Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide, and Importance of Individual Male Variability

Contents Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H 2 O 2 ) 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 200 μ m H 2 O 2 for 2 h at 37°C. Intracellular reactive oxygen species (H 2 DCFDA‐CM) increased with H 2 O 2 concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μ m . Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μ m H 2 O 2 and above. In a second experiment, samples from seven males were submitted to 0 and 200 μ m H 2 O 2 for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H 2 O 2 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 H 2 O 2 or after incubation with H 2 O 2 . Red deer spermatozoa are relatively resilient to H 2 O 2 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.