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ADOPTION is widely recognised as being stressful for a puppy, because it involves major changes. The puppy's maternal bond is broken and it is moved to a new social and physical environment with new rules (Elliot and Scott 1961, Pettijohn and others 1977, Serpell and Jagoe 1995). When a puppy has been bought from a pet shop, it may have experienced conditions that have had adverse effects on its behavioural development. For example, it may have had a shorter than normal period with its mother, which can affect the behaviour and increase the mortality of puppies (Slabbert and Rasa 1993), and may have been subjected to periods of transport and readjustment when it was moved first to the pet shop and then to its new home. Increasing the number of stressors, and particularly repeated ‘rehoming’ of this kind, may intensify stress for a puppy at a time that is critical for its development. Signs of stress may appear during its period in the pet shop and after it has been adopted.
In the new home, signs of stress often appear during periods of social isolation, such as when people are at work during the day or when human contact is not possible during the night. The main sign of social stress is vocalisation (Elliot and Scott 1961, Scott 1970, Hetts and others 1992), which can become annoying for neighbours during the day and for the owners at night, and may impair the integration of the puppy into the family. In order to be able to sleep, people often resort to letting the dog into their bedroom at night, so breaking the rules that they intended to maintain to prevent future behavioural problems.
Dog-appeasing pheromone (dap) is released by glands in the intermammary sulcus of bitches during the first few weeks after parturition. Studies conducted in a wide range of stressful situations, for example, firework phobia (Sheppard and Mills 2003), separation-related disorders (Gaultier and others 2005), anxiety-related behaviour of problem dogs in veterinary clinics (Mills and others 2006), stress and fear-related behaviour in shelter dogs (Tod and others 2005) or during transport (Gaultier and Pageat 2003, Gandia Estellés and Mills 2006) have shown that a synthetic analogue of dap (dap; ceva Animal Health) is effective in calming adult pet dogs. Naturally occurring dap is involved in chemical communication between the bitch and its pups, and the synthetic analogue would therefore be expected to have a calming effect on puppies stressed by being separated from their mother and sold from a pet shop to new owners.
This triple-blind controlled study was designed to assess the potential effectiveness of the analogue of dap in ameliorating the effects of stress that might delay the integration of puppies into new families in the first few days after their purchase from a pet shop.
MATERIALS AND METHODS
Recruitment of puppies and test procedure
Owners who had bought a puppy from a pet shop in Rouen and who agreed to participate in the study were recruited. They were informed that the study was designed to assess the effect of a collar impregnated with dap in facilitating the puppy's integration into its new home. Most of the puppies were of small breeds and eight to 10 weeks old when they arrived at the pet shop. They had typically travelled for 15 to 20 hours from their breeder.
The study was divided into two phases: the ‘pet shop phase’ and the ‘adoption phase’. The first phase was concerned only with the selection and randomisation of the puppies and with the start of their treatment, but it was important that the treatment began during this phase to ensure that its effectiveness in ameliorating the stress of the adoption could be tested. The adoption phase of the study was the test phase, as it involved many stressors. The puppy's adoption involved its transport from the pet shop to the new owner's home, with a change of location and change of social group.
Pet shop phase
After 24 to 48 hours in the pet shop, the puppies were examined by a veterinary surgeon. Those that were healthy went through a programme of vaccination and anthelmintic treatment, but the puppies that were unhealthy or did not conform to the breed standard were returned to the breeder. The selected puppies were fitted with a collar according to the randomisation procedure described below. Until they were sold, the puppies were fed a commercial dry diet and were kept alone or with one or two other puppies in one of six large top-lit glass cages (1·5 m2 and 1 m high). On average, the puppies remained in the pet shop for two to three weeks before being adopted, with a range from three days to two months.
As the study was designed to assess a potential preventive treatment, none of the puppies was excluded on the basis of behavioural criteria.
