View article

Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols and has previously been studied in the pig species using annexin‐V detection of phosphatidylserine translocation. In the present study we designed a new assay to detect these changes in boar spermatozoa, based on the slight increase of sperm membrane permeability occurring during the early stages of cryoinjury, using the combination of three fluorescent probes, SNARF‐1, YO‐PRO‐1 and ethidium homodimer. Four ejaculates from five different boars were frozen–thawed and flow cytometrically (FC) evaluated as paired samples. One of the samples was assayed using the annexin‐V/propidium iodide staining and the other sample was evaluated using the new triple staining. Using this combination of probes, four sperm subpopulations were easily detected: viable, with …