Dietary urolithin a improves hepatic antioxidant function and laying performance in aging hens via Nrf2 signaling pathway activation

The decline in productivity and increased immune stress associated with hepatic oxidative stress in aging laying hens limit the efficiency of egg production. Urolithin A (UA), a natural metabolite, has been shown to activate the Nrf2 antioxidant pathway in mammals; however, its effects and underlying mechanisms in laying hens remain unclear. This study aimed to systematically evaluate the dose-response effects of dietary UA on the health and production performance of laying hens, with an emphasis on its role in activating the Nrf2 pathway. Fifty (45-week-old) Roman Pink laying hens were randomly assigned to one of five dietary treatments: a basal diet supplemented with 0, 50, 200, 400, or 800 mg/kg UA for 6 weeks. The production performance, organ indices, serum and liver lipid profiles, antioxidant status, immune markers, liver histology, and Nrf2 pathway-related mRNA and protein expression were assessed. An in vitro model of primary chicken hepatocytes was also used to validate the direct effects of UA. Compared with the control group (0 mg/kg UA), 200 mg/kg UA supplementation showed significantly improved the laying rate and egg weight, reduced the feed-to-egg ratio, and lowered the serum total cholesterol and triglyceride levels (P < 0.05). This optimal dose also enhanced the total antioxidant capacity and superoxide dismutase and glutathione peroxidase activities in both the serum and liver, decreased the malondialdehyde levels, and increased the immunoglobulin (IgY and IgA) concentrations (P < 0.05). UA at 400 and 800 mg/kg doses did not enhance egg laying (P > 0.05), but significantly reduced liver and abdominal fat indices (P < 0.05). All UA doses lowered hepatic TC content (P < 0.05), but only the higher doses (400 and 800 mg/kg) significantly decreased liver VLDL-C levels (P < 0.05). At the molecular level, 200 mg/kg UA significantly upregulated the mRNA and protein expression of Nrf2 and its key downstream targets in the liver (P < 0.05). These effects were corroborated in vitro, where 5 μmol/L UA significantly promoted hepatocyte viability and upregulated Nrf2 and downstream mRNA expression (P < 0.05). This study is the first to provide systematic evidence that supplementing with 200 mg/kg UA may enhance late-phase laying performance by boosting antioxidant capacity, regulating lipid metabolism, and improving immune function, likely through activation of the hepatic Nrf2 pathway, suggesting its potential as a natural feed additive for sustained peak production.