The effect of cryoprotectants and antioxidants on the motility of fresh zebrafish sperm.

<p>Each sample consisted of fresh, squeezed sperm pooled from 3 males that was treated with cryoprotectants alone or with cryoprotectants and CAT to examine the effect on the motility of the sample. X-axis categories and sample sizes were: Control, no treatment, N = 16; CAT (200 U/ml for all treatments), N = 16; 8% methanol, N = 16; 8% methanol + CAT, N = 16, 10% dimethylformamide (DMF), N = 8; 10% DMF + CAT, N = 8; 10% dimethylacetamide (DMA), N = 8; and, 10% DMA + CAT, N = 8. Of all the treatments, the 8% methanol and 10% DMA reduced the motility over the control value (P<0.5, ANOVA and Tukey’s Multiple Comparison Test, F = 15.4), whereas the other cryoprotectants (with or without CAT) did not (P>0.05). When CAT was added to the cryoprotectants, all the mean values increased, but the 10% DMF + CAT produced the highest mean motility at 90%. Bars with the same letter are not significantly different (P>0.05), but bars with different letters are (P<0.05). Thus, the addition of catalase had a beneficial effect on the motility of zebrafish sperm.</p

Evaluation of the xanthine-xanthine oxidase (X-XO) system and methods to identify DHE production (general oxidative stress) during ROS in zebrafish spermatozoa.

<p>In a concentration dependent manner, induced oxidative stress (X-XO): (<b>A</b>) did not alter the membrane integrity or viability (as assayed by propidium iodide) from the control values (P>0.05; Kruskal-Wallis test and Dunn’s Multiple Comparison test); (<b>B</b>) increased stress as assayed by DHE-positive cells (P<0.05; Kruskal-Wallis test and Dunn’s Multiple Comparison test); and, (<b>C</b>) reduced sperm motility as assayed by visual inspection with phase microscope (P<0.05; Kruskal-Wallis test and Dunn’s Multiple Comparison test). Bars with the same letter are not significantly different (P>0.05), but bars with different letters are (P<0.05). Low X-XO exposure  =  0.1 mMX + 0.01 units/ml XO; Medium X-XO exposure  =  0.1 mMX + 0.01 units/ml XO; High X-XO exposure  =  0.3 mM X + 0.025 units/ml XO. In all treatments, N = 10 samples and each sample consisted of pooled sperm from 3 to 5 males.</p

Evaluation of the intracellular ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility.

<p>(<b>A</b>) Overall, cryopreservation decreased the viability of zebrafish sperm by approximately 50%, but the addition of CAT to the methanol improved its post-thaw viability 16% (P<0.05, ANOVA and Tukey’s Multiple Comparison test, F = 60.5). (<b>B</b>) Cryopreservation increased ROS over 2.7-fold (P<0.05, ANOVA, and Tukey’s Multiple Comparison test F = 6.2), but the addition of CAT did not mitigate it (P>0.5, ANOVA). (<b>C</b>) Motility demonstrated the greatest sensitivity in our treatments. Prior to cryopreservation, the 8% methanol treatment reduced motility by ∼50%, and the addition of CAT to the 8% methanol returned the motility to control levels. In parallel, the cryopreserved sperm (8% methanol) decreased the pot-thaw motility of zebrafish sperm from 83% (control) to 17%, however the addition of CAT doubled this post-thaw motility (P<0.05, ANOVA and Tukey’s Multiple Comparison test, F = 22.6). 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 = 13; CAT (200 U/ml, concentration used in all CAT treatments), N = 9; 8% Methanol, N = 12; 8% Methanol + CAT 200, N = 12; Cryo 8% Methanol  =  sperm cryopreserved according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039397#pone.0039397-Yang1" target="_blank">[6]</a> with 8% methanol, N = 13; Cryo 8% Methanol + CAT, N = 13.</p

Evaluation of the xanthine-xanthine oxidase (X-XO) system and the impact of antioxidants on sperm viability, ROS and motility.

