小鼠胚胎冻前和冻后分割效果的比较研究
摘要
本文主要就不同分割液、培养液、分割处理方法及不同的冷冻保护液和的冷冻处理方法对小鼠桑椹胚和囊胚的影响进行了系统的研究。结果如下:
1首先研究了不同的分割液和不同胚胎发育阶段对分割效果的影响,发现在12.5%蔗糖分割液中分割桑椹胚时,分割成功率显著高于m-PBS处理组(60.83% vs 51.67%,P<0.05 ),而分割囊胚时两分割液之间无显著性差异(P >0.05 );在m-PBS中分割囊胚和桑椹胚,其成功率前者显著高于后者(70.37%vs 51.67%,P<0.01),但是在12.5%蔗糖分割液中分别对囊胚和桑椹胚进行分割时,两者无显著性差异(P>0.05)。
2无论是桑椹胚还是囊胚,其半胚经不同培养液m-PBS和TCM-199-Hepes培养后存活率差异均不显著(62.50% vs 63.64%;80.36% vs 82.76%,P>0.05);桑椹胚和囊胚分割后的半胚经m-PBS培养液培养后,所得半胚存活率两者间差异显著(62.50% vs 80.36%,P<0.05),经TCM-199-Hepes培养液培养后,所得半胚存活率两者间差异显著(63.64% vs 82.76%,P<0.05)。
3桑椹胚去致密化后,其分割成功率显著高于未处理组(73.08% vs 51.92%,P<0.01),半胚培养后的囊胚发育率也显著高于未处理组(71.15% vs 46.15%,P<0.01)。
4半胚经琼脂包埋后,利用VSⅠ和VSⅡ冷冻液进行OPS法玻璃化冷冻,解冻后其存活率均显著高于未处理组(90.16% vs48.28%;88.52% vs 44.83%,P<0.01),培养48h后的半胚发育率,处理组也均显著高于未处理组(61.82% vs 25.86%;61.11% vs 24.13%,P<0.01)。琼脂包埋组解冻后的半胚存活数和发育率,VSⅠ和VSⅡ之间差异均不显著(90.16% vs 88.52%;61.82% vs 61.11%,P>0.05);未包埋组解冻后VSⅠ和VSⅡ之间差异也均不显著(48.28% vs 44.83%;25.86% vs 24.13%,P>0.05)。
5桑椹胚和囊胚分别经两种不同保护液(VSⅠ和VSⅡ)冷冻后形态良好率差异均不显著(96.94% vs 95.96%;86.87% vs 84.38%,P>0.05);经VSⅠ保护液冷冻后,桑椹胚和囊胚之间形态良好率差异非常显著(96.94% vs 86.87%,P<0.01),经VSⅡ保护液冷冻后,两者之间形态良好率差异也极显著(95.96% vs 84.38%,P<0.01)。
6小鼠胚胎经分割后冷冻,其平均存活率为40.48%,经冷冻后分割的平均存活率为81.63%,两者之间差异非常显著(P<0.01)。
In this paper, the effect of different splitting-solutions, medium, splitting method, different refrigerating fluid and freezing method on the morulae and blastulae of mice were studied. The main results were as follows:
1 First studied the effect of different splitting-solutions and different stage of embryo on the splitting. The success rate of morula splitted in 12.5% sucrose was higher than splitted in m-PBS(60.83% vs 51.67%,P<0.05), while there’s no significant difference when the blastulae were splitted(P>0.05). Splitted the blastulae and morulae in m-PBS, the success rate showed significant difference (70.37%vs 51.67%,P<0.01), but there’s no significant difference when they were splitted in 12.5% sucrose (P>0.05).
2 No matter morulae or blastulae, the livabilily of half-embryos between the m-PBS and TCM-199-Hepes showed no significant difference (62.50% vs 63.64%;80.36% vs 82.76%,P>0.05); while cultured in m-PBS, there was significant difference between morulae and blastulae(62.50% vs 80.36%,P<0.05), and so as TCM-199-Hepes(63.64% vs 82.76%,P<0.05).
3 The success rate of decompacted morulaes was higher than untackled(73.08% vs 51.92%,P<0.01), and there was significant difference in the development rate of demi-morulae (71.15% vs 46.15%,P<0.01)
4 Demi-embryos were vitrified with VSⅠand VSⅡby the way of the OPS, after thawed, the livability of demi-embryos embed by agar was higher than untreated group in both two vitrified medium(90.16% vs48.28%;88.52% vs 44.83%, P<0.01), after cultured 48h, the developmental rate of demi-embryo about treated group was higher than that untreated group (61.82% vs 25.86%;61.11% vs 24.13%,P<0.01). In the group of agar , there was no significant difference between the VSⅠand VSⅡof livability and developmental rate(90.16% vs 88.52%;61.82% vs 61.11%,P>0.05), so as the group without tackled (48.28% vs 44.83%;25.86% vs 24.13%,P>0.05).
5 No matter VSⅠor VSⅡ, after vitrification, there was no significant difference between the rate of morphologically of morulae and blastulae(96.94% vs 95.96%;86.87% vs 84.38%,P>0.05).Vitrifide with VSⅠ, there was significant difference between the morphologically rate of morulae and blastulae (96.94% vs 86.87%,P<0.01),and so as the VSⅡ(95.96% vs 84.38%,P<0.01).
6 Vitrified the splitted embryos of mice, the average livability was 40.48%, and the average livability of splitted embryo after vitrification was 81.63%, there showed significant difference between them (P<0.01).
