The Southern Flounder Paralichthys lethostigma is a high-value species and a promising aquaculture candidate. Because sperm volume can be limited in this species (<500 μL), new sperm cryopreservation methods need to be evaluated. Vitrification is an alternative to conventional slow-rate freezing, whereby small volumes are cryopreserved at high cooling rates (>1,000°C/min). The goal of this work was to develop a standardized approach for vitrification of Southern Flounder sperm. The specific objectives were to (1) evaluate thawing methods and vitrification solutions, (2) evaluate the postthaw membrane integrity of sperm vitrified in different cryoprotectant solutions, (3) examine the relationship between membrane integrity and motility, and (4) evaluate the ability of vitrified sperm to fertilize eggs. From the vitrification solutions tested, the highest postthaw motility (28 ± 9% [mean ± SD]) and membrane integrity (11 ± 4%) was observed for 20% ethylene glycol plus 20% glycerol. There was no significant difference in postthaw motility of sperm thawed at 21°C or at 37°C. Fertilization from vitrified sperm in one trial yielded the same fertilization rate (50 ± 20%) as the fresh sperm control, while the sperm from the other two males yielded 3%. This is the first report of fertilization by vitrified sperm in a marine fish. Vitrification can be simple, fast, inexpensive, performed in the field, and, at least for small fishes, offers an alternative to conventional cryopreservation. Because of the minute volumes needed for ultrarapid cooling, vitrification is not presently suited as a production method for large fishes. Vitrification can be used to reconstitute lines from valuable culture species and biomedical models, conserve mutants for development of novel lines for ornamental aquaculture, and transport frozen sperm from the field to the repository to expand genetic resources.

中文翻译：

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玻璃化冷冻作为海洋鱼类精子冷冻保存的替代方法


南方比目鱼Paralichthys lethostigma 是一种高价值物种，也是一个有前途的水产养殖候选物种。因为该物种的精子数量有限（<500 id=0>1,000°C/min）。这项工作的目标是开发南方比目鱼精子玻璃化的标准化方法。具体目标是（1）评估解冻方法和玻璃化溶液，（2）评估在不同冷冻保护剂溶液中玻璃化的精子解冻后膜的完整性，（3）检查膜完整性和运动性之间的关系，以及（4）评估能力玻璃化精子使卵子受精。从测试的玻璃化溶液中，20% 乙二醇加 20% 甘油观察到最高的解冻后活力 (28 ± 9% [平均值 ± SD]) 和膜完整性 (11 ± 4%)。 21℃和37℃解冻的精子解冻后活力没有显着差异。在一项试验中，玻璃化精子的受精率与新鲜精子对照的受精率相同（50±20%），而其他两只雄性的精子的受精率仅为 3%。这是海鱼玻璃化精子受精的首次报道。玻璃化冷冻可以简单、快速、便宜，可以在现场进行，并且至少对于小鱼来说，提供了传统冷冻保存的替代方案。由于超快速冷却所需的体积极小，因此玻璃化目前不适合作为大型鱼类的生产方法。玻璃化可用于从有价值的养殖物种和生物医学模型中重建品系，保存突变体以开发用于观赏水产养殖的新品系，以及将冷冻精子从田间运输到储存库以扩大遗传资源。