Applied Animal Behaviour Science 104 (2007) 71–84

www.elsevier.com/locate/applanim

Assessing fear of novel and startling stimuli in

domestic dogs
Jacqueline Ley a, Grahame J. Coleman b, Robert Holmes c,
Paul H. Hemsworth d,*
a
Animal Behaviour Consultations, Beaumaris, Vic. 3193, Australia
b
Animal Welfare Science Centre, Department of Psychology, Monash University, Caulfield Campus, Vic. 3145, Australia
c
Animal Behaviour Clinics, Camberwell, Vic. 3124, Australia
d
Animal Welfare Science Centre, University of Melbourne and Department of Primary Industries,
University of Melbourne, Vic. 3010, Australia
Accepted 31 March 2006
Available online 19 June 2006

Abstract
Dog attacks on humans are a community issue and, while defensive and offensive aggression are
implicated, little is known about the motivational basis of these attacks. Defensive aggression arising from
fear may be implicated in some attacks and thus research on measuring fear in dogs is clearly required. The
aim of the present study was to examine the validity of several measures of fear of novel and startling stimuli
previously identified in the authors’ laboratory. Anxiolytic drugs have been shown to reduce fear behaviour
in rats in novel situations and thus an anxiolytic drug, clomipramine, was used in the present study to
examine the validity of these previously identified measures of fear. Twenty-four dogs of varying breed, age
and sex were used in a cross-over design, involving a placebo treatment and a clomipramine treatment. It
was found that when the dogs were medicated with clomipramine for 6 weeks they were quicker to approach
and spent more time near the stimulus (P < 0.05) in a novel object test. Furthermore, there was a tendency
(P = 0.06) for a treatment by order interaction in which treatment with clomipramine in the first period but
not the second period was associated with reduced latency to approach and increased time spent near the
stimulus in the startling test. There was no treatment effect on latencies and entries to areas in a light/dark
test and an elevated plus maze test. These results indicate that a number of behavioural variables in two of
the tests, the novel object and startling test, are measures of fear of novel and startling stimuli, respectively,
in dogs and furthermore provide a useful methodology to study fear-induced aggression in dogs.
# 2006 Elsevier B.V. All rights reserved.

Keywords: Dogs; Fear; Behaviour; Stress; Clomipramine

* Corresponding author. Tel.: +61 9742 0444.

E-mail address: phh@unimelb.edu.au (P.H. Hemsworth).

0168-1591/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.applanim.2006.03.021
72 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84