<p>In all the various treatments, the addition of CAT (black bars) performed the best. (<b>A</b>) Viability was not altered by most of the treatments as their mean values were not different than the control values (P>0.05), except CAT 200 U/ml, X-XO + CAT 100 U/ml X-XO + CAT 200 U/ml which were slightly higher than controls (P<0.05, Kruskal-Wallis and Dunn’s Multiple Comparison test). (<b>B</b>) Only the combination of X-XO + CAT mitigated the oxidative stress as their values were not different than the control values (P>0.05), whereas the combinations of X-XO + SOD and X-XO + vitamin E did not (P<0.05, Kruskal-Wallis and Dunn’s Multiple Comparison test). (<b>C</b>) Increased stress was correlated with decreased sperm motility that was mitigated by X-XO + CAT (P>0.05), but not by X-XO + SOD or X-XO + vitamin E (P<0.05; ANOVA and Tukey’s Multiple Comparison test, F = 39.5). Bars with the same letter are not significantly different (P>0.05), but bars with different letters are different (P<0.05). The X- axis categories and sample sizes for all tests were: No X-XO  =  control (N = 4); X-XO (in all treatments)  = 0.2 mM X and 0.0175 U/ml XO (N = 11); SOD 200 U/ml (N = 4); X-XO + SOD 100 U/ml (N = 12); X-XO + SOD 200 U/ml (N = 12); 100 µM vitamin E (N = 9); X-X0 + 100 µM vitamin E (N = 9); X-X0 + 200 µM vitamin E (N = 9); CAT 200 U/ml (N = 4); X-XO + CAT 100 U/ml (N = 12); X-XO + CAT 200 U/ml (N = 12). In all trials, a medium X-XO exposure was used when X-XO was added. In all treatments, each sample consisted of pooled sperm from at least 3 males.</p

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.

<p>(<b>A</b>) 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>B</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). (<b>C</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039397#pone.0039397-Yang1" target="_blank">[6]</a> with 8% methanol, N = 12; Cryo 8% Methanol + CAT, N = 12.</p

Adult and larval forms of <i>A. palmata</i> and <i>F. scutaria</i>.

<p><b>A</b>) Adult and developing <i>A. palmata</i> larvae (inset) at the cornflake stage at ∼24 h. Scale bar = 50 µm. Adult photo by R. Williams, Smithsonian Institution. <b>B</b>) Adult and developing <i>F. scutaria</i> larvae at the swimming stage. Scale bar = 50 µm. Embryos that reached these stages were scored as successfully developed.</p

The effect of cooling rate for successful <i>F. scutaria</i> spermatozoa cryopreservation (no sperm exposed to freezing in any of these treatments).

<p>Two freezing ranges were examined and two cryoprotectant solutions (<i>F. scutaria</i>, n = 3–7 pooled sperm donors/cooling range and n = 16 egg donors) and then the influence of cooling rate on IVF success. Only the 10% DMSO at a cooling rate greater than 20°C produced reasonable post-thaw fertilization. Bars with the same letter were not different (<i>P</i>>0.05; ANOVA), whereas bars with different letters were different (<i>P</i><0.05; ANOVA).</p

Experiment II: <i>In vitro</i> fertilization after exposing fresh sperm to various cryoprotectant treatments.

1<p>Sigma-Aldrich, St Louis, MO, USA.</p>2<p>Acros Organics, Fair Lawn, NJ, USA.</p>*<p>Thirty to 50 fresh eggs were used per pooled sperm sample.</p>***<p>An inseminate concentration of 10⁶ sperm/ml was used for each species.</p

Cryopreservation of coral sperm (all sperm exposed to freezing in these treatments).

<p><b>A</b>) <i>A. palmata</i> sperm were cryopreserved at cooling rates 20 to 30°C/min using 10% DMSO and PG, and IVF success was assessed and averaged over the single spawning period. This averaged graph revealed no difference between the two cryoprotectants and a mean fertilization success of ∼18%. <b>B</b>) However, if the <i>A. palmata</i> fertilization success during the spawning period was graphed by day, a 65% fertilization success occurred on Day 3 with 10% DMSO, whereas it was 25, 0 and 3% on Day 1, 2 and 4, respectively. For the first 3 nights, the control with fresh sperm held at ∼90%, then fell to 76% on the fourth evening. <b>C</b>) <i>F. scutaria</i> sperm were cryopreserved at rates 20 to 30°C/min using 10% DMSO versus PG, and fertilization success was assessed and averaged over two spawning periods (July and August 2010). Averaging indicated that 10% DMSO was the preferred cryoprotectant, and (as in <i>A. palmata</i>) there was no variability in time in terms of physiological responses during a spawning season. <b>D</b>) Variability in <i>F. scutaria</i> IVF success after cryopreservation over two nights of a single representative month (August 2010). Fresh sperm IVF success held steady at 65%, but sperm cryopreserved with 10% DMSO varied from 52 to 13% on the two evenings. Bars with the same letters were not different (<i>P</i>>0.05; ANOVA), whereas bars with different letters were different (<i>P</i><0.05; ANOVA).</p

Embryonic <i>F. scutaria</i> cells after cryopreservation (all cells exposed to freezing in these treatments).

<p>Mean post-thaw viability of <i>F. scutaria</i> cells was ∼50% for all cryoprotectants tested. Ten thousand events were measured for each sample. Controls (the three left bars) were live-stained and unstained cells and 100% dead cells that produced control data for the flow cytometer (i.e., 100% intact versus 100% dead). Bars with the same letters were not different, whereas bars with different letters were different (P<0.05; Kruskal-Wallis test).</p