The consumption of oxygen, uptake of pyruvate and glucose and production of lactate were determined for groups of bovine embryos produced in vitro from the one-cell to the blastocyst stage (day 0\p=n-\6of culture). Measurements were made in Hepes-buffered synthetic oviduct fluid medium supplemented with 1.0 mmol pyruvate l \ m=-\ 1, 10 mmol d,l-lactate l \m=-\1 and 1.5 mmol glucose l \m=-\1and also 3 mg BSA ml \m=-\1and, from day 5 of development, 10% (v/v) fetal calf serum. The amount of ATP production was determined from oxygen consumption and the proportion of glucose taken up that could be accounted for by lactate production. The data revealed that oxygen consumption was relatively constant from days 0\p=n-\4of culture (0.24\p=n-\0.27 nl per embryo h \ m=-\ 1) , but increased with the initiation of compaction (0.39 nl per embryo h\m=-\1) and continued to increase with the formation and expansion of the blastocoel (0.9 nl per embryo h \m=-\1). Both pyruvate and glucose uptake followed similar patterns. Furthermore, when plotted against oxygen consumption, both pyruvate and glucose uptake increased significantly (P < 0.001) in a linear relationship (R2 = 0.61 and 0.49, respectively). Lactate production also increased with development and accounted for 40% of glucose uptake 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 onwards. ATP production followed a similar pattern to that of oxygen consumption (60\p=n-\85 pmol per embryo h \ m=-\ 1 from day 0 to day 4) increasing with compaction (124 pmol per embryo h \ m=-\ 1) and blastulation (221 pmol per embryo h\m=-\1). For precompaction stages, 93\p=n-\96%of ATP production was derived from oxidative phosphorylation, decreasing to 82% with compaction. 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 exogenous substrates are present in the culture medium. The data revealed that bovine embryos were dependent on oxidative phosphorylation for energy (ATP) production at all stages of pre-elongation development, with perhaps a shift in dependence towards glycolysis in conjunction with compaction. It follows that oxidizable substrates, such as pyruvate and certain amino acids, are preferred in embryo culture medium during development in vitro.

Vascularly perfused Fallopian tubes have been used to study the formation and composition of human tubal fluid and the response to adrenergic agents. An artery serving the tube was cannulated and perfused with Medium 199 supplemented with bovine serum albumin (BSA) and antibiotics. A second cannula was attached to the fimbriated end for native tubal fluid collection. The preparation was viable for up to 2 h. Tubal fluid was only obtained in tubes removed in the proliferative and early secretory phases of the ovarian cycle. Isoproterenol (1 mM) added to the perfusate stimulated fluid production, whereas dibutyryl cyclic AMP (1 mM) reduced fluid formation by 66%. Glucose, pyruvate and lactate concentrations in tubal fluid, measured by microfluorescence assays, were 1.11, 0.14 and 5.4 mM respectively. The concentrations of 17 amino acids in tubal fluid were measured by high performance liquid chromatography following fluorescence derivatization. Arginine (0.19 mM) > alanine (0.11 mM) > glutamate (0.09 mM) were present in highest concentration in all phases of the cycle. All 17 amino acid concentrations in tubal fluid were below those in the vascular perfusate. These data provides the basis for a culture medium whose composition mimics the physiological environment to which early human embryos are exposed.

-The quality of in vitro-produced bovine embryos remains variable.

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