Thermal manipulation and heat stress: The transcriptomic landscape of the spleen in broiler chickens.

Embryonic thermal manipulation (TM) has emerged as a promising strategy to enhance broiler thermotolerance, yet the transcriptional mechanisms by which it impacts immune organ responses to post-hatch thermal challenge remain poorly understood. Particularly given that heat stress (HS) compromises immune competence, suppresses lymphocyte proliferation, promotes apoptosis, and causes tissue damage, reducing organ size and functionality, leading to increased susceptibility to pathogens in heat-stressed chickens. Herein, we characterize the splenic transcriptomic profiles of broiler chickens subjected to embryonic TM (38.5°C, 18h/day, embryonic days 10-18) and post-hatch acute heat stress (AHS; 35°C, 12 h, day 22) or post-hatch chronic heat stress (CHS; 35°C, 8 days, days 14-22). TM did not impair hatchability and increased body weight measurements near marketing age. TM induced temporally dynamic transcriptional programs in the spleen across development, transitioning from immune activation and proliferative processes at embryonic and early post-hatch stages to ion homeostasis and transmembrane transport regulation by post-hatch day 22. Differential expression analysis across all experimental groups revealed that TM, AHS, and CHS each induced largely non-overlapping transcriptional profiles, indicating group-specific thermal responses. AHS elicited a proteostasis-driven response dominated by heat shock protein induction and glucose metabolism, whereas CHS produced markedly fewer DEGs. TM chickens under AHS exhibited recurrent upregulation of NAD, carbohydrate, phosphate, and DNA metabolism pathways, along with enrichment in hydrogen peroxide catabolism, suggesting that embryonic TM altered metabolic and antioxidant pathways. The identified genes and functional pathways reflected coordinated crosstalk among proteostatic, metabolic, and immunological networks that chickens use to maintain homeostasis under thermal challenge. Ultimately, understanding the molecular basis of thermoacquisition and heat adaptation is critical for improving breeding strategies that support poultry welfare and production in hot climate regions.