Vajta G, Holm P, Greve T, Callesen H
Embryo Technology Center, Danish Institute of Animal Science, Denmark.
Anim Reprod Sci. 1996 Dec 16;45(3):191-200. doi: 10.1016/s0378-4320(96)01583-7.
The aim of this work was to investigate the possibilities of simplification, and to outline the limits of application, of a vitrification method for cow embryos. Morulae and blastocysts were produced by in vitro fertilization of slaughterhouse-derived, in vitro matured oocytes with frozen-thawed bull semen, and subsequent culture on a granulosa cell monolayer. Vitrification was performed by equilibration of embryos with 12.5% ethylene glycol and 12.5% dimethylsulphoxide at 20-22 degrees C for 60 s, then with 25% ethylene glycol and 25% dimethylsulphoxide at 4 degrees C for another 60 s. Embryos were then loaded in straws, placed in liquid nitrogen vapour for 2 min, and then plunged. Straws were thawed in a 22 degrees C water-bath, the embryos were directly rehydrated and further incubated in straw, and were then expelled and cultured in vitro for 72 h. In the first experiment, embryos of different age and developmental stage (Day 5 compacted morulae, Day 6 early blastocysts, Days 6 and 7 blastocysts, Day 7 expanded blastocysts and Day 8 hatched blastocysts) as well as Days 7 and 5 blastocysts previously subjected to partial zone dissection were vitrified. After thawing, the re-expansion rates of blastocysts and zona-dissected embryos did not differ (67 and 87%, respectively), and hatching was more frequent for blastocysts frozen in advanced developmental stages (34, 47 and 63% for early blastocysts, blastocysts and expanded blastocysts, respectively). The re-expansion rate of morulae was lower (10%) and no hatching of these embryos was observed. In the second experiment, Day 7 expanded blastocysts were vitrified using PBS, PBS+albumin, TCM199 and TCM199+calf serum as holding media. No differences in re-expansion and hatching rates were seen. However, when incubation with the concentrated cryoprotectant solution was performed at 20-22 degrees C, the embryo survival rate decreased (PBS+albumin) or no embryo survived (TCM199+calf serum) the vitrification procedure. In the third experiment, Day 7 expanded blastocysts were vitrified, thawed, cultured for 1 day, and then re-expanded embryos were again vitrified and thawed. Out of the 87% that survived the first cycle, 73% re-expanded and 47% hatched following the second vitrification and thawing. These observations prove that the vitrification procedure described is relatively harmless, that it can be used for blastocysts of different developmental stages and that an intact zona is not required to obtain high survival rates.
本研究旨在探讨简化牛胚胎玻璃化方法的可能性，并概述其应用局限性。通过用冷冻解冻的公牛精液对屠宰场来源的体外成熟卵母细胞进行体外受精，并随后在颗粒细胞单层上培养，获得桑椹胚和囊胚。玻璃化操作如下：将胚胎在20 - 22℃下用12.5%乙二醇和12.5%二甲基亚砜平衡60秒，然后在4℃下用25%乙二醇和25%二甲基亚砜再平衡60秒。然后将胚胎装入细管，置于液氮蒸气中2分钟，随后投入液氮。细管在22℃水浴中解冻，胚胎直接复水并在细管中进一步培养，然后挤出并体外培养72小时。在第一个实验中，对不同年龄和发育阶段的胚胎（第5天的致密桑椹胚、第6天的早期囊胚、第6天和第7天的囊胚、第7天的扩张囊胚和第8天的孵化囊胚）以及先前进行过部分区域切割的第7天和第5天囊胚进行玻璃化处理。解冻后，囊胚和经区域切割的胚胎的再扩张率没有差异（分别为67%和87%），并且在发育晚期冷冻的囊胚孵化更为频繁（早期囊胚、囊胚和扩张囊胚的孵化率分别为34%、47%和63%）。桑椹胚的再扩张率较低（10%），且未观察到这些胚胎孵化。在第二个实验中，使用PBS、PBS +白蛋白、TCM199和TCM199 +小牛血清作为保存液对第7天的扩张囊胚进行玻璃化处理。再扩张率和孵化率没有差异。然而，当在20 - 22℃下与浓缩冷冻保护剂溶液孵育时，胚胎存活率下降（PBS +白蛋白）或没有胚胎在玻璃化过程中存活（TCM199 +小牛血清）。在第三个实验中，对第7天的扩张囊胚进行玻璃化、解冻、培养1天，然后将再扩张的胚胎再次玻璃化和解冻。在第一个周期存活的87%的胚胎中，第二次玻璃化和解冻后，73%再扩张，47%孵化。这些观察结果证明，所描述的玻璃化程序相对无害，可用于不同发育阶段的囊胚，并且获得高存活率不需要完整的透明带。
