Effect of pre-weaning nutrition on the epigenetic and transcriptomic regulation of mammary gland development in Holstein heifers

Dairy calves require a balanced diet that ensures adequate nutrient intake to support optimal growth and physiological development, which may subsequently influence future lactational performance and reproductive capacity through favourable epigenetic modifications in body tissues. This study aimed to investigate the impact of pre-weaning nutritional strategies on the epigenome and transcriptome of the mammary gland tissues in Holstein heifers. Twelve Holstein-Friesian heifer calves were included in the experiment. During their first two weeks of life, all calves received a milk replacer (MR) that met their nutritional requirements, along with unlimited access to concentrate feed and water. At 14 ± 1 days of age, the calves were randomly assigned to one of two feeding treatments. The applied diets involved restricted milk replacer intake (R, n = 6) and ad libitum milk replacer intake (AL, n = 6). Feeding treatments lasted for 48 days, after which all calves were euthanised at 62 ± 1 days of age. Mammary parenchyma (PAR) and mammary fat pad (MFP) tissues were collected and analysed using RRBS, miRNA-seq, and RNA-seq approaches. When comparing two diets, 268 differentially methylated sites (DMSs) were identified in the PAR, and 288 DMSs were found in MFP tissue. The obtained data allowed identification of four differentially expressed miRNAs (DE miRNAs) in PAR and 35 DE miRNAs in MPF tissue. Furthermore, transcriptomic analysis revealed that mammary fat pad tissue reacted to varying diets with a higher number of differentially expressed genes, indicating that parenchyma tissue is less responsive to nutritional changes. Based on integrative omics analysis, specific genes were identified in MFP tissue, whose expression levels were correlated with the level of CpG methylation and miRNA expression, revealing enriched biological processes. However, several important limitations should be considered when interpreting these results, including the relatively small sample size and the absence of long-term follow-up. These findings suggest that pre-weaning feeding strategies have a significant influence on the epigenetic regulation of gene expression, potentially determining the future lactation potential of dairy heifers.