Restoration of tiger (Panthera tigris) follicles from frozen-thawed ovarian tissues

Abstract

Ovarian tissue cryopreservation has been proposed as a tool to conserve the valuable genetics of endangered
species. However, sustaining the viability of follicles and oocytes enclosed within the frozen-thawed tissues is
important for the application of this technology. This study evaluated the impact of eCG supplementation on the
viability and growth of tiger follicles and oocytes within frozen-thawed ovarian tissues during in vitro culture. Six
frozen-thawed ovarian fragments from a tiger obtained post-ovariohysterectomy were included. Twelve antral follicles
were mechanically isolated from the ovarian fragments and randomly allocated to two culture conditions (control and
0.05 IU/mL eCG supplementation) and cultured in an alginate hydrogel for 3 days. Follicle growth was evaluated on
Day 0 and 3 using Image J software. At the end of the culture, follicles were removed from the hydrogel and stained
with neutral red to assess follicle viability. The oocytes were thereafter recovered and evaluated for nuclear status using
Hoechst 33258 dye. Follicle diameter in the control group significantly decreased (P < 0.05), whereas that of the eCG
supplementation group increased their relative growth (P < 0.05). The oocyte recovery rate from cultured follicles was
33% and 67% in the control and eCG groups respectively (P > 0.05). All recovered oocytes remained in the germinal
vesicle phase. The present study showed that tiger frozen-thawed antral follicles could maintain their viability and
structure when eCG was supplemented in the culture medium. In conclusion, viable oocytes within follicles can be
rescued from the tiger ovarian tissues post-mortem.