In vitro survival rate of bovine oocytes following vitrification in glass capillary micropipette [GCM]

ABSTRACT

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-l99 + 10% FBS HM]] for I 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 [LN [2]] 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 micro l of 0.15 M sucrose in HM for another 5 mm and then washed with HM twice. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50 micro 1 droplet of maturation medium [TCM-l99 + 10% FBS - 10 IU/ml PMSG + 15 IU/ml HCG] covered with paraffin oil in a CO [2] incubator at 38.5°C 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