Cryopreservation of sheep ovarian tissue: Effect of different cryoprotectants and equilibration regimen on ovarian follicles structure

This study compared the efficiency of some cryoprotectant agents (CPAs), including both of exposure time and temperature aiming to determine the effect of cryopreservation on structural integrity of ovine ovarian tissue. This study was carried out at the Laboratory of Reproduction and Biotechnology, Sheep and Goats Research Department, Animal Production Research Institute. A number of 118 sheep ovaries were collected from the local slaughter- house and transported to the laboratory in 0.9% saline solution. The ovarian cortex was cut into fragments. The selected ovarian fragments (156 fragments) were randomly distributed over the experimental treatments; 12 fragments (as control) for routine histological and electron microscopy examination. The remained fragments (144) were divided into three equal groups according to the studied cryoprotectant agents; ethylene glycol (EG), propylene glycol (PROH) or dimethyl sulfoxide (DMSO). Then they submitted to the vitrification procedure. The obtained results of this study showed that vitrification and freezing/thawing processes negatively affected on integrity of the follicles in the ovarian fragments, where it achieved the highest (P<0.05) integrity percentage (85.4%), when EG was used after 30 min equilibration at room temperature, compared with using DMSO or PROH (75 and 45.7%, respectively). Whereas, longer exposure time up to 60 min. decreased the integrity of all the groups. Equilibration of ovarian tissue fragments under cooling condition (5oC) for 30 min, achieved significantly (P<0.05) lower proportion of integrity in the frozen/thawed ovarian follicle compared to equilibration for 60 min. The percentage of normal preantral follicles (primordial and primary follicles) was 79.3% in control fragments of ovarian sheep, this percentage was significantly (P<0.05) reduced after exposure of ovarian tissue to cryopreservation in the presence of EG, DMSO or PROH, under different equilibration periods and temperatures. It can be concluded that EG followed DMSO is the most suited cryoprotectant among these freezing/thawing procedures, equilibration of ovarian tissues for 30 min. resulted in high percentage of intact follicle at room temperature compared to those at 5oC. Meanwhile, when extending the period to 60 min, the percentage of intact follicle was higher at 5oC. Finally, the observed follicular damage was rather due to the cryoprotectant than the cryopreservation process itself.