Reactive oxygen species-mediated loss of bovine sperm motility in egg yolk Tris extender: protection by pyruvate, metal chelators and bovine liver or oviductal fluid catalase.

Improvement of bovine semen cryopreservation requires a better understanding of the properties of the currently used extenders. At present, about half of the spermatozoa die or become immotile following cryopreservation. The implication of an oxidative stress during or following the process of cryopreservation has been suspected to alter sperm functions. However, insufficient information is available on the effect of oxidative stress on sperm functions in their surrounding environment, the extender, such as the one based on egg yolk, Tris and glycerol. In this study, we investigated the effects of hydrogen peroxide (H2O2) and superoxide anion (O2*-) on bovine sperm motility in a widely used egg yolk Tris glycerol (EYTG) extender in comparison to a reference medium, the Tyrode's albumen lactate pyruvate (TALP). Bovine sperm were incubated for 6 h with or without concentrations of H2O2 ranging from 12.5 microM to 1.25 mM and with the hypoxanthine/xanthine oxidase system (X/XOD) that generates O2*-. Sperm motility was established by computer assisted semen analysis (CASA) in four similar experiments using the same frozen pool of semen. We have found that sperm motility was reduced significantly by H2O2 concentrations 20-fold lower in EYTG than in TALP medium. The differential resistance of the two media was explained by pyruvate present in TALP that acts as an antioxidant and metals ions, chelated by ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DETAPAC), found in egg yolk that might react with H2O2. Addition of only 5 U/ml of bovine liver catalase or oviductal fluid catalase (OFC) were sufficient to overcome the loss of sperm motility caused by 100 microM H2O2 in both EYTG and TALP. However, OFC was the most effective of the two catalases in EYTG. In addition to maintain sperm motility, catalase (5 U/ml) and pyruvate (5 mM) increased the intracellular sperm ATP level in comparison to sperm incubated alone for 6 h at 38.5 degrees C in EYTG. Moreover, EDTA, pyruvate and catalase prevented sperm ATP loss in presence of 100 mM of H2O2 in EYTG. These results indicated that EYTG has a very limited capacity to neutralize H2O2, and the addition of low amounts of catalase and millimolar concentrations of pyruvate greatly improved the antioxidant properties of a commonly used extender.