Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C(11) (B581) and BODIPY 665/676 C(11) (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H(2)O(2) ) 0.1 mM or 1 mM, or tert-butyl hydroperoxide (TBH) 0.1 mM or 1 mM. LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mM of TBH or H(2)O(2). Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.

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 μm 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 μm. Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H(2)O(2) and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm 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.