Department of Biotechnology, Mingdao University, Peetow, Chang Hua, Taiwan.
Theriogenology. 2010 Mar 15;73(5):605-11. doi: 10.1016/j.theriogenology.2009.10.016. Epub 2009 Dec 16.
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 5.0M) for 20 min at room temperature (25 degrees 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 1.0M 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.
本研究旨在探讨不同浓度的冷冻保护剂（0.25-5.0M）对硬珊瑚卵子（Echinopora spp.）的影响，以期为冷冻保存程序提供基础理论知识。卵子在室温（25°C）下暴露于不同浓度的冷冻保护剂中 20 分钟。采用两种方法评估卵巢滤泡活力：荧光素二乙酸酯（FDA）+碘化丙啶（PI）染色和三磷酸腺苷（ATP）测定。FDA+PI 染色和 ATP 测定均表明，卵子对冷冻保护剂的毒性按甲醇、二甲基亚砜（DMSO）、丙二醇（PG）和乙二醇（EG）的顺序递增。当采用 FDA+PI 染色时，Echinopora spp.卵子的无观察效应浓度分别为 1.0、0.5、0.25 和 0.25M。ATP 测定比 FDA+PI 染色更敏感（P<0.05）。室温下，1.0M 甲醇、DMSO、EG 或 PG 处理 20 分钟后，采用 FDA+PI 试验和 ATP 测定评估的卵子存活率分别为 88.9+/-3.1%和 72.2+/-4.4%、66.2+/-5.0%和 23.2+/-4.9%、58.9+/-5.4%和 1.1+/-0.7%、49.1+/-5.1%和 0.9+/-0.5%。我们推断，ATP 测定是评估冷冻保护剂孵育后细胞损伤的一种有价值的方法。本研究结果为开发珊瑚卵子冷冻保存方案提供了依据。
