Melatonin Mitigates Vitrification-Induced Cryoinjury in Mouse Embryos by Alleviating Metabolic Alterations

Vitrification is a vital tool for the long-term preservation of animal genetic resources, yet cryoinjury-manifesting as oxidative stress, structural damage, and metabolic disorders-severely compromises its efficacy. Here, we investigated the protective effects of melatonin (MT) supplementation on the cryotolerance of mouse morulae. First, mouse morulae were assigned to four groups treated with vitrification and thawing media containing MT (0, 10^-3, 10^-5, and 10^-7 M) to determine optimal MT concentration. Subsequently, embryos treated with the optimal MT concentration were evaluated for developmental competence, oxidative stress, apoptosis, and mitochondrial function. Furthermore, transcriptome sequencing was performed to elucidate MT-regulated molecular pathways. The results demonstrated that MT supplementation at 10^-5 M significantly enhanced developmental competence, as evidenced by increased blastocyst rate, hatched blastocyst rate, total cell number and the inner cell mass (ICM)-to-total cell ratio compared to the MT-free group (p < 0.05). Consequently, embryo transfer outcomes showed higher live births and weaned pups in the 10^-5 M MT group versus those in controls (p < 0.05), achieving levels comparable to fresh embryos (p > 0.05). Mechanistically, MT reversed cryoinjury-induced mitochondrial dysfunction by elevating membrane potential(MMP) and Adenosine Triphosphate(ATP) production while reducing Reactive Oxygen Species (ROS) accumulation (p < 0.05). Transcriptomic analysis further revealed that vitrification perturbed metabolic pathways, including amino acid/fatty acid degradation and glucose/pyruvate metabolism. MT downregulated cryoinjury-induced overexpression of Rela and Nfkb1, inhibiting excessive NF-κB activation and alleviating metabolic dysfunction. Additionally, MT restored expression of nucleotide synthesis genes (Ctps2, Nme4, Gmps, Nudt2, Ppat, Impdh2) critical for cell proliferation, and reversed downregulation of mitochondrial genes Sucla2 and Timm17a, confirming restoration of mitochondrial homeostasis. In conclusion, melatonin alleviates vitrification-induced cryoinjury by restoring mitochondrial function, which rescues nucleotide synthesis and partially reverses associated metabolic dysfunction. These findings advance MT-mediated cryoprotection and underscore its translational value for embryo cryopreservation in animal genetic resource conservation.