Integrated multi-omics analysis reveals coordinated microbial, metabolic, and ovarian immune responses to dietary quercetin in aged laying chickens

Ovarian aging is associated with declining laying performance, but the short-term physiological and molecular responses of aged laying hens to dietary quercetin remain incompletely characterized. In a 30-d feeding trial, 180 Hy-Line White hens (480 d old) received diets supplemented with 0, 200, or 600 mg/kg quercetin (6 cages per treatment; 10 hens per cage). Egg production was recorded by cage, and ovarian, biochemical, and molecular endpoints were assessed at day 30. Cecal microbiota, serum metabolomics, and ovarian transcriptomics were compared between the control and 600 mg/kg groups (n = 6 hens per group). Compared with controls, hens receiving 600 mg/kg quercetin had a higher four-week mean laying rate (90.67% vs. 87.74%), more small white follicles, and increased hepatic and ovarian antioxidant enzyme activities. ANCOM supported a difference in only one genus-level taxon (DEV114). Untargeted metabolomics selected 552 exploratory features based on nominal significance criteria, including elevated signals for features putatively annotated as tryptophan- and indole-related metabolites. Ovarian analyses showed increased CYP1B1 expression, reduced NLRP3 protein abundance, altered immune- and apoptosis-related transcripts, and lower TUNEL positivity in SWFs. Cross-omics associations did not remain significant after false discovery rate correction, limiting inference about independent microbiota–metabolite–ovary relationships. Together, these findings provide an integrated characterization of short-term reproductive and molecular responses to dietary quercetin in aged laying hens and highlight candidate metabolic and immune signatures for targeted mechanistic investigation.