Dietary energy levels modulate rumen metabolites and function in sheep by regulating the rumen microbiome

In intensive ruminant production, high-energy diets are commonly used to enhance animal productivity, as dietary formulation significantly influences rumen fermentation and microbial communities. This study investigated the effects of varying dietary energy levels on the rumen microbial community structure, function, and metabolic profiles in Small-tailed Han (STH) sheep. Thirty 6-month-old sheep were randomly assigned to three groups: high-energy (HE), conventional-energy (CE), and low-energy (LE). All groups were fed iso-nitrogenous diets formulated to provide high-, conventional-, and low-energy levels of 10.8, 9.5, and 8.2 MJ/kg of digestible energy (DE), respectively. Rumen content was collected post-slaughter and analyzed via metagenomic sequencing to assess microbial composition and function, alongside non-targeted metabolomics to characterize the rumen fluid metabolome. Results revealed that Bacteroidota and Bacillota were the dominant phyla. High-energy feeding significantly reduced the relative abundance of Bacteroidota while increasing that of Bacillota, leading to a markedly higher Bacillota-to-Bacteroidota ratio. Functional analysis indicated significant enrichment of carbohydrate metabolism pathways in the HE group, whereas the LE group exhibited enrichment in fundamental cellular processes such as ABC transporters and ribosome, indicating a "survival mode". Metabolomic analysis demonstrated that dietary energy levels substantially reshaped the rumen metabolomic profile. Metabolites in the HE group were enriched in pathways including steroid hormone biosynthesis and the prolactin signaling pathway, while the LE group showed enrichment in histidine metabolism and the TCA cycle. Several aromatic amino acid metabolic pathways were commonly enriched across comparisons. These findings indicate that while the composition of the dominant phyla (Bacteroidota and Bacillota) was conserved across diets with different digestible energy levels, this dietary variation altered community diversity, structure, functional potential, and profoundly reshaped the rumen metabolic environment. This study provides scientific evidence regarding the impact of dietary energy on rumen fermentation and production performance in fattening sheep.