Quan Guobo, Lv Chunrong, Liang Jiachong, Zhao Xueming, Wu Guoquan
Yunnan Animal Science and Veterinary Institute, Kunming, China.
Institute of Animal Sciences (IAS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, China.
Biopreserv Biobank. 2022 Apr;20(2):113-122. doi: 10.1089/bio.2021.0013. Epub 2021 May 26.
The effects of the equilibration time, the vitrification procedure, and the warming procedure on the quality of goat oocytes vitrified by Cryotop were assessed. In the first part of the study, oocytes were exposed to 10% dimethyl sulfoxide (DMSO) and 10% ethylene glycol (EG) for 1, 3, 5, or 10 minutes, respectively, followed by vitrification. In the second part, after equilibration in 7.5% DMSO +7.5% EG for 3 minutes, 10% DMSO +10% EG for 3 minutes, or 4% EG for 10 minutes, oocytes were equilibrated in 15% DMSO +15% EG, 20% DMSO +20% EG, or 35% EG for 30 seconds before vitrification. The vitrification procedures were designated as first vitrification procedure (VPI), second vitrification procedure (VPII), and third vitrification procedure (VPIII), respectively. In the third part, oocytes vitrified using VPIII were warmed by the three procedures (first warming procedure [TPI], second warming procedure [TPII], or third warming procedure [TPIII]) containing different concentrations of trehalose. The results showed that after equilibration for 1 or 3 minutes in 10% DMSO and 10% EG, the viability and developmental capability of vitrified oocytes were significantly superior to the groups after equilibration for over 5 minutes ( < 0.05). With the VPIII procedure, the frequencies with normal morphology, cleavage, and blastocyst formation of vitrified oocytes were 91.87% ± 4.14%, 76.51% ± 4.37%, and 39.84% ± 2.91%, respectively, demonstrating a significant increase compared to the VPI or VPII group ( < 0.05). The rates of vitrified oocytes with normal morphology and cleavage in the TPI group were higher than the TPII or TPIII group ( < 0.05). In conclusion, equilibration in 10% DMSO and 10% EG for <3 minutes benefits the viability of vitrified oocytes. EG may be more efficient for vitrification of goat oocytes compared to DMSO. Higher concentrations (more than 1 M) of trehalose enhance cryosurvival of goat oocytes when warming.
评估了平衡时间、玻璃化程序和复温程序对使用Cryotop法玻璃化的山羊卵母细胞质量的影响。在研究的第一部分,将卵母细胞分别暴露于10%二甲基亚砜(DMSO)和10%乙二醇(EG)中1、3、5或10分钟,随后进行玻璃化处理。在第二部分中,卵母细胞在7.5%DMSO + 7.5%EG中平衡3分钟、在10%DMSO + 10%EG中平衡3分钟或在4%EG中平衡10分钟后,在玻璃化处理前于15%DMSO + 15%EG、20%DMSO + 2%EG或35%EG中再平衡30秒。玻璃化程序分别指定为第一玻璃化程序(VPI)、第二玻璃化程序(VPII)和第三玻璃化程序(VPIII)。在第三部分中,使用VPIII玻璃化的卵母细胞通过包含不同浓度海藻糖的三种程序(第一复温程序[TPI]、第二复温程序[TPII]或第三复温程序[TPIII])进行复温。结果表明,在10%DMSO和10%EG中平衡1或3分钟后,玻璃化卵母细胞的活力和发育能力显著优于平衡超过5分钟的组(P < 0.05)。采用VPIII程序时,玻璃化卵母细胞正常形态、卵裂和囊胚形成的频率分别为91.87%±4.14%、76.51%±4.37%和39.84%±2.91%,与VPI或VPII组相比有显著增加(P < 0.05)。TPI组中具有正常形态和卵裂的玻璃化卵母细胞率高于TPII或TPIII组(P < 0.05)。总之,在10%DMSO和10%EG中平衡<3分钟有利于玻璃化卵母细胞的活力。与DMSO相比,EG对山羊卵母细胞的玻璃化可能更有效。复温时,较高浓度(超过1 M)的海藻糖可提高山羊卵母细胞的冷冻存活率。