绵羊精子胞质内显微受精技术的研究
摘要
本研究着重探讨和研究了绵羊胞质内显微受精(ICSI)各技术环节当中一些因素对绵羊ICSI整个过程的影响,初步建立了一套比较完善的操作体系。研究结果如下:
实验一,对精子DTT处理后进行ICSI,结果如下:(1)采用DTT处理与不处理的精子比较,卵裂率(79.6%、81.6%;P>0.05)没有明显差异,囊胚发育率(31.2%、20.9%;P<0.05)差异显著。实验二,分别采用活精子和死精子进行ICSI,卵裂率(80.0%、83.1%)和囊胚发育率(27.6%、20.2%)均没有显著影响(P>0.05)。实验三,为了研究激活方法对绵羊胞质内显微受精的影响,分别对卵子采用:(1)假注射,不激活;(2)注射精子,不激活;(3)假注射,Ionomysin +6-DMAP激活;(4)注射精子, Ethanol+6-DMAP激活;(5)注射精子,Ionomysin+6-DMAP激活。结果:第1、2组无论注射精子与否,都不激活,卵裂率(7.5%、5.3%)和囊胚发育率(0.94%、1.8%)都很低,差异均不显著。第3组与第4、5组比较,卵裂率(65.7%、71.6%、75.6%;P>0.05)没有明显差异,但囊胚发育率(5.7%、15.6%、26.0%;P<0.01)却明显低于4、5组。采用Ethanol+6-DMAP和Ionomysin+6-DMAP两种方法处理时,卵裂率差异不显著(71.6%、75.6%;P>0.05),但囊胚发育率前者要显著低于后者(15.6%、26.0%;P<0.05)。实验四,本实验对显微注射时对第一极体位置进行分析,发现第一极体位于6点和12点位置,卵裂率(82.4%%、85.8%;P>0.05)和囊胚发育率(28.8%、26.9%;P>0.05)没有显著影响。实验五,精子操作液中PVP浓度分别为10%、8%和5%时,卵裂率(80.2、83.6%、72.0%;P<0.05)和囊胚发育率(27.8%、30.5%、16.0%;P<0.05)均差异显著。实验六,在培养液中添加VEGF和不添加,卵裂率(78.7%%、83.9%;P>0.05)和囊胚发育率(27.7%、23.6%;P>0.05)没有显著影响。实验七,操作液中添加CB和不添加CB,卵裂率(77.5%、74.7%;P>0.05)没有显著影响,但添加CB的囊胚发育率(29.2%、14.3%;P<0.05)却显著高于对照组。实验八,对显微注射时的温度进行分析,恒温和室温条件下,卵裂率(80.1%、90.6%;P<0.05),差异显著,囊胚发育率(31.3%、28.2%;P>0.05)没有显著影响。实验九,显微注射时间在4h以前注射卵裂率(84.7%、86.0%、80.6%;P>0.05)没有显著影响。0~1h注射的囊胚发育率要显著高于1~2h注射的,并极显著高于3-4h的(33.6%、18.3%、13.0%;P<0.05-0.01)。1-2h与3~4h注射的囊胚发育率(18.3%、13.0%;P>0.05没有明显差异。
The aim of this study was to establish an steady and efficient ICSI system for sheep oocytes maturation in vitro in our lab. Experiments were carried out to analyze the diffetent factors which been reported influence the results of ICSI and to optimize the technology of ICSI in sheep and establish a preliminary IVF system. Results:
1.The sperm were treatment by DTT and untreated.after ICSI, The Cleavage rate (79.6%、81.6%;P>0.05)were not significantly different between the two group;whereas,The blastocys rate(31.2%、20.9%;P<0.05)was higher for treatment than untreated. 2.ICSI with live and dead status of the sperm, There was no significant difference of Cleavage rate((80.0%、83.1%)and blastocys rate(27.6%、20.2%) between the two group( P>0.05).3. After ICSI ,the oocytes were activated by different method:(1) Sham-injected, no activated. (2) Sperm-Injected, no activated. (3) Sham-injected, oocytes were activated with Ionomysin+6-DMAP.(4)Sperm-injected, oocytes were activated with Ethanol+6-DMAP.(5) Sperm-injected, oocytes were activated with Ionomysin+6-DMAP. Results : In Sham-injected and Sperm-injected oocytes,but no activated, both the cleavage rate(7.5%、5.3%)and blastocyst rate(0.94%、1.8%)were very low. The cleavage rate(65.7%、71.6%、75.6%;P>0.05)of 3、4、5 groud was no significant difference,but blastocyst rate(5.7%、15.6%、26.0%;P<0.01) of the third groud was lower than others. There was no significant difference of cleavage rate(71.6%、75.6%;P>0.05)of the sperm-injected groud treated with Ethanol+6-DMAP and Ionomysin+6-DMAP,while blastocyst rate (15.6%、26.0%;P<0.05)was lower than latter. 4. This study was to evaluate the Effects of first polar body position on fertilization of sheep oocytes by intracytoplasmic sperm injection .There was no significant difference of cleavage rate (82.4%%、85.8%;P>0.05)and blastocyst rate (28.8%、26.9%;P>0.05 ) to first polar body positioned at 12 o’clock or 6 o’clock. 5. The different concentration(10%、8%、5%) PVP were added to manipulation medium ,The results showed that cleavage rate (80.2%、83.6%、72.0%; P<0.05)and blastocyst rate (27.8%、30.5%、16.0%;P<0.05) was higher for 10% and 8%PVP than 5%PVP . (6) Added VEGF in culture medium , There was no significant difference of cleavage rate (78.7%、83.9%;P>0.05)and blastocyst rate (27.6%、23.6%;P>0.05 ) in the same condition with or without VEGF. (7) There was no significant difference of cleavage rate (77.5%、74.7%;P>0.05)in the same condition with or without CB,but blastocyst rate (30.5%、16.0%;P<0.05) was higher for with than without CB. (8) Injection eggs in room temperature and 37.0℃, respectively. the cleavage rate(90.6%、80.1%;P<0.05)was significantly higher for room temperature than that of latter, but the blastocyst rate(31.3%、28.2%;P>0.05) was not significantly different between the two group. (9) Oocytes were randomly injection for 0-1h、1-2h and 3-4h , cleavage rate were 46.07%、43.33%、30.56% (P>0.05),respectively.The blastocyst rate was higher for oocytes injection for 0~1 h than 1~2 h、3~4 h(33.6%、18.3%、13.0%;P<0.05-0.01), There was no significant difference of blastocyst rate between oocytes injection for 1-2h and 3-4h (P>0.05)
