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Behavioral and cortisol responses of shelter dogs to a cognitive bias test after olfactory enrichment with essential oils

Authors:
  • Odisee University College

Abstract and Figures

A shelter environment tends to present different types of stressors dogs need to cope with. Recent work has shown that olfactory enrichment with essential oils might be able to modify the affective states of certain species (dogs, cats, horses, zoo animals…). In these studies, the welfare measurements included physiological indicators, such as corticosteroid levels, and/or behaviors related to chronic stress. The olfactory effects of 9 essential oils (Cananga od-orata,Cistus ladaniferus, Citrus aurantium, Cupressus sempervirens, Juniperus communis var. montana, Lavandula an-gustifolia, Laurus nobilis, Litsea citrata, Pelargonium graveolens) and a blend of these oils were explored on a cognitive bias test, cortisol levels and the behaviors of 110 shelter dogs (n = 10 dogs within each group). Olfactory enrichment with the blend resulted in a reduced latency to the ambiguous cue, indicating a more optimistic bias and improved welfare. The results of this study suggest that olfactory enrichment with essential oils can have specific effects on the affective states and behaviors of shelter dogs, and could therefore be useful for shelter management. In addition, as not all of the essential oils tested individually were effective, more research should be conducted to better understand the effects of each individual essential oils on dogs.
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Behavioral and cortisol responses of shelter dogs
to a cognitive bias test after olfactory enrichment
with essential oils
Stefania Uccheddu1, Chiara Mariti2, Adinda Sannen3, Hilde Vervaecke3,
Heidi Arnout3,4, Jara Gutierrez Rufo2, Angelo Gazzano2, Anouck Haverbeke1,*
1 Vet Ethology, Leemveldstraat44, 3090 Overijse, Belgium
2 Department of Veterinary Sciences, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy
3 Odisee University College, Agro- & Biotechnology, Ethology & Animal Welfare, Hospitaalstraat 23,
9100 Sint-Niklaas, Belgium
4 University of Antwerp, Departmentof Engineering Management, Prinsstraat 13, 2000 Antwerp, Belgium
Abstract: A shelter environment tends to present dierent types of stressors dogs need to cope with. Recent work
has shown that olfactory enrichment with essential oils might be able to modify the aective states of certain species
(dogs, cats, horses, zoo animals…). In these studies, the welfare measurements included physiological indicators, such
as corticosteroid levels, and/or behaviors related to chronic stress. e olfactory eects of 9 essential oils (Cananga od-
orata,Cistus ladaniferus, Citrus aurantium, Cupressus sempervirens, Juniperus communis var. montana, Lavandula an-
gustifolia, Laurus nobilis, Litsea citrata, Pelargonium graveolens) and a blend of these oils were explored on a cognitive
bias test, cortisol levels and the behaviors of 110 shelter dogs (n = 10 dogs within each group). Olfactory enrichment
with the blend resulted in a reduced latency to the ambiguous cue, indicating a more optimistic bias and improved
welfare. e results of this study suggest that olfactory enrichment with essential oils can have specic eects on the
aective states and behaviors of shelter dogs, and could therefore be useful for shelter management. In addition, as not
all of the essential oils tested individually were eective, more research should be conducted to better understand the
eects of each individual essential oils on dogs.
Key Words: behavior; cognitive bias; cortisol; dogs; essential oils; olfactory enrichment; shelter; welfare.
* Corresponding Author: anouck.haverbeke@vetethology.be
Introduction
A shelter environment tends to present dierent types of stressors dogs need to cope with: social
stressors (reduced intraspecic and/or interspecic social contacts), environmental stressors
(restraint for medical procedures, separation from a caretaker or handler) or psychogenic stressors
(separation anxiety, use of aversive training methods by a previous owner/lack of ethological
knowledge in caretakers). Moreover, stressors are known to cause activation of metabolic and
endocrine responses in sheltered animals (Titulaer et al., 2013).
Recent work has shown that essential oils might be able to modify the aective states of
certain species (dogs, cats, horses and zoo animals: Wells, 2004; Graham et al., 2005; Ferguson
et al., 2013; Wells & Egli, 2015; Binks et al., 2018). In these studies, the welfare measurements
included physiological indicators, such as corticosteroid levels (Beerda et al., 1998) or behaviors
related to chronic stress, such as repetitive behaviors, nosing, paw-liing, increased locomotion,
displacement behavior or excessive drinking (Beerda et al., 1998; Haverbeke et al., 2008).
However, interpretation of these indicators can be dicult (Titulaer et al., 2013). erefore
the detection of a cognitive bias might be a complementary solution. A recent and innovative
approach utilizes the inuence of aective states on the interpretation of current experience.
e resulting aect-induced cognitive biases can be measured (Mendl et al., 2009) through
cognitive bias tests as indicators of the animal’s psychological well-being (Mendl et al., 2009; Paul
et al., 2005). A cognitive bias test in this context refers to the propensity of a subject to show
Dog Behavior, 2-2018, pp. 1-14
doi 10.4454/db.v4i2.87
Submitted, 07/29/2018
Accepted, 09/12/2018
behavior indicating the anticipation of either relatively positive or relatively negative outcomes in
response to aectively ambiguous stimuli (Mendl et al., 2009). Changes in cognitive bias reect an
individual’s experience of positive and negative events and thus its aective valence and welfare
(Mendl et al., 2010). e eects of environmental enrichment have been already tested through
cognitive bias test in dierent species such as rats (Brydges et al., 2011), pigs (Douglas et al., 2012)
and European starling (Bateson & Matheson, 2007).