1首先研究了不同的分割液和不同胚胎发育阶段对分割效果的影响,发现在12.5%蔗糖分割液中分割桑椹胚时,分割成功率显著高于m-PBS处理组(60.83% vs 51.67%,P<0.05 ),而分割囊胚时两分割液之间无显著性差异(P >0.05 );在m-PBS中分割囊胚和桑椹胚,其成功率前者显著高于后者(70.37%vs 51.67%,P<0.01),但是在12.5%蔗糖分割液中分别对囊胚和桑椹胚进行分割时,两者无显著性差异(P>0.05)。
2无论是桑椹胚还是囊胚,其半胚经不同培养液m-PBS和TCM-199-Hepes培养后存活率差异均不显著(62.50% vs 63.64%;80.36% vs 82.76%,P>0.05);桑椹胚和囊胚分割后的半胚经m-PBS培养液培养后,所得半胚存活率两者间差异显著(62.50% vs 80.36%,P<0.05),经TCM-199-Hepes培养液培养后,所得半胚存活率两者间差异显著(63.64% vs 82.76%,P<0.05)。
3桑椹胚去致密化后,其分割成功率显著高于未处理组(73.08% vs 51.92%,P<0.01),半胚培养后的囊胚发育率也显著高于未处理组(71.15% vs 46.15%,P<0.01)。
4半胚经琼脂包埋后,利用VSⅠ和VSⅡ冷冻液进行OPS法玻璃化冷冻,解冻后其存活率均显著高于未处理组(90.16% vs48.28%;88.52% vs 44.83%,P<0.01),培养48h后的半胚发育率,处理组也均显著高于未处理组(61.82% vs 25.86%;61.11% vs 24.13%,P<0.01)。琼脂包埋组解冻后的半胚存活数和发育率,VSⅠ和VSⅡ之间差异均不显著(90.16% vs 88.52%;61.82% vs 61.11%,P>0.05);未包埋组解冻后VSⅠ和VSⅡ之间差异也均不显著(48.28% vs 44.83%;25.86% vs 24.13%,P>0.05)。
5桑椹胚和囊胚分别经两种不同保护液(VSⅠ和VSⅡ)冷冻后形态良好率差异均不显著(96.94% vs 95.96%;86.87% vs 84.38%,P>0.05);经VSⅠ保护液冷冻后,桑椹胚和囊胚之间形态良好率差异非常显著(96.94% vs 86.87%,P<0.01),经VSⅡ保护液冷冻后,两者之间形态良好率差异也极显著(95.96% vs 84.38%,P<0.01)。
6小鼠胚胎经分割后冷冻,其平均存活率为40.48%,经冷冻后分割的平均存活率为81.63%,两者之间差异非常显著(P<0.01)。
In this paper, the effect of different splitting-solutions, medium, splitting method, different refrigerating fluid and freezing method on the morulae and blastulae of mice were studied. The main results were as follows:
1 First studied the effect of different splitting-solutions and different stage of embryo on the splitting. The success rate of morula splitted in 12.5% sucrose was higher than splitted in m-PBS(60.83% vs 51.67%,P<0.05), while there’s no significant difference when the blastulae were splitted(P>0.05). Splitted the blastulae and morulae in m-PBS, the success rate showed significant difference (70.37%vs 51.67%,P<0.01), but there’s no significant difference when they were splitted in 12.5% sucrose (P>0.05).
2 No matter morulae or blastulae, the livabilily of half-embryos between the m-PBS and TCM-199-Hepes showed no significant difference (62.50% vs 63.64%;80.36% vs 82.76%,P>0.05); while cultured in m-PBS, there was significant difference between morulae and blastulae(62.50% vs 80.36%,P<0.05), and so as TCM-199-Hepes(63.64% vs 82.76%,P<0.05).
3 The success rate of decompacted morulaes was higher than untackled(73.08% vs 51.92%,P<0.01), and there was significant difference in the development rate of demi-morulae (71.15% vs 46.15%,P<0.01)
4 Demi-embryos were vitrified with VSⅠand VSⅡby the way of the OPS, after thawed, the livability of demi-embryos embed by agar was higher than untreated group in both two vitrified medium(90.16% vs48.28%;88.52% vs 44.83%, P<0.01), after cultured 48h, the developmental rate of demi-embryo about treated group was higher than that untreated group (61.82% vs 25.86%;61.11% vs 24.13%,P<0.01). In the group of agar , there was no significant difference between the VSⅠand VSⅡof livability and developmental rate(90.16% vs 88.52%;61.82% vs 61.11%,P>0.05), so as the group without tackled (48.28% vs 44.83%;25.86% vs 24.13%,P>0.05).
5 No matter VSⅠor VSⅡ, after vitrification, there was no significant difference between the rate of morphologically of morulae and blastulae(96.94% vs 95.96%;86.87% vs 84.38%,P>0.05).Vitrifide with VSⅠ, there was significant difference between the morphologically rate of morulae and blastulae (96.94% vs 86.87%,P<0.01),and so as the VSⅡ(95.96% vs 84.38%,P<0.01).
6 Vitrified the splitted embryos of mice, the average livability was 40.48%, and the average livability of splitted embryo after vitrification was 81.63%, there showed significant difference between them (P<0.01).
