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.

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.

Resveratrol (RES) and β-hydroxy-β-methylbutyric acid (HMB) have antioxidant, anti-inflammatory, and other beneficial properties. Here, we hypothesize that supplementation with RES and HMB could affect the rumen function in Tibetan sheep. This study aims to explore the effects of RES and HMB supplementation at different protein levels on the rumen microbial and metabolite compositions of Tibetan sheep. Four treatments (n = 30) were prepared according to a 2 × 2 factorial arrangement, with two dietary protein levels (12% and 14%) and two feed additives (RES 1.50 g/day and HMB 1.25 g/day). The experimental treatments were fed diets with 12% CP level non-supplemented (L), 12% protein with RES and HMB (L-RES-HMB), 14% CP level non-supplemented (H), and 14% protein with RES and HMB (H-RES-HMB), respectively. Our results indicated that the trypsin, chymotrypsin, and lipase were significantly increased in the H-RES-HMB group (p < 0.05), while the lipopolysaccharide (LPS) concentration was significantly reduced (p < 0.05). The alpha diversity analysis found that the ACE indices of the L-RES-HMB, and H-RES-HMB groups was significantly higher than that of the L group (p < 0.05). Additionally, compared with the L, L-RES-HMB, and H groups, it was found that the abundance of Euryarchaeota, Spirochaeota, and Metanobrevibacter was significantly increased in the H-RES-HMB group, while the abundance of Proteobacteria was significantly decreased (p < 0.05). A total of 745 significantly different metabolites were identified, of which 14 metabolites were common among the three comparative groups. Differential metabolites were mainly enriched in pathways including the pyrimidine metabolism, the glycine, serine, and threonine metabolisms, and ABC transporters. Overall, CP level and RES/HMB exhibited positively interaction effect on digestive enzyme activity and antioxidant capacity. Dietary RES and HMB supplementation on 14% CP level improved the ruminal digestive enzyme activity and antioxidant capacity through modulating the microbial community and regulating the metabolism.

This study investigated the effects of dietary resveratrol (RES) and β-Hydroxy-β-methyl butyric acid (HMB) on immune, oxidative, and morphological changes in the livers of Tibetan sheep using transcriptomics and metabolomics. One hundred and twenty male Tibetan lambs of a similar initial weight (15.5 ± 0.14 kg) were randomly divided into four groups with thirty lambs per treatment: (1) H group (basal diet without RES or HMB); (2) H-RES group (1.5 g/day of RES); (3) H-HMB group (1250 mg/day of HMB); (4) H-RES-HMB group (1.5 g/day of RES and 1250 mg/day of HMB). The experiment was conducted for 100 days, including a pre-test period of 10 days and a formal period of 90 days. The results showed significantly increased concentrations of glutathione peroxidase, superoxide dismutase, and IgM in the H-RES-HMB group (p < 0.05), while the malondialdehyde levels were significantly decreased (p < 0.05). The glycolytic indices including creatinine kinase (CK), malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) were significantly increased in the H-RES-HMB group compared with the others (p < 0.05). A histological analysis showed that the hepatic plate tissue in the H-RES-HMB group appeared normal with multiple cells. The transcriptomic analysis showed that the expression of genes associated with the calcium signaling pathway (MYLK2, CYSLTR2, ADCY1, HRH1, ATP2B2, NOS2, HRC, ITPR1, and CAMK2B) and the NF-κB signaling pathway (BCL2 and CARD14) in the H-RES-HMB group were upregulated. The key differential metabolites (d-pyroglutamic acid, DL-serine, DL-threonine, fumarate, and glyceric acid) were enriched in the pathways associated with D-amino acid metabolism, the citrate cycle (TCA cycle), and carbon metabolism. The combined transcriptomic and non-targeted metabolomic analyses showed the co-enrichment of differential genes (NOS2 and GLUD1) and metabolites (fumarate) in arginine biosynthesis-regulated glycolytic activity, whereas the differential genes (ME1, SCD5, FABP2, RXRG, and CPT1B) and metabolites (Leukotriene b4) co-enriched in the PPAR signaling pathway affected the immune response by regulating the PI3K/AKT and cGMP/PKG signaling. In conclusion, the dietary RES and HMB affected the hepatic antioxidant capacity, immune response, and glycolytic activity through modulating the transcriptome (BCL2, CAMK2B, ITPR1, and IL1R1) and metabolome (DL-serine, DL-threonine, fumaric acid, and glycolic acid).

With global protein prices on the rise, lowering protein levels in animal feed, together with balancing diet composition and reducing nitrogen emissions, can both reduce the environmental impact of agriculture and save on feed costs. However, the formulation of an ideal amino acid (AA) composition is crucial for better protein utilization by livestock. This study aimed to investigate the effects of different lysine to methionine ratios on the antioxidant capacity and immune function of the rumen in Tibetan sheep. Ninety male Tibetan sheep, weaned at 2 months of age, were randomly divided into three groups (1:1, 2:1 and 3:1 lysine ratios) and subjected to a 100‐day feeding trial. RNA sequencing (RNA‐seq) was utilized to analyse the impact of different AA ratios on gene expression in rumen tissue, whereas the levels of antioxidant enzymes (total antioxidant capacity [T‐AOC], superoxide dismutase [SOD], glutathione peroxidase [GSH‐Px] and catalase [CAT]) and immunoglobulins (immunoglobulin A [IgA], immunoglobulin G [IgG] and immunoglobulin M [IgM]) were evaluated. The results indicated that the 1:1 group significantly upregulated the expression of PTGS2, PLA2G12A and PLA2G4 genes, enhancing antioxidant enzyme activity, reducing free radical production and modulating systemic immune responses. COL16A1 and KCNK5 were highly expressed in the protein digestion and absorption pathway, maintaining the structural integrity and function of the rumen epithelium. BMP4 and TGFBR2 were significantly enriched in the cytokine–cytokine receptor interaction pathway and positively correlated with CAT and T‐AOC. ITGA8 was upregulated in the 1:1 group, participating in the regulation of various cellular signalling pathways. ATP2B1 was enriched in the cyclic guanosine monophosphate (cGMP)– protein kinase G (PKG) signalling and mineral absorption pathways, primarily influencing oxidative stress and immune responses by regulating intracellular calcium ion concentration. This study demonstrates that a 1:1 lysine to methionine ratio is most beneficial for enhancing the antioxidant capacity and immune function of the rumen in Tibetan sheep.