猪胚胎玻璃化冷冻保存技术的优化

Optimization of porcine embryo vitrification

摘要:

在以往工作的基础上本研究对现有猪胚胎玻璃化冷冻保存技术进行了优化。以巴马小型猪为供体, 采用超数排卵技术, 采集5~6日龄的胚胎(囊胚/桑椹胚), 比较冷冻方法、胚胎承载工具胚胎、透明带处理和冷冻胚胎移植受体对猪胚胎冷冻效果的影响。结果表明, 冷冻方法I[胚胎首先在冷冻液1 (TCM199+20% FBS+10% EG+10% DMSO)中平衡 3 min, 然后立即转入冷冻液2 (TCM199+20%FBS+20%EG+20%DMSO+0.4 mol/L SUC)中并在1 min内装管(每管含2~6枚胚胎), 直接投入液氮保存]与冷冻方法II[透明带完整的胚胎在NCSU23(含7.5 mg/mL CB)培养液中平衡25 min, 13 000 ×g离心12~13 min, 然后在含2 mol/L EG的NCSU23中平衡5 min, 再在含8 mol/L EG+7% PVP的NCSU23中快速漂洗, 装进OPS/GMP管, 放入液氮保存]冷冻效果没有显著差异; GMP法能显著提高冷冻胚胎存活率(83.8% vs 77.6%, P < 0.05)和囊胚细胞数(47.5 vs 53.1, P < 0.05); 以0.5%链蛋白酶(Pronase)处理透明带10 s, 虽然对猪胚胎存活率没有显著影响, 但能显著提高囊胚细胞数(60.1 vs 46.6, P < 0.01); 以地方猪种(枫泾母猪)为冷冻胚胎移植受体能显著提高妊娠率和胚胎效率(P<0.01)。

Abstract:

The purpose was to optimize the vitrification for porcine embryos cryopreservation. Blastocyst/Morula(5-6th day-embryos) were collected from superovulated Bama mini-pigs(sows/gilts). We compared different cryopreservation methods, cryopreservation tools, thining of zona pellucida(ZP) and recipient breeds on the efficiency of porcine embryo cryopreservation. The results showed that: in embryo survival rate and blastocyst cell number, there were no significant differences between cryopreservation method I [embryos were vitrified by two step method with open pulled straw (OPS) and glass micropipette (GMP) in solution 1(TCM199 + 20% FBS + 10% EG + 10% DMSO) for 3 min, and solution 2(TCM199 + 20% FBS + 20% EG + 20% DMSO + 0.4 mol/L SUC)for 1 min, stored in liquid nitrogen] and method II[Blastocysts were cultured for 25 min in NCSU23 + 7.5 mg/mL cytochalasin B, centrifuged at approximately 13 000 ×g for 12-13 min, and recovered back into pNCSU23. They were then equilibrated for 5 min in 2 mol/L ethylene glycol in pNCSU23, washed quickly in the vitrification medium, 8 mol/L ethylene glycol, 7% polyvinylpyrrolidone (PVP) in pNCSU23, loaded into OPS/GMP, and plunged into liquid nitrogen). GMP vitrification method was more suitable and efficient than OPS method (P<0.05) in embryo survival rate (83.8% vs 77.6%) and blastocyst cell number (53.1 vs 47.5) after thawing. Thining of ZP did not increase the survival rate, but significantly improved blastocyst cell number in the survival blastcysts (60.1 and 46, P<0.01). Local pig breeds (Fengjing sows) were more suitable as recipients for embryo transfer of vitrified/warmed blastcysts, which can improve pregnant rate and embryo efficiency.