猪卵泡卵母细胞玻璃化冷冻与体外培养的研究
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摘要
本实验以屠宰厂采集的猪卵巢为材料，从中收集猪卵母细胞，进行玻璃化冷冻、体外培养及培养过程中NO含量变化以及不同大小卵泡NO含量测定研究，结果表明：
1．不同发育时期卵母细胞的冷冻效果有一定差异，未成熟的猪卵巢卵母细胞冷冻后形态正常率和存活率都低于正在成熟(培养24h)和已经成熟(培养44h)的卵母细胞，已经成熟的卵母细胞冷冻后损伤最小(形态正常率83.93％)，存活率最高(69.64％)。卵母细胞的NO含量随着成熟而逐渐降低。每个卵母细胞NO含量由0.480±0.026umol/L降到0.378±0.061umol/L。
2．卵泡φ＞6mm的卵母细胞冷冻后形态正常率，卵丘细胞扩展率，第一极体排出率比φ＜3mm的卵泡卵母细胞高。φ＞6mm卵泡卵母细胞形态完整率，卵丘细胞扩展率，第一极体排出率分别为84.78％、53.26％、35.70％，而φ＜3mm卵泡卵母细胞相应为60.19％、41.67％、9.26％，两者差异极显著(P＜0.01)。
3．不同载体对卵巢卵母细胞玻璃化冷冻效果不同，本实验表明麦管组不及GMP组，两者解冻后形态正常率分别为71.12％、50.04％；存活率分别为83.16％、62.50％，解冻后形态正常率差异极显著(P＜0.01)，存活率差异不显著(P＞0.05)。
4．用GMP法比较10％EG与10％DMSO预处理液，玻璃化冷冻液EFS30、EFS40对猪GV期卵母细胞冷冻效果，研究发现其解冻后形态正常率、培养24h卵丘细胞扩展率有不同，但其统计差异不显著(P＞0.05)。
5．在研究卵泡液对猪卵母细胞体外成熟的影响中发现，成熟液中添加10％PFF可以促进猪卵母细胞的体外成熟，使成熟率由60.06％提高到82.89％。
6．不同直径卵泡卵母细胞体外培养成熟44h后发现其卵丘细胞扩展率，第一极体排出率不同，φ为3—6mm最高，φ＞6mm次之，φ＜3mm最小，分别为73.26％、50％)；(62.79％、38.37％)；(54.48％、30.56％)，三者差异显著(P＜0.05)。大卵泡含NO最少，每个为2.618±0.341umol/l、中卵泡含NO次之，每个为3.840±0.321umol/L、小卵泡含NO最多，每个为4.597±0.685 umol/L、三者差异极显著(P＜0.01)。
This experiment, with the discarded Porcine ovaries from the slaughterhouse as the experimental materials by carrying on the cryoperservation and raising Porcine Follicular Oocytes in vitro ,then measuring the content change of NO during the process and NO content of the follicles in different sizes ,indicates :
1. The effect of freezing the oocytes in difffernt growth periods is different: the complete shape rates of unmatvire oocytes of porcine ovaries and the livability after frozen ,compared with the oncoming mature(raised for 24 hours )and already mature occytes (raised for 44 hours)are both lower ,while the mature oocytes will be damaged the least :the shape integrity rate is 83.90%;the livability is the highest (69.64%). the NO content of FoUicular Oocytes decreases gradually with increasing maturity of oocyte from 0.480±0.026 umol/L untile to 0.378±0.061umol/L for each FoUicular Oocytes.
2. The rates of the shape integrity of oocytes of over 6mm in diameter and the oviferous tubercle cell expansion and First polocyte discharge, compared with those of occytes under 3mm in diameter are higher. when 0>6 mm, the mentioned -above rates of the FoUicular Oocytes are 84.78%、 53.26%、 35.70% respectively; when (?)<3 mm, it will be 60.19%、 41.67%、9.26% respectively . the difference is very significant(P<0.01).
3. Different carriers for freezing ovary oocytes in glass will see different effects shown in this experiment, better in group strawpipe than the group GMP. After defrostation, the rates of shape integrity are 71.12% 50.04% ,while the livability are 83.16%、 62.50% respectively. The differences between are significant(P<0.01), the liabilities are not (P<0.05).
4. Comparing the effects of the 10% EG and 10% DMSO pre-treatment liquids and the frozen glass liquids EFS30、 EFS40 on freezing oocytes in the period of Porcine GV with the method of GMP, we find after defrostation, the rates of shape
1．不同发育时期卵母细胞的冷冻效果有一定差异，未成熟的猪卵巢卵母细胞冷冻后形态正常率和存活率都低于正在成熟(培养24h)和已经成熟(培养44h)的卵母细胞，已经成熟的卵母细胞冷冻后损伤最小(形态正常率83.93％)，存活率最高(69.64％)。卵母细胞的NO含量随着成熟而逐渐降低。每个卵母细胞NO含量由0.480±0.026umol/L降到0.378±0.061umol/L。
2．卵泡φ＞6mm的卵母细胞冷冻后形态正常率，卵丘细胞扩展率，第一极体排出率比φ＜3mm的卵泡卵母细胞高。φ＞6mm卵泡卵母细胞形态完整率，卵丘细胞扩展率，第一极体排出率分别为84.78％、53.26％、35.70％，而φ＜3mm卵泡卵母细胞相应为60.19％、41.67％、9.26％，两者差异极显著(P＜0.01)。
3．不同载体对卵巢卵母细胞玻璃化冷冻效果不同，本实验表明麦管组不及GMP组，两者解冻后形态正常率分别为71.12％、50.04％；存活率分别为83.16％、62.50％，解冻后形态正常率差异极显著(P＜0.01)，存活率差异不显著(P＞0.05)。
4．用GMP法比较10％EG与10％DMSO预处理液，玻璃化冷冻液EFS30、EFS40对猪GV期卵母细胞冷冻效果，研究发现其解冻后形态正常率、培养24h卵丘细胞扩展率有不同，但其统计差异不显著(P＞0.05)。
5．在研究卵泡液对猪卵母细胞体外成熟的影响中发现，成熟液中添加10％PFF可以促进猪卵母细胞的体外成熟，使成熟率由60.06％提高到82.89％。
6．不同直径卵泡卵母细胞体外培养成熟44h后发现其卵丘细胞扩展率，第一极体排出率不同，φ为3—6mm最高，φ＞6mm次之，φ＜3mm最小，分别为73.26％、50％)；(62.79％、38.37％)；(54.48％、30.56％)，三者差异显著(P＜0.05)。大卵泡含NO最少，每个为2.618±0.341umol/l、中卵泡含NO次之，每个为3.840±0.321umol/L、小卵泡含NO最多，每个为4.597±0.685 umol/L、三者差异极显著(P＜0.01)。
This experiment, with the discarded Porcine ovaries from the slaughterhouse as the experimental materials by carrying on the cryoperservation and raising Porcine Follicular Oocytes in vitro ,then measuring the content change of NO during the process and NO content of the follicles in different sizes ,indicates :
1. The effect of freezing the oocytes in difffernt growth periods is different: the complete shape rates of unmatvire oocytes of porcine ovaries and the livability after frozen ,compared with the oncoming mature(raised for 24 hours )and already mature occytes (raised for 44 hours)are both lower ,while the mature oocytes will be damaged the least :the shape integrity rate is 83.90%;the livability is the highest (69.64%). the NO content of FoUicular Oocytes decreases gradually with increasing maturity of oocyte from 0.480±0.026 umol/L untile to 0.378±0.061umol/L for each FoUicular Oocytes.
