牛卵母细胞的体外成熟以及活性化处理对孤雌发育的影响
|
摘要
本研究分析了牛卵巢保存温度、外源激素（LH、17β-E2）和不同培养时间对牛卵母细胞体外成熟的影响。结果表明，卵巢保存温度为25～29℃、30～35℃、36～40℃时，牛卵母细胞的体外成熟率分别为55.3%、70.1%、60.7%。经统计分析牛卵巢保存温度对牛卵母细胞体外成熟的影响不大（P >0.05）；成熟培养基中未添加激素组、只添加17β-E2和添加LH+17β-E2组时，成熟率分别为57.8%、64.3%和70.1%。经统计分析添加LH+17β-E2与未添加激素组差异显著（P <0.05）；牛卵母细胞体外培养不同时间22～25 h、26～30 h、31～34 h时，牛卵母细胞的体外成熟率分别为56.3%、65.4%和68.4%，三组间差异不显著（P >0.05）。
本研究还对成熟分裂中期（MⅡ）的牛卵母细胞分别用不同浓度的乙醇和不同浓度钙离子载体A23187进行处理，观察其激活率；筛选出激活卵子最佳乙醇浓度和A23187浓度，再与亚胺环己酮复合活性化处理，观察其激活率和孤雌发育能力。结果表明，用不同浓度的乙醇5%、7%、9%、11%、13%活性化处理后，所得的激活率分别为41.2%、47.2%、53.1%、39.7%、23.4%。经统计分析7%和9%组与其他组差异显著（P<0.05），两组之间差异不显著（P >0.05），因此选用乙醇浓度为9%组做下一步试验；用不同浓度（2.5 μM、5μM、7.5 μM）的钙离子载体A23187活性化处理后，所得的激活率为分别为29.3%、51.8%、45.4%。钙离子载体A23187浓度 5μM与7.5μM组分别与2.5μM组差异显著（P<0.05），但5μM和7.5 μM组之间差异不显著（P>0.05），因此选用5 μM组做进一步实验；9%的乙醇和5 μM
的钙离子载体单一活性化处理和分别与亚胺环己酮复合活性化处理时，所得的单一活性化处理（53.1%，51.8%）低于复合活性化处理的激活率（77.3%，76.8%），经统计分析差异显著（P<0.05），并且乙醇单一活性化处理和复合活性化处理时2PN形成率为44.1%和72.1%，差异显著（P<0.05）；A23187单一活性化处理和复合活性化处理时2PN形成率为40.2%和 72.9%，差异显著（P<0.05）。9%的乙醇和5μM的钙离子载体分别与亚胺环己酮复合活性化处理卵子后，进行了孤雌发育能力的比较，卵子发育为2-8细胞的胚胎占71.0%和62.9%，两种处理方法差异不显著（P>0.05）。
The effects of preserving ovary temperature, exogenous hormone（LH、17β-E2）, and various culturing time （22-25h,26-30h, and 31-34h）to the bovine oocytes maturating in vitro were analyzed in this research . The outcome suggested that , the effects of bovine ovary which maturated in vitro preserving temperature（25-29℃,30-35℃, and 36-40℃） to oocytes were not very clear（P>0.05）,after statistically analyzed, and the bovine oocytes maturating rates were 55.3%,70.1%,and 60.7%,respectively;the maturity rates of the bundles which were not added hormone, added 17β-E2 , and added LH+17β-E2 were 57.8%,64.3% and 70.1% respectively, and the difference between the bundle which was added LH+17β-E2 and the bundle not added hormone was significance（P<0.05）,after statistically analyzed; the mature rate of bovine oocytes which had been cultured in vitro for 31-34h was 68.4%, which was higher than the two bundles which had been cultured for 22-26h(56.3%) and 27-30h (65.4%) respectively, but the differences among the three bundles were not significance（P>0.05）,after statistically analyzed.
