Histological and biological assessment of vitrified ovarian follicles from large animals

Mammalian ovaries contain mixed populations of follicles at different developmental stages. A combination of vitrification and growth culture of ovarian follicles could provide the desired number of mature eggs from a preserved small amount of ovarian tissues. Secondary and primordial follicles from porcine and bovine ovaries were vitrified in solutions containing ethylene glycol, dimethyl sulfoxide and different concentrations of sucrose, and assessed via histological examination, viability staining, xenografting to immunodeficient mice, and in vitro culturing. Histological examination revealed the damage to oocytes and the damage to follicle components separately. The effects of sucrose in vitrification solutions on the follicles were different depending on the developmental stage of the follicle, oocyte size, cell type in the follicle, and species. Viability staining with fluorescein diacetate was useful to assess the damage to oocytes in secondary follicles. In the xenografts, vitrified bovine primordial and secondary follicles developed to the antral stage, and vitrified porcine primordial follicles developed to the secondary stage. Furthermore, bovine secondary follicles formed antrum-like structures in culture. These results suggest that histological examination and viability staining are valuable for assessing the direct effects of vitrification and warming conditions on follicles and oocytes, while xenografting and in vitro culturing can be useful for evaluating the developmental ability of vitrified follicles and oocytes.