Integrating 16S rRNA Sequencing and LC-MS-Based Metabolomics to Evaluate the Effects of Dietary Crude Protein on Ruminal Morphology, Fermentation Parameter and Digestive Enzyme Activity in Tibetan Sheep

Abstract

The dietary crude protein level could affect ruminal fermentation parameters and the microflora of ruminants. The present study's aim was to investigate the effects of different protein level diets on ruminal morphology, fermentation parameters, digestive enzyme activity, microflora and metabolites of Tibetan sheep. Ninety weaned lambs (initial weight of 15.40 ± 0.81 kg, 2 months old) were selected and randomly divided into three groups (six pens/treatment, five rams/pen). Dietary treatments were formulated with 13.03% (high protein, HP), 11.58% (moderate protein, MP) and 10.20% (low protein, LP), respectively. Compared with LP, both papillae length and papillae width were significantly promoted in HP and MP (p < 0.05). The concentrations of ammonia nitrogen, total VFAs, propionic acids and butyric acids in HP were significantly increased compared to those in MP and LP (p < 0.05). The activities of protease and α-amylase in HP were significantly greater than those of LP (p < 0.05). For the ruminal microbial community, higher proportions of phylum Prevotella 1 and Succiniclasticum and genus Rikenellaceae RC9 gut group and Ruminococcus 1 were observed in HP (p < 0.05). A total of 60 differential metabolites (DMs) (28 up, 32 down) between HP and MP; 73 DMs (55 up, 18 down) between HP and LP; and 65 DMs (49 up, 16 down) between MP and LP were identified. Furthermore, four pathways of the biosynthesis of unsaturated fatty acids, tryptophan metabolism, bile secretion and ABC transporters were significantly different (p < 0.05). The abundance of phylum Prevotella 1 was negatively associated with stearic acid and palmitic acid but positively associated with the taurine. The abundance of genus Ruminococcus 1 was negatively associated with stearic acid, oleic acid, erucic acid, Indole-3-acetamide and palmitic acid but positively associated with 6-hydroxymelatonin. In conclusion, a 13.03% CP level improved ruminal morphology, fermentation parameters and digestive enzyme activities through modulating the microbial community and regulating metabolism in Tibetan sheep.

Keywords: Tibetan sheep; metabolites; microbiota; protein level; rumen development.

Figure 1

Effect of dietary protein level on ruminal phenotypes. (A) Representative histological images of ruminal slide stained with hematoxylin–eosin (original magnification 200× μm). (B) The papillae height, papillae width and stratum corneum of rumen. * p < 0.05.

Figure 2

Effect of dietary protein level on bacterial community. (A) Alpha diversity analysis. (B) Beta diversity analysis. * p < 0.05.

Figure 3

Effect of dietary protein level on bacterial composition. (A) Bacterial composition at the phylum. Significantly different bacterial phylum (B) and genus (C) between groups. * p < 0.05.

Figure 4

Orthogonal partial least squares discriminant analysis (OPLS-DA) plot of metabolites in comparisons. (A) Positive ion mode. (B) Negative ion mode.

Figure 5

Metabolome view map of the differentially expressed metabolites identified. The deep color shows higher pathway impact values; the larger size demonstrates higher pathway enrichment. The important pathways selected for this experiment have been labelled in the figure.

Figure 6

Spearman correlation analysis. (A) Association analysis of microbial diversity with rumen fermentation parameters and digestive enzyme activity. (B) Association analysis of rumen metabolites with rumen fermentation parameters and digestive enzyme activity. (C) Association analysis of rumen papillae length with rumen fermentation parameters, digestive enzyme activity and microbial diversity. (D) Association analysis of rumen papillae width with rumen fermentation parameters, digestive enzyme activity and microbial diversity. The color intensity of the circle and line are proportional to the correlation values. * p < 0.05, ** 0.05 > p < 0.01, *** p < 0.01.

Figure 7

The Mantel correlation analysis indicated the correlation between rumen microorganisms and metabolites. The edge width corresponds to the distance correlation corresponding to the Mantel’s r statistic, and the edge color indicates statistical significance.

Figure 8

The mechanism diagram shows that the composition of rumen microbiota, rumen metabolites, fermentation parameters and digestive enzyme activity directly or indirectly alter rumen morphology at high protein levels. Red arrows indicate significant increases; green arrows indicate significant decreases.