引文
[1] Lovelook J E. The protective action of neutral solutes against haemolysis by freezing and thawing[J]. Biochem J, 1954, 56:260
[2] Mervman H T. Modified model for the mechanism of freezing injury in erythrocytes[J]. Nature, 1986, 218: 333
[3] Levitt J. A sulfhydryl-disulfide hypothesis of frost injury and resistance in plants[J]. Theoretical Biology,1962, 3(3): 355~391
[4] 朱士恩,张忠诚,葛西孙一郎. 扩张囊胚玻璃化超低温冷冻保存及移植技术的研究[J]. 中国畜牧杂志,1996, 4(32):5~8
[5] 刘海军,候蓉,张美佳,等. 山羊卵母细胞冷冻保存及其对发育效果的影响[J]. 畜牧兽医学报,2003,34(1):28~32
[6] Visntin J A, Martins J F P, Bevilacqua E M et al.. Cryopreservation of Bos Taurus VS. Bos indicus embryos: are they really different[J]. Theriogenology ,2002; 57: 345~360
[7] 阳年生,卢克焕.玻璃化冷冻对保存牛体外受精胚胎[J]. 广西农业大学学报,1992,11 (3):102~107
[8] 朱捷,张忠诚,刘泽泉.猪卵母细胞在玻璃化冷冻液中的培养效果[J]. 中国畜牧杂志,1995,5(31):8~9
[9] 张秀芳,黄石军,黄芬香,等.水牛体外受精胚胎的玻璃化冷冻[J]. 草食家畜,2003,1:23~24
[10] Vantini R. Cryopreservation study of bovine in vitro produced embryos using different cryoprotectant solution and at different ages[J]. Theriogenology ,2002,57: 487abstr
[11] Amorin C A. Viability of ovine primordial follicles after cryopreservation with different DMSO concentrations[J]. Theriogenology, 2002,57: 460abstr
[12] Magnusson V. In vitio development of bovine oocyles exposed to twovitiifrication solution[J]. Theriogenology ,2002,57:469 abstr
[13] Pribenszky Cs. In vitro survival of hatching and hatched mouse blastocysts cryopreserved byrapid freezing[J]. Theriogenology, 2002,57: 476abstr
[14] 朱淑文,吉泽绿,李培昌.牛体外受精胚的玻璃化保存及细胞遗传学研究[J].黑龙江畜牧兽医,2001,81:6~7
[15] Boggio Devincenzi J C. Survival of ovine embryos vitrified in 5.5M ethyleneglycol + l.0 Msucrose or 1.0M trehalose. First report in Uruguay[J]. Theriogenology, 2002, 57:489abstr
[16] Vieira A J. Cryopreservahon of immature bovineoocyts treated with cytochalasin D and vitrified in OPS[J]. Theriogenology ,2002,57: 488abstr
[17] Mezzalira A. Vitrification of matured bovine oocytes treated with cytochalasin B[J]. Theriogenology ,2002,57:472abstr
[18] Beebe L F S. Piglets born from centrifuged and vitrified early and peri-hatchring blastocysts[J]. Theriogenology , 2002, 57: 2155~2165
[19] Uaws, Siqueira E S Cs. Vitrification of bovine oocytes in presence of follicular fluid using open pulled[J]. Theriogenology, 2002,57: 482abstr
[20] Leibo s. A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos[J]. Theriogenology,1984, 21:767~790
[21] Whittingham D G, et al.. Survival of Mouse Embryo Frozen to -196℃ and -206℃[J]. Science, 1972, 178:411~414.
[22] Wilnut I , Rowson L. Experiments on the low-temperature preservation of cow embryos[J]. Vet. Rec.,1973, 92:686~690
[23] Willadsen S M, Polge C, Rpwson S, et al.. Deepfreezing of sheep embryos[J]. Journal of Reproduction and Fertilization,1976, 46:151~154
[24] Chemineau P,Procuruer R,Cognie Y,et al.. Production, freezing and transfer of embryos from a bluetongue-infected goat herd without bluetongue tranmission[J]. Theriogenology,1986, 26:179~290
[25] Slade N, Takeka T, Elsden R, et al.. A new procedure for the crvopreservation of equine embryos[J]. Theriogenology, 1985, 24:45~58
[26] 冯书堂,刘殿魁. 猪冷冻(-20℃)胚胎在我国移植成功[J].畜牧兽医学报,1993, 24(1):41~44
[27] Zeilnaker G.,Alberda A,Van G.,et al.. Two pregnancies following tranfers of intact frozen-thawed embryos[J]. Fertil. Steril.,1984,42:293~296
[28] Willadsen s. m. Factors affecting the survival of sheep embryos during deep freezing and thawing[J]. Ciba Foundation Symposium, 1977,52 :175~201
[29] Bouysson B, Chupin D. Two step freezing of cattle blastocvsts with dimethylsulfoxyde or glycerol[J]. Theriogenology, 1982, 17:159~166
[30] Takeda t, Elsden R, Seidel G.. Cryopreservation of mouse embryos by direct plunging into liquid nitrogen[J]. Theriogenology, 1984,21:266
[31] Takagashi Y, Kanagawa H. Effects of equilibration period on the viability of frozen thawed mouse morulae after rapid-freezing[J]. Mol. Reprod. Develop.,1990, 26:105~110
[32] Ral1 W F, Fahy G. W. Ice-free cryopreservationof mouse embryos at -196℃ by vitrification[J]. Nature, 1985, 313:373~575
[33] 华泽钊,任禾盛. 低温生物医学技术[M]. 科学出版社,1991,117
[34] Boutron P, Pevridien J. Reduction in toxicity for red blood cells in buffered solutions containing high concentrition[J]. Cryobiology, 1994, 31:367~373
[35] Zhu S E,Kadai M,Otoge H.,et al.. Crvopreservation of expanded mouse blastocysts by vitrification in ethylene-based solution[J]. Journal of Reproduction and Fertilization, 1993, 98:139~145
