Skip to main content Accessibility help

Blastocyst production by in vitro maturation and development of porcine oocytes in defined media following intracytoplasmic sperm injection

  • M. Kobayashi (a1), S. Asakuma (a2) and Y. Fukui (a1)


The present study was carried out to establish porcine defined IVP. In Experiments 1 and 2, we investigated the efficacy of additional 0.6 mM cystine and/or 100 µM cysteamine (Cys) to a defined TCM199 maturation medium with regard to the intracellular glutathione (GSH) concentration and the developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The control medium was a modified TCM199 containing 0.05% (w/v) polyvinyl alcohol (PVA). Cys and/or cystine were added to the control medium. The control group and immature oocytes (presumptive germinal vesicle oocytes; GV) were prepared for GSH assay. In Experiment 3, the efficacy of epidermal growth factor (EGF) addition to a modified porcine zygote medium (mPZM) for in vitro culture (IVC) medium was investigated on embryonic development and the mean cell number of blastocysts following ICSI. As a positive or negative control, 0.3% BSA (mPZM-3) or 0.3% PVA (mPZM-4), respectively, was added to the base medium. The defined IVC medium was supplemented with 5 or 10 ng/ml EGF. In Experiment 1, no significant difference was found in the rates of cleavage (31.4–64.3%) and blastocyst formation (6.5–22.9%) among the treatment and control groups. The mean cell numbers per blastocyst ranged from 30 to 48 among the groups without significant differences. However, in Experiment 2, the intracellular GSH concentrations in the oocytes cultured in the medium supplemented with 100 µM Cys (9.6 pmol/oocyte) or Cys + cystine (9.9 pmol/oocyte) were significantly (p < 0.05) higher than the control (2.5 pmol/oocyte) and 0.6 mM cystine (6.5 pmol/oocyte) groups, but not different from the GV group (9.0 pmol/oocyte). The GSH concentration in the cystine group was also significantly (p < 0.05) higher than that in the control group, but not different from the GV group. In Experiment 3, the rates of cleavage and blastocyst formation and the mean cell numbers of blastocysts were not significantly different among the groups. However, the addition of 5 ng/ml EGF into the mPZM-4 resulted in a significantly (p < 0.05) higher blastocyst rate per cleaved embryo than the other two defined groups (mPZM-4 + 5 ng/ml: 48.6%, mPZM-4 and mPZM-4 +10 ng/ml: 23.4% and 23.1%, respectively).

The present results indicate that the addition of Cys to a defined medium for in vitro maturation (IVM) of porcine oocytes increases intracellular GSH concentration. Further addition of cystine into the IVM medium containing 100 µM Cys is not necessary and TCM199 plus Cys (100 µM) could be used as a defined IVM medium for porcine oocytes. The addition of 5 ng/ml EGF to a defined IVC medium has enhanced subsequent development after ICSI. This study shows that porcine blastocysts can be produced by defined media throughout the steps of IVP (IVM, ICSI and IVC).


Corresponding author

All correspondence to: Y. Fukui, Laboratory of Animal Reproduction, Department of Animal Production Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro 080–8555, Japan. Tel: +81 155 49 5415. Fax: +81 155 49 5593. e-mail:


