小鼠胚胎冷冻保存的研究
|
摘要
本研究采用程序冷冻、直接冷冻、玻璃化冷冻和开放式拉管法(OPS)法对小鼠桑葚胚和早期囊胚进行了冷冻保存，探讨了不同冷冻方法对胚胎存活率的影响，并从冷冻保护剂、植冰温度等不同角度加以研究，以胚胎冷冻后的体外培养和移植来鉴定其冷冻效果。
结果表明：1．将6～8周龄的昆明种雌鼠15只随机分为三组，分别用PMSG和HCG以不同的处理剂量(5IU，10IU，15IU)进行超排处理，每只平均获得胚胎数分别为16.2、23.2和19.0枚，正常胚数分别为12.6、18.8和13.4枚，正常胚的比率分别为：77.78％、81.03％和70.53％，均达到了超排效果。结果表明10IU的处理剂量的超排效果较好(p＞0.05)。2．将超排获得的桑葚胚和早期囊胚随机分为(1)20％乙二醇(EG)+20％DMSO处理30秒，再经0.3M蔗糖处理1 min；(2)20％甘油(Gly)+20％1，2—丙二醇(1，2-Pro)处理30秒，再经0.3M蔗糖(Suc)处理1min；(3)经0.3M蔗糖处理(解冻液处理组)1 min；(4)不经任何冷冻液和解冻液处理(对照组)四组。分别24h培养表明：扩张囊胚率为91.23％、80.65％、89.58％和90.74％；48h后的孵化率为64.91％、51.61％、83.33％和85.19％。(1)和(2)组间差异不显著，二者与(3)和(4)相比，差异均显著(p＜0.01)。3．分别用F1、F2和F3三种冷冻保护剂，行常规程序冷冻方法冷冻，解冻后培养结果表明：扩张囊胚率分别为78.18％、73.58％和57.14％；孵化囊胚率为45.45％、41.51％和26.53％，F1和F2显著高于F3(p＜0.05)。4．将获得的桑葚胚和早期囊胚放入冷冻液F1中平衡5min，冷冻室内停留2min后植冰，再行程序冷冻。证明：-5.5℃、-6.0℃、-6.5℃植冰时，胚胎解冻后体外囊胚孵化率分别为40.38％、45.45％、12.50％，-5.5℃和-6.0℃组的显著高于-6.5℃组(p＜0.01)。5．分别用程序冷冻、常规玻璃化冷冻、OPS、直接冷冻法对小鼠桑葚胚进行冷冻保存。解冻后存活率分别为78.18％、70.37％、81.36％和44.92％；48h后的孵化率分别为45.45％、44.44％、50.85％和27.54％。前三种方法组间差异不显著，但直接冷冻法和其他各组相比差异极显著(p＜0.01)。6．将上述四种方法冷冻保存的83、68、75和71枚胚胎解冻后分别移植给8、6、6和6只受体，平均每只10～13枚，结果表明：妊娠率为12.5％(1/8)、16.67％(1/6)、16.67％(1/6)和16.67％(1/6)。
The present research adopted four methods to cryopreserve the mouse morula and early blastocysts. They were programming freezing, quick cooling, vitrification and OPS. Probed into the effect of different Cryo-preservation on the development of mouse embryos, studied from the cryoptectant and seeding temperature etc. In vitro culture and the embryos surgical transfer were used to appraise the effect of mouse embryos cryopreservation.
The results showed: 1. The experiments were divided into 3 groups according to three different dosages (5 IU, 10 IU andl5 IU). For the dosage of PMSG treatment, 10IU injection was better than both 5 IU and 10 IU groups(p<0.05).
2. The super-ovulated embryos were exposed in four operation solutions as follows:(1) (20%Gly+20%1, 2-Pro) X 30sec.+0.3M Suc. X 1min; (2) (20%EG +20%DMS0) X 30sec. +0.3 M Suc. X 1min; (3) 0.3M Suc. X 1min; (4) Control (no treatment). The survival rates and hatching rates of groups (1) and (2) were
lower than that of groups (3) and (4) (P<0.01). 3. Adopted the programming freezing with three cryoprotectant:F1,F2 and F3. percentage of hatching embryos of F1 and F2 were45. 45%, 41. 51%. they both had significantly higher developmental abilities than F3(26. 53%). 4. Mouse embryos were exposed to the cryoprotectant F1 and seeded at -5.5℃ and -6.0℃ followed lauching into liquid nitrogen at -35℃,after thawing and in vitro culture, percentage of hatching embryos were40.38%, 45. 45%. they both had higher developmental
结果表明：1．将6～8周龄的昆明种雌鼠15只随机分为三组，分别用PMSG和HCG以不同的处理剂量(5IU，10IU，15IU)进行超排处理，每只平均获得胚胎数分别为16.2、23.2和19.0枚，正常胚数分别为12.6、18.8和13.4枚，正常胚的比率分别为：77.78％、81.03％和70.53％，均达到了超排效果。结果表明10IU的处理剂量的超排效果较好(p＞0.05)。2．将超排获得的桑葚胚和早期囊胚随机分为(1)20％乙二醇(EG)+20％DMSO处理30秒，再经0.3M蔗糖处理1 min；(2)20％甘油(Gly)+20％1，2—丙二醇(1，2-Pro)处理30秒，再经0.3M蔗糖(Suc)处理1min；(3)经0.3M蔗糖处理(解冻液处理组)1 min；(4)不经任何冷冻液和解冻液处理(对照组)四组。分别24h培养表明：扩张囊胚率为91.23％、80.65％、89.58％和90.74％；48h后的孵化率为64.91％、51.61％、83.33％和85.19％。(1)和(2)组间差异不显著，二者与(3)和(4)相比，差异均显著(p＜0.01)。3．分别用F1、F2和F3三种冷冻保护剂，行常规程序冷冻方法冷冻，解冻后培养结果表明：扩张囊胚率分别为78.18％、73.58％和57.14％；孵化囊胚率为45.45％、41.51％和26.53％，F1和F2显著高于F3(p＜0.05)。4．将获得的桑葚胚和早期囊胚放入冷冻液F1中平衡5min，冷冻室内停留2min后植冰，再行程序冷冻。证明：-5.5℃、-6.0℃、-6.5℃植冰时，胚胎解冻后体外囊胚孵化率分别为40.38％、45.45％、12.50％，-5.5℃和-6.0℃组的显著高于-6.5℃组(p＜0.01)。5．分别用程序冷冻、常规玻璃化冷冻、OPS、直接冷冻法对小鼠桑葚胚进行冷冻保存。解冻后存活率分别为78.18％、70.37％、81.36％和44.92％；48h后的孵化率分别为45.45％、44.44％、50.85％和27.54％。前三种方法组间差异不显著，但直接冷冻法和其他各组相比差异极显著(p＜0.01)。6．将上述四种方法冷冻保存的83、68、75和71枚胚胎解冻后分别移植给8、6、6和6只受体，平均每只10～13枚，结果表明：妊娠率为12.5％(1/8)、16.67％(1/6)、16.67％(1/6)和16.67％(1/6)。