Several studies have found correlations between cognitive biases and aective states in a wide
range of species, including mammals (Mendl et al., 2009; Doyle et al., 2010) and birds (Matheson
et al., 2008; Salmeto et al., 2011). e aim of the current study was to assess whether olfactory
enrichment through essential oils inuences the aective states of sheltered dogs. To do that, the
possible eects of 9 dierent of essential oils (Cananga odorata, Cistus ladaniferus, Citrus auran-
tium, Cupressus sempervirens, Juniperus communis var. montana, Lavandula angustifolia, Laurus
nobilis, Litsea citrata, Pelargonium graveolens) and a blend of these oils on a cognitive bias test,
cortisol levels and behavior of 110 shelter dogs were explored.
Materials and Methods
Participants
One hundred ten dogs ranging in age from 1 to 10 years, of both sexes, and of either pure or
mixed breed, were enrolled in the study and randomly allocated to one of 11 dierent groups
(Table 1). e dogs lived in groups of three in kennels with an indoor section measuring 1.5
meters x1.5 meters and an outdoor run measuring 1.5 meters x 2 meters, joined by a metal door
operated by sta. Water was available ad libitum, and food was provided twice per day, at 8 am
and 6 pm.
Dogs were selected based on the following criteria: (a) success at the training phase, (b) no
previous diagnosis of anxiety or aggressive behavior, (c) some socialization prerequisites, such as
the ability to deal with people without fear, (d) the veterinarians agreement and (e) ability to walk
on leash (f) good medical health.
Table 1. Description of the study protocol.
Group Number
of dogs
Pre-test
training Cognitive test 1 Exposure to collar
for 3 hours Cognitive test 2
1 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
No addition
(control group)
Aer exposure to collar and aer
collection of saliva at T1
2 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
e blend Aer exposure to collar and aer
collection of saliva at T1
3 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
Litsea citrata Aer exposure to collar and aer
collection of saliva at T1
4 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
Cupressus
sempervirens
Aer exposure to collar and aer
collection of saliva at T1
5 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
Citrus aurantium Aer exposure to collar and aer
collection of saliva at T1
6 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
Pelargonium
graveolens
Aer exposure to collar and aer
collection of saliva at T1
2
3
7 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
Lavandula
angustifolia
Aer exposure to collar and aer
collection of saliva at T1
8 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
Cananga odorata Aer exposure to collar and aer
collection of saliva at T1
9 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
Juniperus
communis var.
Montana
Aer exposure to collar and aer
collection of saliva at T1
10 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
Cistus ladaniferus Aer exposure to collar and aer
collection of saliva at T1
11 10 Ye s
Before exposure to collar
and aer collection of
saliva at T0
Laurus nobilis Aer exposure to collar and aer
collection of saliva at T1
Olfactory enrichment
A blend of 9 essential oils (Arhomani, Belgium) and each separate oil of the blend (Flora s.r.l.,
Pisa, Italy) were tested, for a total of 10 treatments and a control group (Table 1). Essential oils were
diused through a cotton collar worn by the dogs for 3 hours before starting the second cognitive
test procedure (see below). e collar, just before being applied to the dog, had 1 drop of an indi-
vidual oil or of the blend added to it. e control group, as the experimental groups, wore a cotton
collar for 3 hours but without any oils or other addition.
During this part of the experiment, the dogs stayed in the pen where they routinely spent time.
Dogs were allowed to mix in the same pen only if they were allocated to the same essential oil
group. In order to avoid odour contamination, there was a distance of 500m between the dierent
pens.
Test protocol
Cognitive test
All dogs of the 11 groups were subjected to two cognitive bias (CB) tests (modied from
(Mendl et al., 2010), one prior to (CB 1) and one aer essential oil exposure (CB 2). CB 2 was
performed 3 hours aer T0. To avoid more stress caused by a dierent routine in the shelter,
we could not control for order eects. All the dogs were tested on the second cognitive bias test
following olfactory enrichment.e protocol of (Mendl et al., 2010) and (Owczarczak-Garstecka
& Burman, 2016) was modied in this study based on a pilot study we carried out, in which we
observed that dogs in the shelter were unable to maintain attention during the original cognitive
test as proposed in (Mendl et al., 2010) and (Owczarczak-Garstecka & Burman, 2016). For this
reason, we used a shortened version. During the training session, all dogs received a minimum
of 8 training trials instead of 15. During the test, we used just one ambiguous location instead of
three ambiguous locations. e test phase involved 6 trials (instead of the 32 proposed by (Mendl
et al., 2010).
In addition, during the pilot study, we realized that shelter dogs were much more interested
in humans than in food, so we changed the original protocol by having the researcher behind the
camera rather than behind the bowl put on the ground, in order to avoid the dog choosing that
bowl for its closeness with a person (the researcher).
