Cryopreservation of Oocytes Using Slow Cooling,Ultrapid Cooling and Nitrification Protocols

Unfertilized Kunming mouse oocytes obtained 14 h after hCG were cryopreserved employing four freezing protocols:1)SL,the oocytes were equilibrated in 0.5,1.0,1.5 mol／L 1,2-propanediol for 5,5 and 10 min at room temperature respectively;slow cooling at a rate of -0.3 ℃／min to -30 ℃.The frozen eggs were thawed in 37 ℃ water and moved into fresh medium after equilibration in 1.0 mol／L 1,2-propanediol -0.2 mol／L sucrose／PB1,0.5 mol／L 1,2-propanediol／PB1,0.2 mol／L sucrose／PB1 for 5 min,respectively,2)Q1,using a freezing medium of 3.5 mol／L DMSO in Hepes-HTF,and cooling ultrarapidly by simply plunging into liquid nitrogen.The frozen eggs were thawed in 37℃ water and moved into fresh medium after equilibration in 0.5 mol／L sucrose,0.25 mol／L sucrose Hepes-HTF for 5 min respectively;3)R-FVM,vitrifying oocytes in 90% VS1 and thawed in 4℃ bath and moved into fresh medium after equilibration in 50% VS1 and 25% VS1 for 10 min,respectively;4)MVM,vitrifying oocytes in Massips vitrification solution and thawing in 20℃ bath.The frozen eggs equilibration in 1 mol／L sucrose for 10 min and washed 3 times by PBS.The results showed that the survival rate and fertilization rate of SL and R-FVM were similar to those of the control,but significant higher than those of Q1 and MVM (P<0.05).Our results suggested that:1) Both slow cooling and vitrification can preseve Kunming mouse oocytes well;2)The vitrification solution containing both permeating and nonpermeating cryoprotectants (such as VS1) was suitable for Kunming mouse oocyte vitrification.