1. Introduction

Dog attacks on people cause concern to the general community. However, little is known
about the behavioural characteristics of dogs that bite or their motivation to bite humans.
Aggressive behaviour is the most serious behaviour problem seen in dogs (Borchelt and Voith,
1996). It can lead to euthanasia or surrender of a pet (Guy et al., 2001). The present estimate of
the annual cost of hospital treatment for dog bites in Victoria, Australia is about $2.4 million
(Ashby, 2003) or $49,000 per 100,000 people, making dog bites a financial and political problem.
Aggression is a normal part of the behavioural repertoire of dogs and can be used offensively
to obtain or maintain access to resources or defensively to protect the individual (McFarland,
1981). While the aggressive behaviour of dogs has been described (Borchelt and Voith, 1996;
Overall, 1997; Houpt, 1998), little is known about the motivational basis of dog aggression.
Defensive aggression arising from fear appears to be implicated in some dog bite cases: fear-
induced aggression may account for between 10% and 26% of canine aggression cases (Galac
and Knol, 1997; Overall, 1997). While fear in dogs has been studied by a number of researchers
and implicated in many behavioural problems (Wells and Hepper, 2000), reliable behavioural
measures of fear in dogs have not been developed. Without such methodology, the ability to study
the role of fear in dog aggression to humans is limited.
Fear can be considered as an undesirable emotional state of suffering (Jones and Waddington,
1992) that gives rise to defensive behaviour or escape (Toates, 1980). It is a normal, adaptive
response, developed to protect the individual from injury (Toates, 1980; Voith and Borchelt,
1996) and is recognized in all vertebrate species (Tasman et al., 1997). Fear may be triggered by
environmental stimuli which are novel, have high intensity, for example being loud or large, have
special evolutionary dangers such as heights, isolation and darkness, or arise from social
interaction such as contagious learning or that have been previously paired with aversive
experiences (Gray, 1987).
Standard laboratory tests using behavioural measures have been developed that appear to
reliably measure fear in rodents, chickens and pigs (Lister, 1987; Rodgers and Cole, 1993; Erhard
et al., 1999; Andersen et al., 2000a; Marin et al., 2001). These studies have examined the
consistency and appropriateness of a range of behavioural measures in situations that are
intuitively fear-provoking and result in animals responding with either defensive or avoidance
behaviour. The tests used include the Elevated Plus Maze, the light dark box, approach and
avoidance tests with novel objects, and tonic immobility. Recent research using some of these
standard tests modified for dogs have suggested that the Elevated Plus Maze, light dark box and
approach and approach-avoidance tests with a novel object and a startling stimulus may provide
useful behavioural measures of fear and exploration in dogs (King et al., 2003). Moderate to high
correlations were found by King et al. (2003) between some of the variables within individual
tests and between the tests. Principal Component Analysis of the 33 variables measured produced
three components which accounted for 48% of the total variation. Components 1 and 3 contained
high loadings for the latency to approach and time spent near the stimulus in a novel object test
and a startling test, respectively. Component 2 contained variables measuring latencies and
entries to areas in a light/dark test and an elevated plus maze test. It was suggested by the authors
that component 1 may be a measure of a response to novelty and component 3 may be a measure
of a response to startling stimuli, while component 2 may be a measure of exploration.
Components 1 and 3 may be appropriate measures of different aspects of fear. This paper will
refer to component 1 as ‘Novelty’, component 2 as ‘Exploration’ and component 3 as ‘Startling’
components.
 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84 73

The aim of the present study was to examine the validity of these behavioural measures of fear
of novel and startling stimuli previously identified in dogs by King et al. (2003). Anxiolytic
drugs have been shown to reduce fear behaviour in mice (Cole and Rodgers, 1995) and pigs
(Andersen et al., 2000b) in novel situations and thus the anxiolytic drug, clomipramine, was
used in the present study to examine the validity of these previously identified behavioural
measures of fear.
If the measures identified by King et al. (2003) are valid behavioural measures of fear of novel
and startling stimuli, then treatment with an anxiolytic drug, such as clomipramine, should result
in dogs showing reduced latency to approach and increased time spent near the stimulus in the
novel object test and the startling test.

2. Materials and methods

2.1. Animals

Twenty-four entire dogs (n = 9) and entire bitches (n = 15) were acquired from a dog colony
used for research by a commercial company. Most of the dogs were beagle foxhound crosses (17)
and the remaining dogs were a variety of crossbreeds. The dogs were all housed at the Victorian
Institute of Animal Science, Attwood, Vic., Australia in individual pens. Each dog had access to a
sheltered area (9 m  2 m and 3 m  4.7 m in length by width) and a larger, grassed run
(10 m  9.5 m and 12 m  6 m in length by width). They were all fed dry food ad libitum and
had access to water via nipple drinkers.
Each dog was given a full veterinary examination, weighed and randomly assigned to an
individual kennel 24 h after arrival at the experimental site. All the dogs were in good general
health and condition although most of the beagles and one of the crossbred dogs had minor ear
infections. The ear infections were treated with a weekly ear flush with Epi-otic (Virbac), a
commercial ear cleaner designed for dogs, until resolved.

