Antioxidants promote metabolic remodeling in cattle rumen epithelium revealed by single‐cell resolution

Abstract Oxygen signaling is essential for cellular homeostasis and tightly linked to metabolism, growth, and survival. In dairy cows, oxidative stress, arising from an imbalance between reactive oxygen species and antioxidants, is a major postpartum challenge that contributes to disease susceptibility. Using single‐cell transcriptomes from 1,793,854 cells across 59 tissues, we analyzed oxygen signaling states within 1006 cellular clusters. The gastrointestinal tract (GIT) epithelium, particularly the forestomach, exhibits the strongest antioxidant activity, closely coupled to oxidative phosphorylation (OXPHOS) and glycolysis, with OXPHOS levels surpassing those of cardiomyocytes and hepatocytes (Cohen's d > 3.9, p < 0.001). Pseudotime and spatial transcriptomics demonstrated that both OXPHOS and antioxidant capacity increase progressively along the basal‐to‐luminal differentiation axis. Functional experiments in primary rumen epithelial cells showed that antioxidant supplementation or GPX1 modulation enhances mitochondrial respiration, boosts intracellular glutathione, and accelerates epithelial differentiation. Limited proteolysis‐mass spectrometry (LiP‐MS) analysis identified GPX1 , GSTP1 , COX7A2 , and COX6B1 as candidate targets mediating antioxidant‐driven metabolic remodeling. Together, these results reveal a redox‐governed metabolic program in the forestomach epithelium and highlight antioxidant interventions as a potential strategy to support epithelial development and mitigate oxidative stress‐related disorders in dairy cattle.