Abstract
BackgroundCryopreservation of ovarian tissue has the potential to preserve female germ cells of endangered mammals. In the present study, a freezing protocol successfully used for human tissue, was adapted for preserving ovarian tissue of domestic and non-domestic felids. Ovaries from non-domestic felid species were obtained from seven freshly euthanized and two recently deceased wild felids kept in different European Zoos. In addition, ovaries from domestic cats were obtained after ovariectomy from local veterinary clinics for methological adaptations.Ovarian cortex was dissected and uniform sized pieces of 2 mm diameter were obtained. Using a slow freezing protocol (-0.3°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.Results and conclusionDuring 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 huge amount of follicles within ovarian tissue by slow freezing performed in several wild feline species.
Highlights
Cryopreservation of ovarian tissue has the potential to preserve female germ cells of endangered mammals
Our group used BrdU-labeling and Hoechst staining for integrity assessment of mechanical isolated preantral follicles to control culture impacts [21], but it was shown that the procedure of isolation from the ovarian cortex itself can cause a loss of integrity in primordial follicles [22]
In the present study we focused on preserving ovarian tissue by cryopreservation in conjunction with integrity assessment after culture of frozen-thawed pieces by applying standard histology and a novel aspect of follicle counting (40 follicles per piece) for time saving
Summary
Cryopreservation of ovarian tissue has the potential to preserve female germ cells of endangered mammals. A freezing protocol successfully used for human tissue, was adapted for preserving ovarian tissue of domestic and non-domestic felids. Oocytes can tolerate cryopreservation very well irrespective of whether they are frozen within a whole ovary, as ovarian pieces, or as individual, isolated follicles [7]. To utilize the oocyte reserve from female cat gonads, slow freezing of ovarian cortex, similar to that already successfully used for human tissue [9], was tried. Cryopreservation of ovarian cortex might be considered for genome conservation and oocyte retrieval in felids. Bosch et al [14] demonstrated the successful cryopreservation of domestic cat ovarian cortex in conjunction with xenotransplantation into nude mice. Feline oocytes seem to express a higher sensitivity towards cryopreservation protocols [15,16] than other mammalian oocytes
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