2.2. Treatment

The dogs were studied in a cross-over design, involving a control or placebo treatment for 6
weeks and a clomipramine treatment for 6 weeks. The dogs were randomly allotted to one of two
groups (1 and 2) with half of the dogs in each group being randomly assigned to commence the
study under either the placebo treatment or the clomipramine treatment. In order to complete the
battery of behavioural tests on all dogs within a short time-frame, the start of the study was
staggered with Group 1 commencing the study 1 week before Group 2. The dogs were given a 6-
week break after the first series of tests before commencing the second course of treatments.
Clomipramine has a terminal half-life of less than 4 h in dogs (King et al., 2000) but the break
was primarily to allow the dogs to resettle in their shelters after the tests.
Clomipramine was chosen as its effects and side effects are well documented, and it is
registered for use in animals. Clomipramine is a tricyclic antidepressant (TCA) that inhibits the
re-uptake of serotonin and norepinephrine presynaptically (Simpson and Simpson, 1996). It has
been successfully used to treat obsessive compulsive disorders, noise phobias and anxiety states
in dogs (Goldberger and Rapoport, 1991; Overall, 1994; Stein et al., 1994; Voith and Borchelt,
1996; Moon-Fanelli and Dodman, 1998; Seksel and Lindeman, 2001). Side effects reported for
clomipramine in dogs include nausea, vomiting and transient lethargy or sleepiness (King et al.,
2000). Bradycardia is the only cardiovascular effect noted in dogs (Pouchelon et al., 2000).
74 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84

Clomipramine is registered for use in dogs under the product name of Clomicalm (Novartis).
The recommended doses are 1–2 mg/kg bodyweight twice daily. The dogs were dosed using the
product recommendations of either a whole or half tablet, which resulted in a range of doses from
1.0 to 1.9 mg/kg. The medication was given twice daily in a spoonful of commercial tinned dog
food and the dogs observed to ensure the tablet was ingested. Dogs in the placebo treatment were
given the same amount of tinned dog food without the tablet at the same time as the dogs in the
clomipramine treatment. None of the dogs rejected the food or the tablets. The medication was
given twice daily for 6 weeks.

2.2.1. Observations in treatment

During the 5th week in each of the two 6-week treatment periods, dogs in each treatment were
observed over 2 days for general activity in their outdoor runs. It has been reported that clomipramine
may have a transient sedating effect (King et al., 2000), thus these observations were conducted to
determine if there was any observable difference in general activity levels between treated and
control animals. Observers were unaware of treatment allocation and observers sat in cars
approximately 4–6 m from the dog runs to undertake the observations. Kennel staff normally
travelled by car twice daily to the kennels to undertake the routine husbandry and cars were normally
parked near the kennels. The dogs were given 30 min to acclimatize to the presence of the cars before
the observations began. Each observer observed between 1 and 4 dogs and observations of 30 s
duration on each dog were made once every 5 min. Each dog’s location within its run, posture and all
behaviours as listed in Table 1 were recorded in each 30 s set of observations. Observations were
made for 4 h from 1000 to 1430 h with a break of 1 h after 2 h of observation. The dogs were given
another 30 min to acclimatize after this break before observations commenced again.
Each dog had access to an inside or protected area. It was not possible to position observers to
clearly observe dogs indoors. The indoor areas of seven dogs were video recorded over two 12-h
periods in the 5th week of treatment using cameras mounted on an internal wall. It was assumed
that the majority of the time when indoors was spent lying and sitting and these video
observations on posture were used to test this assumption.
From these records, the following variables were calculated: frequency of the postures
standing, sitting and lying, locomotion (walk, trot and run), active behaviour (includes all
behaviours except lying or sitting), grooming (licking, chewing or scratching itself), substrate
directed behaviour (defined as behaviours directed towards the ground) and object directed
behaviour (defined as behaviours directed towards kennel structures or objects such as grasses,
leaves or sticks, within or outside the kennel) and vocalisation (bark and howl).

2.3. Behavioural tests

The behavioural tests were all conducted at another location approximately 20 km from the
kennels. The dogs were transported in groups of 6 (and one group of 5) to the testing location on
the afternoon before the day of testing. The dogs were chosen randomly for transportation and
were housed in individual runs in two rooms in a large experimental facility at the test location.
Each dog was given familiar food and water ad libitum. All medications and placebo treatments
ceased the afternoon that the dogs were transported.
The tests have been detailed by King et al. (2003) and are briefly described below. Each dog
was tested individually and returned to its pen immediately on completion of each test and all
dogs completed each test before the next test was started. The minimum time between the start of
consecutive tests was 30 min. The tests were conducted as follows.
 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84 75

