DNA methylation variation in the brain of laying hens in relation to differential behavioral patterns

Domesticated animals are unique to investigate the contribution of genetic and non-genetic factorsto specific phenotypes. Among non-genetic factors involved in phenotype formation are epigeneticmechanisms. Here we aimed to identify whether DNA methylation changes in the brain (nidopallium)are among the non-genetic factors involved in the emergence of differential behavioral patterns inhens, which lack maternal role (a major confounding factor) once the egg is laid. The nidopallium wasselected due to its important role in complex cognitive function (i.e., decision making) in birds.Naturally emerging behavioral patterns of hens living in a highly controlled environment wereidentified with a unique tracking system that recorded their transitions between pen zones.Behavioral traits and activity levels were characterized through three classification schemes: (i) dailyspecific features of behavioral routines (Entropy), (ii) daily spatio-temporal activity patterns (DynamicTime Warping), and (iii) social leading behavior (Leading Index). Unique differentially methylatedregions (DMRs) were identified between behavioural patterns emerging within classificationschemes, with entropy having the higher number. Functionally, DTW had double the proportion ofaffected 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 tomitochondrial function, and DTW to gene expression regulation. Our study suggests that differentbiological functions within neurons (particularly in nidopallium) could be responsible for theemergence of different behaviour patterns and supports the position that epigenetic variation withinbrain tissues is an essential factor to explain naturally emerging behavioral variation.