Agriculture and Agri-Food Canada, Saskatoon Research Centre, Saskatoon, Saskatchewan, Canada.
Reprod Biol Endocrinol. 2012 Sep 6;10:73. doi: 10.1186/1477-7827-10-73.
The present studies evaluated the effects of cryoprotectants, the vitrification procedure and time in the warming solution containing sucrose on cleavage and embryo development of immature (GV stage) bovine cumulus-oocyte complexes (COCs).
Two experiments were conducted. In Experiment 1, COCs (n = 420) were randomly assigned to four groups: 1) CONTROL GROUP: no treatment; 2) VS1 group: COCs were exposed to vitrification solution 1 (VS1) containing 7.5% ethylene glycol [EG] + 7.5% dimethyl sulfoxide [DMSO] + 20% calf serum [CS] in TCM-199 at 37 C for 5 min; 3) VS1 + VS2 group: COCs were exposed to VS1 for 5 min followed by VS2 (15% EG + 15% DMSO + 17.1% sucrose + 20% CS) at 37 C for 45-60 sec; and 4) Vitrified group: COCs were exposed to VS1 and VS2, loaded on cryotops, vitrified in liquid nitrogen and then warmed in TCM-199 + 17.1% sucrose + 20% CS at 37 C for 1 min. In Experiment 2, COCs (n = 581) were assigned to the same groups, but those in VS1, VS1 + VS2 and Vitrified groups were sub-divided and exposed to the warming solution for either 1 or 5 min. After treatment and/or warming, all COCs in both experiments underwent in vitro maturation, in vitro fertilization and in vitro culture.
Cleavage and blastocyst rates did not differ among Control, VS1 and VS1 + VS2 groups in either experiment. In Experiment 2, there was no effect of time in the warming solution.However, both cleavage and blastocyst rates were lower (P < 0.001) in the Vitrified group than in the Control, VS1 and VS1 + VS2 groups (40.9 and 1.6% vs 92.2 and 34.4%, 79.4 and 25.2%, and 80.2 and 20.8%, respectively in Experiment 1, and 25.0 and 1.7% vs 75.3 and 27.2%, 67.9 and 19.5%, and 62.7 and 22.5%, respectively in Experiment 2).
The permeating cryoprotectants (EG and DMSO) present in VS1 and VS2 solutions and the time in the warming solution containing sucrose had no adverse effects on cleavage and blastocyst rates of immature bovine COCs. However, cleavage rate and early embryo development were reduced following the vitrification and warming.
本研究评估了冷冻保护剂、玻璃化程序和含蔗糖的解冻液中的时间对不成熟（GV 期）牛卵丘-卵母细胞复合物（COCs）的卵裂和胚胎发育的影响。
进行了两项实验。在实验 1 中，将 COCs（n=420）随机分配到四个组：1）对照组：无处理；2）VS1 组：COCs 在 37°C 下暴露于含有 7.5%乙二醇[EG] + 7.5%二甲基亚砜[DMSO] + 20%牛血清[CS]的 VS1 溶液中 5 分钟；3）VS1+VS2 组：COCs 在 37°C 下暴露于 VS1 5 分钟，然后暴露于 VS2（15%EG+15%DMSO+17.1%蔗糖+20%CS）45-60 秒；和 4）玻璃化组：COCs 暴露于 VS1 和 VS2，加载在 cryotops 上，在液氮中玻璃化，然后在 37°C 的 TCM-199+17.1%蔗糖+20%CS 中解冻 1 分钟。在实验 2 中，将 COCs（n=581）分配到相同的组，但 VS1、VS1+VS2 和玻璃化组被细分，并在解冻液中暴露 1 或 5 分钟。处理和/或解冻后，两个实验中的所有 COCs 均进行体外成熟、体外受精和体外培养。
在两个实验中，对照组、VS1 组和 VS1+VS2 组之间的卵裂率和囊胚率没有差异。在实验 2 中，解冻液中的时间没有影响。然而，玻璃化组的卵裂率和囊胚率均低于对照组、VS1 组和 VS1+VS2 组（分别为 40.9%和 1.6%对 92.2%和 34.4%、79.4%和 25.2%、80.2%和 20.8%，实验 1；分别为 25.0%和 1.7%对 75.3%和 27.2%、67.9%和 19.5%、62.7%和 22.5%，实验 2）。
VS1 和 VS2 溶液中的渗透冷冻保护剂（EG 和 DMSO）和含蔗糖的解冻液中的时间对不成熟牛 COCs 的卵裂率和囊胚率没有不良影响。然而，玻璃化和解冻后卵裂率和早期胚胎发育降低。
