Jiménez-Trigos E, Naturil-Alfonso C, Vicente J S, Marco-Jiménez F
Institute of Science and Animal Technology, Laboratorio de Biotecnología de la Reproducción, Universidad Politécnica de Valencia, Valencia, Spain.
Reprod Domest Anim. 2012 Jun;47(3):472-8. doi: 10.1111/j.1439-0531.2011.01906.x. Epub 2011 Oct 3.
Although much progress has been made in oocyte cryopreservation since 1971, live offspring have only been obtained in a few species and in rabbits. The aim of our study was to evaluate the effect of vitrification and slow freezing on the meiotic spindle, cortical granule (CG) distribution and their developmental competence. Oocytes were vitrified in 16.84% ethylene glycol, 12.86% formamide, 22.3% dimethyl sulphoxide, 7% PVP and 1% of synthetic ice blockers using Cryotop as device or slow freezing in 1.5 m PROH and 0.2 m sucrose in 0.25 ml sterile French mini straws. Meiotic spindle and CG distribution were assessed using a confocal laser-scanning microscope. To determine oocyte competence, in vitro development of oocytes from each cryopreservation procedure was assessed using parthenogenesis activation. Our data showed that oocytes were significantly affected by both cryopreservation procedures. In particular, meiotic spindle organization was dramatically altered after cryopreservation. Oocytes with peripheral CG distribution have a better chance of survival in cryopreservation after slow-freezing procedures compared to vitrification. In addition, slow freezing of oocytes led to higher cleavage and blastocyst rates compared to vitrification. Our data showed that, in rabbits, structural alterations are more evident in vitrified oocytes than in slow-frozen oocytes, probably as a consequence of sensitivity to high levels of cryoprotectants. Slow-freezing method is currently the recommended option for rabbit oocyte cryopreservation.
自1971年以来，卵母细胞冷冻保存技术取得了很大进展，但仅在少数物种（包括兔子）中获得了活产后代。我们研究的目的是评估玻璃化冷冻和慢速冷冻对减数分裂纺锤体、皮质颗粒（CG）分布及其发育能力的影响。使用Cryotop装置将卵母细胞在含有16.84%乙二醇、12.86%甲酰胺、22.3%二甲基亚砜、7%聚乙烯吡咯烷酮和1%合成冰阻断剂的溶液中进行玻璃化冷冻，或将其在含有1.5 m丙二醇和0.2 m蔗糖的0.25 ml无菌法国迷你细管中进行慢速冷冻。使用共聚焦激光扫描显微镜评估减数分裂纺锤体和CG分布。为了确定卵母细胞的能力，使用孤雌激活评估每种冷冻保存程序的卵母细胞的体外发育情况。我们的数据表明，两种冷冻保存程序均对卵母细胞有显著影响。特别是，冷冻保存后减数分裂纺锤体的组织发生了显著改变。与玻璃化冷冻相比，在慢速冷冻程序后，具有外周CG分布的卵母细胞在冷冻保存中有更好的存活机会。此外，与玻璃化冷冻相比，卵母细胞的慢速冷冻导致更高的卵裂率和囊胚率。我们的数据表明，在兔子中，玻璃化冷冻的卵母细胞比慢速冷冻的卵母细胞结构改变更明显，这可能是对高水平冷冻保护剂敏感的结果。目前，慢速冷冻方法是兔子卵母细胞冷冻保存的推荐选择。