2. The rates of the shape integrity of oocytes of over 6mm in diameter and the oviferous tubercle cell expansion and First polocyte discharge, compared with those of occytes under 3mm in diameter are higher. when 0>6 mm, the mentioned -above rates of the FoUicular Oocytes are 84.78%、 53.26%、 35.70% respectively; when (?)<3 mm, it will be 60.19%、 41.67%、9.26% respectively . the difference is very significant(P<0.01).
3. Different carriers for freezing ovary oocytes in glass will see different effects shown in this experiment, better in group strawpipe than the group GMP. After defrostation, the rates of shape integrity are 71.12% 50.04% ,while the livability are 83.16%、 62.50% respectively. The differences between are significant(P<0.01), the liabilities are not (P<0.05).
4. Comparing the effects of the 10% EG and 10% DMSO pre-treatment liquids and the frozen glass liquids EFS30、 EFS40 on freezing oocytes in the period of Porcine GV with the method of GMP, we find after defrostation, the rates of shape
引文
[1]李喜龙，季维智．动物种质细胞的超低温冷冻保存[J]．动物学研究，2000，21(5)：407～411
[2]付永伦．人类卵母细胞的冷冻保存[J]．生殖与避孕，1996，3：23～36
[3]Vanuem JF, Schuh B, Koch, S. et al. Birth after cryopreservation of unfertilized oocytes[J], lancet, 2001, 2:752～753
[4]Byrd. Cryopreservation, thawing and transfer of human Embryos[J]. Seminars in Reproductive Medicine, 2002,20(1):37～43
[5]Boiso I, Marti M, Santalo J, et al.A confocal microscopy analysis of the spindle and chromo some configurations of human oocytes cryopreservated at the germinal vesicle and metaphaseⅡ stage[J].Human Reproduction, 2002,17(7):1885～1891
[6]Rienzi L, Martinez F, Ubaldi F, et al. Polscope analysis of meiotic spindle changes in living metaphase Ⅱ human oocytes during the freezing and thawing procedures[J].Hum Reprod, 2004,19(3):655～659
[7]Men H, Monson RL, Parrish JJ, et al. Detection of DNA damage in bovine metaphase Ⅱ oocytes resulting from cryopreservation[J]. Mol Reprod Dev, 2003,64: 245～250
[8]Mandelbaum J, Anastasiou O, Levy R. Effects of cryopreservation the meiotic spindle of humanoocytes[J].Eur J Obstet Gynecol Reprod Biol, 2004, 113:17～23
[9]Obersrein, N. Cryopreservation of equine embryos by open pulled straw. Cryoloop, or conven-tional slow cooling metholds[J]. Therigenology, 2001,55:607～613
[10]Rall WF Fahy GM. Ice-free cryopreservation of mouse embryos at-196℃ by vitrification[J]. Nature 1985, 313:573～575
[11]Mukaida T, Nakamura S, Tomiyama T. et al. Vitrification of human blastocysts using cryoloops, clinical outcome of 223 cycles[J]. Hum Reprod. 2003, 18:384～391
[12] Lane M,Schoolcraft W.B, and Gardner D.K. Vitrification of mouse and human blastocysts using a novel cryoloop technique[J].Fertil. 1999, 72:1073～1078
[13] Chen SU, Lien YR, Cheng YY, et al. Vitrification of mouse oocytes using closed pulled straw(CPS) achieves a highsurvival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws(OPS) and grids[J].Hum Reprod, 2001,16(11):2350～2356
[14] 朱士恩，曾申明，张忠诚．家兔扩张囊胚玻璃化冷冻保存技术的研究[J]．中国养兔杂志，1997，2：16～20
[15] 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[J]．中国农业科学，2002，35(6)：700～704
[16] Kong K.L, Lee S.L, Cho S.K. et al. Comparison of open pulled straw (OPS) VS glass micropipette (GMP) vitrification in mouse blostocysts[J]. Theriogenology,2000,53:1817～1826
[17] Kasai M, Mukaida T. Cryopreservation of animal and human embryos by vitrification[J].Reprod biomed online, 2004, Aug, 9(2): 64～70
[18] Li xh. Study and application of humam oocyte cryopreservation[J]. Beijing Da Xue Xue Bao, 2004, Dec, 36(6):664～667
[19] Papis. K, Arery P, Holm P, et al. The effect of equilibrated or vitrified bovine in vitro matured oocytes[J].Theriogenology, 1995, 43: 293～297
[20] Papis k.,Shimizu M, Iznike Y. Factors affecting the survivability of bovine oocyte vitrified in droplet[J].Theriogenology,2000, 54: 651～658
[21] Misumi k, Suzuki M, Sato S, et al. Successful production of microdrop method [J].Teriogenology,2003,60:253～260
[22] Lane M, Lyons EA, Bavister BD, et al. cryopreservation reduce the ability of hamster 2-cell embryos to regulate in tracellular PH(J).Hum Reprod, 2000,15:389～394
[23] Kouji Misumi, Misae Suzuki, et al. Successful production of piglets derived from vitrified morulae and early blastocysts using a microdroplet method[J]. Theriogenology,2003, 60:253～260
[24] Jiang JY, Umezu M, Sato E. Vitrification of two-cell rat embryos derived from immature hypothyroid rdw rats by in vitro fertilization in ethylene glycol-based solutions[J].Cryobiology, 1999,38:160～164
[25] S.P. Leibo. Cryopreservation of gamctes and embryos of non-domesticspecie [J]. Theriogenology,2002,57:303～326
[26] S.Y. Tomita. The use of in vitro produced cryopreserved for direct transfer to induce twinning in beef cattle [J].Theriogenology, 2002, 57:484-486
[27] Rall WF, Fahy GM. Ice-Free cryoperservation of mouse embryos at-196℃ by vitrification [J].Nature, 1985,313:573～575
[28] Kasai M, Komi JH, Takakamo A, et. al.A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability [J]. Reprod Fertil, 1990,89(1):91～97
[29] Vanderzwalmen P, Bertin G, Debauche CH. Births after vitrification at morula and blastocyst stages:effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod,2002, Mar, 17(3):744～751
[30] Chen SU, Lien YR, Chen HF, Chang LJ, Tsai YY, Yang YS. Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI. Hum Reprod 2005 Mar 24 [Epub ahead of print]
[31] Mukaida T, Takahashi K, Kasai M. Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. Hiroshima Hart Clinic, 2005,34(6):7～10
[32] 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
[33] 刘万勃等．抗冻蛋白的研究进展[J]．生物学杂志，2000，17(8)：7～8
[34] Tomczak MM, Hincha DK, Estrada SD, et al. Antifreeze proteins differentially affect model membranes during freezing. Biochim Biophys Acta, 2001, 1511 (2):255～263
[35] 钟其旺，樊廷俊．鱼类抗冻蛋白的研究进展[J]．生物化学与生物物理学报，2002，34(2)：124～130
[36] Rubinsky B, Arav A, Fletcher GL. Hypothermic Protection-a fundamental property of "antifreeze" proteins. Biochem Biophys protecting liposome membrane integrity depends on phospholipid class. Biochim Biophys Acta, 2001,1511(1):11～16
[37] Luvoni G.C, Pellizzari P. Embryos development of in vitro-matrued cat oocytes cryopreserved by lown and ultrarapid freezing non-domesticcspecie [J].Theriogenology, 2000, 53:260～265