实验一,对精子DTT处理后进行ICSI,结果如下:(1)采用DTT处理与不处理的精子比较,卵裂率(79.6%、81.6%;P>0.05)没有明显差异,囊胚发育率(31.2%、20.9%;P<0.05)差异显著。实验二,分别采用活精子和死精子进行ICSI,卵裂率(80.0%、83.1%)和囊胚发育率(27.6%、20.2%)均没有显著影响(P>0.05)。实验三,为了研究激活方法对绵羊胞质内显微受精的影响,分别对卵子采用:(1)假注射,不激活;(2)注射精子,不激活;(3)假注射,Ionomysin +6-DMAP激活;(4)注射精子, Ethanol+6-DMAP激活;(5)注射精子,Ionomysin+6-DMAP激活。结果:第1、2组无论注射精子与否,都不激活,卵裂率(7.5%、5.3%)和囊胚发育率(0.94%、1.8%)都很低,差异均不显著。第3组与第4、5组比较,卵裂率(65.7%、71.6%、75.6%;P>0.05)没有明显差异,但囊胚发育率(5.7%、15.6%、26.0%;P<0.01)却明显低于4、5组。采用Ethanol+6-DMAP和Ionomysin+6-DMAP两种方法处理时,卵裂率差异不显著(71.6%、75.6%;P>0.05),但囊胚发育率前者要显著低于后者(15.6%、26.0%;P<0.05)。实验四,本实验对显微注射时对第一极体位置进行分析,发现第一极体位于6点和12点位置,卵裂率(82.4%%、85.8%;P>0.05)和囊胚发育率(28.8%、26.9%;P>0.05)没有显著影响。实验五,精子操作液中PVP浓度分别为10%、8%和5%时,卵裂率(80.2、83.6%、72.0%;P<0.05)和囊胚发育率(27.8%、30.5%、16.0%;P<0.05)均差异显著。实验六,在培养液中添加VEGF和不添加,卵裂率(78.7%%、83.9%;P>0.05)和囊胚发育率(27.7%、23.6%;P>0.05)没有显著影响。实验七,操作液中添加CB和不添加CB,卵裂率(77.5%、74.7%;P>0.05)没有显著影响,但添加CB的囊胚发育率(29.2%、14.3%;P<0.05)却显著高于对照组。实验八,对显微注射时的温度进行分析,恒温和室温条件下,卵裂率(80.1%、90.6%;P<0.05),差异显著,囊胚发育率(31.3%、28.2%;P>0.05)没有显著影响。实验九,显微注射时间在4h以前注射卵裂率(84.7%、86.0%、80.6%;P>0.05)没有显著影响。0~1h注射的囊胚发育率要显著高于1~2h注射的,并极显著高于3-4h的(33.6%、18.3%、13.0%;P<0.05-0.01)。1-2h与3~4h注射的囊胚发育率(18.3%、13.0%;P>0.05没有明显差异。
The aim of this study was to establish an steady and efficient ICSI system for sheep oocytes maturation in vitro in our lab. Experiments were carried out to analyze the diffetent factors which been reported influence the results of ICSI and to optimize the technology of ICSI in sheep and establish a preliminary IVF system. Results:
1.The sperm were treatment by DTT and untreated.after ICSI, The Cleavage rate (79.6%、81.6%;P>0.05)were not significantly different between the two group;whereas,The blastocys rate(31.2%、20.9%;P<0.05)was higher for treatment than untreated. 2.ICSI with live and dead status of the sperm, There was no significant difference of Cleavage rate((80.0%、83.1%)and blastocys rate(27.6%、20.2%) between the two group( P>0.05).3. After ICSI ,the oocytes were activated by different method:(1) Sham-injected, no activated. (2) Sperm-Injected, no activated. (3) Sham-injected, oocytes were activated with Ionomysin+6-DMAP.(4)Sperm-injected, oocytes were activated with Ethanol+6-DMAP.(5) Sperm-injected, oocytes were activated with Ionomysin+6-DMAP. Results : In Sham-injected and Sperm-injected oocytes,but no activated, both the cleavage rate(7.5%、5.3%)and blastocyst rate(0.94%、1.8%)were very low. The cleavage rate(65.7%、71.6%、75.6%;P>0.05)of 3、4、5 groud was no significant difference,but blastocyst rate(5.7%、15.6%、26.0%;P<0.01) of the third groud was lower than others. There was no significant difference of cleavage rate(71.6%、75.6%;P>0.05)of the sperm-injected groud treated with Ethanol+6-DMAP and Ionomysin+6-DMAP,while blastocyst rate (15.6%、26.0%;P<0.05)was lower than latter. 4. This study was to evaluate the Effects of first polar body position on fertilization of sheep oocytes by intracytoplasmic sperm injection .There was no significant difference of cleavage rate (82.4%%、85.8%;P>0.05)and blastocyst rate (28.8%、26.9%;P>0.05 ) to first polar body positioned at 12 o’clock or 6 o’clock. 5. The different concentration(10%、8%、5%) PVP were added to manipulation medium ,The results showed that cleavage rate (80.2%、83.6%、72.0%; P<0.05)and blastocyst rate (27.8%、30.5%、16.0%;P<0.05) was higher for 10% and 8%PVP than 5%PVP . (6) Added VEGF in culture medium , There was no significant difference of cleavage rate (78.7%、83.9%;P>0.05)and blastocyst rate (27.6%、23.6%;P>0.05 ) in the same condition with or without VEGF. (7) There was no significant difference of cleavage rate (77.5%、74.7%;P>0.05)in the same condition with or without CB,but blastocyst rate (30.5%、16.0%;P<0.05) was higher for with than without CB. (8) Injection eggs in room temperature and 37.0℃, respectively. the cleavage rate(90.6%、80.1%;P<0.05)was significantly higher for room temperature than that of latter, but the blastocyst rate(31.3%、28.2%;P>0.05) was not significantly different between the two group. (9) Oocytes were randomly injection for 0-1h、1-2h and 3-4h , cleavage rate were 46.07%、43.33%、30.56% (P>0.05),respectively.The blastocyst rate was higher for oocytes injection for 0~1 h than 1~2 h、3~4 h(33.6%、18.3%、13.0%;P<0.05-0.01), There was no significant difference of blastocyst rate between oocytes injection for 1-2h and 3-4h (P>0.05)
引文
[1] HiromotoY. Microinjection of the lives permatozoa into sea urchine ggs[J]. Experimental cell Research,1962, 27 : 416-426
[2] Uehara T , Yanagimachi R. Microsurgically injected of spermatozoa into hamster eggs with subsequent tranformation of sperm nuclei into male pronuclei[J]. Biol Repord, 1976, 15: 467-470
[3] Thadani VM. Injection of sperm heads into immatures mouse oocytes[J]. Exp Zool ,1979, 210: 161-168
[4] Thadani VM. A study of heterospecific sperm-egg interactions in the rat,mouse and deer mouse using in vitro fertilization and sperm injection[J]., Exp Zool ,1980, 212: 435
[5] Perreault SD,Zirkin BR. Sperm nuclear decondensation in mammals:Role of spermassociated Proteinase in vivo[J].Zxp ZooL ,1982,224:253-257
[6] Wei, H., Yutaka, Fukui. Fertilisability of ovine, bovine or minke whale (Balaenoptera acutorostrata) spermatozoa intracytoplasmically injected into bovine oocytes[J]. Zygote , 2000, 8:267-274.
