A Comparison of Cryotop and Solid Surface Vitrification Methods for the Cryopreservation of In Vitro Matured Bovine Oocytes

Abstract

Abstract. The aim of the present study was to compare the efficacies of the cooling systems of the solid surface (SSV) and Cryotop vitrification methods for cryopreservation of bovine oocytes at the metaphase II stage. The effects of vitrification on oocyte viability, in vitro fertilization (IVF), pronucleus formation and subsequent in vitro development were assessed. In vitro matured (IVM) bovine oocytes were subjected to equilibration and vitrification solutions according to the SSV method, and then the oocytes were vitrified either by dropping onto a cold dry metal surface (SSV group) or by plunging into liquid nitrogen on Cryotop sheets (Cryotop group). Warming was conducted according to the SSV method. Some oocytes were subjected to the cryoprotectants and warming regimen without cooling (Solution control group). The live/dead status of oocytes was evaluated by fluorescein diacetate staining. Live oocytes were subjected to IVF, and the resultant embryos were cultured in vitro. The rates of live oocytes were similar among the Fresh control, Solution control, SSV and Cryotop groups. There was no difference in the rates of fertilization, pronuclear formation and monospermy among these groups. The cleavage rates in the SSV and Cryotop groups (41.6 and 53.2%, respectively) were significantly lower than those in the Fresh control and Solution control groups (65.9 and 61.3%, respectively). The blastocyst rates in SSV and Cryotop groups did not differ (10.3 and 12.8%, respectively); however, they were significantly lower than those in the Fresh control and Solution control groups (36.4 and 24.8%, respectively). The inner cell mass, trophectoderm and total cell numbers in blastocysts did not differ significantly among the Fresh control, Solution control, SSV and Cryotop groups. Our results indicate that IVM bovine oocytes could be cryopreserved successfully using the cooling systems of the Cryotop and Solid Surface Vitrification methods with similar efficacy. Key words: Bovine, Cryotop, Oocyte, Solid surface vitrification, Vitrification (J. Reprod. Dev. 56: [176][177][178][179][180][181] 2010) ryopreservation of bovine oocytes is of great importance in the preservation of female genetic resources. Cryopreserved oocytes can be used for in vitro fertilization (IVF), nuclear transfer and genetic manipulation. Rall and Fahy [1] were the first to report vitrification, a glass-like solidification, by ultra-rapid cooling of mouse embryos, showing an alternative to traditional freezing methods to avoid chilling injury and ice crystal formation. Vitrification has been widely applied to cryopreserve oocytes in other mammalian species including cattle [2] In general, oocytes are more susceptible to cooling damage than zygotes because metaphase spindle microtubule integrity is disrupted during cooling and the high concentration of cryoprotectants affects oocytes during equilibration [10]. Also, differences in the membrane structures seem to make oocytes more sensitive to chilling compared with zygotes [11]. Although the tox...

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