[38] Nagashima H, Kuwayam M, Grupen CG. Vitrification of Porcine early cleavage stage embryos and oocytes after removal of cytoplasmic lipid dropts[J].Theriogenology, 1996, 45:180～185
[39] Lim JM, Fukui Y, Ono H Developmental conpetence of bovine oocytes frozen at various matration stages followed by in vitro matratoin and fertilization[J].Theriogenology,1992, 37:351～371
[40] Lim JM, Fukui Y, Ono H.The post-thaw developmental capacity of frozen bovine oocytes following in vitro maturation and fertilization[J]. Theriogenology, 1991, 35:1225～1235
[41] Lim JM, Ko JJ, Hwang WS, et al. Development of invitro matured bovine oocytes after cryopreservation with different cryorotectants[J]. Theriogenology,1999, 51:1303～1310
[42] Hongsheng Mcn, Rick L. Monson, Jack J Rutledgc. Effect of meiotic stages and maturation Protocols on bovine oocytes resistance to cryopreservation [J]. Theriogenology, 2002, 57:1095～1103
[43] 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
[44] Imoedemhe GG, sugu AB. Suvival of human oocytes cryopreserved with or without the cumulus in 1, 2-Propandeil[J].Assist. Reprod Genet, 1992, 323—327
[45] Gook C. A, Osborm S. M, Johnston W. I. H. Cryopreservation of mouse and human oocytes using 1, 2 Propanedieland the configuration of the meiotic spidle[J].Hum Repro, 1993, 8:1101 — 1109
[46] Ashwood smith. MJ, Morris GW, Fowler R, et al. Physical factors are involved in the destruction of embryos and oocytes during freezing and thawing Procedure[J]. Hum Repro ,1988, 3:795—802
[47] Balou Jonathan D. Potential contribution of cryoperserved germ Plasm to the Preservation of genetic diversity and conservation of endanged species in captivity [J]. Cryobiology, 1992, 29:19—25
[48] Li Z, Jiang Q, Zhang Y, et al. In vitro maturation of ferret oocytes [J]. Theirogenology, 2001, 55 : 482—485
[49] Whittingham DG, Fertilization in vitro and developments to term of unfertilized mouse oocytes Previously stored at -196°C [J]. Reprod Fertil. 1977, 49:89—94
[50] Chen C. Pregnancy after human oocyte cryopreservation[J]. Lancet, 1986, 2:884—886
[51] Fuku E, Kojima T, Shioya Y, et al. In vitro fertilization and development of frozen-thawed bovine oocytes[J]. Cryobiology, 1992, 29:485—492
[52] Vajta G, Holm P, Kuwayama M, 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
[53] Suzuki T, Nishikata Y. Fertilization and cleavage of frozen thawed bovine oocytes by one step dilution method in vitro[J]. Theriogenology, 1992, 37:306 abstr
[54] Yoshida M. Effects of genadotropins and estradiol-17β on the timing of nuclear maturation and cumulus mass expansion in Pig oocyte cultured in vitro. Japan. [J].Anim Reprod. 1989, 35: 86—91
[55] Naito K. Fukuda Y, Effects of Porcine follicular fluid on male Pronuclelus formation in Porcine oocyte matured in vitro[J].Gamete Research, 1988, 21:289～295
[56] Mattioli M. Follicular factors influence oocyte by modulating the intercellular cooperation between cumulus cells and oocyte[J].Gamete Res. 1988, 21:223～232
[57] Eng A, et al. Effect of incubation temperature and bicarbonate on maturation of Pig oocytes in vitlro[J].Reprod Fertil. 1988, 76:657～662
[58] Martino A., Palomo MT, Mogas T, et aL. Influence of the collection technique of Prepubertal goat oocytes on. in vitro maturation and fertilization[J].Theriogenology, 1994, 42:859～873
[59] 陈大元主编．受精生物学-受精机制与生殖工程[M]．科学出版社．2000
[60] 张绣．牛卵母细胞激活体外受精[D]．西北农林科技大学2002届攻读硕士学位研究生学位论文．2002
[61] Downs SM. Specifcity of epidermal gowth factor action on maturation of the murine oocytes and cumulus oophorus in vitro[J].Boil Reprod, 1989, 41:371～379
[62] De Loos, F. Maurck P. et al. Structural aspects of bovine oocytes maturation in vitro[J].Mol Reprod. Dev, 1989, 31:208～214
[63] Lonergan P, Vergos. The effect of recovery method on the type of bovine oocyte obtained for in vitro maturation[J].Theriogenology, 1991, 35:231～234
[64] 冯怀亮，孙青原，李子义等，牛体外成熟卵母细胞冷冻保存的研究[J]．畜牧兽医学报，1995，26(3)：481～486
[65] Gook DA, Osbom SM, Johson WI. Cryopreservation of mouse and human oocytes using 1-2 Propandiol and the configuration of the meiotic spindle[J].Hum Reprod. 1993, 8:1101～1109
[66] 薛庆善主编．体外培养的原理与技术[M]．北京科学技术出版社，2001，2(1)，342～345
[67] Fukui Y, Fukuishima M, Terawaki Y, et al. Effects of gonadotropins, steroids and culture media on bovine oocytes maturation in vitro[J]. Theriogenology, 1989, 18:161～175
[68] Mattioli M, Bacci G, Seren E. Effects of LH and FSH on the maturation of Pig oocytes in vitro[J]. Theriogenology,1991, 36:95～105
[69] 卢晨盛，卢可焕．不同血清种类及其浓度对牛卵母细胞体外受精的影响[J]．广西农业大学学报，1994，13(1)：21～26
[70] Fukui Y, Ono H. Effects of sera, hormones and granulose cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes[J].Reprod Fertil. 1989, 86(2):501～506
[71] 旭日干，张锁链，薛晓先等．屠宰母牛卵巢卵母细胞的体外受精与早期发育[J]．内蒙古大学学报(自然版)，1989，20(3)：407～414
[72] Mochizuki H, Fukui Y, Ono H. Effects of the number of granulose cells added to culture medium for in vitro maturation, fertilization and development of bovine oocytes [J].Theriogenology, 1991, 36:973～986
[73] Otoi T, Fujii M, Tanaka M, et al. Effect of serum on the in vitro maturation of canine oocytes[J], Reprod Fertil. Dev, 1999, 11(7):387～390
[74] Funahashi H, Day BN. Effects of different serum supplement in maturation medium on meiotic and cytoplasmic of Pig Ooeytes[J]. Theriogenology,1993, 39:965～973
[75] Wang S, Liu Y, Holyosak GR, et al. Effects of supplement with fluids from bovine follicles of varied sizes in in vitro maturation on subsequent embryo development in vitro[J].Theriogenology, 1999,51:394～397
[76] Choi YH, Takagi M, Kamishita H, et al. Development capcity of bovine oocytes matured in two kinds of follicular fluid and fertilized in vitro[J].Anim Rprod Sci, 1998, 50(1):27～33
[77] Sirard MA, Biledeau R. Granulosa cells inhibit the resumption of meiosis in bovine Oocyte in vitro[J].Biol Reprod, 1990, 43:777～783
[78] Erickon RP. Recent advances in developmental genetics:Growth factors and morphogens[J].Mol Reprod. Dev. 1995, 41:109～125
[79] Crupen CG, Nottle MB.A simple modification of the in vitro fertili- zation Procedure improves the effciency of in vitro Pig embryo Producion[J].Theriogenology, 2000, 53:422 abstr