[7] Kim, Nam-Hyung., Soo, Hyu. Jun., Jung, Tae D.O. et al. Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes[J]. Molecular Reproduction and Development, 1999; 53:84-91.
[8] Markert CL. Fertilization of mammalian eggs by sperm injection[J]. The Journal of Experimental Zoology 1983, 228: 195 -201.
[9] Yanagimachi R. Intracytoplasmic sperm injection experiments using the mouse as a model[J]. Hum Repord,1998, 13(11): 87-98
[10] Ogura A , Yanagimachi R. Round spermatid nuclei injected into hamster oocyters from pronuclei and participate in syngamy[J]. Biol Reprod, 1993, 48:219-225
[11] Goto K, Kinoshita A, Takuma Y, et al. Fertilization of bovine oocyters by the injection of immobilized ,Killed spermatozoa[J]. Vet Res, 1990 ,127 : 517-520
[12] Goto, Kazufumi., Akihiro, Kinoshita., Yoshihiko, Nakanishi. et al. Blastocyst formation following intracytoplasmic injection of in-vitro derived spermatids into bovine[J]. Hum.Reprod,1996; 11:824-829.
[13] Kim, N.H., Shin, J.S., Kim, C., et al. Fertilization and in vitro development of porcine oocytes following intracytoplasmic injection of round spermatid or round spermatid nuclei[J]. Theriogenology, 1999, 51(8): 1441-1449
[14] Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse[J]..Biol Repord, 1995,52: 709-720.
[15] 卢圣忠. 牛和猪精子注射法受精研究[D]。中国农业科学院硕士学位论文 ,1992,6
[16] 罗军, 范必勤. 家兔显微受精的研究[J].江苏农业学报,1993,8(2):1-4
[17] 黄振勇, 张忠诚, 陈大元. 哺乳动物生精细胞显微受精研究现状与展望[J]. Animal science abroad. Feb,1998 Vol.25 No.1: 30-33
[18] 顾文艺, Kotaras P J,Seamark R F, 郭志勤. 精子提取物对绵羊显微受精受精率及胚胎发育影响的研究[J]. 西北农业学报, 1996,5(4):1-4
[19] 李子义, 谭景和, 周琪等..小鼠精子注入兔卵母细胞受精研究[J]. 实验生物学报,1999,32:102-111
[20] 李子仪, 谭景和, 孙兴参等. 小鼠精子和球形精子细胞显微注射受精研究[J]. 解剖学报, 1999,4 Vol.30,No.2: 147-151
[21] 何方方, 廖瑞年, 黄顺才等. 比较金黄地鼠人卵浆内单精子注射技术受精率[J]. 生殖医学杂志,1998,7(4):207-211
[22] 牛向丽. 水牛和黄牛同种内及种间显微受精的初步研究[D]。广西大学硕士学位论文,2003,6
[23] 刘玉良. 大熊猫(Ailuropoda melanoleuca)精子异卵胞质内显微受精初步研究[D]. 四川农业大学硕士学位论文,2003,6
[24] 商微,元蓉,孟繁玉等. 单精子显微注射体外受精治疗不育症 40 例分析[J]. 中国优生与与遗传杂志,2002,10(2):104
[25] 曾俐琴, 黄小金, 唐海勋等.常规体外受精-胚胎移植与卵泡浆内胞质内单精子注射临床结果比较[J]. 实用临床学,2003 ,2(1):93-95
[26] 周锦棠,赵建军,蔡志明等. 单精子卵母细胞显微注射治疗无精子症[J]. Journal of Clinical Urology,2003,18(1):36
[27] 邓春华, 庄广伦, 李满等. 附睾取精卵浆内显微注射受精治疗阻塞性无精子症[J]. Chin J Uorl, Apr,1998,19(4):242-244
[28] 徐珉,冒韵东,殷长军等. 睾丸细针抽吸取精行 ICSI 治疗阻塞性无精子症[J]. 中华男科学,2001,7(3):157-159
[29] 徐建亚,霍山,吴曰然等. 卵泡浆内单精子显微注射治疗男性不育的临床应用[J]. 中国优生与与遗传杂志,2003,11(4):95-96
[30] 罗海宁,刘群,朱桂金. 第一极体位置对卵母细胞显微注射受精后受精和胚胎发育的影响[J].潍坊医学院学报,2002,24(2):91-93
[31] Liu DY, Bourne H, Baker HWG. Fertilization and pregnancy with acrosome intact sperm by intracytoplasmic sperm injection in patients with disordered zona pellucida-induced acrosome reaction[J]. Fert Steril,1995,64:116-121
[32] S.H.Chen and G.E.Seidel,Jr. Effects of oocyte activation and treatment of spermatozoa on embryonic development following intracytoplasmic sperm injection in cattle[J]. Theriogenology,1997, 48(8): 1265-73.
[33] Hosoi, Y., Iritani, A. Rabbit microfertilization[J]. Mol Reprod,1993,36:282-284
[34] Yanagida K , Yanagimachi R, Perreauult SD, et al. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes[J]. Biol Repord, 1991,44: 440-447
[35] Keffer C. Fertilization by sperm injection in the rabbit[J]. Gamete Res, 1989, 22: 59-69
[36] 周曾娣,黎曼侬,陈云鹤等. 超生断尾精子头显微受精-输卵管内移植研究的初步报告[J]. 中国优生与与遗传杂志,2003,11(1):72-73
[37] Kim, N.H., Lee, J.W., Jun, S.H., et al. Fertilization of porcine oocytes following intracytoplasmicspermatozoon or isolated sperm head injection[J]. Mol Reprod 1998; 51(4): 436-44.
[38] Sofikitis N,Zavos P, Koutselimis A, et al. Achievement of pregnancy after injection into round spermatid nuclei into rabbit and embryo transfer: a possible mode of treatment for men with spermatogenetic arrest at the spermatid stage[J]. Urol, 1994, 151: 311
[39] Kim, N-H., Shin, J.S., Kim, C. et al. Fertilization and invitro development of porcine oocytes following intracytoplasmic injection of round spermatid or round spermatid nuclei[J]. Theriogenology,1999, 5(18):1441-1449.
