Abstract
The transition period in dairy cows involves profound metabolic adaptations that challenge energy balance and liver function. This study evaluated the effects of rumen-protected methionine (RPM) and choline (RPC) on hepatic and skeletal muscle metabolism. Twenty-one multiparous Holstein cows from a 2 × 2 factorial design (CON, RPM, RPC) underwent liver and semitendinosus biopsies at -10, +7, and +20 d relative to parturition. Untargeted LC-MS metabolomics detected 2288 and 1454 molecular features in liver and muscle. Data were analyzed using mixed-model ANOVA (FDR ≤ 0.05), complemented by multivariate approaches including sparse PLS-DA and PERMANOVA to assess global metabolic variation. Metabolite annotation was performed using HMDB (±0.005 Da). Dietary supplementation significantly affected 105 hepatic metabolites, whereas time influenced 552 metabolites, generally reflecting increases or decreases in concentration from the prepartum to early postpartum periods. Network analysis identified nine hepatic co-expression modules associated with RPM and RPC. Hub metabolites included glucose-6-phosphate, mannose-6-phosphate, and sphingomyelins, indicating modulation of carbohydrate and lipid metabolism. In muscle, treatment effects were modest, with PERMANOVA and PLS-DA confirming limited discrimination among groups and a predominant temporal effect. Overall, RPM and, to a lesser extent, RPC modulated key hepatic metabolic pathways, supporting energy and redox homeostasis during early lactation. These findings highlight the potential of methyl-donor supplementation to enhance metabolic resilience at the tissue level in transition cows.