Detection of reactive oxygen species (ROS) level

ROS level assay was conducted as previously described [45]. For evaluating testicular intracellular superoxide production using In Situ Dihydroethidium (DHE, Sigma-Aldrich, USA) fluorescence, optimal cutting temperature media-embedded tissues were sectioned (5 μm) at -20 °C. After being fixed, tissues were incubated with 1 μm DHE in a light-protected, humidified chamber at room temperature for 30 min. The level of ROS by tissue was observed under a fluorescent microscope (Eclipse Ti-SR, Nikon Co., Japan). The density of the images was detected using a fluorescence spectrophotometer in arbitrary units per millimetre square field.