Bovine oocytes which were in maturation mephase （MⅡ）were treated by various concentration of ethanol (5%, 7%, 9%, 11%, and 13%) or calcium ionophore A23187 （2.5μM , 5μM, and 7.5μM）in this research , and observed its activating rate. The best concentrations of ethanol and A23781, which were required to activate ova ,were screened . The bovine oocytes were treated by the compound activity of cycloheximide , and observed their activity rates and the ability of parthenogenesis.The outcome suggested that , the bundles which contained the ethanol 7%, and 9% had differences with other bundles ,and the difference between the two bundles was not significance （P>0.05）
after statistically analyzed .But the activity rate (53.1%) of the bundle which contained 9% ethanol was higher than that of the bundle which contained 7% ethanol , so the bundle which contained 9% ethanol was chosen in next step . The difference between the two bundles whose calcium ionophore A23187 concentrations are 5μM or 7.5μM was not significance （P>0.05）, but the difference between the two bundles and the bundles whose concentration was 2.5μM was significance （P<0.05）after statistically analyzed, and the activity rate (51.8%) of the bundle whose concentration was 5μM was chosen in next step. Not only was the difference between the single activating treatment and compound activating treatment which contained 9% ethanol with cycloheximide or 5μM calcium ionophore significance, but also the activating rates (77.3%, 78.8% ,respectively) of compound treatment was higher than that of single treatment (53.1%, 51.8% ,respectively), and the difference between the formation rates of 2PN which were treated by single activating treatment and that of the 2PN treated by compound activating treatment was significance （P<0.05）(40.2%,72.9% respectively) after statistically analyzed. The ova which had been disposed by 9% ethanol with cycloheximide were compared in the ability of parthenogenesis ,and the embryos which were developed to 2-8 cells were 71.0% , 62.9% respectively, but the difference between two ways was not significance （P>0.05）,after statistically analyzed.
本研究还对成熟分裂中期（MⅡ）的牛卵母细胞分别用不同浓度的乙醇和不同浓度钙离子载体A23187进行处理，观察其激活率；筛选出激活卵子最佳乙醇浓度和A23187浓度，再与亚胺环己酮复合活性化处理，观察其激活率和孤雌发育能力。结果表明，用不同浓度的乙醇5%、7%、9%、11%、13%活性化处理后，所得的激活率分别为41.2%、47.2%、53.1%、39.7%、23.4%。经统计分析7%和9%组与其他组差异显著（P<0.05），两组之间差异不显著（P >0.05），因此选用乙醇浓度为9%组做下一步试验；用不同浓度（2.5 μM、5μM、7.5 μM）的钙离子载体A23187活性化处理后，所得的激活率为分别为29.3%、51.8%、45.4%。钙离子载体A23187浓度 5μM与7.5μM组分别与2.5μM组差异显著（P<0.05），但5μM和7.5 μM组之间差异不显著（P>0.05），因此选用5 μM组做进一步实验；9%的乙醇和5 μM
的钙离子载体单一活性化处理和分别与亚胺环己酮复合活性化处理时，所得的单一活性化处理（53.1%，51.8%）低于复合活性化处理的激活率（77.3%，76.8%），经统计分析差异显著（P<0.05），并且乙醇单一活性化处理和复合活性化处理时2PN形成率为44.1%和72.1%，差异显著（P<0.05）；A23187单一活性化处理和复合活性化处理时2PN形成率为40.2%和 72.9%，差异显著（P<0.05）。9%的乙醇和5μM的钙离子载体分别与亚胺环己酮复合活性化处理卵子后，进行了孤雌发育能力的比较，卵子发育为2-8细胞的胚胎占71.0%和62.9%，两种处理方法差异不显著（P>0.05）。
The effects of preserving ovary temperature, exogenous hormone（LH、17β-E2）, and various culturing time （22-25h,26-30h, and 31-34h）to the bovine oocytes maturating in vitro were analyzed in this research . The outcome suggested that , the effects of bovine ovary which maturated in vitro preserving temperature（25-29℃,30-35℃, and 36-40℃） to oocytes were not very clear（P>0.05）,after statistically analyzed, and the bovine oocytes maturating rates were 55.3%,70.1%,and 60.7%,respectively;the maturity rates of the bundles which were not added hormone, added 17β-E2 , and added LH+17β-E2 were 57.8%,64.3% and 70.1% respectively, and the difference between the bundle which was added LH+17β-E2 and the bundle not added hormone was significance（P<0.05）,after statistically analyzed; the mature rate of bovine oocytes which had been cultured in vitro for 31-34h was 68.4%, which was higher than the two bundles which had been cultured for 22-26h(56.3%) and 27-30h (65.4%) respectively, but the differences among the three bundles were not significance（P>0.05）,after statistically analyzed.
