Transgenerational response to the ingestion of an endocrine disruptor: phenotypic, genetic, and epigenetic analyses in quail

Epigenetic mechanisms such as DNA methylation can mediate the effect of the environment on an individual's phenotypes. While parental environments have also been shown to affect offspring phenotypes in several livestock species, evidence for transgenerational inheritance of environmentally induced epigenetic marks remains debated.This study aimed to assess the potential transgenerational inheritance of an environmental effect via epigenetic modifications in Japanese quails (Coturnix japonica). We investigated whether an initial dietary exposure to genistein - a natural phytoestrogen found in soybeans - could induce lasting phenotypic and epigenetic changes in subsequent generations.To disentangle genetic from epigenetic effects, we designed a balanced “mirror-crossing” scheme based on 20 founder families. In each family, two sisters were mated to the same male: one received a 30-day genistein supplementation (epi+ line), while the other was kept as a control, without genistein (epi– line). Their offspring were mated within each 'epiline' for three generations (G0–G2), ensuring a balanced representation of each genetic background in both epilines. Phenotypic data (body and tissue weights, egg production, and behavioral traits) were collected across all generations. DNA methylation profiles and genotypes were obtained from Reduced Representation Bisulfite Sequencing of 1,344 individual blood samples using BISCUIT, which allows jointly methylation and SNP calling. Differential methylation analysis was performed using the DSS package. Methylation heritability and methylation QTLs (metQTLs) were assessed with GCTA.Significant differences between epilines were detected for body weight. Epi+ females were consistently heavier than epi– controls at all ages, with the difference increasing across generations (up to +14 g in G2). A similar, though delayed, pattern was observed in males, becoming significant in G2 (+9 g at slaughter). Other traits, including reproduction and behavior, were not significantly affected. The proportion of phenotypic variance explained by the epiline increased over generations, suggesting either a genetic drift or non-genetic inherited effects.Methylation calling yielded 448,673 CpG sites in 1,267 individuals (mean coverage 34×). Among 111,168 variable CpGs (SD > 0.05), 22 and 72 were differentially methylated between epilines in G1 and G2, respectively, while no significant differences were detected in G0. A large number of CpGs showed significant sex × epiline interactions. The mean methylation heritability (h² = 0.24) supported moderate genetic control of methylation levels, with a high variability between CpGs.Ongoing analyses will contribute to a better understanding of the molecular mechanisms and biological significance of non-genetic inheritance in avian models.This project is funded by the European Union’s Horizon 2020 program GEroNIMO N°101000236, part of EuroFAANG (https://eurofaang.eu).