DOI QR코드

DOI QR Code

Sexing Goat Embryos by PCR Amplification of X- and Y- chromosome Specific Sequence of the Amelogenin Gene

  • Chen, A-qin ;
  • Xu, Zi-rong ;
  • Yu, Song-dong
  • Received : 2007.02.06
  • Accepted : 2007.06.19
  • Published : 2007.11.01

Abstract

The objective of this study was to develop a simplified, efficient, and accurate protocol for sexing goat embryos. Based on the amelogenin gene located on the conservation region of X- and Y- chromosomes, a pair of primers was utilized and the system of PCR was established to amplify a 262 bp fragment from the X- chromosome in female goats, and a 262 bp fragment from X- chromosome and 202 bp fragment from the Y- chromosome in male goats, respectively. The accuracy and specificity of the primers were assessed using DNA template extracted from goat whole blood sample of known sex. 100% (10/10) concordance was obtained by using the PCR assay. Fifty-one biopsied embryos were transferred into 25 recipient goats on the same day that the embryos were collected and sex of the kid was confirmed after parturition. Eighteen kids of predicted sex were born. The biopsied samples from 51 goat embryos were amplified with 100% efficiency and 94.7% accuracy. In conclusion, our results indicated that PCR sexing protocols based on the amelogenin gene is highly reliable and suitable for sex determination of goats.