Table 1
Description of postures and behaviours observed in the dogs in their runs
Behaviour Description of behaviour
Postures
Stand Upright on all four legs
Sit Front legs straight, rear end lowered and resting on ‘hocks’ and perineum. Hind
legs may be turned to one side
Lie Sternal recumbency (SR): sternum touching ground, hind limbs on either side
(bent or stretched out the back)
Lateral recumbency (LR): side of dog touching the ground fully
Combination: SR in front and LR behind
Dorsal recumbency: back of dog touching the ground
Behaviours
Walk Four beat gait, three feet on the ground at any one time
Trot Two beat gait, diagonally opposite legs move together
Run Canter: 3 beat gait with a moment of suspension. Gallop: 4 beat gait
Rear Stand on hind legs with front paws on person or object
Jump Jump up: push off with and land on hind legs. Jump over: push off with hind legs and
land on forelegs
Stretch Extend either forelegs or hindlegs and hold for 1–2 s
Urinate Squat/leg raise/lean
Defecate Hind end lowered, back arched and tail held out
Scratch Scratching body/head with hind paw or head with front paw
Carry Pick up object and carry back to initial point
Drag/Pull Drag: pull object along the ground using the teeth. Pull: pull on object fixed at one
point using teeth
Dig Alternate use of front legs to move dirt, etc.
Lick/chew Lick: using tongue on own body or other object. Chew: using teeth on own
body or an object
Roll Rub back against the ground, moving body from side to side
Pica Eating something other than food
Bark Head and lips forward, mouth opening and shutting repeatedly
Howl Raise muzzle perpendicular to ground and emit a long, drawl out sound through
semi-closed jaws
Pant Mouth wide open with tongue protruding, often moving in and out of the mouth
Sniff Nose to ground/air/object, sides of body moving rapidly in and out
Shake Rotation of the body starting at the head and moving caudally
Not visible Dog has gone inside/behind barrier

2.3.1. Light/dark test

The light dark box is designed to present novel characteristics of contrasting light and dark
areas to the test subject. It has been used with rodents (Bourin and Hascoet, 2003; Colorado
et al., 2006) and pigs (Andersen et al., 2000a). The box used in the present study was the
same as that used by King et al. (2003). The box was 3 m  1.5 m  1.5 m (L  W  H) and
was divided into two equal sized compartments with an opening 0.8 m  0.4 m (H  W)
between them. One compartment was painted white and was uncovered. The other side was
painted black and covered with dark shade cloth to minimize the amount of light entering the
area. The entrance/exit to the apparatus was located in the black compartment. A video
camera was positioned in the lit compartment so the entire lit area could be observed. The
box was located in a large corridor in the experimental building, opposite the rooms
containing the housing pens.
76 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84

All the dogs, except the greyhound, were individually carried by the same handler to the box.
The greyhound was led on lead to the apparatus. Each dog was quietly placed in the dark
compartment through the hinged doorway facing the opening to the white compartment at time
zero. The door was quietly closed behind the dog and, its behaviour was observed via a video
monitor for the next 5 min.
The following variables were calculated from these video observations:

 Time (s) for the dog’s nose, foreleg and body (all four legs) to enter the white compartment.
 Total time (s) spent in the white compartment (defined as all four legs in the white
compartment).
 Number of entries into the white compartment.

Each dog was immediately returned to its holding pen in the experimental facility once the test
was finished.

2.3.2. Elevated plus maze (EPM)

This test is one of the most popular tests to measure fear and uses time spent on the open arms
relative to total time on the arms as an index of fear (Korte, 2001). The apparatus was adapted
from that used by Lister (1987) for mice and Andersen et al. (2000a,b) for pigs. It was adjusted in
size and height to accommodate a large dog.
The EPM consisted of a central square measuring 1.0 m  1.0 m (L  W) with four arms
forming a ‘‘plus’’ configuration. Each arm was 3.0 m long and 1.0 m wide and divided into 1.0 m
squares by masking tape on the floor. Two of the arms (closed arms) had clear Perspex walls
while the other two arms were open with no walls but 2 cm high edge strips (open arms). The
maze was arranged with the two open arms opposite each other. The EPM was elevated 1.5 m off
the floor with hay and foam mat padding under each arm to prevent injury if dogs jumped off the
EPM. Stairs were located close to the central square of the maze.
At the beginning of the test, each dog, except the greyhound, was carried by the same handler
from its holding pen up the stairs and released in the central square facing a closed arm. The
greyhound was led on lead to the apparatus. A video camera, mounted on the shed wall recorded
the dog’s activity on the maze. If the dog remained on the apparatus for the full three minutes, it
was then removed by the handler and immediately returned to its pen. Some dogs jumped off the
apparatus. The test ceased at the time the dog jumped, the animal was examined for any injuries
and then returned to its pen.
The following variables were directly observed during the 3-min period:

 Time (s) to move off the centre square.

