In vitro Fertilization and Development of Pig Oocytes Inseminated with Boar Sperm by Different Sperm Washing Media after Thawing of the Frozen Straws

  • Yi, Y.J. (Division of Animal Science and Resources, Research Center for Transgenic Cloned Pigs Chungnam National University) ;
  • Ko, H.J. (Division of Animal Science and Resources, Research Center for Transgenic Cloned Pigs Chungnam National University) ;
  • Lee, S.H. (College of Visual Image & Health, Kongju National University) ;
  • Yang, C.B. (National Livestock Research Institute, RDA) ;
  • Son, D.S. (National Livestock Research Institute, RDA) ;
  • Kim, H.K. (National Livestock Research Institute, RDA) ;
  • Park, C.S. (Division of Animal Science and Resources, Research Center for Transgenic Cloned Pigs Chungnam National University)
  • Received : 2003.01.06
  • Accepted : 2003.09.24
  • Published : 2004.02.01


This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.


Supported by : Korea Science & Engineering Foundation


  1. Abeydeera, L. R. and B. N. Day. 1997. In vitro penetration of pig oocytes in a modified Tris-buffered medium: Effect of BSA, caffeine and calcium. Theriogenology 48:537-544.
  2. Abeydeera, L. R., W. H. Wang, T. C. Cantley, A. Rieke and B. N. Day. 1998. Coculture with follicular shell pieces can enhance the developmental competence of pig oocytes after in vitro fertilization: relevance to intracellular glutathione. Biol. Reprod. 58:213-218.
  3. Almlid, T., S. E. Staven and L. A. Johnson. 1987. Fertility evaluation of the straw freezing technique for boar semen under practical artificial insemination conditions. Zuchthygiene 22:193-202.
  4. Almlid, T. and L. A. Johnson. 1988. Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws. J. Anim. Sci. 66:2899-2905.
  5. Almlid, T. L., R. N. Clarke, V. G. Pursel and L. A. Johnson. 1989. Effectiveness of in vitro methods for predicting in vivo fertilizing capacity of boar spermatozoa cryopreserved with 2 or 4% glycerol. Zuchthygiene 24:8-15.
  6. Clarke, R. N. and L. A. Johnson. 1987. Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro. Gamete Res. 16:193-204.
  7. Hofmo, P. O. and T. Almlid. 1991. Recent developments in freezing of boar semen with special emphasis on cryoprotectants. In Boar Semen Preservation, Vol. 2, Proceedings of the 2nd International Congress on Boar Semen Preservation. Reprod. Domest. Anim. 1:111-122.
  8. Wang, W. H., Z. Machaty, L. R. Abeydeera, R. S. Prather and B. N. Day. 1999. Time course of cortical and zona reaction of pig oocytes upon intracellular calcium increase induced by thimerosal. Zygote 7:79-86.
  9. Yamauchi, N. and T. Nagai. 1999. Male pronuclear formation in denuded porcine oocytes after in vitro maturation in the presence of cysteamine. Biol. Reprod. 61:828-833.
  10. Yi, Y. J., Y. A. Kwon, H. J. Ko and C. S. Park. 2002. Effects of diluent component, freezing rate, thawing time and thawing temperature on acrosome morphology and motility of frozenthawed boar sperm. Asian-Aust. J. Anim. Sci. 15(11):1553-1558.
  11. Marchal, R., J. Peláez, M. Terqui and P. Mermillod. 2002. Effect of sperm survival and CTC staining pattern on in vitro fertilization of porcine oocytes. Theriogenology 57:1917-1927.
  12. SAS Institute Inc. 1996. SAS/STAT guide for personal computer 6.12. SAS Inst. Inc., Cary, NC., USA.
  13. Mattioli, M., M. L. Bacci, G. Galeati and E. Seren. 1989. Developmental competence of pig oocytes matured and fertilized in vitro. Theriogenology 31:1201-1207.
  14. Funahashi, H. and T. Nagai. 2001. Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine. Mol. Reprod. Dev. 58:424-431.
  15. Wang, W. H., L. R. Abeydeera, R. S. Prather and B. N. Day. 1998. Morphologic comparison of ovulated and in vitro matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization. Mol. Reprod. Dev. 49:308-316.
  16. Gandhi, A. P., M. Lane, D. K. Gardner and R. L. Krisher. 2001. Substrate utilization in porcine embryos cultured in NCSU23 and G1.2/G2.2 sequential culture media. Mol. Reprod. Dev. 58:269-275.
  17. Rath, D. and H. Niemann. 1997. In vitro fertilization of porcine oocytes with fresh and frozen-thawed ejaculated or frozenthawed epididymal semen obtained from identical boars. Theriogenology 47:785-793.
  18. Weitze, K. F., F. Fazano, I. R. Scheid, I. Wentz and D. Rath. 1986. Fertility of boar spermatozoa frozen in 5 ml macrotubes and 1 ml minitubes. In Proceedings of the 9th Congress of the International Pig Veterinary Society. Barcelona, 80.
  19. Crabo, B. G. and G. D. Dial. 1992. Artificial insemination in swine. Vet. Clin. North Am. Food. Anim. Pract. 8:533-544.
  20. Wang, W. H., L. R. Abeydeera, L. R. Fraser and K. Niwa. 1995. Functional analysis using chlortetracycline fluorescence and in vitro fertilization of frozen-thawed ejaculated spermatozoa incubated in a protein-free chemically defined medium. J. Reprod. Fertil. 104:305-313.
  21. Johnson, L. A. 1985. Fertility results using frozen boar spermatozoa: 1970 to 1985 In: Deep Freezing of Boar Semen (Ed. L. A. Johnson and K. Larsson). Swedish University of Agricultural Science, Uppsala, pp. 199-222.
  22. Courtens, J. L. and M. Paquignon. 1985. Ultrastructural of fresh, frozen-thawed spermatozoa. In: Deep Freezing of Boar Semen (Ed. L. A. Johnson and K. Larsson). Swedish University of Agricultural Science, Uppsala, pp. 61-87.
  23. Kikuchi, K., N. Kashiwazaki, T. Nagai, J. Noguchi, A. Shimada, R. Takahashi, M. Hirabayashi, M. Shino, M. Ueda and H. Kaneko. 1999. Reproduction in pigs using frozen-thawed spermatozoa from epididymal stored at 4$^{\circ}C$. J. Reprod. Dev. 45:345-350.
  24. Martinez, E. A., J. M. Vazquez, C. Matas, J. Gadea, M. I. Alonso and J. Roca. 1996. Oocyte penetration by fresh or stored diluted boar spermatozoa before and after in vitro capacitation treatments. Biol. Reprod. 55(1):134-140.
  25. Watson, P. F. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 7:871-891.
  26. Funahashi, H. and B. N. Day. 1997. Advances in in vitro production of porcine embryos. J. Reprod. Fertil. 52 (Suppl.):271-283.

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