Incidence of Apoptotic Cells After Vitrification in Canine Ovarian Tissues

Abstract

Although vitrification and follicular survival subsequent to xenotransplantation of canine ovarian tissues have been reported, the degree of cryoinjury after the vitrification has not yet been fully examined. Since cryoinjury after vitrification of ovaries has been reported in bovine and humans, this study evaluated canine ovarian tissues after vitrification by immunohistochemistry and TUNEL assay. Ovaries were collected from immature bitches. The ovarian tissue cubes were pretreated with 1 M DMSO. Subsequently, DAP 213 solution (2 M DMSO, 1 M acetamide, 3 M propylene glycol) was added. The cryotubes containing the ovarian tissues were placed on ice for 5 min, then plunged directly into liquid nitrogen. After warming with 0.25 M sucrose, cryopreserved ovarian tissues were fixed and subsequently stained with hematoxylin-eosin, proliferation cell nuclear antigen (PCNA) antibody, or active caspase-3 (AC-3) antibody, and evaluated by the TUNEL assay. There were no differences in the numbers of primordial, primary, secondary and antral follicles/mm 2 between the fresh and vitrified-warmed ovarian tissues. Percentages of PCNA and AC-3 antibody positive cells were similar regardless of the developmental stage of the follicles and experimental group. A few TUNEL positive cells were detected in both experimental groups. These results suggest that vitrification with DAP213 does not induce cryoinjury in ovarian tissues from immature bitches.