Metabolome Combined with 16S rDNA Sequencing Reveals a Novel Mechanistic Insight into the Collaboration of Resveratrol and β-Hydroxy-β-Methylbutyric Acid in Regulating the Meat Quality of Tibetan Sheep Through Altering Rumen Microbiota

Abstract

Resveratrol (RES), a natural polyphenol, and β-hydroxy-β-methylbutyric acid (HMB), a key metabolite of leucine, are emerging as potent feed additives in animal production. This study investigated the individual and combined effects of dietary RES and HMB on gut microbiota, metabolic profiles, and meat quality in Tibetan sheep. A total of 120 two-month-old male lambs were randomly assigned to four experimental groups: control (C, basal diet, n = 6), RES (basal diet + 1.5 g/d RES, n = 6), HMB (basal diet + 1.25 g/d HMB, n = 6), and RES-HMB (basal diet + 1.5 g/d RES + 1.25 g/d HMB, n = 6), with 16S rDNA sequencing and LC-MS/MS analyses performed on rumen fluid and longissimus lumborum (LL). Meat quality improved significantly in all supplemented groups, the RES-HMB co-administration exhibited the most pronounced effects, suggesting a synergistic interaction. These improvements were linked to the activation of amino acid (AA) and unsaturated fatty acid biosynthesis pathways, leading to increased levels of AAs and polyunsaturated fatty acids (PUFAs). Concurrently, dietary RES and HMB supplementation enriched the relative abundance of beneficial gut microbiota, notably Christensenellaceae_R-7_group and Solibacillus, which further promoted the production of short-chain fatty acids, AAs, and PUFAs. The study highlights the role of rumen microbiota in regulating muscle metabolism and meat quality, offering a new scientific basis of strategies for using green feed additives in Tibetan sheep.

Keywords: Tibetan sheep; meat quality; metabolites; rumen microbiota.

Figure 1

Impact of RES and/or HMB supplementation on MyHC gene expression in longissimus lumborum (LL) muscle of Tibetan Sheep. ** p < 0.01.

Figure 2

The Orthogonal partial least squares-discriminant analysis (OPLS-DA) score plots illustrate the distinctions between the C and RES groups (A), the C and HMB groups (B), and the C versus RES-HMB groups (C). Volcano plots were generated to highlight the differential metabolites identified in the C vs. RES (D), C vs. HMB (E), and C vs. RES-HMB (F) comparisons. Top 15 KEGG pathways enriched in comparisons: C vs. RES (G), C vs. HMB (H), C vs. RES-HMB (I).

Figure 3

(A) The optimal soft-thresholding power was determined by analyzing its impact on mean connectivity. The red horizontal line indicates the signed R² (Scale-free Topology Model Fit), and the blue horizontal line represents the threshold for the scale-free topology fit. (B) A hierarchical clustering dendrogram illustrates the grouping of differential metabolites (DMs). (C) A bar plot displays the distribution of DMs across identified modules. (D) A heatmap visualizes the correlations between module eigengenes and key phenotypic traits. (E–J) Correlation analysis between the top six modules with the highest number of metabolites and meat quality-related traits ((E) MM.light cyan, (F) MM.royal blue, (G) MM.bisque 4, (H) MM.gray 60, (I) MM.darkturquoise, (J) MM.dark gray).

Figure 4

Impact of RES and HMB supplementation, both separately and together, on rumen short-chain fatty acids. * p < 0.05, ** p < 0.01.

Figure 5

The Venn diagram depicting the operational taxonomic units (OTUs) of the ruminal microbiota among the four groups (A), followed by the analysis of variance (B) and Principal Coordinate Analysis (PCoA) plots representing the overall rumen microbiota samples (C). The relative abundance of bacterial communities is illustrated at both the phylum (D) and genus (E) levels across the four sample groups. The Comparison of microbial relative abundance at the phylum (F) and genus (G) levels as shown by the violin plot. ** p < 0.01.

Figure 6

Spearman correlation analysis between gene expression and meat traits in the LL muscle of Tibetan sheep (A), meat phenotypes and rumen metabolites (B), ruminal short-chain fatty acids (SCFAs), microbiota composition, and core metabolites (C). * p < 0.05, ** p < 0.01, *** p < 0.001.