牛卵母细胞冷冻保存的研究
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摘要
本实验探讨了冷冻保护剂、冷冻方法、卵母细胞发育阶段、冻前预处理方法等因素对牛卵母细胞冷冻效果的影响，目的在于筛选适合超低温冷冻保存牛卵母细胞的冷冻程序。通过这一途径可望建立卵母细胞种子库，并为体外受精、卵核移植和体细胞克隆研究与应用提供实验材料。研究结果如下：
(1)冷冻保护剂对牛卵母细胞毒性实验表明：四种冷冻保护剂：二甲基亚砜(DMSO)、丙二醇(PROH)、乙二醇(EG)、甘油(GL)在室温(25℃)和培养温度(39℃)条件下与牛GV期卵母细胞作用3min，脱保护剂后进行体外成熟培养。表明：四种冷冻保护剂在低浓度室温(25℃)条件下对牛GV期卵母细胞毒性弱，但随浓度增加毒性增强；39℃时毒性明显增加，其中EG毒性最弱。四种冷冻保护剂的毒性由强至弱是：GL＞PROH＞DMSO＞EG。分别添加1.5mol/L冷冻保护剂(DMSO、PROH、EG、GL)程序化冷冻牛GV期卵母细胞时，EG和PROH对牛GV期卵母细胞的保护比GL、DMSO效果要好一些。
(2)采用程序化冷冻法、细管玻璃化法(Straw)、开放式拉管法(OPS)、毛细玻管法(GMP)冷冻牛GV期卵母细胞，解冻经体外成熟、体外受精后观察2-细胞卵裂率表明：程序化冷冻和Straw玻璃化冷冻的卵裂率分别为3.33％和8.33％，二者差异不显著(P＞0.05)；OPS冷冻和GMP冷冻卵裂率分别为36.84％和36.67％，二者差异不显著(P＞0.05)，但OPS冷冻和GMP冷冻与程序化冷冻、Straw玻璃化冷冻的卵裂率相比差异极显著(P＜0.01)。结果表明：玻璃化冷冻效果优于程序化冷冻，GMP和OPS优于Straw玻璃化冷冻，GMP方法冷冻效果与OPS方法相近。
(3)采用GMP法冷冻GV期、体外培养6h、18h、22h各发育阶段的卵母细胞，解冻后再培养24h、18h、6h和2h，经体外受精分析卵裂率。冷冻后GV期卵母细胞的卵裂率为36.67％，培养6h和18h的卵裂率分别为40.98％和41.27％，三者之间无显著差异(P＞0.05)，但与培养22h卵母细胞(52.38％)和对照(68.42％)差异极显著(P＜0.01)。
于如每一出冷冰际名的研究 搞一
体外成熟（IVM）卵母细胞冷冻后卵裂率为 52．38O，与对照（68．42O）差异不显著
（P＞0刀5）。冷冻不同发育阶段的牛卵母细胞的结果表明：体外成熟培养22h的卵母细
胞冷冻效果好于 GV期、培养 6h、18h的卵母细胞，越接近成熟的卵母细胞冷冻效果越
好。
（4）在卵母细胞玻璃化之前，分别采用 1OEG、3％EG、6oEG、10OEG、l／2 VS
溶液预处理卜！smin，比较玻璃化冷冻后卵母细胞的体外成熟率和卵裂率。结果表明采
用3％的EG稀溶液预处理玻璃化前牛卵母细胞可分别获得较高的卵裂率36．96％。
研究表明，采用GMP玻璃化冷冻法来保存牛GV期卵母细胞是可行的。
The objective of this study is to provide an effective method to cryopreserve bovine GV oocytes for research and practice of gamete bank, in vitro fertilization, nuclear transfer and cell cloning. The study compared the effects of cryopreservation reagent , freezing method, the developmental stages of oocytes and equilibration method in bovine. The results as follows:
Studies on cryoprotectants toxicity for bovine oocytes. Bovine GV oocytes were treated with four 1.5-7.2 mol/L cryoprotectants (DMSO, PROH, EG, GL) at 25 and 39 , then diluted cryoprotectants and compared the ratio of the maturation in vitro. The results demonstrated that the four cryoprotectants' toxicity for bovine oocytes were low and increased depend on the concentration at 25 ; And those toxicity were evident at 39 , but EG's is lowest. The toxicity from the high to low was GL>PROH>DMSO>EG. Using the programmed freezing method to freeze the bovine oocytes with 1.5 mol/L DMSCK PROH, EG, GL, the effects of EG and PROH were better than GL, DMSO.
Using programmed freezing method, Straw vitrification, open-pulled-straw (OPS), glass micropipette (GMP) to freeze the bovine GV oocytes, Freezed and warmed oocytes were matrured and fertilized in vitro . The cleavage ratio was 3.33% and 8.33% respectively in programmed freezing method and Straw vitrification, and the latter was higher by 5.1% than the former, but the results was not different (P>0.05 ); The cleavage ratio using OPS and GMP was similar, 36.84% and 36.67% respectively, and the results was not different (P>0.05) ,but was significant different with programmed freezing method and Straw vitrification
( PO.01 ) .This demonstrated that vitrification was better than programmed freezing method,that GMP and OPS was better than Straw vitrification, that the effect of GMP and OPS was similar.
