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Mitochondrial membrane potential assay
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The d-6 blastocysts were incubated in PZM-5 containing 2.5 µM 5,5′,6,6′‐tetrachloro-1,1′,3,3′-tetraemyl-imidacarbocyanine iodide (JC‐1) (M34152; Thermo Fisher Scientific) at 38.5 °C in 5% CO2 for 30 min. The membrane potential was calculated as the ratio of red fluorescence, which corresponded to activated mitochondria (J-aggregates), to green fluorescence, which corresponded to less-activated mitochondria (J-monomers) [46]. Fluorescence was visualized using an epifluorescence microscope (Nikon).
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