Sexing of Sheep Embryos Produced In vitro by Polymerase Chain Reaction and Sex-specific Polymorphism

  • Saravanan, T. (Molecular Virology Lab, Indian Veterinary Research Institute) ;
  • Nainar, A. Mahalinga (Transgenic lab, Department of Animal Biotechnology, Madras Veterinary College) ;
  • Kumanan, K. (Transgenic lab, Department of Animal Biotechnology, Madras Veterinary College) ;
  • Kumaresan, A. (Division of Animal Reproduction, Indian Veterinary Research Institute)
  • Received : 2002.05.27
  • Accepted : 2002.11.04
  • Published : 2003.05.01


The accuracy of Polymerase chain reaction (PCR) assay in sexing of sheep embryos was assessed in this study. A total of 174 ovine embryos produced in vitro at different stages of development (2, 4-8 cell stages, morula and blastocyst) were sexed. The universal primers (P1-5EZ and P2-3EZ) used in this assay amplified ZFY/ZFX-specific sequences and yielded a 445 bp fragment in both sexes. Restriction enzyme analysis of ZFY/ZFX-amplified fragments with Sac I exhibited polymorphism between sexes, three and two fragments in males and in females, respectively. For verification of accuracy, blood samples of known sex were utilized as positive controls in each test. The mean percentages of sex identification by this method at 2 cell, 4-8 cell, morula and blastocyst were $73.00{\pm}5.72$, $89.77{\pm}3.79$, $3.33{\pm}8.08$ and $79.6{\pm}9.09$, espectively with the over all male to female ratio of 1:0.87. It is concluded that the ZFY/ZFX based method is highly reliable for the sexing of sheep embryos.


  1. Bourhis-Le, D., P. Chesne, M. Nibart, J. Marchal, P. Humblot, J. P. Renard and Y. Heyman. 1998. Nuclear transfer from sexed parent embryos in cattle: efficiency and birth of offspring: J. Reprod. Fertil. 113:343-348.
  2. Ennis, S., L. Vaughan and T. F. Gallagher. 1999. The diagnosis of free martinism in cattle using sex-specific DNA sequences. Res. Vet. Sci. 67:111-112.
  3. Fukui, V., A. M. Glen, F. Ganddfi and R. M. Moor. 1988. Ramspecific effects on in vitro fertilization and cleavage of sheep oocytes matured in vitro. J. Reprod. Fertil. 82:337-340.
  4. Totey, S. M., C. H. Pawshe and G. P. Singh. 1993. In vitro maturation and fertilization of buffalo oocytes (Bubalus bubalis): Effects of media, hormones and sera. Theriogenology 39:1153-1171.
  5. Garcia-Muro, E., M. P. Aznar, C. Rodellar and P. Zaragoza. 1997. Sex-specific PCR-RFLPs in the canine ZFY/ZFX loci. Anim. Genet. 28:156.
  6. Leung, W. Y., V. Lindgren, Y. F. C. Lau and T. L. Yang-Feng. 1990. Regional assignments of zinc finger Y-linked gene (ZFY) and related sequences on human and mouse chromosomes. Cytogenet. Cell. Genet. 54:151-153.
  7. Fry, R. C., E. M. Niall, T. L. Simpson, T. J. Squires and J. Reynolds. 1997. The collection of oocytes from bovine ovaries. Theriogenology, 47:977-987.
  8. Pawshe, C. H., S. M. Totey and S. K. Jain, 1994. Effect of methods of recovery of goat oocytes for in vitro maturation and fertilization. Theriogenology 42:117-125.
  9. Betteridge, K. J. 1984. The folklore of sexing. Theriogenology 21:3-6.
  10. Fajfar-Whetstone, C. J., A. Lane Rayburn III, L. B. Schook and M. B. Wheeler. 1993. Sex determination of porcine preimplantation embryos via- Y- chromosome specific DNA sequence. Anim. Biotechnol. 4:183-193.
  11. Boutonnet, J. P. 1999. The Perspectives for the World Sheep Meat Market and its Influence on Future Production Systems and Trends. A review. Asian-Aust. J. Anim. Sci. 12:1123-1128.
  12. 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. Ind. J. Exp. Biol. 36:1201-1208.
  13. Aasen, E. and J. F. Medrano. 1990. Amplification of the ZFY and ZFX genes for sex identification in humans, cattle, sheep and goats. Bio/Technol. 8:1279-1281.
  14. Ennis, S. and T. F. Gallagher. 1994. A PCR-based sex determination assay in cattle based on the bovine amelogenin locus. Anim. Genet. 25:425-427.
  15. Herr, C. M., K. I. Matthaei, K. V. Petrzak and K. C. Reed. 1990. A rapid Y chromosome-detecting ovine embryo sexing assay. Theriogenology 33:245(abstr.).
  16. Montgomery, G. W. and J. A. Sise. 1990. Extraction of DNA from sheep white blood cells. New Zealand J. Agri. Res. 33:437-441.
  17. Peippo, J., M. Kuhtinen and T. Kotilainen. 1995. Sex diagnosis of equine preimplantation embryos using the PCR. Theriogenology 44:619-627.
  18. Snedecor, G. W. and W. G. Cochran. 1994. Statistical methods. 8th edn. Iowa state University Press. USA.
  19. Carvalho, R. V., M. R. Delcampo, A. T. Palasz, Y. Plante and R. J. Mapletoft. 1996. Survival rates and sex ratios of bovine IVF embryos frozen at differential stages on day 7. Theriogenology 45:487-498.
  20. Innis, M. A. and D. H. Gelfand. 1990. Optimization of PCR. In: PCR protocols: A guide to methods and applications. Innis M. A, D.H. Gelfand, J. J. Sninsky and T. J. White (Ed.). Academic Press, San Diego, pp. 1-2.
  21. Pollevick, G. D., S. Giambiagi, Mancardi, L. DeLuca, O. Burrohe, A. C. C Frasch and R. A. Ugalde. 1992. Sex determination of bovine embryos by restriction fragment polymorphisms of PCR amplified ZFY/ZFX loci. Bio/Technol. 10:805-807.
  22. Matthews, M. E. and K. C. Reed. 1991. A DNA sequence that is present in both sexes of Artiodactyla is repeated on the Ychromosome of cattle, sheep and goats. Cytogenet. Cell Genet. 56:40-44.
  23. Saiki, R. H., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis and H. A. Erlich. 1998. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491.
  24. Crawford, A. M. 1999. Recent Advances in Sheep Genome Mapping. A review. Asian-Aust. J. Anim. Sci. 12:1129-1134.

Cited by

  1. Cell culture, sex determination and single cell cloning of ovine transgenic satellite cells in vitro vol.21, pp.1, 2014,
  2. Exposure to maternal obesity alters gene expression in the preimplantation ovine conceptus vol.19, pp.1, 2018,
  3. A genetic method for sex determination in Ovis spp. by interruption of the zinc finger protein, Y-linked (ZFY) gene on the Y chromosome vol.30, pp.9, 2018,