[40] Tesarik, J. Oocyte activation after intracytoplasmic injection of mature and immature sperm cells[J]. Hum Reprod,1998, 13 (l 1):117-27
[41] Kimmura Y, Yanagimachi R. Development of normal mice from oocytes injected with secondary spermatocyte nuclei[J]. Biol Repord, 1995, 53: 885-862
[42] Sasagawa I, Kuretake S, Eppig JJ, et al. Mouse primary spermatocytes can complete two meiotic divisions within the oocyte cytoplasm[J]. Biol Repord, 1998, 58: 248-254
[43] 刘灵, 孙青原, 段崇文等. 小鼠初级精母细胞带下受精的研究[J]. 科学通报,1997,42(13):1432-1436
[44] 杨素芳,石德顺等. 激活方法对水牛卵母细胞孤雌发育的影响[J]. 中国兽医学报,2002,22(4):325-327
[45] Suttner R, Zakhartchenko V, Stojkovic P, et al. Intracytoplasmic sperm injection in bovine: effects of oocyt activation, sperm pretreatment and injection technique[J]. Theriogenology, 2000,54:935-948.
[46] Greeblatt EM,Meriano JS, Casper RF. Type of stimulation protocol affects oocyte maturity , fertilization rate and cleavage rate after intracytoplasmic sperm injection[J]. Fert Steril, 1995, 64: 557-563
[47] Dimitris Loutradis, M.D., Peter Drakakis, M.D., Konstantinos Kallianidis, M.D., et al. Oocyte morphology correlates with embryo quality and pregnancy rate after intracytoplasmic sperm injection[J]. Fertility and Sterility,1999,72(2):240-244
[48] 卢圣忠. 牛和猪精子注射法受精研究[D]. 北京: 中国农业科学院硕士学位论文,1992,6
[49] Ebner T, Yaman C, Moser M, et al. Prognostic value of first polar body morphology on fertilization rate and embryo quatily in intracytoplasmic sperm injection[J]. Human reproduction, 2000, 15 (2): 427-430
[50] Wei H, Yutaka Fukui. Technical improvement in Intracytoplasmic sperm injection (ICSI) in cattle[J]. Journal of Reproduction and Developmen , 2000; 46:403-407.
[51] Osmanagaoglu K, Tournaye H, Kolibianakis E, et al. Cumulative delivery rates after ICSI in women aged >37 years[J]. Hum Reprod, 2002, 17(4):940-944
[52] Hidekazu Saito,Takakazu Saito, Tomoko Kaneko,et al. Relatively poor oocyte quality is an indication for intracytoplasmic sperm injection[J]. Fertility and Sterility, 2000,73(3):465-469
[53] Manee M.D, Arthur M.D, et al. Lack of correlation between oocyte-corona-cumulus complex morphology and nuclear maturity of oocytes collected in stimulated cycles for intracytoplasmic sperm injection[J]. Fertility and Sterility,1999,71(5):937-940
[54] 张艳玲,牛向丽,石德顺,卢克焕. 水牛精子胞质内显微受精技术的初步研究[J]. 草食家畜,2002年,4(117):
[55] Tesarik J. Human oocyte activation after intracytoplasmic sperm injection[J]. Hum Reprod, 1994, 9:977-988
[56] Goto, K. Bovine microfertilization and embryo transfer[J]. Mol Reprod Dew, 1993, 36:288-290
[57] Koji Nakagawa, M.D., Ph.D.,Shuji Yamano, M.D., Ph.D., et al. Effect of activation with Ca ionophore A23187 and puromycin on the development of human oocytes that failed to fertilize after intracytoplasmic sperm injection[J]. Fertility and Sterility,2001,76(1):148-152
[58] Xihe Li, L. H-A. Morris, W. R. Allen. Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection[J]. Journal of Reproduction and Fertility ,2000 119:253–260
[59] 李蓉,庄广伦,周灿权等. 显微注射受精的卵母细胞质膜穿破特点对受精和卵裂的影响[J]. 生殖医学杂志,1998,7(4):204-206
[60] 罗海宁,刘群,朱桂金. 显微注射受精时第一极体位置和卵母细胞膜穿破类型对受精和胚胎发育的影响[J]. 华中科技大学学报,2002,31(4):430-432
[61] Sai Ma, Basil Ho Yuen. Intracytoplasmic sperm injection could minimize the incidence of prematurely condensed human sperm chromosomes[J]. Fertility and Sterility, 2001,75(6):1096-1101
[62] Eichenlaub-Ritter U, Shen Y, Tinneberg HR. Manipulation of the oocyte: possible damage to the spindle apparatus[J]. Reprod Biomed Online , 2002 , 5(2):117-24
[63] 杨素芳. 水牛卵母细胞孤雌激活与体细胞核移植的研究[D]. 博士学位论文, 2002, 11
[64] WesterlakenL A,H elmerhorstFM ,H ermansJ ,et al .Intracytoplasmic sperm injection: position of the polar body affects pregnancy rate[J ]. Hum Reprod,1999,(1 0):2565-2569.
