School of Environmental Science and Public Health, Wenzhou Medical College, Wenzhou, China.
Cryobiology. 2009 Dec;59(3):351-6. doi: 10.1016/j.cryobiol.2009.09.011. Epub 2009 Sep 26.
In this study, we evaluated various parameters of sperm cryopreservation in two livebearers, guppies (Poecilia reticulata) and black mollies (P. latipinna). Our results suggested a common freezing protocol for the guppies and mollies: suspend sperm in Hanks' balanced salt solution (HBSS) at 300 mOsm/kg, use 14% glycerol as cryoprotectant, cool at 25 degrees C/min, and thaw at 40 degrees C in a water bath for 7s. Live young were produced from females inseminated with frozen-thawed sperm in both species. In guppies, percent fertilization (F) and the number of embryos (N) produced with cryopreserved sperm (F=50%, N=74, from 26 females) were similar to those of fresh controls (F=54%, N=61, from 22 females). Interestingly, this same freezing protocol has been used successfully for sperm cryopreservation in green swordtails Xiphophorus helleri, and platyfish of X. couchianus with post-thaw motility as high as 80%. All these species belong to the family of Poeciliidae, and their sperm are similar in morphology exhibiting the absence of acrosome, elongate sperm head, and the long mitochondrial sheaths. Besides their internal fertilization reproduction mode, these fish are also small in size (2-4 cm) and live in a freshwater environment. Sperm cryopreservation in fish has been generally recognized as species specific, and new protocols are required for new species. However, results presented in this study suggested otherwise. Thus, sperm cryopreservation methods optimized for one species may be applicable to others if they are taxonomical closely related species with similar sperm morphology and reproduction mode. Considering the enormous number of fish species on the planet, development of generalized sperm freezing protocols for species in groups could have additional advantages for genetic conservation.
在这项研究中，我们评估了两种生活在水中的鱼（孔雀鱼和黑玛丽鱼）的精子冷冻保存的各种参数。我们的结果表明，对于孔雀鱼和玛丽鱼，可以使用一种通用的冷冻方案：将精子悬浮在 Hank's 平衡盐溶液（HBSS）中，渗透压为 300mOsm/kg，使用 14%甘油作为冷冻保护剂，以 25℃/分钟的速度冷却，在 40℃的水浴中解冻 7 秒。在这两个物种中，用冷冻和解冻的精子授精的雌性都产下了活鱼苗。在孔雀鱼中，用冷冻精子受精的百分比（F）和胚胎数量（N）与新鲜对照组相似（F=54%，N=61，来自 22 只雌性）。有趣的是，相同的冷冻方案已成功用于绿剑尾鱼和盘丽鱼的精子冷冻保存，解冻后的精子活力高达 80%。所有这些物种都属于花鳉科，它们的精子在形态上相似，都没有顶体，精子头部细长，线粒体鞘较长。除了它们的体内受精繁殖模式外，这些鱼的体型也很小（2-4 厘米），生活在淡水环境中。鱼类的精子冷冻保存通常被认为是物种特异性的，需要为新物种制定新的方案。然而，本研究的结果表明并非如此。因此，如果两个物种在分类上密切相关，具有相似的精子形态和繁殖模式，那么为一个物种优化的精子冷冻保存方法可能适用于其他物种。考虑到地球上鱼类物种的数量巨大，如果为同一类群的物种开发通用的精子冷冻保存方案，可能会对遗传保护有额外的好处。