Keywords

Goat Embryos;Amelogenin Gene;Sexing;PCR

References

  1. Andersib, G. B. 1987. Identification of embryonic sex by detection of H-Y antigen. Theriogenology 27:87-97.
  2. Aasen, E. and J. F. Medrano. 1990. Amplification of the ZFY and ZFX genes for sex identification in human, cattle, sheep, and goats. Bio. Technol. 8:1279-1281. https://doi.org/10.1038/nbt1290-1279
  3. Alves, B. C. A., V. F. M. Hossepian De Lima, C. M. Teixeira and C. A. Moreira-Filho. 2003. Use of primers derived from a new sequence of the bovine Y chromosome for sexing Bos Taurus and Bos indicus embryos. Theriogenology 56:1415-1419.
  4. Bredbacka, P. and J. Peippo. 1992. Sex diagnosis of ovine and bovine embryos by enzymatic amplification and digestion of DNA from the ZFY/ZFX locus. Agric. Sci. Fin. 2:233-238.
  5. El-Gayar, M. and W. Holtz. 2005. Transfer of sexed caprine blastocysts freshly collected or derived from cultured morulae. Small. Rumin. Res. 57:151-156. https://doi.org/10.1016/j.smallrumres.2004.06.022
  6. Chen, C. M., C. L. Hu, C. H. Wang, C. M. Hung, H. K. Wu, K. B. Choo and W. T. K. Cheng. 1999. Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific lymorphic fragments in the amelogenin gene. Mol. Reprod. Dev. 54:20-214.
  7. Dhanda, J. S., D. G. Taylor, P. J. Murray, R. B. Pegg and P. J. Shand. 2003. Goat meat production: present status and future possibilities. Asian-Auat. J. Anim. Sci. 16:1842-1852. https://doi.org/10.5713/ajas.2003.1842
  8. Chang, Z. L., X. Z. Fan, M. J. Luo, Z. Y. Wu and J. H. Tan. 2006. Factors affecting superovulation and embryo transfer in Boer goats. Asian-Aust. J. Anim. Sci. 19:341-346. https://doi.org/10.5713/ajas.2006.341
  9. Huang, J. M., Y. Wei and X. W. Tan. 2007. Use of the nonelectrophoretic method to detect testis specific protein gene for sexing in preimplantation bovine embryos. Asian-Aust. J. Anim. Sci. 20:866-871. https://doi.org/10.5713/ajas.2007.866
  10. Holtz, W., R. J. T. A. Pereira, Suyadi, X. Wang, G. Padilla and B. Sohnrey. 2000. Collection of goat embryos via transcervical route. In: Proc. 7th Int. Conf. Goats, Nouzilly, 490-491.
  11. Gutierrez-Adan, A., E. Behboodi, J. F. Medrano, J. D. Murray and G. B. Anderson. 1996. Nested PCR primers for sex determination across a range of mammalian orders. Theriogenology 45:189. https://doi.org/10.1016/0093-691X(96)84662-0
  12. Herr, C. M. and K. C. Reed. 1991. Micromanipulation of bovine embryos for sex determination. Theriogenology 35:45-54. https://doi.org/10.1016/0093-691X(91)90147-6
  13. Ennis, S. and S. Gallagher. 1994. A PCR-based sex-determination assay in cattle based on the bovine amelogenin locus. Anim. Genet. 25:425-427. https://doi.org/10.1111/j.1365-2052.1994.tb00533.x
  14. Pfeiffer, I. and B. Brenig. 2005. X- and Y-chromosome specific variants of the amelogenin gene allow sex determination in sheep (Ovis aries) and European rea deer (Cervus elaphus). BMC. Genet. 6:16. https://doi.org/10.1186/1471-2156-6-16
  15. Ng, A., K. Sathasivam, S. Laurie and E. Notaria. 1996. Determination of sex and chimaerism in the domestic sheep by DNA amplification using HMG-box and icrosatellite sequences. Anim. Reprod. Sci. 41:131-139. https://doi.org/10.1016/0378-4320(95)01444-6
  16. Moore, S. S., L. L. Sargeant, T. J. King, J. S. Mattick, M. Georges and D. J. Hetzel. 1991. The conservation of dinucleotide microsatellites among mammalian genomes allows the use of heterologous PCR primer pairs in closely related species. Genomics. 10:654-660. https://doi.org/10.1016/0888-7543(91)90448-N
  17. Monk, M. and A. H. Handyside. 1988. Sexing of preimplantation mouse embryos by measurement of X-linked gene dosage in a single blastomere. J. Reprod. Fert. 82:365-368. https://doi.org/10.1530/jrf.0.0820365
  18. Malakar, D., S. K. Das, M. Mukesh, M. Sodhi and S. L. Goswarmi. 2007. Production of kids from in vitro fertilized goat embryos and their parentage assessment using microsatellite markers. Asian-Aust. J. Anim. Sci. 20:842-907. https://doi.org/10.5713/ajas.2007.842
  19. Ju, J. C., Y. C. Chang, W. T. Huang, P. C. Tang and S. P. Cheng. 2001. Superovulation and transplantation of demi- and aggregated embryos in rabbits. Asian-Aust. J. Anim. Sci. 14:455-461. https://doi.org/10.5713/ajas.2001.455
