Departamento de Ecoloxía e Bioloxía Animal, Universidade de Vigo, Estrada Colexio Universitario s/n, 36310 Vigo, Galicia, Spain.
Cryobiology. 2009 Dec;59(3):344-50. doi: 10.1016/j.cryobiol.2009.09.010. Epub 2009 Sep 26.
Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me(2)SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to -12 degrees C at 1 degrees C/min, holding for 2 min for seeding, cooling to -20 degrees C at 0.5 degrees C/min, and then cooling to -35 degrees C at 1 degrees C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.
目前的海洋污染监测策略基于化学和生物技术的整合。海胆胚胎-幼虫生物测定是全球应用最广泛的生物方法之一。海胆早期胚胎的冷冻保存可以提供一种有用的工具，以克服这种生物测定的主要限制之一，即全年都有高质量生物材料的可用性。本研究旨在确定几种渗透（二甲亚砜、Me(2)SO；丙二醇、PG；和乙二醇、EG）和非渗透（海藻糖、TRE；聚乙烯吡咯烷酮、PVP）冷冻保护剂（CPAs）及其组合，对海胆 Paracentrotus lividus 的卵子和胚胎进行冷冻保存的适用性。基于 CPAs 的毒性，PG 和 EG 与 PVP 结合似乎最适合 P. lividus 卵子和胚胎的冷冻保存。还测试了几种冷冻程序。最成功的冷冻方案是在 1°C/min 的冷却速度下从 4°C 降至-12°C，保持 2 分钟用于播种，以 0.5°C/min 的冷却速度降至-20°C，然后以 1°C/min 的冷却速度降至-35°C。冷冻受精卵和冷冻囊胚的正常幼虫百分比最高分别为 41.5%和 68.5%，幼虫生长值最高分别为 42.9%和 60.5%。
