83 CRYOPRESERVATION OF IN VITRO PORCINE OOCYTES BY SOLID SURFACE VITRIFICATION (SSV)
A. Dinnyes A B , T. Somfai A , D. Sage D , M. Marosan C , J. Carnwath D and H. Niemann DA Institute of Animal Biology, Agricultural Biotechnology Center, Godollo, Hungary
B Research Group on Applied Animal Genetics and Biotechnology, Hungarian Academy of Sciences and Szent Istvan University, Godollo, Hungary
C Institute of Animal Breeding, University of West Hungary, Mosonmagyarovar, 9200, Hungary
D Department of Biotechnology, Institute for Animal Science, Mariensee, 31535 Neustadt, Germany. Email: dinnyes@abc.hu
Reproduction, Fertility and Development 17(2) 191-192 https://doi.org/10.1071/RDv17n2Ab83
Submitted: 1 August 2004 Accepted: 1 October 2004 Published: 1 January 2005
Abstract
The effects of the cytosceletal inhibitor cytochalasin in solid surface vitrification (SSV; Dinnyes et al. 2000 Biol. Reprod. 63, 513–518) were investigated on in vitro matured (IVM) porcine oocytes. Cumulus-free IVM oocytes were subjected to one of the following: SSV (equilibration in 4% ethylene glycol (EG) for 10 min followed by vitrification in 35% EG, 5% polyvinyl pirrolidone, and 0.3 M trehalose on a cold (about −150°C) surface; warming in 0.4 M trehalose at 37°C), SSV pre-treatment with 5 μg/mL cytochalasin B (SSV + CB), or the steps of SSV without cooling, i.e. toxicity control (TC). Non-lysed oocytes together with the non-treated controls were subjected to parthenogenetic activation and then in vitro cultured (IVC) for six days. The proportion of non-lysed oocytes was higher when pre-treatment with CB was performed compared to SSV. However, both results were significantly lower than that of the TC. After parthenogenetic activation via a combination of a direct current electric pulse (1 kV/cm for 45 μs) followed by 3 h treatment with 2 mM 6-dimethylaminopurine, the proportion of cleaved embryos was higher in the SSV + CB than in the SSV. Nevertheless, significantly more oocytes cleaved in the TC and control groups. On Day 6 no blastocyst, were determined in the SSV group, and one blastocyst was obtained in the SSV + CB, while significantly more blastocysts developed in both the TC and the control (Table 1). There was no significant difference in blastocyst rates and in the number of blastomere nuclei/embryo between the TC and the control. These results indicate that the high concentration of cryoprotectants per se applied in SSV were not detrimental for in vitro development and that CB pre-treatment increased survival and further development of SSV vitrified pig oocytes resulting in one parthenogenetic blastocyst from vitrified pig oocytes.
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This research was supported by a Bilateral Scientific and Technological Collaboration (TET, No. D6/01) grant between Germany and Hungary and by the Hungarian National Office of Research and Technology (OM-KMUFA; BIO-00086/2002).