  20. Mara, L., S. Pilichi, A. Sanna, C. Accardo, B. Chessa, F. Chessa, M. Dattena, G. Bomboi and P. Cappai. 2004. Sexing of in vitro produced ovine embryos by duplex PCR. Mol.Reprod. Deve. 69:35-42. https://doi.org/10.1002/mrd.20147
  21. Kochhar, H. S., B. C. Buckrell, J. W. Pollard and W. A. King. 2000. Production of sexed lambs after biopsy of ovine blastocyts produced in vitro. Can. Vet. J. 41:398-400.
  22. Leoni, G., F. Schmoll, S. Ledda, L. Bogliolo, G. Marogna, P. Calvia and S. Naitana. 1996. Sex determination in goat embryos. In: Proc. Conf. Adv. Biotechnol. Agriculture, Nutrition and Environment, S5-3-7(Abstr.), Ferrara, Itlay, 8-11.
  23. Lindner, G. M. and R. W. Wright. 1983. Bovine embryo morphology and evaluation. Theriogenology 20:407-416. https://doi.org/10.1016/0093-691X(83)90201-7
  24. King, W. A. 1984. Sexing embryos by cytological methods. Theriogenology 21:7-17. https://doi.org/10.1016/0093-691X(84)90302-9
  25. Phua, A. C. Y., R. B. Abdullah and Z. Mohamed. 2003. A PCRbased sex determination method for possible application in caprine gender selection by simultaneous amplification of the Sry and Aml-X genes. J. Reprod. Dev. 49:307-311. https://doi.org/10.1262/jrd.49.307
  26. Sambook, J. and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual third ed. Cold Spring Harbor Laboratory Press.
  27. Schomoll, F. and K. Schellander. 1996. Sex determination of porcine embryos by restriction fragment length polymorphism analysis of ZFX/ZFY loci. Theriogenology 45:255 (Abstr.). https://doi.org/10.1016/0093-691X(96)84728-5
  28. Rao, K. B. and K. B. Totey. 1992. Sex determination in sheep and goats using bovine Y-chromosome specific primers via polymerase chain reaction: potential for embryo sexing. Indian. J. Exp. Biol. 30:775-777.
  29. Saravanan, T., A. M. Nainar, K. Kumaresan and A. Kumarewsan. 2003. Sexing of sheep embryos produced in vitro by polymerase chain reaction and sex-specific polymorphism. Asian-Aust. J. Anim. Sci. 16:650-654. https://doi.org/10.5713/ajas.2003.650
  30. Pollock, D. P., J. P. Kutzko, E. Birck-Wilson, J. L. Williams, Y. Echelard and H. M. Meade. 1999. Transgenic milk as a method for the production of recombinant antibodies. J. Immun. Meth. 231:147-157. https://doi.org/10.1016/S0022-1759(99)00151-9
  31. Sullivan, K. M., A. Mannucci, C. P. Kimpton and P. Gill. 1993. A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin. Biotechniques. 15:636-641.
  32. Sohn, S. H., C. Y. Lee, E. K. Ryu, J. Y. Han, A. S. Multani and S. Pathak. 2002. Rapid sex identification of chicken by fluorescence in situ hybridization using a W chromosomespecific DNA probe. Asian-Aust. J. Anim. Sci. 15:1531-1535. https://doi.org/10.5713/ajas.2002.1531
  33. Zeleny, R., A. Bernreuther, H. Schimmel and J. Pauwels. 2002. Evaluation of PCR-based beef sexing methods. J. Agric. Food. Chem. 50:4169-4175. https://doi.org/10.1021/jf020156d
  34. Weikard, R., C. Pitra and C. Kuhn. 2006. Amelogenin crossamplification in the family bovidae and its application for sex determination. Mol. Reprod. Dev. 73:1333-1337. https://doi.org/10.1002/mrd.20554
  35. Taneja, M., K. B. C. Appa Rao, S. Gangawane, S. G. Zawar and S. M. Totey. 1998. Rapid sexing of bovine preimplantation embryos using PCR: Production of calves with predetermined sex under field conditions. Indian. J. Exp. Biol. 36:1201-1208.
  36. Suyadi, S. B. and W. Holtz. 2000. Transcervical embryo collection in Boer goats. Small. Rumin. Res. 36:195-200. https://doi.org/10.1016/S0921-4488(99)00164-9
  37. Verlinsky, Y., S. Rechitsky, M. Freidine, J. Cieslak, C. Strom and A. Lifchez. 1996. Birth of a healthy girl after preimplantation gender determination using a combination of polymerase chain reaction and fluorescent in situ hybridization analysis. Preimplantation Genetics Group. Fertil. Steril. 65:358-360. https://doi.org/10.1016/S0015-0282(16)58099-6
  38. Miller, J. R. 1991. Isolation of Y chromosome-specific sequences and their use in embryo sexing. Reprod. Domest. Anim. 26:58-65. https://doi.org/10.1111/j.1439-0531.1991.tb01515.x

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Acknowledgement

Supported by : Zhejiang Provincial Science and Technology Council