Int. J. Environ. Res. Public Health 2010, 7 2037 Embryos were cultured in WM supplemented with CZBt medium containing glucose and growth factors (glutamine and taurine) and 0.3% BSA (CM) at 37ºC in an atmosphere of 5% CO2 in air, for five days. Radioimmunoassay Serum samples were analyzed for E2, using radioimmunoassay previously validated in our laboratory [14]. The sensitivity of E2 assay was 2 pg/tube and the intra-and inter-assay coefficients of variation were 4.9 and 10.8%, respectively. Experiment 2 In vitro Culture of Embryos in Bioavailable HU Folliculogenesis and ovulation were induced in adult female C57BL/6J strain of mice (N = 10) and mated to breeder males as described in experiment 1. Two-cell embryos were recovered at the time line indicated in experiment 1 and washed as detailed in experiment 1. Normal 2-cell embryos were selected based on the criteria listed in experiment 1 and subjected to continuous in vitro culture in CM in the presence or absence of bioavailable HU (steady serum concentration post HU treatment = 18.0 g HU/mL for an hour before significant reduction close to nadir; [15]) at 37 ºC in an atmosphere of 5% CO2 in air, for five days. At the end of the culture period, the percentage of embryos at blastocyst stage of development in the HU-treated and control group was evaluated. Experiment 3 Intermittent Culture in vitro, of Embryos in Bioavailable HU Experiment 3 was essentially a repetition of experiment 2 except that 2-cell embryos in the treatment group were cultured in CM containing HU (18.0 g HU/mL) for an hour followed by two washes in CM prior to in vitro culture in the absence of HU. This experiment was designed to simulate the in vivo exposure of embryos to bioavailable HU. Embryos in the control group were exposed to saline as described for embryos in the HU treatment group prior to being cultured in CM. This process was repeated everyday for the 5 days of in vitro culture. At the end of culture period, the percentage of embryos at blastocyst stage of development in the intermittent HU-treated and control group was evaluated. Statistical Analyses Data on ovarian weight, ovulation rate and serum concentrations of E2 were compared by unpaired “t” test while those on fertilization rate and embryo development were analyzed by Chi-Square.