Effects of Sperm Pretreatments and In vitro Culture Systems on Development of In vitro Fertilized Embryos Derived from Prepubertal Boer Goat Oocytes in China

  • Lv, Lihua (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Yue, Wenbin (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Liu, Wenzhong (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Ren, Youshe (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Li, Fuzhong (College of Animal Science and Technology, Shanxi Agricultural University) ;
  • Lee, Kyung-Bon (Laboratory of Mammalian Reproductive Biology and Genomics, Departments of Animal Science and Physiology, Michigan State University) ;
  • Smith, George W. (College of Animal Science and Technology, Shanxi Agricultural University,Laboratory of Mammalian Reproductive Biology and Genomics, Departments of Animal Science and Physiology, Michigan State University)
  • Received : 2009.01.22
  • Accepted : 2009.05.06
  • Published : 2009.07.01


Use of oocytes from prepubertal animals for in vitro embryo production holds potential application for reducing generation intervals and increasing genetic progress through embryo transfer. The objective of these studies was to compare the effect of three sperm pretreatments (prior to in vitro fertilization) and seven embryo culture protocols on fertilization rate and (or) subsequent development of in vitro fertilized embryos derived from oocytes harvested from ovaries of 1-6 month old prepubertal Boer goats in China. Cleavage rates were highest for embryos fertilized with heparin-treated versus calcium ionophore- or caffeine-treated sperm. Similar rates of blastocyst development were observed using heparin- and ionophore-treated sperm, which were higher than obtained with caffeine-treated sperm. No differences in cleavage or blastocyst rates were observed following embryo culture in basal medias (synthetic oviductal fluid (SOF), Charles Rosenkrans 1 (CR1) or tissue culture medium-199 (TCM-199)) containing 10% fetal bovine serum (FBS). Cumulus or oviductal cell co-culture did not enhance cleavage or blastocyst rates relative to culture in SOF+10% FBS. Replacement of FBS in SOF medium with 0.3% BSA increased cleavage rates, but did not increase rates of blastocyst development. Sequential culture in SOF+0.3% BSA followed by SOF+10% FBS increased blastocyst yield versus continuous culture in SOF+10% FBS and tended to increase blastocyst yield versus continuous culture in SOF+0.3% BSA. These results demonstrate a pronounced effects of sperm pretreatments and in vitro embryo culture systems on rates of blastocyst development and provide a potential protocol (sperm pretreatment with heparin and sequential embryo culture in SOF+0.3% BSA followed by SOF+10% FBS) for generation of the significant numbers of in vitro produced blastocysts from oocytes of prepubertal Boer goats necessary for application of embryo transfer in rural regions of China for distribution of Boer goat genetics.


