Evaluation of the lipid membrane ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility demonstrated the same pattern as in Fig. 3.

(A) Methanol (8%) decreased the viability of zebrafish sperm in fresh and cryopreserved treatments, and the addition of CAT to the methanol improved its viability in pre- and post cryopreservation treatments (P<0.05, ANOVA, F = 19.0). (B) Cryopreservation increased ROS 3.0-fold (P<0.05), but CAT did not mitigate it (P>0.5, ANOVA and Tukey’s Multiple Comparison test, F = 13.4). (C) The addition of 8% methanol decreased zebrafish sperm motility in fresh and cryopreserved treatments (P<0.05), and the addition of CAT to the methanol improved its viability in pre- and post cryopreservation treatments (P<0.05, ANOVA and Tukey’s Multiple Comparison test, F = 19.0). Bars with the same letter are not significantly different (P>0.05), but bars with different letters are (P<0.05). X- axis categories and sample sizes were: Control =  live sperm stained with DHE, N = 12; CAT (200 U/ml, concentration used in all CAT treatments), N = 5; 8% Methanol, N = 12; 8% Methanol + CAT 200, N = 12; Cryo 8% Methanol  =  sperm cryopreserved according to [6] with 8% methanol, N = 12; Cryo 8% Methanol + CAT, N = 12.