Bovine oocytes which were in maturation mephase （MⅡ）were treated by various concentration of ethanol (5%, 7%, 9%, 11%, and 13%) or calcium ionophore A23187 （2.5μM , 5μM, and 7.5μM）in this research , and observed its activating rate. The best concentrations of ethanol and A23781, which were required to activate ova ,were screened . The bovine oocytes were treated by the compound activity of cycloheximide , and observed their activity rates and the ability of parthenogenesis.The outcome suggested that , the bundles which contained the ethanol 7%, and 9% had differences with other bundles ,and the difference between the two bundles was not significance （P>0.05）
after statistically analyzed .But the activity rate (53.1%) of the bundle which contained 9% ethanol was higher than that of the bundle which contained 7% ethanol , so the bundle which contained 9% ethanol was chosen in next step . The difference between the two bundles whose calcium ionophore A23187 concentrations are 5μM or 7.5μM was not significance （P>0.05）, but the difference between the two bundles and the bundles whose concentration was 2.5μM was significance （P<0.05）after statistically analyzed, and the activity rate (51.8%) of the bundle whose concentration was 5μM was chosen in next step. Not only was the difference between the single activating treatment and compound activating treatment which contained 9% ethanol with cycloheximide or 5μM calcium ionophore significance, but also the activating rates (77.3%, 78.8% ,respectively) of compound treatment was higher than that of single treatment (53.1%, 51.8% ,respectively), and the difference between the formation rates of 2PN which were treated by single activating treatment and that of the 2PN treated by compound activating treatment was significance （P<0.05）(40.2%,72.9% respectively) after statistically analyzed. The ova which had been disposed by 9% ethanol with cycloheximide were compared in the ability of parthenogenesis ,and the embryos which were developed to 2-8 cells were 71.0% , 62.9% respectively, but the difference between two ways was not significance （P>0.05）,after statistically analyzed.
引文
[1] Campbell K H S et al,1996.Nature,380:64～66
[2] Wilmut I et al,1997.Nature,385:310～313
[3] 王晨光，邓满齐，孙方臻等，小鼠卵母细胞体外成熟不同阶段对钙的依赖性。实验生物学报，1998，31（2）：147～150
[4] Tsafriri A,Channing C P.Influence of follicular.maturation and culture conditions on the meiosis of pig oocytes in vitro.J Reprod Fert,1975,43:149～152
[5] Motlik J,Crozet N,Fulka J.Meiotic competence in vitro of pig oocytes isolated from early antral follicles.J Reprod Fert,1984,72:323～328
[6] 刘素娟，张涌，李勇等，山羊小腔卵泡卵母细胞的体外成熟。河北农业大学学报，1998，21（2）：66～69
[7] 柏家林，关红梅，牛卵母细胞体外成熟和体外受精技术的研究进展1998，25（4）：27～31
[8] Minamihashi,A.,Watson,A.J.,Watson,P.H.,Church,R.B.and chultz,G.A. Bovine parthenogenetic blastocysts following in vitro maturation and oocyte activation with ethanol.theriogenology.1993,40:63～76
[9] 旭日干，张锁链，薜晓先等，屠宰母牛卵巢卵母细胞体外受精与发育的研究.畜牧兽医学报，1989，20（3）：193～198
