Vitrification of Mouse Oocytes by Open Pulled Straw(OPS) Method

Objective To investigate the effect of different frozen-thawing conditions on mouse oocytes viability as a model using open pulled straw(OPS) method,and to provide theoretical and technical reference for a simple and effective oocyte cryopreservation in livestock,endangered animals up to humans.Method Under room temperature(25 ± 0.5℃),oocytes were vitrified in OPS with cryoprotectants(EFS,EDFS)mainly composed of EG and DMSO.Experiment 1: oocytes were firstly pretreated in 10% EG + 10% DMSO or 10% EG for 30 s,then exposed to EDFS or EFS for 15 to 45 s,respectively,at 37℃ ± 0.5℃ or 25℃ ± 0.5℃ on a hot plate.Experiment 2: When warming,three methods were used to dilute the cryoprotectants from the oocytes vitrified in EDFS30 or EDFS40 at 37℃ ± 0.5℃ on a hot plate.Method A: some oocytes were rinsed at first in 0.5 M sucrose for 2 min and then in 0.3 M sucrose for 5 min.Method B: some oocytes were rinsed at first in 0.3 M sucrose for 5 min and then in 0.15 M sucrose for 2 min.Method C: other oocytes were rinsed in 0.5 M sucrose for 5 min.Experiment 3: The oocytes after warming were cultured in vitro with mCZB then chemically activated with SrCl_2. Results and conclusions The highest rates(92.1% at 25℃ ± 0.5℃ and 92.5% at 37℃ ± 0.5℃) of morphologically normal mouse oocytes among treated groups were similar(P 0.05) to that(98.9%) of control.Also,at 37℃ ± 0.5℃ the highest rates(92.5% in EDFS30 and 67.5% in EDFS40) of morphologically normal oocytes recovered after vitrification were observed when method C was used.The developmental rates of vitrified-warmed oocytes cultured in mCZB for 0.5 h before parthenogenetic activation were higher(cleavage rate 80.0% vs.57.7%(P 0.05);blastocyst rate 18.2% vs.0,(P 0.01)) than that of non-cultured ones.But the cleavage rate(80.0% vs.77.8%) or morula rate(52.8 % vs.61.9%) of cultured oocytes and controls after parthenogenetic activation was similar(P 0.05).