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Efficacy of dog-appeasing pheromone in reducing stress associated with
social isolation in newly adopted puppies
Article  in  The Veterinary record · July 2008
DOI: 10.1136/vr.163.3.73 · Source: PubMed
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The Veterinary Record, July 19, 2008 
Papers & Articles
rating 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 effec-
tiveness 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.
Adoption phase The purchasers were informed by the pet 
shop staff that the puppy they wanted to buy was enrolled 
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 experi-
enced conditions that have had adverse effects on its behav-
ioural 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 peri-
ods of social isolation, such as when people are at work dur-
ing 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 com-
munication 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 amelio-
Efficacy of dog-appeasing pheromone 
in reducing stress associated with social 
isolation in newly adopted puppies
E. Gaultier, L. Bonnafous, D. Vienet-Legué, C. Falewee, L. Bougrat,
C. Lafont-Lecuelle, P. Pageat
This study was designed to determine the potential value of dog-appeasing pheromone (DAP) in reducing 
stress in puppies newly adopted from a pet shop. The trial was triple-blinded and placebo-controlled. After 
their arrival at the pet shop, 32 puppies were fitted with a DAP collar and 34 were fitted with a control collar, 
according to a randomisation protocol. Adopting owners were contacted by telephone, three and 15 days 
after they had adopted a puppy, to obtain information about the puppy’s integration into the family, and 
particularly about any signs of distress shown by the puppy when it was socially isolated. All the isolated 
puppies from the control group vocalised during the first night. Signs of distress, particularly vocalisation, 
were significantly lower in the DAP group on day 3 and throughout the rest of the study, and vocalisation 
during the night ceased significantly sooner in this group. These differences were observed in puppies of 
small, medium and large breeds. The DAP collars had no effect on the incidence of house soiling.
Veterinary Record (2008) 
163, 73-80
E. Gaultier, DVM, MSc, 
DipECVBM-CA,
L. Bonnafous, DVM,
C. Falewee, DVM,
L. Bougrat, MSc,
C. Lafont-Lecuelle, MSc,
P. Pageat, PhD, DVM, 
DipECVBM-CA,
Phérosynthèse 
Research Institute, 
Le Rieu Neuf, 84490 
St Saturnin-les-Apt, 
France
D. Vienet-Legué, DVM,
Clinique Vétérinaire du 
Chêne à Leu, 544 Avenue 
de Buchholz, 76380 
Canteleu, France
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 The Veterinary Record, July 19, 2008
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 ques-
tionnaires, 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 pup-
pies (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, scratch-
ing 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 physi-
cal 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 capac-
ity 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 question-
naire.
Data processing and behavioural analysis
Main criterion 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 com-
posite 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 
in a study designed to assess the potential effectiveness of a 
pheromone-impregnatedcollar 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 sec-
ondly 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 iden-
tical 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 remained unoccupied 
for three days.
Data collection
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 liv-
ing 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-
Papers & Articles
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Papers & Articles
The Veterinary Record, July 19, 2008 
(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.
Statistical analyses
The data were analysed using commercially available com-
puter software (StatView 4.5; Abacus Concepts). Values of 
PThe 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 own-
ers’ attempts to reduce the disturbance, as Taylor and Mills 
TABLE 1: Characteristics of the 32 dog-appeasing pheromone 
(DAP)-treated puppies and 34 control puppies and their 
adoptive homes
Characteristic DAP group Control group
Sex
 Male 18 21
 Female 14 13
Number of breeds* 16 18
Breed size
 Small 21 24
 Medium 7 4
 Large 4 6
Gundogs 7 4
Mean (sd) age at adoption
 (months) 2·9 (0·5) 2·85 (0·6)
Mean (sd) time spent in the 
 pet shop (days) 19·0 (16·3) 20·3 (14·2)
Living place
 With garden 21 17
 Without garden 11 17
Other pets (cat and/or dog) 7 8
Mean (sd) number of children 
 (under 18 years old) 0·88 (1·26) 0·77 (1·08)
Previous ownership of a dog 22 19
* There were 10 breeds in common between groups
TABLE 2: Assessment of nuisance activities by the puppies 
three and 15 days after they were adopted
Nuisance activities
DAP group 
(n=32)
Control group 
(n=34) P
At least one on day 3 7 30DAP Dog-appeasing pheromone, NS Not significant
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Papers & Articles
The Veterinary Record, July 19, 2008 
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 iso-
lated on the first night, and some of those in the DAP group, 
vocalised, and in order to reduce this disturbance, their own-
ers tried a number of different strategies. These included leav-
ing 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 treat-
ment 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 dur-
ing 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 pup-
pies 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 pup-
pies (Table 4).
Giving the puppy free access to the bedroom did not 
appear to be a guarantee of peace. In both groups, six pup-
pies 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 own-
er’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 
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 explora-
tory 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 behav-
iour 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 exces-
sively punitive confrontation with its owners over house 
training; this puppy continued to wake them up by whin-
ing when it wanted to urinate during the night. Owing to 
FIG 3: Numbers of the 34 control puppies and 32 puppies wearing a dog-appeasing 
pheromone (DAP)-impregnated collar that caused different types of disturbances when 
left alone at night on days (d) 0, 3 and 15. NS P>0·01, * 0·05find 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 poten-
tially 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 con-
sistently 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 behav-
iour 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 sta-
tistically significant tendency for separation-related destruc-
tiveness 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 
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 dur-
ing 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 con-
trol 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 
TABLE 5: Periods for which the puppies were left alone during 
the day, and the numbers of 30 of the control puppies and 30 of 
the puppies wearing a dog-appeasing pheromone (DAP) collar 
that caused a disturbance during the day on days 3 and 15
Factor affecting disturbances DAP group Control group P
Time the puppy was left 
 alone during the day
 6 hours 7 14
Number of puppies causing a
 disturbance when alone
 Day 3 6 282005).
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.
ACKNOWLEDGEMENTS
The authors thank Jon Bowen for his great help in providing 
English language corrections.
References
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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 distur-
bance after the third day, and the proportion that caused a 
disturbance decreased throughout the trial. Very few of the 
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Data collected from telephone calls are sometimes criti-
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of establishing their accuracy. To counter such criticism, par-
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The questions were easy to understand and they were focused 
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matic events may also lead puppies to develop more separa-
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isolation in newly adopted puppies
reducing stress associated with social 
Efficacy of dog-appeasing pheromone in
Lafont-Lecuelle and P. Pageat
E. Gaultier, L. Bonnafous, D Vienet-Legué, C. Falewee, L. Bougrat, C.
doi: 10.1136/vr.163.3.73
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