Training and cognitive tests were performed with each dog enrolled in the experiment
individually led to a test area (6 meters x 6 meters) within the shelter, the same for all sessions and
all dogs. e setting is described in gure 1. e bowl was placed at one of three predetermined
locations (two during the training) 4 meters in front of the dog’s xed starting position. e
4
latency to reach the bowl, was dened as the time elapsed between release from the lead and the
dog putting its head into the bowl, or touching the rim of the bowl with its nose (Mendl et al.,
2010). CB tests were video recorded and then analysed as described below.
Figure 1. Experimental setting.
Training
Dogs were rst trained to associate a certain location with a reward. During the training, the
distance between the two bowls (Positive and Negative) was 1meter.When the bowl was placed at
the ‘positive’ location (P) on one side of the test area, it contained food, and when it was placed
at the ‘negative’ location (N) on the opposite side of the test area, it was empty. Two visually
identical bowls were used for rewarded (P) and non-rewarded (N) locations, and both bowls had
a piece of food taped to their bottom sides that were inaccessible to the dogs to control for odour
cues. Training was complete when the dogs reached a pre-set criterion, that is, when the dog ran
to the positive location faster than to the negative one twice consecutively.
Each training session started aer a 10-minute period of habituation with the researchers in the
experimental area (Figure 1). e dog was put on a lead and held by one of the researchers behind
a barrier, while the other researcher stood at the far end of the room and baited (or did not bait,
depending on trial type) a food bowl with 50 gr of commercial dog food. e dog was released
to approach the bowl. Each dog received at least 8 training trials conducted so that no location
was repeated more than twice. Each training session started with two positive (rewarded) trials
to encourage participation, followed by two negative (non-rewarded) trials. e remaining trials
were randomly assigned to be rewarded or non-rewarded. e latency to reach the bowl, dened
as the time elapsed between release from the lead and the dog putting its head into the bowl or
touching the rim of the bowl with its nose, was recorded for each trial using a stopwatch. e
maximum time allowed per trial was 30 seconds. If the dog did not reach the food bowl within
that time, the maximum time was scored.
5
Test
When the training was completed, the test started. Each dog was presented a food bowl in
three locations, positive (P), negative (N) and intermediate (M).e Middle bowl was located
between Positive and Negative bowl. e distance between Positive (or Negative) and Middle
Bowl was 50 cm.e bowl was presented in each location twice (P1, M1, N1 and P2, M2, N2) but
in dierent order. e accessible food was only present in the positive location (P). Negative (N)
and Intermediate (M) locations remained empty but with olfactory control cues. All the tests were
videorecorded. Since in each CB the locations were tested twice, we used mean values for each
location in CB 1 and in CB 2 in further analyses.
Behavioral observations
e observations of dog behaviors were carried out on the videos recorded during the whole
test. Each dog was observed using a continuous sampling method.
e behavioral analysis was conducted using the ethogram reported in Table 2 and 3 (Haverbeke
et al., 2008). Depending on the type of behavior, either the duration (in seconds) or the number
of occurrences was recorded.
Table 2. Behaviors scored in terms of number of occurrences.
Behavior Description
Oral behaviors:
Yawning Mouth open to apparent fullest extent while eyes are closed
Non-directed licking
Snout licking
Tongue out, the tip of the tongue is briey extended
Part of the tongue is shown and moved along the upper lip
No oral behaviors
Paw liing Fore paw lied into a position of approximately 45°
Urinatingsquat Urinating by squatting while keeping both hind limbs on cage oor
Urinating, limbraised Urinating while raising one hind limb
Defecating Excreting the contents of the bowels
Table 3. Behaviors analysed in terms of duration (seconds).
Behaviors
Repetitive or stereotypicbehavior
Pacing Immediately repeating a path just taken and continuing in the repetition in circles, in a
gure eight pattern or fence/wall-line running
Circling Continuous walking in short circles, apparently chasing its tail or hind limbs
Other behaviors Manipulating environment (Stereotypic interactions with elements from the
environment, such as digging (scratching the oor with the forepaws in a way that is
similar to how dogs dig holes), oor licking (licking the oor with the tongue)), Auto
grooming
Notseen Unable to determine behavior of the dog owing to darkness or the position of the dog
Miscellaneousoralbehaviors
Barking loud, rough noise
Roaring loud, deep sound
Growling low, rough sound
Whining long, high sound
Yelping sudden, short, high sound
Panting Increased frequency of inhalation and exhalation oen in combination with the opening
of the mouth
Teethclapping Making short loud noise by hitting teeth together
6
Notseen Unable to determine the behavior of the dog owing to darkness or the position of the
dog
Locomotive states
Prone, head down Trunk of body on oor, chin or side of head in contact with the oor, paws or limbs
Prone, head up Trunk of body on the oor, no part of the head in contact with the paws
Sitting Only hindquarters and front paws in contact with the oor
Standing Upright with at least three paws in contact with the oor without any walk
Walking Takes at least one step, shiing body position
Highly active Any motion across oor faster than a walk, including trotting and jumping
Changing from one state of locomotion to another
Notseen Unable to determine behavior of dog owing to darkness or the position of the dog
Postures
High e breed specic posture as shown by dogs under neutral conditions, but in addition
the tail is positioned higher or the position of the head is elevated, and the ears are
pointed forwards, or the animal is standing extremely erect
Neutral e breed posture shown by dogs under neutral conditions
Halow Two or more of the following three features are displayed: a lowered position of the tail
(compared to the neutral posture), a backward position of the ears and bent legs
Low e position of the tail is lowered, the ears are positioned backwards, and the legs are
bent
Verylow Low posture, but now the tail is curled forward between the hind legs
Notseen Unable to determine the behavior of the dog owing to darkness or the position of the
dog
Cortisol
Saliva samples for the assessment of plasma cortisol concentrations were collected, at the same
time in the day, before the addition of the oils at T0 (to identify the basal cortisol levels) and at T1,
i.e. aer 3 hours exposure to the collar for all groups, including the control one. Collection was
always carried out before the cognitive bias tests at T0 and T1. Saliva samples were collected using
Salivette Cortisol code blue(Sarstedt, Nümbrecht, Germany) and stored at-20 °C until they were
further processed using a commercial ELISA kit (Diametra, Milano, Italy).
