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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Pre-implantation Development of Canine Embryos In vivo
  5. Cryopreservation of Oocytes and Embryos
  6. Non-surgical Embryo Transfer in the Dog
  7. Conclusion
  8. References

The assisted reproductive techniques used in dogs have strictly limited utility when compared with other mammals. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen has been only readily available method for improved breeding for companion and working dogs. Recently, successful cryopreservation of embryos and subsequent embryo transfer with a non-surgical technique in dog was reported. Application of embryo cryopreservation and transfer technology could contribute to breeding management in companion dogs, working dogs including guide dogs and drug-detecting dogs and quarantine dogs. Such technology would also facilitate the transportation and storage of genetic materials and aid in the elimination of vertically transmitting diseases in the dog.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Pre-implantation Development of Canine Embryos In vivo
  5. Cryopreservation of Oocytes and Embryos
  6. Non-surgical Embryo Transfer in the Dog
  7. Conclusion
  8. References

Since the first success for cryopreservation of mouse embryos in the early 1970s (Whittingham et al. 1972), this technique has been extended to embryos of most mammalian species including humans. Similar to other mammals, cryopreservation of embryos and subsequent embryo transfer in dogs would be important for optimal utilization of genetic resources. Successful cryopreservation of embryos depends on tolerance of the embryos to physical and chemical changes during the freezing and thawing procedures. One of the most serious problems for successful cryopreservation of canine embryos seems to be large amount of lipid contents within the cytoplasm. The lipid particles are similarly seen in porcine and bovine embryos; however, the density of the lipids is much higher in canine embryos (Abe et al. 2008b). Although there were a few reports on cryopreservation of oocytes or embryos in dogs (Abe et al. 2008a, 2010), recently, the first successful cryopreservation of embryos and subsequent embryo transfer in dogs have been reported (Abe et al. 2011). Because of the anatomy of the reproductive tract of the bitch and the related inaccessibility of the uterus from the vagina, surgical embryo transfer has proven to be the only adopted option (Tsutsui et al. 2001, 2006). In this regard, successful embryo transfer with a non-surgical technique in dogs has been reported (Abe et al. 2011). This article reviews recent advances for cryopreservation of canine embryos.

Pre-implantation Development of Canine Embryos In vivo

  1. Top of page
  2. Contents
  3. Introduction
  4. Pre-implantation Development of Canine Embryos In vivo
  5. Cryopreservation of Oocytes and Embryos
  6. Non-surgical Embryo Transfer in the Dog
  7. Conclusion
  8. References

The dog is a monoestral polyovulatory non-seasonal species. Canine reproductive physiology is considerably different from other mammalian species. The plasma progesterone concentration of the dog begins to increase a few days before ovulation. Pre-ovulatory luteinization is typical in dogs. The oocytes of dogs are ovulated at the germinal vesicle stage, and they complete meiotic maturation within the oviduct. Thus, canine oocytes and embryos spend a long time prior to implantation in the reproductive tract. To develop methods of transfer and cryopreservation of embryos, it is essential to understand early embryonic development in vivo in all mammals. However, owing to singular reproductive features, the actual situation and mechanisms of early development, such as oocyte maturation, fertilization and embryogenesis, have not been fully elucidated for the dog compared to many other domestic mammalian species (Reynaud et al. 2006). It is understood that fertilized eggs develop to the 2-cell stage on Days 6–10 and migrate to the uterus on Days 11–12 post-LH surge in domestic dogs (Concannon and Lein 1989; Johnston et al. 2001). However, the timing of pre-attachment embryogenesis relative to ovulation has yet to be determined (Senger 2003).

Recently, the pre-implantation development of the Labrador Retriever embryos was examined (Abe et al. 2008b, 2011). A total of 620 embryos were collected from oviducts isthmus, and uteri of 134 Labrador Retriever bitches inseminated with ejaculates, and the developmental stages and localization of the collected embryos were examined. As shown in Fig. 1, embryos at the 16-cell to morula stage migrated from the oviduct into the uterus on Day 10 post-LH surge and likely completed within 24 h. By Day 12 post-LH surge, all of the developing embryos were localized in the uteri. Embryos developed to the morula stage by Days 11–12 and to the blastocyst stage by day 12–13 post-LH surge (Abe et al. 2011; H. Suzuki 2012 unpublished data). On the other hand, Tsutsui et al. (2001) recovered blastocysts earlier (9–10 days after progesterone concentrations exceeded 2 ng/ml, which equates to approximately the time of the LH surge). These results may indicate that there are breed-related differences in the pre-implantation development of embryos in dogs.

