Paternal heat conditioning enhances offspring’s thermal resilience via epigenetic regulation of mir-210a

Early-life exposure to environmental stressors can induce long-term physiological adaptations that extend across generations. This study examines embryonic heat conditioning (EHC) effects on heat stress resilience and paternal transgenerational inheritance in poultry. Fertilized male chicken eggs that underwent EHC (F0-EHC) were raised to maturity before breeding with naïve hens to generate F1 offspring, which were reared under standard conditions. A heat challenge at day 10 post-hatch revealed that both F0-EHC chicks and their offspring (F1-EHC) exhibited significantly greater thermal resilience compared to controls, as reflected by a lower increase in body temperature. To explore molecular mechanisms underlying EHC we focused on microRNAs (miRs), analyzing CpG-DNA differentially methylated regions near miR genes in the preoptic anterior hypothalamus (PO/AH) of F1 chicks and identified miR-210a as a key candidate. Both F0-EHC chicks and their offspring (F1-EHC) displayed increased miR-210a expression at baseline, which was significantly reduced during heat challenge. Intracranial injection of mature miR-210a into naïve chicks confirmed its regulatory role, inducing changes in gene expression in the PO/AH. RNA-Seq analysis identified 57 genes differentially expressed post-injection, including genes involved in chromatin organization, stress responses, and thermogenesis. Overlapping analyses of the RNA-Seq results with RRBS methylation data from F0 sperm and F1 PO/AH implicated ARID5B as a potential mediator of epigenetic inheritance to explore its function, we injected ARID5B sense and antisense oligonucleotides into the third ventricle of chicks and found that antisense treatment significantly increased ARID5B and miR-210a expression in the PO/AH without affecting body temperature. Together, these results reveal how embryonic thermal experience shapes hypothalamic gene regulation to promote transgenerational thermotolerance in chickens. Understanding this epigenetic mechanism may inform strategies to enhance resilience and sustainability in poultry exposed to thermal stress.