Response of thawed epidi dymal red deer spermatozoa to increasing concentrations of hydrogen peroxide, and importance of individual male variability

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H2O2) levels on red deer spermatozoa after cryopreservation, and the role of male-to-male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200μm H2O2 for 2h at 37°C. Intracellular reactive oxygen species (H2DCFDA-CM) increased with H2O2 concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200μm. Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50μm H2O2 and above. In a second experiment, samples from seven males were submitted to 0 and 200μm H2O2 for 2h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H2O2 presence. We found that the kinematic parameters reflected male-to-male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H2O2 or after incubation with H2O2. Red deer spermatozoa are relatively resilient to H2O2 after thawing, but it seems to be a great male-to-male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.This work has been supported by the Spanish Ministry of Education and Science (Project AGL2004-05904 ⁄ GAN) and by the Junta de Comunidades de Castilla-La Mancha (Project PAC06-0047). A.E Domínguez-Rebolledo was supported by Consejo Nacional de Ciencia y Tecnología (CONACyT), México. Rocio Fernandez Santos and Felipe Martinez-Pastor were supported by the Juan de la Cierva and Ramón y Cajal programs (Ministerio de Ciencia y Tecnología,Spain), respectively.Peer Reviewe