We study the effects of the developmental stages of oocytes. GV oocytes and ooctyes cultured 6h, 18h, 22h in vitro were vitrification using GMP. the cleavage ratio of warmed oocytes cultured 24h, 18h, 6h and 2h and fertilized in vitro was compared .The cleavage ratio of warmed GV oocytes was 36.67%, 40.98% and 41.27% after culture 6h and 18h. The three was not different (P>0.05).However, this was significant different compared with oocytes cultured 22h (52.38%) and the control (68.42%) (P>0.05) . The cleavage ratio of IVM freezed and thawed oocytes was 52.38%, and it was not different with the control (68.42%) (P>0.05).This results indicated the cyroperservation effectiveness of oocytes cultured 22h was better than that of GV and those oocytes cultured 6h and 18h. The more maturation of bovine oocytes were, the more cyroperservation effectiveness was.
We study the effects of equilibration for vitrification. Prior to vitrification of bovine oocytes in the vilification solution, the oocytes were exposed to equilibration solution (1%EG, 3%EG, 6%EG, 10%EG, 1/2 VS solution) for 1-1 5min, the results indicated that effectiveness of 3%EG is best than others, whose cleavages and maturation ratio in vitro were high.This suggested the diluted solution equilibrium can improve the cyroperservation effectiveness before vitrification.
The conclusions proved that GMP vitrification procedure can be used to cyro-perserve bovine GV oocytes.
(1)冷冻保护剂对牛卵母细胞毒性实验表明：四种冷冻保护剂：二甲基亚砜(DMSO)、丙二醇(PROH)、乙二醇(EG)、甘油(GL)在室温(25℃)和培养温度(39℃)条件下与牛GV期卵母细胞作用3min，脱保护剂后进行体外成熟培养。表明：四种冷冻保护剂在低浓度室温(25℃)条件下对牛GV期卵母细胞毒性弱，但随浓度增加毒性增强；39℃时毒性明显增加，其中EG毒性最弱。四种冷冻保护剂的毒性由强至弱是：GL＞PROH＞DMSO＞EG。分别添加1.5mol/L冷冻保护剂(DMSO、PROH、EG、GL)程序化冷冻牛GV期卵母细胞时，EG和PROH对牛GV期卵母细胞的保护比GL、DMSO效果要好一些。
(2)采用程序化冷冻法、细管玻璃化法(Straw)、开放式拉管法(OPS)、毛细玻管法(GMP)冷冻牛GV期卵母细胞，解冻经体外成熟、体外受精后观察2-细胞卵裂率表明：程序化冷冻和Straw玻璃化冷冻的卵裂率分别为3.33％和8.33％，二者差异不显著(P＞0.05)；OPS冷冻和GMP冷冻卵裂率分别为36.84％和36.67％，二者差异不显著(P＞0.05)，但OPS冷冻和GMP冷冻与程序化冷冻、Straw玻璃化冷冻的卵裂率相比差异极显著(P＜0.01)。结果表明：玻璃化冷冻效果优于程序化冷冻，GMP和OPS优于Straw玻璃化冷冻，GMP方法冷冻效果与OPS方法相近。
(3)采用GMP法冷冻GV期、体外培养6h、18h、22h各发育阶段的卵母细胞，解冻后再培养24h、18h、6h和2h，经体外受精分析卵裂率。冷冻后GV期卵母细胞的卵裂率为36.67％，培养6h和18h的卵裂率分别为40.98％和41.27％，三者之间无显著差异(P＞0.05)，但与培养22h卵母细胞(52.38％)和对照(68.42％)差异极显著(P＜0.01)。
于如每一出冷冰际名的研究 搞一
体外成熟（IVM）卵母细胞冷冻后卵裂率为 52．38O，与对照（68．42O）差异不显著
（P＞0刀5）。冷冻不同发育阶段的牛卵母细胞的结果表明：体外成熟培养22h的卵母细
胞冷冻效果好于 GV期、培养 6h、18h的卵母细胞，越接近成熟的卵母细胞冷冻效果越
好。
（4）在卵母细胞玻璃化之前，分别采用 1OEG、3％EG、6oEG、10OEG、l／2 VS
溶液预处理卜！smin，比较玻璃化冷冻后卵母细胞的体外成熟率和卵裂率。结果表明采
用3％的EG稀溶液预处理玻璃化前牛卵母细胞可分别获得较高的卵裂率36．96％。
研究表明，采用GMP玻璃化冷冻法来保存牛GV期卵母细胞是可行的。
The objective of this study is to provide an effective method to cryopreserve bovine GV oocytes for research and practice of gamete bank, in vitro fertilization, nuclear transfer and cell cloning. The study compared the effects of cryopreservation reagent , freezing method, the developmental stages of oocytes and equilibration method in bovine. The results as follows:
Studies on cryoprotectants toxicity for bovine oocytes. Bovine GV oocytes were treated with four 1.5-7.2 mol/L cryoprotectants (DMSO, PROH, EG, GL) at 25 and 39 , then diluted cryoprotectants and compared the ratio of the maturation in vitro. The results demonstrated that the four cryoprotectants' toxicity for bovine oocytes were low and increased depend on the concentration at 25 ; And those toxicity were evident at 39 , but EG's is lowest. The toxicity from the high to low was GL>PROH>DMSO>EG. Using the programmed freezing method to freeze the bovine oocytes with 1.5 mol/L DMSCK PROH, EG, GL, the effects of EG and PROH were better than GL, DMSO.
