USING AMINE REACTIVE DYES TO ESTIMATE MEMBRANE INTEGRITY IN FIXED FISH SPERM: A TOOL TO STRENGTHEN THE QUALITY CONTROL OF CRYOPRESERVATION PROTOCOLS

The analysis of sperm membrane integrity in freshly collected cells is generally overlooked during most cryopreservation efforts in aquatic species due to the typical absence of a flow cytometer.  To solve this problem, a fluorescent dye that is reactive with cellular amines and allows for cell fixation was tested.  The objectives of the present study were to determine: (1) a working dye concentration for fish sperm samples; and, (2) if the traditional propidium iodide/SYBR-14 and amine reactive dye methods are comparable at identifying proportions of heat-treated cell (membrane-compromised) populations, as well as at identifying cell populations with intact and compromised membranes after sperm activation, refrigerated storage, and exposure to a cryoprotectant and a surfactant.  Zebrafish (Danio rerio) sperm were obtained by stripping, and pooled samples (in triplicate) were used in all tests.  Six dilutions of the amine dye (ranging from 0.625 to 0.02 µL/mL) were evaluated and compared to the traditional staining protocol.  A concentration of 0.5 µL/mL amine reactive dye was selected to be used in subsequent assays.

Membrane integrity was estimated in proportions of 100, 75, 50, 25 and 0% membrane-intact sperm cells prepared using heat-treated cells.  Sperm suspensions were activated with deionized water to simulate urine contamination.  Activation was stopped in 10-sec intervals, and the procedure was repeated until the sperm remained activated for 120 consecutive sec; membrane integrity was analyzed at each time interval.  For the storage assay, sperm suspensions were prepared in Hanks' balanced salt solution at 302 mOsm/kg osmolality (HBSS302), HBSS354 and HBSS402, and evaluated every 2 hr for 8 hr, and then every 24 hr for 72 hr.  Cryoprotectant toxicity was tested by diluting sperm suspensions in HBSS340 with methanol at 5, 10 and 15% final concentrations.  Surfactant toxicity was tested by diluting sperm suspensions in HBSS354 with Triton X-100 at 0.2, 0.15 and 0.1 mM final concentrations.  In each toxicity assay, membrane integrity was tested every 20 min for 80 min.  The number of membrane-intact cells significantly decreased across time in all treatments (ANOVA/Kruskal-Wallis, P < 0.05).  Significant differences between staining protocols were observed after activation and after exposure to methanol 10 and 15%, and to Triton X-100 (Paired t-test/Wilcoxon signed rank sum test P < 0.05).  The average difference, however, was minor (between 1 and 6% in average).  Results showed that this method can allow reliable preservation of stained samples at the time of collection for later analysis, greatly strengthening the overall quality control process.