The purchasers were informed by the pet shop staff that the puppy they wanted to buy was enrolled in a study designed to assess the potential effectiveness of a pheromone-impregnated collar in reducing weaning stress and increasing the ease of the puppy's integration into the new family. They were also informed that the study was placebo-controlled, so that the collar that their puppy was wearing might contain no active ingredient. If they agreed to participate in the trial, they were asked to allow the puppy to continue to wear the collar for two weeks after its adoption. They also agreed to be contacted by the investigator twice by telephone to answer a short questionnaire about their puppy's behaviour, first at three days (± one day) and secondly at 15 days (± two days) after its adoption. If the owners refused to participate, the collar was removed and the puppy was excluded from the trial.
Exclusion criteria during the adoption phase were, first, any detrimental change in the treatment as a result of the destruction, removal or loss of the collar and, secondly, any lack of data about the adoption phase.
Treatment and study design
The study was a triple-blinded, placebo-controlled, single-centre, parallel-group trial. A 35 cm collar containing 2·5 per cent dap was fitted to the neck of each puppy in the treated group. Puppies in the control group were fitted with an identical collar that contained no dap. Neither the vet who fitted the collars, nor the pet shop staff or purchasers, were allowed to know to which group each puppy had been allocated. If a puppy remained at the pet shop for more than four weeks, its collar was renewed. A new collar was fitted on the day the puppy was adopted.
Detergent products may strip the pheromone from the collar, and so the owners were asked to remove the collar temporarily whenever they washed their puppy.
To avoid any risk of contamination, the puppies housed in the same cage at the pet shop received the same treatment. Each cage had high glass walls and its own ventilation system, so that the treatment groups were well isolated from each other. The randomisation procedure therefore related to the cages and not to the puppies; half the cages were assigned to the treated puppies and the other half to the controls. Each week, between four and six new puppies arrived at the pet shop, and they were assigned to cages according to their breed size and the availability of free places, and given the treatment assigned to the cage in which they were housed. When all the puppies in a cage had been sold, the cage was thoroughly cleaned and decontaminated, and then emained unoccupied for three days.
The questionnaire focused on four main points; first, the characteristics of the puppy and its adopting family; secondly, the expression of nuisance activities during the night and the solutions found by owners to reduce them; thirdly, any signs of distress shown by the puppy when it was left alone during the day; and finally, how well the puppy tolerated the collar.
Characteristics of the puppy and its owners
Information about the characteristics of the puppy when it was adopted, the time it had spent in the pet shop, and the new owners' previous experience with a dog provided information about the comparability of the two treatment groups, especially with respect to confounding factors that might be related to the dependent variables. The composition of the family was of particular interest, because it has been shown that dogs living in large families have fewer separation-related problems when they are left alone (Topal and others 1998, Podberseck and others 1999, Flannigan and Dodman 2001). It has also been observed that some breeds are more likely to show signs of separation-related problems (Bradshaw and others 2002, Taylor and Mills 2007). Some breeds may also express sepa-ration-related problems in different ways from others (Scott and Fuller 1965, Scott 1970, Lund and Jorgensen 1999). In order to take this into account, the puppies were categorised according to breed size (small, medium or large) and the breed's function or type, for example, gundog.
Nuisance activities during the night
The owners indicated where they had intended the puppy to sleep, and where it actually slept. They specified whether the puppy slept alone or with another animal (cat and dog), or whether it had access to the owner's, or a child's, bedroom. They also reported whether and when there had been any change in these arrangements between the first and the second questionnaires, particularly if any such changes were related to an attempt to reduce nuisance behaviour. In a study of the effect of dap on the night-time behaviour of puppies, Taylor and Mills (2007) reported that sleeping with another dog had such a large effect on the intensity of any disturbance that these dogs should be excluded from the analysis of efficacy. In the present study, such puppies were not excluded, in order to stay as close as possible to the intention-to-treat conditions.
The owners reported the number of times the puppy woke them up each night during the two weeks after it was adopted. Each night they recorded the occurrence of any nuisance behaviour from a list of those typically expressed by the puppies (whining, howling, scratching at the door, wandering, moving or destroying objects, interacting with owners and waking them up). An open question allowed the investigator to collect further details of any of these nuisance activities.
Signs of distress when the puppies were left alone during the day
The owners were asked whether the dog was usually left alone during the day and for how long; if it was not usually left alone it was excluded from this part of the analysis. In the other cases, the owner reported whether the dog performed any nuisance behaviours when left alone, and which kind (vocalising, such as whining or howling, scratching at the door, moving or destroying objects). In order to make an assessment of vocalisation and scratching during the day, the owners were asked to wait for two minutes out of sight of their puppy after their departure from the home.