Using programmed freezing method, Straw vitrification, open-pulled-straw (OPS), glass micropipette (GMP) to freeze the bovine GV oocytes, Freezed and warmed oocytes were matrured and fertilized in vitro . The cleavage ratio was 3.33% and 8.33% respectively in programmed freezing method and Straw vitrification, and the latter was higher by 5.1% than the former, but the results was not different (P>0.05 ); The cleavage ratio using OPS and GMP was similar, 36.84% and 36.67% respectively, and the results was not different (P>0.05) ,but was significant different with programmed freezing method and Straw vitrification
( PO.01 ) .This demonstrated that vitrification was better than programmed freezing method,that GMP and OPS was better than Straw vitrification, that the effect of GMP and OPS was similar.
We study the effects of the developmental stages of oocytes. GV oocytes and ooctyes cultured 6h, 18h, 22h in vitro were vitrification using GMP. the cleavage ratio of warmed oocytes cultured 24h, 18h, 6h and 2h and fertilized in vitro was compared .The cleavage ratio of warmed GV oocytes was 36.67%, 40.98% and 41.27% after culture 6h and 18h. The three was not different (P>0.05).However, this was significant different compared with oocytes cultured 22h (52.38%) and the control (68.42%) (P>0.05) . The cleavage ratio of IVM freezed and thawed oocytes was 52.38%, and it was not different with the control (68.42%) (P>0.05).This results indicated the cyroperservation effectiveness of oocytes cultured 22h was better than that of GV and those oocytes cultured 6h and 18h. The more maturation of bovine oocytes were, the more cyroperservation effectiveness was.
We study the effects of equilibration for vitrification. Prior to vitrification of bovine oocytes in the vilification solution, the oocytes were exposed to equilibration solution (1%EG, 3%EG, 6%EG, 10%EG, 1/2 VS solution) for 1-1 5min, the results indicated that effectiveness of 3%EG is best than others, whose cleavages and maturation ratio in vitro were high.This suggested the diluted solution equilibrium can improve the cyroperservation effectiveness before vitrification.
The conclusions proved that GMP vitrification procedure can be used to cyro-perserve bovine GV oocytes.
引文
1． 冯怀亮，孙青原，李子义等．牛的体外成熟卵母细胞冷冻保存的研究．畜牧兽医学报，1995，26(6)：481～486
2． 冯怀亮．哺乳动物胚胎工程．吉林：吉林科学出版社，1994，119～120
3． 付永伦，严敬明．人类卵母细胞的冷冻保存．生殖与避孕，1996，16(3)：163～166
4． 郝志明，张涌，钱菊芬．山羊卵泡卵母细胞低温冷冻保存的研究．西北农业大学学报，1992，20(增刊)：121～124
5． 李善国，邵敬於．人类胚胎和卵子冷冻保存。生殖与避孕，1997，17(1)：3～7
6． 刘海军．山羊卵母细胞冷冻保存的研究．2000博士论文
7． 石德顺，杨素芳，谭世俭等．牛受精卵的玻璃化冷冻保存研究．第十界全国动物繁殖学术交流论文．
8． 孙青原．牛卵母细胞体外成熟、冷冻及受精的研究．东北农业大学博士论文，1994
9． 王光亚，段恩奎．山羊胚胎工程．陕西：天则出版社，1994，61～62
10． 王君辉，黄纯农．抗冻蛋白与细胞的低温和超低温保存．细胞生物学杂志，1996，8(3)：107～111
11． 阳年生，卢克焕．玻璃化冷冻体外成熟牛卵母细胞的尝试．广西农业大学学报，1994，13(1)：73～77
12． 余永生，常万成，李鹏等．不同冷冻方法对牛卵泡卵母细胞发育潜力的影响．黄牛杂志，2002，28(2)：21～23
13． 朱士恩，曾申明，吴通义等．OPS法玻璃化冷冻牛卵母细胞的研究．中国农业科学，2002，35(6)：700～704
14. Agca Y. Yiu J,Peter AT, Crister ES and Criter JK.Effect of developmental stage on bovine oocyte plasm menbrane water and cryoprotentant permeanibility characteristic. Mol Reprod Dev 1998;49:408～515
15. Aleida PA, Bolton VN.The effects of temperature fluctuations on the cytoskeletal organization and chromosomal constitution of human oocyte. Zygoat, Cambrige University Press, 1995,3:357～365.