[80] Prochazka R, Srsen V, Nagyora E, et al. Development regulation of effect of epiderrnal growth factor on Porcine oocyte-cumulus cell complexes nuclear maturation, expansion and Fact in remodeling[J]. Mol Reprod, Dev. 2000, 56:63～73
[81] Abeydeera LR, Wang WH, Cantley TC et al. Epidermal growth factor can enhance the developmental competence of Pig oocytes matured in vitro under Protein-free culture conditions[J].Theriogenology,2000, 54:787～797
[82] Cameiro G, Lorenzo P, Pimentel C, et al.Influence of insulin-like growth factor-1 and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes[J].Biol of Reprod, 2001,65(3):899～905
[83] Ayoub MA，Hunter AG著，朱玉林译，牛卵泡液对牛卵母细胞体外成熟的抑制作用[D]．草食家畜，1995，89(4)：34～37
[84] Wang S, Liu Y, Holyosak GR, et al. Effects of supplementation with fluids from bovine follicules of varied sizes in in vitro maturation on subsequent embryo development in vitro[J].Theriogenology, 1999, 51:394～398
[85] Choi YH, Takagi M, Kamishita H, et al. Development capacity of bovine oocytes matured in two kinds of follicular fluid and fertilized in vitro[J].Anim Reprod Sci, 1998, 50(1):27～33
[86] Sirard MA, Bilodeau S. Granulosa cells inhibit the resumption of meiosis in bovine oocyte in vitro[J]. Biol Reprod, 1990, 43:777～783
[87] Sun FJ, Holm P, Lrvine B, et al. Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro[J].Theriogenology, 1994, 41:981～988
[88] Georgios, Vatzias and Daniel R. Hagen Effects of Porcine follicular fluid and oviduct-conditioned on maturation and fertilization of Porcine oocytes in vitro[J].Boil of Reprod. 1999, 60:424～448
[89] 钱云，师蔚进，朱猛进等．卵泡内环境对猪卵泡卵体外成熟和发育的影响[J]，生命科学研究．2001，21(6)：624～626
[90] Eppig JJ. Mouse oocyte development in vitro with Various culture systems[J].Dev. Boil of Reprod. 1997, 60:371～388
[91] Chesnel F, Wigglesworth K. Acquisition of meiotic competence by denuded mouse oocytes: Participation of somatic-cell Products and camp [J].Dev. Biolof Reprod. 1994, 161:285～295
[92] Mochizuki H, Fukui Y. Ono H. Effect of the number of granulosa cells added to culture medium for in vitro maturation and development of bovine oocytes[J].Theriogenology, 1991, 36:973～986
[93] Fukui Y. Mcgowan L T. James R W. et al. Factors affecting the in vitro development to blastocysts of bovine oocytes matured and fertilized in vitro[J]. Reprod. Fert. 1991, 92:125～131
[94] Coskun S, SanbuisshaA, Liu YC, et al. Fertilizability and subsequent development ability of bovine oocytes matured in medium containing EGF[J]. Theriogenology,1991, 36:485～494
[95] Bavister BD, Rose-Hellekant TA, pinyopummintr T. Development of in vitro matured/ in vitro fertilized bovine embryos into morulae and blastocysts in defined culture media[J].Theriogenology, 1992, 37:127-146
[96] Saeki K, Hoshi M, Leibfried-Rutledge ML, et al. In vitro fertilization of bovine in a Protein-free medium and subsequent development to the blastocyst stage[J].Theriogenology, 1993, 39:302 abstr
[97] 张涌，刘泽隆，李欲强．山羊卵泡卵母细胞体外成熟的研究[J]，西北农业大学学报，1999，27(1)：14～18
[98] Dorado CM, Mclana LM, Miller JA, et al. Nuclear status of bovine oocyte exposed to elevated temperature after resumpte of meiosis[J]. Theriogenology, 2001, 55:469 abstr
[99] Azambuja RM, De Moreno JF, Ktaemer D, et al. Effect of gas at mosphere on in vitro maturation of bovine oocytes[J].Theriogenology, 1993, 39:184-187.
[100] Lee ES, Yoon KW, Lim J, et al. Effect of gas composition during in vitro maturation and culture on in vitro development of Porcine follicular oocytes[J].Theriogenology, 2001, 55(1):480 abstr
[101] Bing YZ, Hirao Y, Takenouchi N, et al. In vitro maturation of denuded Porcine oocytes in the Presence of cysteamine under the conditions of different oxygen environments[J].Theriogenology, 2002, 57:713 abstr
[102] Albina JS, Lisa M, Olson LM, et al. Nitric oxide is essential for optimal meiotic maturation of murine cumlus-oocyte complexes in vitro [J]. Mol Repro. Dev, 2000, 55:412～421
[103] Kazuo S, Naoyuki T. Requirement of nitric oxide for murine oocyte maturation, embryo development and trophoblast out-growth in vitro[J]. Mol Reprod Dev, 2001, 58:262～268
[104] 孙青原，刘国艺，徐立滨等．牛卵泡卵母细胞冷冻保存后发育潜力的研究[J]．中国兽医学报1994，14(4)：34～2345
[105] 孙青原．牛卵母细胞体外成熟、冷冻及受精的研究[D]．东北农业大学博士学位论文，1994．
[106] Hochi S, Ito K, Hirabayashi M, et al. Effect of nuclear stages during IVM on the Survival of vitrified-warmed bovine oocytes[J]. Theriogenology, 1998,49(4):787～796
[107] Lim JM, Fukui Y, Ono H. Dvelopmental competence of bovine oocytes frozen at various maturation stages followed by in vitro maturation and fertilization[J].Theriogenology,1992;37:351～361
[108] 刘海军．山羊卵母细胞冷冻保存研究[D]。西北农林科技大学2000届博士学位研究生论文．
[109] 刘海军，侯蓉，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
[110] Jisachenko V, Isachenkoe, Michelmann HW, et al. hipoysis and ultrastructural changes of intilcellular lipid vesicles after cooling of bovine and porcine GV-oocytes[J].Anat Histol Embryol, 2001,30(6):333～338
[111] Hefler LA Gregg AR. Inducible and endothelial nitric oxide synthase:Genetic bakeground affects ovulation in mice [J]. Mol Reprod, Dev, 2000, 55:412～421
[112] Tao Y, Fu Z, Zhang M. Immunohistochemical localization of inducible and endothelial nitric oxide synthase in porcine ovaries and effects of NO on antrum formation and oocyte meiotic maturation [J].Mol Cell Endocrinol, 2004,222(2):93～103
[113] MotikJ, Crozet N, Fulka J. Meiotic competence in vitro of pig oocytes isolated from early antral follicles[J]. Reprod Fertil, 1984, 72:323～328
[114] Wickramasinge D, Ebert KM, Albertini DF. Meiotic competence acquisition is associated with the appearance of M-Phase characteristics in growing mouse oocytes[J].Dev Biol.1991, 143:162～172
[115] Sehramr RD, Tennier MT, Boatman DE, Bavister BD. Chromatin configurations and meiotic competence of oocytes are related to follicular diameter in nonstimulated rhesus monkeys[J].Biol Reprod, 1993, 48:349～356
[116] De Smedt V, Crozet N, Gall L. Morphological and functional changes accompanying the acquisition of meiotic competence of goat ovarian oocyte[J]. Theriogenology, 2001,269:128～139
[117] 蔡令波．猪卵母细胞体外成熟与冷冻保存的研究[D]．南京农业大学硕士论文，2002．6
[118] Otoi T, Yamamoto K, Koyarna N, et al. Cryopreservation of mature bovine oocytes by vitrification in Straws. Cryobiology[J], 1998,37(1):77～85
