As shown by the birth of the first cloned dog Snuppy, a protocol for producing viable cloned dogs has been established.¹ Dogs are thought to be valuable models for biomedical research because they share many genetic diseases with humans.^2,3 This similarity between dogs and humans attracts a great deal of scientific interest which led to developing techniques for cloning dogs. Despite all of these advances, the application of somatic cell nuclear transfer (SCNT) in dog is still limited by low success rate of cloning.^1,4 In addition, concerns about the health and longevity of resulting cloned animals have been raised due to high rates of abnormal pregnancies during cloning procedure.^5-7 Accordingly, the postnatal characteristics of cloned dogs were investigated to widen the applicability of dog cloning. The growth and hematologic parameters were assessed in three genetically identical female cloned dogs and the reproductive characteristic of cloned male and female dogs were evaluated. After then, we evaluated the influence of genetic factors on phenotypic variation, since variations among genetically identical cloned dogs can be a useful tool to elucidate mechanisms underlying phenotypic differences.

Growth and Hematologic Characteristics of Cloned Dogs

The effects of SCNT on postnatal development have not been well studied; hence we assessed growth data of the cloned dogs describe if all clones share similar characteristics.⁸ The several key aspects of growth were evaluated, including overall measurements of body weight and height, radiographic assessment, blood type, and measurements of hematology and serum biochemistry. The increase in body weight and height of the cloned dogs was similar to one another, and mature body weight and heights were similar between each cloned dog and the donor dog. Also, we performed radiographic assessment by X-ray images derived from the skull, the lumbar vertebrae, and the pelvic region in order to evaluate skeletal maturity. The results demonstrated that the bone growth patterns were similar among the cloned dogs and the donor dog at the fully grown stage. All measured parameters increased rapidly during the 8 to 16 weeks of age and then plateaued, which was similar to the increasing patterns of the body weight and height. Then, the blood typing by dog erythrocyte antigen was performed. The blood phenotype of cloned dogs was identical, which may be due to equal genetic information that the cloned dogs and the donor dog share. However, cloned dogs and the donor dog demonstrated variation in the dentition. As a result, three genetically identical cloned dogs showed similar characteristics, and further studies about the genetic effects on dental growth should be encouraged. Finally, hematologic and serum biochemical values were collected from 7-week to 1-year old female cloned dogs and age-matched control dogs to evaluate if apparently normal cloned dogs share similar clinical characteristics with normal controls produced by natural breeding. An age-specific pattern was identified on hematologic and serum biochemical measurements in both cloned dogs and age-matched controls, and the parameters examined were within the normal reference ranges of healthy dogs. In summary, three genetically identical cloned dogs showed similar growth characteristics and had normal hematological and serum biochemical parameters.

Reproductive Characteristics of Cloned Dogs

The first cloned male dog, Snuppy (an Afghan hound), which was generated at our laboratory, is currently a healthy 3 yr. 7 mo. old.¹ Three cloned Afghan hound females that were generated later have displayed signs of estrus initiation recently⁹, indicating that they may have reached reproductive maturity. Subsequently, the reproductive characteristics of cloned male and female dogs were evaluated to assess the effect of cloning procedure on reproductive abilities of cloned dogs. We investigated reproductive activity of cloned dogs by 1) performing sperm analysis using computer-assisted sperm analysis and early embryonic development, 2) assessing reproductive cycling by measuring serum progesterone (P4) levels and performing vaginal cytology, and (3) breeding cloned dogs using artificial insemination.¹⁰ Results showed that most parameters of sperm motility in a cloned male dog were within the reference range and that three cloned female dogs displayed normal patterns of P4 levels and morphological changes of the vaginal epithelium. Two cloned female dogs became pregnant with semen from the cloned male dog and successfully delivered ten puppies by natural labor. The birth weights of puppies born from cloned dogs were within the reference ranges and showed consistent increase without any disorders. These data demonstrated that both cloned male and female dogs are fertile, as apparently healthy cloned dogs have no serious reprogramming errors on their reproductive organs. This discovery may allow the application of cloning technology for the purpose of expanding an elite canine gene pool in dogs.

Hormonal Regulation in Cloned Dogs and the Epigenetic Perspective

Phenotypic variation among organisms with identical genetic information has been attributed to the effects of epigenetic differences and epigenetic interpretations using twin model systems. These systems have been suggested as a way to better understand the mechanisms of variation in complex phenotypes as well as the origins of complex diseases.^11-13 The term 'epigenetics' is classically used to explain phenotypic events that cannot be described by genetic mechanisms alone and are known to play a critical role in regulating a variety of genomic functions.¹⁴ To explore the relative contributions of genes, environment and other factors to phenotypic variance, genetic sources of variations were minimized by using cloned animals derived from identical somatic cell donors. Since cloned dogs also have genetically identical information, variations among these animals with virtually identical chromosomal DNA sequences can be a good tool to elucidate the underlying mechanism of phenotypic differences. Thus, another potential use of cloned dogs is the production of genetically identical animals for research to study the influence of genetic and environmental factors on phenotypic variation and to elucidate the mechanism of epigenetic regulation. To evaluate the relationship between genetic information and phenotypic differences, changes in concentration patterns of growth hormone (GH), insulin like growth factor-1 (IGF-1), and IGF binding protein 3 (IGFBP-3) were compared among clones and age-matched controls.¹⁵ In addition, the concentrations of GH and IGF-1 following the administration of GH releasing hormone (GHRH) and somatostatin (SRIF) were measured in both groups. In comparing hormone profiles, the control dogs had larger standard deviations from the means for GH, IGF-1, and IGFBP-3 than the clones. Also, the mean concentration of IGFBP-3 was significantly lower in clones than in controls between 7 to 12 months of age, whereas the IGFBP-3 changes in both clones and controls followed the same pattern. Injecting GHRH increased the serum GH concentration in both clones and controls. However, the concentration of IGF-1 was lower in clones than in controls, and larger standard variations were noted in the control group. This notes that the measured traits were more homogeneous in cloned animals than in controls, and that cloned animals could be valuable in future assessments of effects of genotype and environment interactions.

Conclusions

In this study, postnatal growth, reproductive ability and hormonal regulation of cloned dogs were assessed. Three genetically identical cloned dogs showed similar growth characteristics and had normal hematological and serum biochemical parameters, and all cloned male and female dogs proved to be fertile. The results demonstrated that cloned dogs with same genomic information have no abnormalities, hormonal regulation profiles of clones were similar to those of controls, and larger standard variations were noted in the control group. Thus, genetically identical clones can be a model system for studies on the mechanism of genetic and epigenetic regulation by controlling the effects of genotype and environment interactions. Furthermore, this model system will widen the opportunity of using SCNT techniques for research in human disease and biomedical science.

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