Statistical analysis
e statistical analysis was performed using IBM SPSS Statistics for Windows, version 22.0
(Armonk, NY: IBM Corp). For each of the oils under study, the dierence in the variables
measured before and aer exposure was tested using a Wilcoxon signed-rank test. is paired
dierence test was used because each subject is measured twice, resulting inpairsof observations.
is reduces the eect of confounders like individual dierences (e.g. in pace length or in interest
in food) between dogs.e test statistics (sum of positive ranks) as well as the two-sided p-values
are reported in the results below. P values ≤ 0.05 were deemed statistically signicant.
We additionally tested for T0 as well as T1whether the dogs’ responses during the cognitive
bias tests were appropriate (i.e. dogs were slower to approach the ‘negative’ location N when
compared to the ‘positive’ location P) by using a one-sided paired t-test comparing latency to
approach N versus latency to approach P. Statistical p values ≤ 0.05 were deemed statistically
signicant.
7
Results
Cognitive test
We explored the dogs’ latency to approach P and N, just to make sure dogs’ response to the CB
test 1 (before exposure) and CB test 2 (aer exposure) was appropriate (i.e. animals were slower
to approach N than P). e results are reported in Table 4.
Table 4. Statistical results of the comparison between latency to reach positive and negative locations be-
fore exposure and after exposure to essential oils (CB 1: Cognitive test before exposure; CB 2: Cognitive test
after exposure).
Mean (seconds) N Standard. Deviation Standard Error Mean
CB 1 Latency before
exposure P location 19.32 110 17.01 1.62
Latency before
exposure N location 24.18 110 27.28 2.60
CB 2 Latency aer
exposure P location 2.64 110 0.48 0.05
Latency aer
exposure N location 13.81 110 13.53 1.29
e analysis revealed a significant eect of the blend “e blend” in reducing the latency
to reach the intermediate position (test statistic=3; n=10; p=0.039). We also observed a trend
towards reducing the latency to reach the intermediate position (test statistic=5; n=10; p=0.078)
for Litsea citrata oil (Table 5).
Table 5. Latency (mean ± Standard Deviation in seconds before and after 3 hours of exposure) and cortisol
values (mean ± Standard Deviation in ng/ml before and after 3 hours of exposure) to each essential oil or
after 3 hours without any exposure in the control group (P < 0.05, *).
Before
exposure
Aer 3 hours
of exposure
(T1)
Statistical
results
Before
exposure
Aer 3 hours
of exposure
(T1)
Statistical
results
Latency value
(seconds)
Cortisol value
(ng/ml)
Control group
(no exposure) 20.60±11.00 15.95±10.98 P=0.38 2.406 ± 0.30* 1.762 ± 0.435 P=0.03*
Cananga odorata 18.65±7.84 16.92±9.35 P=0.84 1.923 ± 0.70 1.512 ± 0.111 P=0.08
Cistus
ladaniferus 18.77±11.78 14.98±11.93 P=0.54 1.538 ± 0.22 1.424 ± 0.132 P=0.18
Citrus aurantium 17.22±12.48 11.21±11.98 P=0.35 1.642 ± 0.21 1.507 ± 0.196 P=0.43
Cupres
sussempervirens 24.47±7.14 18.46±11.32 P=0.19 1.766 ± 0.58 2.175 ± 0.424 P=0.12
Juniperus
communis var.
