Lahnsteiner F
Department for Organismic Biology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
Theriogenology. 2008 Feb;69(3):384-96. doi: 10.1016/j.theriogenology.2007.10.007. Epub 2007 Nov 26.
The study investigated the effects of internal (DMSO, 1,2-propanediol, glycerol, ethylene glycol, methanol, N,N-dimethylacetamide) and external cryoprotectants (glucose, sucrose) on the viability and on morphometric parameters of zebrafish embryos. From the tested internal cryoprotectants, DMSO had the lowest toxicity, followed by 1,2-propanediol and glycerol. The external cryoprotectants were less toxic then the internal ones. Early ontogenetic stages were more sensible to cryoprotectant exposure than advanced stages. Two-step incubation procedures in increasing concentrations of internal and external cryoprotectants were superior to multiple-step exposure procedures. All tested vitrification solutions exceeded the tolerance limit of embryos. The tolerance of zebrafish embryos to cryoprotectants was highly variable in a concentration range causing approximately 50% embryo mortality. The width of the perivitelline space showed significant morphometrical changes due to cryoprotectant exposure. In the germinative tissue non-significant changes occurred. The yolk did not change morphometrically after exposure to internal cryoprotectants and showed no sign of dehydration after exposure to external cryoprotectants. Based on these results the study comes to the following conclusions: as yolk dehydration was impossible and as vitrification solutions were over the tolerance limit it seems unlikely that successful vitrification of zebrafish embryos can be achieved. Under these considerations slow freezing methods would be a better option as lower cryoprotectant concentrations can be used and embryos can be dehydrated during freezing.
该研究调查了内部冷冻保护剂（二甲基亚砜、1,2 - 丙二醇、甘油、乙二醇、甲醇、N,N - 二甲基乙酰胺）和外部冷冻保护剂（葡萄糖、蔗糖）对斑马鱼胚胎活力和形态测量参数的影响。在测试的内部冷冻保护剂中，二甲基亚砜毒性最低，其次是1,2 - 丙二醇和甘油。外部冷冻保护剂的毒性低于内部冷冻保护剂。早期发育阶段比晚期发育阶段对冷冻保护剂暴露更敏感。采用内部和外部冷冻保护剂浓度递增的两步孵育程序优于多步暴露程序。所有测试的玻璃化溶液都超过了胚胎的耐受极限。在导致约50%胚胎死亡的浓度范围内，斑马鱼胚胎对冷冻保护剂的耐受性差异很大。由于冷冻保护剂暴露，卵周隙宽度出现了显著的形态测量变化。在生殖组织中未发生显著变化。暴露于内部冷冻保护剂后，卵黄形态测量上没有变化，暴露于外部冷冻保护剂后也没有脱水迹象。基于这些结果，该研究得出以下结论：由于卵黄不可能脱水，且玻璃化溶液超过了耐受极限，斑马鱼胚胎成功玻璃化似乎不太可能实现。基于这些考虑，慢速冷冻方法可能是更好的选择，因为可以使用较低浓度的冷冻保护剂，并且胚胎在冷冻过程中可以脱水。
