Takeshima T, Nakagata N, Ogawa S
Imamichi Institute for Animal Reproduction, Ibaraki, Japan.
Jikken Dobutsu. 1991 Oct;40(4):493-7. doi: 10.1538/expanim1978.40.4_493.
The spermatozoa of cauda epididymis of mature mice were suspended in 3% skim milk in distilled water supplemented with 12, 15, 18 or 21% (W/V) raffinose. The suspension of spermatozoa were frozen in liquid nitrogen gas for 10 min, then stored in liquid nitrogen (-196 degrees C). The frozen suspensions of spermatozoa were thawed by rapid warming in water bath at room temperature. For removing the cryopreservative solution, a pair of syringes connected with a three stop cock and a filter unit (pore size 0.45 mu) were used. Highest sperm motility was obtained after 1 hr of thawing from the cryopreservative solution containing 18% raffinose and 3% skim milk. These cryopreserved spermatozoa were used for fertilization in vitro. The proportion of pronuclear oocytes was 35.9% (74/206) 6 hr after insemination, and the proportion of 2-cell embryos was 33.6% (42/125) 28 hr after insemination. All 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 45.2% (19/42) developed to normal young.
将成熟小鼠附睾尾部的精子悬浮于蒸馏水中含12%、15%、18%或21%（W/V）棉子糖的3%脱脂乳中。将精子悬液在液氮气体中冷冻10分钟，然后储存在液氮（-196℃）中。冷冻的精子悬液通过在室温的水浴中快速复温进行解冻。为去除冷冻保护剂溶液，使用了一对与三通旋塞和过滤单元（孔径0.45μm）相连的注射器。从含有18%棉子糖和3%脱脂乳的冷冻保护剂溶液中解冻1小时后，精子活力最高。这些冷冻保存的精子用于体外受精。授精6小时后，原核卵母细胞的比例为35.9%（74/206），授精28小时后，2-细胞胚胎的比例为33.6%（42/125）。所有2-细胞胚胎都被转移到假孕受体的输卵管中，45.2%（19/42）发育为正常幼崽。