16. Arakawa, T., Carpenter, JF, and Crowe, JH. Basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology 1990, 27:401-415
17. Arav. A,Zeron. Y, Vitrification of bovine oocytes using modified minimum drop size technicque (MDS) is effected by the composition and the concentration of the vitrification solution and by the cooling condition .Theriogenology 1997;47:341 absr
18. Arav. A.B.,Rubinskky. E,Seren.I.F, Roche. M, Bolany. P. The role of thermal hysteresis protins during cryopreservation of oocytes and embryos Theriogenology 1994,41: 107～112
19. Ashwood smith.MJ,Morris. GW, Fowler. R,Appleton.TC,Ashorn.R Physical factors are involved in the destruction of embryos and oocytes during freezing and thawing procedures .Hum Repro 1988;3:795～802
20. Barnes FL, Damiani.P, Looney CR, Duby R.T. The meiotic stage affects subsequent development of cooled bovine oocytes. Theriogenology 1997;47:183
21. Bouquet M,Selva J,Aurox M.The incidence of chromosomal abnormalities in frozen-thawed mouse oocytes after in-vitro fertilization. Hum. Reprod, 1992,7:76～80
22. Carrol J,Wood MJ,Wittingham DG.Normal fertilization and development of frozen-thawd mouse oocytes:protective action of certain macromoleculars. Biol.Reprod, 1993,48:606～612
23. Carrol.J,Warnes G.M,Matthews. D. Increase in giggny explains polyploidy after in vivo fertilization of fozen-thawed mouse oocytes.J.Reprod Fertil 1989;85:489～494
24. Chen C.Pregancy after human oocyte cryopreservation. Lancet, 1986, ⅱ:884～886
25. Chen SU,Lien YR,Cheng YY, Chen HF, Hohn Yang YS. Vitrification of mouse oocytes using closed pulled straw(CPS) achieves a high survival and preserves good patterns of meiotic spindle, compared with conventional open-pulled straw(OPS) and grid. Hum Repro 2001 Nov; 16(11):2350～2356
26. Fabrbri R, Porcu E,Marsella M.R, et al. Technical aspects of oocytes cryopreservation. Mol and Cell Endo 2000;169:39～42
27. Fiedler S,Giudice LC,Lamb EG.Cryopreservation of embryo and ova. Fertil Steril,1988,49:743～764
28. Fuku E,Kojima T, Shioya Y, Marcus G.L, Downey B.R. In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology 1992;29:485～492
29. Fuku E,Xia L,Downey BR. Ultrastructural changes in bovine ooeytes cryopreservation by vitrification. Cryobilogy,1995,32: 139～156
30. Glenister. P.H,Wood. M.J,Kirby. C.Whittingham D.G. Incidenceof chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thrawed oocytes fertilized in vitro .Gamete. Res 1987; 16:205～216
31. Gook C.A, Osborm SM,Johnston W.I.H. Cryopreservation of mouse and human oocytes using 1,2 propanediel and the configuration of the meiotic spidle. Hum Repro 1993;8:1101～1109
32. Hiroshi N,Ranad D,Massashige K.Survival of porcine delipided oocyte and embros after cryopreservation by freezing or vitrification. Reprod. Dev, 1999,45(2):1676
33. Hochi S,Fujimoto,Choi YH et al. Cryopreservation of equine oocytes by 2-step freezing. Theriogenology 1994;42:1085～1094
34. Hochi S,Itok, Hirabayashi M,Ueda M,Kimura K, Hanada A. Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes. Theriogenology 1998;49:787～796
35. Hochi S,Kozawam,Fujimoto T, Honda E,Yamada J,Qguri N.In vitro maturation and transmission electron microscope observation of horse oocytes after vitrification. Cryobiology 1996;33:300～310
36. Hochi Shinichi ,Mitsubiro Akiyama ,Gen Minagawa, Ken Kimura, Akira Hanada .Effect of cooling and warming rates
during vitrification of in vitro-matured bovine oocytes .Cryobiogy 2001;41:69～73
37. Hongsheng Men,Rick L.Monson,Jack J.Rutledge. Effect of meiotic stages and maturation protocols on bovine oocytes resistance to cryopreservation. Theriogenology 2002;57:1095～1103
38. Hunter.J.E,Bernard A, Fuller BJ.Measurements of the membrane perneabukutues and its temperature dependece in human fresh and failed to fertilized oocyte. Cryobiology, 1992,29:240～249
39. Hurtt. A.E.,.FLandim-ALvaronga. G.E, Squiires. E.L Vitrification of immatrueand mature equine and bovine oocytes in an ethylene glycol,Ficoll and sucrose solution using open-pulled straw. Therioganology 2000;54:119～128
40. Hyttel P, Vajta G,Callesen H. Vitrification of bovine oocytes with the open-pulled straw method:ultrastructural consequences. Mol Reprod Dev 2000;56(1):80～88
41. Im KS,Kang JK, Kim HS.Effects of cumulus cell,different cryoprotectant,various maturation stages and preincubation before insemination on development capacity of frozen-thrawed bovine oocyte. Theriogenology 1997;47:881～891
42. Imoedemhe GG,sugu AB.Survival of human oocytes cryopreserved with or without the cumulus in 1,2-propandeil.Assist. Reprod. GENET, 1992,323～327
43. James J,Stachecki,Jacques Cohen, Steea Willadsen. Detrimental effects of sodium during mouse oocyte cryopreservation. Biology of Reproduction 1998;59:395～400
44. Joe Wolfe, Gary Bryant. Freezing,drying, and/or vitrification of membrane-solute-water systems. Cryobiology1999, 39:103～129
45. Jonson MH, Pikering SJ. The effects of dimethysulphoxide on the microtubular system of mouse oocyte. Development1987,100: 313～324
46. Kasai M,Komi J.H,Takakamo A,Tsunoda H,Sakurai T, Machida T.A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution,without appreciable loss of viability.J.Reprod Fertil 1990;89:;91～97
