It is possible to propagate animals with specific genetics or elite genetics through somatic cell nuclear transfer (SCNT). A number of breeds of dogs have been produced by SCNT [9, 10, 11, 13, 16, 17], and it has been shown that both male and female cloned animals have normal reproductive characteristics and can reproduce normally [12, 15, 21]. For example, one report demonstrated that cloned females have normal reproductive hormone levels and ovarian follicle development [8]. Another study reported that healthy offspring were produced when cloned females were artificially inseminated using semen of cloned males, both cloned males and females are derived from adult somatic cells [18]. However, no studies have investigated the behavioral characteristics of the offspring of cloned dogs to date.

Behavioral traits and aptitude for various roles in dogs are also likely to be influenced by genetic factors [14]. Indeed, it has been shown that genetic factors contribute significantly to several behavioral traits such as fearfulness, aggressiveness, reactivity and general activity [2, 20]. In particular, it was reported that behavioral traits such as courage, nervous stability, hardness and affability have positive genetic correlations [19]. Accordingly, dogs exhibiting superior detection ability are recommended for breeding to increase the production of such animals, thereby reducing the burdens associated with training and selection of detection dogs. Because of its high efficiency, SCNT using donor cells from dogs with superior talent represents an excellent approach for producing drug detection dogs [4].

In this study, we investigated the reproductive ability of a cloned male dog by natural breeding. We also investigated the behavioral traits of these puppies and their performance in the selection test to evaluate their normality.

We examined the reproductive ability of a male cloned dog and the behavioral traits of his offspring. Several previous studies reported that cloned animals had normal reproductive ability [3, 5, 18]. In this study, ten healthy puppies were produced by natural mating between a cloned male dog and a normal female dog in the DDTC. It was previously reported that although the first cloned male dog exhibited normal libido, natural mating could not be performed because of his inexperience in the clasping method [18]. However, we found that the cloned male dog had a normal libido along with other normal mating behavior and reproductive ability.

His puppies showed normal growth and matured well without congenital defects or infectious disease until final selection. In our report, seven of the ten puppies showed mature weights above 25 kg, which is similar to that of their parents. All of these offspring had similar mature weights that were within the normal reference range [6, 7]. Among the four puppies that failed the final selection test, two showed lower weights than the other dogs. However, we could not find any correlation between final test score and mature weight in puppies because the population was too small to lead to a solid conclusion. Behavioral traits of the puppies were monitored to evaluate their trainability and compare similarities between the cloned parent dog and his puppies because it was assumed that behavioral traits of the puppies were inherited from the cloned male dog. To determine the behavioral traits of puppies, Volhard tests were performed. As in our previous report, a cloned male dog scored subtype 2 in a modified Campbell test [4]. However, only one of the puppies, P3, scored subtype 2, which was identical to his father's score. Interestingly, six puppies showed the 2 or 3 subtype in the Volhard test, indicating suitability for training. Notably, none of the puppies receiving a 2 or 3 subtype score in the Volhard test were among the final selected dogs.

Furthermore, the cloned dog's offspring achieved a 60% success rate in the final selection tests in the present study. When compared with the approximately 30% success rate of the Japan detector dog training program [14], as well as the 0–40% success rate of the US customs service (unpublished data), our 60% training outcome success rate was remarkable.

The cloned detector dog received an excellent grade in training; therefore, it was concluded that cloning elite dogs using the SCNT technique was a better approach for producing qualified detector dogs [4]. We showed that cloned dogs could breed normally, which led us to hypothesize that this method will allow generation of better candidate dogs with higher potential to pass the training course. Furthermore, while the behavioral traits of puppies correlate with the genetic factors of a cloned male dog, and although only one litter was investigated for this study, it is generally accepted that genetic factors are also important in training dogs.

In conclusion, a cloned male dog had a normal libido and could mate with females naturally. The pairs could produce puppies that later successfully completed the training course for drug detection with a greater than 60% success selection rate. It is expected that breeding cloned dogs with excellent abilities will be an optimal method to produce more candidate dogs for unique purposes.