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Bulletine of UASVM Animal Science and Biotechnologies
Year: 2010  |  Volume: 67  |  Issue: 1  |  Page No.: 487 - 487

The Influence of Exposure Time to Ethylene Glycol on Immature Swine Oocytes Vitrification

Andrea HETTIG, Vasile MICLEA, Marius ZAHAN, Iulian ROMAN and Ileana MICLEA    

Abstract: Porcine oocytes are particularly difficult to cryopreserv, and there are no reports of live offspring production after thawing via in vitro fertilization and embryo transfer. This may be partially due to the large size of the oocyte which has a low surface to volume ratio, making it more difficult for water and cryoprotectants to move across the cell plasma membrane. (Guang-Bin Zhou, 2009). This study was design to investigate the effect of the exposure time of the immature swine oocytes to the vitrification media. Vitrification procedure was performed stepwise: preequilibration medium (PM) with 15% ethylene glycol (EG) and 0.25M trehalose in basic medium (DPBS, supplemented with FCS 20%), and vitrification medium (VM) containing 45% EG and 0.5 M trehalose in basic medium. A total number of 1138 were vitrified in two experimental designs. In the first experiment (n=586) the cells were kept 4 min. in PM and 40 seconds in the VM. In the second experiment (n=552) the oocytes were kept 4 minute in the PM and 1 min in the VM. The eggs were loaded with the vitrification medium in the super fine open pull straws and plunged into the liquid nitrogen. After thawing the oocytes were stained with florescent dyes FDA (fluorescent diacetate) and PI, two complementary dyes and examined at the microscope’s UV light to test their viability. Each experimental design had been done in 5 repetitions. The results shows very significant differences (p< 0.0001) between the 2 experiments. Oocytes exposed to cryoprotectants in the VM for 40 sec presents a higher viability (average: 46,30%) vs. oocytes exposed to VM for 1 min (average: 7,19%). These differences show the importance of the exposure time to high concentrations of cryoprotectors. It is worldwide accepted that the higher the cryoprotectant concentration is the better the result is, but a high concentration also could lead to toxicity and cell death if the exposure time exceed the critical point. It means that 1min exposure to a 45% EG in BM is critical to swine oocytes. An acceptable protocol in vitrification can be made only with repeated exploratory regarding to both exposure time to cryoprotectants and their concentration.

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