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.

Simple Summary: Dietary protein satisfies growth performance requirements and contributes to the maintenance of the rumen environment. High protein intake increased the total volatile fatty acids (VFAs), propionic acids and butyric acids by regulating the microbiome (Prevotella 1, Rikenellaceae RC9 gut group, Candidatus Saccharimonas, Ruminococcus 1, Coprostanoligenes group, Ruminococcaceae UCG-014, Lachnospiraceae NK3A20 group and Succiniclasticum) and metabolome (stearic acid, oleic acid, palmitic acid, erucic acid, 6-hydroxymelatonin, indole-3-acetamide and taurine), thereby altering the papillae length and papillae width in the rumen of Tibetan sheep. 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. [ABSTRACT FROM AUTHOR]