The present research adopted four methods to cryopreserve the mouse morula and early blastocysts. They were programming freezing, quick cooling, vitrification and OPS. Probed into the effect of different Cryo-preservation on the development of mouse embryos, studied from the cryoptectant and seeding temperature etc. In vitro culture and the embryos surgical transfer were used to appraise the effect of mouse embryos cryopreservation.
The results showed: 1. The experiments were divided into 3 groups according to three different dosages (5 IU, 10 IU andl5 IU). For the dosage of PMSG treatment, 10IU injection was better than both 5 IU and 10 IU groups(p<0.05).
2. The super-ovulated embryos were exposed in four operation solutions as follows:(1) (20%Gly+20%1, 2-Pro) X 30sec.+0.3M Suc. X 1min; (2) (20%EG +20%DMS0) X 30sec. +0.3 M Suc. X 1min; (3) 0.3M Suc. X 1min; (4) Control (no treatment). The survival rates and hatching rates of groups (1) and (2) were
lower than that of groups (3) and (4) (P<0.01). 3. Adopted the programming freezing with three cryoprotectant:F1,F2 and F3. percentage of hatching embryos of F1 and F2 were45. 45%, 41. 51%. they both had significantly higher developmental abilities than F3(26. 53%). 4. Mouse embryos were exposed to the cryoprotectant F1 and seeded at -5.5℃ and -6.0℃ followed lauching into liquid nitrogen at -35℃,after thawing and in vitro culture, percentage of hatching embryos were40.38%, 45. 45%. they both had higher developmental
引文
[1] 张德福，张忠明，徐平．动物胚胎生物技术发展概况上海实验动物科学．2003，23(2)：119～122
[2] 李勇，窦忠英，杨炜峰．胚胎工程技术及其在畜牧业中的应用[J]．西北农林科技大学学报(自然科学版)．2004，32(2)：104～109
[3] 严云勤．发育生物学原理与胚胎工程[M]．哈尔滨：黑龙江科学出版社．1995：278
[4] 马新武．哺乳动物胚胎冷冻原理及研究进展[J]．动物医学进展．2000，21(4)：112～116
[5] 李广武，郑从义，唐兵．低温生物学[M]．湖南科学技术出版社．1998，8
[6] 陈北亨，王建辰．兽医产科学[M]．北京：中国农业出版社．2001，4：339～355
[7] 徐勇，张嘉宝．哺乳动物胚胎超低温冷冻保存的研究进展[J]．黑龙江动物繁殖．2001，9(1)：15～17
[8] 动物胚胎冷冻保存技术的研究应用[J]．中国草食动物．2003，23(4)：42～43
[9] 朱士恩，曾申明，张忠诚．哺乳动物胚胎玻璃化冷冻技术研究进展[J]．中国畜牧杂志．1998，3：2～4
[10] Zhu S.E, et al. Cryopreservation of mouse zona—free blastocysts developed from—two cell embryos' blastomeres by vitrification. 动物生物技术国际会议论文集．1997
[11] Whittingham DG. Survial of mouse embryos frozen to-196℃ and-269℃[J]. Science, 1972, 178: 411
[12] L. J. Maclellan. et al.[M]. Therigenology, Abstract. 2001: 300～318
[13] N. Oberstein, et al. Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling metholds[J]. Therigenology, 2001, (55): 607～613
[14] Levent keskintepe, Yuksel Agca, Gabriela A, et al. Use of cryopreserved pronuclear embryos for the production of transgenic mice[J]. 2001, 65: 407～411
[15] 包华琼，李跃民．哺乳动物胚胎冷冻的研究进展[J]．草食家畜(季刊)．2001，12(4)：1～5
[16] 阳年生．脱防冻剂方法对牛体外受精胚胎冷冻后成活率的影响[J]．中国兽医学报．1998，18(3)：291～294
[17] 张嘉宝，周虚．动物繁殖学[M]．吉林：吉林科技出版社．1999：230～257
[18] 孙新明．小鼠胚胎一步冷冻试验[D]．陕西杨陵：西北农业大学．1993
[19] Miyake T, Kasai M, Zhu S E, etal. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method[J]. Theriogenology. 1993, 40: 121～134.
