P-86: Determining The Most Oocyte Challenging Step during Vitrification-Warming in Sheep

Background: Oocyte cryopreservation is of utmost importance in the current repertories of assisted reproductive technology (ART). Despite this, oocyte vitrification is yet a problematic issue with limited successful pregnancies has been arisen after transfer of embryos derived from vitrified-warmed oocytes. In this sense, great attention has been paid to investigate possible mechanisms underlying low developmental competence of vitrifiedwarmed oocytes. In this study, we provide a sequential and detailed observation on early and delayed responses of in vitro matured sheep oocytes to different steps involved in a routine oocyte vitrification process to define the most critical stage of vitrification-warming process and to clarify the essentiality of post-warming interval. Materials and Methods: This study was carried out to investigate how different steps of a common vitrificationwarming process affect the early (immediately after warming) and delayed (2hours after warming) aspects of the treated oocytes including; volume changes, microtubule and chromosome organization, distribution of cortical granules (CGs), mRNA abundances, ultrastructure, and in vitro embryo development. Results: Cellular, molecular and ultrastructural results indicated that the first moments of oocyte exposure to cryoprotectants (CPAs) are the most challenging stage with comparable features to oocytes endured a complete process of oocyte vitrification-warming. However, developmental competence of oocytes exposed only to CPAs in response to parthenogenetic activation (PA), in vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) were quite comparable to control oocytes. Whereas, developmental competence of vitrified-warmed oocytes dramatically decreased compared to control oocytes, and only immediately activated (PA) oocytes had reasonable competency for development compared to control oocytes. Time-dependent assessment of CPAs and cryo -shocks indicated that although some of the injuries are reversible, the overall advantages and disadvantages of this time rest are not in favor of the oocyte developmental competence. Conclusion: The first set of cellular, molecular and ultrastructural evidences obtained in this study provided some indications to suggest that the most challenging step of current routine process of vitrification-warming maybe the first step in which oocyte is equilibrated in a mix solution of DMSO and EG cryoprotectants (ES step).