After fertilization, presumptive zygotes were cultured in mSOF for 0, 12, 22, 40, 70, 100, or 150 h to obtain embryos at the 1-cell, 2-cell, 3- to 4-cell, 5- to 8-cell, 9- to 16-cell, morula, or blastocyst stages, respectively. Then embryos at each developmental stage were finally cultured in mSOF containing activin A or follistatin at concentrations of 10 ng/ml or no addition for up to 220 h postinsemination. The numbers of embryos that developed to the blastocyst and hatched blastocyst stages were recorded at 175 and 220 h postinsemination, respectively. buy flovent inhaler
Each experiment was replicated on several different days using a microdrop of presumptive zygotes or embryos per treatment on each day. Data were analyzed with the StatView (Abacus Concepts Inc., Berkeley, CA) software package. For analysis of development, each microdrop was considered to be an experimental unit, and the percentages of cleavage, morulae, blastocysts, and hatched blastocysts were calculated within each microdrop. Differences in the mean percentages of cleavage, morulae, blastocysts, and hatched blastocysts among the experimental groups were analyzed by one-way ANOVA. When ANOVA revealed a significant effect of the treatments, treatments were compared by Dunnett’s procedure as a multiple comparison procedure. The total numbers of cells in morulae and blastocysts were subjected to logarithmic transformation and then assigned by one-way ANOVA. Differences of p < 0.05 were taken as significant.