93 Phosphoenolpyruvate carboxykinase supports bovine embryo development in the absence of carbohydrates

Carbohydrates, including glucose and fructose, are considered important metabolic substrates during early embryo development; however, bovine embryos can be produced in vitro using carbohydrate-free media. We have shown that inhibition and knockdown of phosphoenolpyruvate carboxykinase (PEPCK), a rate limiting enzyme of gluconeogenesis, impairs early embryonic development in vitro, although its function has not been fully defined. Since glycolytic intermediates are required for important metabolic pathways, such as the pentose phosphate pathway, we hypothesised that PEPCK-mediated gluconeogenic activity sustains pre-implantation embryo growth in the absence of carbohydrates. We used hydrazine sulfate (HS), a PEPCK inhibitor, to determine the effects of reduced PEPCK activity during cleavage stage and blastocyst development in standard and carbohydrate-free conditions. Bovine cumulus–oocyte complexes (COC) were matured for 23 h and fertilized using female sex-sorted sperm (Day 0). At 18 h after fertilization, potential zygotes (n = 1310) were separated into four groups for 8 days of culture in a defined 2-step culture system (CDM1/2; De La Torre-Sanchez et al. 2006 Rep. Fertil. Dev. 18, 597) at 38.5°C, 5% CO₂, 5% O₂, and 90% N₂): (1) control, using standard media containing fructose as the carbohydrate source, (2) control + 0.25 mM HS, (3) fructose-free medium, and (4) fructose-free medium + 0.25 mM HS. Four replicates were performed to compare developmental endpoints including: cleavage rate, cell number at ~72 h, and blastocyst rate on Day 8. Cell numbers at 72 h were characterised as normal (6–10c), delayed (4–5c), and very delayed (2–3c). Logistic regression and Dunnett’s test were used for statistical comparisons. Control and carbohydrate-free medium resulted in similar cleavage rates, cell numbers, and blastocyst development rates 18% and 21%, respectively). The addition of 0.25 mM hydrazine sulfate to both culture conditions was detrimental to blastocyst formation (control +HS, 0.5% and carbohydrate-free +HS, no blastocysts), although HS exposure did not affect the percentage of cleaved embryos in either group. However, at 72 h, cleaved embryos cultured in carbohydrate-free medium +HS resulted in fewer normal developing embryos (P < 0.0001) and an increased number of delayed and very delayed embryos (P < 0.0001) when compared to embryos cultured in control medium +HS. Therefore, PEPCK inhibition did not affect cell numbers at 72 h when media contained fructose (control). Our study demonstrated that cleavage-stage bovine embryos are more sensitive to PEPCK inhibition by HS in the absence of carbohydrates, suggesting a specific function of PEPCK to support energy production, biosynthesis, or redox balance from noncarbohydrate sources. Further understanding of this metabolic adaptation could lead to future selection of metabolically superior embryos to improve embryo transfer success rates.