The effect of inhibiting ATP production via oxidative phosphorylation during pericompaction of in vitro produced bovine embryos was investigated. This was achieved by: (i) varying the atmospheric O2 concentration (0, 1, 2, 4 and 7%); (ii) addition of oxidative phosphorylation inhibitors, NaN3 and antimycin A; and (iii) addition of 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation from electron transport. The development of embryos under various O2 concentrations from day 5 to day 7 of development indicated that an optimal concentration occurred at about 2%. Addition of NaN3 revealed that doses above 100 mumol l-1 were toxic to embryo development, but that concentrations of 5-10 mumol l-1 stimulated embryo development by 10-25%. A similar result was observed after addition of 2,4-dinitrophenol, whereas antimycin A was inhibitory at doses as low as 1 mumol l-1. At concentrations of NaN3 or 2,4-dinitrophenol that stimulated embryo development, the number of cells of the resulting blastocysts was also significantly increased. Addition of NaN3 from day 1 of development inhibited subsequent development. Metabolic data of NaN3-treated embryos revealed that O2 uptake was significantly lower at inhibitory doses (100 mumol l-1). A significant (P < 0.05) log linear increase in glucose uptake was measured between the three concentrations of NaN3 (0, 10 and 100 mumol l-1). These results demonstrate that ATP production via oxidative phosphorylation is essential for bovine embryo development in vitro. However, transient (subacute) inhibition appears to be beneficial to embryo development and the number of cells, perhaps by creating a more favourable intracellular environment.

The effects of media (TCM199 vs. synthetic oviduct fluid, SOF), sera (foetal calf serum, FCS vs. human serum, HS), gas atmosphere (5% CO2 in air vs. 5% CO2, 5% O2 and 90% N2) and coculture with bovine oviduct epithelial cells (cells vs. no cells) on the in-vitro development of in-vitro matured and fertilized bovine oocytes were examined. Immature oocytes surrounded with compacted cumulus cells were cultured for 24 h in TCM199 supplemented with 10% FCS, 10 micrograms follicle-stimulating hormone (FSH)/ml and 10 micrograms luteinizing hormone (LH)/ml, 1 microgram oestradiol/ml, and 1 x 10(6) granulosa cells at 39 degrees C under 5% CO2 in air. In-vitro fertilization was performed with frozen-thawed, heparin-treated (100 micrograms/ml, 15 min) spermatozoa from 2 bulls. Oocytes were incubated with 2.5 x 10(6) spermatozoa/ml for 24 h and then cultured in one of 16 treatments for 7 days. Cleavage (2-8-cell) and development to blastocysts were recorded on Days 2 and 7, respectively, after the start of culture. SOF was superior to TCM199 for cleavage (P less than 0.01), development to blastocysts (P less than 0.001) and for proportion of cultured ova resulting in blastocysts with at least 60 or at least 100 nuclei (P less than 0.001). FCS was superior to HS for development to blastocysts (P less than 0.001) and 5% oxygen was superior to air for the proportion of ova reaching at least 60 cells (P less than 0.01). For cleavage and development to blastocysts, there was an interaction between serum and cells (P less than 0.01). In the presence of cells, ova preferred FCS, in their absence, serum had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)

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