47. Kathryn M,Saunders,John E.Park. Effect of cryopreservation procedures on the cytology and fertilization rate of in vitro-matured bovine oocytes. Biology of Reproduction. 1999;61:178～187
48. Kong I.K,.Lea S.I,.ChoS.G, Cho S.K. Comparision of open-pulled straw(OPS) vs glassmicropitte(GMP) vitrification in mouse blastocytes. Theriogenology 2000;53: 1817～1826
49. Kuleshove, L.L,.Macfarlane. D.R,Trounson. A.O..Sugers exert a major influncc on the vitrification propertis. Cryobiology, 1999,38:119～130
50. Lane M,Forest KT, lyons EA,Bavister BD,Live births following virtrification of hamster embryos using a novel container-lesstechnique Thetiohenology 1999,51:167
51. Leboeuf B,Maxwell WMC,Evans G. Survival of mouse morulac vitrified in media containing antifreeze protein
I. Theriogenology, 1997,47:349(Abstract)
52. Lim JM ,Fukui Y, Ono h Developmental conpetence of bovine oocytes frozen at various matration stages followed by in vitro matratoin and fertilization Theriogenology 1992;37:351～371
53. Lim JM, Fukui Y, Ono h .The post-thaw developmental capacity of frozen bovine oocytes following in vitro maturation and fertilization. Theriogenology 1991,35(6):1225～1235
54. Lim JM, Ko JJ,Hwang WS,Chung HM,Niwa K.Development of in vitro matured bovine oocytes after cryopreservation with different cryoprotectants. Theriogenology 1999;51(7):1303～1310
55. Luvoni G.C, Pellizzari P, Battocchio M.Effects of slow and ultrarapid freezing on morphology and resumption of meiosis immature cate oocytes.J.Reprod Fertil, 1997,51:93～99
56. Luvoni.G.C,.Pellizzari. P. Embryos development of in vitro-matrued cat oocytes cryopreserved by lown and ultrarapid freezing. Theriogenology 2000;53:260
57. Martino A, Songsasen N,Leibo SP. Development into blastocyts of bovine oocytes cryopreserved by ultra-rapid cooling .Biol Reprod 1996;54:1059～1069
58. Mazur P, Rall WF, Laibo SP. Kinetics of water loss and the likehood of intraceellar freezing in mouse ova. Cell Biophys, 1984,6:197～203
59. Micheal J,Tucker, Graham Wright ,Paula C,Morton ,Joe B,Massey. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation .fertility amd sterility 1998;.70(.3.): ,578～579
60. Nagashima H,Kuwayama M,Grupen CG.Vitrification of porcine early cleavage stage embryos and oocytes after removal of cytoplasmic lipid dropts. Theriogenology 1996;45: 180
61. Oberstein N,Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methold. Therigenology,2001,55:607～613
62. Otoi T, Tachikawa s, Kondo S.Development capacity of bovine oocytes frozen in different cryoproprotectants. Theriganology, 1993,40:801～807
63. Otoi T, Yamamoto K, Koyama N.Cryopreservation of mature bivine oocytes folloeing centrifugation treatment. Cryobiology, 1997,34:36～41
64. Papis. K, Arery P, Holm P, Challesen H,Greve T. The effect of equilibrated or vitrified bovine in vitro matured oocytes. Theriogenology 1995;43:293
65. Papis. K.,.Shimizu. M, Izaike. Y. Factors affecting the survivability of bovine oocytes vitrifid in droplet. Theriogenology 2000;54:651～658
66. Parks, JE; Ruffing, NA .Factors affecting low temperature survival of mammalian oocytes. Theriogenology,
1992,.37:.59～73.
67. Rall W.F, Fahy GM.Ice-free cryopreservation of mouse embryos at-196 by vitrification.Nature, 1985,313:573～575
68. Rubinsky B,Arav A,Mattiolim et al. The effect of antifreeze glycopeptides on membrane potential changes at hypothermic temperatures. Biochem Biophys Res Comm, 1990,173:1369～1347
69. Rzucidlo ST ,Gibbons J,Stice SL .Comparision by retriction fragment differential display RT-PCR gene expression pattern in bovine oocytes matured in the presence or absence of fetal calf serm .Mol Reprod Dev 2002;59:90～96
70. Schalkoff M.E,Oskowitz SP, Powers PD.Ultrastructural observation of human and mouse oocytes treated: with cryopreservation.Ⅰ: BioI.Reprod,1989,40: 379～393
71. Schellander K,Peli J,Schmoll F. Effects of different cryoprotectants and carbohydrates on freezing of matured and un matured bovine oocytes. Therigenology, 1994,42:909～915
72. Stepon.K.P,Myers.SP,Lynch.DV,Gardner.L,Bronshteyn.V,Leibo.SP,Rall.WF,Pitt.RE.Cryopreservation of drosophila melanogaster embryos .Nuture 1990;345:170～172
73. Suzuki T, Boediono A,Takagi M .Saha S,Sumantri.C. Fertiliaztion and development of frozen-thawed germinal vesicle bovine oocytes by a one step dilution method in vitro .Cryobiology 1996;33:515～524
74. Tahomasl ,Susan E,Bruce A ,Waleed A.Cryopreservation of human prophasa Ⅰoocytes collcted from unstimulated follicles .Ferility and sterilily 1994;61(6):1077～1082
75. Trounson A,Kirby C.Problem in the cryoprotectants of unfertilized eggs by slowing in dimethysulfoxide. Fertil Steril, 1989,52:778～786
76. Vajta G, Holm p,Kuwayama M, Booth PJ,Jacobsen H,Grcve T, Calleson H.Open pulled straw (OPS) vitrification :a new way to reduce cryoinjuries of bovine ova and embryos Mol Rprod Dev 1998;51:53～58
77. Vajta G,Kuwayama M,Booth PJ,Holn P, Greve T ,Callesen H.Open pulled straw (OPS) vitrification of cattle oocyes. Theriogenology 1998;49:176
78. Vajta G,Lewiszm,Kuwayama M,Greve T, Callesen H,Sterile application of the open pulled straw vitrification Cryo-letters 1998b;19:389～392.
