Human sperm samples were prepared on a 30% Percoll gradient and reactive oxygen species (ROS) production was measured. In samples that generated ROS incubation under 95%O 2 :5%CO 2 for 30 min decreased the proportion of spermatozoa capable of the stimulated acrosome reaction by 40% in comparison to samples incubated under 95%N 2 :5%CO 2 (P < 0.001, repeated measures analysis of variance), but the degree of inhibition did not increase after more prolonged incubation periods (up to 6 h). The

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... ion of the antioxidants catalase and superoxide dismutase prevented the inhibitory effect of 95%O 2 :5%CO 2 . Leukocyte removal from samples prior to 95%O 2 :5%CO 2 incubation preserved the ability of the spermatozoa to acrosome react. Sperm motility parameters were less affected by 95%O 2 :5%CO 2 but track velocity was 64.1 µm/s ⍨ 1.96 after 2 h incubation under 95%N 2 :5%CO 2 compared with 54.7 µm/s ⍨ 1.41 after 2 h incubation under 95%O 2 :5%CO 2 (P < 0.05, repeated measures analysis of variance). Sperm samples that did not generate detectable ROS were not affected by 95%O 2 :5%CO 2 . The toxic effects of incubation under 95%O 2 :5%CO 2 on human spermatozoa result from increased endogenous ROS production, mostly from leukocytes. High ROS levels inhibit sperm function, with the stimulated acrosome reaction being more susceptible than motility parameters.