非电泳法与电泳法牛胚胎PCR性别鉴定的研究与对比
摘要
为选出更高效、实用的牛胚胎性别鉴定方法，本实验根据已经报道的牛性染
色体上DNA序列，选择并合成了8对扩增性染色体上DNA序列的引物。采集
晋南牛(中国黄牛品种)、荷斯坦牛(奶牛品种)血液样本，标记，提取基因组DNA
并测得浓度。用PCR法应用8对引物分别对所有的成年牛血液提取的模板DNA
进行扩增，使用电泳方法或非电泳法(引物DYZ-1)分析产物。经过对PCR条件
的摸索和优化，非电泳法对血液模板DNA扩增的准确率达到了100％。
为了把非电泳法应用于牛胚胎性别鉴定生产中，实验通过FSH法对雌晋南
牛超数排卵，非手术法冲胚。胚胎评级后装管，使用乙二醇常规法冷冻后放入液
氮罐中保存至使用。胚胎解冻后，使用玻璃针法取样，裂解。每一个取样胚胎的
样本数为3-4份不等，取其中一份样本用SE引物扩增，如扩增有效，则其余的
样本都用来做DYZ-1扩增；否则再取一份样本用SE扩增，直至扩增有效。以
SE扩增结果为参照，验证DYZ-1的判定结果。血液模板DNA扩增结果与胚胎
样本扩增结果都表明非电泳法在比SE引物电泳法节约1小时以上的同时，具有
比SE更高的灵敏度，在模板浓度低至1-2拷贝时仍能达到90％的扩增有效率。
本研究在我国首次成功地把非电泳PCR法应用到牛胚胎性别鉴定之中，减少了
胚胎性别鉴定的操作步骤，并且与国外研究相比，进一步缩短了性别鉴定所需要
的时间，从而使胚胎移植现场性别鉴定更加可行。在5对未见报道用于性别鉴定
的微卫星DNA多态性引物中，筛选出2对具有性别特异性的引物，经过进一步
的研究可能用于胚胎性别鉴定及品种鉴定。
In order to develop a more efficient and applicable method of embryo sexing, 8 pairs of primers for sex chromosome DNA sequences were chosen and synthesized basing on some reported DNA sequences on XY chromosome. Blood samples of Jin-nan cattle and Hostem cattle were labeled to extract genome DNA, concentration of which was gauged. Both electrophoretic and nonelectrophoretic PCR sexing was used in the research and nonelectrophoretic PCR sexing both had reached an accuracy of 100% through a series of PCR condition system ameliorating.
FSH was injected to cause superovulation of Jin-nan female cattle, followed by insemination procedure; then on day 7, embryos were flushed and collected for examining the applicability of nonelectrophoretic method in embryo sexing. After being graded, embryos were put in pipes for routine freezing in cryoprotectant with ethylene glycol. Pipes with embryos were stored in liquid nitrogen until been used. After thawing, embryos were sampled by needle and then there was a DNA releasing procedure right before PCR. Each embryo had 2-4 samples; one was amplified by SE first. In case of successful amplification, other samples were all amplified by DYZ-1; otherwise another sample was amplified until a successful amplification occurred. The result of DYZ-1 was compared with result of SE for the same embryo to confirm if it was right. Nonelectrophoretic PCR sexing seems more sensitive to low DNA concentration than SE, meanwhile it takes about 1 hour less than SE. It reached an efficiency of 90% even when the template c
oncentration was only 1-2 copies.
This study successfully applied nonelectrophoretic PCR to bovine embryo sexing for the very first time in our country, it also reduced a traditional step in embryo sexing. Furthermore, compared with abroad research, it further shortened the time for sexing, therefore made PCR sexing in field more applicable. In 5 pairs of microsatellite polymorphic DNA primers used in this research, 2 pairs being tested sex specific showed potential usability for bovine embryo sexing.
