Cryopreservation Extenders Affect Calcium Flux in Bovine Spermatozoa During a Temperature Challenge

Abstract

ABSTRACT: 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°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 Ca2+‐free PBS over a continuous temperature gradient of 25°C (15 minutes), cooling to 5°C (32 minutes), at 5°C (15 minutes), rewarming to 25°C (25 minutes), and at 25°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 immediately increased internal Ca2+. The initial rate of Ca2+ uptake at 25°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°C. During rewarming to 25°C, the internal Ca2+ in all spermatozoa declined. The rate of decline of Ca2+ in control exceeded that of EYT‐exposed spermatozoa. Addition of 1 mM Ca2+ during the final 25°C incubation caused internal Ca2+ to increase for 1 minute in all samples. The rate of increase in the milk samples was greater than that of its control (P < 0.05). There was no further change over the subsequent 35 minutes except for an increase of free intracellular Ca2+ in the EYT control sample. The percentage of either viable or intact spermatozoa had decreased in all samples at the end of the temperature gradient. The brief exposure of spermatozoa to milk and egg yolk increased free intracellular Ca2+ in spermatozoa, perhaps initiating the early stages of capadtation. Milk and EYT differed in their effect on Ca2+ flux; this result may have implications for fertility.