Relationship between Developmental Ability and Cell Number of Day 2 Porcine Embryos Produced by Parthenogenesis or Somatic Cell Nuclear Transfer

  • Uhm, Sang Jun (Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University) ;
  • Gupta, Mukesh Kumar (Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University) ;
  • Chung, Hak-Jae (Hanwoo Experiment Station, National Institute of Animal Science) ;
  • Kim, Jin Hoi (Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University) ;
  • Park, Chankyu (Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University) ;
  • Lee, Hoon Taek (Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University)
  • Received : 2008.06.30
  • Accepted : 2008.12.20
  • Published : 2009.04.01


In vitro produced porcine embryos have potential application in reproductive biotechnology. However, their development potential has been very low. This study evaluated the in vitro developmental ability and quality of cloned and parthenogenetic porcine embryos having 2-4 cells or 5-8 cells on Day 2 of in vitro culture. Analysis of results showed that 2 to 4 cell embryos had higher ability to form blastocysts than 5 to 8 cell embryos (p<0.05). Blastocysts produced from culture of 2 to 4 cell embryos also contained higher cell numbers and had lower BAX:BCLxL transcript ratio than those produced from 5 to 8 cell embryos (p<0.05), thereby suggesting 2 to 4 cell embryos have higher development potential. Further investigation revealed that 5 to 8 cell embryos had higher incidence (100${\pm}$0.0%) of blastomeric fragmentation than 2 to 4 cell embryos (15.2${\pm}$5.5% for parthenogenetic and 27.7${\pm}$7.1% for cloned embryos). This suggests that low development potential of 5 to 8 cell embryos was associated with blastomeric fragmentation. In conclusion, we have shown that morphological selection of embryos based on cell number on Day 2 of in vitro culture could offer a practical and valuable non-invasive means to select good quality porcine embryos.


Supported by : RDA


  1. Barcroft, L. C., A. Hay-Schmidt, A. Caveney, E. Gilfoyle, E. W. Overstrom, P. Hyttel and A. J. Watson. 1998. Trophectoderm differentiation in the bovine embryo: characterization of a polarized epithelium. J. Reprod. Fertil. 114:327-339
  2. Hao, Y., L. Lai, J. Mao, G. S. Im, A. Bonk and R. S. Prather. 2003. Apoptosis and in vitro development of preimplantation porcine embryos derived in vitro or by nuclear transfer. Biol. Reprod. 69:501-507
  3. Kikuchi, K., N. Kashiwazaki, J. Noguchi, A. Shimada, R. Takahashi, M. Hirabayashi, M. Shino, M. Ueda and H. Kaneko. 1999. Developmental competence, after transfer to recipients, of porcine oocytes matured, fertilized, and cultured in vitro. Biol. Reprod. 60:336-340
  4. Liu, L. and D. L. Keefe. 2000. Cytoplasm mediates both development and oxidation-induced apoptotic cell death in mouse zygotes. Biol. Reprod. 62:1828-1834
  5. Meirelles, F., A. R. Caetano, Y. F. Watanabe, P. Ripamonte, S. F. Carambula, G. K. Merighe and S. M. Garcia. 2004. Genome activation and developmental block in bovine embryos. Anim. Reprod. Sci. 82-83:13-20
  6. Puissant, F., M. Van Rysselberge, P. Barlow, J. Deweze and F. Leroy. 1987. Embryo scoring as a prognostic tool in IVF treatment. Hum. Reprod. 2:705-708
  7. Tanghe, S., A. Van Soom, L. Duchateau, H. Nauwynck and A. de Kruif. 2004. Carbohydrates and glycoproteins involved in bovine fertilization in vitro. Mol. Reprod. Dev. 68:492-499