79. Vincent C,Garnier V, Heyman Y, et.al. Solvent effects on cytoskeletal organization and in vivo survival after of rabbit oocytes.J.Reprod. Fertil, 1989,87: 809～820
80. Vincent C,Johnson MH.Cooling cryoprotectants and the cytokeleton of the mammalian oocytes. Oxford Rev. Reprod. Biol, 1992,14:73～100
81. Whittingham D.G Fertilization in vitro and developments to term of unfertilized mouse oocytes previousely stored at -196℃.J.Reprod. Fertil. 1977,49:89～94
82. Whittingham D.G.Leibo S.P, Mazur P. Survival of mouse embryos ftozen to -196℃ and -296℃. Scince(N.Y), 1972,178:411～414
83. Whittingham D.G.Survival of mouse embryos after freezing and thawing .Nature(Lond),1971,233:125～126
84. Zeron. Y, Tomczak M.,J.Crowe. Electrofusion of bovine oovytes with different changes the membrane thermobehaviory and reduces chilling sensitivity .Theriogenology 2000;53:26
2． 冯怀亮．哺乳动物胚胎工程．吉林：吉林科学出版社，1994，119～120
3． 付永伦，严敬明．人类卵母细胞的冷冻保存．生殖与避孕，1996，16(3)：163～166
4． 郝志明，张涌，钱菊芬．山羊卵泡卵母细胞低温冷冻保存的研究．西北农业大学学报，1992，20(增刊)：121～124
5． 李善国，邵敬於．人类胚胎和卵子冷冻保存。生殖与避孕，1997，17(1)：3～7
6． 刘海军．山羊卵母细胞冷冻保存的研究．2000博士论文
7． 石德顺，杨素芳，谭世俭等．牛受精卵的玻璃化冷冻保存研究．第十界全国动物繁殖学术交流论文．
8． 孙青原．牛卵母细胞体外成熟、冷冻及受精的研究．东北农业大学博士论文，1994
9． 王光亚，段恩奎．山羊胚胎工程．陕西：天则出版社，1994，61～62
10． 王君辉，黄纯农．抗冻蛋白与细胞的低温和超低温保存．细胞生物学杂志，1996，8(3)：107～111
11． 阳年生，卢克焕．玻璃化冷冻体外成熟牛卵母细胞的尝试．广西农业大学学报，1994，13(1)：73～77
12． 余永生，常万成，李鹏等．不同冷冻方法对牛卵泡卵母细胞发育潜力的影响．黄牛杂志，2002，28(2)：21～23
13． 朱士恩，曾申明，吴通义等．OPS法玻璃化冷冻牛卵母细胞的研究．中国农业科学，2002，35(6)：700～704
14. Agca Y. Yiu J,Peter AT, Crister ES and Criter JK.Effect of developmental stage on bovine oocyte plasm menbrane water and cryoprotentant permeanibility characteristic. Mol Reprod Dev 1998;49:408～515
15. Aleida PA, Bolton VN.The effects of temperature fluctuations on the cytoskeletal organization and chromosomal constitution of human oocyte. Zygoat, Cambrige University Press, 1995,3:357～365.
16. Arakawa, T., Carpenter, JF, and Crowe, JH. Basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology 1990, 27:401-415
17. Arav. A,Zeron. Y, Vitrification of bovine oocytes using modified minimum drop size technicque (MDS) is effected by the composition and the concentration of the vitrification solution and by the cooling condition .Theriogenology 1997;47:341 absr
18. Arav. A.B.,Rubinskky. E,Seren.I.F, Roche. M, Bolany. P. The role of thermal hysteresis protins during cryopreservation of oocytes and embryos Theriogenology 1994,41: 107～112
19. Ashwood smith.MJ,Morris. GW, Fowler. R,Appleton.TC,Ashorn.R Physical factors are involved in the destruction of embryos and oocytes during freezing and thawing procedures .Hum Repro 1988;3:795～802
20. Barnes FL, Damiani.P, Looney CR, Duby R.T. The meiotic stage affects subsequent development of cooled bovine oocytes. Theriogenology 1997;47:183
21. Bouquet M,Selva J,Aurox M.The incidence of chromosomal abnormalities in frozen-thawed mouse oocytes after in-vitro fertilization. Hum. Reprod, 1992,7:76～80
22. Carrol J,Wood MJ,Wittingham DG.Normal fertilization and development of frozen-thawd mouse oocytes:protective action of certain macromoleculars. Biol.Reprod, 1993,48:606～612
23. Carrol.J,Warnes G.M,Matthews. D. Increase in giggny explains polyploidy after in vivo fertilization of fozen-thawed mouse oocytes.J.Reprod Fertil 1989;85:489～494
24. Chen C.Pregancy after human oocyte cryopreservation. Lancet, 1986, ⅱ:884～886
25. Chen SU,Lien YR,Cheng YY, Chen HF, Hohn Yang YS. Vitrification of mouse oocytes using closed pulled straw(CPS) achieves a high survival and preserves good patterns of meiotic spindle, compared with conventional open-pulled straw(OPS) and grid. Hum Repro 2001 Nov; 16(11):2350～2356
26. Fabrbri R, Porcu E,Marsella M.R, et al. Technical aspects of oocytes cryopreservation. Mol and Cell Endo 2000;169:39～42
27. Fiedler S,Giudice LC,Lamb EG.Cryopreservation of embryo and ova. Fertil Steril,1988,49:743～764
28. Fuku E,Kojima T, Shioya Y, Marcus G.L, Downey B.R. In vitro fertilization and development of frozen-thawed bovine oocytes. Cryobiology 1992;29:485～492
29. Fuku E,Xia L,Downey BR. Ultrastructural changes in bovine ooeytes cryopreservation by vitrification. Cryobilogy,1995,32: 139～156
30. Glenister. P.H,Wood. M.J,Kirby. C.Whittingham D.G. Incidenceof chromosome anomalies in first-cleavage mouse embryos obtained from frozen-thrawed oocytes fertilized in vitro .Gamete. Res 1987; 16:205～216