The owners were also asked whether the puppy urinated in the house during the day or during the night. The ability to retain urine in an unstressed state is related to the physical capacity of the bladder, and urination at night or during the day should therefore not necessarily be considered a sign of stress. This behaviour was recorded but not classified as a stress-related nuisance activity. To complete information about urination, the puppy's acquisition of house training at night and during the day was evaluated twice, to provide data relevant to hypotheses about the urine retention capacity of puppies and investigate the potential benefit of dap in improving house training.
Toleration of the collar
How well the puppy tolerated the collar was evaluated twice: first, by the pet shop staff, who regularly examined each puppy's neck, and secondly by the owners, who reported their experience through the questionnaire.
Data processing and behavioural analysis
The efficacy of the dap collar was evaluated from a composite parameter that combined the incidence of nuisance behaviours performed by the puppy during the first period after its adoption together with the presence of at least one kind of diurnal and/or nocturnal disturbance (except urination) on the third day after its adoption. This composite parameter was chosen as the main criterion for two reasons: first, it was a sensible indicator of the general level of stress the puppy experienced in the first period after its adoption; secondly, because of the results of a similar study of the night-time behaviour of puppies adopted directly from local breeders by Taylor and Mills (2007), who found that the third day seemed to be of special significance. They showed that puppies with a tendency to disturb their owners at night during the first three days in the new home stopped when dap was used.
The potential influence of breed as a confounding factor was also investigated. The data were analysed on the basis of the size of breed of the dog and whether it was a gundog type, including labradors, weimaraners and spaniels. First, the potential influence of the size of the breed on the tendency to show nuisance activities on day 3 was assessed by measuring the proportions of the control and treated puppies in each of the three size categories (small, medium and large) that showed at least one sign. This would indicate whether the puppies in any size category responded differently at first to dap than the others, and this information would be amplified when the overall effect of the dap collar on puppies of each size category was assessed. Secondly, the same kind of analysis was applied to the gundog types.
Two other criteria were defined to assess the overall effect of the treatment on the puppies' nuisance activities: first, the occurrence of at least one nuisance activity (any kind of diurnal and/or nocturnal disturbance except urination) during the two weeks after the puppy's adoption, and secondly, a reassessment of the main criterion on day 5.
Other secondary criteria concerned nocturnal and diurnal nuisance activities, with each kind (whining, howling, scratching at the door, and so on) considered separately. The distribution of the puppies in the two treatment groups that showed at least one kind of nocturnal nuisance activity (other than urination) was then compared on a nightly basis during the two weeks after they were adopted, and the same comparison was made for each kind of nocturnal nuisance activity. The same comparisons were also applied to diurnal nuisance activities (total and each kind of activity) on day 3 and day 15. Differences in the consecutive mean number of night-time disturbances involving the puppy waking someone up were assessed by a two-way repeated analysis of variance.
There was a particular interest in assessing the owners' attempts to reduce the disturbance, as Taylor and Mills (2007) reported a large reduction in disturbance when the puppy slept with another dog in the same bed. A daily record was made of the number of puppies that benefited from a given strategy. The effect of these strategies on reducing night-time nuisance activities in the control group was also compared with the effect of the dap treatment on the puppies that slept alone.
The data were analysed using commercially available computer software (StatView 4.5; Abacus Concepts). Values of P<0·05 were considered significant. Quantitative values were analysed using a t test. When variances differed, a non-parametric test, the Mann-Whitney U test, was used. The changes in the quantitative measurements were analysed with an analysis of variance for repeated values. Qualitative assessments were analysed using a chi-squared test of independence. When theoretical expected values were less than 5, Fisher's exact test was used.
Puppies that were sold were considered as the ‘full analysis set’, except those for which no follow-up data could be collected, either because the owners could not be contacted or because the puppies had not been correctly exposed to the treatment because the collar had been destroyed or lost. Consistent with the intention-to-treat principle, when there was a partial lack of data, because data had been collected from the first questionnaire but not from the second, rather than exclude the case, the results collected on day 3 were applied on day 15, in accordance with the carrying forward imputation technique (ICH 1999). In that way, if the treatment was effective, the analysis would not give it a biased advantage.
