Zona pellucida binding ability and responsiveness to ionophore challenge of cryopreserved dog spermatozoa after different periods of capacitation in vitro

The present study was aimed to evaluate the functional status of cryopreserved dog spermatozoa after different periods (2, 8 and 24 h) of capacitation in vitro. Sperm motility, viability and binding capacity to the zona pellucida of canine oocytes derived from frozen-thawed ovaries were evaluated at each time point. Sperm viability was assessed by flow cytometry using the Ca2+-sensitive indicator Fluo 3 AM and PI, to simultaneously detect the proportion of live spermatozoa and the existence of live sperm subpopulations with different intracellular Ca2+ content. In addition, the acrosome reaction frequency in ionophore-treated aliquots of spermatozoa incubated in capacitating (CCM) versus non-capacitating (NCM) medium, were evaluated by using eosin-nigrosin staining at the same time intervals. The number of spermatozoa bound to the zona pellucida decreased in about 50% (from 18.61±14.40 to 7.7±6.97) when sperm incubation was prolonged from 2 to 8 h, however, sperm motility, viability and the subpopulation of live spermatozoa with higher intracellular Ca2+ concentration decreased in lower extent (10–15%). In CCM-incubated samples, the rate of acrosomal exocytosis in response to ionophore challenge was high (>80%), independently of the evaluation period. NCM-incubated sperm were not affected by ionophore treatment, however, their intracellular Ca2+ concentration was no different than that observed in CCM-incubated spermatozoa. It was concluded that, after being capacitated, motile and viable spermatozoa seem to lose their ability to bind to the zona pellucida, but this loss is not accompanied by a reduced response to ionophore challenge and it may not be related with changes in the intracellular Ca2+ concentration of spermatozoa.