Tissue-Specific Mitochondrial Functionality and Mitochondrial-Related Gene Profiles in Response to Maternal Nutrition and One-Carbon Metabolite Supplementation During Early Pregnancy in Heifers

Background: Mitochondria are essential for fetal development, regulating energy metabolism and metabolic programming. This study examined how maternal nutrition and one-carbon metabolite (OCM) supplementation during early gestation affect mitochondrial function in fetal liver and muscle at day 161 of gestation in beef heifers. Methods: Twenty-nine crossbred Angus heifers were assigned to one of four treatments in a 2 × 2 factorial design: control (CON; 0.45 kg/day ADG) or restricted gain (RES; −0.23 kg/day), with or without OCM supplementation. Treatments were applied from breeding to day 63 of gestation, after which all heifers received a common diet. Fetal liver and muscle tissues were collected at day 161. Mitochondrial respiration (Seahorse assay), mtDNA copy number (qPCR), and mitochondria-related gene expression (RNA-seq) were assessed. Results: In fetal liver, state 3 respiration was highest in CON + OCM, while state 4o respiration was lowest in RES + OCM (p ≤ 0.05). mtDNA copy number was greater in RES and +OCM groups. In fetal muscle, mtDNA copy number was influenced by gain, but respiration was unaffected. Transcriptomic analysis revealed more mitochondria-related differentially expressed genes (mtDEGs) in fetal muscle than liver (90% versus 10% of total mtDEG), with most genes downregulated in the RES and +OCM groups compared to the CON and −OCM groups (FDR ≤ 0.10). Conclusions: OCM supplementation enhanced mitochondrial respiration and biogenesis in fetal liver, likely via post-translational mechanisms. In contrast, fetal muscle showed downregulation of mitochondria-related genes without functional changes, indicating transcriptional reprogramming with potential effects on later metabolic function. These results underscore early gestation as a critical window for OCM-based nutritional interventions to improve metabolic outcomes in livestock.