[20] Tada N, Sato M, Amann E, et al A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: beneficial effect of sucrose and raffinose on their cryosurvival rate[J]. Theriogenology, 1993, 40: 333～344
[21] M. A Nowshari, G. Brem. Effect of freezing rate and expose time to cryopretectant on the development of mouse pronuclear stage embryos[J]. Human Reproduction 2001, 16 (11): 2368～2373
[22] Park SE, Chung HM, Cha KY. cryopreservation of ICR mouse oocytes: improved post-thawed, preimplantation development after vitrification using Taxol, a cytoskeleton stabilizer[J]. Fertil steril. 2001, 75 (6): 1177～1184
[23] Jun-hui wang. A comprehensive Evaluation of the Effects and mechanisms of Antifreeze proteins during low-Temperature Preservation [J]. cryobiology. 2000, 41: 1～9
[24] J. Aki, J. N. Shelton. Design of Vitrification solutions for the cryopreservation of Embryos[J]. Journal of Reproduction and fertility. 1993, 99: 471～477
[25] 丁志丽，桑林华，王杏龙．哺乳动物胚胎冷冻保存的研究进展[J]．河北畜牧兽医．2003，19(12)：24～26
[26] 曲玉秀，刘铁铮，汤家铭等．小鼠的胚胎冷冻保存研究[J]．四川畜牧兽医．2002，29(12)：28～29
[27] 朱士恩，王海彦．生物频谱对小鼠桑葚胚玻璃化冷冻体内外发育影响的研究[J]．农业生物技术学报．1998，3
[28] 史洪才．家畜胚胎冷冻保存的研究进展[J]．草食家畜(增刊) 1999，102：31～34
[29] S. Ohboshi, N. Fujihara. Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts [Z]. Animal reproducation Science, 1997, 48: 27～36
[30] Michelle Lane. Vitrification of embryos Using a Nylon Loop[Z]. The Embryologists' Newsletter. 2000, winter
[31] S. Cseh, W. Horlacher, G. Brem. Vitrification of mouse embryos in two cryoprotectant solutions[J]. Theriogenology. 1999, 52: 103～113
[32] Joe Wilfe, Gary Bryant. Cellular cryobiology: thermodynamic and mechanical effects[Z]. International Journal of Refrigeration. 2001, 438～450
[33] Thatcher W W, Moreira F, Santos J E P. Effects of hormonal treatments on reproductive performance and embryo production[J]. Theriogenology 2001, 55: 75～89
[34] 王锋，蔡令波，王启风等．小鼠胚胎超低温冷冻保存方法的比较研究[J]．草食家畜(增刊)．2001：136～139
[35] 朱士恩，张忠诚．小鼠桑椹胚简易玻璃化冷冻技术再讨论[J]．中国畜牧杂志．1997，33(3)：14～17
[36] Schiewe MC, Rall WF, Stuart LD. In situ dilution of ovine embryos cryopreserved by conventional freezing or vitrification [J]. Theriogenology. 1990, 33: 321
[37] Vajta G, Booth P J, KuwayamaM, et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos [J]. Mol Reprod Dev, 1998, 51: 53～58
[38] Booth P J, Vajta G, H oj A. Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts: work in progress[J]. Theriogenology. 1999, 51(5): 999～1006
[39] Vajta G. Vitrification of the oocytes and embryos of domestic animals[J]. Anim Reprod S ci, 2000, 61: 357～364
[40] U shijima H, Yamakawa H, Nagashima H. Cryopreservation of bovine pre-morula-stage in vitro matured/in vitro fertilized embryos after delipidation and before use in nucleus transfer[J]. BiolReprod, 1999, 60: 534～539
[41] 刘丽均，徐平．小鼠胚胎玻璃化冷冻的研究进展[J]．中国实验动物学杂志 2001，11(2)：115～117
[42] Emiliant S, Van Den Bergh M, Vannin A S, et al. Comparison of ethylene glycol, 1, 2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts[J]. Hum Reprod, 2000, 15 (4): 905～910
[43] 曹佐武，潘善培，刘学高．影响玻璃化法冷冻保存胚胎的一些因素[J]．暨南大学学报(自然科学与医学报)．19(12)：48～50
[44] 努尔江，史洪才，黄俊成等．小鼠早期桑椹胚和致密桑椹胚玻璃化冷冻保存试验[J]．草食家畜 2000，1(3) 27～29
[45] 朱士恩，曾申明，张忠诚．液氮气熏蒸法玻璃化冷冻小鼠扩张囊胚的研究[J]．农业生物技术学报．1999，7(2)：163～166
[46] Liu J, Van Den Abbeel E, Van Steirteghem A C. Assessment of ultrarapid and slow freezing procedures for 1-cell and 4-cell mouse embryos[J]. Hum Reprod. 1993, 8(7): 1115～1119
[47] K Nakao, N Nakagata, M Katsuki. Simple and efficient procedure for cryopreserservaton of mouse embryos by simple vitrification[J]. Exp Anim. 1997, 46: 231～134
