Cryopreservation extenders affect calcium flux in bovine spermatozoa during a temperature challenge

Bovine spermatozoa are commercially cryopreserved by diluting the cells in media, known as extenders, followed by slow cooling and freezing. Previous work has shown that this process of cryopreservation alters the cells' ability to control divalent calcium (Ca2+) movement. This study evaluated the effect of a brief exposure to common extenders on bovine spermatozoa during subsequent cooling and rewarming. Three fresh ejaculates from each of three bulls were each split and incubated for 30 minutes at 25 degrees C in milk extender or phosphate-buffered saline (PBS) (control); three other fresh ejaculates from each of three bulls were similarly incubated in egg yolk-Tris extender (EYT) or PBS. Spermatozoa were washed and the fluorescent Ca2+ indicator, indo-1 acetoxymethyl ester, was used to monitor the internal Ca2+ in the spermatozoa in Ca(2+)-free PBS over a continuous temperature gradient of 25 degrees C (15 minutes), cooling to 5 degrees C (32 minutes), at 5 degrees C (15 minutes), rewarming to 25 degrees C (25 minutes), and at 25 degrees C (15 minutes). Milk exposure reduced the initial percentage of missing acrosomes and EYT exposure improved the initial viability and acrosome morphology compared to the controls; only milk immediatetly increased internal Ca2+. The initial rate of Ca2+ uptake at 25 degrees C was greater for milk or EYT-exposed spermatozoa than controls (P < 0.05). During cooling, the rate of Ca2+ uptake in all spermatozoa increased (P < 0.01), and it continued to increase during the 15 minutes at 5 degrees C. During rewarming to 25 degrees C, the internal Ca2+ in all spermatozoa declined.(ABSTRACT TRUNCATED AT 250 WORDS)