During the study, the pet shop received 100 puppies but 21 were returned for medical reasons. Of the remaining 79 puppies, 40 were allocated to the dap group and 39 to the control group; 39 of the dap group and 37 of the controls were sold. Thirty-four of the owners in each group could be contacted, but two of the puppies in the dap group lost their collars soon after they were adopted. As a result, 32 dap puppies and 34 control puppies fulfilled all the criteria necessary for them to be included in and followed throughout the study. One owner of a puppy in the dap group could not be contacted on day 15.
Characteristics of the puppies and the owners
The characteristics of the puppies are shown in Table 1. There were 39 male dogs and 27 females. There were 24 different breeds, 10 of which were common to both groups. Forty-five of the puppies belonged to small breeds, 11 to medium breeds and 10 to large breeds. There were 11 gundogs, six cocker spaniels (four in the dap group and two in the control group) and five labrador retrievers (three in the dap group and two in the control group). The puppies remained in the pet shop for an average of 19 days and were nearly three months old when they were adopted.
For one-third of the owners, the puppy was their first experience of dog ownership. One-quarter of the puppies joined a family where there was at least one child and another pet cat or dog. Two-thirds of the puppies went to a house that had a garden. The median age range of the owners was 36 to 45 years old.
There were no significant differences between the dap and control groups in terms of the characteristics of the puppies, the composition of their adoptive families or the owners' previous experience of dog ownership.
Assessment of efficacy
Assessment of nuisance behaviour
At three and 15 days after their adoption, significantly fewer of the dap puppies were performing at least one nuisance behaviour than the control puppies (P<0·001), and a similar difference was recorded for the occurrence of at least one nuisance behaviour during the two weeks after their adoption (Table 2).
There was no significant difference between the three breed-size groups in terms of the expression of nuisance activities on day 3 in either the dap-treated puppies or the control puppies, but a significant difference was observed between DAP-treated and control puppies for all the size groups (Table 3).
Similar results were observed for the breed types. For the main criterion, there was no difference between gundogs and other breeds in either treatment group, but dap significantly reduced the incidence of nuisance activities on day 3 in both the gundogs and the other breeds.
Sleeping location and disturbances at night
On the first night, 19 of the puppies (29 per cent) had the benefit of social contact; 12 of them had access to the owner's bedroom and the other seven slept with a cat or dog (Fig 1). Fourteen of these 19 puppies performed a nuisance behaviour on the first night, seven in the placebo group and seven in the dap group.
In the control group as a whole significantly more of the dogs performed nuisance behaviours. They woke their owners more often during the night and continued to disturb them for a greater proportion of the two weeks after their adoption (Figs 2, 3, 4, Table 4).
Gundogs in the control group woke their owners during the night for a median of 11 nights compared with one night for the same breed type in the dap group (P=0·02). As previously observed, gundogs were no more likely to disturb their owners at night than other breeds.
The most common night-time nuisance behaviour was whining, and puppies that vocalised constituted more than 91 per cent of the dogs that caused a disturbance at night (Fig 3, Table 4). Because all the dogs that howled also whined, these two nuisances were combined to form a parameter called ‘vocalisations’.
‘Scratching at the door’ and ‘direct contact’ could be considered as alternative expressions of the same ‘contact-seeking behaviour’ that was defined in two situations — when puppies left in a separate room scratched at the door, and when puppies with free access to their owners' bedroom woke them up by interacting directly with them. Contact-seeking behaviour was common, with 63 per cent of the dap group and 82 per cent of the control group of puppies that caused night-time disturbances showing this behaviour during the first night (Fig 3).
‘Walking up and down’, ‘moving’ and/or ‘destroying objects’ could be considered as different forms of exploratory behaviour, and these were also combined for statistical analysis. Exploratory behaviour was less common, with less than 27 per cent of night-time disturbances resulting from it (Fig 3).
