Cryopreservation of canine ovarian and testicular fibroblasts

To derive a practical procedure to store canine somatic cells, fibroblasts isolated from testicular or ovarian tissues were cryopreserved in 1.2 M ethylene glycol or in 1.2 M dimethylsulfoxide prepared in Dulbecco's Modified Eagle Medium as cryoprotectants, and were frozen either in plastic straws or vials. Thawed cells were cultured for 24 hr at 38.5°C in a humidified atmosphere of 5% CO₂ + 95% air, and then their membrane integrity was assayed with a double fluorescent stain, Fertilight®. In addition, frozen-thawed fibroblasts were cultured for 4 days, and then their functional survival was measured after staining small colonies with trypan blue. After freezing and thawing, membrane integrity of testicular fibroblasts was 55-70% and functional survival ranged from 20-40%. With frozen-thawed ovarian cells, the average membrane integrity was 55- 75% and the average functional survival was 35-40%. When frozen in ethylene glycol, functional survival of ovarian fibroblasts was significantly higher than that of testicular cells (P<0.05). These methods should prove useful to preserve cells collected from canids in the wild.