image

Figure 1. Localization of the developing canine embryos in reproductive tracts. A total 620 embryos were recovered from 134 bitches after insemination at Days 7–15 post-LH surge. B = blastocyst, M = morula

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Cryopreservation of Oocytes and Embryos

  1. Top of page
  2. Contents
  3. Introduction
  4. Pre-implantation Development of Canine Embryos In vivo
  5. Cryopreservation of Oocytes and Embryos
  6. Non-surgical Embryo Transfer in the Dog
  7. Conclusion
  8. References

Recently, cryopreservation of oocytes and embryos in dogs has been attempted. Canine oocytes are similar to porcine oocytes in diameter and in the characteristic that they have a high cytoplasmic lipid content; the procedure of cryopreservation in porcine oocytes may therefore be suitable for canine oocytes. When dog cumulus-oocyte complexes (COCs) were collected from ovaries and were vitrified in DMSO-based (DAP213) or ethylene glycol-based (E30S) medium in a cryotube or on a cryotop sheet, respectively, there were no significant differences in morphological normality (59–62%) post-warming (Abe et al. 2008a). However, integrity of the plasma membrane, assessed by propidiun iodide staining, in the vitrified-warmed COCs in the DAP213 in the cryotube (5%) was lower than that in the E30S on the cryotop sheet (18%). To improve the warming and cooling rates, when COCs were vitrified and warmed by DAP213 on a cryotop sheet, the plasma membrane integrity of the oocytes increased to 44% (Abe et al. 2010).

For cryopreservation of canine embryos, Abe et al. (2011) examined the effects of vitrification on the viability of embryos at pre-implantation development. The embryos at 1-cell to blastocyst stages were exposed to PB1 containing 5%, 10% and 20% ethylene glycol, and then 30% ethylene glycol plus 0.5 m sucrose (E30S) for 5, 2, 2 and 1 min, respectively, at room temperature. They were then placed on a cryotop sheet, which was immediately plunged into liquid nitrogen. To warm the cryopreserved embryos, the cryotop with embryos was soaked in PB1 containing 0.5 m sucrose at 37°C. To remove the cryoprotectants, the embryos recovered from the cryotop were exposed to PB1 with a sequential series of 0.5, 0.25, and 0.125 m sucrose for 1 min in each solution at 37°C. As a result, over 80% of the 1-cell to morula stages embryos exhibited a normal morphology. However, the majority of cryopreserved blastocysts showed abnormal morphology after warming. The viability of the morphologically normal embryos at 1- to 16-cell, morula and blastocyst stages was 90–100%, 50% and 40%, respectively, which was assessed by the staining with propidium iodide (Abe et al. 2011). These results suggest that differentiation of the canine embryos during pre-implantation influences their susceptibility to vitrification. In addition, the sensitivity of canine oocytes to cryopreservation may be related to their high lipid content; if so, tolerance to cryopreservation might increase if their lipid content is reduced. In porcine, the removal of cytoplasmic lipid droplets improved the survival of oocytes and embryos (Ushijima et al. 2004; Hara et al. 2005). Further studies seem to be required to develop an optimal cryopreservation method for canine oocytes and embryos.

Non-surgical Embryo Transfer in the Dog

  1. Top of page
  2. Contents
  3. Introduction
  4. Pre-implantation Development of Canine Embryos In vivo
  5. Cryopreservation of Oocytes and Embryos
  6. Non-surgical Embryo Transfer in the Dog
  7. Conclusion
  8. References

Although there are a few reports on surgical embryo transfer in dogs (Tsutsui et al. 2001, 2006), non-surgical embryo transfer has not been reported until 2010. The surgical method might be more commonly applied because of the difficulty of non-surgical embryo transfer owing to the unique morphology of the vagina and cervix in the dog. However, non-surgical embryo transfer techniques would enhance application to clinical and field situations. In 2011, successful transcervical embryo transfer using was reported by Abe et al. (2011). When the four-cell to morula stage of cryopreserved embryos were non-surgically transferred into the uteri of nine recipient bitches using a catheter equipped with a cystoscope for human use, five recipients became pregnant and four of them delivered a total seven puppies. In addition, paternity testing based on the microsatellite genotypes by using 23 microsatellite markers demonstrated that those delivered puppies were derived from donor embryos but not from the recipient bitch (Abe et al. 2011). This embryo transfer system was originally developed for artificial insemination for large breeds such as Labrador Retrievers (Wilson 2001).

