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The effect of vitrification of immature bovine oocytes to the subsequent in vitro development and gene expression

  • Marwa S. Faheem (a1) (a2), E. Baron (a1), I. Carvalhais (a1), A. Chaveiro (a1), K. Pavani (a1) and F. Moreira da Silva (a3)...


Immature bovine oocytes were vitrified using the cryotop method and their post-warming survivability and capability to undergo in vitro maturation, fertilization and subsequent embryonic development were evaluated. In addition throughout the embryonic 2-cell, 4-cell, morula and blastocyst stages, the expression of four developmentally important genes (Cx43, CDH1, DNMT1 and HSPA14) was analysed using the real-time polymerase chain reaction (PCR). Immature oocytes (n = 550) were randomly assigned to non-vitrified (fresh) or cryotop vitrification groups using ethylene glycol (EG) with 1,2 propanediol (PROH) or dimethylsulphoxide (DMSO). After warming, oocytes survivability, embryo cleavage and embryonic developmental rates were not statistically different between the two cryoprotectants groups. However, the DMSO group had a lower (P < 0.05) oocyte maturation rate compared with the fresh and PROH groups. For morula and blastocyst rates, the DMSO group achieved a lower (P < 0.05) morula rate compared with the fresh group, while at the blastocyst stage, there were no differences between fresh and both cryoprotectants groups. For molecular analysis, at the 4-cell stage, most studied genes showed an inconsistent pattern of expression either from the PROH or DMSO groups. Noteworthily, these differences were limited at the morula and blastocyst stages. In conclusion, the cryotop method is sufficient for vitrification of immature bovine oocytes, both for embryonic developmental competence and at the molecular level. Moreover, PROH showed some advantage over DMSO as a cryoprotectant.


Corresponding author

All correspondence to: Fernando Moreira da Silva. Animal Reproduction, Department of Agrarian Sciences, University of the Azores, CITA-A, 9700–042 Angra do Heroísmo, Portugal. Tel: +351 295 402420. Fax: +351 295 402421. e-mail:


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Borges, E.N., Silva, R.C., Futino, D.O., Rocha-Junior, C.M., Amorim, C.A., Bao, S.N. & Lucci, C.M. (2009). Cryopreservation of swine ovarian tissue: effect of different cryoprotectants on the structural preservation of preantral follicle oocytes. Cryobiology 59, 195200.
Cetin, Y. & Bastan, A. (2006). Cryopreservation of immature bovine oocytes by vitrification in straws. Anim. Reprod. Sci. 92, 2936.
Chian, R.C., Kuwayama, M., Tan, L., Tan, J., Kato, O. & Nagai, T. (2004). High survival rate of bovine oocytes matured in vitro following vitrification. J. Reprod. Dev. 50, 685–96.
Dean, W., Santos, F., Stojkovic, M., Zakhartchenko, V., Walter, J., Wolf, E. & Reik, W. (2001). Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos. Proc. Natl. Acad. Sci. USA 98, 13734–8.
Dutta, D.J., Dev, H. & Raj, H. (2013). In vitro blastocyst development of post-thaw vitrified bovine oocytes, Vet. World 6 (10): 730–3.
Ebrahimi, B., Valojerdi, M.R., Eftekhari-Yazdi, P. & Baharvand, H. (2010). In vitro maturation, apoptotic gene expression and incidence of numerical chromosomal abnormalities following cryotop vitrification of sheep cumulus–oocyte complexes. J. Assist. Reprod. Genet. 27, 239–46.
Faheem, M.S., Carvalhais, I., Chaveiro, A. & Moreira da Silva, F. (2011). In vitro oocyte fertilization and subsequent embryonic development after cryopreservation of bovine ovarian tissue, using an effective approach for oocyte collection. Anim. Reprod. Sci. 125, 4955.
Fleming, T.P., Sheth, B. & Fesenko, I. (2001). Cell adhesion in the preimplantation mammalian embryo and its role in trophectoderm differentiation and blastocyst morphogenesis. Front. Biosci. 6, D1000–7.
Golding, M.C. & Westhusin, M.E. (2003). Analysis of DNA (cytosine 5) methyltransferase mRNA sequence and expression in bovine preimplantation embryos, fetal and adult tissues. Gene Expr. Patterns 3, 551–8.
Goodall, H. & Johnson, M.H. (1984). The nature of intercellular coupling within the preimplantation mouse embryo. J. Embryol. Exp. Morphol. 79, 5376.
Goossens, K., Van Poucke, M., Van Soom, A., Vandesompele, J., Van Zeveren, A. & Peelman, L.J. (2005). Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos. BMC Dev. Biol. 5:27. doi:10.1186/1471-213X-5-27.
Gurtovenko, A.A. & Anwar, J. (2007). Modulating the structure and properties of cell membranes: the molecular mechanism of action of dimethyl sulfoxide. J. Phys. Chem. B. 111, 10453–60.
Habibi, A., Farrokhi, N., Moreira da Silva, F., Bettencourt, B.F., Bruges-Armas, J., Amidi, F. & Hosseini, A. (2010). The effects of vitrification on gene expression in mature mouse oocytes by nested quantitative PCR. J. Assist. Reprod. Genet. 27, 599604.
Hajarian, H., Wahid, H., Rosnina, Y., Daliri, M., Dashtizad, M., Mirzapour, T., Yimer, N., Bukar, M.M., Iswadi, M.I. & Abas Mazni, O. (2011). Cryotop device enhances vitrification outcome of immature bovine oocytes. J. Anim. Vet. Advan. 10, 2541–5.
Houghton, F.D. (2005). Role of gap junctions during early embryo development. Reproduction 129, 129–35.
Hyafil, F., Morello, D., Babinet, C. & Jacob, F. (1980). A cell surface glycoprotein involved in the compaction of embryonal carcinoma cells and cleavage stage embryos. Cell 21, 927–34.
Johnson, M.H. & Pickering, S.J. (1987). The effect of dimethylsulfoxide on the microtubular system of the mouse oocyte. Development 100, 313–24.
Khatir, H., Anouassi, A. & Tibary, A. (2004). Production of dromedary (Camelus dromedarius) embryos by IVM and IVF and co-culture with oviductal or granulosa cells. Theriogenology 62, 1175–85.
Kuwayama, M. (2007). Highly efficient vitrification for cryopreservation of human oocytes and embryos: the cryotop method. Theriogenology 67, 7380.
Levy, R. (2001). Genetic regulation of preimplantation embryo survival. Int. Rev. Cytol. 210, 137.
Liebermann, J. & Tucker, M.J. (2002). Effect of carrier system on the yield of human oocytes and embryos as assessed by survival and developmental potential after vitrification. Reproduction 124, 483–9.
Liebermann, J. & Tucker, M.J. (2006). Comparison of vitrification and conventional cryopreservation of day 5 and day 6 blastocysts during clinical application. Fertil. Steril. 86, 20–6.
Modina, S., Leoni, G.G., Lodde, V., Naitana, S., Pirani, S., Succu, S., Berlinguer, F. & Luciano, A.M. (2010). Involvement of E-cadherin in early in vitro development of adult and juvenile sheep embryos. Reprod. Fertil. Dev. 22, 468–77.
Morato, R., Izquierdo, D., Paramio, M.T. & Mogas, T. (2008). Cryotops versus open-pulled straws (OPS) as carriers for the cryopreservation of bovine oocytes: effects on spindle and chromosome configuration and embryo development. Cryobiology 57, 137–41.
Neuer, A., Spandorfer, S.D., Giraldo, P., Dieterle, S., Rosenwaks, Z. & Witkin, S.S. (2000). The role of heat shock proteins in reproduction. Hum. Reprod. Update 6, 149–59.
Ogawa, H., Oyamada, M., Mori, T., Mori, M. & Shimizu, H. (2000). Relationship of gap junction formation to phosphorylation of connexin43 in mouse preimplantation embryos. Mol. Reprod. Dev. 55, 393–8.
Pavani, K.C. (2012). Optimization of the messenger RNA (mRNA) extraction protocol from fresh and vitrified bovine oocytes for gene expression studies. MSc. thesis. University of Skövde.
Pfaffl, M.W., Horgan, G.W. & Dempfle, L. (2002). Relative Expression Software Tool (REST©) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 30, 110.
Riethmacher, D., Brinkmann, V. & Birchmeier, C. (1995). A targeted mutation in the mouse E-cadherin gene results in defective preimplantation development. Proc. Natl. Acad. Sci. USA 92, 855–9.
Rozen, S. & Skaletsky, H. (2000). Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–86.
Santos, R.R., Tharasanit, T., Van Haeften, T., Figueiredo, J.R., Silva, J.R. & Van den Hurk, R. (2007). Vitrification of goat preantral follicles enclosed in ovarian tissue by using conventional and solid-surface vitrification methods. Cell Tissue Res. 327, 167–76.
Sharma, G.T., Dubey, P.K. & Chandra, V. (2010). Morphological changes, DNA damage and developmental competence of in vitro matured, vitrified-thawed buffalo (Bubalus bubalis) oocytes: A comparative study of two cryoprotectants and two cryodevices. Cryobiology 60, 315–21.
Shi, L. & Wu, J. (2009). Epigenetic regulation in mammalian preimplantation embryo development. Reprod. Biol. Endocrinol. doi:10.1186/1477-7827-7-59.
Stinshoff, H., Wilkening, S., Hanstedt, A., Bruning, K. & Wrenzycki, C. (2011). Cryopreservation affects the quality of in vitro produced bovine embryos at the molecular level. Theriogenology 76, 1433–41.
Succu, S., Bebbere, D., Bogliolo, L., Ariu, F., Fois, S., Leoni, G.G., Berlinguer, F., Naitana, S. & Ledda, S. (2008). Vitrification of in vitro matured ovine oocytes affects in vitro pre-implantation development and mRNA abundance. Mol. Reprod. Dev. 75, 538–46.
Telford, N.A., Watson, A.J. & Schultz, G.A. (1990). Transition from maternal to embryonic control in early mammalian development: a comparison of several species. Mol. Reprod. Dev. 26, 90100.
Tesfaye, D., Lonergan, P., Hoelker, M., Rings, F., Nganvongpanit, K., Havlicek, V., Besenfelder, U., Jennen, D., Tholen, E. & Schellander, K. (2007). Suppression of connexin 43 and E-cadherin transcripts in in vitro derived bovine embryos following culture in vitro or in vivo in the homologous bovine oviduct. Mol. Reprod. Dev. 74, 978–88.
Vajta, G. & Kuwayama, M. (2006). Improving cryopreservation system. Theriogenology 65, 236–44.
Varghese, A.C., Nagy, Z.P. & Agarwal, A. (2009). Current trends, biological foundations and future prospects of oocyte and embryo cryopreservation. Reprod. Biomed. Online 19, 126–40.
Vestweber, D. & Kemler, R. (1984). Rabbit antiserum against a purified surface glycoprotein decompacts mouse preimplantation embryos and reacts with specific adult tissues. Exp. Cell. Res. 152, 169–78.
Yang, X.G., Wang, X.W., Zuo, E.W., Hao, C.L., Lu, S.S., Liu, H.B., Xu, H.Y., Lu, Y.Q. & Lu, K.H. (2012). cDNA cloning of porcine CDH1 and its expression profile in porcine early parthenotes. Anim. Sci. Pap. Rep. 30, 249–59.



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