P-121: Assessment of Microtubule and Nuclear Status at Different Intervals of Bovine In Vitro Oocyte Maturation

Background: Mammalian oocyte undergoes a series of structural nuclear modulations during maturation in order to obtain full competence to support fertilization and early embryonic development. Microtubules are major cytoskeleton components and have pivotal modulators for chromosomal movement, and it has been shown that partial or immature spindle organization may compromise correct ploidy of the matured oocytes in mammalian oocytes. Therefore, this study was carried out to investigate the optimum time of in vitro maturation in terms of nuclear and spindle organization in bovine oocytes. In this study we investigated the effect of various maturation time point on nuclear status and microtubular organization in order to introduce the best time of maturation for parthenogenetic activation. Materials and Methods: Oocytes were cultured in maturation medium for 14-32 hours. At 2 hours intervals, cultured oocytes were removed from IVM, fixed and immunostained using a monoclonal anti-β-tubulin antibody to investigate the microtubular organization and counterstained with H33342 to detect the nuclear status. Results: In this study we observed that: the percentages of MI oocytes were i) increased during 14 to 18 hours after maturation, ii) decreased during 18-32 hr after maturation in bovine oocyte. The percentage of MII oocytes from 14-22 hours (14 hours: 24.1, 16 hours: 28.1, 18 hours: 27.6, 20 hours: 74.3, and 22 hours: 87 %) increased in bovine oocyte. The oocytes were arrested at 22 hours after maturation until 32 hours in MII stage. The best development and organization of microtubules and arrangement of chromosomes on spindle were observed at 22 hours post of maturation. Conclusion: Our results demonstrated that, the best time for full development of MII spindle and arrangement of MII chromosomes is 22 hours post maturation and increasing time of IVM resulted in increasing degrees of spindle anomalies.