Integrated metabolome and microbiome analysis deciphers the effects of resveratrol and β-hydroxy-β-methylbutyric acid on jejunal function under different protein levelsin Tibetan sheep

Research has demonstrated that resveratrol (RES) and β-hydroxy-β-methylbutyric acid (HMB) promote gastrointestinal health by altering the intestinal microbiome. The present study aimed to evaluate the impacts of RES and HMB on the bacterial community composition and metabolomic profiles of the jejunum in Tibetan sheep. A total of 120 male Tibetan lambs, with an average initial body weight of 16.87 ± 0.31 kg, were randomly assigned to four experimental groups according to a 2×2 factorial design. The experimental diets contained 11.19% protein diet, or 12.69% protein diet supplemented with RES/HMB (H-RES-HMB and L-RES-HMB, respectively), and 11.19% protein diet, or 12.69% protein diet without supplemented with RES/HMB (LCP and HCP, respectively). The findings indicated a significant increase in jejunal digestive enzyme activities (α-amylase and chymotrypsin), antioxidant capacity (catalase, glutathione peroxidase, and superoxide dismutase), and immune responses (including immunoglobulins A, G, and M) in the H-RES-HMB group, in contrast to a notable decrease in markers of oxidative stress and pro-inflammatory cytokines (interleukin 1β, interleukin 6, and tumor necrosis factor alpha) (<i>P</i> < 0.05). Morphological assessments revealed a significant reduction in crypt depth within the H-RES-HMB group (<i>P</i> < 0.05). Among the short-chain fatty acids, butyric acid concentration was significantly elevated in the H-RES-HMB group (<i>P</i> < 0.05). Additionally, mRNA expression levels of barrier-associated genes, including <i>OCLN, Muc-2,</i> and <i>ZO-1</i> in the jejunum, were significantly enhanced in the H-RES-HMB group. Microbial community analysis demonstrated that the inclusion of RES and HMB in the 14% protein diet resulted in an increase in the relative abundance of <i>Firmicutes</i>, <i>Bacillus</i>, and <i>Methanobrevibacter</i>, while simultaneously reducing the abundance of <i>Proteobacteria, Escherichia,</i> and <i>Shigella</i>. Notably, the increased abundance of these beneficial bacteria was positively correlated with butyric acid concentrations. Furthermore, metabolomic analysis indicated higher levels of L-Arginine and His-Lys in the H-RES-HMB group, which also showed a positive correlation with butyric acid concentration, whereas isovaleric acid, D-xylose, and diacetyl concentrations were lower in this group. In conclusion, the findings suggest that a 14% protein diet is more effective in enhancing jejunal morphology and barrier function. Supplementation with RES and HMB in this diet resulted in elevated concentrations of volatile fatty acids, particularly butyric acid, by modulating microbial community composition (notably <i>Firmicutes, Bacillus,</i> and <i>Methanobrevibacter</i>) and metabolite profiles (including Gamma-aminobutyric acid, Succinate, L-Arginine, and His-Lys). A synergistic effect was found between the crude protein level (14%) and dietary RES/HMB on villus phenotype, digestive enzyme capacity, and antioxidant activity.IMPORTANCETo thrive in harsh, high-altitude environments, Tibetan sheep require efficient nutrient absorption. Our study shows that a diet with optimal protein (12.69%) supplemented with natural additives-resveratrol and β-hydroxy-β-methylbutyric acid-significantly enhances gut health. This dietary regimen strengthened the intestinal barrier, suppressed harmful inflammation, and boosted local immunity. Furthermore, it enriched the gut microbiome with beneficial butyric acid-producing bacteria, a key nutrient for intestinal cells. This research establishes a practical nutritional strategy to improve intestinal function and overall resilience in Tibetan sheep, supporting sustainable livestock production in challenging plateau regions.