When analysing the two treatment groups separately, the results were consistent, regardless of the category of nuisance behaviour considered. In the control group, the number of puppies exhibiting nuisance behaviours decreased during the study but more than 30 per cent of them were still causing a disturbance at night on day 15. In the dap group, there was a more rapid reduction in the frequency of nuisance behaviour at night, and on night 5 none of them performed any of the recorded nuisance behaviours. However, one puppy relapsed on night 6, after three quiet nights, after an excessively punitive confrontation with its owners over house training; this puppy continued to wake them up by whining when it wanted to urinate during the night. Owing to the circumstances of this relapse its cause cannot strictly be linked to the stress of adoption. However, as these kinds of events are a common cause of stress for newly adopted puppies, the data from this puppy were kept in the analysis.
Importance of social contact and free access to the bedroom
All the puppies in the control group that were isolated on the first night, and some of those in the dap group, vocalised, and in order to reduce this disturbance, their owners tried a number of different strategies. These included leaving a radio on in the kitchen (one control puppy), applying advice from the vet (one control puppy), exclusion from the owner's bedroom (three dap puppies), giving free access to the owner's bedroom (10 control puppies) and allowing the puppy to sleep with another animal (two control puppies). More of the owners of puppies in the control group tried some kind of strategy, and the nature of the strategy differed between the two groups. Owners of puppies in the dap group most often excluded the puppy from the bedroom, whereas the owners of the control puppies tended to give them free access to the bedroom. The strategy adopted by the owners to gain some peace was significantly associated with the treatment the puppy received (Table 4).
The efficacy of these strategies varied widely from one puppy to another. The combined analysis of the changes in the number of dogs benefiting from the strategies (Fig 1) and the changes in the number of dogs performing nuisance activities (Fig 2) provided information on the potential efficacy of these strategies. In the control group, 19 of the 29 puppies causing night-time disturbances stopped during the study; of these, eight stopped after the owners let them have free access to their bedroom, two stopped after the owners let the puppy sleep with another animal, and nine stopped spontaneously. In the dap group, all 11 puppies that performed at least one nuisance activity at night stopped during the study; two stopped after being excluded from the owner's bedroom and nine stopped spontaneously. Considering only the puppies that stopped spontaneously, the puppies in the dap group stopped causing a disturbance at night in less than half the time taken by the control puppies (Table 4).
Giving the puppy free access to the bedroom did not appear to be a guarantee of peace. In both groups, six puppies were given free access to the bedroom on the first night, and four of the puppies in each group woke their owners up. In the dap group, one puppy stopped spontaneously on night 5 and the other three stopped because the owner excluded them from the bedroom; the exclusion did not lead to the puppy performing another nuisance behaviour. In the control group, one puppy stopped spontaneously on night 3, but the other three continued to cause a disturbance until the end of the study, despite being kept in the owner's bedroom.
The social contact strategy (providing access to the owner's bedroom or sleeping with another pet) appeared not to have a reliable impact on the disturbance, regardless of whether the strategy was introduced on the first night or on subsequent nights. Of the 22 puppies in the control group that were given social contact overnight, seven out of 10 caused a nuisance when the strategy was introduced on the first night, but only one puppy out of 12 caused a disturbance at night when it was introduced on the following nights; this difference was significant (P=0·006). A comparison between the 10 puppies in the control group that had the opportunity to maintain social contact during the first night and the 23 puppies in the dap group that slept alone shows that wearing a dap collar reduced first-night disturbance more effectively than social contact (P=0·006). In the control group, of the six puppies that were allowed to sleep with another pet, five stopped nuisance behaviour after one night, but six of the 12 control puppies that slept alone were still causing a nuisance at the end of the study. Sleeping with another pet appeared to control nuisance at night (P=0·05).