[10] 旭日干，张锁链，薜晓先等.绵羊体外成熟、体外受精卵的体外发育及移植后的产羔.内蒙古大学学报（自然科学版），1989，20（4）：560～569
[11] Yutaka F，Kei I,Noel F，et al.Follicle culture enhances fertilizability and cleavage of bovine oocytes matured in vitro，J Anim Sci，1987，64：935～941
[12] Cross PC,Brinster RL.In vitro development of mouse oocytes. Biology of Reproduction,1970 3:298-307
[13] Leibfried-Rutledge M L,Critser E S, First N L. Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes.Biology of Reproduction,1986,35:850-857
[14] San Buissho A,Threlfall W R. The effects of oestrous cow serum on the in vitro maturation and fertilization of bovine follicular oocytes. Theriogenology,1989,31:300-309
[15] Lu K H.Studies related to the in vitro maturation and fertilization of ovarian oocytes in cattle:[Ph D Thesis].National University of Ireland. 1990.247～253
[16] 寇全安等。小鼠卵母细胞的孤雌激活及体外发育。西北农业大学学报，1997，25（6）：82～86
[17] 谭景和，杨婵云，周 琪等。小鼠不同卵龄卵母细胞电剌激后第二极体形成和原核发育的研究。细胞生物学杂志，1994，16:42～44
[18] 李裕强等.乙醇和电场激活小鼠早期卵母细胞的研究.西北农业大学学报，1999，27（2），58～61
[19] Stice S，James M R.Activation lf mammalian oocytes by a factor obtained from rabbit sperm.Mol Peprod Dev，1990，25：272～280
[20] 朱洗，王幽兰等。蟾蜍体外成熟卵裂球的人工单性发育及卵球成熟程度之不同与单性发育的关系。实验生物学报，1959，6（4），276～297
[21] 梁桂霞，司克媛，李荔，陈永龙等.移植非洲爪蟾胚胎细胞核引起花背蟾蜍成熟卵单性发育的研究.动物学报，2000，46（2）183～189
[22] 刘林，张忠诚，王永胜等，绵羊体外成熟卵母细胞的电激活及其在体外孤雌发育的研究。动物学报，1998，44（1）74～81
[23] 郑行编著.动物生殖生理学.北京:北京农业大学出版社，1994，45～49
[24] 卢晟盛，谭世俭，石德顺等，不同成熟时间对黄牛小腔卵泡卵母细胞成熟率的影响.广西畜牧兽医，2000，16（1）12～13
[25] Fulka J Jr,Motlik J;Fulka J,Jilek F.Effect of cycloheximide on nuclear maturation of Pig and Mouse oocytes.J Reprod Fertil,1986,77:281～285
[26] Hashimoto N,Kishimoto T.Regulation of meiotic metaphase by a cytoplamic maturation-promoting factor during mouse oocyte maturation ,Dev Biol,1988,126:242～252
[27] Kubiak J Z,Weber M,De Pennart H,Winston N J ,Maro B.The metaphase Ⅱ arrest in mouse oocytes is controlled through microtuble-dependent destruction of cyclin Bin the presence of CSF.EMBO J,1993,12:3773～3778
[28] Clarke H J,Masni Y.The induction of reversible and irrerersible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes.Dev Biol,1983,97:291～301
[29] Goto K,Ishida M,Ookutsu S,et al.Activation of unaged bovine oocytes varicus parthenogenetic stimuli. Theriogenology, 1994, 41:207
[30] Thibault C.Szollosi D,Gerard M.Mammalian oocyte maturation. Reprod.Num.Dev.27:865～896.1987
[31] Minamihashi A,Watson A.J,Watson P.H,Church R.B and Schultz G.A.Bovine parthenogenetic blastocyts following in vitro maturation and oocyte activation with ethanol. Theriogenology, 1993,40:63～76
[32] Romano J.E.Effect of service on estrus duration in dairy goats.Theriogenology,1993,40:77～84
[33] ??? ???? ?????? ????? ?????? ??? ?? 1998,????
[34] 安部茂樹。ウシ卵巣の生理食塩水中での保存温度および保存時間がウシ体外受精胚発生に及ぼす影響。日畜会報、１９９６、６７（７）：６３３～６３８
[35] 安部茂樹。ウシ卵巣の輸送温度が体外成熟、体外受精卵子の胚発生の及ぼす影響。日畜会報、１９９３、６４：３２～３６
[36] 青柳敬人、古館 誠。牛のならびに卵胞内卵子の非凍結低温下での短期保存について。家畜繁殖誌。１９８６、３２：１３８～１４２
[37] 邓满齐等，小鼠卵激活过程中胞质游离Ca2+变化及孤雌发育研究。实验生物学报，1994，27（3）：289～294
