Cryopreservation of Mexican fruit flies by vitrification: Stage selection and avoidance of thermal stress
Cryopreservation of Mexican fruit flies by vitrification: Stage selection and avoidance of thermal stress
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DOI：
10.1016/j.cryobiol.2006.10.192
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发表时间：
2007-02-01
期刊：
影响因子：
2.7
通讯作者：
Leopold, R. A.
中科院分区：
文献类型：
--
作者：
Rajamohan, A.;Leopold, R. A.
本报告介绍了一种冷冻保存墨西哥果蝇胚胎的玻璃化方法的细节。综合数据表明，选择合适的发育阶段进行冷冻保存是最重要的标准。选择正确阶段的关键是平衡肠道蛋黄含量的消耗和胚胎角质层的发育。在21.7℃条件下孵育90 ~ 120 h，将胚胎发育分为4个阶段，根据肠道蛋黄含量和下颌-上颌复合体的初始发育情况进行分类。发现中肠蛋黄含量低和口钩外观的阶段最适合冷冻保存。在30℃下发育的胚胎相对于肠道发育有较早的角质层形成，冷冻保存后的孵化率显著降低。在液氮中直接淬火的胚胎玻璃化效果不如在液氮蒸汽中退火的胚胎玻璃化效果好。以1,2-乙二醇为主要成分的玻璃化溶液的淬火样品出现断裂。当溶液经过退火和含有聚乙二醇时，破裂发生的频率较低。在液氮中保存超过12个月的玻璃化胚胎的孵化率与仅保存15分钟的胚胎没有统计学差异。我们的方案产生了高达61%的标准化孵化率。在30分钟的时间内，从产卵雌性收集的胚胎群体中选择冷冻保存的确切阶段，近80%的胚胎孵化成幼虫。Elsevier Inc.出版。
This report presents details of a vitrification methodology for the cryopreservation of embryos of the Mexican fruit fly, Anastrepha ludens. The overall summary of the data indicates that selecting the correct developmental stage for cryopreservation is the most important criterion. The key aspect in selection of the correct stage is to balance depletion of the gut yolk content against development of the embryonic cuticle. Embryogenesis was divided into four stages between 90 and 120 h after incubation at 21.7 degrees C. The classification was based on the intestinal yolk content and the initial development of mandibular-maxillary complex. Stages having low mid-gut yolk content and the appearance of mouth hooks were found to be the most suitable for cryopreservation. Embryos developing at 30 degrees C had premature cuticle formation relative to gut development and significantly lower hatching after cryopreservation. Vitrification of embryos by direct quenching in liquid nitrogen was less effective than quenching after annealing the samples in liquid nitrogen vapor. Quenched samples of vitrification solutions containing 1,2-ethanediol as the major component exhibited fractures. Fracturing occurred less frequently when the solutions were annealed and when containing polyethylene glycol. Hatching of vitrified embryos stored in liquid nitrogen for over 12 months was not statistically different from those held for only 15 min. Our protocol yielded normalized hatching rates that ranged as high as 61%. Selecting the exact stage for cryopreservation from a population of embryos obtained by collection from ovipositing females during a span of just 30 min resulted in nearly 80% of the embryos hatching into larvae. Published by Elsevier Inc.