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

Abstract

Summary 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.