Figure 1: (a–f) Representative images of spermatozoa labeled with fluorescent probes against ROS/RNS. In the first row, (a) MitoSOX™ Red (1 μmol l−1) and (b) DHE (50 μmol l−1) showed red fluorescence in the sperm head; (c) RedoxSensor™ Red CC-1 (3 μmol l−1) stained mostly the midpiece of the cell with red fluorescence, with some cases labeling the head as well; (d) CellROX® Orange Reagent (1 μmol l−1) labeled only the midpiece with red fluorescence, while (e) MitoPY1 (10 μmol l−1) and (f) DAF-2 DA (1 μmol l−1) labeled the same area in green fluorescence. Sperm nucleus were labeled with Hoechst (blue fluorescence, middle row). Phase contrast images in gray show the entire sperm cell (bottom row). Scale bars = 5 μm. (g–k) Percentage of labeled cells obtained through flow cytometry (FC) versus fluorescence microscopy (FM). Percentage of labeled cells for the fluorescent probes: (g) MitoSOX™ Red (n = 5), (h) DHE (n = 4), (i) RedoxSensor™ Red CC-1 (n = 4), (j) CellROX® Orange Reagent (n = 5) and (k) MitoPY1 (n = 5). All data present a Gaussian distribution; therefore, the Student's t-test was the statistical test performed. Data are presented as mean ± standard deviation. *P < 0.05, **P < 0.01. (l) Dot plot representation of the gate set to exclude nonsperm-specific events. ROS: reactive oxygen species; RNS: reactive nitrogen species.