Stage-specific requirement of phosphate for development of rat 1-cell embryos in a chemically defined medium

Rat 1-cell embryos, recovered from naturally mated females, were cultured in a chemically defined medium (mR1ECM). When examined after 24, 56, 64 and 80 h of culture, embryos developed to the 2-cell (100%), 4-cell (93%), 4-cell to 8-cell (97%) and > or = 8-cell (95%) stages, respectively. When 0.4 M phosphate (NaH2PO4) was added to the medium after 0, 24, 56 and 64 h of culture, percentages (0-67%) of embryos that developed to the blastocyst stage after 115 h of culture were lower than that (86%) in the medium without phosphate. However, addition of phosphate after 80 h of culture accelerated blastocyst formation; a significantly higher percentage (94%) of blastocysts was obtained after 110 h of culture in this medium compared with when phosphate was not added (67%). When phosphate was added to the medium after 64 h, almost all (97%) 8-cell embryos developed to the blastocyst stage by 100 h of culture but development of 4-cell to 7-cell embryos was inhibited (22-63%). Acceleration of blastocyst formation was caused by addition of phosphate rather than by the exchange of the medium. The stimulatory effect of phosphate on embryo development was observed at concentrations of 0.1-1.2 mM. The mean numbers of cells (54.5-60.9 cells) in blastocysts examined after 115 h of culture were increased by the addition of 0.4-1.6 mM phosphate at 80 h as compared with blastocysts from cultures without phosphate (46.6 cells).