色体上DNA序列，选择并合成了8对扩增性染色体上DNA序列的引物。采集
晋南牛(中国黄牛品种)、荷斯坦牛(奶牛品种)血液样本，标记，提取基因组DNA
并测得浓度。用PCR法应用8对引物分别对所有的成年牛血液提取的模板DNA
进行扩增，使用电泳方法或非电泳法(引物DYZ-1)分析产物。经过对PCR条件
的摸索和优化，非电泳法对血液模板DNA扩增的准确率达到了100％。
为了把非电泳法应用于牛胚胎性别鉴定生产中，实验通过FSH法对雌晋南
牛超数排卵，非手术法冲胚。胚胎评级后装管，使用乙二醇常规法冷冻后放入液
氮罐中保存至使用。胚胎解冻后，使用玻璃针法取样，裂解。每一个取样胚胎的
样本数为3-4份不等，取其中一份样本用SE引物扩增，如扩增有效，则其余的
样本都用来做DYZ-1扩增；否则再取一份样本用SE扩增，直至扩增有效。以
SE扩增结果为参照，验证DYZ-1的判定结果。血液模板DNA扩增结果与胚胎
样本扩增结果都表明非电泳法在比SE引物电泳法节约1小时以上的同时，具有
比SE更高的灵敏度，在模板浓度低至1-2拷贝时仍能达到90％的扩增有效率。
本研究在我国首次成功地把非电泳PCR法应用到牛胚胎性别鉴定之中，减少了
胚胎性别鉴定的操作步骤，并且与国外研究相比，进一步缩短了性别鉴定所需要
的时间，从而使胚胎移植现场性别鉴定更加可行。在5对未见报道用于性别鉴定
的微卫星DNA多态性引物中，筛选出2对具有性别特异性的引物，经过进一步
的研究可能用于胚胎性别鉴定及品种鉴定。
In order to develop a more efficient and applicable method of embryo sexing, 8 pairs of primers for sex chromosome DNA sequences were chosen and synthesized basing on some reported DNA sequences on XY chromosome. Blood samples of Jin-nan cattle and Hostem cattle were labeled to extract genome DNA, concentration of which was gauged. Both electrophoretic and nonelectrophoretic PCR sexing was used in the research and nonelectrophoretic PCR sexing both had reached an accuracy of 100% through a series of PCR condition system ameliorating.
FSH was injected to cause superovulation of Jin-nan female cattle, followed by insemination procedure; then on day 7, embryos were flushed and collected for examining the applicability of nonelectrophoretic method in embryo sexing. After being graded, embryos were put in pipes for routine freezing in cryoprotectant with ethylene glycol. Pipes with embryos were stored in liquid nitrogen until been used. After thawing, embryos were sampled by needle and then there was a DNA releasing procedure right before PCR. Each embryo had 2-4 samples; one was amplified by SE first. In case of successful amplification, other samples were all amplified by DYZ-1; otherwise another sample was amplified until a successful amplification occurred. The result of DYZ-1 was compared with result of SE for the same embryo to confirm if it was right. Nonelectrophoretic PCR sexing seems more sensitive to low DNA concentration than SE, meanwhile it takes about 1 hour less than SE. It reached an efficiency of 90% even when the template c
oncentration was only 1-2 copies.
This study successfully applied nonelectrophoretic PCR to bovine embryo sexing for the very first time in our country, it also reduced a traditional step in embryo sexing. Furthermore, compared with abroad research, it further shortened the time for sexing, therefore made PCR sexing in field more applicable. In 5 pairs of microsatellite polymorphic DNA primers used in this research, 2 pairs being tested sex specific showed potential usability for bovine embryo sexing.