[36] Palasz A,Grustafsson H,Grusta, et al.. Vitrification of bovine IVF blastocysts in and ethylene glycol !sucrose solution and heat stable plant-extracted protein[J]. Theriogenology, 1997, 47: 865~879
[37] Kasai M,Komi,Takakamo A,et al.. A simple method for mouse embryocryopreservation in a low toxicity vitrification solution, without appreciable loss of viablity[J]. Journal of Reproduction and Fertilization, 1990, 89:91~97
[38] Komo T, Tsunda Y. Ovicidal effects of vitrication solution and the vitrification warming cycle and establishment of the proportion of toxic effects on nuclei and cytoplasm of mouse embryos[J]. Cryobiology, 1988, 25:197~202
[39] Smorage Z,Gajda B,Wieczorek B, et al.. Stage-dependent viability of vitrified rabbit embryos[J]. Theriogenology, 1989, 31:1221~1231
[40] Saha S, Otoi T, Suzuki T. The efficiency of ethylene glycol, trehalose and polyvivyrrolidone for successful vitrification of IVF bovine embryos[J]. Journal of Reproduction and Development,1996, 42(3):163~169
[41] Nai tana S,Ledda S,Cappai P,et al.. Effect of biopsy and vitrification cn in vitro of ovine embryos at different stages of development[J]. Theriogenology, 1996, 46(5):813~824
[42] Hoch i S,Fujimoto T,Braun J, et al.. Pregnancies following of equine embryos cryopreserved by vitrification[J]. Theriogenology, 1994, 42:483~488
[43] Dobrinsky J, Johnson L. Cryopreservation of porcine embryos by vitrification:A study of in vitro development[J]. Theriogenlogy, 1994, 42(1):25~35
[44] Ohta N,Kojimahara T, Ito M. Embryos by vitrification method[J]. Journal of Fertilization and Sterilation, 1993, 30:413~422
[45] 谭世俭,Henrik Callesen. OPS 法玻璃化冷冻牛卵母细胞和囊胚[J]. 广西农业生物科学,2002,23(1):1~7
[46] Von Baer A. Vitrification and cold storage of Llama hatched blastocysts[J]. Theriogenology,2002,57: 489abstr
[47] Siqueira E S C. Vitrification of bovine oocytes in presence of follicular fluid using open pulled[J]. Theriogenology ,2002;57: 482abstr
[48] 朱士恩,权国波. 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
[49] Naik B R, Rao B S, Vagdevi R, et al.. Conventional slow freezing,vitrification and open pulled straw(OPS) vitrification of rabbit embryos [J]. Animal Reproducation Science, 2005,86:329~338
[50] Ei-Gayar M, Holm P, Holtz W. Successful transfer of vitrified goat blastocysts with the open pulled straw(ops) method[J]. Theriogenology ,2001,55(1):305
[51] Rizos D, Lonergan P, Ward F, et al.. Survival of in vitro produced ovine and bovine blastocysts following open pulled straw vitrification[J]. Theriogrnology, 2001, 55(1):315
[52] Holm P, Vajta G, Machaty Z, et al.. Open pulled straw(ops) vitrification of porcine blastocysts: Simple procedure yielding excellent in vitro survival, but so far no piglets following transfer[J]. Cryo-Letters, 1999,20:307~310
[53] 周剑利. 大鼠卵巢组织冷冻保存和自体移植后形态与功能的研究[M]. 河北省:河北省人民医院,2002
[54] Nicholas J S, Hall BV. Experiments on developing rats. II. The development of isolated blastomeres and fused eggs[J]. J Exp Zool,1942, 90:441
[55] Tarkowski, Andrzej K. Experiments on the development of isolated blastomeres of mouse eggs[J]. Nature,1959, 184:1286~1287
[56] Willadsen S M A. Method for culture of micromanipulated sheep embryos and itsuse to produce monozygotic twins[J]. Nature, 1979, 227(25):298~300
[57] Ozil J P, Heyman Y. Renard JP Production of monozygotic twins by Micromanipulation and cervical transfer in the cow[J]. Vet. Rec., 1982, 110:126~127
[58] Allen W R, Pashen R L. Production of monozygotic(identical) horse twins by embryo Micromanipulation[J]. J Reprod. Ferti1.,1984, 71:607~613
[59] Nagashima H, Katoh Y. Production of normal piglets from microsurgical split morulae and blastocysts[J]. Theriogenology, 1988, 29(2) :485
[60] Handyside A H, Kontogianni E H, Hardy K, et al.. Pregnancies from biopsied human preimplantation embryos sexed by Y specific DNA amplification[J]. Nature, 1990, 344:768~770
[61] Moore N W, Adams C E, Rowson L E A. Developmental potential of single blastomeres of the rabbit eggs[J]. J Reprod Fertil., 1968, 17:527~531
[62] Saito S, Niemann H. Effects of extracellular matrices and growth factors on the development of isolated porcine blastomeres[J]. Biol. Reprod., 1991, 44: 927~936
[63] Willadsen S M, Polge C. Attempts to produce monozygotic quadruplets in cattle by blastomere separation[J]. Vet. Rec., 1981, 108:211~213
[64] Willadsen S M. Nuclear transplantation in sheep embryos[J]. Nature, 1986, 320: 63~65
[65] Willadsen S M. The viability of early cleavage stages containing half the normal number of blastomeres in the sheep[J]. J Reprod. Fert., 1980, 59:357~362
