Use of an adenosine triphosphate assay, and simultaneous staining with fluorescein diacetate and propidium iodide, to evaluate the effects of cryoprotectants on hard coral (Echinopora spp.) oocytes

The objective was to examine the effects of cryoprotectants on oocytes of hard corals (Echinopora spp.) to obtain basic knowledge for cryopreservation procedures. Oocytes were exposed to various concentrations of cryoprotectants (0.25 to 5.0 M) for 20 min at room temperature (25 °C). Two tests were used to assess ovarian follicle viability: fluorescein diacetate (FDA) + propidium iodide (PI) staining, and adenosine triphosphate (ATP) assay. Both FDA + PI staining and ATP assay indicated that cryoprotectant toxicity to oocytes increased in the order methanol, dimethyl sulfoxide (DMSO), propylene glycol (PG), and ethylene glycol (EG). The no observed effect concentrations for Echinopora spp. oocytes were 1.0, 0.5, 0.25, and 0.25 M for methanol, DMSO, PG, and EG, respectively, when assessed with FDA + PI. The ATP assay was more sensitive than FDA + PI staining (P < 0.05). Oocyte viability after 1.0 M methanol, DMSO, EG, or PG treatment for 20 min at room temperature assessed with FDA + PI tests and ATP assay were 88.9 ± 3.1% and 72.2 ± 4.4%, 66.2 ± 5.0% and 23.2 ± 4.9%, 58.9 ± 5.4% and 1.1 ± 0.7%, and 49.1 ± 5.1% and 0.9 ± 0.5%, respectively. We inferred that the ATP assay was a valuable measure of cellular injury after cryoprotectant incubation. The results of this study provided a basis for development of protocols to cryopreserve coral oocytes.