Untersuchungen zur Vitrifikation von immaturen und In-vitro ...

vmf.uni.leipzig.de

Untersuchungen zur Vitrifikation von immaturen und In-vitro ...

101either with cumulus cells or partially denuded. The success of vitrification methods was assessed bysurvival rate and developmental competence of vitrified-warmed oocytes after DNA-staining withHoechst dye, IVF or induced parthenogenetic activation. Maturation rates, cleavage- and blastocystrates of vitrified and unvitrified control oocytes were recorded and compared. A total amount of1300 GV-oocytes were used in 24 trials, additionally processed were more than 1550 maturedoocytes in 31 trials and the same amount of oocytes for controls.The aseptic vitrification method was suitable for cryopreservation of immature and maturedbovine oocytes. In comparison to controls developmental competence of treated oocytes wassignificantly reduced regardless of the vitrification method used. Aseptic vitrification proved topreserve developmental competence of partially denuded oocytes better than OPS-vitrificationwhen 3-steps equilibration method was performed. GV-oocytes cleaved but none reached theblastocyst stage no matter which vitrification method was used. The presence of cumulus cellsduring cryopreservation of MII-oocytes negatively affected further ability to develop in vitro. When3-steps and 1-step equilibration procedures were compared, the latter proved to be superior. Resultsof OPS-vitrification experiments showed evidence that the use of the 3-steps equilibration methodlead to inefficient cryoprotection of cells, toxic or osmotic damage or a combination of all theseevents compromising developmental competence in vitro. Dilution of CPAs with the titration-likemethod supported further cleavage and embryonic development better than the stepwise procedure.No difference in cryoprotective efficiency could be observed between media D+E and EG, mediumURF, which held the lowest concentration of CPAs, was not suitable for aseptic vitrificationmethod.Results of these investigations state, that the aseptic method can provide the tool for a safeand suitable future cryopreservation method for bovine oocytes. However, further investigationsneed to focus on the problems associated with equilibration procedures and high intracellular CPAconcentration.

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