Embryo Culture;In vitro Fertilization;Oocyte;Prepubertal Goat;Sperm Pretreatment


  1. Baldassarre, H., B. Wang, C. L. Keefer, A. Lazaris and C. N. Karatzas. 2004. State of the art in the production of transgenic goats. Reprod. Fertil. Dev. 16:465-470
  2. Cognie, Y., N. Poulin, Y. Locatelli and P. Mermillod. 2004. Stateof-the-art production, conservation and transfer of in-vitroproduced embryos in small ruminants. Reprod. Fertil. Dev. 16:437-445
  3. Cox, J. F., F. Saravia, M. Briones and A. S. Maria. 1995. Dosedependent effect of heparin on fertilizing ability of goat spermatozoa. Theriogenology 44:451-460
  4. Gardner, D. K. 2008. Dissection of culture media for embryos: the most important and less important components and characteristics. Reprod. Fertil. Dev. 20:9-18
  5. Gutierrez-Adan, A., P. Lonergan, D. Rizos, F. A. Ward, M. P. Boland, B. Pintado and J. De La Fuente. 2001. Effect of the in vitro culture system on the kinetics of blastocyst development and sex ratio of bovine embryos. Theriogenology 55:1117-1126
  6. Keskintepe, L., G. M. Darwish, A. T. Kenimer and B. G. Brackett. 1994a. Term development of caprine embryos derived from immature oocytes in vitro. Theriogenology 42:527-535
  7. Pinyopummintr, T. and B. D. Bavister. 1994. Development of bovine embryos in a cell-free culture medium: Effects of type of serum, timing of its inclusion and heat inactivation. Theriogenology 41:1241-1249
  8. Rizos, D., A. Gutierrez-Adan, S. Perez-Garnelo, J. De La Fuente, M. P. Boland and P. Lonergan. 2003. Bovine embryo culture in the presence or absence of serum: implications for blastocyst development, cryotolerance, and messenger RNA expression. Biol. Reprod. 68:236-243
  9. Salicioni, A. M., M. D. Platt, E. V. Wertheimer, E. Arcelay, A. Allaire, J. Sosnik and P. E. Visconti. 2007. Signaling pathways involved in sperm capacitation. Soc. Reprod. Fertil. Suppl. 65: 245-259
  10. Thompson, J. G., N. W. Allen, L. T. McGowan, A. C. Bell, M. G. Lambert and H. R. Tervit. 1998. Effect of delayed supplementation of fetal calf serum to culture medium on bovine embryo development in vitro and following transfer. Theriogenology 49:1239-1249
  11. Mishra, O. P., J. N. Pandey and P. G. Gawande. 2003. Study on biochemical constituents of caprine ovarian follicular fluid after superovulation. Asian-Aust. J. Anim. Sci. 16(12):1711-1715
  12. Younis, A. I., K. A. Zuelke, K. M. Harper, M. A. Oliveira and B. G. Brackett. 1991. In vitro fertilization of goat oocytes. Biol. Reprod. 44:1177-1182
  13. Chang, Z. L., X. 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(3):341-346
  14. Duby, R. T., P. Damiani, C. R. Looney, R. A. Fissore and J. M. Robl. 1996. Prepuberal calves as oocyte donors: Promises and problems. Theriogenology 45:121-130
  15. Pereira, R. J., R. K. Tuli, S. Wallenhorst and W. Holtz. 2000. The effect of heparin, caffeine and calcium ionophore A23187 on in vitro induction of the acrosome reaction in frozen-thawed bovine and caprine spermatozoa. Theriogenology 54:185-192
  16. Cox, J. F., J. Avila, F. Saravia and A. Santa Maria. 1994. Assessment of fertilizing ability of goat spermatozoa by in vitro fertilization of cattle and sheep intact oocytes. Theriogenology 41:1621-1629
  17. Park, H. S., S. Y. Jung, T. S. Kim, J. K. Park, T. S. Moon, S. P. Hong, J. I. Jin, J. S. Lee, J. H. Lee, S. H. Sohn, C. Y. Lee and Y. S. Moon. 2007. Production of cloned Korean native goat (Capra hircus) by somatic cell nuclear transfer. Asian-Aust. J. Anim. Sci. 20(4):487-495
  18. Yonghong, H. 2002. Utilization and development of Boer goats in China. 2001 Conference on Boer goats in China. pp. 21-24
  19. Young, L. E., K. D. Sinclair and I. Wilmut. 1998. Large offspring syndrome in cattle and sheep. Rev. Reprod. 3:155-163
  20. Yadav, P. S., A. Saini, A. Kumar and G. C. Jain. 1998. Effect of oviductal cell co-culture on cleavage and development of goat IVF embryos. Anim. Reprod. Sci. 51:301-306
