Amine reactive dyes: an alternative to estimate membrane integrity in fish sperm cells

Fluorescent dyes that binds irreversibly to cellular amines, come in several available emission spectra, and do not poses health concerns were used to evaluate membrane integrity in fish sperm cells. 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 staining combination was comparable with the amine reactive dye (ARD) methods at identifying cell populations with intact and compromised membranes after sperm activation, refrigerated storage, and exposure to cryoprotectant and 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 with the traditional staining protocol. A concentration of 0.5 μL/mL ARD was selected to be used in subsequent assays. Sperm suspensions were activated with deionized water to simulate urine contamination. After 10 sec, osmolality was increased to stop activation, and the procedure was repeated in 10-sec intervals 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 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 (p < 0.05). Significant differences between staining protocols were observed after activation and after exposure to methanol at 10 and 15%, and to Triton X-100 (p < 0.05). The average difference, however, was minor (between 1 and 6% in average) in relation to the typical values used for decision making based on this assay. Results showed that this method has the potential to contribute greatly to the standardization of cryopreservation in aquatic species.