Transcriptomic Insights into Thermal Manipulation and Heat Stress in Skeletal Muscles of Broiler Chickens

Heat stress (HS) poses a substantial burden to poultry production, compromising muscle integrity, growth performance, and overall welfare of broiler chickens. Elevated ambient temperatures disrupt energy metabolism, impair muscle function, and activate cellular stress responses. Embryonic thermal manipulation (TM) has emerged as a promising strategy to enhance thermotolerance through developmental reprogramming; however, the molecular basis underlying TM, acute, and chronic HS effects in skeletal muscle remains incompletely understood. In this study, bulk RNA sequencing of the pectoralis major muscle was performed in broilers using a factorial experimental design. Eggs were incubated under thermoneutral (37.8°C, 56% RH) or thermal manipulation (38.5°C, 65% RH, 18 hours per day) conditions, and then the chickens were later subjected to either acute heat stress (35 ± 0.5°C for 12 h on day 22 post-hatch) or chronic heat stress (35 ± 0.5°C for 5 consecutive days from day 18 to 22 post-hatch). Transcriptomic analysis identified distinct molecular profiles associated with TM and HS. Acute HS resulted in pronounced transcriptional changes, including activation of immune signaling, stress-responsive pathways, and suppression of metabolic and energy-related processes, while chronic HS affected subtle transcriptional changes related to immune and inflammatory response. Also, TM induced persistent modulation, altering the muscle response to acute HS, partially preserving metabolism and energy generation with enriched pathways related to cellular organization, ion homeostasis, and muscle maintenance. Integrative functional analyses using over-representation analysis (ORA) and gene set enrichment analysis (GSEA) revealed that TM reshaped the transcriptional landscape by promoting potentially adaptive responses rather than stress-induced disruption. Collectively, our findings demonstrate that embryonic TM impacts long-lasting transcriptional plasticity in skeletal muscle and that acute and chronic HS induced both shared and different expression profiles. This work provides molecular insight into muscle-specific heat adaptation and supports the incorporation of TM-based strategies into sustainable poultry production systems facing warming climates.