Changes in Rumen Microbiology and Metabolism of Tibetan Sheep with Different Lys/Met Ratios in Low-Protein Diets

Abstract

In ruminants, supplementing appropriate amounts of amino acids improves growth, feed utilization efficiency, and productivity. This study aimed to assess the effects of different Lys/Met ratios on the ruminal microbial community and the metabolic profiling in Tibetan sheep using 16S rDNA sequencing and non-target metabolomics. Ninety-two-month-old Tibetan rams (initial weight = 15.37 ± 0.92 kg) were divided into three groups and fed lysine/methionine (Lys/Met) of 1:1 (LP-L), 2:1 (LP-M), and 3:1 (LP-H) in low-protein diet, respectively. Results: The T-AOC, GSH-Px, and SOD were significantly higher in the LP-L group than in LP-H and LP-M groups (p < 0.05). Cellulase activity was significantly higher in the LP-L group than in the LP-H group (p < 0.05). In the fermentation parameters, acetic acid concentration was significantly higher in the LP-L group than in the LP-H group (p < 0.05). Microbial sequencing analysis showed that Ace and Chao1 indicators were significantly higher in LP-L than in LP-H and LP-M (p < 0.05). At the genus level, the abundance of Rikenellaceae RC9 gut group flora and Succiniclasticum were significantly higher in LP-L than in LP-M group (p < 0.05). Non-target metabolomics analyses revealed that the levels of phosphoric acid, pyrocatechol, hydrocinnamic acid, banzamide, l-gulono-1,4-lactone, cis-jasmone, Val-Asp-Arg, and tropinone content were higher in LP-L. However, l-citrulline and purine levels were lower in the LP-L group than in the LP-M and LP-H groups. Banzamide, cis-jasmone, and Val-Asp-Arg contents were positively correlated with the phenotypic contents, including T-AOC, SOD, and cellulase. Phosphoric acid content was positively correlated with cellulase and lipase activities. In conclusion, the Met/Lys ratio of 1:1 in low-protein diets showed superior antioxidant status and cellulase activity in the rumen by modulating the microbiota and metabolism of Tibetan sheep.

Keywords: Ovis aries; amino acids; antioxidants; digestive enzymes; low-protein diets; off-target metabolomics; rumen microbes.

Figure 1

Rumen microbial diversity among the three groups. Venn diagram showing the number of common or unique OTUs (A). Principal component analysis (PCA) by the weighted Unifrac of beta diversity (B). Anosim analysis (C). Shannon index box plot (D). Ace index box plot (E). Chao1 index box plot (F). LP-H, LP diet supplemented with the Lys/Met ratio at 3. LP-M, LP diet supplemented with the Lys/Met ratio at 2. LP-L, LP diet supplemented with the Lys/Met ratio at 1.

Figure 2

Composition and differences of the highly abundant microbial community at different Lys/Met ratios in the rumen. Percentage composition of the predominant phyla (A). The relative abundance of Firmicutes (B), Bacteroidetes (C), Proteobacteria (D), and Acidobacteria (E). Percentage composition of the predominant genus (F). The relative abundance of uncultured rumen bacterium (G), Prevotella 1 (H), Rikenellaceae RC9 gut group (I), and Succiniclasticum (J). ns represent no significant difference. ** p < 0.05. LP-H, LP diet supplemented with the Lys/Met ratio at 3. LP-M, LP diet supplemented with the Lys/Met ratio at 2. LP-L, LP diet supplemented with the Lys/Met ratio at 1.

Figure 3

The circles radiating from the inside out in the cladistic diagram represent taxonomic levels from phylum to genus (or species). The red, blue, and green areas represent different groups, and the red, blue, and green nodes in the branches represent microbial groups that play an important role in each group, while the yellow nodes represent microbial groups that play no important role in any of the three groups (A). Significantly different bacterial taxa identified by the linear discriminant analysis effect size (B). LP-H, LP diet supplemented with the Lys/Met ratio at 3. LP-M, LP diet supplemented with the Lys/Met ratio at 2. LP-L, LP diet supplemented with the Lys/Met ratio at 1.

Figure 4

Volcano diagram of three groups of metabolites. Positive ion mode (A). Negative ion mode (B). LP-H, LP diet supplemented with the Lys/Met ratio at 3. LP-M, LP diet supplemented with the Lys/Met ratio at 2. LP-L, LP diet supplemented with the Lys/Met ratio at 1.

Figure 5

Principal component analysis (PCA) model score scatter plot, orthogonal partial least-squares discriminant analysis (OPLS-DA) model, and permutation test of rumen metabolic profiling. Principal component analysis (PCA) in positive and negative ion modes (A). Three sets of OPLS-DA diagrams and three sets of permutation test diagrams for positive ion mode (B). Three sets of OPLS-DA diagrams for negative ion mode and three sets of permutation test diagrams (C). LP-H, LP diet supplemented with the Lys/Met ratio at 3. LP-M, LP diet supplemented with the Lys/Met ratio at 2. LP-L, LP diet supplemented with the Lys/Met ratio at 1.

Figure 6

Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis between treatment groups. LP-H vs. LP-M (A). LP-H vs. LP-L (B). LP-M vs. LP-L (C). LP-H, LP diet supplemented with the Lys/Met ratio at 3. LP-M, LP diet supplemented with the Lys/Met ratio at 2. LP-L, LP diet supplemented with the Lys/Met ratio at 1.

Figure 7

Microbiome–metabolome network heat map (A,B). Heat map of the correlation between microbiome and phenotype (C). Heat map of correlation between metabolomics and phenotype (D). * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001.