Short-term culture of ovarian cortex pieces to assess the cryopreservation outcome in wild felids for genome conservation

Ovarian cortex was dissected and uniform sized pieces of 2 mm diameter were obtained. Using a slow freezing protocol (-0.3[degree sign]C per min) in 1.5 mol/L ethylene glycol, 0.1 mol/L sucrose, the pieces were cultured for up to 14 days both before and after cryopreservation. The integrity of primordial follicles was assessed by histology, and the impact of different protein sources (FCS or BSA) and Vitamin C was determined during two weeks of culture.During culture the number of primordial follicles decreased within the ovarian pieces (p < 0.05). This effect was less pronounced when FCS was used as the protein source instead of BSA. Supplementation with Vitamin C had a detrimental effect on follicle survival. Since the procedure of cryopreservation had no effect on the follicle survival after one week of culture we conclude that the freezing protocol was suitable for felids. This is the first report of preserving a hugh amount of follicles within ovarian tissue by slow freezing performed in several wild feline species.