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1 生物工程学报 Chin J Biotech 2009, July 25; 25(7): journals.im.ac.cn Chinese Journal of Biotechnology ISSN cjb@im.ac.cn 2009 Institute of Microbiology, CAS & CSM, All rights reserved 生物技术与方法 张德福 1,2, 戴建军 1,2, 吴彩凤 1,2, 吴华莉 1,2, 刘东 1,2, 杨宇 3, 张廷宇 3, 刘伟 4, 殷方芝王少兵 4 4, 王昭凯 1, , , , : 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 μg/ml CB) 培养液中平衡 25 min, 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) : 猪, 胚胎, 玻璃化冷冻 4, Optimization of porcine embryo vitrification Defu Zhang 1,2, Jianjun Dai 1,2, Caifeng Wu 1,2, Huali Wu 1,2, Dong Liu 1,2, Yu Yang 3, Tingyu Zhang 3, Wei Liu 4, Fangzhi Yin 4, Shaobing Wang 4, and Shaokai Wang 4 1 Animal Husbandry and Veterinary Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai , China 2 Division of Animal Genetic Engineering, Shanghai Municipal Key Laboratory of Agri-genetic and Breeding, Shanghai , China 3 Animal Sciences & Technology College, Sichuan Agricultural University, Ya an , China 4 Animal Sciences& Technology College, Nanjing Agricultural University, Nanjing , China 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 Received: November 17, 2008; Accepted: April 28, 2009 Supported by: Shanghai Agriculture Committee (Nos , , ), Science and Technology Commission of Shanghai (No. 04DZ05611), Shanghai Municipal Key Laboratory of Agri-genetic and Breeding of China (No. Shagb ), Youth Fundation from Shanghai Academy of Agricultural Sciences (No ). Corresponding author: Defu Zhang. Tel: ; zhangdefu10@yahoo.com.cn 上海市农委项目 (Nos , , ), 上海市科学技术委员会科技攻关项目 (No. 04DZ05611), 上海市农业遗传育种重点实验室课题 (No. Shagb ), 上海农业科学院青年基金 (No ) 资助

2 1096 ISSN CN /Q Chin J Biotech July 25, 2009 Vol.25 No.7 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(TCM % FBS + 10% EG + 10% DMSO) for 3 min, and solution 2(TCM % FBS + 20% EG + 20% DMSO mol/l SUC)for 1 min, stored in liquid nitrogen] and method II[Blastocysts were cultured for 25 min in NCSU μg/ml cytochalasin B, centrifuged at approximately g for 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. Keywords: pig, embryos, vitrification Whittingham [1] 1972 ( ), [1] ; 1977 Willadson [2],, 16, 1985 Rall [3], 8-,,,,,, ( ), 1989 ( 35 o C) [4], 1990 ( 196 o C) [5] ;, [6] 20 o C, [7] [8], [9] OPS, ( 196 o C) 1997 Vajta OPS, [10], (Glass micropipette, GMP) (Microdroplet) (Electron microscopic grids, EMG) (Cryoloop vitrification, CLV) (Solid-surface vitrification, SSV),, [11] ;, [12 14],, [12,15],,, 1 材料与方法 1.1 试验动物与试剂 (BM), 40~50 kg 8~10, ( ),, Sigma TCM199 Gibco ; (PG) ; (PMSG); (hcg) 1.2 超排处理 PG+PMSG+hCG : 16~17 d 0.2 mg PG 1000 IU PMSG,, hcg 500 IU 1.3 胚胎的手术回收 0, 5~ % 30~50 ml 8% ( 5% )50~100 ml,

3 : ~8 cm,,, 1/2~2/3 ( ) ; 60 ml ( 2% ),,, 50 ml, o C, 10 min min,, 1.4 猪胚胎冷冻承载工具的制备 (Open pulled straw; OPS) 0.25 ml (France) 0.7 mm (Glass micropipette, GMP) 0.25~0.40 cm,,, 200~300 μm, 1.5 胚胎冷冻 I: Berthelot [16], 1(TCM199+20% FBS+10% EG+ 10% DMSO) 3 min, 2(TCM199+20% FBS+20% EG+20% DMSO+0.4 mol/l Suc) 1 min, 1 min, OPS/GMP ( 2~6 ),, 3 37 o C, TCM199+20% FBS, OPS 0.13 mol/l Suc, 1 min Suc 5 min, mol/l Suc 5 min, 37 o C 5% CO 2 II: Cameron [13] NCSU23( 7.5 μg/ml CB) 25 min, g 12~13 min; 2 mol/l EG NCSU23 5 min, 8 mol/l EG+ 7% PVP NCSU23, OPS/GMP, ; 1.6 胚胎死活鉴定及囊胚细胞计数 24 h ; 5 μg/ml Hoechst33342, 1.7 胚胎移植 6,, 1.8 实验设计 1: I, ( ), OPS GMP 2: GMP, ( ), I II 3: GMP I, ( ) 0.5% (Pronase, 10 s),, pronase ;, 4: 3, 10, 数据处理 SPSS, 2 结果 2.1 实验 1: 承载工具对猪胚胎冷冻效果的影响 I, ( ), OPS GMP ( 1) GMP OPS

