Spermatozoa ROS level was assessed using a fluorescent probe with DCFH-DC46 (link). Briefly, an aliquot of thawed semen was diluted in freezing extender I without egg yolk (1:12) and 50 µL were mixed with 1 µL DCFH-DC (10 mM). After 30 min incubation at 37 °C in the dark, add PBS to wash the sample. The sample was added to 400 µL PBS and analyzed using a multifunctional microplate reader (PerkinElmer, Waltham, MA, USA) at excitation/emission = 488/525 nm and the ROS level was determined based on the fluorescence intensity.
Free full text: Click here
