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Production animals are constantly subjected to early adverse environmental conditions that influence the adult phenotype and produce epigenetic effects. CpG dinucleotide methylation in red blood cells (RBC) could be a useful epigenetic biomarker to identify animals subjected to chronic stress in the production environment. Here we compared a reduced fraction of the RBC methylome of chickens exposed to social isolation to non-exposed. These experiments were performed in two different locations: Brazil and Sweden. The aim was to identify stress-associated DNA methylation profiles in RBC across these populations, in spite of the variable conditions to which birds are exposed in each facility and their different lineages. Birds were increasingly exposed to a social isolation treatment, combined with food and water deprivation, at random periods of the day from weeks 1–4 after hatching. We then collected the RBC DNA from individuals and compared a reduced fraction of their methylome between the experimental groups using two bioinformatic approaches to identify differentially methylated regions (DMRs): one using fixed-size windows and another that preselected differential peaks with MACS2. Three levels of significance were used ( P  ≤ 0.05,  P  ≤ 0.005, and  P  ≤ 0.0005) to identify DMRs between experimental groups, which were then used for different analyses. With both of the approaches more DMRs reached the defined significance thresholds in BR individuals compared to SW. However, more DMRs had higher fold change values in SW compared to BR individuals. Interestingly, ChrZ was enriched above expectancy for the presence of DMRs. Additionally, when analyzing the locations of these DMRs in relation to the transcription starting site (TSS), we found three peaks with high DMR presence: 10 kb upstream, the TSS itself, and 20–40 kb downstream. Interestingly, these peaks had DMRs with a high presence (>50%) of specific transcription factor binding sites. Three overlapping DMRs were found between the BR and SW population using the most relaxed  p -value ( P  ≤ 0.05). With the most stringent  p -value ( P  ≤ 0.0005), we found 7 and 4 DMRs between treatments in the BR and SW populations, respectively. This study is the first approximation to identify epigenetic biomarkers of long-term exposure to stress in different lineages of production animals.

Putative Epigenetic Biomarkers of Stress in Red Blood Cells of Chickens Reared Across Different Biomes