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A serum-free culture system for efficient in vitro production of bovine blastocysts with improved viability after freezing and thawing

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Abstract

The aim of this study was to evaluate whether two completely serum-free media (IVMD101 and IVD101) could improve the yield and quality of bovine blastocysts from in vitro matured and fertilized oocytes. The media were evaluated in the presence (IVMD101) or absence (IVD101) of bovine cumulus/granulosa cell (BCGC) cocultures. The proportion of embryos developing to the blastocyst stage in IVMD101 medium with BCGC cocultures (36.5%) and IVD101 medium without BCGC cocultures (37.1%) was significantly higher than in serum-supplemented medium (TCM199 + 5% calf serum) with BCGC cocultures (25.1%). Furthermore, the mean cell numbers per blastocyst on Day 7 developed in IVMD101 medium (179.5 cells) and IVD101 medium (177.1 cells) were greater than in the serum-supplemented medium (145.7 cells). The survival rates of blastocysts derived in IVMD101 medium (73.3%) and IVD101 medium (60.0%) based on hatching after 72 h of post-thaw culture were superior to that of blastocysts derived in the serum-supplemented medium (48.1%). Under microscopic observation, bovine blastocysts derived in the serum-supplemented medium showed abundant lipid droplets, largely into the trophectoderm cells. This morphological difference may partly explain the sensitivity of serum-derived embryos after freezing and thawing. In conclusion, these new serum-free culture media are useful, not only to study the mechanisms of early embryogenesis, but also for mass production of good quality embryos for embryo transfer, cloning and transgenesis.

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References

  • Adamson ED (1993) Activities of growth factors in preimplantation embryos. J Cell Biochem 53: 280–287.

    Article  CAS  Google Scholar 

  • Barnes D and Sato G (1980) Methods for growth of cultured cells in serum-free medium. Anal. Biochem 102: 255–270.

    Article  CAS  Google Scholar 

  • Bavister BD (1995) Culture of preimplantation embryos: facts and artifacts. Human Reprod Update 1: 91–148.

    CAS  Google Scholar 

  • Brackett BG and Oliphant G (1975) Capacitation of rabbit spermatozoa in vitro. Biol Reprod 12: 260–274.

    Article  CAS  Google Scholar 

  • Eyestone WH and First NL (1989) Co-culture of early cattle embryos to the blastocyst stage with oviductal tissue or in conditioned medium. J Reprod Fertil 85: 715–720.

    Article  CAS  Google Scholar 

  • Freeman MR, Whitworth CM and Hill GA (1995) Granulosa cell co-culture enhances human embryo development and pregnancy rate following in-vitro fertilization. Human Reprod 10: 408–414.

    CAS  Google Scholar 

  • Fukuda Y, Ichikawa M, Naito K and Toyoda Y (1990) Birth of normal calves resulting from bovine oocytes matured, fertilized and cultured with cumulus cells in vitro up to blastocyst stage. Biol Reprod 42: 114–119.

    Article  CAS  Google Scholar 

  • Fukui Y (1989) Effects of sera and steroid hormones on development of bovine oocytes matured and fertilized in vitro and co-cultured with bovine oviduct epithelial cells. J Anim Sci 67: 1318–1323.

    CAS  Google Scholar 

  • Fukui Y, McGowan LT, James RW, Pugh PA and Tervit HR (1991) Factors affecting the in-vitro development to blastocysts of bovine oocytes matured and fertilized in vitro. J Reprod Fertil 92: 125–131.

    Article  CAS  Google Scholar 

  • Goto K, Iwai N, Ide K, Takuma Y and Nakanishi Y (1994) Viability of one-cell bovine embryos cultured in vitro: comparison of cell-free culture with co-culture. J Reprod Fertil 101: 257–264.

    Article  Google Scholar 

  • Greve T, Avery B and Callesen H (1993) Viability of in vivo and in vitro produced bovine embryos. Reprod Dom Anim 28: 164–169.

    Google Scholar 

  • Herrler A, Lucas-Hahn A and Nieman H (1992) Effects of insulin-like growth factor-I on in vitro production of bovine embryos. Theriogenology 37: 1213–1224.

    Article  CAS  Google Scholar 

  • Johnson MH and Nasr-Esfahani MH (1994) Radical solutions and cultural problems: Could free oxygen radicals be responsible for the impaired development of preimplantation mammalian embryos in vitro? BioEssays 16: 31–38.

    Article  CAS  Google Scholar 

  • Kobayashi K, Takagi Y, Satoh T, Hoshi H and Oikawa T (1992) Development of early bovine embryos to the blastocyst stage in serum-free conditioned medium from bovine granulosa cells. In Vitro Cell Dev Biol 28A: 255–259.

    CAS  Google Scholar 

  • Kobayashi K, Yamashita S and Hoshi H (1994a) Influence of epidermal growth factor and transforming growth factor-α on in vitro maturation of cumulus cell-enclosed bovine oocytes in a defined medium. J Reprod Fertil 100: 439–446.

    Article  CAS  Google Scholar 

  • Kobayashi K, Yamashita S, Satoh T and Hoshi H (1994b) Low oxygen and glucose improves the development of fertilized bovine oocyte in defined medium without somatic cells. In Vitro Cell Dev Biol 30A: 556–558.

