Effects of embryonic thermal manipulation on jejunal and cecal microbiome in response to post-hatch acute heat stress challenge

Thermal manipulation (TM) during embryogenesis has been proposed as a sustainable strategy to enhance thermotolerance and resilience in broiler chickens. However, its long-term influence on gut microbiota composition, particularly under post-hatch acute heat stress (AHS), remains unclear. This study investigated the effects of TM on the jejunal and cecal microbiota of Ross broilers following AHS. Fertile eggs were incubated under standard conditions (37.8 C, 56% RH) for controls or thermally manipulated at 39 C and 65% RH for 18 h/day during embryonic days 10-18. On day 35 post-hatch, birds were exposed to 35 C for three hours to induce AHS, and jejunal and cecal contents were analyzed by 16S rRNA gene sequencing (V3-V4 region, Illumina MiSeq). Alpha diversity indices (Shannon, evenness) did not differ significantly between treatments, although the cecum consistently exhibited higher richness and evenness than the jejunum. Beta diversity analysis revealed strong separation between intestinal segments but no treatment-driven clustering. Firmicutes and Bacteroidota dominated all groups, with regional enrichment of Lactobacillus and Ligilactobacillus in the jejunum, and Faecalibacterium in the cecum. Differential abundance analysis confirmed that 30 taxa differed between organs, while no significant taxa were detected between TM, control, or AHS groups after FDR correction. Collectively, these results demonstrate that the intestinal segment exerts a greater influence on microbial composition than embryonic or post-hatch thermal exposure. The stability of microbial diversity under acute thermal challenge suggests that TM's beneficial effects on thermotolerance are likely mediated through host physiological or epigenetic mechanisms rather than microbiome remodeling.