In vitro survival rate of bovine oocytes following vitrification in glass capillary micropipette (GCM)

The purpose of this study was to evaluate the use of glass capillary micropipette (GCM) as a vessel for vitrification of bovine oocytes. Cumulus-oocyte complexes (COCs) were obtained from slaughter-house and washed 5 to 6 times in the washing medium (TCM-199 + 20% FBS) and randomly assigned to treatment and control group. In the first step of vitrification, COCs were exposed to first vitrification solution (VS1) (10% ethylene glycol (EG), 10% DMSO in holding medium (TCM-199 + 10% FBS: HM)) for 1 min at room temperature and then placed in VS2 solution (20% EG, 20% DMSO in HM) for 25 sec and immediately were loaded into the GCM vessel. The filled portion of GCM vessels were placed in liquid nitrogen (LN2) for 3 to 5 sec and then completely immersed and stored there. The oocytes were thawed by immersing the capillary end of the straw in 1 ml of 0.25 M sucrose in HM and gently expelling the contents. After 1 min the oocytes were transferred into 100 μl of 0.15 M sucrose in HM for another 5 min and then washed with HM twice. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50 μl droplet of maturation medium (TCM-199 + 10% FBS + 10 IU/ml PMSG + 5 IU/ml HCG) covered with paraffin oil in a CO2 incubator at 38.5ºC for 24 hrs. A high proportion of morphologically normal oocytes (90%) was recovered after vitrification-warming. The percentage of live oocytes after 24 hrs when tested with trypan blue in GCM group was 85.18%, significantly did not differ from control group (90%). The proportion of oocytes which were found to have undergone nuclear maturation did not show statistical difference between the control and GCM group (61.29% vs 40%, respectively). The results of present study demonstrated that vitrification of immature bovine oocytes in the GCM vessels and EG + DMSO solution have high survival rate.