P-92: Specific Activation Requirements of In Vitro Matured Sheep Oocytes following Vitrification-Warming

Background: Oocyte vitrification and assisted oocyte activation have increasingly important implications in assisted reproductive technology. However, an important area of concern with matured oocyte cryobiology is that elements of oocytes intimately involved in metaphase-II arrest may be modified by cryopreservation. In a comparison between different cellular characteristics of unvitrified, vitrified-warmed and unvitrified-activated oocytes, the present study investigated how vitrification-warming process may affect developmental competence of in vitro matured sheep oocytes following parthenogenetic activation. Materials and Methods: At 20-22 hours post-maturation (hpm), sheep oocytes were randomly divided for use as follows: 1. unvitrified, 2. vitrified and 3. unvitrifiedactivated. At half an hour after vitrification or parthenogenetic activation, the categorized oocytes were used for assessment of: 1. meiotic spindle and chromosomal organization, 2. zona-dissolution time, 3. DNA-fragmentation, 4. ultrastructural organization and 5. quantitative assessment of gene expression. Based on the results obtained during the first part of this study, further experiments were carried out to investigate if, and so how, different activation protocols affect in vitro development of vitrified-warmed vs. unvitrified oocytes. Accordingly, unvitrified (group i) and vitrified (group ii) oocytes were activated with nine different activation protocols, and then cultured for 8 days for assessment of embryonic development. Results: Structural, ultrastructural and molecular analyses indicated that the characteristics of vitrified-warmed oocytes vastly differed from fresh oocytes, yet resembled unvitrified-activated oocytes. For unvitrified oocytes, the highest blastocyst yield (41.8 ± 0.6%) was achieved using maximum ionomycin concentration (5 μM), and importantly, the duration of ionomycin treatment was not of utmost importance at this concentration. In contrast, the maximum blastocyst yield of vitrified-warmed oocytes (28.4 ± 1.4%) was achieved at minimal duration of ionomycin treatment (1 minute), and further increase in duration dramatically reduced developmental potential of vitrified-warmed oocyte. Conclusion: The obtained results suggested that vitrified- warmed oocytes may need a specific activation protocol different from unvitrified oocytes. In this respect, unvitrified oocytes were more sensitive to the concentration rather than the duration of ionomycin treatment when compared with vitrified oocytes, which were sensitive to the treatment duration. These results may provide a platform to improve the potential applications of vitrified oocytes in medicine and agriculture.