Epigenetic insights into mastitis in Mediterranean Italian River buffalo using nanopore sequencing

Mastitis represents one of the main challenges in dairy buffalo farming, with significant implica- tions for animal health, milk quality, and farm profitability. Among its different forms, subclinical mastitis is difficult to detect due to the absence of standardised diagnostic tools. Epigenetic mechanisms, such as DNA methylation, have been associated with immune response and dis- ease susceptibility in cattle, but remain unexplored in buffaloes. This study explores genome- wide DNA methylation profiles in Mediterranean Italian River Buffaloes, using nanopore sequenc- ing technology, to identify potential epigenetic signatures associated with mastitis resistance. CpG sites showed a unimodal distribution, with most sites exhibiting high methylation levels. A total of 22 differentially methylated cytosines (DMCs) were identified, with 68% showing hypo- methylation in the control group and 32% showing hypermethylation. Genomic annotation revealed that hypermethylated DMCs were predominantly located in intronic regions, while hypomethylated DMCs were largely enriched in distal intergenic regions. This study is the first to investigate DNA methylation changes associated with mastitis in Mediterranean Italian River Buffalo using nanopore sequencing. Distinct epigenetic patterns were identified between healthy and mastitic animals. This study provides a first epigenetic overview of mastitis in buffa- loes and lays the groundwork for future investigations with larger cohorts to validate and extend these observations. Given the small sample size, these findings should be considered exploratory, but offer insights into the molecular basis of mastitis and may support the develop- ment of new diagnostic tools based on validated epigenetic signatures.