[38] Collas P,Balise J.J,Hofmann G.A and Robl J.M.Electrical activation of mouse oocytes.Theriogenology,1989,5:835～844
[39] Kono T,Iwasaki S and Nakahara T.Parthenogenetic activation by electric stimulus of bovine oocytes matured in vitro.Theriogenology,1989,4:569～576
[40] Nagai T.Development of bovine in vitro-matured folloicular oocytes activated with ethanol.Theriogenology,1992,37:869～875
[41] Onodera M and Tsunoda Y.Parthenogenetic activation of mouse and Rabbit Eggs by Electric Stimulation In vitro.Gamete Research,1989,22:277～283
[42] Ware C.B,Barnes F.L,Maiki-Laurila M and First N.L.Gamete Research,1989,22:265～275
[43] Ball BA,Thomas PGA,Brinsko SP,Miller PG and Ellington JE.Theriogenoloy,1992:37:189～198
[44] Naito K and Toyoda Y.Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes. J.Reprod.Fert.1991,93:467～473
[45] 王永胜，张忠诚等，哺乳动物胚胎细胞核移植克隆技术的研究进展.国外畜牧科技，1997，24（4）：42～48
[46] Kaufam M H,Barton S C and Surani M A H.Normal postimplantation development of mouse parthenogenetic embryos to the forelimb bud stage.Nature,1977,265:53～55
[47] Kaufman M H.Parthenogenesis:a system facilitating understanding of factors that influence early mammalian development.In:R J Harrison and R L Holmes.ed.Progress in Anatomy.Vol.1.Cambridge University Press,1981,1～34
[48] Fulka J Jr,Motlik J;Fulka J,Jilek F.Effect of cycloheximide on nuclear maturation of Pig and Mouse oocytes.J Reprod Fertil,1986,77:281～285
[49] 刘红林，范必勤，汪河海，陈宜峰等，亚胺环己酮对小鼠卵母细胞成熟和人工激活的影响。农业生物技术学报，1995，3（4）：53～56
[50] 董 伟主编，家畜繁殖学。北京，北京农业大学出版社，2000，97～111
[51] 阎隆飞，张玉麟主编，分子生物学。北京，中国农业大学出版社，1997，437～444
[52] Yang NS Lu KH.Go,con I.In vitro fertiliaction(IVF) and culture(IVC) of bovine oocytes from stored ovaries.Theriogenology,1990,33:352
[53] 青柳敬人、小西正人 。ウシ体外成熟卵子の人為的活性化処理による胚盤胞への発育に関する研究―直流パルスおよぴＣａイオノホアサイクロヘキシマドを用いた複合活性処理についてーJ.Reprod.1994,Dev.40:5-11
[54] 青柳敬人、小西正人、和田貴子。ウシ体外成熟卵子の複合活性処理による活性化卵子およぴ核移植由来胚の胚盤胞への発育―J.Reprod.1994,Dev.40（５）：２４）
[55] 秦应和，卵裂球培养的研究进展。国外畜牧科技，1999，26（3）：31～33
[56] Fukuda Y,Ichikawa K,Naito K,Tyoda Y.Birth of normal calves
resulting from bovine oocytes matured,fertilized and cultured with cumulus cells in vitro up to the blastocyst stage.Biol Reprod,1990,42:114～119
[57] 刘红林，范必勤，汪河海，陈宜峰，王公金等，6-DMAP对小鼠卵母细胞减数分裂启动及孤雌发育作用。1997，30（4）：403～407
[58] Johnson et al.The Veterinary Record,1995,137,15～16
[59] 颜淑卿，中国畜牧学会会志，1994，24：391～405
[60] M.A.Handel著，李子义编译，配子发生中成熟分裂的调控。国外畜牧科技，1999，26（2）：29～33
[2] Wilmut I et al,1997.Nature,385:310～313
[3] 王晨光，邓满齐，孙方臻等，小鼠卵母细胞体外成熟不同阶段对钙的依赖性。实验生物学报，1998，31（2）：147～150
[4] Tsafriri A,Channing C P.Influence of follicular.maturation and culture conditions on the meiosis of pig oocytes in vitro.J Reprod Fert,1975,43:149～152
[5] Motlik J,Crozet N,Fulka J.Meiotic competence in vitro of pig oocytes isolated from early antral follicles.J Reprod Fert,1984,72:323～328
[6] 刘素娟，张涌，李勇等，山羊小腔卵泡卵母细胞的体外成熟。河北农业大学学报，1998，21（2）：66～69
[7] 柏家林，关红梅，牛卵母细胞体外成熟和体外受精技术的研究进展1998，25（4）：27～31
