Ca2+ regulation by cryopreserved bull spermatozoa in response to A23187

The regulation of intracellular Ca2+ by fresh and cryopreserved bull spermatozoa from the same ejaculates (n = 5) was investigated using the fluorescent Ca2+ indicator, indo-1. Relative internal Ca2+ levels of the spermatozoa were monitored for 30 min prior to the addition of phosphate-buffered saline (PBS), 1 mM Ca2+, the Ca2+ ionophore A23187 (0.1 microM), or Ca2+ + A23187; during the additions the levels of intracellular Ca2+ were observed in detail. After these additions, changes in internal Ca2+ levels were monitored for 120 min. The initial intracellular Ca2+ levels in the cryopreserved spermatozoa were greater than those in fresh (1.05 +/- 0.03 vs 0.97 +/- 0.03 Ca2+ units, P = 0.0001). The addition of Ca2+ + A23187 induced elevated cellular Ca2+ in both fresh (P = 0.0177) and cryopreserved spermatozoa (P < or = 0.0588) within 5 s. Ca2+ alone did not differ from Ca2+ + A23187 in increasing Ca2+ in cryopreserved spermatozoa (P = 0.2225); fresh spermatozoa were slower to respond to exogenous Ca2+ alone (P = 0.0438). At the start of the post-treatment 120 min, cryopreserved spermatozoa had more internal Ca2+ than the corresponding fresh samples (P < or = 0.0088) except for those exposed to both Ca2+ + A23187 (P = 0.2918). All spermatozoa increased internal Ca2+ over the post-treatment time except for the cryopreserved PBS controls, and these cryopreserved controls differed from the fresh controls in accumulation of Ca2+ (P = 0.0620). None of the Ca2+/A23187 treatments induced different rates of change in internal Ca2+ over time in fresh cells (P = 0.3142). The rate of Ca2+ accumulation by cryopreserved spermatozoa in the presence of exogenous Ca2+ exceeded controls (23.12 +/- 6.75 vs 8.11 +/- 6.75, P = 0.0005), but A23187, with or without Ca2+, had no effect (P > or = 0.0907). Following Ca2+ measurements, the viability of the cryopreserved samples was reduced (P < or = 0.015) in all but the Ca(2+)-treated spermatozoa (P = 0.1474); no viability differences were noted for fresh spermatozoa (P > or = 0.2298). The cryopreservation process did not affect acrosomal morphology of the indo-1-exposed spermatozoa (P > or = 0.1147). These data indicate that Ca2+ regulation by Ca(2+)- and A23187-challenged bull spermatozoa differs following cryopreservation procedures, possibly relating to the reduced fertilization capacity of commercially cryopreserved semen.