 First arm entered.
 Number of entries into open arms.
 Number of entries into closed arms.
 Total arm entries.
 Time (s) spent in open arms.
 Time (s) spent in closed arms.
 Areas entered along each arm.
 Whether the dogs jumped off the apparatus.
The observers remained in the same room as the maze, with one observer standing stationary
about 3 m from the end of an open arm and the other 3 m from the end of a closed arm.
 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84 77

2.3.3. Novel object test

Different authors have used different novel objects in their research with a variety of species to
study response to novelty (Goddard and Beilharz, 1984; Boissy and Boissou, 1995; Visser et al.,
2001). The test used in this study and by King et al. (2003) was based on previous studies that
have used mechanical toys to assess fear of novelty in dogs (Plutchik, 1971; Goddard and
Beilharz, 1984).
This test was conducted in an open-roofed race 3.5 m  1.0 m with solid metal walls and a
mesh gate. The floor was non-slip concrete. The race was in a vacant room off the main corridor.
A video camera mounted on the wall allowed the dogs to be filmed in the entire race. Discrete
marks were made on the floor starting 10 cm from the gate to allow measurement from the video
footage.
Each dog was carried from its holding pen to the race (the greyhound was led). The dog was
placed in the race and allowed to move around freely. The handler moved out of sight during this
time. After 30 s the handler re-appeared at 3.5 m from the gate holding a remote controlled car.
The handler said ‘‘hello’’ in a neutral voice to the dog and placed the car on the floor, directed
towards the gate. When the dog approached within 10 cm of the gate or after 30 s if the dog did
not return to the gate, the handler drove the car at maximum speed (0.6 m/s) towards the gate. The
test commenced once the car first made contact with the gate. The handler remained stationary,
3.5 m from the gate, for the duration of the test.
The time (s) for the dog to approach within 100, 50 and 10 cm of the novel object were
calculated from direct observations. The following variables were calculated from video footage:

 Maximum withdrawal distance (m) after 10, 30 and 50 s.

 Closest approach (m) to the novel object.
 Time (s) spent within 100, 50 and 10 cm of the novel object.
 Number of entries within 100, 50 and 10 cm of the novel object.

2.3.4. Startling test

Dogs tend to exhibit avoidance behaviour towards sudden movement (Melzack, 1952). The
test in the present study used an opening umbrella to create a sudden movement. Previous studies
have used an opening umbrella to elicit fearful and aggressive behaviour in dogs (Goddard and
Beilharz, 1984; Netto and Planta, 1997). The test in the present study was conducted in the same
raceway as the novel object test. Behavioural responses, salivary cortisol concentrations and
heart rate were measured in this test. The pre-test salivary cortisol samples were collected a
minimum of 30 min before the commencement of the behavioural tests for the day and all dogs
were sampled at the same time. Prior to this sampling, the dogs had not been handled since the
previous day. To ensure a baseline cortisol concentration unaffected by handling stress, the dogs
were sampled in their pens and samples were collected from all the dogs within 4 min of entry to
the accommodation room to avoid a corticosteroid release associated with handling (Broom and
Johnson, 1993).
After the pre-test cortisol samples were collected, any long haired dogs were clipped on both
sides of the chest to allow good skin contact with the heart rate monitor electrodes. Each dog was
removed from its enclosure by the handler and fitted with a heart rate monitor (Polar Sport
Tester1) before being carried to the raceway. This was the same model as used by King et al.
(2003) and was set to record the dog’s heart rate every 5 s. The two electrodes were coated with
conducting gel then strapped on the ventrolateral chest approximately over the heart using the
supplied chest band. Due to the variety of chest sizes and shapes, it was necessary to use a Velcro
78 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84