  21. Yi, Y. J., S. H. Lee and C. S. Park. 2004. Comparison of semen characteristics, frozen-thawed sperm viability, testosterone concentration and embryo development between Yorkshire boar A and B. Asian-Aust. J. Anim. Sci. 17(5):612-616
  22. Marquez, B. and S. S. Suarez. 2004. Different signaling pathways in bovine sperm regulate capacitation and hyper activation. Biol. Reprod. 70:1626-1633
  23. Rinaudo, P. and R. M. Schultz. 2004. Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos. Reprod. 128: 301-311
  24. Lonergan, P. and T. Fair. 2008. In vitro-produced bovine embryos: dealing with the warts. Theriogenology 69:17-22
  25. Van Langendonckt, A., I. Donnay, N. Schuurbiers, P. Auquier, C. Carolan, A. Massip and F. Dessy. 1997. Effects of supplementation with fetal calf serum on development of bovine embryos in synthetic oviduct fluid medium. J. Reprod. Fertil. 109:87-93
  26. Jang, H. Y., H. S. Kong, J. D. Oh, B. K. Park, B. K. Yang, G. J. Jeon and H. K. Lee. 2008. Maintenance of sperm characteristics and in vitro developmental rate of embryos against oxidative stress through antioxidants in pig. Asian-Aust. J. Anim. Sci. 21(3):340-345
  27. Prichard, J. F., J. K. Thibodeaux, S. H. Pool, E. G. Blakewood, Y. Menezo and R. A. Godke. 1992. In-vitro co-culture of early stage caprine embryos with oviduct and uterine epithelial cells. Hum. Reprod. 7:553-557
  28. Kane, M. T., E. W. Carney and J. E. Ellington. 1992. The role of nutrients, peptide growth factors and co-culture cells in development of preimplantation embryos in vitro. Theriogenology 38:297-313
  29. Keskintepe, L., G. M. Darwish, A. I. Younis and B. G. Brackett. 1994b. In vitro development of morulae from immature caprine oocytes. Zygote. 2:97-102
  30. Sakkas, D., P. A. Batt and A. W. Cameron. 1989. Development of preimplantation goat (Capra hircus) embryos in vivo and in vitro. J. Reprod. Fertil. 87:359-365
  31. Izquierdo, D., P. Villamediana, M. J. Palomo, T. Mogas and M. T. Paramio. 1998. Effect of sperm capacitation and fertilization media on IVF and early embryo development of prepubertal goat oocytes. Theriogenology 49:1501-1513
  32. Mishra, O. P., P. G. Gawande, R. K. Nema and S. K. Tiwari. 2004. Estrus behavior and superovulatory response in black bengal goats (Capra hircus) following administration of prostaglandin and gonadotropins. Asian-Aust. J. Anim. Sci. 17(10):1374-1377
  33. Brackett, B. G. and G. Oliphant. 1975. Capacitation of rabbit spermatozoa in vitro. Biol. Reprod. 12:260-274
  34. Parrish, J. J., J. Susko-Parrish, M. A. Winer and N. L. First. 1988. Capacitation of bovine sperm by heparin. Biol. Reprod. 38: 1171-1180
  35. Visconti, P. E. and G. S. Kopf. 1998. Regulation of protein phosphorylation during sperm capacitation. Biol. Reprod. 59:1-6
  36. Cognie, Y., G. Baril, N. Poulin and P. Mermillod. 2003. Current status of embryo technologies in sheep and goat. Theriogenology 59:171-188
  37. Katska-Ksiazkiewicz, L., B. Rynska, B. Gajda and Z. Smorag. 2004. Effect of donor stimulation, frozen semen and heparin treatment on the efficiency of in vitro embryo production in goats. Theriogenology 62:576-586
  38. Vajta, G. and M. Gjerris. 2006. Science and technology of farm animal cloning: state of the art. Anim. Reprod. Sci. 92:211-230
  39. Dapino, D. G., P. E. Marini and M. O. Cabada. 2006. Effect of heparin on in vitro capacitation of boar sperm. Biol. Res. 39: 631-639
  40. Ho, H. C. and S. S. Suarez. 2001. An inositol 1,4,5-trisphosphate receptor-gated intracellular Ca(2+) store is involved in regulating sperm hyperactivated motility. Biol. Reprod. 65: 1606-1615

Cited by

  1. Effect of capacitating agents on sperm pretreatment during in vitrofertilization for blastocyst production in caprines vol.40, pp.13036181, 2016,