Dietary chlorogenic acid enhances intestinal barrier function and modulates cecal microbiota in laying hens under H2O2-induced oxidative stress

Oxidative stress is a well-known challenge in poultry production. While chlorogenic acid (CGA) is recognized for its antioxidant, anti-inflammatory, antibacterial, and lipid-lowering properties, its protective role against oxidative stress-induced intestinal damage in laying hens has not been extensively explored. This study investigated the effects of dietary CGA on the intestinal structure, barrier integrity, and cecal microbiota in laying hens exposed to hydrogen peroxide (H₂O₂). A sum of 240 Hy-Line Brown hens (43-wk-old) was divided into 4 groups (n = 6 replicates/group, 10 hens/replicate). Groups were fed a basal diet with CGA at 0 mg/kg (control, H₂O₂) or 600 mg/kg (600 mg/kg CGA, 600 mg/kg CGA + H₂O₂) for 12 weeks. Intraperitoneal injections of 10 % H₂O₂ were performed on days 64 and 78 in the H₂O₂ and 600 mg/kg CGA + H₂O₂ groups. The results showed that CGA pretreatment prevented the H₂O₂-induced oxidative stress, as evidenced by reducing MDA and H₂O₂ levels, along with enhanced activities of T-SOD, CAT, and GSH-Px (P < 0.05). CGA administration significantly countered the decline in productivity, villus height (VH), and the VH-to-crypt depth (CD) ratio, as well as the increase in CD caused by H₂O₂ (P < 0.05). Furthermore, CGA ameliorated the adverse effects of H₂O₂ on intestinal barrier function, inflammation, and immune response, including preventing H₂O₂-induced downregulation of occludin, TGFβ, IL-10, IgA, cluster of differentiation 3D (CD3D), and CD4 gene expression, and upregulation of TLR4 and TNFα genes and MyD88 protein expression in the gut tissues. Additionally, H₂O₂ was found to disrupt the structural composition of the cecal microbiota, resulting in an increased relative abundance of potential pathogenic taxa such as unclassified_f_Synergistaceae and uncultured_bacterium_Treponema. This disruption was accompanied by a decrease in the relative abundance of potentially beneficial bacterial species, including Bacteroides_caecicola, Collinsella_massiliensis_g_collinsella, unclassified_g_Oscillibacter, and uncultured_bacterium_g_Turicibacter. Furthermore, elevated serum levels of diamine oxidase (DAO) and lipopolysaccharide (LPS) were observed. More importantly, pre-treatment with CGA ameliorated the microbiota structure and serum DAO and LPS levels in laying hens subjected to H₂O₂. Our findings collectively indicate that CGA may mitigate H₂O₂-induced oxidative stress, barrier dysfunction, and immune impairment, while enhancing egg production and gut microbiota in laying hens. These findings highlight the novel role of CGA in mitigating oxidative stress-induced intestinal damage and microbiota dysbiosis in laying hens, providing new insights into its potential as a feed additive for improving poultry health and productivity.