Montana
21.93±9.30 14.06±13.71 P=0.20 1.397 ± 0.30 1.497 ± 0.364 P=0.74
Laurus nobilis 20.80 ±11.35 15.45±9.59 P=0.10 1.082 ± 0.45 1.435 ± 0.198 P=0.14
Lavandula
angustifolia 22.19±9.60 16.70±14.08 P=0.29 1.821 ± 0.39*1.549 ± 0.245 P=0.03*
Litsea citrate 21.97±9.34 14.70±10.39 P=0.078 1.467 ± 0.30 1.919 ± 0.313 P=0.078
Pelargonium
graveolens 20.74±9.58 15.48±10.70 P=0.10 1.287 ± 0.33 1.596 ± 0.504 P=0.10
e blend 23.83±9.80 13.46±11.28 P=0.039* 1.557 ± 0.49 1.316 ± 0.119 P=0.25
8
Behavioral observations
Only the olfactory enrichment with Laurus nobilis induced a signicantly longer duration of
high posture among these dogs (test statistic=26.5; n=10; p=0.047).
e analysis revealed non-signicant trends for dierent oils: Cananga odorata reduced the
“nosing” time (test statistic=9; n=10; p=0.064), Citrus aurantium (test statistic=46; n=10; p=0.064)
and Cupressus sempervirens (test statistic=39; n=10; p=0.055) increased the time spent in “tail
wagging”, and Pelargonium graveolens (test statistic=3; n=10; p=0.078) reduced the time spent
in “non-oral stress behaviors” (circling, pacing, manipulation of environment, autogrooming).
Cortisol
Olfactory enrichment with Lavandula angustifolia induced a signicant reduction in saliva
cortisol levels (test statistic=3; n=8; p=0.039). A similar signicant reduction was also found in the
control group (test statistic= 0; n=6; p=0.031) (Table 5).
Discussion
Cognitive test
In the present study, authors applied a cognitive test to evaluate the eectiveness of olfactory
enrichment with essential oils in reducing the level of stress in sheltered dogs. Olfactory enrichment
with the blend of oils resulted in a reduced latency to the ambiguous cue in the cognitive test,
indicating a more optimistic bias and, consequently, an improved welfare (Mendl et al., 2010).
ese results provide support for the idea that the interactions between compounds oen result in
biological activity that is greater than the activity of the isolated compounds(Galindo et al., 2010).
Many domestic dogs are kept in rescue and rehoming shelters which are frequently stressful
and impoverished environments. Dog’s welfare is oen compromised within these environments
and there is a need to determine new practical and eective methods to improve the welfare
of these kenneled dogs (Binks et al., 2018).e development of objective methods to assess the
aective states of non-human animals is a crucial step in improving animal welfare (e.g. Dawkins,
2008.). Mendl (Mendl et al., 2009) enumerated several potential advantages of the cognitive bias
test, including the ability to make a priori predictions for dierent species: mammals (Mendl
et al., 2009; Doyle et al., 2010), birds (Matheson et al., 2008; Salmeto et al., 2011) and insects
(Salmeto et al., 2011). Douglas et al. (2012) support the hypothesis that an enriched environment
induces a more optimistic cognitive bias indicative of a more positive aective state and better
welfare in pigs.
Negative eects from inadequate environmental manipulations have been investigated by
several researchers. Environmental manipulations chosen to induce negative eect produce
pessimistic cognitive biases in animals’ responses to ambiguous stimuli (reviewed in (Mendl et
al., 2009)). Rats show pessimistic responses when housed in impoverished cages but switch to
optimistic responses when moved to enriched cages (Brydges et al., 2011).
To our knowledge, this is the rst time that a cognitive bias test has been applied to assess the
eect of olfactory enrichment with essential oils. Although this is a rst study on this topic and
the number of dogs tested in each experimental group was relatively low, it is remarkable that
the statistical analysis revealed some signicant dierences. In particular, the results regarding
the blend of oils are in line with previous studies that reported improved optimism through
environmental enrichment (Douglas et al., 2012) in pigs.
However, we should take into account that the medium latency for reaching the positive location
in the cognitive bias test 2 (P2- aer exposure) is signicantly lower than the medium latency
9
for reaching the positive location in test 1 (P1- before exposure). So although the dogs respond
appropriate to each CB-test (i.e. approaching N signicantly slower than P), they approach both
P and N signicantly faster during CB2 than during CB1, which might suggest some eagerness to
participate in the test.
Behavioral observations
e results of the present study indicate that olfactory enrichment with Laurus nobilis induced
high posture among dogs. In volatiles, Laurus nobilis has been reported to improve vigilance
performance in a discrimination task (Matsubara et al., 2011), which could be interpreted
positively as increased self-condence (Fatjó et al., 2007) or alternatively as a heightened alertness
due to a negative state. In humans, a high individual variability in response to olfactory exposure
to 1,8-cineol (major component of Laurus nobilis), jasmine absolute ether, linalyl acetate and
peppermint essential oil has been observed on vigilance (Heuberger & Ilmberger, 2010). In the
present study, dog’s high posture aer olfactory exposure with Laurus nobilisis not accompanied
by other signs of alertness and can therefore be interpreted as a sign of improved self-condence
in line with Haverbeke et al. (2008).
Some essential oils had a slight eect on behavior. Olfactory enrichment with Cananga odorata
reduced the “nosing” time. is could indicate a decrease of stress among the dogs (Beerda et al.,
1998). In line with these interpretations, Hongratanaworakit and Buchbauer (2004) showed that
in humans Cananga odorata decreases blood pressure and pulse rate and increases subjective
attentiveness and alertness. Olfactory enrichment with Citrus aurantium and Cupressus
sempervirens increased the time spent in “tail wagging”. Tail wagging can be seen in the interactive
social context or to facilitate interaction and could have ambivalent interpretations going from an
increase to a decrease in condence in dogs (Gasci et al., 2005). In the present study, the exposure
of dogs to Citrus aurantium and Cupressus sempervirens are not accompanied by other changes.
erefore it is likely that in this study tail wagging is a sign of relaxation. is is in line with
previous results that have demonstrated anti-anxiety eects of both Citrus aurantium (in humans
(Akhlaghi et al., 2011; Carvalho-Freitas et al., 2002; De Moraes Pultrini et al., 2006) and rats (Leite
et al., 2008) and Cupressus sempervirens(in humans Bouguenoun et al., 2006).
