Exploring the effects of resveratrol and β-hydroxy-β-methylbutyric acid under different protein levels on the ileal health of tibetan sheep

Abstract

Introduction: Resveratrol (RES) and β-hydroxy-β-methylbutyric acid (HMB) have been shown to improve growth performance in Tibetan sheep by regulating the gut microbiota. This study explored the effects of RES and HMB supplementation on the microbial composition and metabolite levels in the ileum of Tibetan sheep receiving diets with different protein levels.

Methods: In a 2 × 2 factorial arrangement, consisting of dietary protein levels (12% and 14%) and feed additive levels (no addition; and RES 1.50 g/d and HMB 1.25 g/d addition). A total of 120 healthy two-month-old male Tibetan lambs (16.87 ± 0.31) were randomly divided into 4 groups (n = 6): 12% protein level group (L), the 12% protein level supplemented with RES and HMB group (L-RES-HMB); the 14% protein level group (H); and the 14% protein level supplemented with RES and HMB group (H-RES-HMB).

Results: The results demonstrated that the activities of digestive enzymes (β-amylase, trypsin, lipase, and cellulase), the levels of antibodies (IgA and IgM), and the contents of short-chain fatty acid (SCFA) (butyric acid) were significantly increased in the H-RES-HMB group (P < 0.05). Additionally, RES and HMB supplementation affected the morphology of ileum tissue, improving the villus height, crypt depth, and mucosal thickness (P < 0.05). Microbial analysis revealed that compared with the L-RES-HMB group, the H-RES-HMB group had a higher abundance of Planctomycetota, Solibacillus, and Paenibacillus (P < 0.05). Metabolomics analysis revealed a total of 229 significantly different metabolites, of which Irinotecan, Erdosteine thioacid, 4,4'-diaminodiphenylmethane, and Morphine N-oxide emerged as the key up-regulated metabolites. These differential metabolites were mainly enriched in pathways such as protein digestion and absorption, metabolic pathways, and mineral absorption.

Discussion: Overall, when the dietary protein content was 14%, digestive enzyme activities, immune responses, and SCFAs levels in the ileum were improved, and the mucosal morphology of the ileum was enhanced. When the 14% protein diet was supplemented with RES and HMB, the concentration of butyric acid was increased. This increase was due to the regulation of the ileum microbiota (Firmicutes and Clostridium_sensu_stricto_1) and metabolites (xanthine and uric acid), which promoted the activities of digestive enzymes and immune responses and improved mucosal morphology in the ileum.

Keywords: Tibetan sheep; metabolomics; microbiota; resveratrol; β-hydroxy-β-methylbutyric acid.

FIGURE 1

Effects of resveratrol and β-hydroxy-β-methylbutyric acid under different protein levels on the digestive enzyme activity (A) and immune status (B) of ileal contents. L: diet with 12% protein level. L-RES-HMB: diet with 12% protein level supplemented with 1.50 g/d RES and 1.25 g/d HMB. H: diet with 14% protein level. H-RES-HMB: Diet with 14% protein level supplemented with 1.50 g/d RES and 1.25 g/d HMB. IgA: Immunoglobulin A. IgM: Immunoglobulin M. IgG: Immunoglobulin G. TNF-α: tumor necrosis factor-α. IL-1β: Interleukin-1β. Different letters on the shoulder mark indicate significant difference (P < 0.05), the same letter or no letter indicates that the difference is not significant (P ≥ 0.05).

FIGURE 2

Effects of resveratrol and β-hydroxy-β-methylbutyric acid under different protein levels on the ileum morphology. Representative histological images of ileum slides stained with hematoxylin-eosin (200×).

FIGURE 3

Effects of resveratrol and β-hydroxy-β-methylbutyric acid under different protein levels on the ileal microbiology. (A) OTU Venn diagram of the overlap of ileum microbiota. (B) Principal coordinate analysis (PCoA) of bacterial communities. (C) Anosim analysis in the ileum contents of Tibetan sheep. (D) Relative abundance of microbial community proportion at the phylum and genus levels. (E) Different bacterial phylum and genus between groups, green for L-RES-HMB group, red for H-RES-HMB group. *P < 0.05, **P < 0.01. (F) Linear discriminant analysis effect size (LEfSe). (G) KEGG function prediction.

FIGURE 4

Effects of resveratrol and β-hydroxy-β-methylbutyric acid under different protein levels on the ileal metabolite. (A) Positive ion OPLS-DA score plots. (B) Negative ion OPLS-DA score plots. (C) Permutation test plots of positive ions. (D) Permutation test plots of negative ions. (E) Volcano plots of differential metabolites. (F) Analysis of KEGG pathway. The size of the bubbles indicates the number of differential metabolites enriched in the pathway, while the color of bubbles indicates the significance of enrichment in the pathway. The larger the value, the more significant the enrichment. up: significantly upregulated metabolites. nodiff: no significantly different metabolites. down: significantly downregulated metabolites.

FIGURE 5

Correlation analysis. (A) Spearman correlation heat map of ileal bacteria and digestive enzyme activity, immune activity, ileal mucosal morphology and short-chain fatty acid concentration. (B) Spearman correlation heat map between ileal metabolome and digestive enzyme activity, immune activity, ileal mucosal morphology and short-chain fatty acid concentration. (C) Spearman correlation heat map of ileal bacteria and metabolome.