Disturbances during the day
Two of the puppies in the dap group and four in the control group were never left alone during the day, and these puppies were excluded from the analysis of the incidence of nuisance activities during the day, leaving 30 puppies in each group. More of the puppies in the dap group (30 of 32 [94 per cent]) than in the control group (30 of 34 [88 per cent]) were left alone. Seven of the puppies in the dap group and four of these in the control group were left alone for periods exceeding six hours. However, the lack of a significant difference between the two distributions (Table 5) made it possible to compare the two groups. Significantly more of the puppies in the control group performed a nuisance behaviour on day 3 and on day 15 (Table 5). Vocalisation (whining and/or howling) was the most frequent kind of nuisance, with 20 of the dap group and 29 of the control group showing this behaviour (Fig 5). On day 3, 12 of the dap group and 13 of the control group urinated when left alone, and on day 15, nine of the dap group and 11 of the control group did so (Fig 5). Data about the development of house soiling problems gave the same results (Fig 6). Treatment seemed to have no influence on non-stress-related house soiling, which confirmed that the puppies were not mature enough to have full control over urine retention.
Assessment of tolerability and compliance
One puppy developed a minor skin reaction to the collar while it was in the pet shop, but none of the owners indicated that they had removed the collar for any reason.
The results of this study show that dap collars can quickly and effectively reduce several of the stress-related nuisance behaviours of puppies encountered during the days after they have been adopted.
One of the first stressful situations experienced by a puppy is when it is put to bed at night on its own. In this study, the puppies that wore a dap collar behaved consistently in the same way, whereas the puppies in the control group behaved in three different ways. The puppies with a dap collar all consistently stopped nuisance activities — mainly vocalising or scratching at the door — before the third night, indicating that they all responded equally to the pheromone. In contrast, only one-third of the puppies in the control group stopped nuisance activities spontaneously, and on average two days later than the dap puppies; a further one-third stopped only when the owners allowed the puppies to sleep with another dog or have free access to their bedroom; the remaining one-third continued to disturb the owners until the end of the study. Providing social contact overnight appears to be moderately effective in stopping nuisance behaviour, but it can also have negative consequences. For example, many owners find the habit very difficult to break when the dog reaches adulthood. Moreover, Guy and others (2001) reported that being able to sleep on someone's bed during the first two months of a dog's ownership could be a significant risk factor for it going on to bite its owners; the habit is therefore potentially dangerous and not to be encouraged. It also appeared to be rather less effective than wearing a dap collar because it did not guarantee the owners sleep at night. The use of the dap collar reduced night-time nuisance behaviour more consistently and more effectively without these potential risks. In addition, of the owners whose puppy was fitted with a dap collar, those who let it sleep in their bedroom for the first night decided that for the subsequent nights the puppy would sleep in a separate room, because its playful behaviour repeatedly woke them up. When these puppies were left alone, they did not show any sign of distress and did not wake their owners.
The second stressful situation encountered by puppies is when they are left alone during the day, most often when people are at work. Serpell and Jagoe (1995) observed a statistically significant tendency for separation-related destructiveness in adult dogs to increase in parallel with the time they were left alone as puppies. They also observed that an increased prevalence of excessive barking was associated with being left alone as a puppy for periods of either up to two hours or six to eight hours. Although more of the puppies in the dap group were left alone for longer than six hours during the day, the great majority of them did not cause any disturbance after the third day, and the proportion that caused a disturbance decreased throughout the trial. Very few of the control group puppies improved, and the majority continued to vocalise when left alone during the day until the end of the trial.
Data collected from telephone calls are sometimes criticised because of their lack of precision and the impossibility of establishing their accuracy. To counter such criticism, particular care was paid to the construction of the questionnaire. The questions were easy to understand and they were focused on specific, objective points. They were cross-checked to detect any potentially misleading or inconsistent answers. The relatively short period between the two telephone calls would have helped the owners to recall accurately the events that occurred during the two weeks of the trial. Data relating to the disturbance caused by dogs when nobody is present for example, when the owners are away from the home tend to underestimate the disturbance caused, owing to a lack of reliable reporting. The questions therefore focused on whether the owners could hear the dogs vocalising or scratching at the door during the first two minutes immediately after their departure. Whether they had been destructive, moved objects about or urinated could be assessed on the owner's return. The demographic data suggest that the distribution of confounding variables within the two groups of puppies was similar, allowing the two sets of data to be compared. This is of particular importance for factors such as ‘composition of the family’, ‘breed’, ‘age when adopted’, and ‘time spent in the pet shop’, which are known to affect the development of nuisance behaviours linked to social isolation (Topal and others 1998, Podberseck and others 1999, Flannigan and Dodman 2001, Taylor and Mills 2007).
