Preservation of bovine preantral follicle viability and ultra-structure after cooling and freezing of ovarian tissue

Bovine preantral follicles within ovarian fragments were exposed and cryopreserved in absence or presence of 1.5 M glycerol (GLY), ethylene glycol (EG), propanediol (PROH) or dimethyl sulfoxide (DMSO), undergoing a previous cooling at 20 °C for 1 h (protocol 1) or at 4 °C for 24 h (protocol 2) in 0.9% saline solution. At the end of each treatment, preantral follicles were classified as non-viable/viable when they were stained/not stained with trypan blue, respectively. To confirm viability staining, ultra-structure of the follicles was evaluated by transmission electronic microscopy (TEM). Data were compared by Chi-square test (P < 0.05). The storage of the ovaries at 20 °C for 1 h (78%) and 4 °C for 24 h (80%) did not reduce significantly the percentage of viable preantral follicles when compared to the control (75%). Similar results were obtained when ovarian fragments, respectively, for protocols 1 and 2, were exposed to MEM (78 and 77%), 1.5 M EG (78 and 71%), as well as frozen in 1.5 M EG (74 and 77%). Percentages of viable follicles in control were similar to those observed after exposure (75%) and freezing (76%) in presence of 1.5 M DMSO only when protocol 1 was used. The increase of the concentration from 1.5 to 3.0 M, for all cryoprotectants, reduced significantly the percentage of viable preantral follicles after freezing. Ultra-structural analysis has confirmed trypan blue results, showing that not only basement membrane, but also organelles, were intact in viable preantral follicles. In conclusion, ovarian tissue cooling at 4 °C for 24 h before cryopreservation (protocol 2) does not affect the viability of bovine preantral follicles when 1.5 M EG is present in the cryopreservation medium.