Effect of Dietary Nitrogen-to-Sulfur Ratio on Rumen Microbiota, Metabolites, and Ruminal Antioxidant Status in Tibetan Sheep

Optimizing the dietary nitrogen-to-sulfur (N:S) ratio is crucial for enhancing ruminal microbial fermentation and nutrient digestibility. This study investigated the effects of different dietary N:S ratios on ruminal antioxidant indices, digestive enzyme activities, immune parameters, volatile fatty acid (VFA) profiles, microbial communities, and metabolomic profiles in plateau-type Tibetan sheep. Ninety 2-month-old male Tibetan sheep (initial body weight, 15.55 ± 0.20 kg) were randomly assigned to three dietary treatments formulated with N:S ratios of 9.5:1 (HP-H), 8.5:1 (HP-M), and 7.5:1 (HP-L). Sheep fed the HP-H diet exhibited improved ruminal antioxidant indices compared with the other treatments, with CAT and T-AOC activities being higher than those of both the HP-M and HP-L groups, while SOD and GSH-Px activities were significantly higher only compared with those in the HP-L (p < 0.05). Furthermore, ruminal cellulase activity, as well as propionate and butyrate levels, were significantly higher in the HP-H group (p < 0.05). Microbiome analysis revealed that the HP-H diet enriched the relative abundances of Candidatus Saccharimonas, the Rikenellaceae RC9 gut group, and uncultured rumen bacteria. Correspondingly, untargeted metabolomics identified higher signal intensities of 3-hydroxyphenylacetic acid, nervonic acid, arachidonic acid (peroxide-free), and calciferol in the ruminal fluid of the HP-H group relative to the other treatments. In conclusion, when the nitrogen-to-sulfur ratio was 9.5:1, ruminal antioxidant enzyme activities were increased, and the concentrations of propionic acid and butyric acid were increased. The differential microorganisms and metabolites were mainly associated with lipid metabolism, as well as cofactor and vitamin metabolism.