CRYOPRESERVATION ALTERS THE CA(2+) FLUX OF BOVINE SPERMATOZOA

Capacitation and the acrosome reaction are Ca2+-dependent events that must be properly timed for successful fertilization to occur. To test the hypothesis that the cryopreservation procedures alter the ability of bovine spermatozoa to regulate Ca2+, the ability of Percoll-washed fresh and cryopreserved spermatozoa obtained from the same ejaculate (four ejaculates from each of six bulls) to regulate Ca2+ over time was monitored using the fluorescent Ca2+ chelator indo-1. The ability of spermatozoa to control Ca2+ levels varied among ejaculates from a bull, and among bulls; these effects were statistically accounted for in the analysis of the effects of cryopreservation. Initially, cryopreserved spermatozoa had lower viability, motility, and normal acrosome scores and more intra- and extra-cellular Ca2+ than fresh spermatozoa. Intra- and extra-cellular Ca2+ was monitored for 150 min, with 1 mM exogenous Ca2+ or buffer being added at 30 min to the spermatozoa being used to monitor intracellular Ca2+. By 30 min, extracellular Ca2+ was higher for fresh cells and then remained constant. Intracellular Ca2+ of fresh and cryopreserved spermatozoa in Ca2+-free media slowly increased. While both fresh and cryopreserved cells in Ca2+-supplemented media accumulated Ca2+ more rapidly, cryopreserved spermatozoa did so faster than fresh. Post-experimental viability was lower in cryopreserved spermatozoa that had been exposed to exogenous Ca2+. In conclusion, cryopreservation affects initial intra- and extra-cellular Ca2+ levels of bovine spermatozoa, and their ability to control subsequent rates of Ca2+ accumulation.