I-11: Cryopreservation of Canine Embryos

Background: The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer and cryopreservation of gametes have considerably contributed to the development of biomedical sciences in addition to improved breeding in domestic animals and infertility treatment in humans. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen is only available for the improved breeding and reproduction for companion and working dogs. The first mammalian species to have embryos successfully cryopreserved was the mouse in the early 1970s, and over the next three decades this has been extended to embryos of most species including humans. However, cryopreservation of canine embryos has not progressed far enough. Similar to the other mammals, embryo cryopreservation and subsequent transfer in canines would be centrally important in reproductive technologies by allowing optimal utilization of genetic resources in the development of breeding programs in animals by permitting genetic banking (for example, of rare or economically and socially important breeds), and by enhancing the clinical approach to infertility and sexually transmitted disease treatment. We describe here successful cryopreservation of embryos and subsequent embryo transfer in dogs. Materials and Methods: Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. The 4-cell to morula stage of cryopreserved embryos were non-surgically transferred into the uteri of recipient bitches by using a cystoscope. The pregnant bitches allowed to delivery by natural labor or cesarean section. Paternity testing based on microsatellite polymorphisms for the delivered pups was performed by using 23 micro-satellite markers. Results: Of nine embryo transfer experiments five recipients became pregnant and four of them delivered seven pups in total. Percentages of development to newborn after the transfer of cryopreserved canine embryos were 9.1%. A very lower percentage of embryos disappeared after detection of fetal sacs in recipients. The suitable combination of stages between transferring embryo and estrus of recipient might be when 8-cell to 16-cell stage of embryos were transferred to recipients on day 8-9 post LH surge. The microsatellite genotypes clearly demonstrated that those delivered pus were derived from donor embryos but not from the recipient animal. These results clearly indicate that cryopreserved canine embryos are able to develop to term, even if developmental rates after the transfer were lower.