Received January 21, 2014
Accepted February 20, 2014

Objective: The aim of this study was to determine the most efficient protocol for cryopreservation of ovarian tissue using the automatic Freeze Control® system to test two different cooling curves combined with two different cryoprotectants: dimethyl sulfoxide (DMSO) and ethylene glycol (EG). Methods: In this study, 20 female Wistar rats underwent bilateral oophorectomy. The ovaries were divided into two parts: one cryopreserved in DMSO 1.5M and the other in EG 1.5M. Two cooling curves, slow (1h50min) and rapid (35min) were analyzed. Tissue samples were frozen, thawed, fixed, and processed for hematoxylin and eosin staining to analyze oocyte integrity. Follicular analysis was performed under optical microscopy (400x magnification) and preantral follicles were classified as primordial or primary according development stage. ANOVA was performed and Tukey’s test was used for comparison between means, with P<0.05 defined as significant. Results: In cryopreserved tissue, the follicles with preserved integrity in each ovary were 79% primordial and 29% primary. In non-frozen (control) tissue, all follicular types were observed (primordial, primary, secondary, preantral, and antral). Reversible changes included cytoplasmic vacuolization and irregular cell outline, and pyknosis occurred as an irreversible alteration. EG was more efficient as then DMSO, preserving a greater number of viable primordial and primary follicles. Nevertheless, comparison of both cooling curves revealed no statistically significant differences. Conclusion: EG is indicated as the best cryoprotectant to obtain higher viable numbers of primordial and primary follicles from rat ovary tissue. Extra evaluations should be indicated to demonstrate ovarian functionality, as hormone levels detections.