Figure 4

Effects of slow freezing and vitrification on mitochondrial activity, intracellular ROS levels and mt/ROS colocalization in single mouse morulae and blastocysts. In each group, energy status and ROS intracellular levels are expressed as mean ± SD of Mitotracker Orange CMTM Ros (Panel a) and DCF (Panel b) fluorescence intensity of individual embryos in arbitrary densitometric units (ADU) and mt/ROS colocalization is expressed as mean ± SD of Pearson’s correlation coefficient of individual embryos (Panel c). In embryos at the morula stage, energy status, expressing embryonic mt activity, was reduced by cryopreservation (P < 0.05) whereas in embryos at the blastocyst stage, it did not change. In embryos at the morula stage, ROS levels increased after vitrification (P < 0.05) whereas no changes were observed in embryos at the blastocyst stage. Vitrified morulae and blastocysts showed significantly higher Pearson’s correlation coefficient than controls (P < 0.05). Numbers of analyzed embryos per group are indicated on the bottom of each histogram. One-way ANOVA followed by Multiple Comparison Dunn’s method: comparisons among morula stage embryos: a,b P < 0.05; comparisons among blastocyst stage embryos: c,d P < 0.05.