The demographic data also suggest that the sample size was sufficient to avoid the overrepresentation of either sex, or dogs of any age or breed. Although small-breed puppies constituted 70 to 80 per cent of the sample population, this proportion is similar to that in pet shops. However, previous studies have recorded variations in the rate of distress vocalisation with breed (Scott and Fuller 1965, Scott 1970, Bradshaw and others 2002, Taylor and Mills 2007). In the present trial neither the breed type (whether the puppy was a gundog or not) nor size had any effect on the occurrence of distress signs on day 3. Moreover, dap produced a significant effect on the main criterion assessed in the study — the presence of at least one kind of diurnal and/or nocturnal disturbance during the first three days after adoption — regardless of breed size or type. However, caution should be exercised when generalising this aspect of the results. It can be concluded only that there was no observable difference relating to breed type or size in the population studied. The power of the statistical tests may not have been adequate to detect one. The population included few puppies from medium and large breeds, thus increasing the likelihood of type 2 statistical errors. However, the apparent overrepresentation of small-breed puppies seems to have had no influence on the parameters assessed, because the puppies in the dap group consistently caused fewer disturbances on day 3 irrespective of their size or whether they were gundogs. The beneficial effects of dap observed may therefore reasonably be generalised to other kinds of sizes of puppy.
Puppies bought from a pet shop are known to have a high risk of developing various behavioural problems (Serpell and Jagoe 1995), owing to the succession of stresses they undergo: weaning at under six weeks (Slabbert and Rasa 1993) and early exposure to traumatic handling and transportation (Guy and others 2001). The repetition of such early traumatic events may also lead puppies to develop more separation-related problems just after they have been adopted, and results in puppies from pet shops often being considered as a special, at-risk population. However, the data do not seem to confirm this hypothesis: the puppies in this study did not appear to disturb their owners any more than those in a previous study by Taylor and Mills (2007), which involved puppies adopted from local private dog breeders. The commercial nature of the pet shop studied here ensured that the puppies came from France, that they were transported only moderate distances, and that they were not separated from their mothers before the minimum legal age of weaning at eight weeks. These factors may explain the lack of differences between the behaviour of the puppies in these two studies, even though they were from different sources.
The positive results observed in this study appeared to be more pronounced than those observed by Taylor and Mills (2007), in which the efficacy of dap was also assessed in newly adopted puppies. However, the two studies differ substantially, because Taylor and Mills (2007) administered the dap through a diffuser, only after the puppies had arrived at their new home on the day they were adopted. It is the authors' clinical experience that collars are more effective in certain situations, and that the time lag between adoption and the initial administration of dap was also be important. In the present study the puppies were fitted with dap collars for a period before they were adopted, and this might be expected to produce a greater effect.
The results of this study provide further evidence that being able to sleep with another dog effectively reduces nighttime nuisance behaviour and that vocalisation is the main sign of distress shown by puppies at night (Taylor and Mills 2007). Although howling was not the predominant type of vocalisation, it appears from a comparison with data from a study by Lund and Jorgensen (1999) that puppies may howl more often than adult dogs. The results also show that ‘contact-seeking behaviours’ constitute a significant part of the nuisance behaviour of puppies, and that destructive behaviours occur less frequently than in adult dogs suffering from separation-related problems (Flannigan and Dodman 2001, Gaultier and others 2005). With regard to urination, no clear conclusion can be drawn, because it was impossible to determine whether the urine found by returning owners was due to isolation distress, the limited capacity of the puppy's bladder, the lack of house training, or a combination of these factors. The lack of a difference between the two treatment groups is consistent not only with the hypothesis that the urine found by returning owners was not the product of isolation distress alone, but also with the hypothesis that the main factor for the efficient learning of house training is the use of appropriate training techniques and not dap.
Although the expression of distress linked to separation seems to differ between puppies and adult dogs, the results of this study were similar to the results of observations on adult dogs concerning the efficiency of dap in reducing problem behaviours (Gaultier and others 2005).
The results of this study provide further evidence that dap helps to calm and settle puppies as soon as three days after they have been adopted.
The authors thank Jon Bowen for his great help in providing English language corrections.
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