Conclusion

  1. Top of page
  2. Contents
  3. Introduction
  4. Pre-implantation Development of Canine Embryos In vivo
  5. Cryopreservation of Oocytes and Embryos
  6. Non-surgical Embryo Transfer in the Dog
  7. Conclusion
  8. References

Although the factors impede vitrified embryos from developing to term after transfer are not well understood, vitrification is one suitable method for cryopreservation of canine embryos, and pregnancy can be obtained following transcervical embryo transfer using a cystoscope. These techniques may contribute to the control of genetic diversity in various canid species and dog breeds. One of the most effective applications of cryopreservation of canine embryos would be for an improved breeding system of guide dogs for the blind as well as other working dogs. Although guide dogs make a remarkable contribution to the quality of life of the blind people in the world, many countries suffer from an acute shortage of guide dogs. In part, this is because only a certain percentage of available dogs meet the requisite aptitude standards. Even among Labrador Retrievers, which are particularly suited to the role, only 30–40% of the dogs that are trained ultimately work out as guide dogs in Japan. In addition, a further problem in breeding system for the guide dogs is that guide dogs have to be spayed before training starts, which means the dogs who do succeed in becoming guide dogs can never be used for breeding. Cryopreservation of embryos could overcome, at least in a part, these difficulties.

Conflicts of interest

None of the authors have any conflicts of interest to declare.

Author contributions

HS is responsible for the conception and design of this review article.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Pre-implantation Development of Canine Embryos In vivo
  5. Cryopreservation of Oocytes and Embryos
  6. Non-surgical Embryo Transfer in the Dog
  7. Conclusion
  8. References
  • Abe Y, Lee DS, Kim SK, Suzuki H, 2008a: Vitrification of canine oocytes. J Mamm Ova Res 25, 3236.
  • Abe Y, Suwa Y, Ueta YY, Suzuki H, 2008b: Preimplantation development in labrador retrievers. J Reprod Dev 54, 135137.
  • Abe Y, Asano T, Ali M, Suzuki H, 2010: Vitrification of canine cumulus-oocyte complexes in DAP213 with a cryotop holder. Reprod Med Biol 9, 115120.
  • Abe Y, Suwa Y, Asano T, Ueta YY, Kobayashi N, Ohshima N, Shirasuna S, Abdel-Ghani MA, Oi M, Kobayashi Y, Miyoshi M, Miyahara K, Suzuki H, 2011: Cryopreservation of canine embryos. Biol Reprod 84, 363368.
  • Concannon PW, Lein DH, 1989: Hormonal and clinical correlates of ovarian cycles, ovulation, pseudopregnancy and pregnancy in dogs. In: Krik R (ed.), Current Veterinary Therapy (Small Animal Practice), vol. 10. WB Saunders Company, Philadelphia, pp. 12691282.
  • Hara K, Abe Y, Kumada N, Aono N, Kobayashi J, Matsumoto H, Sasada H, Sato E, 2005: Extrusion of removal of lipid from the cytoplasm of porcine oocytes at the germinal vesicle stage: centrifugation under hypotonic conditions influences vitrification. Cryobiology 50, 216222.
  • Johnston SV, Root Kustritz MV, Olson PNS, 2001: Canine pregnancy. In: Johnston SV (ed.), Canine and Feline Theriogenology. WB Saunders Company, Philadelphia, pp. 66104.
  • Reynaud K, Fontbonne A, Marseloo N, de Lesegno CV, Saint-Dizier M, Chastant-Mailard S, 2006: In vitro canine oocyte maturation, fertilization and early embryogenesis: a review. Theriogenology 66, 16851693.
  • Senger PL, 2003: Early embryogenesis and maternal recognition of pregnancy. In: Senger PL (ed.), Pathway to Pregnancy and Parturition, 2 edn. Current Conceptions Inc, Pullman, pp. 284303.
  • Tsutsui T, Hori T, Okazaki H, Tanaka A, Shiono M, Yokosuka M, Kawakami E, 2001: Transfer of canine embryos at various developmental stages recovered by hysterectomy or surgical uterine flushing. J Vet Med Sci 63, 401405.
  • Tsutsui T, Hori T, Endo S, Hayama A, Kawakami E, 2006: Intrauterine transfer of early canine embryos. Theriogenology 66, 17031705.
  • Ushijima H, Yoshioka H, Esaki R, Takahashi K, Kuwayama M, Nakane T, Nagashima H, 2004: Improved survival of vitrified in vivo-derived porcine embryos. J Reprod Dev 50, 481486.
  • Whittingham DG, Leibo SP, Mazur P, 1972: Survival of mouse embryos frozen to −196°C and −269°C. Science 178, 411414.
  • Wilson MS, 2001: Transcervical insemination techniques in the bitch. Vet Clin North Am Small Anim Pract 31, 291304.