Effects of 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). In each group, mt/ROS colocalization is expressed as mean±SD of Pearson’s correlation coefficient of individual embryos (panel c). Representative mt/ROS colocalization scatterplots graph of a fresh (panel d) and a vitrified (panel e) blastocyst are shown. In embryos at the morula stage, mt activity did not change after vitrification whereas in embryos at the blastocyst stage, mt activity was significantly reduced (P<0.05). ROS levels significantly increased in vitrified embryos at morulaand blastocyst stages (P<0.001). Mt/ROS colocalizationsignificantly increased in vitrifiedmorulae. Numbers of analyzed embryos per group are indicated on the top of each histogram. Student’s t-Test: a,b P<0.05; c,d P<0.001.