Effect of low-temperature stress on sperm DNA methylation and oxidative damage during cryopreservation of Tibetan pig semen

Background and Aim: Cryopreservation of boar semen is widely used for genetic conservation and artificial insemination; however, it induces oxidative stress and molecular alterations that compromise sperm function. Epigenetic modifications, particularly DNA methylation, play a critical role in sperm integrity and fertilization capacity. This study aimed to investigate genome-wide DNA methylation changes and their association with oxidative stress during cryopreservation of Tibetan pig semen. Materials and Methods: Semen samples were collected from healthy Tibetan pigs and cryopreserved using a standard freeze–thaw protocol. Sperm quality parameters, including motility, viability, and membrane integrity, were assessed post-thaw. Oxidative stress markers, including reactive oxygen species (ROS) levels and antioxidant enzyme activities, were evaluated. Genome-wide DNA methylation profiling was performed using methylated DNA immunoprecipitation sequencing (MeDIP-Seq). Differentially methylated regions (DMRs) were identified, and functional enrichment analyses, including Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways, were conducted. Results: Cryopreservation significantly reduced sperm motility, viability, and membrane integrity, accompanied by elevated ROS levels and disrupted antioxidant defense systems. MeDIP-Seq analysis revealed widespread alterations in DNA methylation patterns between fresh and cryopreserved sperm, with numerous DMRs identified across the genome. These methylation changes were predominantly associated with genes involved in oxidative stress response, apoptosis, energy metabolism, and signal transduction pathways. Functional enrichment analysis highlighted significant involvement of pathways related to mitochondrial function, reactive oxygen species metabolism, and cellular stress responses, indicating a strong link between epigenetic modifications and cryoinjury mechanisms. Conclusion: Cryopreservation induces substantial oxidative stress and genome-wide alterations in DNA methylation in Tibetan pig sperm. The identified epigenetic changes are closely associated with pathways regulating oxidative stress and cellular integrity, suggesting that DNA methylation plays a crucial role in sperm cryodamage. These findings provide novel insights into the molecular mechanisms underlying cryopreservation-induced sperm dysfunction and may contribute to the development of improved cryopreservation strategies for enhanced reproductive performance.