Impact of Lysine to Methionine Ratios on Antioxidant Capacity and Immune Function in the Rumen of Tibetan Sheep: An RNA‐Seq Analysis

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.