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Bulletine of UASVM Animal Science and Biotechnologies
Year: 2010  |  Volume: 67  |  Issue: 1  |  Page No.: 486 - 486

Results Concerning Immature Swine Oocytes Vitrification using Different Concentration of Cryoprotectant

Andrea HETTIG, Vasile MICLEA, Marius ZAHAN, Iulian ROMAN, Florin VARO-GHIURU and Alexandru RUSU    

Abstract: The aim of this study was to establish a protocol with a higher number of viable oocytes after the vitrification procedure. The oocytes were collected by the follicular punction method from swine prepuberal ovaries. A total of 1792 immature oocytes were cryopreserved stepwise in previtrification (PV) and vitrification (VM) media, containing ethylene glycol (EG) permeating agent, combined with trehalose a non-permeating agent. The cells were preserved in three experimental ways, with three different concentration of cryoprotectant in the VM respectively 30, 40 and 45% EG. The exposure time of the cells to the cryoprotectants was the same in each experimental design, 4 minutes in the PV and 40 seconds in the VM. The concentration of the trehalose and the EG in the PM were remained the same. The vitrification procedure was performed by the superfine open pull straw method. For each experiment, there were 5 repetitions. After tawing the cells were stained with fluoresceine diacetate (FDA) and propidium iodide (PI), and there were exposed to the microscope UV light to test their viability. The viable oocytes had a florescent green color because of the FDA staining, which binds the esterase present only in the viable cells. The PI penetrates only dead cells, with destroyed membrane, binding the DNA, and giving them a red color. The results show an increase of percentage of viable cells once the concentration of the cryoprotectant is increased. With a 30% of EG in the VM, the medium percentage of viable cells is 10,26%, with a standard deviation of 1, 06. For 40% EG in the VM, the median is 33, 81%, with a standard deviation of 0,96, and 46,30% for a concentration of 45% EG in the VM with a standard deviation of 0,50. The statistical analysis shows significant differences (p< o, ooo1) between the experiments. Vitrification is the solidification of a solution, glass formation, at low temperatures without ice crystal formation. The phenomenon can be regarded as an extreme increase of viscosity and requires either rapid cooling rates or the use of cryoprotectant solutions, which depress ice crystal formation and increase viscosity at low temperatures. (Vajta G., 2000). These results sustain the theory according to which the higher the cryoprotectant is, the better the result is but limited by the exposure time.

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