Bagis Haydar, Mercan H Odaman, Cetin S, Sekmen S
TUBITAK, Research Institute for Genetic Engineering and Biotechnology (RIGEB), Transgenic Core Facility, Kocaeli, Turkey.
Mol Reprod Dev. 2005 Dec;72(4):494-501. doi: 10.1002/mrd.20263.
The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage (PN) mouse embryos. A novel vitrification technique (solid surface vitrification, SSV) was compared with a convential one in straws both for cryosurvival and obtaining progeny from cryopreserved PN mouse embryos. In the SSV method, 15-20 PN embryos were exposed to vitrification solutions for approximately 20 sec after equilibration, and then they were dropped in 2 microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. In the straws method, groups of 5-10 PN embryos were loaded in a single straw after equilibration. In experiment I, it was compared the effect of the vitrification solutions alone, without vitrification. No reduction was detected in survival, cleavage and blastocysts rates and the lowest development rate was obtained from hatched blastocyst for 20 min equilibration (24.5%). In experiment II, SSV method resulted in significantly higher survival and cleavage rates than that of in-straw vitrified 15-20 min group (87% vs. 60%, 83% vs. 67%, respectively; P < 0.05). There were no statistical differences among any of the blastocyts groups. However, there was a statistical difference in hatched blastocysts between 15 to 5, 10, and 20 min (P < 0.05). In experiment III, it was found no major effect among equilibration time periods in toxicity groups according to the mean cell number of blastocysts developed from PN embryos. But, there was a significant differences between 15 min SSV and 10 min in straw vitrified according to the mean cell number of blastocysts developed from PN embryos following vitrification (P < 0.05). The good results were obtained from 15 min equilibration group for SSV and 10 min equilibration group for straw vitrification. In the last experiment, embryo transfer after vitrification and toxicity was investigated. There were significant differences between SSV and straw just on the rate of pups born (30% and 20.5% respectively; P < 0.05). In conclusion, vitrification of PN mouse embryos by SSV can result in high rates of in vitro development to expanded and hatched blastocyst stage and in vivo development to live pups.
本研究的目的是提高原核期（PN）小鼠胚胎的冷冻保存效率。将一种新型玻璃化技术（固体表面玻璃化，SSV）与传统的细管玻璃化技术在冷冻存活率以及从冷冻保存的PN小鼠胚胎获得后代方面进行比较。在SSV方法中，15 - 20个PN胚胎平衡后暴露于玻璃化溶液中约20秒，然后将它们滴入2微升液滴中，滴到预冷（-150至-180摄氏度）的金属表面上。在细管方法中，5 - 10个PN胚胎平衡后装入单个细管中。在实验I中，比较了仅使用玻璃化溶液而不进行玻璃化的效果。未检测到存活率、分裂率和囊胚率降低，并且对于20分钟平衡的孵化囊胚获得了最低的发育率（24.5%）。在实验II中，SSV方法导致的存活率和分裂率显著高于细管玻璃化15 - 20分钟组（分别为87%对60%，83%对67%；P < 0.05）。任何囊胚组之间均无统计学差异。然而，15至5、10和20分钟的孵化囊胚之间存在统计学差异（P < 0.05）。在实验III中，根据从PN胚胎发育而来的囊胚的平均细胞数，发现在毒性组的平衡时间段之间没有主要影响。但是，根据玻璃化后从PN胚胎发育而来的囊胚的平均细胞数，15分钟SSV和10分钟细管玻璃化之间存在显著差异（P < 0.05）。对于SSV，15分钟平衡组和细管玻璃化10分钟平衡组获得了良好的结果。在最后一个实验中，研究了玻璃化和毒性后的胚胎移植情况。仅在出生幼崽率方面SSV和细管之间存在显著差异（分别为30%和20.5%；P < 0.05）。总之，通过SSV对PN小鼠胚胎进行玻璃化可导致体外发育到扩张和孵化囊胚阶段以及体内发育到活幼崽的高比率。