  8. Uhm, S. J., H. M. Chung, C. Kim, H. Shim, N. H. Kim, H. T. Lee and K. S. Chung. 2000a. In vitro development of porcine enucleated oocytes reconstructed by the transfer of porcine fetal fibroblasts and cumulus cells. Theriogenology 54:559-570
  9. Wheeler, M. B., S. G. Clark and D. J. Beebe. 2004. Developments in in vitro technologies for swine embryo production. Reprod. Fertil. Dev. 16:15-25
  10. Yadav, B. R., W. A. King and K. J. Betteridge. 1993. Relationships between the completion of first cleavage and the chromosomal complement, sex, and developmental rates of bovine embryos generated in vitro. Mol. Reprod. Dev. 36:434-439
  11. Yoshioka, K., C. Suzuki, A. Tanaka, I. M. Anas and S. Iwamura. 2002. Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol. Reprod. 66:112-119
  12. Zhang, Y. H., E. S. Song, E. S. Kim, P. Q. Cong, S. H. Lee, J. W. Lee, Y. J. Yi and C. S. Park. 2009. Effects of donor cell passage, size and type on development of porcine embryos derived from somatic cell nuclear transfer. Asian-Aust. J. Anim. Sci. 22:194-200
  13. Comizzoli, P., B. Marquant-Le Guienne, Y. Heyman and J. P. Renard. 2000. Onset of the first S-phase is determined by a paternal effect during the G1-phase in bovine zygotes. Biol. Reprod. 62:1677-1684
  14. Wang, W. H., L. R. Abeydeera, Y. M. Han, R. S. Prather and B. N. Day. 1999. Morphologic evaluation and actin filament distribution in porcine embryos produced in vitro and in vivo Biol. Reprod. 60:1020-1028
  15. Mittwoch, U. 1989. Sex differentiation in mammals and tempo of growth: probabilities vs. switches. J. Theor. Biol. 137:445-55
  16. Park, M. R., S. K. Cho, S. Y. Lee, Y. J. Choi, J. Y. Park, D. N. Kwon, W. J. Son, S. S. Paik, T. Kim, Y. M. Han and J. H. Kim. 2005. A rare and often unrecognized cerebromeningitis and hemodynamic disorder: a major cause of sudden death in somatic cell cloned piglets. Proteomics 5:1928-1939
  17. Uhm, S. J., M. K. Gupta, J. H. Yang, S. H. Lee and H. T. Lee. 2007b. Selenium improves the developmental ability and reduces the apoptosis in porcine parthenotes. Mol. Reprod. Dev. 74:1386-1394
  18. Yin, X. J., S. K. Cho, M. R. Park, Y. J. Im, J. J. Park, B. Jong Sik, D. N. Kwon, S. H. Jun, N. H. Kim and J. H. Kim. 2003. Nuclear remodelling and the developmental potential of nuclear transferred porcine oocytes under delayed-activated conditions. Zygote. 11:167-174
  19. Han, Y. M., W. H. Wang, L. R. Abeydeera, A. L. Petersen, J. H. Kim, C. Murphy, B. N. Day and R. S. Prather. 1999. Pronuclear location before the first cell division determines ploidy of polyspermic pig embryos. Biol. Reprod. 61:1340-1346
  20. McKiernan, S. H. and B. D. Bavister. 1994. Timing of development is a critical parameter for predicting successful embryogenesis. Hum. Reprod. 9:2123-2129
  21. Brussow, K. P., H. Torner, W. Kanitz and J. Ratky. 2000. In vitro technologies related to pig embryo transfer. Reprod. Nutr. Dev. 40:469-480
  22. Kim, J. Y., S. B. Kim, M. C. Park, H. Park, Y. S. Park, H. D. Park, J. H. Lee and J. M. Kim. 2007. Addition of macromolecules to PZM-3 culture medium on the development and hatching of in vitro porcine embryos. Asian-Aust. J. Anim. Sci. 20:1820-1826
  23. Uhm, S. J., N. H. Kim, T. Kim, H. M. Chung, K. H. Chung, H. T. Lee and K. S. Chung. 2000b. Expression of enhanced green fluorescent protein (EGFP) and neomycin resistant (Neo(R)) genes in porcine embryos following nuclear transfer with porcine fetal fibroblasts transfected by retrovirus vector. Mol. Reprod. Dev. 57:331-337<1::AID-MRD1>3.0.CO;2-X
