Moussa M, Bersinger I, Doligez P, Guignot F, Duchamp G, Vidament M, Mermillod P, Bruyas J-F
INRA, Physiologie de la Reproduction et des Comportements, UMR INRA-CNRS-Université de Tours-Haras Nationaux, 37380 Nouzilly, France.
Theriogenology. 2005 Oct 15;64(7):1619-32. doi: 10.1016/j.theriogenology.2005.04.001.
Vitrification using open pulled straw (OPS) has provided encouraging results with embryos from other species. The aim of this study was to compare the survival of 6.5- and 6.75-day-old equine embryos after OPS vitrification and slow-cooling. Eighteen embryos were frozen using a slow-cooling method. Embryos were placed in modified PBS with increasing glycerol concentration (2.5%, 5%, 7.5% and 10% (v/v) 5 min each). Embryos were loaded into 0.25 ml straws then placed in a programmable freezer and subsequently plunged into liquid nitrogen. After thawing, cryoprotectant was removed by five steps with decreasing glycerol and sucrose concentrations. Twenty embryos were vitrified using the OPS method. Embryos were exposed to 7.5% dimethyl-sulfoxide (DMSO)+7.5% ethylene glycol (EG) for 3 min and in 18% DMSO+18% EG+0.4M sucrose for 1 min, loaded in OPS and plunged into liquid nitrogen. After warming, embryos were placed in decreasing sucrose concentrations. All embryos were cultured in synthetic oviduct fluid (SOF) medium for 3h and evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining. The percentage of cells entering in S-phase (%SC) was evaluated by incorporation of BrdU. No significant differences were observed for mean diameter, morphological grade and percentage of degenerate embryos after 3h of culture for slow-cooling and OPS methods. The percentage of dead cells per embryo was similar for the two procedures (42+/-6 versus 46+/-9). The percentage of cells entering in S-phase did not differ significantly between the two procedures (27+/-5 versus 26+/-6). OPS vitrification may be as efficient as slow-cooling for the cryopreservation of equine embryos. However, these results should be confirmed by the transfer of OPS vitrified embryos to recipient mares.
使用开放式拉长细管（OPS）玻璃化冷冻技术在其他物种的胚胎上已取得了令人鼓舞的成果。本研究的目的是比较OPS玻璃化冷冻和慢速冷冻后6.5日龄和6.75日龄马胚胎的存活率。18枚胚胎采用慢速冷冻法进行冷冻。将胚胎置于添加了递增甘油浓度（2.5%、5%、7.5%和10%（v/v），各5分钟）的改良PBS中。胚胎被装入0.25毫升的细管中，然后放入程序降温仪，随后投入液氮中。解冻后，通过五个步骤降低甘油和蔗糖浓度来去除冷冻保护剂。20枚胚胎采用OPS法进行玻璃化冷冻。胚胎先在7.5%二甲基亚砜（DMSO）+7.5%乙二醇（EG）中暴露3分钟，然后在18%DMSO+18%EG+0.4M蔗糖中暴露1分钟，装入OPS并投入液氮中。复温后，将胚胎置于递减的蔗糖浓度中。所有胚胎在合成输卵管液（SOF）培养基中培养3小时，并使用4',6-二脒基-2-苯基吲哚（DAPI）染色进行评估。通过掺入溴脱氧尿苷（BrdU）评估进入S期的细胞百分比（%SC）。对于慢速冷冻和OPS法，培养3小时后，胚胎的平均直径、形态学等级和退化胚胎百分比均未观察到显著差异。两种方法中每个胚胎的死细胞百分比相似（42±6对46±9）。两种方法之间进入S期的细胞百分比也没有显著差异（27±5对26±6）。对于马胚胎的冷冻保存，OPS玻璃化冷冻可能与慢速冷冻一样有效。然而，这些结果应通过将OPS玻璃化冷冻胚胎移植到受体母马中得到证实。