4 1098 ISSN CN /Q Chin J Biotech July 25, 2009 Vol.25 No.7 表 1 承载工具对猪胚胎冷冻效果的影响 Table 1 Effect of two different vitrification tools (OPS and GMP) on pig embryo cryopreservation Group % viable after 24 h in vitro(n) Cell number S.E.M (n) OPS 77.6 (23) (20) GMP 83.8 (21) * (19) * * : P < 0.05; ** : P < 0.01, the same as below. 2.2 实验 2: 冷冻方法对猪胚胎冷冻效果的影响 GMP, ( ), I II ( 2) (P > 0.05) 2.3 实验 3: 透明带处理对猪胚胎存活率的影响 GMP I, ( ), ( 3) (0.5% 10 s), (P < 0.01) 表 2 不同冷冻方法对猪胚胎冷冻效果的影响 Table 2 Effect of two different freezing methods on pig embryo cryopreservation Group % viable after 24 h in vitro(n) Cell number S.E.M (n) Freezing method I 84.6 (22) (18) Freezing method II 80.8 (21) (18) 表 3 透明带薄化处理对猪胚胎存活率的影响 Table 3 Effect of thining of ZP on pig embryo cryopreservation Group % viable after 24 h in vitro(n) Cell number S.E.M (n) Zp thining 84.8 (28) (22) ** Control 81.8 (27) (22) ** : P < 实验 4: 不同受体母猪对猪冷冻胚胎移植的影响 3, 10 (LS) (FJ), ( 4) (P < 0.01) 3 讨论,, [17,18], OPS, [8], [19],,,,,,, (IETS), [20],, 5~6 2 : [12,16], TCM199+20% FBS, EG DMSO SUC ; [13 15], CB NCSU23,, EG,, CB, I, ( 196 o C) [17] 1.5 mol/l DMSO EG,,,,, EG DMSO, [21], 1 min,,,,,,

5 : 1099 表 4 不同受体母猪对猪冷冻胚胎移植的影响 Table 4 Effect of two different recipients on pig cryopreservation embryo transfer Breeds Number of recipients Total number of transferred blastcyst Pregnant rate (%) Number of piglets born per farrowing Total number of live-born piglets Survival rate LS %(12/200) FJ ** %(31/200) LS: Landrace Shanghai White pig. ** : P < [3],,, [22,23] EG (62.07), ; DMSO 78.14,,,, 2,, DMSO, [21],,, (Assisted hatching), pronase,,,, Beebe LFS [15], 10 OPS, 10 [24], / 1 min,,, (Straw) GMP [25,26] GMP (< 0.8 mm),,, ; OPS, OPS 0.8 mm,, ; GMP OPS,, OPS, GMP ; GMP OPS [27],,,,,, [16], GMP I, ( ), ( ), REFERENCES [1] Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to 196 degrees and 269 degrees C. Science, 1972, 178(59): [2] Willadsen SM. Factors affecting the survival of sheep embryos during deep-freezing and thawing. In The Freezing of Mammalian Embryos. London: Butterworths Press, 1977: [3] Rall WF, Fahy GM. Ice-free cryopreservation of mouse embryos at 196 o C by vitrification. Nature, 1985, 313: [4] Hayashi S, Kobayashi K, Mizuno T, et al. Birth of piglets from frozen embryos. Vet Rec, 1989, 125(2): [5] Kashiwasaki N, Ohtani S, Miyamoto K, et al. Production of normal piglets from hatched blastocysts frozen at 196 o C. Vet Rec, 1991, 128: [6] Feng ST, Liu DK. Piglets delivered from frozen ( 20 o C) embryos. Acta Vet Zootech Sin, 1993, 24(1): ,. ( 20 o C)., 1993, 24(1): [7] Wang ZK, Zhang SQ. Study on collection of swine hatched-embryos and freezing methods. J Guangdong Anim Husb Vet Med, 1998, 23(4): ,.., 1998, 23(4): [8] Xu MB, Matusmoto T. Some factors affecting efficiency

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