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Background:L-proline is a natural,nontoxic cryoprotectant that helps cells and tissues to tolerate freezing in a variety of plants and animals.The use of L-proline in mammalian oocyte cryopreservation is rare.In this study,we explored the cryobiological characteristics of L-proline and evaluated its protective effect in mouse oocyte cryopreservation.Methods:The freezing property of L-proline was detected by Raman spectroscopy and osmometer.Mature oocytes obtained from 8-week-old B6D2F 1 mice were vitrified in a solution consisting various concentration of L-proline with a reduced proportion of dimethyl sulfoxide (DMSO) and ethylene glycol (EG),comparing with the control group (15％ DMSO and 15％ EG without L-proline).The survival rate,5-methylcytosine (5-mC) expression,fertilization rate,two-cell rate,and blastocyst rate in vitro were assessed by immunofluorescence and in vitro fertilization.Data were analyzed by Chi-square test.Results:L-proline can penetrate the oocyte membrane within 1 min.The osmotic pressure of 2.00 mol/L L-proline mixture is similar to that of the control group.The survival rate of the postthawed oocyte in 2.00 mol/L L-proline combining 7.5％ DMSO and 10％ EG is significantly higher than that of the control group.There is no difference of 5-mC expression between the L-proline combination groups and control.The fertilization rate,two-cell rate,and blastocyst rate in vitro from oocyte vitrified in 2.00 mol/L L-proline combining 7.5％ DMSO and 10％ EG solution are similar to that of control.Conclusions:It indicated that an appropriate concentration of L-proline can improve the cryopreservation efficiency of mouse oocytes with low concentrations of DMSO and EG,which may be applicable to human oocyte vitrification.