引文
[1] 朱晓华，赵万里，王志跃．家畜性别决定的机制及其控制的研究进展．动物科学与动物医学，2001，18(1)：22-24
[2] 王建辰，章孝荣．动物生殖调控[M]．安徽科学技术出版社，1998．8，第1版
[3] 李馨，杨隽，郭志光．家畜性别控制研究现状与展望[J]．内蒙古畜牧科学，1997(2)：15-18
[4] G.E.Seidel. Sexed Semen Applications in Dairy Cattle[C].Western Dairy Managemnet Conference.1999,April 8-10:183-186
[5] M. Thibier and M.Nibart. The Sexing of Bovine Embryos in the Field[J]. Theriogenology, 1995, 43:71-80
[6] 葛宝生，李善如，张忠诚等．哺乳动物的性别控制．内蒙古畜牧科学，1998(2)：14-17
[7] Stephen S. Wachtel. H-Y antigen in the Study of Sex Determination and Control of Sex Ratio[J]. Theriogenology, 1984, 21:18-27
[8] T.J.Williams. A Technique for Sexing Mouse Embryos by a Visual Colorimeteric Assay of the X-linked Enzyme, Glucose 6-phsphate Dehydrogenase[J]. Theriogenology, 1986, 25:733-738
[9] Peura T et al. A Reliable Sex Determination Assay for Bovine Preimplantation Embryos Using the PCR[J]. Theriogenology, 1991, 35(3): 547-555
[10] B.F. Shea. Determining the Sex of Bovine Embryos Using PCR Results :A 6-year Retrospective Study[J]. Theriogenology, 1999, 51:841-854
[11] K.Roschlau, D.Roschlau, R.Roselius et al. Over 5 Years Experience in Sexing of Bovine Morulae and Blastocysts During Routine Embryo Transfer[J]. Theriogenology, 1997, 47:273 (Abstract)
[12] Lechniak D. the Use of Amelogenin Gene Polymorphism in PCR Embryo Sexing in Bovine IVF Embryos[J]. Journal of Applied Genetics, 1997,38(1):45-49
[13] G.Carbonneau, N.Morin, J.Durocher et al. Viability of Bovine IVF Embryos Biopsied with Microsection or Microaspiration Technique for Sexing[J]. Theriogenology, 1996, 45: 266(abstract)
[14] J.F.Hasler, E.Cardey, J.E.Stokes et al. Nonelectrophoretic PCR-sexing of Bovine Embryos in a Commercial Environment[J]. Theriogenology, 2002, 58:1457-1469
[15] P.Chrenek, L.Boulanger, Y.Heyman et al. Sexing and Multiple Genotype Analysis from a Single Cell of Bovine Embryo[J]. Theriogenology, 2001, 55:1071-1081
[16] 黄淑帧，陈美珏，黄英等．试管牛和试管羊胚胎性别的鉴定[J]．遗传，2000，22：65-68
[17] Yindee Kitiyanant, Jumnian Saikhun, Boripat Siriaroonrat et al. Sex Determination by PCR and Karyotyping of Bovine Embryos at First Cleavage in Vitro[J]. ScienceAsia, 2000, 26:9-13
[18] K.Kameyama et al. Direct Transfer of Bovine Frozen-thawed Embryos Sexed with a Rapid Y-chromosome-detection Assay[J]. Theriogenology, 1996, 45: 227(Abstract)
[19] K.Makondo, G.S.Amiridis, I.A.Jeffcoate et al. Use of the PCR to Sex the Bovine Fetus Using
Cells Recovered by Ultrasound-guided Fetal Fluid Aspiration[J]. Animal Reproduction Science, 1997, 49:125-133
[20] Peippo-J. Detection of Bovine Fetal DNA from Amniotic Fluid Using the PCR. Agricultural and Food Science in Finland, 1996,5(5): 541-546
[21] Taneja-M et al. Rapid Sexing of Bovine Preimplantation Embryos Using PCR: Production of Calves with Predetermined Sexunder Field Conditions[J]. Indian Journal of Experimental Biology, 1998, 36(12): 1201-1208
[22] J.F.Garcia, M.F.G.Nogueira, F.Pupim et al. Pregnancy Rates of Blastomere Biopsied Bovine Embryos Frozen in Ethylene Glycol[J]. Theriogenology, 1997,47: 268(Abstract)
[23] 吕碧文，俞颂东，金水仙．牛早期胚胎性别鉴定的PCR方法研究[J]．武汉大学学报
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[28] P.Bredbacka, A.Kankaanp and J.Peippo. PCR-sexing of Bovine Embryos: A Simplified Protocol[J]. Theriogenology, 1995, 44:167-176
[29] S.Ennis, T F Gallagher. A PCR-based Sex-determination Assay in Cattle Based on the Bovine Amelogenin Locus[J]. Animal Genetics, 1994, 25:6,425-427
[30] Machaty Z. Biopsy and Sex Determination by PCR of IVF Bovine Embryos[J]. Journal of Reproduction and Fertility, 1993, 98(2): 467-470
[31] Lehn Jenson,H et al. Deep-freezing of Cow Half and Quarter Embryos[J]. Theriogenology, 1983(15):427-432.