[65] Stoddart, N.R., Fleming, S.D. Orientation of the first polar body of the oocyte at 6 or 12 o'clock during ICSI does not affect clinical outcome[J]. Hum Reprod, 2000, 15(7): 1580-1585
[66] Blake, M., Garrisi, J., Tomkin, G., et al. Sperm deposition site during ICSI affects fertilization and development[J]. Fertil Steril , 2000 Jan; 73(1): 31-7
[67] Jacob Ashkenazi, Itai Bar-Hava, Jacob Farhi, et al. The role of purified follicle stimulatinghormone therapy in the male partner before intracytoplasmic sperm injection[J]. Fertility and sterility,1999,72(4):670-673
[68] Y H Choi, L B Love, D D Varner, et al. Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection[J]. Reproduction ,2004,127: 187–194
[69] 黄敏珍,陈士岭,邢福祺. 单精子注入卵泡浆过程对胚胎发育的影响[J]. 第一军医大学学报, 2001,21(5):363-364
[70] Van Steirteghem A , Liu J, Nagy Z, et al. Intracytoplasmic sperm injection[J]. Pionerrs in Vitro Fertilization, 1995,,3:103
[71] Edwards PG. Cell cycle factors in the human oocyte and the Intracytoplasma injection of sperm atozoa[J]. Repord Fertil, 1995, 7: 14
[72] Hanna Aboulghar, Mohamed Aboulghar, Ragaa Mansour,et al. A prospective controlled study of karyotyping for 430 consecutive babies conceived through intracytoplasmic sperm injection[J].Fertility and Sterility,2001,76(2):249-253
[73] Bonduelle1 M., Wennerholm U.-B, Loft A,et al. A multi-centre cohort study of the physical health of 5-year-old children conceived after intracytoplasmic sperm injection, in vitro fertilization and natural conception[J]. Human Reproduction, 2005, 20(2):413–419
[74] 陈大元主编. 受精生物学[M]. 北京: 科学出版社,2000
[75] 孟凡丽. 水牛卵母细胞显微受精研究[D]. 广西大学硕士学位论文,2004,5
[76] 张艳玲. 水牛精子胞质内显微受精技术的初步研究[D]. 广西大学硕士学位论文,2002,6
[77] Wei, H., Fukui, Y. Effects of bull, sperm type and sperm pretreatment on male pronuclear formation after intracytoplasmic sperm injection in cattle[J]. Reprod Fertil Dev 1999; 11(1): 59-65
[78] Palermo GD, Schlegle PN, Colombero LT, et al, Aggerssive sperm in mobilization in ICSI using epididymal and testicular sperm atozoa improves fertilization and pregnancy rates[J]. Hum Reprod, 1997, 12: 336
[79] Svalander P, Porsberg AS,Jakobosson AH,et al .Factors of importance for the establishment of a successful program of intracytoplasmic sperm injection treatment for male infertility[J]. Fertil Steril, 1996,52(l):112
[80] Arie Berkovitz, Fina Eltes, Shlomit Yaari,et al. The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm[J]. Human Reproduction, 2005, 20(1):185–190
[81] García et al,. Influence of sperm pretreatment on the efficiency of Intracytoplasmic Sperm Injection (ICSI) in pigs[J]. Journal of Andrology,2005,22:1-23
[82] 周家勃. 山羊体外受精和显微受精及相关问题的研究[D]。东北农业大学博士学位论文,2003,6
[83] Keefer, Carol. L., Abdelmoneim, I., et al. Cleavage development of bovine oocytes fertilized by sperm injection[J]. Molecular Reproduction and Development 1990; 25:281-285.
[84] Jang-Won Lee, X. Cindy Tian, and Xiangzhong Yang. Failure of Male Pronucleus Formation Is the Major Cause of Lack of Fertilization and Embryo Development in Pig Oocytes Subjected to Intracytoplasmic Sperm Injection1[J].Biology of reproduction, 2003, 68: 1341–1347
[85] Fissor RA ,Robl JM .Intracellular Ca2+ response of rabbit oocytes to electrical stimulation[J]. Mol Reprod , 1992,32:9 -16.
[86] Miyazaki S. Repetitive calcium transients in hamster oocyte[J]. Cell Calcium,1991,12 :20 5-216.
[87] Rho, G.J., Wu, B., Kawarsky, S., et al. Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection[J]. Mol Reprod Dev 1998, 50(4): 485-92
[88] F. J′?lek, R. Hu¨ttelov′, J. Petr,et al. Activation of pig oocytes using calcium ionophore:effect of protein synthesis inhibitor cycloheximide[J]. Animal Reproduction Science, 2000, 63:101–111
[89] Li X, Hamano K, Qian X, et al .Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection[J]. Zygote,1999,7:233–237
[90] Th.Hardarson,K.L undin,Hamberger, et al. The position of the metaphase II spindle cannot be predicted by the lacation of the first polar body in the human oocyte[J]. Human reproduction, 2000,15(6):1372-1376
[91] 潘欣.血管内皮细胞生长因子的研究进展。国外医学临床生物化学检验学分册[J]。2000,21(2):66-69
[92] 罗海玲,木村康二,青木真理等.血管内皮生长因子对牛胚胎体外发育影响的最适添加浓度筛选实验[J]。云南畜牧兽医,2002,4:3-4
[93] Dildi N,Bassan M,Fusl F M,et al. In controlled ovarian hyperstimulation ,steroid production oocyte retrieval and pregnancy rate correlate with gene expression of vascular endothelilial growth factor[J].J Assist Reprod Genet,1997,14(10):589-592
[94] 孙贻娟,冯云,张爱军.卵泡液中三种相关因子与胚胎质量的关系[J]。上海第二医科大学学报,2003,23(6):505-507
[95] 李峰.山羊卵母细胞显微受精(ICSI)及早期胚胎体外培养的研究[D]。扬州大学硕士学位论文。2003,5
[96] Wang WH, Meng L, Hackett RJ, et al. Rigorous thermal control during intracytoplasmic sperm injection stabilizes the meiotic spindle and improves fertilization and pregnancy rates[J]. Fertility and Sterility,2002,77: 1274–1277
[97] Wakayama T,Yanagimachi R . Development of normal mice from oocytes injectedwith freeze-dried spermatozoa[J]. Nature Biotechnology ,1998,16:639–641.
[98] 李子义,周琪,邹啸环等. 家兔显微受精影响因素及显微受精胚胎超微结构[J]。中国兽医学报,1997,17(6):593-599
[99] Peura TT,Hyttimen JM,Tolvanen M, et al. Effects of microinjection related treatment on the subsequent development of in vitro produced bovine oocyees[J]. Theriogenology,1994,42:433-443
[100] Y H Choi, L B Love, D D Varner,et al. Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection[J]. Reproduction,2004,127:187–194
[101] Marleen Jacobs, Annette M.StoIwijk, AIex M.M.Wetzeis. The effect of insemination/injection time on the results of IVF and ICSI[J]. Human reproduction, 2001,16(8):1708-1713
[102] 李满,庄广伦,李蓉等. 用射出精子、附睾精子、睾丸精子显微授精治疗男性不育[J]。中华外科杂志,2000,38(4):280-281
[103] Fishel S , Green S , Bishop M, et al . Pregnancy after intracytoplasmic injection of spermatid [J ] . Lancet , 1995 , 345 (8965) : 164121642.
[104] Keskintepe, L., Morton, P.C., Smith, S.E., et al. Caprine blastocyst formation following intracytoplasmic sperm injection and defined culture[J]. Zygote, 1997 ,5(3): 261-265
[105] Gomez, M.C., Catt, S.L., Gillan, L., et al. Time course of pronuclear formation and fertilisation after insemination in vitro and intracytoplasmic sperm injection of in vitro matured sheep oocytes[J]. Zygote, 1998 , 6(3): 261-270
[106] Gomez, M.C., Catt, J.W., Evans, G., et al. Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization[J]. Theriogenology, 1998, 15; 49(6): 1143-1154
[2] Uehara T , Yanagimachi R. Microsurgically injected of spermatozoa into hamster eggs with subsequent tranformation of sperm nuclei into male pronuclei[J]. Biol Repord, 1976, 15: 467-470
[3] Thadani VM. Injection of sperm heads into immatures mouse oocytes[J]. Exp Zool ,1979, 210: 161-168
[4] Thadani VM. A study of heterospecific sperm-egg interactions in the rat,mouse and deer mouse using in vitro fertilization and sperm injection[J]., Exp Zool ,1980, 212: 435
[5] Perreault SD,Zirkin BR. Sperm nuclear decondensation in mammals:Role of spermassociated Proteinase in vivo[J].Zxp ZooL ,1982,224:253-257
[6] Wei, H., Yutaka, Fukui. Fertilisability of ovine, bovine or minke whale (Balaenoptera acutorostrata) spermatozoa intracytoplasmically injected into bovine oocytes[J]. Zygote , 2000, 8:267-274.