[119] Papis K, Shimizu M, Izaike Y. Factors affecting the survivability of bovine oocytes vitrified in droplets[J].Theriogenology, 2000, 54: 651～658
[120] Vajta G, Holm P, Greve T, et al. Vitrification of porcine embryos using the Open Pulled Straw(OPS) [J]. Theriogenology, 1997,38:349～352
[121] Beginl, Bhatia B, Baidassajrre H, et al. Cryopreservation of goat oocytes and in vivo derived 2-to 4-cell embryos using the cryoloop(CLV) and Solid-surfce vitrification(SSV)methods[J].Theriogenology, 2003, 59:1839～1850
[122] Vajta G, Holm P, Kuwayama M, et al. Open Pulled Straw(OPS) vitrification:a new way to reduce cryo injuries of bovine ova and embryos[J].Mol Reprod, Dev, 1998, 51:53～58
[123] Luvoni. GC, Pellizzari P. Embryos development of in vitro-matrued cat oocytes cryopreserved by lown and ultrarapid freezing[J]. Theriogenology, 2000,53:260～267
[124] Hyttel P, Vajtao, Callesen H. Vitrification of bevine oocytes with the open Pulled straw method:Ult Structural consequences[J].Mol Reprod, Dev, 2000,56(1): 88～91
[125] 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[J]，中国农业科学，2002，35(6)：7007～7014
[126] Remero-Arredondo A, Seidel FE, Jr. Effects of follicular fluid on maturation of bovine oocytes[J]. Theriogenology,1994, 41:383～394
[127] Naito K, Fukuda Y, Toyoda Y. Effect of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro[J]. Gamete Research, 1988, 21: 289～295
[128] Rath D, Niemann H, Tao T. In vitro maturation of porcine oocytes in follicular fluid with subsequent effects on fertilization and embryo yield in vitro[J]. Theriogenology, 1995, 44: 529～538
[129] 刘红林，汪河海，范必勤等．猪卵母细胞的体外成熟[J]．江苏农学院学报，1994，15(3)：37～41
[130] 秦鹏春，谭景和，吴光明等．猪卵巢卵母细胞体外成熟与体外受精的研究[J]．中国农业科学，1995，28(3)：58～66
[131] Marchal R, Feugang JM, Perreau C, et al. Meiotic and developmental competence of prepubenal and aduh swine oocytes[J]. Theriogenology, 2001,56: 17～29
[132] 孟庆刚，张成林，张永忠等．猪小腔卵泡卵母细胞体外成熟研究[J]．畜
牧兽医学报, 2001, 32(3): 213-219
[133] Rath D, Niemann H, Tao T. In vitro maturation of porcine oocytes in follicular fluid with subsequent effects on fertilization and embryo yield in vitro[J]. Theriogenology, 1995, 44: 529—538
[134] 陈大元.受精生物学[D].北京科学出版社, 1999, 154-155
[135] Sirard MA. Effects of fresh or cultured follicular follicular fractions on meiotic resumption in bovine oocytes. Theriogenology, 1992,37:39—47
[136] Moor RM. Translational capacity of sheep oocytes microinje- cted with messenger RNA [J].Reprod Fert, 1989, 86:289—295
[137] Tan SJ, Lu KH. Effects of different oestrous cycle stages of ovaries and sizes of follicles on generation of IVF early embryos. Theriogenology 2001, 33:335(abstract)
[139] Mccaffrey C et al. Factors involved in the in vitro developmeni of IVF cattle ova. Proceeding of the Iirish orassland and Animal Production Association[J], 18[th] Annual Research meeting. Dubbin:0' kiely 1992, 33-34
[140] Lonergan P. Effect of follicle size on bovine oocyte quality and developmenial competence following maturation, fertilization, and culture in vitro[J].Mol Reprod dev, 1994 37:48—53
[141] Lim JM, Pukui Y, Ono H. Developmental competence of bovine oocytes frozen ant various maturation stages followed by in vitro maturation and fertiliZation[J]. Theriogenology, 1992, 37:351-361
[142] Albina JS, Lisa M. Nitric oxideis essential for optimal meiotic maturation of murine cumulus oocyte complexesinvitro[J]. Mol Reprod Dev, 2000, 55 241—421
[143] Kazuo S , Naoyuki T. Requirement of nitric oxide for murine oocyte maturation , embryo development and tropho blastout growth in vitriol [J] .Mol Reprod Dev, 2001, 58 262-268
[2]付永伦．人类卵母细胞的冷冻保存[J]．生殖与避孕，1996，3：23～36
[3]Vanuem JF, Schuh B, Koch, S. et al. Birth after cryopreservation of unfertilized oocytes[J], lancet, 2001, 2:752～753
[4]Byrd. Cryopreservation, thawing and transfer of human Embryos[J]. Seminars in Reproductive Medicine, 2002,20(1):37～43
[5]Boiso I, Marti M, Santalo J, et al.A confocal microscopy analysis of the spindle and chromo some configurations of human oocytes cryopreservated at the germinal vesicle and metaphaseⅡ stage[J].Human Reproduction, 2002,17(7):1885～1891
[6]Rienzi L, Martinez F, Ubaldi F, et al. Polscope analysis of meiotic spindle changes in living metaphase Ⅱ human oocytes during the freezing and thawing procedures[J].Hum Reprod, 2004,19(3):655～659
[7]Men H, Monson RL, Parrish JJ, et al. Detection of DNA damage in bovine metaphase Ⅱ oocytes resulting from cryopreservation[J]. Mol Reprod Dev, 2003,64: 245～250
[8]Mandelbaum J, Anastasiou O, Levy R. Effects of cryopreservation the meiotic spindle of humanoocytes[J].Eur J Obstet Gynecol Reprod Biol, 2004, 113:17～23
[9]Obersrein, N. Cryopreservation of equine embryos by open pulled straw. Cryoloop, or conven-tional slow cooling metholds[J]. Therigenology, 2001,55:607～613
[10]Rall WF Fahy GM. Ice-free cryopreservation of mouse embryos at-196℃ by vitrification[J]. Nature 1985, 313:573～575
[11]Mukaida T, Nakamura S, Tomiyama T. et al. Vitrification of human blastocysts using cryoloops, clinical outcome of 223 cycles[J]. Hum Reprod. 2003, 18:384～391
[12] Lane M,Schoolcraft W.B, and Gardner D.K. Vitrification of mouse and human blastocysts using a novel cryoloop technique[J].Fertil. 1999, 72:1073～1078
[13] Chen SU, Lien YR, Cheng YY, et al. Vitrification of mouse oocytes using closed pulled straw(CPS) achieves a highsurvival and preserves good patterns of meiotic spindles, compared with conventional straws, open pulled straws(OPS) and grids[J].Hum Reprod, 2001,16(11):2350～2356
[14] 朱士恩，曾申明，张忠诚．家兔扩张囊胚玻璃化冷冻保存技术的研究[J]．中国养兔杂志，1997，2：16～20
[15] 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[J]．中国农业科学，2002，35(6)：700～704
[16] Kong K.L, Lee S.L, Cho S.K. et al. Comparison of open pulled straw (OPS) VS glass micropipette (GMP) vitrification in mouse blostocysts[J]. Theriogenology,2000,53:1817～1826
[17] Kasai M, Mukaida T. Cryopreservation of animal and human embryos by vitrification[J].Reprod biomed online, 2004, Aug, 9(2): 64～70
[18] Li xh. Study and application of humam oocyte cryopreservation[J]. Beijing Da Xue Xue Bao, 2004, Dec, 36(6):664～667
[19] Papis. K, Arery P, Holm P, et al. The effect of equilibrated or vitrified bovine in vitro matured oocytes[J].Theriogenology, 1995, 43: 293～297
