Domesticated animals are unique to investigate the contribution of genetic and non-genetic factors to specific phenotypes. Among non-genetic factors involved in phenotype formation are epigenetic mechanisms. Here we aimed to identify whether relative DNA methylation differences in the nidopallium between groups of individuals are among the non-genetic factors involved in the emergence of differential behavioral patterns in hens. The nidopallium was selected due to its important role in complex cognitive function (i.e., decision making) in birds. Behavioral patterns that spontaneously emerge in hens living in a highly controlled environment were identified with a unique tracking system that recorded their transitions between pen zones. Behavioral activity patterns were characterized through three classification schemes: (i) daily specific features of behavioral routines (Entropy), (ii) daily spatio-temporal activity patterns (Dynamic Time Warping), and (iii) social leading behavior (Leading Index). Unique differentially methylated regions (DMRs) were identified between behavioral patterns emerging within classification schemes, with entropy having the higher number. Functionally, DTW had double the proportion of affected promoters and half of the distal intergenic regions. Pathway enrichment analysis of DMR-associated genes revealed that Entropy relates mainly to cell cycle checkpoints, Leading Index to mitochondrial function, and DTW to gene expression regulation. Our study suggests that different biological functions within neurons (particularly in the nidopallium) could be responsible for the emergence of distinct behavior patterns and that epigenetic variation within brain tissues would be an important factor to explain behavioral variation.
