Integrated multi-omics reveals genistein-mediated protection of broiler intestine against heat stress via metabolic-microbial-barrier crosstalk.

Heat stress severely impairs intestinal health in broilers, highlighting the urgent need for effective nutritional interventions. This study examined whether dietary genistein (150 mg/kg) mitigates heat stress-induced intestinal injury in Ningdu Yellow chickens. A total of 288 one-day-old chicks were assigned to a 2 × 2 arrangement of thermal environment (thermoneutral or intermittent heat stress at 37 ± 2°C) and diet (basal diet or basal diet plus 150 mg/kg genistein), forming four groups: control, genistein, heat stress, and heat stress plus genistein. Duodenal morphology, antioxidant indices, and tight-junction and inflammatory proteins were measured; duodenal transcriptomes, cecal microbiota, and serum metabolites were profiled and integrated by correlation analysis. Phenotypic analyses demonstrated that genistein supplementation significantly (P < 0.05) alleviated heat stress-induced duodenal morphological damage, oxidative stress, barrier injury, and inflammatory responses. RNA sequencing showed that heat stress downregulated duodenal genes in protein processing, metabolic pathways, and tight junction signaling, while genistein reversed suppression of the tight junction pathway, modulated co-expression modules linked to barrier genes, and restored protein levels of CLDN1, CLDN3, CLDN5, and CDC42. Cecal 16S rRNA sequencing showed that genistein partially restored the abundance of beneficial genera (Akkermansia and Lactobacillus) and β-diversity disrupted by heat stress. Serum metabolomic analysis indicated that genistein regulated amino acid metabolism, fatty acid biosynthesis and AMP-activated protein kinase signaling pathways. Multi-omics integration constructed a "metabolic-microbial-intestinal barrier" crosstalk axis, with key correlations including a putative Arg-Trp-Arg feature correlated with Lactobacillus and CLDN3, CLDN11, and JAM3. Collectively, these findings demonstrate that genistein exerts multi-target protective effects against heat stress together with improved duodenal antioxidant and inflammatory indices and changes in microbial-metabolic correlations, thereby providing a theoretical basis for its application as a functional feed additive to mitigate heat stress in poultry.