Epigenetic changes in red blood cells during the life of chickens exposed to social isolation stress or an enrich environment

Epigenetic mechanisms offer a promising approach to understanding long-term effects of early-life stress. Stress during early development can alter DNA methylation patterns and red blood cells (RBCs) may retain such signatures. This study explores: i) whether specific stressors leave distinct epigenetic marks in RBCs, and ii) how long these epigenetic alterations persist. We exposed 4-day-old male chickens to social isolation stress and enriched environment incrementally for 3.5 weeks and control group in barren condition. RBCs were collected immediately and six months later. DNA methylation analysis using GBS-MeDIP revealed that stress exposure led to progressive hypermethylation with age, while enrichment induced age-related hypomethylation and resulted in fewer overall DMRs. In contrast, the control group showed a balanced mix of hyper- and hypomethylated sites between old and young individuals. Notably, DMRs were significantly overrepresented on chrZ and immune-related genes. Pathway enrichment analysis further revealed that stress was associated with broad transcription factor activation and immune-metabolic pathways, while enrichment was linked to changes involving microRNAs. Overall, these findings demonstrate that early environmental quality leaves lasting epigenetic mark in chicken RBCs, with stress and enrichment causing opposite methylation trends and candidate biomarkers of long-term welfare.