  24. Gupta, M. K., S. J. Uhm and H. T. Lee. 2007c. Differential but beneficial effect of phytohemagglutinin on efficiency of in vitro porcine embryo production by somatic cell nuclear transfer or in vitro fertilization. Mol. Reprod. Dev. 74:1557-1567
  25. Gupta, M. K., S. J. Uhm and H. T. Lee. 2008a. Sexual maturity and reproductive phase of oocyte donor influence the developmental ability and apoptosis of cloned and parthenogenetic porcine embryos. Anim. Reprod. Sci. 108:107-121
  26. Gupta, M. K., S. J. Uhm, S. H. Lee and H. T. Lee. 2008b. Role of nonessential amino acids on porcine embryos produced by parthenogenesis or somatic cell nuclear transfer. Mol. Reprod. Dev. 75:588-597
  27. Kawarsky, S. J., P. K. Basrur, R. B. Stubbings, P. J. Hansen and W. A. King. 1996. Chromosomal abnormalities in bovine embryos and their influence on development. Biol. Reprod. 54:53-59
  28. Gupta, M. K., S. J. Uhm, D. W. Han and H. T. Lee. 2007a. Embryo quality and production efficiency of porcine parthenotes is improved by phytohemagglutinin. Mol. Reprod. Dev. 74:435-444
  29. Jang, H. Y., H. S. Kong, S. S. Lee, K. D. Choi, G. J. Jeon, B. K. Yang, C. K. Lee and H. K. Lee. 2004. Expression of the antioxidant enzyme and apoptosis genes in in vitro maturation/in vitro fertilization porcine embryos. Asian-Aust. J. Anim. Sci. 17:33-38
  30. Leese, H. J., I. Donnay and J. G. Thompson. 1998. Human assisted conception: a cautionary tale. Lessons from domestic animals. Hum. Reprod. 13(Suppl 4):184-202
  31. Lindner, G. M. and R. W. Wright, Jr. 1983. Bovine embryo morphology and evaluation. Theriogenology 20:407-416
  32. Verma, P. J., Z. T. Du, L. Crocker, R. Faast, C. G. Grupen, S. M. McIlfatrick, R. J. Ashman, I. G. Lyons and M. B. Nottle. 2000. In vitro development of porcine nuclear transfer embryos constructed using fetal fibroblasts. Mol. Reprod. Dev. 57:262-9<262::AID-MRD8>3.0.CO;2-X
  33. Ziebe, S., K. Petersen, S. Lindenberg, A. G. Andersen, A. Gabrielsen and A. N. Andersen. 1997. Embryo morphology or cleavage stage: how to select the best embryos for transfer after in-vitro fertilization. Hum. Reprod. 12:1545-1549
  34. Iwasaki, S., N. Yoshiba, H. Ushijima, S. Watanabe and T. Nakahara. 1990. Morphology and proportion of inner cell mass of bovine blastocysts fertilized in vitro and in vivo. J. Reprod. Fertil. 90:279-284
  35. Im, G. S., B. S. Yang, L. Lai, Z. Liu, Y. Hao and R. S. Prather. 2005. Fragmentation and development of preimplantation porcine embryos derived by parthenogenetic activation and nuclear transfer. Mol. Reprod. Dev. 71:159-165
  36. Ferry, L., P. Mermillod, A. Massip and F. Dessy. 1994. Bovine embryos cultured in serum-poor oviduct-conditioned medium need cooperation to reach the blastocyst stage. Theriogenology 42:445-453
  37. Kim, D. Y. and S. L. McElroy. 2008. Optimization of procedure for efficient gene transfer into porcine somatic cells with lipofection. Asian-Aust. J. Anim. Sci. 21:648-656
  38. Eid, L. N., S. P. Lorton and J. J. Parrish. 1994. Paternal influence on S-phase in the first cell cycle of the bovine embryo. Biol. Reprod. 51:1232-1237
  39. Mateusen, B., A. Van Soom, D. G. Maes, I. Donnay, L. Duchateau and A. S. Lequarre. 2005. Porcine embryo development and fragmentation and their relation to apoptotic markers: a cinematographic and confocal laser scanning microscopic study. Reprod. 129:443-452
