Green forage impacts on the DNA methylation in the ruminal wall of Italian mediterranean dairy buffaloes

Nutrition impacts the epigenetic signature, including DNA methylation. The aim of this study was to identify genomic regions differentially methylated in the rumen of Italian Mediterranean dairy buffaloes fed green forage [Total Mixed Ration (TMR) + ryegrass green feed (30% of diet)] compared to those receiving a standard TMR diet, through Reduced Representation Bisulfite Sequencing. We found 6571 differentially methylated genomic regions (DMRs), 51.73% hypomethylated and 48.27% hypermethylated. DMRs were uniformly dispersed in genes and intergenic regions and along chromosomes. Genes-associated DMRs were mainly hypomethylated, while intergenic DMRs were mostly hypermethylated. We highlighted 4648 genes associated with DMRs (differentially methylated genes, DMGs), mostly protein-coding genes. Gene Ontology study performed with hypermethylated or hypomethylated DMGs highlighted categories related to response to oxidative stress and inflammation, as well as rumen functionality. The integration of our results with differential expression data identified genes whose expression varies as a function of DNA methylation. This subset of genes included those involved in immune system functioning, inflammation, fatty acid metabolism, and stress response. Our findings highlighted the impact of green forage on rumen DNA methylation, which potentially influences molecular mechanisms relevant to rumen functionality and, then, animal welfare and production, through the modulation of gene expression.