Cryopreservation of zebrafish (Danio rerio) oocytes using improved controlled slow cooling protocols
Cryopreservation of zebrafish (Danio rerio) oocytes using improved controlled slow cooling protocols
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DOI：
10.1016/j.cryobiol.2008.03.001
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发表时间：
2008-06-01
期刊：
影响因子：
2.7
通讯作者：
Zhang, T.
中科院分区：
文献类型：
--
作者：
Guan, M.;Rawson, D. M.;Zhang, T.
配子的超低温保存为鱼类遗传物质的保存提供了一种很有前途的方法。在此之前，我们报道了一些初步的研究结果，斑马鱼（Danio rerio）卵母细胞冷冻保存使用控制缓慢冷却，并确定了最佳的冷冻保护介质和冷却速度为第III阶段斑马鱼卵母细胞。在本研究中，两种不同的冷冻保存介质，冷冻保护剂的去除方法，最终样品冷冻前液氮投入温度，升温速率，解冻后孵育时间对卵母细胞活力的影响进行了研究。常用的冷冻保护剂为甲醇和葡萄糖。将第III阶段斑马鱼卵母细胞冷冻在标准培养基50%L-15或无钠KCl缓冲液培养基中。采用台盼蓝染色和ATP测定法评价卵母细胞活力。在KCl缓冲液中冻存的卵母细胞的存活率显著高于在L-15培养基中冻存的卵母细胞。结果还显示，快速解冻和逐步去除冷冻保护剂显著提高了卵母细胞存活率，当通过台盼蓝染色评估时，快速解冻后立即获得最高存活率88.0 +/- 1.7%。然而，在22 ℃下孵育2小时后，冻融卵母细胞的存活率下降至29.5 +/-5.1%。结果还表明，卵母细胞在解冻后即刻ATP水平显著下降。所有卵母细胞在冷冻后变得半透明，这使GVBD试验的使用复杂化（卵母细胞的体外成熟，随后观察到生殖囊泡破裂，这导致卵母细胞变得半透明）。因此，迫切需要新的卵母细胞存活力评估方法。(C)2008年爱思唯尔公司All rights reserved.
Cryopreservation of gametes provides a promising method to preserve fish genetic material. Previously we reported some preliminary results on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and determined the optimum cryoprotective medium and cooling rate for stage III zebrafish oocytes. In the present study, the effects of two different cryopreservation media, cryoprotectant removal method, final sample freezing temperature before LN2 plunge, warming rate, and the post-thaw incubation time on oocyte viability were investigated. Commonly used cryoprotectant methanol and glucose were used in this study. Stage III zebrafish oocytes were frozen in standard culture medium 50% L-15 or in a sodium-free KCl buffer medium. Oocyte viability was assessed using trypan blue staining and ATP assay. The viability of oocytes frozen in KCl buffer was significantly higher than oocytes frozen in L-15 medium. The results also showed that fast thawing and stepwise removal of cryoprotectant improved oocyte survival significantly, with highest viability of 88.0 +/- 1.7% being obtained immediately after rapid thawing when assessed by trypan blue staining. However, after 2 h incubation at 22 degrees C the viability of freeze-thawed oocytes decreased to 29.5 +/- 5.1%. Results also showed that the ATP level in oocytes decreased significantly immediately after thawing. All oocytes became translucent after freezing which complicated the use of GVBD test (in vitro maturation of oocytes followed by observation of germinal vesicle breakdown which results in oocytes becoming translucent). New oocyte viability assessment methods are urgently needed. (C) 2008 Elsevier Inc. All rights reserved.