OXYGEN-UPTAKE AND CARBOHYDRATE-METABOLISM BY IN-VITRO DERIVED BOVINE EMBRYOS

The consumption of oxygen, uptake of pyruvate and glucose and producti on of lactate were determined for groups of bovine embryos produced in vitro from the one-cell to the blastocyst stage (day 0-6 of culture). Measurements were made in Hepes-buffered synthetic oviduct fluid medi um supplemented with 1.0 mmol pyruvate l(-1), 10 mmol D,L-lactate l(-1 ) and 1.5 mmol glucose l(-1) and also 3 mg BSA ml(-1) and, from day 5 of development, 10% (v/v) fetal calf serum. The amount of ATP producti on was determined from oxygen consumption and the proportion of glucos e taken up that could be accounted for by lactate production. The data revealed that oxygen consumption was relatively constant from days 0- 4 of culture (0.24-0.27 nl per embryo h(-1)), but increased with the i nitiation of compaction (0.39 nl per embryo h(-1)) and continued to in crease with the formation and expansion of the blastocoel (0.9 nl per embryo h(-1)). Both pyruvate and glucose uptake followed similar patte rns. Furthermore, when plotted against oxygen consumption, both pyruva te and glucose uptake increased significantly (P < 0.001) in a linear relationship (R(2) = 0.61 and 0.49, respectively). Lactate production also increased with development and accounted for 40% of glucose uptak e at day 0 of culture (putative zygotes), increasing to 70% by day 2 ( eight-cell stage) and 100% of glucose uptake from day 4 of culture onw ards. ATP production followed a similar pattern to that of oxygen cons umption (60-85 pmol per embryo h(-1) from day 0 to day 4) increasing w ith compaction (124 pmol per embryo h(-1)) and blastulation (221 pmol per embryo h(-1)). For precompaction stages, 93-96% of ATP production was derived from oxidative phosphorylation, decreasing to 82% with com paction. ATP produced by oxidative phosphorylation could be accounted for by the uptake of pyruvate, suggesting that bovine embryos produced in vitro utilize little endogenous substrates when appropriate exogen ous substrates are present in the culture medium. The data revealed th at bovine embryos were dependent on oxidative phosphorylation for ener gy (ATP) production at all stages of pre-elongation development, with perhaps a shift in dependence towards glycolysis in conjunction with c ompaction. It follows that oxidizable substrates, such as pyruvate and certain amino acids, are preferred in embryo culture medium during de velopment in vitro.