31. Gook C.A, Osborm SM,Johnston W.I.H. Cryopreservation of mouse and human oocytes using 1,2 propanediel and the configuration of the meiotic spidle. Hum Repro 1993;8:1101～1109
32. Hiroshi N,Ranad D,Massashige K.Survival of porcine delipided oocyte and embros after cryopreservation by freezing or vitrification. Reprod. Dev, 1999,45(2):1676
33. Hochi S,Fujimoto,Choi YH et al. Cryopreservation of equine oocytes by 2-step freezing. Theriogenology 1994;42:1085～1094
34. Hochi S,Itok, Hirabayashi M,Ueda M,Kimura K, Hanada A. Effect of nuclear stages during IVM on the survival of vitrified-warmed bovine oocytes. Theriogenology 1998;49:787～796
35. Hochi S,Kozawam,Fujimoto T, Honda E,Yamada J,Qguri N.In vitro maturation and transmission electron microscope observation of horse oocytes after vitrification. Cryobiology 1996;33:300～310
36. Hochi Shinichi ,Mitsubiro Akiyama ,Gen Minagawa, Ken Kimura, Akira Hanada .Effect of cooling and warming rates
during vitrification of in vitro-matured bovine oocytes .Cryobiogy 2001;41:69～73
37. Hongsheng Men,Rick L.Monson,Jack J.Rutledge. Effect of meiotic stages and maturation protocols on bovine oocytes resistance to cryopreservation. Theriogenology 2002;57:1095～1103
38. Hunter.J.E,Bernard A, Fuller BJ.Measurements of the membrane perneabukutues and its temperature dependece in human fresh and failed to fertilized oocyte. Cryobiology, 1992,29:240～249
39. Hurtt. A.E.,.FLandim-ALvaronga. G.E, Squiires. E.L Vitrification of immatrueand mature equine and bovine oocytes in an ethylene glycol,Ficoll and sucrose solution using open-pulled straw. Therioganology 2000;54:119～128
40. Hyttel P, Vajta G,Callesen H. Vitrification of bovine oocytes with the open-pulled straw method:ultrastructural consequences. Mol Reprod Dev 2000;56(1):80～88
41. Im KS,Kang JK, Kim HS.Effects of cumulus cell,different cryoprotectant,various maturation stages and preincubation before insemination on development capacity of frozen-thrawed bovine oocyte. Theriogenology 1997;47:881～891
42. Imoedemhe GG,sugu AB.Survival of human oocytes cryopreserved with or without the cumulus in 1,2-propandeil.Assist. Reprod. GENET, 1992,323～327
43. James J,Stachecki,Jacques Cohen, Steea Willadsen. Detrimental effects of sodium during mouse oocyte cryopreservation. Biology of Reproduction 1998;59:395～400
44. Joe Wolfe, Gary Bryant. Freezing,drying, and/or vitrification of membrane-solute-water systems. Cryobiology1999, 39:103～129
45. Jonson MH, Pikering SJ. The effects of dimethysulphoxide on the microtubular system of mouse oocyte. Development1987,100: 313～324
46. Kasai M,Komi J.H,Takakamo A,Tsunoda H,Sakurai T, Machida T.A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution,without appreciable loss of viability.J.Reprod Fertil 1990;89:;91～97
47. Kathryn M,Saunders,John E.Park. Effect of cryopreservation procedures on the cytology and fertilization rate of in vitro-matured bovine oocytes. Biology of Reproduction. 1999;61:178～187
48. Kong I.K,.Lea S.I,.ChoS.G, Cho S.K. Comparision of open-pulled straw(OPS) vs glassmicropitte(GMP) vitrification in mouse blastocytes. Theriogenology 2000;53: 1817～1826
49. Kuleshove, L.L,.Macfarlane. D.R,Trounson. A.O..Sugers exert a major influncc on the vitrification propertis. Cryobiology, 1999,38:119～130
50. Lane M,Forest KT, lyons EA,Bavister BD,Live births following virtrification of hamster embryos using a novel container-lesstechnique Thetiohenology 1999,51:167
51. Leboeuf B,Maxwell WMC,Evans G. Survival of mouse morulac vitrified in media containing antifreeze protein
I. Theriogenology, 1997,47:349(Abstract)
52. Lim JM ,Fukui Y, Ono h Developmental conpetence of bovine oocytes frozen at various matration stages followed by in vitro matratoin and fertilization Theriogenology 1992;37:351～371
53. Lim JM, Fukui Y, Ono h .The post-thaw developmental capacity of frozen bovine oocytes following in vitro maturation and fertilization. Theriogenology 1991,35(6):1225～1235
54. Lim JM, Ko JJ,Hwang WS,Chung HM,Niwa K.Development of in vitro matured bovine oocytes after cryopreservation with different cryoprotectants. Theriogenology 1999;51(7):1303～1310
55. Luvoni G.C, Pellizzari P, Battocchio M.Effects of slow and ultrarapid freezing on morphology and resumption of meiosis immature cate oocytes.J.Reprod Fertil, 1997,51:93～99
56. Luvoni.G.C,.Pellizzari. P. Embryos development of in vitro-matrued cat oocytes cryopreserved by lown and ultrarapid freezing. Theriogenology 2000;53:260