[8] Minamihashi,A.,Watson,A.J.,Watson,P.H.,Church,R.B.and chultz,G.A. Bovine parthenogenetic blastocysts following in vitro maturation and oocyte activation with ethanol.theriogenology.1993,40:63～76
[9] 旭日干，张锁链，薜晓先等，屠宰母牛卵巢卵母细胞体外受精与发育的研究.畜牧兽医学报，1989，20（3）：193～198
[10] 旭日干，张锁链，薜晓先等.绵羊体外成熟、体外受精卵的体外发育及移植后的产羔.内蒙古大学学报（自然科学版），1989，20（4）：560～569
[11] Yutaka F，Kei I,Noel F，et al.Follicle culture enhances fertilizability and cleavage of bovine oocytes matured in vitro，J Anim Sci，1987，64：935～941
[12] Cross PC,Brinster RL.In vitro development of mouse oocytes. Biology of Reproduction,1970 3:298-307
[13] Leibfried-Rutledge M L,Critser E S, First N L. Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes.Biology of Reproduction,1986,35:850-857
[14] San Buissho A,Threlfall W R. The effects of oestrous cow serum on the in vitro maturation and fertilization of bovine follicular oocytes. Theriogenology,1989,31:300-309
[15] Lu K H.Studies related to the in vitro maturation and fertilization of ovarian oocytes in cattle:[Ph D Thesis].National University of Ireland. 1990.247～253
[16] 寇全安等。小鼠卵母细胞的孤雌激活及体外发育。西北农业大学学报，1997，25（6）：82～86
[17] 谭景和，杨婵云，周 琪等。小鼠不同卵龄卵母细胞电剌激后第二极体形成和原核发育的研究。细胞生物学杂志，1994，16:42～44
[18] 李裕强等.乙醇和电场激活小鼠早期卵母细胞的研究.西北农业大学学报，1999，27（2），58～61
[19] Stice S，James M R.Activation lf mammalian oocytes by a factor obtained from rabbit sperm.Mol Peprod Dev，1990，25：272～280
[20] 朱洗，王幽兰等。蟾蜍体外成熟卵裂球的人工单性发育及卵球成熟程度之不同与单性发育的关系。实验生物学报，1959，6（4），276～297
[21] 梁桂霞，司克媛，李荔，陈永龙等.移植非洲爪蟾胚胎细胞核引起花背蟾蜍成熟卵单性发育的研究.动物学报，2000，46（2）183～189
[22] 刘林，张忠诚，王永胜等，绵羊体外成熟卵母细胞的电激活及其在体外孤雌发育的研究。动物学报，1998，44（1）74～81
[23] 郑行编著.动物生殖生理学.北京:北京农业大学出版社，1994，45～49
[24] 卢晟盛，谭世俭，石德顺等，不同成熟时间对黄牛小腔卵泡卵母细胞成熟率的影响.广西畜牧兽医，2000，16（1）12～13
[25] Fulka J Jr,Motlik J;Fulka J,Jilek F.Effect of cycloheximide on nuclear maturation of Pig and Mouse oocytes.J Reprod Fertil,1986,77:281～285
[26] Hashimoto N,Kishimoto T.Regulation of meiotic metaphase by a cytoplamic maturation-promoting factor during mouse oocyte maturation ,Dev Biol,1988,126:242～252
[27] Kubiak J Z,Weber M,De Pennart H,Winston N J ,Maro B.The metaphase Ⅱ arrest in mouse oocytes is controlled through microtuble-dependent destruction of cyclin Bin the presence of CSF.EMBO J,1993,12:3773～3778
[28] Clarke H J,Masni Y.The induction of reversible and irrerersible chromosome decondensation by protein synthesis inhibition during meiotic maturation of mouse oocytes.Dev Biol,1983,97:291～301
[29] Goto K,Ishida M,Ookutsu S,et al.Activation of unaged bovine oocytes varicus parthenogenetic stimuli. Theriogenology, 1994, 41:207
[30] Thibault C.Szollosi D,Gerard M.Mammalian oocyte maturation. Reprod.Num.Dev.27:865～896.1987
[31] Minamihashi A,Watson A.J,Watson P.H,Church R.B and Schultz G.A.Bovine parthenogenetic blastocyts following in vitro maturation and oocyte activation with ethanol. Theriogenology, 1993,40:63～76
[32] Romano J.E.Effect of service on estrus duration in dairy goats.Theriogenology,1993,40:77～84
[33] ??? ???? ?????? ????? ?????? ??? ?? 1998,????
