Integrated DNA Methylation and Transcriptome Analysis Reveals Epigenetic Mechanisms of Lactation Performance Differences in Cloned Buffalo

Buffalo milk plays a vital role in the dairy industry, with milk yield regulated by both transcriptomic and epigenetic mechanisms. While previous studies have primarily focused on differences among individuals or breeds, the epigenetic basis underlying milk yield variation in genetically identical animals remains poorly understood. In this study, we employed a cloned buffalo model and integrated whole-genome bisulfite sequencing (WGBS) with RNA sequencing (RNA-seq) to investigate how DNA methylation and transcriptional regulation contribute to milk yield variation. Results tentatively revealed that low-yielding buffalo exhibited globally reduced DNA methylation in mammary tissues, with distinct distribution patterns across genomic features and regulatory regions. Differentially methylated genes were enriched in PI3K-Akt, HIF-1, and immune-related pathways, whereas hypomethylated genes were associated with calcium signaling, cAMP pathways, and metabolic processes. Transcriptome analysis showed that high-yielding buffalo upregulated genes involved in lipid metabolism and cell proliferation, while low-yielding buffalo displayed enrichment in immune stress and amino acid metabolism. Integrative analysis identified 126 hypo-upregulated genes and highlighted hub regulators such as KLF6, NR4A1, ESR1, KCNQ1. Collectively, this study outlines a preliminary multi-omics regulatory landscape of milk yield variation in cloned buffalo, suggests the interplay between DNA methylation and transcription, provides preliminary insights into the potential interplay between DNA methylation and transcription, and suggests potential connections that merit further investigation.