Ghasem Saki, Negar Karimian
Physiology Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran.
J Pak Med Assoc. 2013 Apr;63(4):486-9.
To compare the effect of using open and closed carriers on count of inner cell mass in vitrified mouse blastocyst after warming.
The experimental study was conducted at Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran, from April to September 2010. Forty female NMRI (Naval Medical Research Institute, USA) mice were injected with pregnant mare's serum gonadotropin and human chorionic gonadotropin in order to induce superovulation. Following the latter injection, two or three females were caged with the same-breed male mice. The presence of vaginal plug was examined the following morning. To collect blastocyst embryos, the pregnant females were sacrificed by cervical dislocation at 88-90 hours after the injection and dissected. Blastocysts were collected in phosphate-buffered saline and allocated to four groups: vitrification in conventional straw, closed pulled straw, open pulled straw and cryoloop. The vitrification solution was ethylene glycol, Ficol and sucrose (EFS) 20% and 40%. After storage for 1 month in liquid nitrogen, the blastocysts were thawed in 0.5 M sucrose then cultured in M16 medium. After 6 hours of culture, the number of expanded blastocysts was recorded and stained by double-dye technique. After staining, the number of total cell and inner cell mass was calculated.
The re-expansion rate of blastocysts in the cryoloop group (n = 90; 78.26%) was significantly higher (p < 0.05) than open pulled straw (n = 83; 69.16%), closed pulled straw (n = 68; 54.83%) and conventional straws (n = 63; 51.21%) groups. Significant differences (p < 0.05) in the number of inner cell mass in blastocysts vitrified in open pulled straws, closed straws and cryoloop with blastocysts cryopreserved in conventional straws.
The re-expansion rate and total cell number of mouse blastocysts vitrified using open system had a better result compared with the closed system. The value of cryoloop and open pulled straws as carriers in vitrification of blastocysts may also merit more investigation.
比较使用开放式和封闭式载体对玻璃化保存的小鼠囊胚复温后内细胞团数量的影响。
实验研究于2010年4月至9月在伊朗阿瓦士的阿瓦士军迪沙普尔医科大学进行。40只雌性NMRI（美国海军医学研究所）小鼠注射孕马血清促性腺激素和人绒毛膜促性腺激素以诱导超排卵。在第二次注射后，将两三只雌性小鼠与同品种雄性小鼠关在一个笼子里。次日早晨检查是否有阴道栓。为了收集囊胚胚胎，在注射后88 - 90小时通过颈椎脱臼处死怀孕的雌性小鼠并进行解剖。将囊胚收集在磷酸盐缓冲盐水中并分为四组：常规细管玻璃化、封闭式拉制细管、开放式拉制细管和冷冻环。玻璃化溶液为20%和40%的乙二醇、菲可和蔗糖（EFS）。在液氮中保存1个月后，将囊胚在0.5M蔗糖中解冻，然后在M16培养基中培养。培养6小时后，记录扩张囊胚的数量并用双重染色技术染色。染色后，计算总细胞数和内细胞团数量。
冷冻环组（n = 90；78.26%）囊胚的再扩张率显著高于开放式拉制细管组（n = 83；69.16%）、封闭式拉制细管组（n = 68；54.83%）和常规细管组（n = 63；51.21%）（p < 0.05）。开放式拉制细管、封闭式细管和冷冻环玻璃化保存的囊胚与常规细管冷冻保存的囊胚在内细胞团数量上存在显著差异（p < 0.05）。
与封闭式系统相比，使用开放式系统玻璃化保存的小鼠囊胚的再扩张率和总细胞数有更好的结果。冷冻环和开放式拉制细管作为囊胚玻璃化载体的价值也可能值得进一步研究。