Olfactory enrichment with Pelargonium graveolens reduced the time spent in “non-oral
stress behaviors” (circling, pacing, manipulation of environment, autogrooming). is observed
reduction of stress behaviors (Haverbeke et al., 2008) is in line with the ndings of Rashidi Fakari
et al., 2015, who observed an anxiolytic and sedative eect of Pelargonium graveolens in humans.
Cortisol
e observed reduction in saliva cortisol with Lavandula angustifoliais in line with Atsumi
& Tonosaki who have observed a decrease of salivary cortisol level on humans aer smelling
lavender essential oil (Atsumi & Tonosaki, 2007). In addition, a previous study using olfactory
enrichment with Lavandula angustifolia on sheltered dogs showed a change in dogs’ activities
(resting time) suggestive of relaxation (Graham et al., 2005).
We also observed a reduction of cortisol levels in the control group. is nding is in line with
previous research in dogs (Shiverdecker et al., 2013; Cobb et al., 2016). One possible explanation
is that, the mere application of a cognitive test can result in a stress relieving factor, being a sort
of cognitive enrichment for sheltered dogs. However, this does not explain why the cognitive test
with essential oil exposure had no eect on cortisol levels except in the Lavandula angustifolia
group. Another explanation is that essential oils (except Lavandula angustifolia) has increased
neophobia (i.e. the fear of novelty, which can be sometimes observed in captive animals that
have received little or no previous novel sensory stimulation (Mason et al., 1991) as observed in
Goeldi’s monkeys exposed to peppermint oil (Boon, 2003) and in a young tiger exposed to catnip
10
(Todd, 2015). However, as dogs belong to a species who tends to be very neophilic (Kaulfuss
& Mills, 2008) this explanation might probably not be considered for the canine species.As the
interpretation remains open, further studies are required in order to demonstrate through a more
detailed and rigorous analysis the eects of Lavandula angustifolia essential oil on cortisol levels
versus the eects of the other essential oils.
Although saliva collection was carried out at dierent times of the day, it is unlikely that
the dierences we observed were inuenced by this. In fact, previous research has not found
a circadian rhythm in the HPA (Hypothalamic Pituitary Adrenal) activity of dogs: neither in
laboratory dogs at 30 minutes intervals over a period of 28 hours (Takahashi et al., 1981) nor
at 20 minutes intervals over a period of 25 hours (Kemppainen & Sartin, 1994), nor in working
dogs exposed to defense training and trailing tasks at 90-180 minutes intervals over a period of 24
hours (Kolevska et al., 2003).
e saliva cortisol collected aer T1 could not have reected an earlier emotional state (pre-
olfactory enrichment), because cortisol concentrations rise approximately 20 minutes aer a dog
encounters a stressor (Vincent & Michell, 1992). Moreover, previous authors (Kirschbaum &
Hellhammer, 2000) have shown that changes in plasma and salivary cortisol levels are closely
synchronized: aer injections of cortisol, salivary levels increase within 1 minute and peak
concentrations in blood are seen 2-3 minutes later in saliva.
Some methodological limitations have been encountered during this study. Firstly, we used
a short version of the cognitive bias test because the sheltered dogs were not able to perform
the longer test (author’s observations in an unpublished, pilot study). eir limited performance
might be due to the fact that these dogs were not accustomed to be involved in cognitive activities
in their actual environment (presence of physical and social stimuli).
Secondly, being a study carried out in the eld and not in a laboratory setting, many factors
could not be controlled. For instance, there is a potential risk of olfactory confounding eects.
However, in order to reduce the risk of crossed stimulation among dierent essential oils groups,
a distance of 500 meters from one pen to another was set. Further, in a shelter environment the
quantity of olfactory stimuli is high and similar for all dogs. us a possible eect of odours other
than essential oils should be equally distributed for all dogs, which is not the case in the present
study. Lastly, even if we might consider any olfactory confounding eect, the main olfactory
eect should still remain the one obtained by the tested essential oil as it is the most proximate
odour from the dog’s nose. In order to conrm our results, further research should investigate the
maximum or minimum distance necessary to create an olfactory eect with essential oils.
irdly, the ndings should be interpreted with caution because it is possible that the dogs’
behavior was inuenced by a learning eect and a decreased interest because the cognitive bias
test was repeated twice. However, each time that a cognitive bias test is being used, information
processing, including attention, learning, memory and decision-making is being addressed
(Mendl et al., 2009).
