The impact of lettuce-derived polyphenols on laying hens: Production performance, antioxidant status, immune function, and cecal microbiota profiles

This study aimed to evaluate the effects of dietary supplementation with polyphenol-rich lettuce extract (PRLE) derived from the high-polyphenol lettuce cultivar Binfen-1 (BF-1) on production performance, antioxidant capacity, immune function, and cecal microbiota composition of Hyline Brown laying hens. A total of 140 healthy pre-laying hens (15 days before egg production) were randomly assigned to 4 groups: a control group fed a corn-soybean basal diet, and three experimental groups (TR1, TR2, TR3) fed the basal diet supplemented with 0.05%, 0.1%, and 0.15% PRLE, respectively. Diets were fed for 12 wk, afterwards 10 bird per group were euthanized for sampling blood and cecal digesta. Production performance monitoring showed that compared with the control group, 0.05% and 0.15% PRLE supplementation significantly increased the laying rate during Weeks 1-4 (p < 0.05), but no significant effects on laying rate were observed in Weeks 5-8 or 9-12 (p > 0.05). For serum antioxidant indices, PRLE supplementation significantly reduced malondialdehyde (MDA) content and increased glutathione peroxidase (GSH-Px) activity across all experimental groups (p < 0.05). In terms of immunity, 0.1% PRLE significantly elevated serum immunoglobulin (Ig) G and IgM concentrations (p < 0.05). 16S rRNA gene sequencing revealed that PRLE supplementation increased the relative abundances of Firmicutes, Desulfobacterota, and Actinobacteriota while decreasing Proteobacteria at the phylum level(p < 0.05). At the genus level, it reduced the abundance of the conditional pathogen Peptococcus and increased the abundances of beneficial bacteria (e.g., Bacteroides, Lachnospiraceae, Butyricicoccus, Romboutsia) (p < 0.05). PICRUSt2 functional prediction indicated that PRLE enriched pathways related to infectious bacterial diseases, cell motility, and the immune system, while reducing pathways associated with drug resistance and glycan metabolism. Non-targeted metabolomics showed that PRLE significantly altered the cecal metabolite profile and modulated key pathways including arachidonic acid metabolism, bile secretion, and steroid hormone biosynthesis. In conclusion, dietary PRLE supplementation enhances the antioxidant capacity and immune function of Hyline Brown laying hens, improves cecal microbiota composition and metabolic profiles, but exerts only transient (Weeks 1-4) positive effects on laying rate under the tested conditions. This study provides a scientific basis for the application of PRLE in commercial laying hen production, with future research needed to optimize its dosage, supplementation duration, and age-specific efficacy.