiling Reveals the Synergistic Effects of Astaxanthin and Melatonin on the Inhibition of Cryoinjuries in Ram Sperm.

The present study aimed to investigate the protective effects of astaxanthin (AST) and melatonin (MLT) on ram sperm quality-associated indicators during cryopreservation, and to explore the molecular effects of the two cryoprotectants on the protein profile of cryopreserved sperm. First, ram sperm were diluted with freezing media supplemented with different concentrations of AST and MLT, and then cryopreserved. The effects of different concentrations of AST and MLT were assessed by evaluating the motility and motile parameters, functional and structural indicators, antioxidant capacity, and associated indicators of post-thawed sperm. The concentrations of 50 μM AST (AST group), 0.5 mM MLT (MLT group), and 50 μM AST and 0.5 mM MLT (AST.MLT group) were the most effective, as compared to the control (CON group). Furthermore, alterations in the sperm proteomic profile among the three comparisons (AST vs CON, MLT vs CON, AST.MLT vs CON) were determined using an Orbitrap Astral coupled with a data-independent acquisition (DIA) proteomic approach. After comprehensive bioinformatics analysis, significantly differentially abundant proteins (DAPs) among the three comparisons (32 DAPs in AST vs CON, 34 DAPs in MLT vs CON, 116 DAPs in AST.MLT vs CON) and their enriched molecular pathways were discovered, suggesting that AST and MLT may have synergistic effects on frozen sperm by regulating the abundance changes in proteins. In particular, certain positive proteins like ACAT2, PRDX6, G6PD, HSP90a, HSPA8, HSL, ACTN1, and Ezrin, and negative proteins like Cyt c, CTSF, and AFU, which are tightly associated with the regulation of sperm motility, oxidative stress, and other metabolic processes, may serve as novel potential biomarkers for evaluating sperm quality after freeze-thawing. In conclusion, this study provides valuable insights into the protective effects of AST and MLT on ram semen quality and replenishes information for further clarifying the molecular mechanism based on the modification of the sperm proteome during cryopreservation.