Zhang Defu, Dai Jianjun, Wu Caifeng, Wu Huali, Liu Dong, Yang Yu, Zhang Tingyu, Liu Wei, Yin Fangzhi, Wang Shaobing, Wang Shaokai
Animal Husbandry and Veterinary Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai 201106, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Jul;25(7):1095-100.
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 microg/mL cytochalasin B, centrifuged at approximately 13 000 xg 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.
目的是优化猪胚胎冷冻保存的玻璃化方法。从超排的巴马小型猪(母猪/后备母猪)中收集囊胚/桑葚胚(5-6日龄胚胎)。我们比较了不同的冷冻保存方法、冷冻保存工具、透明带(ZP)变薄处理以及受体品种对猪胚胎冷冻保存效率的影响。结果表明:在胚胎存活率和囊胚细胞数量方面,冷冻保存方法I[胚胎在溶液1(TCM199 + 20%胎牛血清 + 10%乙二醇 + 10%二甲基亚砜)中用开放式拉长细管(OPS)和玻璃微吸管(GMP)两步法玻璃化3分钟,然后在溶液2(TCM199 + 20%胎牛血清 + 20%乙二醇 + 20%二甲基亚砜 + 0.4 mol/L蔗糖)中玻璃化1分钟,储存于液氮中]和方法II[囊胚在NCSU23 + 7.5μg/mL细胞松弛素B中培养25分钟,以约13000×g离心12 - 13分钟,然后回收到pNCSU23中。接着在pNCSU23中2 mol/L乙二醇中平衡5分钟,在玻璃化溶液(pNCSU23中8 mol/L乙二醇、7%聚乙烯吡咯烷酮(PVP))中快速洗涤,装入OPS/GMP,投入液氮]之间没有显著差异。解冻后,在胚胎存活率(83.8%对77.6%)和囊胚细胞数量(53.1对47.5)方面,GMP玻璃化方法比OPS方法更合适且更有效(P < 0.05)。ZP变薄并未提高存活率,但显著增加了存活囊胚中的囊胚细胞数量(60.1和46,P < 0.01)。本地猪品种(枫泾母猪)更适合作为玻璃化/解冻后囊胚胚胎移植的受体,可提高妊娠率和胚胎效率。