Fourthly, in our study all the dogs were tested on the second cognitive bias test following
olfactory enrichment. Unfortunately, we could not control for any order eects, because a dierent
protocol would have caused changes in the shelter routine and therefore additional stress for the
shelter dogs and the sta. Nevertheless, as the order was the same for the tested dogs, the results
of a potential order eect should be the same for all groups. e dierent ndings observed in sub
samples suggest that essential oils have dierent eects: this could be a combination of essential
oils’ stimulation and repetition of the test. Further research should investigate the eects of
single essential oils in dierent conditions.e tendencies or signicant decreases that are found
in dierent behaviors in various groups could be caused by an increase in condence the dogs
experienced in the second CB (they were familiar with the CB and might have been eager to
11
participate and enjoy human contact or the enrichment).If a design would be applied in which
50% of the dogs start with essential oils (group 1) and 50% of the dogs without essential oils
(group 2), it is not possible to conduct the control-CB within the same day as the essential oil-
CB for the rst group. It is quite likely that aer 3 hours of exposure to essential oils, an eect of
essential oils would still be present during the control-CB. Conducting the control-CB at another
day would generate a confounding eect of day.
Lastly, we should take into account that Galindo (Galindo et al., 2010) armed that eects
of essential oils can vary considerably depending on the dosage. In our study, we used the same
dosage for each oil. Further studies will need to focus on the eects which obtained by diusing
dierent concentrations of essential oils.
Conclusions
ese preliminary results suggest that olfactory enrichment with essential oils can inuence the
aective states and behaviors of shelter dogs. More research is needed to understand the impact of
each individual essential oil and its eect on dog’s welfare, considering possible factors aecting
their inuence, including individual factors or dierent concentrations of the essential oils.
Acknowledgments
We would rstly like to thank AHVMA for having supported this research. anks to Giorgia
Ascheri and the sta of the shelter Shardana (Sardinia, Italy) for their help during data collection.
We appreciate the contribution of Stijn Schoelynck during the data analysis.
Author Contributions
e idea for the paper was conceived by Haverbeke A. and Uccheddu S. e experimental
protocol was designed by Uccheddu S., Haverbeke A. and Mariti C. e data were statistically
analysed by Arnouts H. and Sannen A. and discussed by all authors. e videos were analysed by
Gutierrez Rufo J. e cortisol concentration in the saliva was analysed by Mariti C. and Gazzano
A.. e paper was written by Uccheddu S. and Haverbeke A. and discussed by all authors.
Conicts of Interest
ere could be a potential conict of interest because Haverbeke A. has selected the composition
of the oils of the blend. e funding sponsors had no role in the design of the study; in the
collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision
to publish the results.
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14
Risposte comportamentali e del cortisolo di cani di canile sottoposti ad un “Cognitive bias test”
dopo arricchimento olfattivo con olii essenziali
Stefania Uccheddu1, Chiara Mariti2, Adinda Sannen3, Hilde Vervaecke3, Heidi Arnout3,4,
Jara Gutierrez Rufo2, Angelo Gazzano2, Anouck Haverbeke1
1 Vet Ethology, Leemveldstraat44, 3090 Overijse, Belgium
2 Department of Veterinary Sciences, University of Pisa, Viale delle Piagge 2, 56124 Pisa, Italy
3 Odisee University College, Agro- & Biotechnology, Ethology & Animal Welfare, Hospitaalstraat 23,
9100 Sint-Niklaas, Belgium
4 University of Antwerp, Departmentof Engineering Management, Prinsstraat 13, 2000 Antwerp, Belgium
Sintesi
L’ambiente di canile comporta per gli animali diverse forme di stress a cui i cani devono adattarsi. Recenti ricerche
hanno dimostrato che l’arricchimento con olii essenziali potrebbe essere in grado di modicare lo stato emozionale di
certe specie animali (cani, gatti, animali di zoo..). In questi studi la valutazione del welfare includeva indicatori siologici,
come ad esempio le concentrazioni di corticosteroidi e/o comportamenti correlati allo stress cronico.
L’eetto olfattorio di 9 olii essenziali (Cananga odorata, Cistus ladaniferus, Citrus aurantium, Cupressus sempervirens,
Juniperus communis var. montana, Lavandula angustifolia, Laurus nobilis, Litsea citrata, Pelargonium graveolens) e di una
miscela di questi olii è stato valutato sui risultati di un “Cognitive bias test”, sui livelli di cortisolo e sul comportamento
di 110 cani di canile (n= 10 cani per ogni gruppo).
L’arricchimento olfattivo con la miscela di olii ha ridotto la latenza della scelta dello stimolo ambiguo, indicando un
pregiudizio ottimistico ed un miglioramento del welfare.
I risultati di questo studio suggeriscono che l’arricchimento olfattivo con olii essenziali può avere un eetto specico
sullo stato emozionale e sul comportamento dei cani di canile e potrebbe perciò essere utile nel management di queste
strutture.
Inoltre, poiché non tutti gli olii testati singolarmente si sono dimostrati ecaci, ulteriori ricerche dovrebbero essere
eettuate per comprendere meglio gli eetti dei singoli olii sul cane.
... Also, in animals, the use of aromatherapy as environmental enrichment has been studied in e.g. zoo animals [8][9][10] , kenneled dogs [11][12][13] , kenneled cats [14] , and horses [15][16] . ...
