In vitro maturation of canine oocytes co-cultured with bovine and canine granulosa cell monolayers.

The present study investigated the effects of bovine granulosa cell monolayers (BGML) and canine granulosa cell monolayers (CGML) on nuclear maturation of canine oocytes with and without cumulus cells. Cumulus-oocyte complexes (COCs) or cumulus-free oocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM, control group), DMEM with BGML (BGML group), or DMEM with CGML (CGML group) for 72 h at 38.5 °C in 5% CO(2), 5% O(2,) and 90% N(2). All media were supplemented with 10% of FCS, 50 ng/mL of EGF, 2 µg/mL of estradiol-17ß, 0.1 IU/mL of hCG, 0.1 IU/mL of FSH, 0.25 mM of pyruvic acid, 100 µM of ß-mercaptoethanol, 100 IU/mL of penicillin, and 100 µg/mL of streptomycin. In cumulus-enclosed oocytes retrieved from ovaries at estrus and/or diestrus, the highest percentage of M-II oocytes (P < 0.05) was present in the BGML group (27.0%) compared with the CGML group (7.9%) and the control group (3.5%). In cumulus-free oocytes collected from ovaries at estrus and/or diestrus, the proportions of M-II oocytes co-cultured with the CGML were low (3.0%) and similar (P > 0.05) to proportions achieved with control (3.0%). However, the presence of BGML improved (P < 0.05) the ability of denuded oocytes to develop into M-II (10.2%). The BGML group had the highest overall meiotic resumption (P < 0.05), and least oocyte degeneration (P < 0.05) among experimental groups. In conclusion, BGML had a positive impact on the in vitro maturation system, as well as meiotic resumption of canine oocytes.

Cryopreservation of canine embryos.

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. 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 to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.

Effect of graft site and gonadotrophin treatment on follicular development of canine ovarian grafts transplanted to NOD-SCID mice.

PURPOSE: To investigate the effect of graft site and gonadotrophins administration on the number and survival rate of follicles of canine ovarian grafts transplanted to NOD-severe combined immune deficiency (SCID) mice. METHODS: Fresh ovarian cortex slices obtained from immature bitches were grafted subcutaneously (SC), under kidney capsule (KC) or into ovarian bursa (OB) in NOD-SCID mice. Two months after surgery, the mice allocated into non-treated and treated gonadotrophins groups that injected with porcine follicle stimulating hormone during 7 days and human chorionic gonadotrophin 48 h later. Ovarian grafts were collected after 10 h of last injection and processed for histology. RESULTS: The number of transitional and preantral follicles under KC and into OB was significantly higher in gonadotrophins-treated mice than those who received saline. Furthermore, the survival rates of primary, transitional and preantral follicles under KC and into OB grafts were significantly higher than those placed SC in the treated gonadotrophins group, and in the non-treated gonadotrophins group; the proportion of primary and preantral follicle survival was significantly higher under KC and into OB than SC grafts. CONCLUSIONS: In canine ovarian xenografting, administration of gonadotrophin could be effective for improvement of survival of transplanted ovary. Furthermore, the grafting into OB appeared to be better than grafting under KC, which in turn is better than SC.