[48] A Very S M. Embryo cryopreservation[M]. Brinsden PR. A Textbook of In Vitro Fertilization and Assisted Reproduction, 2nd ed. USA: The Parehenon Publishing group Inc, 1999: 211～217
[49] 左琴，岳秉飞，刘双环等．近交系小鼠胚胎玻璃化冷冻技术研究[J]．中国实验动物学杂志 2001年6月，11(2)：82～84
[50] 王俊霞，朱桂金．三种不同玻璃化冷冻方法对小鼠2—细胞期胚胎冷冻效果的比较[J]．生殖与避孕．2004，24(2)：214～217
[51] Senn A, Vozzic, Chanson A, et al. prospective randomized study of two cryopreservation polices avoiding embryo selection: the pronucleate stage leads to a higher cumulative delivery rate than the early cleavage stage[J]. Fertil and steril. 2000, 74(5): 946～952
[52] Mukaida T, Wada S, Takahashi K, et al. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos[J]. Hum Reprod. 1998, 13(10): 2874～2879
[53] Cocero M J, Diaz De la, Espina S M, et al. Ultrastructural characteristics of fresh and frozen-thawded ovine embryos using two cryoprotectants[J]. Biol Reprod. 2002, 66(5): 1244～1258
[54] 赵小徽，岳天孚，随笑琳．玻璃化法冻融小鼠桑葚胚期和囊胚期胚胎的效果观察[J]．中华妇产科杂志．2004，39(8)：526～528
[55] 孙艳香，姜国诚．冷冻液中甘油对小鼠胚胎保护效果的研究[J]．广西师范大学学报(自然科学版)．2000，18(1)：92～94
[56] 王果，马燕琳，吕祁峰等平皿-微滴法玻璃化冷冻保存小鼠胚胎．湖南医科大学学报．2002，27：229～301
[57] Cardner DK, Schoolcraft WB, Wagley L, et al. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization[J]. Hum Reprod. 1998, 13: 3434～3440
[58] 孙艳香，姜国诚．乙二醇对速冻中小鼠胚胎发育的作用[J]．广西师范大学学报(自然科学版)．2001，19(3)：69～71
[59] 高建明，彭邵华．植冰前后平衡时间对小鼠胚胎一步细管冷冻解冻效果的影响[J]．北京农学院学报．15(4)：30～32
[60] 努尔江，张家心，黄俊成等．不同冷冻保护液和不同植冰温度对小鼠胚胎冷冻效果的影响[J]．草食家畜(季刊)．2000，3：31～33
[61] 张嘉宝，赵志辉，周虚等．小鼠胚胎快速冷冻的研究[J]．黑龙江动物繁殖．6(2)：8～9
[62] Vabderzwalmen P, Bertin G, Debauche CH, et al. births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before virtrification[J]. Hum Reprod. 2002, 17: 744～751
[63] Tao J, Tamis R, Fink K. Cryopreservation of mouse embryos at morula/compact stage[J]. J Assust Reorid Genet, 2001, 18: 235～243
[64] 董雅娟．牛胚胎冷冻保存的现状及展望[J]．草食家畜，1995，(2)：20～22
[65] 安志兴，李向臣，王超等．不同植冰温度下冷冻液的结晶变化及其对小鼠胚胎冷冻的效果[J]．西北农林科技大学学报(自然科学版)．2003，31(3)：11～14
[66] 管彤，王凤山，于国德等．实验小鼠胚胎冷冻技术实验研究[J]．实验动物科学与管理．18(2)：14～17
[67] 王燕蓉．体外授精与胚胎移植[M]．北京：人民卫生出版社，1990
[68] 马保华．动物胚胎工程[M]．北京：中国农业出版社，2001
[69] 高建明，姜晓鹃，焦占海等冷冻液平衡时间和复温时间对小鼠胚胎冷冻解冻效果的影响[J]．北京农学院学报．2000，15(3)：27～30
[70] Desai N, Lawson j, Goldfarb J, et al. Assessment of growth factou effects on post-thaw development of cryopreserved mouse mourlae to the blastocyst stage[J]. Hum Reprod. 2000, 15: 410～418
[71] Han MS, Niwa K, Kasai M. Vitrification of rat embryos at various developmental stages[J]. Theriogenology. 2003, 59: 1851～1863
[72] YuswiatiE, Hiltz W. Work in progress: Successful transfer of Vetrified goat embryos[J]. Theriogenology 1990, 34: 629～632
[73] 刘伟信，黄萍，王丽等．不同冷冻保护剂对早期卵裂期小鼠胚胎冷冻后成活率和发育的影响[J]．细胞生物学杂志．2004，226(3)：317～319
[74] 姜国诚，孙艳香．速冻过程中低温保护剂对小鼠胚胎发育的影响[J]．浙江农业大学学报．1995，21(2)：208～210
[75] 徐平，川野佳代，刘丽均等，实验大鼠、小鼠胚胎的缓慢冷冻和玻璃化冷冻的比较[J]．上海交通大学学报(农业科学报)．2002，20(4)：261～266
[76] 李瑞利等．不同冷冻保护剂和投入液氮时温度对冷冻兔胚存活的影响[J]．畜牧兽医学报，1988，19(1)：1～6
[77] Jiang Guocheng, Sun Yanxiang, Yang Zhonghua, et al. The differences of osmotic behaviors bovine embryos from in vitro and in vivo and their quality evaluation[J]. Theriogenology. 1997, 47(1): 307
[78] Rall W F. Cryopreservation of Oocytes and embryos: Methods and Applications[J]. Animal Reproduction Science, 1992, 28: 237～245
[79] Glenister P H, W F Rall. Cryopreservation and rederivation of embryos and gametes. Genetics & Transgenics: A Prac-ticalApproach (2ndEdition) [M]. Oxford: Oxford Univ. Press, 1999: 27～59
