Abstract/Summary

Background: Perilla frutescens seeds (PFS) are gaining recognition as a natural alternative to antibiotics in livestock, supporting sustainable farming and animal health. However, the underlying molecular mechanisms through which PFS influence host immune function and antioxidant capacity, especially via the gut-liver-muscle axis, remain largely unknown. This study employed an integrative multi-omics approach to elucidate how PFS supplementation modulates the microbiota–gut–liver–muscle axis and enhances immune and antioxidant functions in lambs. Results: PFS supplementation markedly improved immune and antioxidant profiles, demonstrated by elevated serum levels of IL-10, IgM, IgG, GSH-PX, and SOD, and reductions in IL-1β, TNF-α, and MDA. Microbial analysis revealed elevated abundances of ruminal and intestinal taxa commonly associated with gut homeostasis and metabolic health (Christensenellaceae_R-7_group) and reduced levels of species with pathogenic or pro-inflammatory potential (Bacillus cereus and Clostridioides) in the ileum. Transcriptomic and metabolomic profiling of liver tissue indicated modulation of key inflammatory and bile acid signaling pathways, including the downregulation of TLR4, NLRP3, ATF3, CYP2J2, and LXR-α. PFS also increased hepatic concentrations of anti-inflammatory metabolites such as chlorquinaldol and indole-3-carboxaldehyde, while reducing levels of LysoPC(20:4) and phosphatidic acid. Correlation and mediation analyses revealed strong interconnections among gut microbiota, hepatic gene expression, lipid metabolites in liver and muscle, and systemic immune-antioxidant markers. Conclusion: These findings highlight the microbiota–gut–liver–muscle axis as a central mechanism through which PFS enhances immune function and antioxidant capacity in lambs. PFS supplementation represents a promising nutritional strategy to improve healthy lamb production, supporting the development of antibiotic-free and sustainable livestock systems.