Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution
Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution
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DOI：
10.1016/j.cryobiol.2007.08.011
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发表时间：
2007-12-01
期刊：
影响因子：
2.7
通讯作者：
Sato, Eimei
中科院分区：
文献类型：
--
作者：
Yamashiro, Hideaki;Han, Young-Joon;Sato, Eimei
本研究的目的是建立一种理想的大鼠附睾精子冷冻液和冷冻方法。将从30只Wistar雄性收集的附睾精子冷冻，并进行实验以研究其解冻后的特性，当用不含棉子糖的缓冲液或溶解在蒸馏水和去离子水中的各种浓度的棉子糖和蛋黄、PBS或基于改良Krebs-Ringer碳酸氢盐（mKRB）的扩展剂冷冻时。还测试了溶解在PBS或含有mKRB的蛋黄中的不同浓度的甘油、Equex STM或十二烷基硫酸钠（SDS）。基于这些实验的数据，进一步的实验测试了不同的糖如棉子糖、海藻糖、乳糖、果糖和葡萄糖溶解在含有Equex STM、SDS和蛋黄补充剂的mKRB中如何影响冷冻保存精子的解冻后特征。通过显微镜下评估精子活力指数（SMI）判断冷冻-解冻精子的冷冻存活率，并使用FITC-PNA染色测量顶体完整性。对解冻的精子进行3小时的耐热性试验。当将0.1 M棉子糖的mKRB溶液与0.75% Equex STM、0.05% SDS和20%蛋黄一起使用时，获得了对精子解冻后存活的有益效果。与在不同浓度的其他冷冻保护剂和海藻糖、乳糖、果糖、葡萄糖中冷冻的精子相比，用这种处理冷冻保存的精子表现出更高的运动指数，并在整个孵育过程中保持更大的SMI和顶体完整性。总之，在含有Equex STM、SDS和蛋黄的rnKRB中溶解棉子糖的稀释液中冷冻保存可大大提高大鼠附睾精子的冷冻能力。(C)2007年爱思唯尔公司All rights reserved.
The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs-Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in rnKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm. (C) 2007 Elsevier Inc. All rights reserved.