[20] Papis k.,Shimizu M, Iznike Y. Factors affecting the survivability of bovine oocyte vitrified in droplet[J].Theriogenology,2000, 54: 651～658
[21] Misumi k, Suzuki M, Sato S, et al. Successful production of microdrop method [J].Teriogenology,2003,60:253～260
[22] Lane M, Lyons EA, Bavister BD, et al. cryopreservation reduce the ability of hamster 2-cell embryos to regulate in tracellular PH(J).Hum Reprod, 2000,15:389～394
[23] Kouji Misumi, Misae Suzuki, et al. Successful production of piglets derived from vitrified morulae and early blastocysts using a microdroplet method[J]. Theriogenology,2003, 60:253～260
[24] Jiang JY, Umezu M, Sato E. Vitrification of two-cell rat embryos derived from immature hypothyroid rdw rats by in vitro fertilization in ethylene glycol-based solutions[J].Cryobiology, 1999,38:160～164
[25] S.P. Leibo. Cryopreservation of gamctes and embryos of non-domesticspecie [J]. Theriogenology,2002,57:303～326
[26] S.Y. Tomita. The use of in vitro produced cryopreserved for direct transfer to induce twinning in beef cattle [J].Theriogenology, 2002, 57:484-486
[27] Rall WF, Fahy GM. Ice-Free cryoperservation of mouse embryos at-196℃ by vitrification [J].Nature, 1985,313:573～575
[28] Kasai M, Komi JH, Takakamo A, et. al.A simple method for mouse embryo cryopreservation in a low toxicity vitrification solution, without appreciable loss of viability [J]. Reprod Fertil, 1990,89(1):91～97
[29] Vanderzwalmen P, Bertin G, Debauche CH. Births after vitrification at morula and blastocyst stages:effect of artificial reduction of the blastocoelic cavity before vitrification. Hum Reprod,2002, Mar, 17(3):744～751
[30] Chen SU, Lien YR, Chen HF, Chang LJ, Tsai YY, Yang YS. Observational clinical follow-up of oocyte cryopreservation using a slow-freezing method with 1,2-propanediol plus sucrose followed by ICSI. Hum Reprod 2005 Mar 24 [Epub ahead of print]
[31] Mukaida T, Takahashi K, Kasai M. Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. Hiroshima Hart Clinic, 2005,34(6):7～10
[32] 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
[33] 刘万勃等．抗冻蛋白的研究进展[J]．生物学杂志，2000，17(8)：7～8
[34] Tomczak MM, Hincha DK, Estrada SD, et al. Antifreeze proteins differentially affect model membranes during freezing. Biochim Biophys Acta, 2001, 1511 (2):255～263
[35] 钟其旺，樊廷俊．鱼类抗冻蛋白的研究进展[J]．生物化学与生物物理学报，2002，34(2)：124～130
[36] Rubinsky B, Arav A, Fletcher GL. Hypothermic Protection-a fundamental property of "antifreeze" proteins. Biochem Biophys protecting liposome membrane integrity depends on phospholipid class. Biochim Biophys Acta, 2001,1511(1):11～16
[37] Luvoni G.C, Pellizzari P. Embryos development of in vitro-matrued cat oocytes cryopreserved by lown and ultrarapid freezing non-domesticcspecie [J].Theriogenology, 2000, 53:260～265
[38] Nagashima H, Kuwayam M, Grupen CG. Vitrification of Porcine early cleavage stage embryos and oocytes after removal of cytoplasmic lipid dropts[J].Theriogenology, 1996, 45:180～185
[39] Lim JM, Fukui Y, Ono H Developmental conpetence of bovine oocytes frozen at various matration stages followed by in vitro matratoin and fertilization[J].Theriogenology,1992, 37:351～371
[40] Lim JM, Fukui Y, Ono H.The post-thaw developmental capacity of frozen bovine oocytes following in vitro maturation and fertilization[J]. Theriogenology, 1991, 35:1225～1235
[41] Lim JM, Ko JJ, Hwang WS, et al. Development of invitro matured bovine oocytes after cryopreservation with different cryorotectants[J]. Theriogenology,1999, 51:1303～1310
[42] Hongsheng Mcn, Rick L. Monson, Jack J Rutledgc. Effect of meiotic stages and maturation Protocols on bovine oocytes resistance to cryopreservation [J]. Theriogenology, 2002, 57:1095～1103
[43] 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
[44] Imoedemhe GG, sugu AB. Suvival of human oocytes cryopreserved with or without the cumulus in 1, 2-Propandeil[J].Assist. Reprod Genet, 1992, 323—327
[45] Gook C. A, Osborm S. M, Johnston W. I. H. Cryopreservation of mouse and human oocytes using 1, 2 Propanedieland the configuration of the meiotic spidle[J].Hum Repro, 1993, 8:1101 — 1109
[46] Ashwood smith. MJ, Morris GW, Fowler R, et al. Physical factors are involved in the destruction of embryos and oocytes during freezing and thawing Procedure[J]. Hum Repro ,1988, 3:795—802
[47] Balou Jonathan D. Potential contribution of cryoperserved germ Plasm to the Preservation of genetic diversity and conservation of endanged species in captivity [J]. Cryobiology, 1992, 29:19—25
[48] Li Z, Jiang Q, Zhang Y, et al. In vitro maturation of ferret oocytes [J]. Theirogenology, 2001, 55 : 482—485
[49] Whittingham DG, Fertilization in vitro and developments to term of unfertilized mouse oocytes Previously stored at -196°C [J]. Reprod Fertil. 1977, 49:89—94
[50] Chen C. Pregnancy after human oocyte cryopreservation[J]. Lancet, 1986, 2:884—886
[51] Fuku E, Kojima T, Shioya Y, et al. In vitro fertilization and development of frozen-thawed bovine oocytes[J]. Cryobiology, 1992, 29:485—492
[52] Vajta G, Holm P, Kuwayama M, 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
[53] Suzuki T, Nishikata Y. Fertilization and cleavage of frozen thawed bovine oocytes by one step dilution method in vitro[J]. Theriogenology, 1992, 37:306 abstr
[54] Yoshida M. Effects of genadotropins and estradiol-17β on the timing of nuclear maturation and cumulus mass expansion in Pig oocyte cultured in vitro. Japan. [J].Anim Reprod. 1989, 35: 86—91
[55] Naito K. Fukuda Y, Effects of Porcine follicular fluid on male Pronuclelus formation in Porcine oocyte matured in vitro[J].Gamete Research, 1988, 21:289～295
[56] Mattioli M. Follicular factors influence oocyte by modulating the intercellular cooperation between cumulus cells and oocyte[J].Gamete Res. 1988, 21:223～232
[57] Eng A, et al. Effect of incubation temperature and bicarbonate on maturation of Pig oocytes in vitlro[J].Reprod Fertil. 1988, 76:657～662
[58] Martino A., Palomo MT, Mogas T, et aL. Influence of the collection technique of Prepubertal goat oocytes on. in vitro maturation and fertilization[J].Theriogenology, 1994, 42:859～873
[59] 陈大元主编．受精生物学-受精机制与生殖工程[M]．科学出版社．2000
