2.7. H2DCFDA Assay (Reactive Oxygen Species (ROS) Detection)

ROS was measured through 2,7,dichlorodihydrofluorescein diacetate (H₂DCFDA) as described previously [38]. In brief, the live mature (MII) oocytes (30 to 35 used per each group/experiment) or day-8 blastocysts (10 to 15 used per each group/experiment) were incubated in 1 × PBS containing 10 μM of H₂DCFDA for 30 min in a humidified atmosphere of 5% (v/v) CO₂ in air at 38.5 °C. When the incubation time was finished, the COCs were washed three times with PBS, mounted onto glass slides, and examined under an epifluorescence microscope (Olympus IX71, Tokyo, Japan) under 490 nm excitation and 525 emission.