[32] Jaakma-U et al.Sex determination of Embryos Before Transfer[J]. Agraarteadus, 1998,9(3):172-175
[33] Rorie,RW et al. Viability of Demi-Embryos Produced before vs after Freezing[J].Theriogenology, 1986,25:192(Abstract)
[34] Gao JM et al. Simplified Method of Sampling Embryo Cells for Bovine Embryo Sexing and Result of Sexed Embryo Transfer
[35] Herr CM. Rapid,Accurate Sexing of Livestock Embryos[J]. Proceedings of the 4th World Congress on Genetics Applied to Livestock Production, 1990, 334-343
[36] Li-X et al. Studies on Sexing of Bovine Embryo by PCR Method with Double Primer[J]. Journal of Reproduction and Development, 1995, 41(6):J97-J101
[37] Thibier M et al. Bovine Embryo Sexing by a DNA Probe in the Field[J]. Reproduction in Domestic Animals, 1992,27(1):29-33
[38] Eric Aasen and Juan Fernando Medrano. Amplification of the ZFY and ZFX Genes for Sex Identification in Humans, Cattle, Sheep and Goats[J]. Bio/technology, 1990(8):1279-1281
[39] Bredbacka-P. Survival of Biopsied and Sexed Bovine Demi-embryos[J]. Theriogenology, 1994, 41(5): 1023-1031
[40] Pollevick-GD et al. Sex Determination of Bovine Embryos by Restriction Fragment Polymorphisms of PCR Amplified ZFX/ZFY Loci[J]. Bio/technology, 1992,10(7): 805-807
[41] P.Bredbacka et al. A Simplified Protocol for PCR-sexing of Bovine Embryos: A field Trial[J]. Theriogenology, 1996, 45:218 (Abstract)
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[2] 王建辰，章孝荣．动物生殖调控[M]．安徽科学技术出版社，1998．8，第1版
[3] 李馨，杨隽，郭志光．家畜性别控制研究现状与展望[J]．内蒙古畜牧科学，1997(2)：15-18
[4] G.E.Seidel. Sexed Semen Applications in Dairy Cattle[C].Western Dairy Managemnet Conference.1999,April 8-10:183-186
[5] M. Thibier and M.Nibart. The Sexing of Bovine Embryos in the Field[J]. Theriogenology, 1995, 43:71-80
[6] 葛宝生，李善如，张忠诚等．哺乳动物的性别控制．内蒙古畜牧科学，1998(2)：14-17
[7] Stephen S. Wachtel. H-Y antigen in the Study of Sex Determination and Control of Sex Ratio[J]. Theriogenology, 1984, 21:18-27
[8] T.J.Williams. A Technique for Sexing Mouse Embryos by a Visual Colorimeteric Assay of the X-linked Enzyme, Glucose 6-phsphate Dehydrogenase[J]. Theriogenology, 1986, 25:733-738
[9] Peura T et al. A Reliable Sex Determination Assay for Bovine Preimplantation Embryos Using the PCR[J]. Theriogenology, 1991, 35(3): 547-555
[10] B.F. Shea. Determining the Sex of Bovine Embryos Using PCR Results :A 6-year Retrospective Study[J]. Theriogenology, 1999, 51:841-854
[11] K.Roschlau, D.Roschlau, R.Roselius et al. Over 5 Years Experience in Sexing of Bovine Morulae and Blastocysts During Routine Embryo Transfer[J]. Theriogenology, 1997, 47:273 (Abstract)
[12] Lechniak D. the Use of Amelogenin Gene Polymorphism in PCR Embryo Sexing in Bovine IVF Embryos[J]. Journal of Applied Genetics, 1997,38(1):45-49
[13] G.Carbonneau, N.Morin, J.Durocher et al. Viability of Bovine IVF Embryos Biopsied with Microsection or Microaspiration Technique for Sexing[J]. Theriogenology, 1996, 45: 266(abstract)
[14] J.F.Hasler, E.Cardey, J.E.Stokes et al. Nonelectrophoretic PCR-sexing of Bovine Embryos in a Commercial Environment[J]. Theriogenology, 2002, 58:1457-1469
[15] P.Chrenek, L.Boulanger, Y.Heyman et al. Sexing and Multiple Genotype Analysis from a Single Cell of Bovine Embryo[J]. Theriogenology, 2001, 55:1071-1081
