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Dairy cattle with high (HM) versus low muscle (LM) reserves as determined by longissimus dorsi muscle depth (LDD) in late gestation exhibit differential muscle mobilization related to subsequent milk production. Moreover, branched-chain volatile fatty acid (BCVFA) supplementation increased blood glucose levels. We hypothesized that differences in HM and LM reflect distinct muscle metabolism and that BCVFA supplementation altered metabolic pathways. At 42 days before expected calving (BEC), Holstein dairy cows were enrolled in a 2 × 2 factorial study of diet and muscle reserves, by assignment to control (CON)- or BCVFA-supplemented diets and LDD of HM (>4.6 cm) or LM (≤4.6 cm) groups: HM-CON (n = 13), HM-BCVFA (n = 10), LM-CON (n = 9), and LM-BCVFA (n = 9). Longisumus dorsi muscle was biopsied at 21 days BEC, total RNA was isolated, and protein-coding gene expression was measured with RNA sequencing. Between HM and LM, 713 genes were differentially expressed and 481 between BCVFA and CON (P < 0.05). Transcriptional signatures indicated differential distribution of type II fibers between groups, with MYH1 greater in LM cattle and MYH2 greater in HM cattle (P < 0.05). Signatures of LM cattle relative to HM cattle indicated greater activation of autophagy, ubiquitin-proteasome, and Ca2+-calpain pathways. HM cattle displayed greater expression of genes that encode extracellular matrix proteins and factors that regulate their proteolysis and turnover. BCVFA modified transcriptomes by increasing expression of genes that regulate fatty acid degradation and flux of carbons into the tricarboxylic acid cycle as acetyl CoA. Molecular signatures support distinct metabolic strategies between LM and HM cattle and that BCVFA supplementation increased substrates for energy generation. NEW & NOTEWORTHY: Muscle biopsies of the longissimus dorsi of prepartum dairy cattle indicate that molecular signatures support distinct metabolic strategies between low- and high-muscle cattle and that branched-chain volatile fatty acid supplementation increased substrates for energy generation. [ABSTRACT FROM AUTHOR]