Faragó Bernadett, Kollár Tímea, Szabó Katalin, Budai Csilla, Losonczi Eszter, Bernáth Gergely, Csenki-Bakos Zsolt, Urbányi Béla, Pribenszky Csaba, Horváth Ákos, Cserepes Judit
Applied Cell Technology Ltd., Budapest, Hungary.
Szent István University, Department of Aquaculture, Gödöllő, Hungary.
PLoS One. 2017 Feb 6;12(2):e0171520. doi: 10.1371/journal.pone.0171520. eCollection 2017.
Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked.
In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested.
Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90-120 days) and were able to reproduce, resulting in offspring in expected quantity and quality.
Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development.
尽管存在市场需求，并且为了保存众多现有品系之间的遗传变异付出了巨大努力，但斑马鱼胚胎的冷冻保存仍是一个未解决的问题。胚胎的冷藏可能是朝着成功的冷冻保存迈出的一步，但迄今为止尚无有效的方法。
在本研究中，我们应用了一种新策略，通过对胚胎进行预处理静水压力处理来提高斑马鱼胚胎的耐冷性。在我们的实验中，26体节期和Prim-5期斑马鱼胚胎在预处理后于0°C冷藏24小时。测试了胚胎的存活率、成熟能力和受精能力。
我们的结果表明，应用的预处理技术使冷藏胚胎能够正常发育直至成熟，并像正常情况一样产生健康的后代，从而成功传递其遗传物质。处理后的胚胎具有显著更高的存活率和更好的发育率，此外，处理组在持续发育过程中正常形态的比例更高。虽然所有冷藏胚胎的对照组在受精后30天死亡，但处理组达到成熟（约90-120天）并能够繁殖，产生了预期数量和质量的后代。
基于我们的结果，我们得出结论，预处理技术在斑马鱼胚胎耐冷性方面有显著提高，从而实现长期存活。此外，由于胚胎发育在冷藏期间停止，该技术还提供了同步或延迟发育的解决方案。