[34] 安部茂樹。ウシ卵巣の生理食塩水中での保存温度および保存時間がウシ体外受精胚発生に及ぼす影響。日畜会報、１９９６、６７（７）：６３３～６３８
[35] 安部茂樹。ウシ卵巣の輸送温度が体外成熟、体外受精卵子の胚発生の及ぼす影響。日畜会報、１９９３、６４：３２～３６
[36] 青柳敬人、古館 誠。牛のならびに卵胞内卵子の非凍結低温下での短期保存について。家畜繁殖誌。１９８６、３２：１３８～１４２
[37] 邓满齐等，小鼠卵激活过程中胞质游离Ca2+变化及孤雌发育研究。实验生物学报，1994，27（3）：289～294
[38] Collas P,Balise J.J,Hofmann G.A and Robl J.M.Electrical activation of mouse oocytes.Theriogenology,1989,5:835～844
[39] Kono T,Iwasaki S and Nakahara T.Parthenogenetic activation by electric stimulus of bovine oocytes matured in vitro.Theriogenology,1989,4:569～576
[40] Nagai T.Development of bovine in vitro-matured folloicular oocytes activated with ethanol.Theriogenology,1992,37:869～875
[41] Onodera M and Tsunoda Y.Parthenogenetic activation of mouse and Rabbit Eggs by Electric Stimulation In vitro.Gamete Research,1989,22:277～283
[42] Ware C.B,Barnes F.L,Maiki-Laurila M and First N.L.Gamete Research,1989,22:265～275
[43] Ball BA,Thomas PGA,Brinsko SP,Miller PG and Ellington JE.Theriogenoloy,1992:37:189～198
[44] Naito K and Toyoda Y.Fluctuation of histone H1 kinase activity during meiotic maturation in porcine oocytes. J.Reprod.Fert.1991,93:467～473
[45] 王永胜，张忠诚等，哺乳动物胚胎细胞核移植克隆技术的研究进展.国外畜牧科技，1997，24（4）：42～48
[46] Kaufam M H,Barton S C and Surani M A H.Normal postimplantation development of mouse parthenogenetic embryos to the forelimb bud stage.Nature,1977,265:53～55
[47] Kaufman M H.Parthenogenesis:a system facilitating understanding of factors that influence early mammalian development.In:R J Harrison and R L Holmes.ed.Progress in Anatomy.Vol.1.Cambridge University Press,1981,1～34
[48] Fulka J Jr,Motlik J;Fulka J,Jilek F.Effect of cycloheximide on nuclear maturation of Pig and Mouse oocytes.J Reprod Fertil,1986,77:281～285
[49] 刘红林，范必勤，汪河海，陈宜峰等，亚胺环己酮对小鼠卵母细胞成熟和人工激活的影响。农业生物技术学报，1995，3（4）：53～56
[50] 董 伟主编，家畜繁殖学。北京，北京农业大学出版社，2000，97～111
[51] 阎隆飞，张玉麟主编，分子生物学。北京，中国农业大学出版社，1997，437～444
[52] Yang NS Lu KH.Go,con I.In vitro fertiliaction(IVF) and culture(IVC) of bovine oocytes from stored ovaries.Theriogenology,1990,33:352
[53] 青柳敬人、小西正人 。ウシ体外成熟卵子の人為的活性化処理による胚盤胞への発育に関する研究―直流パルスおよぴＣａイオノホアサイクロヘキシマドを用いた複合活性処理についてーJ.Reprod.1994,Dev.40:5-11
[54] 青柳敬人、小西正人、和田貴子。ウシ体外成熟卵子の複合活性処理による活性化卵子およぴ核移植由来胚の胚盤胞への発育―J.Reprod.1994,Dev.40（５）：２４）
[55] 秦应和，卵裂球培养的研究进展。国外畜牧科技，1999，26（3）：31～33
[56] Fukuda Y,Ichikawa K,Naito K,Tyoda Y.Birth of normal calves
resulting from bovine oocytes matured,fertilized and cultured with cumulus cells in vitro up to the blastocyst stage.Biol Reprod,1990,42:114～119
[57] 刘红林，范必勤，汪河海，陈宜峰，王公金等，6-DMAP对小鼠卵母细胞减数分裂启动及孤雌发育作用。1997，30（4）：403～407
[58] Johnson et al.The Veterinary Record,1995,137,15～16
[59] 颜淑卿，中国畜牧学会会志，1994，24：391～405
[60] M.A.Handel著，李子义编译，配子发生中成熟分裂的调控。国外畜牧科技，1999，26（2）：29～33