Hide All
Abeydeera, L.R., Wang, W.H., Prather, R.S. & Day, B.N. (1998a). Presence of β-mercaptoethanol can increase the glutathione content of pig oocytes matured in vitro and the rate of blastocyst development after in vitro fertilization. Theriogenology 50, 747–56.
Abeydeera, L.R., Wang, W.H., Cantley, T.C., Pieke, A., Prather, R.S. & Day, B.N. (1998b). Presence of epidermal growth factor during in vitro maturation of pig oocytes and embryo culture can modulate blastocyst development after in vitro fertilization. Mol. Reprod. Dev. 51, 395401.
Abeydeera, L.R., Wang, W.H., Cantley, T.C., Rieke, A., Murphy, C.N., Prather, R.S. & Day, B.N. (2000). Development and viability of pig oocytes matured in a protein-free medium containing epidermal growth factor. Theriogenology 54, 787–97.
Bing, Y.Z., Hirao, Y., Iga, K., Che, L.M., Takenouchi, N., Kuwayama, M., Fuchimoto, D., Rodriguez-Martinez, H. & Nagai, T. (2002). In vitro maturation and glutathione synthesis of porcine oocytes in the presence or absence of cysteamine under different oxygen tensions: role of cumulus cells. Reprod. Fertil. Dev. 14, 125–31.
Brad, A.M., Bormann, C.L., Swain, J.E., Durkin, R.E., Johnson, A.E., Clifford, A.L. & Krisher, R.L. (2003). Glutathione and adenosine triphosphate content of in vivo and in vitro matured porcine oocytes. Mol. Reprod. Dev. 64, 492–8.
Chang, S.C.S., Jones, J.D., Ellefson, R.D. & Ryan, R.J. (1976). The porcine ovarian follicle: I. Selected chemical analysis of follicular fluid at different developmental stages. Biol. Reprod. 15, 321–8.
de Matos, D.G., Furnus, C.C. & Moses, D.F. (1997). Glutathione synthesis during in vitro maturation of bovine oocytes: role of cumulus cells. Biol. Reprod. 57, 1420–5.
de Matos, D.G. & Furnus, C.C. (2000). The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development: effect of β-mercaptoethanol, cysteine and cystine. Theriogenology 53, 761–71.
de Matos, D.G., Gasparrini, B.Pasqualini, S.R. & Thompson, J.G. (2002). Effect of glutathione synthesis stimulation during in vitro maturation of ovine oocytes on embryo development and intracellular peroxide content. Theriogenology 57, 1443–51.
Gasparrini, B., Boccia, L., Marchanidise, J., Palo, R.D., George, F., Donnay, I. & Zicarelli, L. (2006). Enrichment of in vitro maturation medium for buffalo (Bubalus bubalis) oocytes with thiol compounds: effects of cystine on glutathione synthesis and embryo development. Theriogenology 65, 275–87.
Gonzales, E.R., Bejar, M.L., Mertens, M.J. & Paramio, M.T. (2003). Effects on in vitro embryo development and intracellular glutathione content of the presence of thiol compounds during maturation of prepubertal goat oocytes. Mol. Reprod. Dev. 65, 446–53.
Grazul-Bilska, A.T., Choi, J.T., Bilski, J.J., Weigel, R.M., Kirsch, J.D., Kraft, K.C., Reynolds, L.P. & Redmer, D.A. (2003). Effects of epidermal growth factor on early embryonic development after in vitro fertilization of oocytes collected from ewes treated with follicle stimulating hormone. Theriogenology 59, 1449–57.
Grupen, G.G., Nagashima, H. & Nottle, M.B. (1995). Cysteamine enhances in vitro development of porcine oocytes matured and fertilized in vitro. Biol. Reprod. 53, 173–8.
Guerin, P., El Mouatassim, S. & Menézo, Y. (2001). Oxidative stress and protection against reactive oxygen species in the preimplantation embryo and its surroundings. Hum. Reprod. Update 7, 175–89.
Hagen, D.R., Prather, R.S., Sims, M.M. & First, N.L. (1991). Development of one-cell porcine embryos to the blastocyst stage in simple media. J. Anim. Sci. 69, 1147–50.
Holm, P., Booth, P.J., Schmidt, M.H., Greve, T. & Callesen, H. (1999). High bovine blastocyst development in a static in vitro production system using SOFaa medium supplemented with sodium citrate and myo-inositol with or without serum proteins. Theriogenology 52, 683700.
Iwasaki, T., Kimura, E. & Totsukawa, K. (1999). Studies on a chemically defined medium for in vitro culture on in vitro matured and fertilized porcine oocytes. Theriogenology 51, 709–20.
Kishida, R., Lee, E.S. & Fukui, Y. (2004). In vitro maturation of porcine oocytes using a defined medium and developmental capacity after intracytoplasmic sperm injection. Theriogenology 62, 1663–76.
Kitagawa, Y., Suzuki, K., Yoneda, A. & Watanabe, T. (2004). Effects of oxygen concentration and antioxidants on the in vitro developmental ability, production of reactive oxygen species (ROS), and DNA fragmentation in porcine embryos. Theriogenology 62, 1186–97.
Kobayashi, M., Lee, E.S. & Fukui, Y. (2006). Cysteamine or β-mercaptoethanol added to a defined maturation medium improves blastocyst formation of porcine oocytes after intracytoplasmic sperm injection. Theriogenology 65, 1191–9.
Lee, E.S. & Fukui, Y. (1995). Effect of various growth factors in a defined culture medium on in vitro development of bovine embryos matured and fertilized in vitro. Theriogenology 44, 7183.
Lee, G.S., Kim, H.S., Hyun, S.H., Jeon, H.Y., Nam, D.H., Jeong, Y.W., Kim, S., Kim, J.H., Kang, S.K., Lee, B.C. & Hwang, W.S. (2005). Effect of epidermal growth factor in preimplantation development of porcine cloned embryos. Mol. Reprod. Dev. 71, 4551.