[66] Willadsen S M, Godke RA. A simple procedure for the production of identical sheep twins[J]. Veterinary Record., 1984, 114(3):240~243
[67] 张涌,钱菊芬. 山羊胚胎分割及同卵双生试验[J]. 畜牧兽医学报,1989, 20 (2 ): 97~101
[68] Gray K R, Bondioli, Betts C L. The commercial application of embryo splitting in beef cattle[J]. Theriogenology, 1991,35(1):37~44
[69] 窦忠英,王新庄. 奶牛胚胎一分为四移植试验[J]. 西北农业大学学报,1992,20 (2):1~4
[70] 谭丽玲,李乐义. 奶牛四分胚分割研究[J]. 畜牧兽医学报,1992, 23 (4): 289~294
[71] Rho G J, Walter H J. Cellular composition and viability of demi-and quarter-embryos made from bisected morulae and blastocysts[J]. Theriogenology, 1998, 50:885~895
[72] Bredbacka P, Velmala R. Survival of biopsied and sexed bovine demi-embryos[J]. Theriogenology, 1994,41:1023~1031
[73] Chan A, Dominko T, Luetjens C, et al.. Clonal propagation of primate offspring by embryo splitting[J]. Science, 1999, 287 (2000) :317~318
[74] Le Bourhis D, Chesne P, Nibart M, et al.. Nuclear transfer from sexed parent embros in cattle; efficiency and birth of offspring[J]. J Reprod Fertil, 1998, 113: 343~348
[75] Meng L, Ely J J, Stouffer R L, et al.. Rhesus monkeys produced by nuclear transfer[J]. Biol Reprod., 1997,57:454~459
[76] 毕春明,文瑞成,陈大元,等. 小鼠囊胚内细胞团置换[J]. 科学通报,2003, 48(10): 1064~1068
[77] Watanabe Y, Nonaka K, Sasaki Y, et al.. A longitudinal observation of the postnatal craniofacial growth in artificial monozygotic twin mice[J]. J Craniofac Genet Dev Biol,1998, 18:107~118
[78] Yoshizawa M, Konno H, Zhu S, et al.. Chromosomal diagnosis in each individual blastomere of 5- to10-cell bovine embryos derived from in vitro fertilization[J]. Theriogenology, 1999, 51:1239~1250
[79] Wood C. Embryo splitting : a role in infertility ? [J]. Reprod Fertil Dev , 2001, 13:91~93
[80] Williams T J, Elsden R P, Seidel G E. Pregnancy rates with bisected bovine embryos[J]. Theriogenology,1984, 22(5):521~531
[81] Mcevoy T G, Sreenan J M. The survival of bisected cattle embryos without Zonae pellucidae[J]. Theriogenology, 1987, 27(1):257
[82] Beatrice Mintz. Experimental study of the developing mammalian egg: Removal of the zona pellucida[J]. Science, 1962, 138(2):594~595
[83] Jiang shie, Zhang Li. A simple technique of embryo bisection[J]. Theriogenology, 1988, 29(1):305
[84] Sotomaru Y, Kato Y, Tsunoda Y, et al.. A comparativeinvestigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocyst to producechimeras[J]. Theriogenology, 1997 , 48:977~984.
[85] Sotomaru Y, Kato Y, Tsunoda Y. Production of monozygotic twins afterfreezing and thawing of bisected mouse embryos[J]. Cryobiology, 1998, 37:139~145
[86] Wang M K, KatoY, Tsunoda Y. Effects of several factors on the monozygotic twin production in the mouse[J]. J Reprod Dev., 1997, 43:91~95
[87] Tominaga K, Yoneda K, Utsumi K. Influence of “Solcosery”during culture on the sex-dependent repaire of bodemi-embryos[J]. Mol Reprod Dev, 1996, 43:331~335
[88] King W A , Picard L , Bousquet D , et al.. Sex-dependent loss of bected bovine morulae culture and freezing[J]. J Reprod Fertil , 1992 ,96:453~459
[89] Niemann H, Bren G, Sacher B. An approch to successful freezing of demi-embryos derived from Day-7 bovime embryos[J]. Theriogenology,1986, 25(4): 519~524
[90] Fiser P S, Macpherson J W. Development of enbryonic structures from isolated mouse blastomeres Can[J]. J Anim. Sci., 1976, 56:33~36
[91] Wang M K, KatoY, Tsunoda Y. Effects of several factors on the monozygotic twin production in the mouse[J]. J. Reprod Dev., 1997, 43:91~95
[92] Suzuki T, Shimohira I. Viability of frozen-thawed bovine embryos bisected in sucrose: A preliminary report[J]. Theriogenology, 1986, 26(3):333~339
[93] 张涌,王建辰. 小鼠冻胚分割试验[J]. 西北农业大学学报,1992,20(1): 1~4
[94] 张勇,钱菊汾,王建辰. 小鼠胚胎分割方法及同卵双生试验[J]. 西北农业大学学报,1987,15(2):10
[94] Warfield S J, Seidel G E Jr, Elsden R P. Transfer of bovine demi-embryos with and without pellucidae[J]. J Anim Sci., 1987, 65(3):756~761
[95] 侯世忠,陶涛. 奶牛和小鼠半胚低温冷冻的研究[J]. 山东农业大学学报,1994, 25 (3): 330~334
[96] Fuku E, Fiser P S, Marcus G. J, et al.. Development of Whole and Demi-embryos of Mice in Culture and in Vivo after Supercooled Storage[J]. Cryobiology, 1993, 30( 6): 604~608
[97] Gajda B, Smorag Z. Factors affecting the survival of one and two cell rabbitembryos cryopreserved by vitrification[J]. Theriogenology, 1993, 39(2): 499~506
[98] Valdez C A. Effect of equilibration time, precooling and developmental stage on the survival of mouse embryos cryopreserved by vitrification[J]. Theriogenology, 1990, 3(3): 627~636
[99] Van Der Zwalmen P. Some factors affection successful vitrification of mouse blastocysts[J]. Theriogenology, 1988, 30: 1177~1183