girth strap over the electrode strap to ensure the electrodes had good contact with the skin. Heart
rate recordings started as the dog was released into the raceway and the handler moved out of
sight.
Following release in the raceway, dogs were allowed to settle for 30 s with the handler out of
sight. The handler then returned to the gate on the raceway and slid a bowl (21 cm diameter) of a
familiar commercial dry dog food under the gate and placed it in the centre of the raceway about
5 cm from the gate. The handler remained crouching, 0.3 m from the gate, and the dog was
allowed to feed for 3 s (feeding was defined as head over bowl). After 3 s, the handler rapidly
opened a brightly coloured umbrella close to the gate and adjacent to the dog’s nose. If feeding
had not commenced within 30 s of introducing the food, the umbrella was opened regardless of
the dog’s position and orientation. The test began at the opening of the umbrella and lasted 60 s.
The umbrella remained open for the entire test and dogs were observed via a monitor.
At the end of the test, the heart rate monitor was removed and the dog was returned to it
kennel.
The post-test saliva sample was collected 10 min after the commencement of the startling test.
These samples were collected from individual dogs in the main corridor out of sight of other
dogs. Two people collected the pre- and post-test saliva samples; one person restrained the dog
while the other collected the sample. Citric acid swabs were rubbed over the dog’s tongue, gums
and the roof of the mouth to stimulate the flow of saliva. Clean cotton wool swabs were wiped
over the same areas and under the tongue to collect the saliva. The samples were stored in ice
before being centrifuged at 2000  g for 15 min to extract the saliva. The saliva from each dog
was pipetted into a single labelled container and stored at 21 8C until assayed. The
concentrations of cortisol in saliva were measured by a commercial radioimmunoassay kit for
cortisol (Orion Diagnostica, Turku, Finland) according to the protocol for salivary samples. Due
to assay insensitivity, samples with low values were allocated the lower limit of detection of
0.25 nmol l 1 rather than zero.
The heart rate data were stored in the monitor device and data were downloaded at the
completion of the day’s testing onto a computer using the Polar Advisor Software. Readings were
set at 5 s intervals, with each reading providing the mean heart rate for the preceding 5 s. A video
camera mounted on the shed wall allowed the dogs to be filmed in the entire race. The dog was
given 30 s to acclimatize in the raceway. The time (s) for the dog to approach within 100, 50 and
10 cm of the umbrella were calculated from direct observations. The following variables were
calculated from video footage:

 Maximum withdrawal distance (m) after 10, 30 and 50 s.

 Closest approach (m) to the umbrella.
 Time (s) spent within 100, 50 and 10 cm of the umbrella.
 Number of entries within 100, 50 and 10 cm of the umbrella.

Physiological variables collected from the heart rate measurements and the cortisol assay
included:

 Maximum heart rate.

 Maximum heart rate post stimulus.
 Mean heart rate.
 Mean heart rate 45 s pre-test.
 Mean heart rate 30 s pre-test.
 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84 79

 Mean heart rate 30 s post-test.

 Saliva cortisol concentration pre-test.
 Saliva cortisol concentration post-test.
 Difference between saliva cortisol concentration pre- and post-test.

2.4. Statistical analysis

The effects of treatment on the observed frequency (number of observations in which the
variable was observed) of individual postures (standing, sitting and lying), locomotion, active
behaviour, vocalisation, grooming, substrate directed behaviour and object directed behaviour
were examined by analysis of variance.
Three components identical to those identified by King et al. (2003), ‘Novelty’, ‘Exploration’
and ‘Startling’ (variables contained in these components are presented in Table 2), were
compiled from the observations in the behaviour tests. An analysis of variance using GLM
procedure of SPSS (v12.0.1) with the treatment (control and clomipramine) and order (periods 1
and 2) was used to examine the effect of treatment and order on the three component scores.
Due to problems of obtaining accurate placement of the heart rate monitor in the short period
available before testing and then maintaining this placement during testing, heart rate records
were not obtained from many of the dogs (8 dogs in the first period of tests, 12 in the second) and
these limited data were not analysed. Many of the dogs used in the study had not previously worn
dog collars and variation in chest sizes and shapes together with attempts to dislodge the girth
strap holding the heart rate monitor resulted in these difficulties in obtaining heart rate
recordings.

