The impact of embryonic thermal manipulation on the microbiome of the jejunum and cecum in response to post-hatch acute heat stress

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 (n = 182) were incubated under standard conditions (37.8°C and 56 % relative humidity), while the thermally manipulated group (n = 182) was incubated at 38.5°C and 65 % relative humidity for 18 h/day during embryonic days 10-18. On day 35 post-hatch, birds were exposed to 35°C and 60-65 % RH 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 (P > 0.05), although the cecum consistently exhibited higher richness and evenness than the jejunum (P < 0.05). 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 revealed 30 taxa that differed significantly (q < 0.05) at the family level across intestinal segments. In contrast, comparing the TM, AHS, and control groups within any single segment yielded no significant taxonomic differences after false discovery rate (FDR) correction. These results indicate that anatomical location within the intestine exerts a more substantial influence on microbial community composition than either embryonic or post-hatch heat exposure. The stability of microbial diversity under acute thermal challenge suggests that the beneficial effects of TM on thermotolerance, as reported in the literature, are likely mediated through host physiological or epigenetic mechanisms rather than microbiome remodeling.