[80] PeterH, Glenister, ClaireEThornton. Crypreservation-archiving forthefuture[J]. Mamm. Ceno. 2000, 11, 565～571
[81] Mahmoudzadeh AR, Van Soom A, Bols P, et al. Optimization of a simple vitrification procedure for bovine embryos produced in vitro: effect of developmental stage, two-step addition of cryoprotectant and sucrose dilution on embryonicsurvival[J]. Reprod Fertil. 1995, 103～139
[82] Dinnyes A, Wallace GA, Rall WF. Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing method. Molecular Reproduction and development. 1995, 40: 429～435
[83] Pomp D, Eisen EJ. Gentic control of survival of frozen mouse embryos [J]. Biol Reprod. 1990, 42: 775～786
[84] Rall WF, Wood MJ. High in vitro and in vivo survival of day 3 mouse embrys vitrified or frozen in a non-toxic solution of glycerol and albumin. Journal of Reproduction and Fertility. 1994, 101: 681～688
[85] 朱士恩，左琴，曾申明等．乙二醇为主体的玻璃化溶液对小鼠桑椹胚的超低温保存[J]．中国实验动物学杂志．2000，10(3)：150～157
[86] 朱士恩，曾申明，张忠诚．小鼠胚胎玻璃化冷冻保存及保存时间对其体内外发育率的影响[J]．农业生物技术学报．1999，7(2)：163～166
[87] Yeoman RR, Gerami-Naini B, MitalipovS, et al. Cryoloop vitrification yields superior survival of Rhesus monkey blatocysts[J]. Hum Reprod, 2001, 16(9): 1965～1969
[88] 袁红花，朱孝荣，李厚达．Lats基因敲除小鼠胚胎玻璃化冷冻的研究[J]．实验动物科学与管理，20(2)：47～49
[89] 朱士恩，权国波，吴通义等．小鼠原核胚OPS法冷冻保存技术[J]．中国兽医学报．2002，22(4)：362～364
[90] 刘丽均，顾建忠，朱东琴等．不同品系小鼠胚胎玻璃化冷冻保存的比较研究[J]．中国实验动物学报．2002，10(2)：77～80
[91] Yokota Y, Sato S, Yokota M, et al. successful pregnancy following blastocyst vitrification[J]. Hum Reprod, 2000, 15: 1802～1803
[92] Boiso I, Marti M, Santalo J, et al. A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle andmetaphase Ⅱ stage[J]. Hum Reprod. 2002, 17(7): 1885～1891
[93] Tetsunori MD, Nakamura BS, Tatsuhiro MD, et al. Cryoloop vitrification yields superior survival of Rhesus monkey blatocysts[J]. Fertil Steril. 2001, 76 (3): 618～620
[94] 孙艳香，姜国诚．玻璃化冷冻保存小鼠受精卵的研究[J]．广西师范大学学报(自然科学报)．1995，21(3)：77～79
[95] 朱士恩，李玉欣，曾申明等．小鼠桑椹胚简易玻璃化冷冻技术再探讨[J]．中国畜牧杂志．1997，33(3)：14～16
[96] 王燕蓉，刘斌．甘油蔗糖液对哺乳动物桑椹胚的渗透反应及冻存效果研究[J]．解剖学报．1998，29(3)：307～311
[97] 朱亮，罗琛，黄敏珍等．应用两种麦管系统对小鼠囊胚的玻璃化冷冻[J]．第一军医大学学报．2003，23(9)：992～995
[2] 李勇，窦忠英，杨炜峰．胚胎工程技术及其在畜牧业中的应用[J]．西北农林科技大学学报(自然科学版)．2004，32(2)：104～109
[3] 严云勤．发育生物学原理与胚胎工程[M]．哈尔滨：黑龙江科学出版社．1995：278
[4] 马新武．哺乳动物胚胎冷冻原理及研究进展[J]．动物医学进展．2000，21(4)：112～116
[5] 李广武，郑从义，唐兵．低温生物学[M]．湖南科学技术出版社．1998，8
[6] 陈北亨，王建辰．兽医产科学[M]．北京：中国农业出版社．2001，4：339～355
[7] 徐勇，张嘉宝．哺乳动物胚胎超低温冷冻保存的研究进展[J]．黑龙江动物繁殖．2001，9(1)：15～17
[8] 动物胚胎冷冻保存技术的研究应用[J]．中国草食动物．2003，23(4)：42～43
[9] 朱士恩，曾申明，张忠诚．哺乳动物胚胎玻璃化冷冻技术研究进展[J]．中国畜牧杂志．1998，3：2～4
[10] Zhu S.E, et al. Cryopreservation of mouse zona—free blastocysts developed from—two cell embryos' blastomeres by vitrification. 动物生物技术国际会议论文集．1997
[11] Whittingham DG. Survial of mouse embryos frozen to-196℃ and-269℃[J]. Science, 1972, 178: 411
[12] L. J. Maclellan. et al.[M]. Therigenology, Abstract. 2001: 300～318
[13] N. Oberstein, et al. Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling metholds[J]. Therigenology, 2001, (55): 607～613
[14] Levent keskintepe, Yuksel Agca, Gabriela A, et al. Use of cryopreserved pronuclear embryos for the production of transgenic mice[J]. 2001, 65: 407～411
[15] 包华琼，李跃民．哺乳动物胚胎冷冻的研究进展[J]．草食家畜(季刊)．2001，12(4)：1～5
[16] 阳年生．脱防冻剂方法对牛体外受精胚胎冷冻后成活率的影响[J]．中国兽医学报．1998，18(3)：291～294
[17] 张嘉宝，周虚．动物繁殖学[M]．吉林：吉林科技出版社．1999：230～257
[18] 孙新明．小鼠胚胎一步冷冻试验[D]．陕西杨陵：西北农业大学．1993
[19] Miyake T, Kasai M, Zhu S E, etal. Vitrification of mouse oocytes and embryos at various stages of development in an ethylene glycol-based solution by a simple method[J]. Theriogenology. 1993, 40: 121～134.