Lee, M.S., Kang, S.K., Lee, B.C. & Hwang, W.S. (2005). The beneficial effects of insulin and metformin on in vitro developmental potential of porcine oocytes and embryos. Biol. Reprod. 73, 1264–8.
Luberda, Z. (2005). The role of glutathione in mammalian gametes. Reprod. Biol. 5, 517.
Merlo, B., Iacono, E., Zambelli, D., Prati, F. & Belluzzi, S. (2005). Effect of EGF on in vitro maturation of domestic cat oocytes. Theriogenology 63, 2032–9.
Miyoshi, K., Rzucidlo, S.J., Pratt, S.L. & Stice, S.L. (2002). Utility of rapidly matured oocytes as recipients for production of cloned embryos from somatic cells in the pig. Biol. Reprod. 67, 540–5.
Mori, T., Amano, T. & Shimizu, H. (2000). Roles of gap junctional communication of cumulus cells in cytoplasmic maturation of porcine oocytes cultured in vitro. Biol. Reprod. 62, 913–9.
Nagai, T. (2001). The improvement of in vitro maturation systems for bovine and porcine oocytes. Theriogenology 55, 1291–301.
Nagashima, H., Grupen, C.G., Ashman, R.J. & Nottle, M.B. (1996). Developmental competence of in vivo and in vitro matured porcine oocytes after subzonal sperm injection. Mol. Reprod. Dev. 45, 359–63.
O'Neill, C. (1997). Culture of preimplantation embryos: facts and artifacts. Biol. Reprod. 56, 229–37.
Oyamada, T., Iwayama, H. & Fukui, Y. (2004a). Additional effect of epidermal growth factor during in vitro maturation for individual bovine oocytes using a chemically defined medium. Zygote 12, 143–50.
Oyamada, T. and Fukui, Y. (2004b). Oxygen tension and medium supplements for in vitro maturation of bovine oocytes cultured individually in a chemically defined medium. J. Reprod. Dev. 50, 107–17.
Paria, B.C. & Dey, S.K. (1990). Preimplantation embryo development in vitro: cooperative interactions among embryos and role of growth factors. Proc. Natl. Acad. Sci. USA 87, 4756–60.
Petters, R.M. & Wells, K.D. (1993). Culture of pig embryos. J. Reprod. Fertil. Suppl. 48, 6173.
Pujol, M., Bejar, M.L. and Paramio, M.T. (2004). Developmental competence of heifer oocytes selected using the brilliant cresyl blue (BCB) test. Theriogenology 61, 735–44.
Rui-Hua, L., Yong-Hai, L., Li-Hong, J., Xiao-Ning, W., Hong, W. & Wei-Hua, W. (2002). Extracellular and intracellular factors affecting nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles. Zygote 10, 253–60.
Sawai, K., Funahashi, H. & Niwa, K. (1997). Stage-specific requirement of cysteine during in vitro maturation of porcine oocytes for glutathione synthesis associated with male pronuclear formation. Biol. Reprod. 57, 16.
Sirisathien, S., Fonseca, H.J.H. & Brackett, B.G. (2003). Influences of epidermal growth factor and insulin-like growth factor-I on bovine blastocyst development in vitro. Anim. Reprod. Sci. 77, 2132.
Sturmey, R.G. & Leese, H.J. (2003). Energy metabolism in pig oocytes and early embryos. Reproduction 126, 197204.
Takahashi, M., Nagai, T., Hamano, S., Kuwayama, M., Okamura, N. & Okano, A. (1993). Effect of thiol compounds on in vitro development and intracellular glutathione content of bovine embryos. Biol. Reprod. 49, 228–32.
Tatemoto, H., Muto, N., Sunagawa, I., Shinjo, A. & Nakada, T. (2004). Protection of porcine oocytes against cell damage caused by oxidative stress during in vitro maturation: role of superoxide dismutase activity in porcine follicular fluid. Biol. Reprod. 71, 1150–7.
Wei, Z., Park, K.W., Day, B.N. & Prather, R.S. (2001). Effect of epidermal growth factor on preimplantation development and its receptor expression in porcine embryos. Mol. Reprod. Dev. 60, 457–62.
Yang, X-Z., Han, M., Niwa, K. & Iannaccone, P.M. (2004). Factors required during preculture of rat oocytes soon after sperm penetration for promoting their further development in a chemically defined medium. J. Reprod. Dev. 50, 533–40.
Yong, H.W., Pyo, B.S., Hong, J.Y., Kang, S.K., Lee, B.C., Lee, E.S. & Hwang, W.S. (2003). A modified method for ICSI in the pig: injection of head membrane-damaged sperm using a 3–4 µM diameter injection pipette. Hum. Reprod. 18, 2390–6.
Yoshioka, K., Suzuki, C., Tanaka, A., Anas, I.M.-K. & Iwamura, S. (2002). Birth of piglets derived from porcine zygotes cultured in a chemically defined medium. Biol. Reprod. 66, 112–9.
Yoshioka, K., Suzuki, C., Itoh, S., Kikuchi, K., Iwamura, S. & Rodiguez-Martinez, H. (2003). Production of piglets derived from in vitro-produced blastocysts fertilized and cultured in chemically defined media: effects of theophylline, adenosine, and cysteine during in vitro fertilization. Biol. Reprod. 69, 2092–9.
Zuelke, K.A., Jeffay, S.C., Zucker, R.M. & Perreault, S.D. (2003). Glutathione (GSH) concentrations vary with the cell cycle in maturing hamster oocytes, zygotes, and preimplantation stage embryos. Mol. Reprod. Dev. 64, 106–12.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

  • ISSN: 0967-1994
  • EISSN: 1469-8730
  • URL: /core/journals/zygote
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed