Flow Cytometry and Confocal Microscopy for ROS Evaluation in Fish and Human Spermatozoa

Reactive oxygen species (ROS) could have a negative impact on sperm cellular function and viability. This chapter describes a protocol for oxidative stress evaluation using dichlorofluorescein (DCF) which can specifically reveal intracellular reactive oxygen species. The protocol described here has been used in human and teleost species sperm samples. The method can be used with two approaches: (1) flow cytometry, for quantification of DCF⁺ cells, or (2) confocal microscopy, for the localization of ROS within the cells.