Effects of different freezing parameters on the morphology and viability of preantral follicles after cryopreservation of doe rabbit ovarian tissue

ObjectiveTo compare the effects of dimethyl sulfoxide (DMSO), 1,2-propanediol (PROH), sucrose, trehalose, concentration of cryoprotectants, equilibration method, and postseeding freezing rate on doe rabbit ovarian tissue preservation after freezing, using fractional experimental design.DesignExperimental prospective study.SettingResearch institute in veterinary and agronomic colleges.Animal(s)Californian doe rabbits.Intervention(s)Ovarian cortices were prepared from ovaries collected in slaughterhouse. Fractional experimental design was used to evaluate simultaneously five chemophysical factors influencing the cryopreservation of ovarian tissue.Main Outcome Measure(s)Follicle viability by Live/Dead® viability/cytotoxicity kit and histologic evaluation of the ovarian tissue.Result(s)Experimental design suggests that equilibration method and cryoprotectant concentration have no effect on the proportion of normal follicles. Penetrating and nonpenetrating cryoprotectants seems to influence the preservation of the follicles with advantage for PROH and trehalose. The follicular preservation seems to be highly influenced by the postseeding freezing rate. Freezing rate of 0.3°C/min seems to be less deleterious than 2°C/min. Morphologic preservation ratio reaches 85% using PROH and trehalose.Conclusion(s)Cryopreservation of doe rabbit ovarian tissue using conventional cryoprotectant and 0.3°C/min as freezing rate seems to be a promising technique and could be used as a model for women.