Successful pregnancy following transfer of feline embryos derived from vitrified immature cat oocytes using 'stepwise' cryoprotectant exposure technique
Successful pregnancy following transfer of feline embryos derived from vitrified immature cat oocytes using 'stepwise' cryoprotectant exposure technique
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DOI：
10.1016/j.theriogenology.2011.06.014
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发表时间：
2011-11-01
期刊：
影响因子：
2.8
通讯作者：
Techakumphu, Mongkol
中科院分区：
文献类型：
--
作者：
Tharasanit, Theerawat;Manee-In, Sukanya;Techakumphu, Mongkol
卵母细胞冷冻保存是“长期”保存雌性遗传潜力的理想工具，特别是对于濒危/珍贵物种。本研究旨在探讨不同冷冻保护剂（CPA）和CPA暴露技术对猫未成熟卵母细胞玻璃化冷冻损伤的保护能力。未成熟卵母细胞接受不同的CPA暴露技术：1）2步DMSO，2）4步DMSO，3）2步EG，4）4步EG，5）2步EG加DMSO和6）4步EG加DMSO。非CPA处理的非玻璃化冷冻卵母细胞作为对照。然后将卵母细胞进行体外成熟（实验1，n = 334）或玻璃化/加温（实验2，n = 440）。随后确定了核成熟的阶段。在实验3中，玻璃化冷冻的未成熟卵母细胞（n = 254）体外成熟和受精，并评估其发育能力。将424枚玻璃化冷冻后的未成熟卵母细胞移植到6只受体雌猫的输卵管中（试验4），玻璃化冷冻显著降低了未成熟卵母细胞的减数分裂和发育能力。EG单独或EG和DMSO的组合产生比DMSO更高的成熟率，无论使用的CPA平衡技术。4步EG玻璃化冷冻的成熟率最高（37.6%），但卵裂和囊胚率最低。结果显示，玻璃化冷冻组的存活率分别为24.8%和30.2%，而非玻璃化冷冻组的存活率分别为62.5%和49.3%。在接受来自非玻璃化和玻璃化/温热的未成熟卵母细胞的胚胎的受体中建立妊娠。可以得出结论，逐步CPA曝光技术可以成功地应用于玻璃化冷冻未成熟的猫卵母细胞，在体外发育，但它可能会影响在子宫内的发展。(C)2011 Elsevier Inc. All rights reserved.
Oocyte cryopreservation is the desired tool for the 'long-term' storage of female genetic potential especially for endangered/valuable species. This study aims at examining the ability of 'different cryoprotectant (CPA) and CPA exposure techniques to protect immature feline oocytes against cryoinjury during vitrification. Immature oocytes were submitted to different CPA exposure techniques: 1) 2-step DMSO, 2) 4-step DMSO, 3) 2-step EG, 4) 4-step EG, 5) 2-step EG plus DMSO and 6) 4-step EG plus DMSO. Non-CPA treated, non-vitrified oocytes served as controls. The oocytes were then submitted either to in vitro maturation (Experiment 1, n = 334) or to vitrification/warming (Experiment 2, n = 440). The stage of nuclear maturation was subsequently determined. In Experiment 3, the vitrified immature oocytes (n = 254) were matured and fertilized in vitro, and their developmental competence was assessed. A total of 424 embryos derived from vitrified immature oocytes were transferred into the oviduct of 6 recipient queens (Experiment 4).Vitrification reduced significantly the meiotic and developmental competence of immature cat oocytes compared with the non-vitrified controls. The EG alone or a combination of EG and DMSO yielded higher maturation rates than DMSO, irrespective of the CPA equilibration techniques used. The 4-step EG vitrification resulted in the highest maturation rate (37.6%) but cleavage and blastocyst. rates were significantly lower than the non-vitrified controls (24.8% and 30.2% vs 62.5% and 49.3%, respectively). Pregnancy was established in recipients receiving embryos derived from non-vitrified and vitrified/warmed immature oocytes. It is concluded that the stepwise CPA exposure technique can be successfully applied for vitrification of immature cat oocytes, in terms of in vitro development but it is likely to affect in utero development. (C) 2011 Elsevier Inc. All rights reserved.