Cryopreservation may reduce sperm fertility due to cryodamage including physical-chemical and oxidative stress damages. As a powerful antioxidant, melatonin has been reported to improve cryoprotective effect of sperm. However, the molecular mechanism of melatonin on cryopreserved ram sperm hasn't been fully understand. Give this, this study aimed to investigate the postthaw motility parameters, antioxidative enzyme activities and lipid peroxidation, as well as proteomic, metabolomic changes of Huang-huai ram spermatozoa with freezing medium supplemented with melatonin. Melatonin was firstly replenished to the medium to yield five different final concentrations: 0.1, 0.5, 1.0, 1.5, and 2.0 mM. A control (NC) group without melatonin replenishment was included. Protective effects of melatonin as evidenced by postthaw motility, activities of T-AOC, T-SOD, GSH-Px, CAT, contents of MDA, 4-HNE, as well as acrosome integrity, plasma membrane integrity, with 0.5 mM being the most effective concentration (MC group). Furthermore, 29 differentially abundant proteins involving in sperm functions were screened among Fresh, NC and MC groups of samples (n = 5) based on the 4D-LFQ, with 7 of them upregulated in Fresh and MC groups. 26 differentially abundant metabolites were obtained involving in sperm metabolism among the three groups of samples (n = 8) based on the UHPLC-QE-MS, with 18 of them upregulated in Fresh and MC groups. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the abundance changes of vital proteins and metabolites related to sperm function. Particularly, several proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine may be potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa. In conclusion, a dose-dependent replenishment of melatonin to freezing medium protected ram spermatozoa during cryopreservation, which can improve motility, antioxidant enzyme activities, reduce levels of lipid peroxidation products, modify the proteomic and metabolomic profiling of cryopreserved ram spermatozoa through reduction of oxidative stress, maintenance of OXPHOS and microtubule structure. SIGNIFICANCE: Melatonin, a powerful antioxidant protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner, with 0.5 mM being the most effective concentration. Furthermore, sequencing results based on the 4D-LFQ combined with the UHPLC-QE-MS indicated that melatonin modifies proteomic and metabolomic profiling of ram sperm during cryopreservation. According to the bioinformatic analysis, melatonin may have positive effects on frozen ram spermatozoa by regulating the expression changes of vital proteins and metabolites related to sperm metabolism and function. Particularly, several potential novel biomarkers for evaluating the postthaw quality of ram spermatozoa were acquired, proteins such as PRCP, NDUFB8, NDUFB9, SDHC, DCTN1, TUBB6, TUBA3E, SSNA1, as well as metabolites like L-histidine, L-targinine, ursolic acid, xanthine.
Keywords: Cryopreservation; Melatonin; Metabolome; Proteome; Ram; Spermatozoa.
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