El-Shalofy A S, Ismail S T, Badawy A A B, Darwish G M, Badr M R, Moawad A R
Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University, PO BOX 12211, Giza, Egypt.
Department of AI and ET, Animal Reproduction Research Institute, Agriculture Research Centre, Giza, Egypt.
Cryo Letters. 2020 Nov-Dec;41(6):351-357.
Cryopreservation of immature oocyte is a potential strategy for preserving the female germline, providing a non-seasonal, easily accessible source for reproduction and science. Exposure of oocytes to high concentrations of cryoprotectants during vitrification is toxic and can negatively impact the fertilization ability and development of vitrified/warmed oocytes.
Cumulus oocyte complexes (COCs) obtained at slaughter from mature buffalo ovaries were randomly assigned into five groups: control - directly subjected to IVM); VS1 group - exposed to 20% ethylene glycol (EG) + 20% glycerol (GLY) + 0.5 M sucrose; VS2 group - exposed to 20% EG + 20% GLY; VS3 group - subjected to 20% EG+20% dimethyl sulfoxide (DMSO) + 0.5 M sucrose; and VS4 group - subjected to 20% EG+20% DMSO. Following cryoprotectant dilution, viable oocytes were matured in vitro for 22 h; cumulus expansion and nuclear maturation were then evaluated (Experiment 1). COCs were vitrified by solid surface vitrification (SSV) in a solution composed of 20% EG + 20% DMSO (VS4). Following vitrification, COCs were warmed in a solution composed of either sucrose or trehalose in decreasing concentrations (1 M, 0.5 M and 0.25 M). Morphologically viable oocytes were matured, fertilized and cultured in vitro. Cleavage and blastocyst rates were evaluated at 30 h and day 7 post-insemination (p.i.), respectively (Experiment 2).
Exposure of GV-buffalo oocytes to different cryoprotectant combinations did not significantly affect cumulus expansion following IVM. However, nuclear maturation rate (oocytes at MII) was significantly higher (P<0.05) in the groups exposed to sucrose-free vitrification solutions (VS2 and VS4) and not significantly different from the control. Compared with the control group, the cleavage and blastocyst rates were significantly (P<0.05) lower in oocytes vitrified and then warmed in a solution containing trehalose; whilst this was not the case when sucrose was present in the solution.
Our results suggest that exposure of buffalo GV-oocytes to sucrose-free vitrification solutions improved nuclear maturation after IVM. Moreover, warming of vitrified buffalo oocytes in sucrose-based solution improved preimplantation development following IVM and IVF compared to trehalose based media.
未成熟卵母细胞的冷冻保存是保存雌性生殖系的一种潜在策略，可为生殖和科研提供非季节性、易于获取的资源。卵母细胞在玻璃化过程中暴露于高浓度冷冻保护剂是有毒的，会对玻璃化/解冻卵母细胞的受精能力和发育产生负面影响。
1）评估水牛生发泡（GV）期卵母细胞暴露于添加或不添加蔗糖的不同玻璃化溶液（VS）对体外成熟（IVM）后卵丘扩展和核成熟的影响；2）比较解冻液中蔗糖和海藻糖对GV期玻璃化水牛卵母细胞发育能力的影响。
从屠宰的成熟水牛卵巢中获取的卵丘卵母细胞复合体（COCs）随机分为五组：对照组——直接进行IVM；VS1组——暴露于20%乙二醇（EG）+20%甘油（GLY）+0.5 M蔗糖；VS2组——暴露于20%EG+20%GLY；VS3组——暴露于20%EG+20%二甲基亚砜（DMSO）+0.5 M蔗糖；VS4组——暴露于20%EG+20%DMSO。冷冻保护剂稀释后，存活的卵母细胞体外成熟22小时；然后评估卵丘扩展和核成熟情况（实验1）。COCs通过固体表面玻璃化（SSV）在由20%EG+20%DMSO组成的溶液（VS4）中进行玻璃化。玻璃化后，COCs在由浓度递减的蔗糖或海藻糖（1 M、0.5 M和0.25 M）组成的溶液中解冻。形态学上存活的卵母细胞进行成熟、受精和体外培养。分别在授精后30小时和第7天评估卵裂率和囊胚率（实验2）。
GV期水牛卵母细胞暴露于不同冷冻保护剂组合对IVM后的卵丘扩展没有显著影响。然而，暴露于无蔗糖玻璃化溶液（VS2和VS4）的组中，核成熟率（处于MII期卵母细胞）显著更高（P<0.05），且与对照组无显著差异。与对照组相比，玻璃化后在含海藻糖溶液中解冻的卵母细胞的卵裂率和囊胚率显著更低（P<0.05）；而溶液中存在蔗糖时情况并非如此。
我们的结果表明，水牛GV期卵母细胞暴露于无蔗糖玻璃化溶液可提高IVM后的核成熟率。此外，与基于海藻糖的培养基相比，在基于蔗糖的溶液中解冻玻璃化水牛卵母细胞可改善IVM和IVF后的植入前发育。
