Integrated Transcriptomic and Metabolomic Analysis Reveals Gut Microbiota–Mediated Regulation of Muscle Fiber Composition and Metabolism in Germ‐Free Pigs

The gut microbiota is a key regulator of host metabolism and skeletal muscle physiology, yet its role in muscle fiber-type transformation in pigs remains unclear. Here, we used germ-free (GF) and specific pathogen-free (SPF) Rongcheng piglets to explore how microbial absence influences muscle development. GF piglets showed significantly reduced body weight and an increased proportion of fast-twitch fibers compared with SPF controls. Transcriptome sequencing identified 1332 differentially expressed genes (DEGs), notably those involved in cGMP-PKG, Thyroid hormone, HIF-1, VEGF, and Butanoate metabolism signaling pathways, which are essential for myofiber specification. Non-targeted metabolomic profiling revealed 320 differentially expressed metabolites (DEMs), with major alterations in Bile secretion. Integrated transcriptomic and metabolomic uncovered 10 KEGG pathways co-enriched with DEGs and metabolites, including Bile secretion, Ferroptosis, Inflammatory mediator regulation of TRP channels, and HIF-1 signaling pathway. Correlation analysis revealed that strong positive correlations were observed between fast-twitch fiber genes (e.g., PRKCA, MYOZ3, NFATC1) and fatty acid-related metabolites (e.g., Dodecanoic acid, Valproic Acid, and Decanoic acid), whereas negative correlations were detected between these genes and bile acid-related metabolites (e.g., Cholic acid and 7-Ketolithocholic acid), suggesting microbial metabolites is associated with muscle fiber phenotype via metabolic and signaling crosstalk. Collectively, our findings demonstrate that gut microbial absence in pigs disrupts bile acid metabolism and fatty acid metabolism, which in turn drives the shift of skeletal muscle fiber composition toward fast-twitch glycolytic fibers. These metabolites are identified as key microbial mediators linking the gut microbiota to the transcriptional regulatory function of porcine muscle fiber type specialization, providing new insights into the gut-muscle axis and highlighting the important role of microbiota-derived metabolites in maintaining porcine muscle fiber composition and metabolic balance.