3. Results

3.1. Observations in treatment

All the dogs were monitored for side effects from the clomipramine treatment. One dog
became aggressive and was euthanized. No other side effects were noticed and in general all the
dogs were in good health throughout the treatment and behavioural testing. Analysis of variance
comparing the clomipramine treated and control dogs on their behaviour in their runs revealed
few differences. There was a tendency for the treatment dogs to be observed less often displaying

Table 2
Components and variables
Component Variables
Novelty Time spent within 10 cm of the novel object, time spent within 100 cm of the novel object,
latency to enter within 10 cm of the novel object, latency to enter within 100 cm of the novel
object, number of entries made within 10 cm of the novel object, distance from the novel object
at 50 s after the start of the test
Exploration Number of entries into the open arms, time spent on the open, time spent on the closed arms,
latency to move from the central square of the EPM, total number of entries into all areas of the
EPM, number of entries into the lit compartment, time spent in the lit compartment, latency
to put the nose into the lit compartment, latency to put the body into the lit compartment
Startling Time spent within 10 cm of the startling stimulus, time spent within 100 cm of the startling
stimulus, latency to move within 10 cm of the startling stimulus, latency to move within 100 cm
of the startling stimulus, number of entries within 100 cm of the startling stimulus
80 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84

both substrate directed activity, defined as behaviours directed towards the ground (mean number
of observations (treatment) = 0.65, S.E.M. (treatment) = 0.15; mean number of observations
(control) = 1.74, S.E.M. (control) = 0.57) and object directed behaviour, defined as behaviours
directed towards kennel structures or objects such as grasses, leaves or sticks, within or outside
the kennel (mean number of observations (treatment) = 1.96, S.E.M. (treatment) = 0.43; mean
number of observations (control) = 3.30, S.E.M. (control) = 0.72). Analysis of the results from
the video footage of the dogs inside their kennels showed that the majority of time inside was
spent either sitting or lying (mean of 75.88% and S.E. of 6.62) or standing (mean of 24.12% and
S.E. of 1.64).

3.2. Behavioural tests

There was a significant treatment effect on the novelty component items (F 1,20 = 4.71,
P = 0.04). As the novelty component contains items relating to latency to approach and time
spent near the novel object, these results indicate dogs under the clomipramine treatment showed
a decreased latency to approach and increased time spent near the novel object in comparison to
the control animals. There was no treatment by order effect on this measure. The exploration
component items were not affected by treatment or treatment by order interaction (F 1,20 = 0.54,
P = 0.48). There was a tendency for the startling component items to be affected by treatment
(F 1,20 = 3.00, P = 0.10) and the treatment by order interaction (F 1,20 = 4.02, P = 0.06) (see
Table 3). The former indicates that treatment with clomipramine decreases the time to approach

Table 3
The means and standard errors of three component scores for the main effects
Main factor Mean (estimated) S.E.
Novelty component score
Treatment
Clomipramine 0.233 0.186
Control 0.266 0.238
Order
1 0.049 0.255
2 0.082 0.255

Exploration component score

Treatment
Clomipramine 0.122 0.655
Control 0.069 0.266
Order
1 0.267 0.306
2 0.214 0.324

Startling component score

Treatment
Clomipramine 0.177 0.148
Control 0.192 0.247
Order
1 0.173 0.240
2 0.189 0.251
 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84 81

the startling stimulus and increases time spent near the startling stimulus. The tendency for the
treatment by order interaction indicates that animals which received the treatment in the second
period had a higher scores, that is longer latencies to approach and spent less time close to the
startling stimulus than those that received the treatment in the first period.
There were no significant treatment effects (P < 0.05) on salivary cortisol concentrations
[mean salivary cortisol pre-test (treatment) = 6.68, S.E. (treatment) = 5.83 nmol l 1; salivary
cortisol post-test (treatment) = 6.14, S.E. (treatment) = 2.86; mean salivary cortisol pre-test
(control) = 7.72, S.E. (control) = 5.94; mean salivary cortisol post-test (control) = 6.86, S.E.
(control) = 2.41].