[7] Kim, Nam-Hyung., Soo, Hyu. Jun., Jung, Tae D.O. et al. Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes[J]. Molecular Reproduction and Development, 1999; 53:84-91.
[8] Markert CL. Fertilization of mammalian eggs by sperm injection[J]. The Journal of Experimental Zoology 1983, 228: 195 -201.
[9] Yanagimachi R. Intracytoplasmic sperm injection experiments using the mouse as a model[J]. Hum Repord,1998, 13(11): 87-98
[10] Ogura A , Yanagimachi R. Round spermatid nuclei injected into hamster oocyters from pronuclei and participate in syngamy[J]. Biol Reprod, 1993, 48:219-225
[11] Goto K, Kinoshita A, Takuma Y, et al. Fertilization of bovine oocyters by the injection of immobilized ,Killed spermatozoa[J]. Vet Res, 1990 ,127 : 517-520
[12] Goto, Kazufumi., Akihiro, Kinoshita., Yoshihiko, Nakanishi. et al. Blastocyst formation following intracytoplasmic injection of in-vitro derived spermatids into bovine[J]. Hum.Reprod,1996; 11:824-829.
[13] Kim, N.H., Shin, J.S., Kim, C., et al. Fertilization and in vitro development of porcine oocytes following intracytoplasmic injection of round spermatid or round spermatid nuclei[J]. Theriogenology, 1999, 51(8): 1441-1449
[14] Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse[J]..Biol Repord, 1995,52: 709-720.
[15] 卢圣忠. 牛和猪精子注射法受精研究[D]。中国农业科学院硕士学位论文 ,1992,6
[16] 罗军, 范必勤. 家兔显微受精的研究[J].江苏农业学报,1993,8(2):1-4
[17] 黄振勇, 张忠诚, 陈大元. 哺乳动物生精细胞显微受精研究现状与展望[J]. Animal science abroad. Feb,1998 Vol.25 No.1: 30-33
[18] 顾文艺, Kotaras P J,Seamark R F, 郭志勤. 精子提取物对绵羊显微受精受精率及胚胎发育影响的研究[J]. 西北农业学报, 1996,5(4):1-4
[19] 李子义, 谭景和, 周琪等..小鼠精子注入兔卵母细胞受精研究[J]. 实验生物学报,1999,32:102-111
[20] 李子仪, 谭景和, 孙兴参等. 小鼠精子和球形精子细胞显微注射受精研究[J]. 解剖学报, 1999,4 Vol.30,No.2: 147-151
[21] 何方方, 廖瑞年, 黄顺才等. 比较金黄地鼠人卵浆内单精子注射技术受精率[J]. 生殖医学杂志,1998,7(4):207-211
[22] 牛向丽. 水牛和黄牛同种内及种间显微受精的初步研究[D]。广西大学硕士学位论文,2003,6
[23] 刘玉良. 大熊猫(Ailuropoda melanoleuca)精子异卵胞质内显微受精初步研究[D]. 四川农业大学硕士学位论文,2003,6
[24] 商微,元蓉,孟繁玉等. 单精子显微注射体外受精治疗不育症 40 例分析[J]. 中国优生与与遗传杂志,2002,10(2):104
[25] 曾俐琴, 黄小金, 唐海勋等.常规体外受精-胚胎移植与卵泡浆内胞质内单精子注射临床结果比较[J]. 实用临床学,2003 ,2(1):93-95
[26] 周锦棠,赵建军,蔡志明等. 单精子卵母细胞显微注射治疗无精子症[J]. Journal of Clinical Urology,2003,18(1):36
[27] 邓春华, 庄广伦, 李满等. 附睾取精卵浆内显微注射受精治疗阻塞性无精子症[J]. Chin J Uorl, Apr,1998,19(4):242-244
[28] 徐珉,冒韵东,殷长军等. 睾丸细针抽吸取精行 ICSI 治疗阻塞性无精子症[J]. 中华男科学,2001,7(3):157-159
[29] 徐建亚,霍山,吴曰然等. 卵泡浆内单精子显微注射治疗男性不育的临床应用[J]. 中国优生与与遗传杂志,2003,11(4):95-96
[30] 罗海宁,刘群,朱桂金. 第一极体位置对卵母细胞显微注射受精后受精和胚胎发育的影响[J].潍坊医学院学报,2002,24(2):91-93
[31] Liu DY, Bourne H, Baker HWG. Fertilization and pregnancy with acrosome intact sperm by intracytoplasmic sperm injection in patients with disordered zona pellucida-induced acrosome reaction[J]. Fert Steril,1995,64:116-121
[32] S.H.Chen and G.E.Seidel,Jr. Effects of oocyte activation and treatment of spermatozoa on embryonic development following intracytoplasmic sperm injection in cattle[J]. Theriogenology,1997, 48(8): 1265-73.
[33] Hosoi, Y., Iritani, A. Rabbit microfertilization[J]. Mol Reprod,1993,36:282-284
[34] Yanagida K , Yanagimachi R, Perreauult SD, et al. Thermostability of sperm nuclei assessed by microinjection into hamster oocytes[J]. Biol Repord, 1991,44: 440-447
[35] Keffer C. Fertilization by sperm injection in the rabbit[J]. Gamete Res, 1989, 22: 59-69
[36] 周曾娣,黎曼侬,陈云鹤等. 超生断尾精子头显微受精-输卵管内移植研究的初步报告[J]. 中国优生与与遗传杂志,2003,11(1):72-73
[37] Kim, N.H., Lee, J.W., Jun, S.H., et al. Fertilization of porcine oocytes following intracytoplasmicspermatozoon or isolated sperm head injection[J]. Mol Reprod 1998; 51(4): 436-44.
[38] Sofikitis N,Zavos P, Koutselimis A, et al. Achievement of pregnancy after injection into round spermatid nuclei into rabbit and embryo transfer: a possible mode of treatment for men with spermatogenetic arrest at the spermatid stage[J]. Urol, 1994, 151: 311
[39] Kim, N-H., Shin, J.S., Kim, C. et al. Fertilization and invitro development of porcine oocytes following intracytoplasmic injection of round spermatid or round spermatid nuclei[J]. Theriogenology,1999, 5(18):1441-1449.