  40. Kure-bayashi, S., M. Miyake, K. Okada and S. Kato. 2000. Successful implantation of in vitro-matured, electro-activated oocytes in the pig. Theriogenology 53:1105-1119
  41. Uhm, S. J., M. K. Gupta, T. Kim and H. T. Lee. 2007a. Expression of enhanced green fluorescent protein in porcine- and bovinecloned embryos following interspecies somatic cell nuclear transfer of fibroblasts transfected by retrovirus vector. Mol. Reprod. Dev. 74:1538-1547
  42. Vandaele, L., B. Mateusen, D. Maes, A. de Kruif and A. Van Soom. 2006. Is apoptosis in bovine in vitro produced embryos related to early developmental kinetics and in vivo bull fertility? Theriogenology 65:1691-1703
  43. Kane, M. T., P. M. Morgan and C. Coonan. 1997. Peptide growth factors and preimplantation development. Hum. Reprod. Update. 3:137-157
  44. Racowsky, C., K. V. Jackson, N. A. Cekleniak, J. H. Fox, M. D. Hornstein and E. S. Ginsburg. 2000. The number of eight-cell embryos is a key determinant for selecting day 3 or day 5 transfer. Fertil. Steril. 73:558-564
  45. Ebner, T., C. Yaman, M. Moser, M. Sommergruber, W. Polz and G. Tews. 2001. Embryo fragmentation in vitro and its impact on treatment and pregnancy outcome. Fertil. Steril. 76:281-285
  46. Hong, S. B., S. J. Uhm, H. Y. Lee, C. Y. Park, M. K. Gupta, B. H. Chung, K. S. Chung and H. T. Lee. 2005. Developmental ability of bovine embryos nuclear transferred with frozenthawed or cooled donor cells. Asian-Aust. J. Anim. Sci. 18:1242-1248
  47. Lee, S. Y., J. Y. Park, Y. J. Choi, S. K. Cho, J. D. Ahn, D. N. Kwon, K. C. Hwang, S. J. Kang, S. S. Paik, H. G. Seo, H. T. Lee and J. H. Kim. 2007. Comparative proteomic analysis associated with term placental insufficiency in cloned pig. Proteomics 7:1303-1315
  48. Lonergan, P., H. Khatir, F. Piumi, D. Rieger, P. Humblot and M. P. Boland. 1999. Effect of time interval from insemination to first cleavage on the developmental characteristics, sex ratio and pregnancy rate after transfer of bovine embryos. J. Reprod. Fertil. 117:159-167
  49. Betthauser, J., E. Forsberg, M. Augenstein, L. Childs, K. Eilertsen, J. Enos, T. Forsythe, P. Golueke, G. Jurgella, R. Koppang, T. Lesmeister, K. Mallon, G. Mell, P. Misica, M. Pace, M. Pfister-Genskow, N. Strelchenko, G. Voelker, S. Watt, S. Thompson and M. Bishop. 2000. Production of cloned pigs from in vitro systems. Nat. Biotechnol. 18:1055-1059
  50. Viuff, D., P. J. Hendriksen, P. L. Vos, S. J. Dieleman, B. M. Bibby, T. Greve, P. Hyttel and P. D. Thomsen. 2001. Chromosomal abnormalities and developmental kinetics in in vivo-developed cattle embryos at days 2 to 5 after ovulation. Biol. Reprod. 65:204-208
  51. Lai, L., D. Kolber-Simonds, K. W. Park, H. T. Cheong, J. L. Greenstein, G. S. Im, M. Samuel, A. Bonk, A. Rieke, B. N. Day, C. N. Murphy, D. B. Carter, R. J. Hawley and R. S. Prather. 2002. Production of alpha-1,3-galactosyltransferase knockout pigs by nuclear transfer cloning. Sci. 295:1089-1092
  52. Miyoshi, K. and E. Sato. 2000. Recent advances in cloning technology in the pig. Asian-Aust. J. Anim. Sci. 13:258-265
  53. Gupta, M. K., S. J. Uhm and H. T. Lee. 2007b. Cryopreservation of immature and in vitro matured porcine oocytes by solid surface vitrification. Theriogenology 67:238-248

Cited by

  1. Maturation on the Developmental Competence of Cloned Bovine Embryos vol.50, pp.2, 2015,