[2] Mervman H T. Modified model for the mechanism of freezing injury in erythrocytes[J]. Nature, 1986, 218: 333
[3] Levitt J. A sulfhydryl-disulfide hypothesis of frost injury and resistance in plants[J]. Theoretical Biology,1962, 3(3): 355~391
[4] 朱士恩,张忠诚,葛西孙一郎. 扩张囊胚玻璃化超低温冷冻保存及移植技术的研究[J]. 中国畜牧杂志,1996, 4(32):5~8
[5] 刘海军,候蓉,张美佳,等. 山羊卵母细胞冷冻保存及其对发育效果的影响[J]. 畜牧兽医学报,2003,34(1):28~32
[6] Visntin J A, Martins J F P, Bevilacqua E M et al.. Cryopreservation of Bos Taurus VS. Bos indicus embryos: are they really different[J]. Theriogenology ,2002; 57: 345~360
[7] 阳年生,卢克焕.玻璃化冷冻对保存牛体外受精胚胎[J]. 广西农业大学学报,1992,11 (3):102~107
[8] 朱捷,张忠诚,刘泽泉.猪卵母细胞在玻璃化冷冻液中的培养效果[J]. 中国畜牧杂志,1995,5(31):8~9
[9] 张秀芳,黄石军,黄芬香,等.水牛体外受精胚胎的玻璃化冷冻[J]. 草食家畜,2003,1:23~24
[10] Vantini R. Cryopreservation study of bovine in vitro produced embryos using different cryoprotectant solution and at different ages[J]. Theriogenology ,2002,57: 487abstr
[11] Amorin C A. Viability of ovine primordial follicles after cryopreservation with different DMSO concentrations[J]. Theriogenology, 2002,57: 460abstr
[12] Magnusson V. In vitio development of bovine oocyles exposed to twovitiifrication solution[J]. Theriogenology ,2002,57:469 abstr
[13] Pribenszky Cs. In vitro survival of hatching and hatched mouse blastocysts cryopreserved byrapid freezing[J]. Theriogenology, 2002,57: 476abstr
[14] 朱淑文,吉泽绿,李培昌.牛体外受精胚的玻璃化保存及细胞遗传学研究[J].黑龙江畜牧兽医,2001,81:6~7
[15] Boggio Devincenzi J C. Survival of ovine embryos vitrified in 5.5M ethyleneglycol + l.0 Msucrose or 1.0M trehalose. First report in Uruguay[J]. Theriogenology, 2002, 57:489abstr
[16] Vieira A J. Cryopreservahon of immature bovineoocyts treated with cytochalasin D and vitrified in OPS[J]. Theriogenology ,2002,57: 488abstr
[17] Mezzalira A. Vitrification of matured bovine oocytes treated with cytochalasin B[J]. Theriogenology ,2002,57:472abstr
[18] Beebe L F S. Piglets born from centrifuged and vitrified early and peri-hatchring blastocysts[J]. Theriogenology , 2002, 57: 2155~2165
[19] Uaws, Siqueira E S Cs. Vitrification of bovine oocytes in presence of follicular fluid using open pulled[J]. Theriogenology, 2002,57: 482abstr
[20] Leibo s. A one-step method for direct nonsurgical transfer of frozen-thawed bovine embryos[J]. Theriogenology,1984, 21:767~790
[21] Whittingham D G, et al.. Survival of Mouse Embryo Frozen to -196℃ and -206℃[J]. Science, 1972, 178:411~414.
[22] Wilnut I , Rowson L. Experiments on the low-temperature preservation of cow embryos[J]. Vet. Rec.,1973, 92:686~690
[23] Willadsen S M, Polge C, Rpwson S, et al.. Deepfreezing of sheep embryos[J]. Journal of Reproduction and Fertilization,1976, 46:151~154
[24] Chemineau P,Procuruer R,Cognie Y,et al.. Production, freezing and transfer of embryos from a bluetongue-infected goat herd without bluetongue tranmission[J]. Theriogenology,1986, 26:179~290
[25] Slade N, Takeka T, Elsden R, et al.. A new procedure for the crvopreservation of equine embryos[J]. Theriogenology, 1985, 24:45~58
[26] 冯书堂,刘殿魁. 猪冷冻(-20℃)胚胎在我国移植成功[J].畜牧兽医学报,1993, 24(1):41~44
[27] Zeilnaker G.,Alberda A,Van G.,et al.. Two pregnancies following tranfers of intact frozen-thawed embryos[J]. Fertil. Steril.,1984,42:293~296
[28] Willadsen s. m. Factors affecting the survival of sheep embryos during deep freezing and thawing[J]. Ciba Foundation Symposium, 1977,52 :175~201
[29] Bouysson B, Chupin D. Two step freezing of cattle blastocvsts with dimethylsulfoxyde or glycerol[J]. Theriogenology, 1982, 17:159~166
[30] Takeda t, Elsden R, Seidel G.. Cryopreservation of mouse embryos by direct plunging into liquid nitrogen[J]. Theriogenology, 1984,21:266
[31] Takagashi Y, Kanagawa H. Effects of equilibration period on the viability of frozen thawed mouse morulae after rapid-freezing[J]. Mol. Reprod. Develop.,1990, 26:105~110
[32] Ral1 W F, Fahy G. W. Ice-free cryopreservationof mouse embryos at -196℃ by vitrification[J]. Nature, 1985, 313:373~575
[33] 华泽钊,任禾盛. 低温生物医学技术[M]. 科学出版社,1991,117
[34] Boutron P, Pevridien J. Reduction in toxicity for red blood cells in buffered solutions containing high concentrition[J]. Cryobiology, 1994, 31:367~373
[35] Zhu S E,Kadai M,Otoge H.,et al.. Crvopreservation of expanded mouse blastocysts by vitrification in ethylene-based solution[J]. Journal of Reproduction and Fertilization, 1993, 98:139~145
[36] Palasz A,Grustafsson H,Grusta, et al.. Vitrification of bovine IVF blastocysts in and ethylene glycol !sucrose solution and heat stable plant-extracted protein[J]. Theriogenology, 1997, 47: 865~879
