Cryopreservation affects bovine sperm intracellular parameters associated with capacitation and acrosome exocytosis

Abstract

Although semen cryopreservation is widely and commonly used in the bovine breeding industry, half the spermatozoa do not survive and most of those that do survive undergo numerous physiological changes that affect their fertilising ability. The aim of the present study was to determine how cryopreservation affects the intracellular events involved in sperm capacitation and acrosome reaction. Immediately after thawing and washing, almost 50% of spermatozoa were capacitated and more than 20% had lost their acrosome. The sperm cAMP concentration was lower than that in freshly ejaculated spermatozoa, but the cytosolic pH (pH_cyt) was in the expected range. The free cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) was higher than in fresh spermatozoa and cryopreserved spermatozoa had internally stored Ca²⁺. Phenylarsine oxide increased pH_cyt and both cytosolic and stored Ca²⁺ concentrations, whereas orthovanadate enhanced acrosome loss and protein tyrosine phosphorylation (P-Tyr). Heparin increased the percentage of spermatozoa expressing the B (capacitated) chlortetracycline binding pattern, pH_cyt, P-Tyr and Ca²⁺ storage. Moreover, positive correlations exist between capacitation, cAMP, P-Tyr and stored Ca²⁺, whereas the acrosome reaction is positively correlated with pH_cyt and [Ca²⁺]_cyt. These results demonstrate that sperm regulatory mechanisms may be affected by the cryopreservation procedure, but frozen–thawed sperm can still regulate their capacitation and acrosome reaction signalling pathways.