57. Martino A, Songsasen N,Leibo SP. Development into blastocyts of bovine oocytes cryopreserved by ultra-rapid cooling .Biol Reprod 1996;54:1059～1069
58. Mazur P, Rall WF, Laibo SP. Kinetics of water loss and the likehood of intraceellar freezing in mouse ova. Cell Biophys, 1984,6:197～203
59. Micheal J,Tucker, Graham Wright ,Paula C,Morton ,Joe B,Massey. Birth after cryopreservation of immature oocytes with subsequent in vitro maturation .fertility amd sterility 1998;.70(.3.): ,578～579
60. Nagashima H,Kuwayama M,Grupen CG.Vitrification of porcine early cleavage stage embryos and oocytes after removal of cytoplasmic lipid dropts. Theriogenology 1996;45: 180
61. Oberstein N,Cryopreservation of equine embryos by open pulled straw, cryoloop, or conventional slow cooling methold. Therigenology,2001,55:607～613
62. Otoi T, Tachikawa s, Kondo S.Development capacity of bovine oocytes frozen in different cryoproprotectants. Theriganology, 1993,40:801～807
63. Otoi T, Yamamoto K, Koyama N.Cryopreservation of mature bivine oocytes folloeing centrifugation treatment. Cryobiology, 1997,34:36～41
64. Papis. K, Arery P, Holm P, Challesen H,Greve T. The effect of equilibrated or vitrified bovine in vitro matured oocytes. Theriogenology 1995;43:293
65. Papis. K.,.Shimizu. M, Izaike. Y. Factors affecting the survivability of bovine oocytes vitrifid in droplet. Theriogenology 2000;54:651～658
66. Parks, JE; Ruffing, NA .Factors affecting low temperature survival of mammalian oocytes. Theriogenology,
1992,.37:.59～73.
67. Rall W.F, Fahy GM.Ice-free cryopreservation of mouse embryos at-196 by vitrification.Nature, 1985,313:573～575
68. Rubinsky B,Arav A,Mattiolim et al. The effect of antifreeze glycopeptides on membrane potential changes at hypothermic temperatures. Biochem Biophys Res Comm, 1990,173:1369～1347
69. Rzucidlo ST ,Gibbons J,Stice SL .Comparision by retriction fragment differential display RT-PCR gene expression pattern in bovine oocytes matured in the presence or absence of fetal calf serm .Mol Reprod Dev 2002;59:90～96
70. Schalkoff M.E,Oskowitz SP, Powers PD.Ultrastructural observation of human and mouse oocytes treated: with cryopreservation.Ⅰ: BioI.Reprod,1989,40: 379～393
71. Schellander K,Peli J,Schmoll F. Effects of different cryoprotectants and carbohydrates on freezing of matured and un matured bovine oocytes. Therigenology, 1994,42:909～915
72. Stepon.K.P,Myers.SP,Lynch.DV,Gardner.L,Bronshteyn.V,Leibo.SP,Rall.WF,Pitt.RE.Cryopreservation of drosophila melanogaster embryos .Nuture 1990;345:170～172
73. Suzuki T, Boediono A,Takagi M .Saha S,Sumantri.C. Fertiliaztion and development of frozen-thawed germinal vesicle bovine oocytes by a one step dilution method in vitro .Cryobiology 1996;33:515～524
74. Tahomasl ,Susan E,Bruce A ,Waleed A.Cryopreservation of human prophasa Ⅰoocytes collcted from unstimulated follicles .Ferility and sterilily 1994;61(6):1077～1082
75. Trounson A,Kirby C.Problem in the cryoprotectants of unfertilized eggs by slowing in dimethysulfoxide. Fertil Steril, 1989,52:778～786
76. Vajta G, Holm p,Kuwayama M, Booth PJ,Jacobsen H,Grcve T, Calleson H.Open pulled straw (OPS) vitrification :a new way to reduce cryoinjuries of bovine ova and embryos Mol Rprod Dev 1998;51:53～58
77. Vajta G,Kuwayama M,Booth PJ,Holn P, Greve T ,Callesen H.Open pulled straw (OPS) vitrification of cattle oocyes. Theriogenology 1998;49:176
78. Vajta G,Lewiszm,Kuwayama M,Greve T, Callesen H,Sterile application of the open pulled straw vitrification Cryo-letters 1998b;19:389～392.
79. Vincent C,Garnier V, Heyman Y, et.al. Solvent effects on cytoskeletal organization and in vivo survival after of rabbit oocytes.J.Reprod. Fertil, 1989,87: 809～820
80. Vincent C,Johnson MH.Cooling cryoprotectants and the cytokeleton of the mammalian oocytes. Oxford Rev. Reprod. Biol, 1992,14:73～100
81. Whittingham D.G Fertilization in vitro and developments to term of unfertilized mouse oocytes previousely stored at -196℃.J.Reprod. Fertil. 1977,49:89～94
82. Whittingham D.G.Leibo S.P, Mazur P. Survival of mouse embryos ftozen to -196℃ and -296℃. Scince(N.Y), 1972,178:411～414
83. Whittingham D.G.Survival of mouse embryos after freezing and thawing .Nature(Lond),1971,233:125～126
84. Zeron. Y, Tomczak M.,J.Crowe. Electrofusion of bovine oovytes with different changes the membrane thermobehaviory and reduces chilling sensitivity .Theriogenology 2000;53:26