[20] Tada N, Sato M, Amann E, et al A simple and rapid method for cryopreservation of mouse 2-cell embryos by vitrification: beneficial effect of sucrose and raffinose on their cryosurvival rate[J]. Theriogenology, 1993, 40: 333～344
[21] M. A Nowshari, G. Brem. Effect of freezing rate and expose time to cryopretectant on the development of mouse pronuclear stage embryos[J]. Human Reproduction 2001, 16 (11): 2368～2373
[22] Park SE, Chung HM, Cha KY. cryopreservation of ICR mouse oocytes: improved post-thawed, preimplantation development after vitrification using Taxol, a cytoskeleton stabilizer[J]. Fertil steril. 2001, 75 (6): 1177～1184
[23] Jun-hui wang. A comprehensive Evaluation of the Effects and mechanisms of Antifreeze proteins during low-Temperature Preservation [J]. cryobiology. 2000, 41: 1～9
[24] J. Aki, J. N. Shelton. Design of Vitrification solutions for the cryopreservation of Embryos[J]. Journal of Reproduction and fertility. 1993, 99: 471～477
[25] 丁志丽，桑林华，王杏龙．哺乳动物胚胎冷冻保存的研究进展[J]．河北畜牧兽医．2003，19(12)：24～26
[26] 曲玉秀，刘铁铮，汤家铭等．小鼠的胚胎冷冻保存研究[J]．四川畜牧兽医．2002，29(12)：28～29
[27] 朱士恩，王海彦．生物频谱对小鼠桑葚胚玻璃化冷冻体内外发育影响的研究[J]．农业生物技术学报．1998，3
[28] 史洪才．家畜胚胎冷冻保存的研究进展[J]．草食家畜(增刊) 1999，102：31～34
[29] S. Ohboshi, N. Fujihara. Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts [Z]. Animal reproducation Science, 1997, 48: 27～36
[30] Michelle Lane. Vitrification of embryos Using a Nylon Loop[Z]. The Embryologists' Newsletter. 2000, winter
[31] S. Cseh, W. Horlacher, G. Brem. Vitrification of mouse embryos in two cryoprotectant solutions[J]. Theriogenology. 1999, 52: 103～113
[32] Joe Wilfe, Gary Bryant. Cellular cryobiology: thermodynamic and mechanical effects[Z]. International Journal of Refrigeration. 2001, 438～450
[33] Thatcher W W, Moreira F, Santos J E P. Effects of hormonal treatments on reproductive performance and embryo production[J]. Theriogenology 2001, 55: 75～89
[34] 王锋，蔡令波，王启风等．小鼠胚胎超低温冷冻保存方法的比较研究[J]．草食家畜(增刊)．2001：136～139
[35] 朱士恩，张忠诚．小鼠桑椹胚简易玻璃化冷冻技术再讨论[J]．中国畜牧杂志．1997，33(3)：14～17
[36] Schiewe MC, Rall WF, Stuart LD. In situ dilution of ovine embryos cryopreserved by conventional freezing or vitrification [J]. Theriogenology. 1990, 33: 321
[37] Vajta G, Booth P J, KuwayamaM, et al. Open pulled straw (OPS) vitrification: a new way to reduce cryoinjuries of bovine ova and embryos [J]. Mol Reprod Dev, 1998, 51: 53～58
[38] Booth P J, Vajta G, H oj A. Full-term development of nuclear transfer calves produced from open-pulled straw (OPS) vitrified cytoplasts: work in progress[J]. Theriogenology. 1999, 51(5): 999～1006
[39] Vajta G. Vitrification of the oocytes and embryos of domestic animals[J]. Anim Reprod S ci, 2000, 61: 357～364
[40] U shijima H, Yamakawa H, Nagashima H. Cryopreservation of bovine pre-morula-stage in vitro matured/in vitro fertilized embryos after delipidation and before use in nucleus transfer[J]. BiolReprod, 1999, 60: 534～539
[41] 刘丽均，徐平．小鼠胚胎玻璃化冷冻的研究进展[J]．中国实验动物学杂志 2001，11(2)：115～117
[42] Emiliant S, Van Den Bergh M, Vannin A S, et al. Comparison of ethylene glycol, 1, 2-propanediol and glycerol for cryopreservation of slow-cooled mouse zygotes, 4-cell embryos and blastocysts[J]. Hum Reprod, 2000, 15 (4): 905～910