4. Discussion

The aim of this study was to validate previously identified indices of fear in the response of
dogs to novel and startling stimuli by using a cross-over design, involving a control or placebo
treatment for 6 weeks and a clomipramine treatment for 6 weeks. Clomipramine has been
extensively studied and tested for its effects on fear and anxiety in animals and people
(Beaumont, 1979; Overall, 1994; Hewson et al., 1998; King et al., 2000). A transient sedating
effect has also been reported (King et al., 2000). One dog in the present study developed
aggressive behaviour after 14 days of clomipramine. It is not clear whether this dog would have
become aggressive without being treated with clomipramine, however aggression after treatment
with clomipramine has been reported in one other study (King et al., 2000). None of the other side
effects were observed in any of the dogs in the study.
In the present study, observation of dogs in their runs revealed that there were no effects of
clomipramine on the amount of time spent lying, sitting, standing or moving. It is therefore
concluded that any differences observed between the two treatments in the behavioural responses
of dogs in the behavioural tests were not due to a sedating or depressing effect of normal
behaviour by the clomipramine.
Treatment with clomipramine resulted in dogs spending more time near and approaching
closer to the novel object in the Novel Object test. This test has been found to reliably provoke
avoidance behaviour in other species (Boissy and Boissou, 1995; Visser et al., 2001). King et al.
(2003) suggested that these variables, time near and time to approach the novel object (that is the
novelty component) are measuring a response to novelty in the dog and the present results
support this contention. The second component (called exploration) identified by King et al.
(2003) involved variables from the light/dark test and the elevated plus maze test such as latencies
and entries to areas in the two tests (see Table 2). The authors suggested this component was
measuring exploration rather than fear. The results of the present study indicate that
clomipramine did not affect the exploration component and thus support the interpretation by
King et al. (2003) that this component does not measure fear. As suggested previously by King
et al. (2003) these two tests, the light/dark test and elevated plus maze test, may not be sufficiently
aversive for the dogs. For example, many dogs appeared to move confidently around the elevated
plus maze and a number even jumped off the apparatus. It was perhaps too wide and not high
enough to evoke a substantial fear response in the dogs.
The present results on the startling component provide support for the contention by King
et al. (2003) that this component is measuring fear of startling stimuli. There was a tendency
(P < 0.10) for treatment with clomipramine to decrease the time to approach the startling
stimulus and increase the time spent near the startling stimulus. There was also a tendency
(P < 0.06) for a treatment by order interaction in which treatment with clomipramine in the first
82 J. Ley et al. / Applied Animal Behaviour Science 104 (2007) 71–84

period but not the second period was associated with reduced latency to approach and increased
time spent near the startling test. A second exposure irrespective of treatment would be expected
to reduce fear responses to startling stimuli.
In comparison to the study by King et al. (2003), incomplete records on heart rate were
obtained in the present study. The authors of the present study considered that large differences in
the size of the dogs and attempts by some dogs to remove the girth strap were responsible for
these difficulties in obtaining heart rate recordings. However, the latter responses are considered
unlikely to markedly affect the validity of the behavioural measures in the startling test. For
example, the girth strap is a less aversive stimulus relative to the sudden appearance of an open
umbrella as 70% of dogs in the control treatment withdrew more than 10 cm from the umbrella.
Furthermore, the behavioural responses of the dogs in this study and in the study by King et al.
(2003) were similar. The mean (and range in) time spent within 10 cm of the startling stimulus
was 7.8  17.86 s and the mean (and range in) latency to move within 10 cm of the startling
stimulus was 47.35  23.65 s in the control treatment in present study and 18.5  26.4 s and
32.9  25.8 s, respectively, in the study by King et al. (2003).

5. Conclusion

These results together with those of King et al. (2003) show that the approach behaviour of
dogs to the stimuli in the novel object test and the startling test are measuring responses to novelty
and startling stimuli, respectively, and thus are appropriate measures of different aspects of fear.
These will be of benefit in future studies examining fear in dogs and its role in dog aggression
directed at humans.

Acknowledgement

The authors would like to acknowledge funding from the Bureau of Animal Welfare,
Department of Primary Industries, Victoria, Australia.

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