Sperm Viability Assay with Inhibitors

Sperm viability was determined following the method described by Roa-Espitia et al. [39 (link)]. Sperm suspensions were incubated in MCM-PL at pH 7.8 and 37 °C for 60 min in the absence and presence of VAS2870 (40 µM) or NSC23766 (100 µM). Once the incubation was finished, we added a PI solution (1 μg/mL) to a sample of spermatozoa to a 1:1 ratio and mixed and incubated the mixture at room temperature for 30 min. The spermatozoa were washed and the number of stained and unstained spermatozoa was counted (500 cell X sample, n = 3) under an epifluorescence microscope (Olympus BX500, Tokyo, Japan). Images were registered and analyzed using the software Nikon Elements 3.1.