[43] 曹佐武，潘善培，刘学高．影响玻璃化法冷冻保存胚胎的一些因素[J]．暨南大学学报(自然科学与医学报)．19(12)：48～50
[44] 努尔江，史洪才，黄俊成等．小鼠早期桑椹胚和致密桑椹胚玻璃化冷冻保存试验[J]．草食家畜 2000，1(3) 27～29
[45] 朱士恩，曾申明，张忠诚．液氮气熏蒸法玻璃化冷冻小鼠扩张囊胚的研究[J]．农业生物技术学报．1999，7(2)：163～166
[46] Liu J, Van Den Abbeel E, Van Steirteghem A C. Assessment of ultrarapid and slow freezing procedures for 1-cell and 4-cell mouse embryos[J]. Hum Reprod. 1993, 8(7): 1115～1119
[47] K Nakao, N Nakagata, M Katsuki. Simple and efficient procedure for cryopreserservaton of mouse embryos by simple vitrification[J]. Exp Anim. 1997, 46: 231～134
[48] A Very S M. Embryo cryopreservation[M]. Brinsden PR. A Textbook of In Vitro Fertilization and Assisted Reproduction, 2nd ed. USA: The Parehenon Publishing group Inc, 1999: 211～217
[49] 左琴，岳秉飞，刘双环等．近交系小鼠胚胎玻璃化冷冻技术研究[J]．中国实验动物学杂志 2001年6月，11(2)：82～84
[50] 王俊霞，朱桂金．三种不同玻璃化冷冻方法对小鼠2—细胞期胚胎冷冻效果的比较[J]．生殖与避孕．2004，24(2)：214～217
[51] Senn A, Vozzic, Chanson A, et al. prospective randomized study of two cryopreservation polices avoiding embryo selection: the pronucleate stage leads to a higher cumulative delivery rate than the early cleavage stage[J]. Fertil and steril. 2000, 74(5): 946～952
[52] Mukaida T, Wada S, Takahashi K, et al. Vitrification of human embryos based on the assessment of suitable conditions for 8-cell mouse embryos[J]. Hum Reprod. 1998, 13(10): 2874～2879
[53] Cocero M J, Diaz De la, Espina S M, et al. Ultrastructural characteristics of fresh and frozen-thawded ovine embryos using two cryoprotectants[J]. Biol Reprod. 2002, 66(5): 1244～1258
[54] 赵小徽，岳天孚，随笑琳．玻璃化法冻融小鼠桑葚胚期和囊胚期胚胎的效果观察[J]．中华妇产科杂志．2004，39(8)：526～528
[55] 孙艳香，姜国诚．冷冻液中甘油对小鼠胚胎保护效果的研究[J]．广西师范大学学报(自然科学版)．2000，18(1)：92～94
[56] 王果，马燕琳，吕祁峰等平皿-微滴法玻璃化冷冻保存小鼠胚胎．湖南医科大学学报．2002，27：229～301
[57] Cardner DK, Schoolcraft WB, Wagley L, et al. A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization[J]. Hum Reprod. 1998, 13: 3434～3440
[58] 孙艳香，姜国诚．乙二醇对速冻中小鼠胚胎发育的作用[J]．广西师范大学学报(自然科学版)．2001，19(3)：69～71
[59] 高建明，彭邵华．植冰前后平衡时间对小鼠胚胎一步细管冷冻解冻效果的影响[J]．北京农学院学报．15(4)：30～32
[60] 努尔江，张家心，黄俊成等．不同冷冻保护液和不同植冰温度对小鼠胚胎冷冻效果的影响[J]．草食家畜(季刊)．2000，3：31～33
[61] 张嘉宝，赵志辉，周虚等．小鼠胚胎快速冷冻的研究[J]．黑龙江动物繁殖．6(2)：8～9
[62] Vabderzwalmen P, Bertin G, Debauche CH, et al. births after vitrification at morula and blastocyst stages: effect of artificial reduction of the blastocoelic cavity before virtrification[J]. Hum Reprod. 2002, 17: 744～751
[63] Tao J, Tamis R, Fink K. Cryopreservation of mouse embryos at morula/compact stage[J]. J Assust Reorid Genet, 2001, 18: 235～243
[64] 董雅娟．牛胚胎冷冻保存的现状及展望[J]．草食家畜，1995，(2)：20～22
[65] 安志兴，李向臣，王超等．不同植冰温度下冷冻液的结晶变化及其对小鼠胚胎冷冻的效果[J]．西北农林科技大学学报(自然科学版)．2003，31(3)：11～14
[66] 管彤，王凤山，于国德等．实验小鼠胚胎冷冻技术实验研究[J]．实验动物科学与管理．18(2)：14～17
[67] 王燕蓉．体外授精与胚胎移植[M]．北京：人民卫生出版社，1990
[68] 马保华．动物胚胎工程[M]．北京：中国农业出版社，2001
[69] 高建明，姜晓鹃，焦占海等冷冻液平衡时间和复温时间对小鼠胚胎冷冻解冻效果的影响[J]．北京农学院学报．2000，15(3)：27～30
[70] Desai N, Lawson j, Goldfarb J, et al. Assessment of growth factou effects on post-thaw development of cryopreserved mouse mourlae to the blastocyst stage[J]. Hum Reprod. 2000, 15: 410～418
[71] Han MS, Niwa K, Kasai M. Vitrification of rat embryos at various developmental stages[J]. Theriogenology. 2003, 59: 1851～1863