[60] 张绣．牛卵母细胞激活体外受精[D]．西北农林科技大学2002届攻读硕士学位研究生学位论文．2002
[61] Downs SM. Specifcity of epidermal gowth factor action on maturation of the murine oocytes and cumulus oophorus in vitro[J].Boil Reprod, 1989, 41:371～379
[62] De Loos, F. Maurck P. et al. Structural aspects of bovine oocytes maturation in vitro[J].Mol Reprod. Dev, 1989, 31:208～214
[63] Lonergan P, Vergos. The effect of recovery method on the type of bovine oocyte obtained for in vitro maturation[J].Theriogenology, 1991, 35:231～234
[64] 冯怀亮，孙青原，李子义等，牛体外成熟卵母细胞冷冻保存的研究[J]．畜牧兽医学报，1995，26(3)：481～486
[65] Gook DA, Osbom SM, Johson WI. Cryopreservation of mouse and human oocytes using 1-2 Propandiol and the configuration of the meiotic spindle[J].Hum Reprod. 1993, 8:1101～1109
[66] 薛庆善主编．体外培养的原理与技术[M]．北京科学技术出版社，2001，2(1)，342～345
[67] Fukui Y, Fukuishima M, Terawaki Y, et al. Effects of gonadotropins, steroids and culture media on bovine oocytes maturation in vitro[J]. Theriogenology, 1989, 18:161～175
[68] Mattioli M, Bacci G, Seren E. Effects of LH and FSH on the maturation of Pig oocytes in vitro[J]. Theriogenology,1991, 36:95～105
[69] 卢晨盛，卢可焕．不同血清种类及其浓度对牛卵母细胞体外受精的影响[J]．广西农业大学学报，1994，13(1)：21～26
[70] Fukui Y, Ono H. Effects of sera, hormones and granulose cells added to culture medium for in-vitro maturation, fertilization, cleavage and development of bovine oocytes[J].Reprod Fertil. 1989, 86(2):501～506
[71] 旭日干，张锁链，薛晓先等．屠宰母牛卵巢卵母细胞的体外受精与早期发育[J]．内蒙古大学学报(自然版)，1989，20(3)：407～414
[72] Mochizuki H, Fukui Y, Ono H. Effects of the number of granulose cells added to culture medium for in vitro maturation, fertilization and development of bovine oocytes [J].Theriogenology, 1991, 36:973～986
[73] Otoi T, Fujii M, Tanaka M, et al. Effect of serum on the in vitro maturation of canine oocytes[J], Reprod Fertil. Dev, 1999, 11(7):387～390
[74] Funahashi H, Day BN. Effects of different serum supplement in maturation medium on meiotic and cytoplasmic of Pig Ooeytes[J]. Theriogenology,1993, 39:965～973
[75] Wang S, Liu Y, Holyosak GR, et al. Effects of supplement with fluids from bovine follicles of varied sizes in in vitro maturation on subsequent embryo development in vitro[J].Theriogenology, 1999,51:394～397
[76] Choi YH, Takagi M, Kamishita H, et al. Development capcity of bovine oocytes matured in two kinds of follicular fluid and fertilized in vitro[J].Anim Rprod Sci, 1998, 50(1):27～33
[77] Sirard MA, Biledeau R. Granulosa cells inhibit the resumption of meiosis in bovine Oocyte in vitro[J].Biol Reprod, 1990, 43:777～783
[78] Erickon RP. Recent advances in developmental genetics:Growth factors and morphogens[J].Mol Reprod. Dev. 1995, 41:109～125
[79] Crupen CG, Nottle MB.A simple modification of the in vitro fertili- zation Procedure improves the effciency of in vitro Pig embryo Producion[J].Theriogenology, 2000, 53:422 abstr
[80] Prochazka R, Srsen V, Nagyora E, et al. Development regulation of effect of epiderrnal growth factor on Porcine oocyte-cumulus cell complexes nuclear maturation, expansion and Fact in remodeling[J]. Mol Reprod, Dev. 2000, 56:63～73
[81] Abeydeera LR, Wang WH, Cantley TC et al. Epidermal growth factor can enhance the developmental competence of Pig oocytes matured in vitro under Protein-free culture conditions[J].Theriogenology,2000, 54:787～797
[82] Cameiro G, Lorenzo P, Pimentel C, et al.Influence of insulin-like growth factor-1 and its interaction with gonadotropins, estradiol, and fetal calf serum on in vitro maturation and parthenogenic development in equine oocytes[J].Biol of Reprod, 2001,65(3):899～905
[83] Ayoub MA，Hunter AG著，朱玉林译，牛卵泡液对牛卵母细胞体外成熟的抑制作用[D]．草食家畜，1995，89(4)：34～37
[84] Wang S, Liu Y, Holyosak GR, et al. Effects of supplementation with fluids from bovine follicules of varied sizes in in vitro maturation on subsequent embryo development in vitro[J].Theriogenology, 1999, 51:394～398
[85] Choi YH, Takagi M, Kamishita H, et al. Development capacity of bovine oocytes matured in two kinds of follicular fluid and fertilized in vitro[J].Anim Reprod Sci, 1998, 50(1):27～33
[86] Sirard MA, Bilodeau S. Granulosa cells inhibit the resumption of meiosis in bovine oocyte in vitro[J]. Biol Reprod, 1990, 43:777～783
[87] Sun FJ, Holm P, Lrvine B, et al. Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro[J].Theriogenology, 1994, 41:981～988
[88] Georgios, Vatzias and Daniel R. Hagen Effects of Porcine follicular fluid and oviduct-conditioned on maturation and fertilization of Porcine oocytes in vitro[J].Boil of Reprod. 1999, 60:424～448
[89] 钱云，师蔚进，朱猛进等．卵泡内环境对猪卵泡卵体外成熟和发育的影响[J]，生命科学研究．2001，21(6)：624～626
[90] Eppig JJ. Mouse oocyte development in vitro with Various culture systems[J].Dev. Boil of Reprod. 1997, 60:371～388
[91] Chesnel F, Wigglesworth K. Acquisition of meiotic competence by denuded mouse oocytes: Participation of somatic-cell Products and camp [J].Dev. Biolof Reprod. 1994, 161:285～295
[92] Mochizuki H, Fukui Y. Ono H. Effect of the number of granulosa cells added to culture medium for in vitro maturation and development of bovine oocytes[J].Theriogenology, 1991, 36:973～986
[93] Fukui Y. Mcgowan L T. James R W. et al. Factors affecting the in vitro development to blastocysts of bovine oocytes matured and fertilized in vitro[J]. Reprod. Fert. 1991, 92:125～131
[94] Coskun S, SanbuisshaA, Liu YC, et al. Fertilizability and subsequent development ability of bovine oocytes matured in medium containing EGF[J]. Theriogenology,1991, 36:485～494
[95] Bavister BD, Rose-Hellekant TA, pinyopummintr T. Development of in vitro matured/ in vitro fertilized bovine embryos into morulae and blastocysts in defined culture media[J].Theriogenology, 1992, 37:127-146
[96] Saeki K, Hoshi M, Leibfried-Rutledge ML, et al. In vitro fertilization of bovine in a Protein-free medium and subsequent development to the blastocyst stage[J].Theriogenology, 1993, 39:302 abstr
[97] 张涌，刘泽隆，李欲强．山羊卵泡卵母细胞体外成熟的研究[J]，西北农业大学学报，1999，27(1)：14～18
[98] Dorado CM, Mclana LM, Miller JA, et al. Nuclear status of bovine oocyte exposed to elevated temperature after resumpte of meiosis[J]. Theriogenology, 2001, 55:469 abstr
[99] Azambuja RM, De Moreno JF, Ktaemer D, et al. Effect of gas at mosphere on in vitro maturation of bovine oocytes[J].Theriogenology, 1993, 39:184-187.
