Tada N, Sato M, Yamanoi J, Mizorogi T, Kasai K, Ogawa S
Section of Experimental Animal Administration, Hoechst Japan Limited, Saitama.
J Reprod Fertil. 1990 Jul;89(2):511-6. doi: 10.1530/jrf.0.0890511.
When mouse epididymal spermatozoa were rapidly frozen in two steps (37 to -70 degrees C for solid CO2 and -70 to -196 degrees C for liquid nitrogen) as pellets, 18% raffinose provided the greatest protection to ICR mouse spermatozoa against cold-shock; sperm motility and fertilizing ability were 43% and 22.4%, respectively. A small proportion of spermatozoa frozen with 10% sucrose was motile but incapable of fertilizing ovulated oocytes. Glycerol and dimethylsulphoxide were less effective at any concentration examined. However, the fertilizing ability of frozen-thawed ICR spermatozoa was significantly improved (35.5%) by addition of glycerol (1.75% final concentration) to medium containing 18% raffinose. Spermatozoa from one outbred (ddY) and 5 inbred (C57BL/6N, C3H/HeN, DBA/2N, BALB/c and kk) strains of mice were successfully frozen in the presence of 18% raffinose and 1.75% glycerol, although the fertilization rates of frozen-thawed spermatozoa varied among strains (13% for C57BL/6N to 64% for DBA/2N). A small fraction of mouse eggs resulting from fertilization by frozen-thawed spermatozoa developed normally in vitro (37% in C57BL/6N to 71% in ICR) to the blastocyst stage and in vivo (19% for C57BL/6N spermatozoa and ddY oocytes) to Day 18 of gestation.
当将小鼠附睾精子制成颗粒，分两步快速冷冻（37℃至-70℃用固态二氧化碳，-70℃至-196℃用液氮）时，18%的棉子糖对ICR小鼠精子的冷休克具有最大的保护作用；精子活力和受精能力分别为43%和22.4%。用10%蔗糖冷冻的一小部分精子有活力，但不能使排卵的卵母细胞受精。在任何检测浓度下，甘油和二甲亚砜的效果都较差。然而，在含有18%棉子糖的培养基中添加甘油（终浓度1.75%），可使冻融后的ICR精子的受精能力显著提高（35.5%）。一只远交（ddY）和5只近交（C57BL/6N、C3H/HeN、DBA/2N、BALB/c和kk）品系小鼠的精子在18%棉子糖和1.75%甘油存在的情况下成功冷冻，尽管冻融后精子的受精率在不同品系间有所不同（C57BL/6N为13%，DBA/2N为64%）。由冻融精子受精产生的一小部分小鼠卵在体外（C57BL/6N为37%，ICR为71%）正常发育到囊胚阶段，在体内（C57BL/6N精子和ddY卵母细胞为19%）发育到妊娠第18天。
