Embryonic Thermal Manipulation Affects Neurodevelopment and Induces Heat Tolerance in Layers

Background/Objectives: The poultry industry faces severe heat-stress challenges that threaten both economic sustainability and animal welfare. Embryonic thermal manipulation (ETM) has been proposed as a thermal programming strategy to enhance chick heat tolerance, yet its efficacy in layers requires verification, and its effects on growth performance and neurodevelopment remain unclear. Methods: White Leghorn embryos at embryonic days 13 to 18 (ED 13–18) were exposed to 39.5 °C (ETM). Hatch traits and thermotolerance were recorded, and morphometric and histopathological analyses were performed on brain sections. Transcriptome profiling of the whole brains and hypothalami was conducted to identify differentially expressed genes (DEGs). Representative pathway genes responsive to ETM were validated by RT-qPCR. Results: ETM reduced hatchability, increased deformity rate, and decreased hatch weight and daily weight gain. During a 37.5 °C challenge, ETM chicks exhibited delayed panting and lower cloacal temperature. Histopathology revealed impaired neuronal development and myelination. Transcriptomic analysis of ED18 whole brains showed DEGs enriched in neurodevelopment, stimulus response, and homeostasis pathways. RT-qPCR confirmed hypothalamic sensitivity to ETM: up-regulation of heat-shock gene HSP70, antioxidant gene GPX1, the inflammatory marker IL-6, and apoptotic genes CASP3, CASP6, CASP9; elevated neurodevelopmental marker DCX, indicative of a stress-responsive neuronal state; and reduced orexigenic neuropeptide AGRP. Conclusions: ETM improves heat tolerance in layers but compromises hatching performance and brain development, with widespread perturbation of hypothalamic stress responses and neurodevelopmental gene networks. These findings elucidate the mechanisms underlying ETM and provide a reference for enhancing thermotolerance in poultry.