Efficiency of CellROX deep red® and CellROX orange® fluorescent probes in identifying reactive oxygen species in sperm samples from high and low fertility bulls.

Detection of reactive oxygen species (ROS) is of great interest in semen analysis since their excess is detrimental to sperm function and male fertility. Fluorescence microscopy has achieved attention for providing broad possibilities of sperm evaluations and also for presenting substantial accessibility. In this context, this study investigated the efficiency of CellROX Deep Red® and Orange® probes in detecting ROS in bovine sperm cells and assessed their relationship with sperm fertility potential. First, 16 ejaculates were assigned in three treatments: T0 (no ROS production induced), T1x (ROS production induced once) and T2x (ROS production induced twice). Samples were incubated with Red and Orange probes and percentages of cells producing ROS were evaluated using fluorescence microscopy. Coefficient of determination was 0.61 for Red and 0.56 for Orange. Afterwards, frozen-thawed semen samples from high and low fertility bulls were evaluated regarding percentages of cells producing ROS detected by Red and Orange. Higher levels of ROS assessed by Red were detected in low fertility bovine samples. In conclusion, CellROX Red® and Orange® are both efficient in detecting ROS in bovine spermatozoa. Furthermore, higher sperm ROS detection by CellROX Red® might be associated with low fertility samples.