冷冻保护剂添加-去除过程对猪MII期卵母细胞损伤研究

Study on Injuries of Porcine MII-stage Oocytes during Cryoprotectants Addition and Removal Processes

在卵母细胞的低温保存过程中,冷冻保护剂的添加与去除是一个必不可少的步骤,但高浓度的冷冻保护剂会对细胞造成渗透损伤和毒性损伤。为研究冷冻保护剂添加与去除过程对猪MII期卵母细胞的损伤,本文采用传统的方法(一步法、两步法)来添加和去除冷冻保护剂,探究冷冻液、解冻液、稀释液对细胞存活率的影响。实验结果表明:在冷冻保护剂添加-去除过程中,卵母细胞的渗透损伤主要发生在卵母细胞从冷冻液转移至解冻液,以及保护剂去除阶段;解冻液浓度与平衡时间对卵母细胞存活率有很大影响;卵母细胞保护剂的去除与添加过程是相关的,解冻液浓度和平衡时间的选择依赖于细胞在冷冻液中的浓度和平衡时间。此研究结果将为猪MII期卵母细胞的低温保存方案提供参考。

Cryoprotectants addition and removal are essential steps during oocytes cryopreservation, however, high concentration of cryoproteetants may cause osmotic and toxic injuries to oocytes. In order to study the injuries of porcine MII-stage oocytes during cryoprotectants addition and removal processes, eryoprotectant was loaded and unloaded to/from oocytes with traditional methods （one-step method, two-step method）, the effects of vitrification solution, thawing solution and dilution solution on the survival rate of oocytes were investigated. The experimental results showed that the osmotic injuries to oocytes happened mainly in two stages ： the transfer process from vitrification solution to thawing solution, and the cryoprotectants removal process. The concentration of thawing solution and equilibrium time had a great influence on the survival rate of oocytes. The cryoprotectants removal process was highly correlated with addition process, and the selection of thawing solution concentration and equilibrium time relied on the vitrification solution concentration and equilibrium time. These results could provide a reference for cryopreservation protocols of porcine MII-stage oocytes.