Cabrita E, Robles V, Chereguini O, Wallace J C, Herráez M P
Center for Marine Sciences-CCMAR, University of Algarve, 8000 Faro, Portugal.
Cryobiology. 2003 Dec;47(3):204-13. doi: 10.1016/j.cryobiol.2003.10.001.
Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, in order to achieve a vitrifiable medium, a high concentration of permeable cryoprotectants must be employed, and the incorporation of high molecular weight compounds should also be considered. The toxicity of these permeable and non-permeable agents has to be assessed, particularly when high concentrations are required. In the present study, permeable and non-permeable cryoprotectant toxicity was determined in turbot embryos at two development stages (F stage-tail bud and G stage-tail bud free). Embryos treated with pronase (2mg/ml, 10 min at 22 degrees C) were incubated in dimethyl sulfoxide (Me2SO), methanol (Meth.) or ethylene glycol (EG) in concentrations ranging from 0.5 to 6M for periods of 10 or 30 min, and in 5, 10, and 15% polyvinylpyrrolidone (PVP), 10, 15, and 20% sucrose or 0.1, 1, and 2% X-1000 for 2 min. The embryos were then washed well and incubated in seawater until hatching. The toxicity of permeable cryoprotectants increased with concentration and exposure time. There were no significant differences between permeable cryoprotectants. However, embryos tolerated higher concentrations of Me2SO than other cryoprotectants. Exposure to permeable cryoprotectants did not affect the hatching rate except at G stage with X-1000 treatment and 20% sucrose. Taking into account the cryoprotectant toxicity and the vitrification ability of cryoprotectant mixtures, three vitrification solutions (V1, V2, and V3), and one protocol for stepwise incorporation were designed. The tested solutions contained 5M Me2SO+2M Meth+1M EG plus 5% PVP, 10% sucrose or 2% X-1000. The hatching rate of embryos that had been exposed to the the vitrification solutions was analyzed and no significant differences were noticed compared with the controls. Our results demonstrate that turbot embryos can be subject to this cryoprotectant protocol without deleterious effect on the hatching rate.
玻璃化可为鱼类胚胎的冷冻保存提供一种有前景的工具。然而，为了获得可玻璃化的培养基，必须使用高浓度的渗透性冷冻保护剂，并且还应考虑加入高分子量化合物。必须评估这些渗透性和非渗透性试剂的毒性，尤其是在需要高浓度时。在本研究中，测定了两种发育阶段（F期——尾芽期和G期——无尾芽期）的大菱鲆胚胎对渗透性和非渗透性冷冻保护剂的毒性。用链霉蛋白酶（2mg/ml，22℃处理10分钟）处理后的胚胎在浓度范围为0.5至6M的二甲基亚砜（Me2SO）、甲醇（Meth.）或乙二醇（EG）中孵育10或30分钟，以及在5%、10%和15%的聚乙烯吡咯烷酮（PVP）、10%、15%和20%的蔗糖或0.1%、1%和2%的X - 1000中孵育2分钟。然后将胚胎充分洗涤并在海水中孵育直至孵化。渗透性冷冻保护剂的毒性随浓度和暴露时间增加。渗透性冷冻保护剂之间没有显著差异。然而，胚胎对Me2SO浓度的耐受性高于其他冷冻保护剂。除了在G期用X - 1000处理和20%蔗糖处理外，暴露于渗透性冷冻保护剂对孵化率没有影响。考虑到冷冻保护剂的毒性和冷冻保护剂混合物的玻璃化能力，设计了三种玻璃化溶液（V1、V2和V3）以及一种逐步加入的方案。测试溶液包含5M Me2SO + 2M Meth + 1M EG加5% PVP、10%蔗糖或2% X - 1000。分析了暴露于玻璃化溶液后的胚胎孵化率，与对照组相比未发现显著差异。我们的结果表明，大菱鲆胚胎可以采用这种冷冻保护剂方案，而对孵化率没有有害影响。
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