[37] Kasai M,Komi,Takakamo A,et al.. A simple method for mouse embryocryopreservation in a low toxicity vitrification solution, without appreciable loss of viablity[J]. Journal of Reproduction and Fertilization, 1990, 89:91~97
[38] Komo T, Tsunda Y. Ovicidal effects of vitrication solution and the vitrification warming cycle and establishment of the proportion of toxic effects on nuclei and cytoplasm of mouse embryos[J]. Cryobiology, 1988, 25:197~202
[39] Smorage Z,Gajda B,Wieczorek B, et al.. Stage-dependent viability of vitrified rabbit embryos[J]. Theriogenology, 1989, 31:1221~1231
[40] Saha S, Otoi T, Suzuki T. The efficiency of ethylene glycol, trehalose and polyvivyrrolidone for successful vitrification of IVF bovine embryos[J]. Journal of Reproduction and Development,1996, 42(3):163~169
[41] Nai tana S,Ledda S,Cappai P,et al.. Effect of biopsy and vitrification cn in vitro of ovine embryos at different stages of development[J]. Theriogenology, 1996, 46(5):813~824
[42] Hoch i S,Fujimoto T,Braun J, et al.. Pregnancies following of equine embryos cryopreserved by vitrification[J]. Theriogenology, 1994, 42:483~488
[43] Dobrinsky J, Johnson L. Cryopreservation of porcine embryos by vitrification:A study of in vitro development[J]. Theriogenlogy, 1994, 42(1):25~35
[44] Ohta N,Kojimahara T, Ito M. Embryos by vitrification method[J]. Journal of Fertilization and Sterilation, 1993, 30:413~422
[45] 谭世俭,Henrik Callesen. OPS 法玻璃化冷冻牛卵母细胞和囊胚[J]. 广西农业生物科学,2002,23(1):1~7
[46] Von Baer A. Vitrification and cold storage of Llama hatched blastocysts[J]. Theriogenology,2002,57: 489abstr
[47] Siqueira E S C. Vitrification of bovine oocytes in presence of follicular fluid using open pulled[J]. Theriogenology ,2002;57: 482abstr
[48] 朱士恩,权国波. 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
[49] Naik B R, Rao B S, Vagdevi R, et al.. Conventional slow freezing,vitrification and open pulled straw(OPS) vitrification of rabbit embryos [J]. Animal Reproducation Science, 2005,86:329~338
[50] Ei-Gayar M, Holm P, Holtz W. Successful transfer of vitrified goat blastocysts with the open pulled straw(ops) method[J]. Theriogenology ,2001,55(1):305
[51] Rizos D, Lonergan P, Ward F, et al.. Survival of in vitro produced ovine and bovine blastocysts following open pulled straw vitrification[J]. Theriogrnology, 2001, 55(1):315
[52] Holm P, Vajta G, Machaty Z, et al.. Open pulled straw(ops) vitrification of porcine blastocysts: Simple procedure yielding excellent in vitro survival, but so far no piglets following transfer[J]. Cryo-Letters, 1999,20:307~310
[53] 周剑利. 大鼠卵巢组织冷冻保存和自体移植后形态与功能的研究[M]. 河北省:河北省人民医院,2002
[54] Nicholas J S, Hall BV. Experiments on developing rats. II. The development of isolated blastomeres and fused eggs[J]. J Exp Zool,1942, 90:441
[55] Tarkowski, Andrzej K. Experiments on the development of isolated blastomeres of mouse eggs[J]. Nature,1959, 184:1286~1287
[56] Willadsen S M A. Method for culture of micromanipulated sheep embryos and itsuse to produce monozygotic twins[J]. Nature, 1979, 227(25):298~300
[57] Ozil J P, Heyman Y. Renard JP Production of monozygotic twins by Micromanipulation and cervical transfer in the cow[J]. Vet. Rec., 1982, 110:126~127
[58] Allen W R, Pashen R L. Production of monozygotic(identical) horse twins by embryo Micromanipulation[J]. J Reprod. Ferti1.,1984, 71:607~613
[59] Nagashima H, Katoh Y. Production of normal piglets from microsurgical split morulae and blastocysts[J]. Theriogenology, 1988, 29(2) :485
[60] Handyside A H, Kontogianni E H, Hardy K, et al.. Pregnancies from biopsied human preimplantation embryos sexed by Y specific DNA amplification[J]. Nature, 1990, 344:768~770
[61] Moore N W, Adams C E, Rowson L E A. Developmental potential of single blastomeres of the rabbit eggs[J]. J Reprod Fertil., 1968, 17:527~531
[62] Saito S, Niemann H. Effects of extracellular matrices and growth factors on the development of isolated porcine blastomeres[J]. Biol. Reprod., 1991, 44: 927~936
[63] Willadsen S M, Polge C. Attempts to produce monozygotic quadruplets in cattle by blastomere separation[J]. Vet. Rec., 1981, 108:211~213
[64] Willadsen S M. Nuclear transplantation in sheep embryos[J]. Nature, 1986, 320: 63~65
[65] Willadsen S M. The viability of early cleavage stages containing half the normal number of blastomeres in the sheep[J]. J Reprod. Fert., 1980, 59:357~362
[66] Willadsen S M, Godke RA. A simple procedure for the production of identical sheep twins[J]. Veterinary Record., 1984, 114(3):240~243
