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Invitro culture (IVC) systems fail to completely recapitulate the invivo environment, resulting in metabolic stress during pre-implantation development and reduced blastocyst quality. We hypothesised that IVC-induced metabolic dysregulation in bovine embryos is mediated by changes in expression and/or activity of protein biomarkers associated with key metabolic pathways. Our objectives were to determine (1) expression of enzymes involved in glycolysis (HK-2, PKM2, LDHA, B and C isoforms), entry into the tricarboxylic acid (TCA) cycle (PDH), energy sensing/fatty acid oxidation (AMPK), and the metabolic signalling pathways (AKT, ERK, STAT3, 4EBP1) at the 1-cell (1C), 8- to 16-cell (8–16 C), and Day 7 blastocyst (d7BL) stage; and (2) evaluate the functional activity of these proteins both invivo (superovulated and flushed) and invitro (IVM/IVF/IVC) produced embryos using capillary Western blot (Protein-Simple, JESS; n=1 embryo/stage; n=3 replicates). For each protein, expression was normalized with total protein abundance in the same capillary and functional activity was determine based on the ratio of phosphorylated (p) to total (t) protein abundance in each sample. Data were analysed using a two-sample t-test. Results demonstrated significantly (P