[72] YuswiatiE, Hiltz W. Work in progress: Successful transfer of Vetrified goat embryos[J]. Theriogenology 1990, 34: 629～632
[73] 刘伟信，黄萍，王丽等．不同冷冻保护剂对早期卵裂期小鼠胚胎冷冻后成活率和发育的影响[J]．细胞生物学杂志．2004，226(3)：317～319
[74] 姜国诚，孙艳香．速冻过程中低温保护剂对小鼠胚胎发育的影响[J]．浙江农业大学学报．1995，21(2)：208～210
[75] 徐平，川野佳代，刘丽均等，实验大鼠、小鼠胚胎的缓慢冷冻和玻璃化冷冻的比较[J]．上海交通大学学报(农业科学报)．2002，20(4)：261～266
[76] 李瑞利等．不同冷冻保护剂和投入液氮时温度对冷冻兔胚存活的影响[J]．畜牧兽医学报，1988，19(1)：1～6
[77] Jiang Guocheng, Sun Yanxiang, Yang Zhonghua, et al. The differences of osmotic behaviors bovine embryos from in vitro and in vivo and their quality evaluation[J]. Theriogenology. 1997, 47(1): 307
[78] Rall W F. Cryopreservation of Oocytes and embryos: Methods and Applications[J]. Animal Reproduction Science, 1992, 28: 237～245
[79] Glenister P H, W F Rall. Cryopreservation and rederivation of embryos and gametes. Genetics & Transgenics: A Prac-ticalApproach (2ndEdition) [M]. Oxford: Oxford Univ. Press, 1999: 27～59
[80] PeterH, Glenister, ClaireEThornton. Crypreservation-archiving forthefuture[J]. Mamm. Ceno. 2000, 11, 565～571
[81] Mahmoudzadeh AR, Van Soom A, Bols P, et al. Optimization of a simple vitrification procedure for bovine embryos produced in vitro: effect of developmental stage, two-step addition of cryoprotectant and sucrose dilution on embryonicsurvival[J]. Reprod Fertil. 1995, 103～139
[82] Dinnyes A, Wallace GA, Rall WF. Effect of genotype on the efficiency of mouse embryo cryopreservation by vitrification or slow freezing method. Molecular Reproduction and development. 1995, 40: 429～435
[83] Pomp D, Eisen EJ. Gentic control of survival of frozen mouse embryos [J]. Biol Reprod. 1990, 42: 775～786
[84] Rall WF, Wood MJ. High in vitro and in vivo survival of day 3 mouse embrys vitrified or frozen in a non-toxic solution of glycerol and albumin. Journal of Reproduction and Fertility. 1994, 101: 681～688
[85] 朱士恩，左琴，曾申明等．乙二醇为主体的玻璃化溶液对小鼠桑椹胚的超低温保存[J]．中国实验动物学杂志．2000，10(3)：150～157
[86] 朱士恩，曾申明，张忠诚．小鼠胚胎玻璃化冷冻保存及保存时间对其体内外发育率的影响[J]．农业生物技术学报．1999，7(2)：163～166
[87] Yeoman RR, Gerami-Naini B, MitalipovS, et al. Cryoloop vitrification yields superior survival of Rhesus monkey blatocysts[J]. Hum Reprod, 2001, 16(9): 1965～1969
[88] 袁红花，朱孝荣，李厚达．Lats基因敲除小鼠胚胎玻璃化冷冻的研究[J]．实验动物科学与管理，20(2)：47～49
[89] 朱士恩，权国波，吴通义等．小鼠原核胚OPS法冷冻保存技术[J]．中国兽医学报．2002，22(4)：362～364
[90] 刘丽均，顾建忠，朱东琴等．不同品系小鼠胚胎玻璃化冷冻保存的比较研究[J]．中国实验动物学报．2002，10(2)：77～80
[91] Yokota Y, Sato S, Yokota M, et al. successful pregnancy following blastocyst vitrification[J]. Hum Reprod, 2000, 15: 1802～1803
[92] Boiso I, Marti M, Santalo J, et al. A confocal microscopy analysis of the spindle and chromosome configurations of human oocytes cryopreserved at the germinal vesicle andmetaphase Ⅱ stage[J]. Hum Reprod. 2002, 17(7): 1885～1891
[93] Tetsunori MD, Nakamura BS, Tatsuhiro MD, et al. Cryoloop vitrification yields superior survival of Rhesus monkey blatocysts[J]. Fertil Steril. 2001, 76 (3): 618～620
[94] 孙艳香，姜国诚．玻璃化冷冻保存小鼠受精卵的研究[J]．广西师范大学学报(自然科学报)．1995，21(3)：77～79
[95] 朱士恩，李玉欣，曾申明等．小鼠桑椹胚简易玻璃化冷冻技术再探讨[J]．中国畜牧杂志．1997，33(3)：14～16
[96] 王燕蓉，刘斌．甘油蔗糖液对哺乳动物桑椹胚的渗透反应及冻存效果研究[J]．解剖学报．1998，29(3)：307～311
[97] 朱亮，罗琛，黄敏珍等．应用两种麦管系统对小鼠囊胚的玻璃化冷冻[J]．第一军医大学学报．2003，23(9)：992～995