[67] 张涌,钱菊芬. 山羊胚胎分割及同卵双生试验[J]. 畜牧兽医学报,1989, 20 (2 ): 97~101
[68] Gray K R, Bondioli, Betts C L. The commercial application of embryo splitting in beef cattle[J]. Theriogenology, 1991,35(1):37~44
[69] 窦忠英,王新庄. 奶牛胚胎一分为四移植试验[J]. 西北农业大学学报,1992,20 (2):1~4
[70] 谭丽玲,李乐义. 奶牛四分胚分割研究[J]. 畜牧兽医学报,1992, 23 (4): 289~294
[71] Rho G J, Walter H J. Cellular composition and viability of demi-and quarter-embryos made from bisected morulae and blastocysts[J]. Theriogenology, 1998, 50:885~895
[72] Bredbacka P, Velmala R. Survival of biopsied and sexed bovine demi-embryos[J]. Theriogenology, 1994,41:1023~1031
[73] Chan A, Dominko T, Luetjens C, et al.. Clonal propagation of primate offspring by embryo splitting[J]. Science, 1999, 287 (2000) :317~318
[74] Le Bourhis D, Chesne P, Nibart M, et al.. Nuclear transfer from sexed parent embros in cattle; efficiency and birth of offspring[J]. J Reprod Fertil, 1998, 113: 343~348
[75] Meng L, Ely J J, Stouffer R L, et al.. Rhesus monkeys produced by nuclear transfer[J]. Biol Reprod., 1997,57:454~459
[76] 毕春明,文瑞成,陈大元,等. 小鼠囊胚内细胞团置换[J]. 科学通报,2003, 48(10): 1064~1068
[77] Watanabe Y, Nonaka K, Sasaki Y, et al.. A longitudinal observation of the postnatal craniofacial growth in artificial monozygotic twin mice[J]. J Craniofac Genet Dev Biol,1998, 18:107~118
[78] Yoshizawa M, Konno H, Zhu S, et al.. Chromosomal diagnosis in each individual blastomere of 5- to10-cell bovine embryos derived from in vitro fertilization[J]. Theriogenology, 1999, 51:1239~1250
[79] Wood C. Embryo splitting : a role in infertility ? [J]. Reprod Fertil Dev , 2001, 13:91~93
[80] Williams T J, Elsden R P, Seidel G E. Pregnancy rates with bisected bovine embryos[J]. Theriogenology,1984, 22(5):521~531
[81] Mcevoy T G, Sreenan J M. The survival of bisected cattle embryos without Zonae pellucidae[J]. Theriogenology, 1987, 27(1):257
[82] Beatrice Mintz. Experimental study of the developing mammalian egg: Removal of the zona pellucida[J]. Science, 1962, 138(2):594~595
[83] Jiang shie, Zhang Li. A simple technique of embryo bisection[J]. Theriogenology, 1988, 29(1):305
[84] Sotomaru Y, Kato Y, Tsunoda Y, et al.. A comparativeinvestigation on the potency of cells from the inner cell mass and trophectoderm of mouse blastocyst to producechimeras[J]. Theriogenology, 1997 , 48:977~984.
[85] Sotomaru Y, Kato Y, Tsunoda Y. Production of monozygotic twins afterfreezing and thawing of bisected mouse embryos[J]. Cryobiology, 1998, 37:139~145
[86] Wang M K, KatoY, Tsunoda Y. Effects of several factors on the monozygotic twin production in the mouse[J]. J Reprod Dev., 1997, 43:91~95
[87] Tominaga K, Yoneda K, Utsumi K. Influence of “Solcosery”during culture on the sex-dependent repaire of bodemi-embryos[J]. Mol Reprod Dev, 1996, 43:331~335
[88] King W A , Picard L , Bousquet D , et al.. Sex-dependent loss of bected bovine morulae culture and freezing[J]. J Reprod Fertil , 1992 ,96:453~459
[89] Niemann H, Bren G, Sacher B. An approch to successful freezing of demi-embryos derived from Day-7 bovime embryos[J]. Theriogenology,1986, 25(4): 519~524
[90] Fiser P S, Macpherson J W. Development of enbryonic structures from isolated mouse blastomeres Can[J]. J Anim. Sci., 1976, 56:33~36
[91] Wang M K, KatoY, Tsunoda Y. Effects of several factors on the monozygotic twin production in the mouse[J]. J. Reprod Dev., 1997, 43:91~95
[92] Suzuki T, Shimohira I. Viability of frozen-thawed bovine embryos bisected in sucrose: A preliminary report[J]. Theriogenology, 1986, 26(3):333~339
[93] 张涌,王建辰. 小鼠冻胚分割试验[J]. 西北农业大学学报,1992,20(1): 1~4
[94] 张勇,钱菊汾,王建辰. 小鼠胚胎分割方法及同卵双生试验[J]. 西北农业大学学报,1987,15(2):10
[94] Warfield S J, Seidel G E Jr, Elsden R P. Transfer of bovine demi-embryos with and without pellucidae[J]. J Anim Sci., 1987, 65(3):756~761
[95] 侯世忠,陶涛. 奶牛和小鼠半胚低温冷冻的研究[J]. 山东农业大学学报,1994, 25 (3): 330~334
[96] Fuku E, Fiser P S, Marcus G. J, et al.. Development of Whole and Demi-embryos of Mice in Culture and in Vivo after Supercooled Storage[J]. Cryobiology, 1993, 30( 6): 604~608
[97] Gajda B, Smorag Z. Factors affecting the survival of one and two cell rabbitembryos cryopreserved by vitrification[J]. Theriogenology, 1993, 39(2): 499~506
[98] Valdez C A. Effect of equilibration time, precooling and developmental stage on the survival of mouse embryos cryopreserved by vitrification[J]. Theriogenology, 1990, 3(3): 627~636
[99] Van Der Zwalmen P. Some factors affection successful vitrification of mouse blastocysts[J]. Theriogenology, 1988, 30: 1177~1183