Vacuum-cooled liquid nitrogen increases the developmental ability of vitrified-warmed bovine oocytes
Vacuum-cooled liquid nitrogen increases the developmental ability of vitrified-warmed bovine oocytes
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DOI：
10.1590/s0103-84782006000500024
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发表时间：
2006-10-01
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影响因子：
--
通讯作者：
Mezzalira, Alceu
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作者：
Santos, Rodrigo Marques dos;Barreta, Marcos Henrique;Mezzalira, Alceu
本研究的目的是确定真空冷却液氮对玻璃化未成熟（生发囊泡期；GV）和成熟（中期II； MII）牛卵母细胞再加热后发育的影响。液氮暴露于常压或真空（300mmhg 45秒）；后者将液氮温度降低至-200℃，将部分剥落的卵母细胞在选择后（GV）或体外成熟（MII） 22小时后在TCM 199培养基+ 10%的发情母马血清中玻璃化。为了玻璃化，卵母细胞首先暴露在中间溶液（10% EG + 10% DMSO）中30秒，然后暴露在玻璃化溶液（20% EG + 20% DMSO + 0.5M蔗糖）中20秒。每组三到四个卵母细胞被装入一根打开的吸管中，并直接注入液氮。卵母细胞随后通过暴露于空气（25℃）中4秒重新加热，然后暴露于浓度降低（0.3和0.15M）的蔗糖中5分钟。受精（第0天）使用2 × 106 mL（-1）精子（通过游泳程序选择）并孵育18至22小时。假定的受精卵在SOFaaci培养基的四孔培养皿中培养，温度为39摄氏度，二氧化碳浓度为5%，湿度为饱和。常压下GV期卵母细胞的裂解率（第2天）和囊胚率（第8天）分别为33.9%和4.2%，真空下GV期卵母细胞为41.2和8.8%，常压下MII期卵母细胞为43.5和6.7%，真空下MII期卵母细胞为53.6和10.6%。综上所述，真空冷却液氮提高了玻璃化解冻牛卵母细胞的发育速度。
The objective of this study was to determine the effects of vacuum-cooled liquid nitrogen on the development of vitrified immature (germinal vesicle stage; GV) and mature (metaphase II; MII) bovine oocytes after re-warming. Liquid nitrogen was exposed to either atmospheric pressure or to a vacuum (300mm Hg for 45sec); the latter decreased the temperature of the liquid nitrogen to -200 degrees C. Partially denuded oocytes were vitrified either just after selection (GV) or after 22 hours of in vitro maturation (MII) in TCM 199 medium + 10% of estrous mare serum. For vitrification, oocytes were firstly exposed to an intermediate solution (10% EG + 10% DMSO) for 30sec, followed by the vitrification solution (20% EG + 20% DMSO + 0.5M sucrose) for 20sec. Groups of three or four oocytes were loaded into an open-pulled-straw and directly plunged into liquid nitrogen. Oocytes were subsequently rewarmed by exposure to air (25 degrees C) for 4sec, followed by 5 min exposure to decreasing concentrations (0.3 and 0.15M) of sucrose. Fertilization (Day 0) was done with 2 x 106 spermatozoa mL(-1) (selected by a swim-up procedure) and incubated for 18 to 22 hours. Presumptive zygotes were cultured at 39 degrees C in four-well dishes with SOFaaci medium, under 5% CO2 and saturated humidity. Cleavage (Day 2) and blastocyst rates (Day 8) were 33.9 and 4.2%, respectively, for GV stage oocytes at atmospheric pressure, 41.2 and 8.8% for GV oocytes under vacuum, 43.5 and 6.7% for MII oocytes at atmospheric pressure, and 53.6 and 10.6% for MII oocytes under vacuum. In conclusion, vacuum-cooled liquid nitrogen improved developmental rates of vitrified-thawed bovine oocytes.