**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** BOVINE IN VITRO FERTILIZATION (IVF) AND SUBSEQUENT EMBRYO CULTURE HAS BECOME THE PRIMARY SOURCE OF EMBRYOS USED FOR TRANSFER INTO SURROGATES; HOWEVER, THE POTENTIAL OF THESE EMBRYOS TO DEVELOP INTO BLASTOCYSTS AND ESTABLISH PREGNANCY IS LOW. RAPID CELL PROLIFERATION IS REQUIRED TO MAINTAIN EMBRYO VIABILITY AND ESTABLISH PREGNANCY, ALTHOUGH THE METABOLIC MECHANISMS THAT SUPPORT THIS GROWTH ARE NOT UNDERSTOOD. PHOSPHOENOLPYRUVATE CARBOXYKINASE (PEPCK) HAS RECENTLY BEEN SHOWN TO PROMOTE CELL PROLIFERATION IN CANCER CELLS BY IMPROVING THE USE OF MULTIPLE SUBSTRATES TO SUPPORT ENERGY AND BIOSYNTHETIC DEMANDS. WE HAVE SHOWN THAT PEPCK IS AN IMPORTANT ENZYME DURING EARLY EMBRYO DEVELOPMENT; HOWEVER, THE SPECIFIC METABOLIC FUNCTION OF PEPCK HAS NOT BEEN ELUCIDATED. THIS PROJECT INVOLVES A SET OF EXPERIMENTS TO DETERMINE THE FUNCTION OF PEPCK, INCLUDING AN ABILITY TO INCREASE BIOSYNTHESIS OF IMPORTANT COMPOUNDS FROM ALTERNATIVE SOURCES AND IMPROVE EMBRYO DEVELOPMENT IN A RANGE OF ENVIRONMENTS.SPECIFIC AIM 1 WILL ASSESS THE ROLE OF PEPCK TO SUSTAIN EMBRYO DEVELOPMENT IN LOW OR ABSENT GLUCOSE CONCENTRATIONS. SPECIFIC AIM 2 WILL DETERMINE THE EFFECTS OF PEPCK TO GENERATE IMPORTANT BIOSYNTHETIC PRODUCTS FROM NON-GLYCOLYTIC SOURCES TO SUPPORT DEVELOPMENT USING STABLE ISOTOPE METABOLIC TRACERS. FOR THE STUDY, BOVINE OOCYTES WILL BE FERTILIZED AND CULTURED TO THE BLASTOCYST STAGE. A PEPCK INHIBITOR WILL BE USED DURING CULTURE TO DETERMINE THE ROLE OF PEPCK TO PROMOTE EMBRYO SURVIVAL IN LOW-GLUCOSE CONDITIONS AND PROMOTE PYRUVATE AND GLUTAMINE METABOLISM TO SUPPORT GROWTH. AN UNDERSTANDING OF THESE METABOLIC ADAPTATIONS COULD LEAD TO FUTURE TARGETED SELECTION OF METABOLICALLY SUPERIOR EMBRYOS AND IMPROVED EMBRYO CULTURE SYSTEMS.