[100] Lee ES, Yoon KW, Lim J, et al. Effect of gas composition during in vitro maturation and culture on in vitro development of Porcine follicular oocytes[J].Theriogenology, 2001, 55(1):480 abstr
[101] Bing YZ, Hirao Y, Takenouchi N, et al. In vitro maturation of denuded Porcine oocytes in the Presence of cysteamine under the conditions of different oxygen environments[J].Theriogenology, 2002, 57:713 abstr
[102] Albina JS, Lisa M, Olson LM, et al. Nitric oxide is essential for optimal meiotic maturation of murine cumlus-oocyte complexes in vitro [J]. Mol Repro. Dev, 2000, 55:412～421
[103] Kazuo S, Naoyuki T. Requirement of nitric oxide for murine oocyte maturation, embryo development and trophoblast out-growth in vitro[J]. Mol Reprod Dev, 2001, 58:262～268
[104] 孙青原，刘国艺，徐立滨等．牛卵泡卵母细胞冷冻保存后发育潜力的研究[J]．中国兽医学报1994，14(4)：34～2345
[105] 孙青原．牛卵母细胞体外成熟、冷冻及受精的研究[D]．东北农业大学博士学位论文，1994．
[106] Hochi S, Ito K, Hirabayashi M, et al. Effect of nuclear stages during IVM on the Survival of vitrified-warmed bovine oocytes[J]. Theriogenology, 1998,49(4):787～796
[107] Lim JM, Fukui Y, Ono H. Dvelopmental competence of bovine oocytes frozen at various maturation stages followed by in vitro maturation and fertilization[J].Theriogenology,1992;37:351～361
[108] 刘海军．山羊卵母细胞冷冻保存研究[D]。西北农林科技大学2000届博士学位研究生论文．
[109] 刘海军，侯蓉，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
[110] Jisachenko V, Isachenkoe, Michelmann HW, et al. hipoysis and ultrastructural changes of intilcellular lipid vesicles after cooling of bovine and porcine GV-oocytes[J].Anat Histol Embryol, 2001,30(6):333～338
[111] Hefler LA Gregg AR. Inducible and endothelial nitric oxide synthase:Genetic bakeground affects ovulation in mice [J]. Mol Reprod, Dev, 2000, 55:412～421
[112] Tao Y, Fu Z, Zhang M. Immunohistochemical localization of inducible and endothelial nitric oxide synthase in porcine ovaries and effects of NO on antrum formation and oocyte meiotic maturation [J].Mol Cell Endocrinol, 2004,222(2):93～103
[113] MotikJ, Crozet N, Fulka J. Meiotic competence in vitro of pig oocytes isolated from early antral follicles[J]. Reprod Fertil, 1984, 72:323～328
[114] Wickramasinge D, Ebert KM, Albertini DF. Meiotic competence acquisition is associated with the appearance of M-Phase characteristics in growing mouse oocytes[J].Dev Biol.1991, 143:162～172
[115] Sehramr RD, Tennier MT, Boatman DE, Bavister BD. Chromatin configurations and meiotic competence of oocytes are related to follicular diameter in nonstimulated rhesus monkeys[J].Biol Reprod, 1993, 48:349～356
[116] De Smedt V, Crozet N, Gall L. Morphological and functional changes accompanying the acquisition of meiotic competence of goat ovarian oocyte[J]. Theriogenology, 2001,269:128～139
[117] 蔡令波．猪卵母细胞体外成熟与冷冻保存的研究[D]．南京农业大学硕士论文，2002．6
[118] Otoi T, Yamamoto K, Koyarna N, et al. Cryopreservation of mature bovine oocytes by vitrification in Straws. Cryobiology[J], 1998,37(1):77～85
[119] Papis K, Shimizu M, Izaike Y. Factors affecting the survivability of bovine oocytes vitrified in droplets[J].Theriogenology, 2000, 54: 651～658
[120] Vajta G, Holm P, Greve T, et al. Vitrification of porcine embryos using the Open Pulled Straw(OPS) [J]. Theriogenology, 1997,38:349～352
[121] Beginl, Bhatia B, Baidassajrre H, et al. Cryopreservation of goat oocytes and in vivo derived 2-to 4-cell embryos using the cryoloop(CLV) and Solid-surfce vitrification(SSV)methods[J].Theriogenology, 2003, 59:1839～1850
[122] Vajta G, Holm P, Kuwayama M, et al. Open Pulled Straw(OPS) vitrification:a new way to reduce cryo injuries of bovine ova and embryos[J].Mol Reprod, Dev, 1998, 51:53～58
[123] Luvoni. GC, Pellizzari P. Embryos development of in vitro-matrued cat oocytes cryopreserved by lown and ultrarapid freezing[J]. Theriogenology, 2000,53:260～267
[124] Hyttel P, Vajtao, Callesen H. Vitrification of bevine oocytes with the open Pulled straw method:Ult Structural consequences[J].Mol Reprod, Dev, 2000,56(1): 88～91
[125] 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[J]，中国农业科学，2002，35(6)：7007～7014
[126] Remero-Arredondo A, Seidel FE, Jr. Effects of follicular fluid on maturation of bovine oocytes[J]. Theriogenology,1994, 41:383～394
[127] Naito K, Fukuda Y, Toyoda Y. Effect of porcine follicular fluid on male pronucleus formation in porcine oocytes matured in vitro[J]. Gamete Research, 1988, 21: 289～295
[128] Rath D, Niemann H, Tao T. In vitro maturation of porcine oocytes in follicular fluid with subsequent effects on fertilization and embryo yield in vitro[J]. Theriogenology, 1995, 44: 529～538
[129] 刘红林，汪河海，范必勤等．猪卵母细胞的体外成熟[J]．江苏农学院学报，1994，15(3)：37～41
[130] 秦鹏春，谭景和，吴光明等．猪卵巢卵母细胞体外成熟与体外受精的研究[J]．中国农业科学，1995，28(3)：58～66
[131] Marchal R, Feugang JM, Perreau C, et al. Meiotic and developmental competence of prepubenal and aduh swine oocytes[J]. Theriogenology, 2001,56: 17～29
[132] 孟庆刚，张成林，张永忠等．猪小腔卵泡卵母细胞体外成熟研究[J]．畜
牧兽医学报, 2001, 32(3): 213-219
[133] Rath D, Niemann H, Tao T. In vitro maturation of porcine oocytes in follicular fluid with subsequent effects on fertilization and embryo yield in vitro[J]. Theriogenology, 1995, 44: 529—538
[134] 陈大元.受精生物学[D].北京科学出版社, 1999, 154-155
[135] Sirard MA. Effects of fresh or cultured follicular follicular fractions on meiotic resumption in bovine oocytes. Theriogenology, 1992,37:39—47
[136] Moor RM. Translational capacity of sheep oocytes microinje- cted with messenger RNA [J].Reprod Fert, 1989, 86:289—295
[137] Tan SJ, Lu KH. Effects of different oestrous cycle stages of ovaries and sizes of follicles on generation of IVF early embryos. Theriogenology 2001, 33:335(abstract)
[139] Mccaffrey C et al. Factors involved in the in vitro developmeni of IVF cattle ova. Proceeding of the Iirish orassland and Animal Production Association[J], 18[th] Annual Research meeting. Dubbin:0' kiely 1992, 33-34
[140] Lonergan P. Effect of follicle size on bovine oocyte quality and developmenial competence following maturation, fertilization, and culture in vitro[J].Mol Reprod dev, 1994 37:48—53
[141] Lim JM, Pukui Y, Ono H. Developmental competence of bovine oocytes frozen ant various maturation stages followed by in vitro maturation and fertiliZation[J]. Theriogenology, 1992, 37:351-361
[142] Albina JS, Lisa M. Nitric oxideis essential for optimal meiotic maturation of murine cumulus oocyte complexesinvitro[J]. Mol Reprod Dev, 2000, 55 241—421
[143] Kazuo S , Naoyuki T. Requirement of nitric oxide for murine oocyte maturation , embryo development and tropho blastout growth in vitriol [J] .Mol Reprod Dev, 2001, 58 262-268