Epifluorescence microscope te2000 s

After 42–44 h of IVM, the matured COCs were denuded and washed several times thoroughly in TALP medium. For measuring the intracellular ROS and GSH levels in oocytes, H2DCFDA (2′, 7′-dichlorodihydrofluorescein diacetates; Invitrogen, Waltham, MA, USA) and CellTracker Blue (4-chloromethyl-6.8-difluoro-7-hydroxycoumarin; CMF2HC; Invitrogen) were used, respectively. The oocytes were then transferred to 10 μM of CellTracker Blue or 10 μM of H2DCFDA in TALP medium and incubated for 30 min at room temperature (Light avoided). Stained oocytes were washed several times in TALP medium, and then they were transferred to a 4 μL droplet of TALP medium and covered with mineral oil. Epifluorescence microscope (TE2000-S; Nikon, Tokyo, Japan) was used for measuring the intensities of the fluorescence, observed through UV filters (460 nm for ROS and 370 nm for GSH), then the images were captured. Analysis was performed by Image J software and the intensities of the control group was standardized to 1. For three independent replications, at least 36 oocytes from each experimental group were used in this experiment.

After 42–44 h of IVM, the matured COCs were denuded and washed several times thoroughly in TALP medium. H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetates; Invitrogen) and CellTracker Blue (4-chloromethyl-6.8-difluoro-7-hydroxycoumarin; CMF2HC; Invitrogen) were used for measuring the intracellular ROS and GSH levels in oocytes, respectively. The oocytes were transferred to 10 μM of CellTracker Blue or 10 μM of H2DCFDA in TALP medium, and incubated for 30 min at room temperature, avoiding light. Stained oocytes were again washed several times in TALP medium, then they were transferred to a 4-μL droplet of TALP medium, covered with mineral oil. Epifluorescence microscope (TE2000-S; Nikon, Tokyo, Japan) was used for measuring the intensities of the fluorescence, observed through UV filters (460 nm for ROS and 370 nm for GSH), then the images were captured. Analysis was performed by Image J software and the intensities of the control group was standardized to 1. For three independent replications, at least 36 oocytes from each experimental group were used in this experiment.