Integrative Multi-Omics Reveal Silymarin Alleviates Heat-Stress-Driven Hepatic Lipid Disruption in Laying Hens

Heat stress (HS) has emerged as a major environmental stressor, inducing oxidative stress, hepatic steatosis and impairing production performance and health in laying hens, with limited evidence-based nutritional interventions available. This study investigated the hepatoprotective effects of dietary silymarin (SIL) against chronic HS. In a 10-week trial, 252 43-week-old Hy-Line Brown hens were exposed to daily HS (32 ± 1℃, temperature-humidity index [THI] > 73) and fed either a basal diet or one supplemented with 100 mg/kg SIL. SIL significantly increased laying rate (P < 0.05) and improved albumen height, Haugh units, and shell strength by week 8 (P < 0.05). Histological analysis showed a 48% reduction in non-alcoholic fatty liver disease (NAFLD) activity score, with significantly decreased hepatic triglyceride content (P < 0.05); Oil Red O staining confirmed reduced lipid droplet accumulation. SIL restored redox balance by increasing plasma and hepatic total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) (P < 0.05), increasing hepatic catalase (CAT) and glutathione (GSH) levels while decreasing malondialdehyde (MDA) (P < 0.05). Untargeted plasma metabolomics identified 11 key metabolites related to 2-oxoglutarate and purine metabolism, while hepatic transcriptomics revealed 835 differentially expressed genes primarily in the PPAR signaling and fatty acid biosynthesis pathways. SIL suppressed de novo lipogenesis via downregulation of ACACA and FASN, and enhanced β-oxidation through upregulation of CPT1A and ACSL1 (P < 0.05). Molecular docking and Western blotting confirmed strong SIL binding to these targets and corresponding protein changes. Correlation networks associated ACSL1 and CPT1A with improved performance and antioxidant indices, while FASN, ACACA, and xanthosine showed inverse relationships. These findings emphasize the potential of SIL as a sustainable animal nutrition antioxidant additive, which can alleviate HS induced lipid disorders in the liver of laying hens and provide insights for livestock applications.