Cryopreservation of the germinal vesicle stage bovine oocytes by using vitrification method in straws and microdrops
Keywords:Cryopreservation, in vitro maturation, in vitro fertilization, vitrification.
This study aimed to examine the in vitro maturation and produce embryos of in vitro fertilization from the germinal vesicle stage bovine oocytes cryopreserved by vitrification method in straws and microdrops. The cumulus-oocyte complexes (COCs) with three or more layers of cumulus cells and homogeneous cytoplasm were selected for cryopreserveation in two steps: oocytes were first equilibrated in the equilibrated solution for 45 seconds, then transferred to the vitrification solution for 25 seconds and loaded on straw or microdrop. Straws and microdrops were plunged directly into liquid nitrogen within 30 seconds. Oocytes were thawed in three steps through media: firstly, oocytes were directly expelled into medium for 1.5 minutes, then in second medium for 1.5 minutes and at last in third medium for 5 minutes. The thawing was performed at 25-28oC. The ratio of survival COCs was evaluated by morphological observation, then some of them were evaluated by AO/PI stained method, the remaining were transferred to maturation medium 1 or 2 at 38,5oC in a humidified atmosphere of 5% CO2 in air for 20-24hrs. The matured COCs were used for in vitro fertilization. The ratios of survival COCs by morphological observation in straws and microdrops were 68.52±1.19% vs 73.53± 1.17% (p<0.05); by AO/PI stained method were 52.69±3.66% vs 48.33±3.72% (p>0.05); Metaphase II stage rates were 20.79±1.38% vs 12.79±1.13% (p<0.05); insemination rates (two-cell embryo) were 15.64±2.72% vs 12.61±3.15%; respectively; 2/28 embryos had developed to blastocyst (from straws).