Evaluation of Intracellular Location of Reactive Oxygen Species in Solea Senegalensis Spermatozoa

Oxidative stress is one of the important factors in decreasing sperm quality. Developing efficient protocols for detecting reactive oxygen species (ROS) in spermatozoa is of high importance in any species, but these methods are rarely used and even less in teleost. Cryopreservation is a useful technique in aquaculture for different purposes, including gene banking and guaranteed sperm availability throughout the year. Freezing/thawing procedures could cause ROS production and damage the sperm cells. Considering the prospective damage that an excess of ROS production could cause in spermatozoa depending on their localization, here a detailed methodology to detect H2O2 and to evaluate its intracellular localization by confocal microscopy is provided. For this purpose, a combination of 3 fluorochromes (2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA), a live mitochondria stain and 4',6-Diamidino-2-phenylindole dihydrochloride (DAPI)) are used to evaluate the colocalization of H2O2 with spermatozoa nuclei or mitochondria in Solea senegalesis sperm samples.