Abstract
Ovarian tissue cryopreservation combined with immature follicle development can preserve female fertility in wildlife, regardless of age or reproductive timing. To investigate the effects of different cryopreservation methods and cryoprotectants on follicular survival and developmental capacity, ovarian cortical pieces from 15 dogs were cryopreserved by slow freezing or vitrification with different additional cryoprotectants as follows: dimethyl sulfoxide (DMSO), polyvinylpyrrolidone (PVP), combined DMSO and PVP (each at half the concentration of when used independently), or none (control). Cryopreserved ovarian tissues were evaluated by neutral red staining, histology, and xenotransplantation assays. Among cryopreservation conditions tested, vitrification with combined DMSO and PVP significantly improved the maintenance of follicular morphology compared to that in control. Furthermore, ovarian tissues vitrified using this condition maintained follicle morphology and developmental capacity 9 weeks after grafting, as shown by an increased percentage of primary and secondary follicles and a significant decrease in the transition stage from primordial to primary stage follicles 5 and 9 weeks after grafting. In contrast, slow freezing and control groups lost intact follicles by 5 weeks after grafting. The described cryopreservation techniques, which preserve canine follicle development, will build the foundation of ovarian tissue cryopreservation to preserve female fertility in wild canids.