Cryopreservation of medaka spermatozoa
Cryopreservation of medaka spermatozoa
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DOI：
10.2108/zsj.14.641
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发表时间：
1997-08-01
影响因子：
0.9
通讯作者：
Ishikawa, Y
中科院分区：
文献类型：
--
作者：
Aoki, K;Okamoto, M;Ishikawa, Y
为了建立一个可靠的和可重复的青鳉精子冷冻保存方法，我们测试了几种程序。采用液氮（LN）的气相、液氮的液相和干冰进行冷冻，并使用二甲基亚砜和N，N-二甲基甲酰胺（DMF）作为冷冻保护剂。使用以下方法获得最佳结果。通过挤压分离的睾丸，将青鳉精子收集在含有50 μ l补充有10%DMF的胎牛血清的塑料管中。通过将试管在LN的气相中保持10或20分钟，在距容器顶部9或10 cm的深度处冷冻精子悬浮液。将冷冻样品浸入并储存在LN中。在储存超过一周后，通过在30 ℃水浴中孵育0.5-1分钟来快速解冻管中的样品，然后立即用2体积的Iwamatsu溶液稀释。用新鲜的未受精卵进行的受精试验表明，储存的精子可以使96-100%的卵受精。受精卵的孵化率为84- 100%。因此，这项研究提供了，第一次，一个实用的方法保存青鳉精子。
In order to establish a reliable and reproducible method for cryopreservation of medaka spermatozoa, we tested several procedures. The vapor phase of liquid nitrogen (LN), the liquid phase of LN, and dry ice were used for freezing, and dimethyl sulfoxide and N, N-dimethylformamide (DMF) were used as cryoprotectants. The best results were obtained using the following method. Medaka spermatozoa were collected in a plastic tube containing 50 mu l of fetal bovine serum supplemented with 10% DMF by squeezing an isolated testis. The sperm suspension was frozen by holding the tube for 10 or 20 min in the vapor phase of LN at a depth of 9 or 10 cm from the top of a container. The frozen sample was immersed and stored in LN. After more than one week of storage, the sample in the tube was rapidly thawed by being incubated in a waterbath for 0.5-1 min at 30 degrees C, and then immediately diluted with 2 volumes of Iwamatsu's solution. Fertilization tests using fresh unfertilized eggs showed that the stored spermatozoa could fertilize 96-100% of the eggs. The hatchability of the fertilized eggs was 84-100%. Thus, this study provides, for the first time, a practical method for preserving medaka spermatozoa.