Multi-omics analysis reveals protein-driven modulation of growth and metabolism in Gangba sheep under different dietary protein levels

This study investigated the effects of different dietary protein concentrations’ influence on Gangba sheep’s growth metrics, nutrient absorption efficiency, carcass characteristics, meat quality via integrated 16S rRNA sequencing, metabolomics and transcriptomic analysis. These multi-omics approaches revealed that moderate protein supplementation coordinately regulated gut microbial composition and host metabolic and immune pathways. Forty one-year-old Gangba sheep were randomly allocated to four dietary treatments (n = 10 per group) receiving protein levels equivalent to 0.9 × (14.25%), 1.0 × (16.12%), 1.1 × (17.84%), and 1.2 × (19.31%) of the standard concentrate feed. Compared with the 0.9 × group, sheep fed higher protein levels exhibited significantly greater final body weight and average daily gain ( p < 0.05). The 0.9 × group also showed significantly higher fecal crude fiber, calcium, phosphorus, neutral detergent fiber, and acid detergent fiber contents, indicating lower nutrient utilization. Antioxidant status improved with increased dietary protein, as evidenced by higher superoxide dismutase and lower malondialdehyde, aspartate aminotransferase, and alanine aminotransferase levels. Carcass weight, dressing percentage, lean weight, and meat-bone ratio were significantly improved in protein-supplemented groups, whereas centrifugal loss and unfavorable meat color parameters were reduced. Higher dietary protein also enhanced gastrointestinal morphology by increasing gastric mucosal thickness and villus height while reducing crypt depth. The 1.1 × protein group showed the most favorable intestinal architecture, with the highest villus height-to-crypt depth ratio. 16S rRNA sequencing revealed increased relative abundance of Firmicutes and reduced Bacteroidota in the 1.1 × group, accompanied by enrichment of beneficial bacteria such as Faecalibaculum and reductions in potentially harmful genera, including Streptococcus, Marvinbryantia, and Eubacterium. Transcriptomic analysis identified 365 differentially expressed genes in the 1.1 × group, mainly enriched in pathways related to immune regulation, protein digestion and absorption, bile secretion, and mineral transport. Correlation analysis further demonstrated strong associations between specific microbial taxa and growth, nutrient utilization, antioxidant status, carcass characteristics, and intestinal morphology. Overall, moderate protein supplementation at 1.1 × the standard level optimally improved growth performance, nutrient utilization, antioxidant capacity, meat quality, gut microbial composition, and metabolic and immune functions in Gangba sheep.