Integrated proteomics and metabolomics profiling reveal mechanisms for the improvement of hoof health, liver function, and oxidative stress in lactating dairy cows fed rumen-protected biotin

Biotin is a vital coenzyme involved in diverse metabolic pathways and plays a key role in hoof health by supporting keratin synthesis and the protective barrier of the hoof. This study systematically investigated the effects of rumen-protected biotin (RPB) on hepatic metabolic networks, redox homeostasis, and hoof health in lactating dairy cows using an integrated multiomics approach. The RPB supplement consisted of 2.1% biotin (purity ≥99%), 62.9% glucose, and 35% hydrogenated palm oil fatty acids. Eighty multiparous Holstein cows were stratified by parity (2.48 ± 0.69), body weight (637.76 ± 55.71 kg), body condition score (3.01 ± 0.20), days in milk (141 ± 16), and average milk yield (32.35 ± 4.36 kg/d). They were assigned for 75-d using a randomized block design to 4 treatment groups: Control (Con; basal diet, n = 20), Low (0.5 g/d RPB, n = 20), Mid (1.0 g/d RPB, n = 20), or High RPB (2.0 g/d RPB, n = 20). Milk and blood samples were collected on d 0, 15, 30, 45, 60, and 75 for analysis of milk composition and serum biochemical parameters. Data were analyzed using mixed models with orthogonal polynomial contrasts to evaluate linear and quadratic effects of RPB. Among the markers of liver function, serum albumin increased but total cholesterol, alanine aminotransferase, aspartate aminotransferase, and total bilirubin decreased in a linear and quadratic fashion with higher doses of RPB. Feeding RPB increased serum total antioxidant capacity, glutathione, and superoxide dismutase in a linear and quadratic fashion, while malondialdehyde decreased. Incremental feeding of RPB decreased linearly the serum type II collagen C-terminal peptide concentration and lameness scores. Further, serum cartilage oligomeric matrix protein concentration decreased in a linear and quadratic fashion, whereas serum procollagen IIA N-terminal propeptide and hoof horn hardness increased in a linear and quadratic fashion. Based on serum biochemical and hoof health results on d 75, the Con and Mid groups were selected for proteomic and metabolomic analyses of serum. Proteomics revealed that RPB upregulated key proteins involved in antioxidant reactions and keratinization, including GSR, GCLC, GPX3, TGM1, and TGM3. Metabolomics identified L-cysteine, glycine, and pyruvate as key metabolites associated with RPB suggesting upregulated glutathione synthesis and flux through the tricarboxylic acid cycle. Integrated proteomics and metabolomics analyses revealed that GSR, GCLC, GPX3, TGM1, and TGM3 were positively correlated with L-cysteine and glycine but negatively correlated with γ-glutamylcysteine and palmitic acid. Overall, feeding RPB reduces oxidative stress and improves liver function in part by enhancing glutathione metabolism while reducing lipid peroxidation. Further, RPB promotes keratinization and limits cartilage degradation, thereby enhancing hoof health. These responses to dietary RPB supplementation provide molecular evidence for its targeted application in dairy herd nutrition management.