Vitrification of germinal vesicle and metaphase II swine oocytes

Abstract

In vitro maturation of oocytes is a technique that implies extended cultivation of immature oocytes, recovered by aspiring the cumulus-oocytes complexes from follicles, their maturation under adequate conditions for 48 hours, until they reach the metaphase II stage, so they can be in vitro fertilized later on. Oocytes, once recovered from the follicles, remain in the germinal vesicle stage, representing the diplotene of the first meiotic division at 0 hours of maturation. After maturation, swine oocytes evolve, passing through several stages, from germinal vesicle breakdown, to the first meiotic division and then achieving the metaphase II stage. Vitrification is one of the most interesting cryopreservation techniques, more and more used in the past few years, due to the advantages this technique has, compared to freezing. Vitrification prevents formation of ice crystals during the cooling process; the liquid gets a glassy solid structure, that stops molecular mobility, without any structural reorganization of the liquid that contains the oocytes. Freezing is a slow process, that can take hours until completed and it is very difficult to establish a balance between the agents that produce osmotic injury, due to ice crystals formation and due to osmotic stress. Assisted reproduction techniques rely also on using gametes stored at very low temperatures, in order to enable unlimited access to them. Whereas vitrification can be performed in different stages of gamete and zygote development, in this research we wanted to see the effectiveness of applying this process at the beginning or during gamete processing. Cultivation of oocytes to maturation and their potential use in assisted reproduction techniques, enabled the oocyte development to metaphase II for 52,63% of those belonging to Group 1 ( vitrification / cultivation) and for 78,46% of the oocytes belonging to Group 2 (maturation / vitrification / cultivation). Obviously, cultivation of oocytes before vitrification brings more advantages for the meiotic resumption. Regardless the time of vitrification induction, after warming, the oocytes resumed their meiotic process. By using both protocols, we gathered satisfying rates (over 50%) of oocytes ready to be used in assisted reproduction techniques.