    CAS  Google Scholar 

  • Larson RC, Ignotz GG and Currie WB (1992) Transforming growth factor β and fibroblast growth factor synergistically promote early bovine embryo development during the fourth cell cycle. Mol Reprod Dev 33: 432–435.

    Article  CAS  Google Scholar 

  • Lee E-S and Fukui Y (1996) Synergistic effect of alanine and glycine on bovine embryos cultured in a chemically defined medium and amino acid uptake by in vitro-produced bovine morulae and blastocysts. Biol Reprod 55: 1383–1389.

    Article  CAS  Google Scholar 

  • Leibo SP and Loskutoff NM (1993) Cryobiology of in vitro-derived bovine embryos. Theriogenology 39: 81–94.

    Article  Google Scholar 

  • Leibo SP, Pollard JW and Martino PA (1995) Chilling and freezing sensitivity of ‘reassembled’ in vitro-derived bovine embryos. Theriogenology 43: 265 (Abstr).

    Article  Google Scholar 

  • Lim JM, Okitsu O, Okuda K and Niwa K (1994) Effect of fetal calf serum in culture medium on the development of bovine oocytes matured and fertilized in vitro. Theriogenology 41: 1091–1098.

    Article  CAS  Google Scholar 

  • Luvoni GC, Keskintepe L and Brackett BG (1996) Improvement in bovine embryo production in vitro by gluthathione-containing culture media. Mol Reprod Dev 43: 437–443.

    Article  CAS  Google Scholar 

  • Massip A, Mermillod P and Dinnyes A (1995) Morphology and biochemistry of in vitro produced bovine embryos: implications for their cryopreservation. Human Reprod 10: 3004–3011.

    CAS  Google Scholar 

  • Nasr-Esfahani MH, Aitken RJ and Johnson MH (1990) Hydrogen peroxide levels in mouse oocytes and early cleavage stage embryos developed in vitro or in vivo. Development 109: 501–507.

    CAS  Google Scholar 

  • Nasr-Esfahani MH and Johnson MH (1992) How does transferrin overcome the in vitro block to development of the mouse preimplantation embryo? J Reprod Fertil 96: 41–48.

    Article  CAS  Google Scholar 

  • Pinyopummintr T and Bavister BD (1991) In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined protein-free culture media. Biol Reprod 45: 736–742.

    Article  CAS  Google Scholar 

  • Rexroad CE (1989) Co-culture of domestic animal embryos. Theriogenology 31: 105–114.

    Article  Google Scholar 

  • Satoh T, Kobayashi K, Yamashita S, Kikuchi M, Sendai Y and Hoshi H (1994) Tissue inhibitor of metalloproteinase (TIMP-1) produced by granulosa cells and oviduct cells enhances in vitro development of bovine embryo. Biol Reprod 50: 835–844.

    Article  CAS  Google Scholar 

  • Schultz GA and Heyner S (1993) Growth factors in preimplantation embryos. Oxf Rev Reprod Biol 15: 43–81.

    CAS  Google Scholar 

  • Semple ME, Betteridge KJ and Leibo SP (1995) Cryopreservation of in vitro — derived bovine embryos produced in a serum-free culture system. Theriogenology 43: 320 (Abstr).

    Article  Google Scholar 

  • Shamsuddin M, Larsson B, Gustafsson H and Rodriguez-Martinez H (1993) In vitro development up to hatching of bovine in vitro-matured and fertilized oocytes with or without support from somatic cells. Theriogenology 39: 1067–1079.

    Article  CAS  Google Scholar 

  • Stadtman TC (1974) Selenium biochemistry. Science 183: 915–922.

    CAS  Google Scholar 

  • Takagi M, Otoi T, Boediono A, Saha S and Suzuki T (1994) Viability of frozen-thawed bovine IVM/IVF embryos in relation to aging using various cryoprotectants. Theriogenology 41: 915–921.

    Article  CAS  Google Scholar 

  • Takahashi Y and First NL (1992) In vitro development of bovine one-cell embryos: influence of glucose, lactate, pyruvate, amino acids and vitamines. Theriogenology 37: 963–978.

    Article  CAS  Google Scholar 

  • Thomas EL, Learn DB, Jefferson MM and Weathered W (1988) Superoxide — dependent oxidation of extracellular reducing agents by isolated neutrophils. J Biol Chem 263: 2178–2186.

    CAS  Google Scholar 

  • Thompson JG, Gardner DK, Pugh PA, McMillan WH and Tervit HR (1995) Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos. Biol Reprod 53: 1385–1391.

    Article  CAS  Google Scholar 

  • Wright RW and Bondioli KR (1981) Aspects of in vitro fertilization and embryo culture in domestic animals. J Anim Sci 53: 702–729.

    CAS  Google Scholar 

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Authors
  1. Shoko Yamashita
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  2. Hiroyuki Abe
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  3. Takehiro Itoh
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  4. Takeshi Satoh
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  5. Hiroyoshi Hoshi
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Yamashita, S., Abe, H., Itoh, T. et al. A serum-free culture system for efficient in vitro production of bovine blastocysts with improved viability after freezing and thawing. Cytotechnology 31, 123–131 (1999). https://doi.org/10.1023/A:1008044827145

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  • DOI: https://doi.org/10.1023/A:1008044827145

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