[40] Tesarik, J. Oocyte activation after intracytoplasmic injection of mature and immature sperm cells[J]. Hum Reprod,1998, 13 (l 1):117-27
[41] Kimmura Y, Yanagimachi R. Development of normal mice from oocytes injected with secondary spermatocyte nuclei[J]. Biol Repord, 1995, 53: 885-862
[42] Sasagawa I, Kuretake S, Eppig JJ, et al. Mouse primary spermatocytes can complete two meiotic divisions within the oocyte cytoplasm[J]. Biol Repord, 1998, 58: 248-254
[43] 刘灵, 孙青原, 段崇文等. 小鼠初级精母细胞带下受精的研究[J]. 科学通报,1997,42(13):1432-1436
[44] 杨素芳,石德顺等. 激活方法对水牛卵母细胞孤雌发育的影响[J]. 中国兽医学报,2002,22(4):325-327
[45] Suttner R, Zakhartchenko V, Stojkovic P, et al. Intracytoplasmic sperm injection in bovine: effects of oocyt activation, sperm pretreatment and injection technique[J]. Theriogenology, 2000,54:935-948.
[46] Greeblatt EM,Meriano JS, Casper RF. Type of stimulation protocol affects oocyte maturity , fertilization rate and cleavage rate after intracytoplasmic sperm injection[J]. Fert Steril, 1995, 64: 557-563
[47] Dimitris Loutradis, M.D., Peter Drakakis, M.D., Konstantinos Kallianidis, M.D., et al. Oocyte morphology correlates with embryo quality and pregnancy rate after intracytoplasmic sperm injection[J]. Fertility and Sterility,1999,72(2):240-244
[48] 卢圣忠. 牛和猪精子注射法受精研究[D]. 北京: 中国农业科学院硕士学位论文,1992,6
[49] Ebner T, Yaman C, Moser M, et al. Prognostic value of first polar body morphology on fertilization rate and embryo quatily in intracytoplasmic sperm injection[J]. Human reproduction, 2000, 15 (2): 427-430
[50] Wei H, Yutaka Fukui. Technical improvement in Intracytoplasmic sperm injection (ICSI) in cattle[J]. Journal of Reproduction and Developmen , 2000; 46:403-407.
[51] Osmanagaoglu K, Tournaye H, Kolibianakis E, et al. Cumulative delivery rates after ICSI in women aged >37 years[J]. Hum Reprod, 2002, 17(4):940-944
[52] Hidekazu Saito,Takakazu Saito, Tomoko Kaneko,et al. Relatively poor oocyte quality is an indication for intracytoplasmic sperm injection[J]. Fertility and Sterility, 2000,73(3):465-469
[53] Manee M.D, Arthur M.D, et al. Lack of correlation between oocyte-corona-cumulus complex morphology and nuclear maturity of oocytes collected in stimulated cycles for intracytoplasmic sperm injection[J]. Fertility and Sterility,1999,71(5):937-940
[54] 张艳玲,牛向丽,石德顺,卢克焕. 水牛精子胞质内显微受精技术的初步研究[J]. 草食家畜,2002年,4(117):
[55] Tesarik J. Human oocyte activation after intracytoplasmic sperm injection[J]. Hum Reprod, 1994, 9:977-988
[56] Goto, K. Bovine microfertilization and embryo transfer[J]. Mol Reprod Dew, 1993, 36:288-290
[57] Koji Nakagawa, M.D., Ph.D.,Shuji Yamano, M.D., Ph.D., et al. Effect of activation with Ca ionophore A23187 and puromycin on the development of human oocytes that failed to fertilize after intracytoplasmic sperm injection[J]. Fertility and Sterility,2001,76(1):148-152
[58] Xihe Li, L. H-A. Morris, W. R. Allen. Effects of different activation treatments on fertilization of horse oocytes by intracytoplasmic sperm injection[J]. Journal of Reproduction and Fertility ,2000 119:253–260
[59] 李蓉,庄广伦,周灿权等. 显微注射受精的卵母细胞质膜穿破特点对受精和卵裂的影响[J]. 生殖医学杂志,1998,7(4):204-206
[60] 罗海宁,刘群,朱桂金. 显微注射受精时第一极体位置和卵母细胞膜穿破类型对受精和胚胎发育的影响[J]. 华中科技大学学报,2002,31(4):430-432
[61] Sai Ma, Basil Ho Yuen. Intracytoplasmic sperm injection could minimize the incidence of prematurely condensed human sperm chromosomes[J]. Fertility and Sterility, 2001,75(6):1096-1101
[62] Eichenlaub-Ritter U, Shen Y, Tinneberg HR. Manipulation of the oocyte: possible damage to the spindle apparatus[J]. Reprod Biomed Online , 2002 , 5(2):117-24
[63] 杨素芳. 水牛卵母细胞孤雌激活与体细胞核移植的研究[D]. 博士学位论文, 2002, 11
[64] WesterlakenL A,H elmerhorstFM ,H ermansJ ,et al .Intracytoplasmic sperm injection: position of the polar body affects pregnancy rate[J ]. Hum Reprod,1999,(1 0):2565-2569.
[65] Stoddart, N.R., Fleming, S.D. Orientation of the first polar body of the oocyte at 6 or 12 o'clock during ICSI does not affect clinical outcome[J]. Hum Reprod, 2000, 15(7): 1580-1585
[66] Blake, M., Garrisi, J., Tomkin, G., et al. Sperm deposition site during ICSI affects fertilization and development[J]. Fertil Steril , 2000 Jan; 73(1): 31-7
[67] Jacob Ashkenazi, Itai Bar-Hava, Jacob Farhi, et al. The role of purified follicle stimulatinghormone therapy in the male partner before intracytoplasmic sperm injection[J]. Fertility and sterility,1999,72(4):670-673
[68] Y H Choi, L B Love, D D Varner, et al. Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection[J]. Reproduction ,2004,127: 187–194
[69] 黄敏珍,陈士岭,邢福祺. 单精子注入卵泡浆过程对胚胎发育的影响[J]. 第一军医大学学报, 2001,21(5):363-364
[70] Van Steirteghem A , Liu J, Nagy Z, et al. Intracytoplasmic sperm injection[J]. Pionerrs in Vitro Fertilization, 1995,,3:103
[71] Edwards PG. Cell cycle factors in the human oocyte and the Intracytoplasma injection of sperm atozoa[J]. Repord Fertil, 1995, 7: 14
[72] Hanna Aboulghar, Mohamed Aboulghar, Ragaa Mansour,et al. A prospective controlled study of karyotyping for 430 consecutive babies conceived through intracytoplasmic sperm injection[J].Fertility and Sterility,2001,76(2):249-253
[73] Bonduelle1 M., Wennerholm U.-B, Loft A,et al. A multi-centre cohort study of the physical health of 5-year-old children conceived after intracytoplasmic sperm injection, in vitro fertilization and natural conception[J]. Human Reproduction, 2005, 20(2):413–419
[74] 陈大元主编. 受精生物学[M]. 北京: 科学出版社,2000
[75] 孟凡丽. 水牛卵母细胞显微受精研究[D]. 广西大学硕士学位论文,2004,5
[76] 张艳玲. 水牛精子胞质内显微受精技术的初步研究[D]. 广西大学硕士学位论文,2002,6
[77] Wei, H., Fukui, Y. Effects of bull, sperm type and sperm pretreatment on male pronuclear formation after intracytoplasmic sperm injection in cattle[J]. Reprod Fertil Dev 1999; 11(1): 59-65
[78] Palermo GD, Schlegle PN, Colombero LT, et al, Aggerssive sperm in mobilization in ICSI using epididymal and testicular sperm atozoa improves fertilization and pregnancy rates[J]. Hum Reprod, 1997, 12: 336
[79] Svalander P, Porsberg AS,Jakobosson AH,et al .Factors of importance for the establishment of a successful program of intracytoplasmic sperm injection treatment for male infertility[J]. Fertil Steril, 1996,52(l):112
[80] Arie Berkovitz, Fina Eltes, Shlomit Yaari,et al. The morphological normalcy of the sperm nucleus and pregnancy rate of intracytoplasmic injection with morphologically selected sperm[J]. Human Reproduction, 2005, 20(1):185–190
[81] García et al,. Influence of sperm pretreatment on the efficiency of Intracytoplasmic Sperm Injection (ICSI) in pigs[J]. Journal of Andrology,2005,22:1-23
[82] 周家勃. 山羊体外受精和显微受精及相关问题的研究[D]。东北农业大学博士学位论文,2003,6
[83] Keefer, Carol. L., Abdelmoneim, I., et al. Cleavage development of bovine oocytes fertilized by sperm injection[J]. Molecular Reproduction and Development 1990; 25:281-285.
[84] Jang-Won Lee, X. Cindy Tian, and Xiangzhong Yang. Failure of Male Pronucleus Formation Is the Major Cause of Lack of Fertilization and Embryo Development in Pig Oocytes Subjected to Intracytoplasmic Sperm Injection1[J].Biology of reproduction, 2003, 68: 1341–1347
[85] Fissor RA ,Robl JM .Intracellular Ca2+ response of rabbit oocytes to electrical stimulation[J]. Mol Reprod , 1992,32:9 -16.
[86] Miyazaki S. Repetitive calcium transients in hamster oocyte[J]. Cell Calcium,1991,12 :20 5-216.
[87] Rho, G.J., Wu, B., Kawarsky, S., et al. Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection[J]. Mol Reprod Dev 1998, 50(4): 485-92
[88] F. J′?lek, R. Hu¨ttelov′, J. Petr,et al. Activation of pig oocytes using calcium ionophore:effect of protein synthesis inhibitor cycloheximide[J]. Animal Reproduction Science, 2000, 63:101–111
[89] Li X, Hamano K, Qian X, et al .Oocyte activation and parthenogenetic development of bovine oocytes following intracytoplasmic sperm injection[J]. Zygote,1999,7:233–237
[90] Th.Hardarson,K.L undin,Hamberger, et al. The position of the metaphase II spindle cannot be predicted by the lacation of the first polar body in the human oocyte[J]. Human reproduction, 2000,15(6):1372-1376
[91] 潘欣.血管内皮细胞生长因子的研究进展。国外医学临床生物化学检验学分册[J]。2000,21(2):66-69
[92] 罗海玲,木村康二,青木真理等.血管内皮生长因子对牛胚胎体外发育影响的最适添加浓度筛选实验[J]。云南畜牧兽医,2002,4:3-4
[93] Dildi N,Bassan M,Fusl F M,et al. In controlled ovarian hyperstimulation ,steroid production oocyte retrieval and pregnancy rate correlate with gene expression of vascular endothelilial growth factor[J].J Assist Reprod Genet,1997,14(10):589-592
[94] 孙贻娟,冯云,张爱军.卵泡液中三种相关因子与胚胎质量的关系[J]。上海第二医科大学学报,2003,23(6):505-507
[95] 李峰.山羊卵母细胞显微受精(ICSI)及早期胚胎体外培养的研究[D]。扬州大学硕士学位论文。2003,5
[96] Wang WH, Meng L, Hackett RJ, et al. Rigorous thermal control during intracytoplasmic sperm injection stabilizes the meiotic spindle and improves fertilization and pregnancy rates[J]. Fertility and Sterility,2002,77: 1274–1277
[97] Wakayama T,Yanagimachi R . Development of normal mice from oocytes injectedwith freeze-dried spermatozoa[J]. Nature Biotechnology ,1998,16:639–641.
[98] 李子义,周琪,邹啸环等. 家兔显微受精影响因素及显微受精胚胎超微结构[J]。中国兽医学报,1997,17(6):593-599
[99] Peura TT,Hyttimen JM,Tolvanen M, et al. Effects of microinjection related treatment on the subsequent development of in vitro produced bovine oocyees[J]. Theriogenology,1994,42:433-443
[100] Y H Choi, L B Love, D D Varner,et al. Factors affecting developmental competence of equine oocytes after intracytoplasmic sperm injection[J]. Reproduction,2004,127:187–194
[101] Marleen Jacobs, Annette M.StoIwijk, AIex M.M.Wetzeis. The effect of insemination/injection time on the results of IVF and ICSI[J]. Human reproduction, 2001,16(8):1708-1713
[102] 李满,庄广伦,李蓉等. 用射出精子、附睾精子、睾丸精子显微授精治疗男性不育[J]。中华外科杂志,2000,38(4):280-281
[103] Fishel S , Green S , Bishop M, et al . Pregnancy after intracytoplasmic injection of spermatid [J ] . Lancet , 1995 , 345 (8965) : 164121642.
[104] Keskintepe, L., Morton, P.C., Smith, S.E., et al. Caprine blastocyst formation following intracytoplasmic sperm injection and defined culture[J]. Zygote, 1997 ,5(3): 261-265
[105] Gomez, M.C., Catt, S.L., Gillan, L., et al. Time course of pronuclear formation and fertilisation after insemination in vitro and intracytoplasmic sperm injection of in vitro matured sheep oocytes[J]. Zygote, 1998 , 6(3): 261-270
[106] Gomez, M.C., Catt, J.W., Evans, G., et al. Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization[J]. Theriogenology, 1998, 15; 49(6): 1143-1154