Paternal Methionine Supplementation Alters DNA Methylation Patterns in Preimplantation Sheep Embryos

Background/Objectives: Parental environmental factors can shape developmental outcomes through epigenetic mechanisms that regulate gene expression. While maternal dietary effects on offspring epigenetics have been well characterized, the impact of paternal diet on embryonic DNA methylation remains poorly understood. Here, we investigated the effect of paternal methionine supplementation on DNA methylation patterns in preimplantation embryos in Polypay sheep. Methods: Four yearling rams (two control and two methionine-supplemented) were bred to twelve ewes following estrus synchronization and superovulation. Embryos were collected after natural mating and analyzed using whole-genome bisulfite sequencing (WGBS). Results: A total of 842 differentially methylated cytosines (DMCs) were identified in embryos derived from methionine-supplemented sires compared to controls, with 835 hypermethylated and 7 hypomethylated. The majority of DMCs were located in intergenic regions, with minimal representation in exonic regions. To assess overlap between parental dietary effects, DMCs identified in this study were compared with those previously reported in embryos derived from methionine-supplemented dams. Eight hypermethylated DMCs were shared between the two datasets, while no hypomethylated DMCs overlapped. To evaluate the functional relevance of differentially methylated genes, we performed siRNA-mediated knockdown of SSU72, a gene associated with multiple DMCs. Knockdown of SSU72 resulted in an average 18% decrease in blastocyst formation rate (p < 0.001). Conclusions: These results demonstrate that paternal methionine supplementation alters embryonic DNA methylation patterns and that affected genes may play critical roles in early embryonic development, contributing to fetal programming.