[16] 黄淑帧，陈美珏，黄英等．试管牛和试管羊胚胎性别的鉴定[J]．遗传，2000，22：65-68
[17] Yindee Kitiyanant, Jumnian Saikhun, Boripat Siriaroonrat et al. Sex Determination by PCR and Karyotyping of Bovine Embryos at First Cleavage in Vitro[J]. ScienceAsia, 2000, 26:9-13
[18] K.Kameyama et al. Direct Transfer of Bovine Frozen-thawed Embryos Sexed with a Rapid Y-chromosome-detection Assay[J]. Theriogenology, 1996, 45: 227(Abstract)
[19] K.Makondo, G.S.Amiridis, I.A.Jeffcoate et al. Use of the PCR to Sex the Bovine Fetus Using
Cells Recovered by Ultrasound-guided Fetal Fluid Aspiration[J]. Animal Reproduction Science, 1997, 49:125-133
[20] Peippo-J. Detection of Bovine Fetal DNA from Amniotic Fluid Using the PCR. Agricultural and Food Science in Finland, 1996,5(5): 541-546
[21] Taneja-M et al. Rapid Sexing of Bovine Preimplantation Embryos Using PCR: Production of Calves with Predetermined Sexunder Field Conditions[J]. Indian Journal of Experimental Biology, 1998, 36(12): 1201-1208
[22] J.F.Garcia, M.F.G.Nogueira, F.Pupim et al. Pregnancy Rates of Blastomere Biopsied Bovine Embryos Frozen in Ethylene Glycol[J]. Theriogenology, 1997,47: 268(Abstract)
[23] 吕碧文，俞颂东，金水仙．牛早期胚胎性别鉴定的PCR方法研究[J]．武汉大学学报
[24] Totey-SM. Healthy Calves Born from Sexed Embryos. Animal Biotechnology-Bulletin, 1995, 5(1): 9-10
[25] Andre T.Palasz and Reuben J.Mapletoft. Cryopreservation of Mammalian Embryos and Oocytes: Recent Advances[J]. Biotechnology Advances, 1996,14(2):127-149
[26] Xiangzhong Yang and Gary B.Anderson. Micromanipulation of Mammalian Embryos: Principles, Progress and future possibilities[J]. Theriogenology, 1992, 38:315-335
[27] C.M.Herr et al. Micromanipulation of Bovine Embryos for Sex Determination[J]. Theriogenology, 1991, 35:45-54
[28] P.Bredbacka, A.Kankaanp and J.Peippo. PCR-sexing of Bovine Embryos: A Simplified Protocol[J]. Theriogenology, 1995, 44:167-176
[29] S.Ennis, T F Gallagher. A PCR-based Sex-determination Assay in Cattle Based on the Bovine Amelogenin Locus[J]. Animal Genetics, 1994, 25:6,425-427
[30] Machaty Z. Biopsy and Sex Determination by PCR of IVF Bovine Embryos[J]. Journal of Reproduction and Fertility, 1993, 98(2): 467-470
[31] Lehn Jenson,H et al. Deep-freezing of Cow Half and Quarter Embryos[J]. Theriogenology, 1983(15):427-432.
[32] Jaakma-U et al.Sex determination of Embryos Before Transfer[J]. Agraarteadus, 1998,9(3):172-175
[33] Rorie,RW et al. Viability of Demi-Embryos Produced before vs after Freezing[J].Theriogenology, 1986,25:192(Abstract)
[34] Gao JM et al. Simplified Method of Sampling Embryo Cells for Bovine Embryo Sexing and Result of Sexed Embryo Transfer
[35] Herr CM. Rapid,Accurate Sexing of Livestock Embryos[J]. Proceedings of the 4th World Congress on Genetics Applied to Livestock Production, 1990, 334-343
[36] Li-X et al. Studies on Sexing of Bovine Embryo by PCR Method with Double Primer[J]. Journal of Reproduction and Development, 1995, 41(6):J97-J101
[37] Thibier M et al. Bovine Embryo Sexing by a DNA Probe in the Field[J]. Reproduction in Domestic Animals, 1992,27(1):29-33
[38] Eric Aasen and Juan Fernando Medrano. Amplification of the ZFY and ZFX Genes for Sex Identification in Humans, Cattle, Sheep and Goats[J]. Bio/technology, 1990(8):1279-1281
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