Cmf2hc

We detected ROS and GSH levels using oocytes that had reached the 4-cell developmental stage. The oocytes were incubated in PBS-PVA, containing 10 µM 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA, Beyotime, Shanghai, China) and 10 µM 4-chloromethyl-6,8-difluoro-7-hydroxycoumarin (CMF2HC, Beyotime, Shanghai, China), in the dark at 37.5 °C for 30 min. Next, we washed the oocytes four times in PBS-PVA and placed them into 3 µL of droplets of PBS-PVA. Finally, pictures were photographed using an inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan), and the fluorescence intensity was analyzed using ImageJ version 8.0.2 software (NIH, Bethesda, MD, USA). We collected 20 oocytes at the 4-cell stage of development each time for ROS and GSH level detection. Each indicator was tested over three replicates.

After 42–44 h, ROS and GSH levels of MII oocytes were detected through H2DCFDA (Beyotime, Shanghai, China) and CMF2HC (Beyotime, Shanghai, China). Each group was stained by H2DCFDA and CMF2HC for 10 μM and 30 min at room temperature, then 4 μL PBS droplets were placed in a 35 mm small dish (Thermo Fisher Scientific, Wilmington, DE, USA), and each group of oocytes was transferred to different dishes in turn. Stained samples were imaged with an inverted fluorescence microscope (Ti2eU; Nikon, Tokyo, Japan) using ImageJ software (version 8.0.2, NIH, Bethesda, MD, USA) to examine fluorescence intensity. For each experimental group, 30 to 60 oocytes were selected for analysis.