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

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. Graphical Single-cell and transcriptomics from 1.79 million cells identify the forestomach epithelium as the dominant site of antioxidant activity and oxidative phosphorylation (OXPHOS) in the bovine gastrointestinal tract. Along the basal-to-luminal differentiation axis, antioxidant capacity and OXPHOS rise in a coordinated metabolic–redox gradient. Primary rumen cell assays show that this gradient is strengthened by Cys-Cys-Cys supplementation or modulation of GPX1 expression, demonstrating a direct causal influence on epithelial metabolism. Key redox-metabolic regulators, including GPX1 and GSTP1 , mediate these responses. Overall, antioxidants actively drive metabolic remodeling and promote epithelial differentiation in the cattle forestomach epithelium. Highlights A single-cell transcriptomic atlas of 1.79 million bovine cells revealed the forestomach epithelium as one of the most antioxidant-active tissues. As the forestomach epithelium differentiates from the basal layer towards the luminal surface, both mitochondrial respiration and antioxidant capacity progressively intensify, establishing a coupled metabolic–redox gradient. Functional assays identified GPX1 as a key antioxidant target mediating OXPHOS and cell differentiation.