... Olfactory enrichment with a blend of 9 anxiolytic essential oils (Cananga odorata, Cistus ladaniferus, Citrus aurantium, Cupressus sempervirens, Juniperus communis var. montana, Lavandula angustifolia, Laurus nobilis, Litsea citrata, Pelargonium graveolens) resulted in a reduced latency to the ambiguous cue (cognitive bias test), indicating a more optimistic bias [13] . A cognitive bias test in this context refers to the propensity of a subject to show behaviour indicating the anticipation of either relatively positive or relatively negative outcomes in response to affectively ambiguous stimuli [17] . ...
... We tested the same blend of Eos applied in [13] . The aim of this study was to evaluate dog's postures before and after EOs diffusion. ...
Article
Full-text available
The influence of essential oils (EOs) on emotions has been widely described among humans and animals. Several studies have investigated the effects and the actions of EOs on behaviour, mood and perception. In this study, shelter dogs (n=23) were exposed to olfactory stimulation through diffusion of 9 anxiolytic essential oils in one blend (olfactory enrichment) for 8 weeks in order to check long-term effects on behaviour. First, dog's postures have been evaluated in both groups before and after exposure. Secondly, in order to collect the preliminary results on the distance necessary to obtain an effect of EOs, dogs were divided in 2 groups according to the distance from the diffuser. Our results indicate that olfactory enrichment with this blend of EOs is related to less time spent by dogs in high posture. More research is needed to investigate a potential gradual effect of distance and concentration of EOs on dog's welfare.
... Notwithstanding, non-verbal paradigms are the only available tool to assess judgement biases in animals, therefore various protocols have been developed and adapted for several animal species (e.g. rats Harding et al. 2004;Rygula et al. 2013Rygula et al. , 2015Hales et al. 2014Hales et al. , 2016, starlings (Matheson et al. 2008), bees (Bateson et al. 2011), sheep (Doyle et al. 2010;Verbeek et al. 2014b, a), macaques (Bethell et al. 2012), pigs (Douglas et al. 2012), horses (Hintze et al. 2018), calves (Neave et al. 2013)), including the domestic dog, Canis familiaris (Mendl et al. 2010a;Wells et al. 2017;Uccheddu et al. 2018; Barnard et al. 2018;Duranton and Horowitz 2019). Despite the large number of studies that have used JBTs to evaluate the welfare and the affective state in animals, doubts remain about its reliability: results are not always in line with predictions, with some studies even leading to null results or to opposite findings to those expected (Iigaya et al. 2016;Roelofs et al. 2016;Raoult et al. 2017). ...
... Finally, we detected a potentially influence of the presence of a researcher, as highlighted also by others (Müller et al. 2012;Kis et al. 2015;Uccheddu et al. 2018): the median latency to reach the M location was extremely short, potentially because dogs were interested in the researcher who stood behind the bowl placed in M, rather than in the bowl itself. ...
Article
Full-text available
The judgement bias test represents one of the most applied tools to evaluate animals’ optimistic/pessimistic attitude and to infer their emotional and welfare state accordingly. The judgement bias test (JBT) has been used several times with dogs (Canis familiaris), in most cases using a spatial test that evaluates the dog’s attitude towards a bowl placed in ambiguous positions (located between two unambiguous trained positions associated with opposite outcomes). Results are contrasting and methodological and statistical caveats emerged: dogs struggled to learn the association between unambiguous positions and their outcomes, they hardly discriminated between adjacent locations and they might be influenced by researchers. Therefore, we propose a novel paradigm, aimed at easing the learning process and at achieving more reliable measures. Improvements of the novel paradigm are the increased difference between payoffs of trained locations, the reduction of the number of trials and of their length and the removal of the potential influence of researchers. Results showed that 98% of dogs reached the learning criterion and that their learning appeared more stable: dogs behaved differently between the two trained stimuli and the variability of responses towards these stimuli was lower than the one towards ambiguous stimuli. Behavioural analyses confirmed that dogs fully learned outcomes associated with trained stimuli and that they were hesitant towards ambiguous stimuli. Furthermore, dogs managed to successfully discriminate between each pair of adjacent locations. These results suggest that this protocol is a promising tool to assess judgement biases in dogs and to evaluate their affective state.
... ;Binks et al. 2018;Hermiston et al. 2018;Uccheddu et al. 2018;Haverbeke et al. 2019).Graham et al. (2005a) investigated lavender, chamomile, rosemary, peppermint along with a no-odor control, each diffused in front of and behind the dogs' kennels for four hours a day over five consecutive days. Exposure to both lavender and chamomile led to increases in dogs' resting and decreases in movement and ...
Chapter
Dogs experience a variety of stressors within the shelter that could negatively impact their welfare. The use of enrichment interventions that provide social interaction, either with a human or canine; object enrichment; and sensory stimulation (auditory, olfactory, or visual) is necessary for dogs living in animal shelters, along with the assessment of engagement and determination of benefits. There are a wide range of sampling and measurement techniques for monitoring enrichment usage and its behavioral effects, and such efforts are only worthwhile if the data being collected are used. Data‐informed decisions about which enrichment types are provided, on both the shelter‐wide and individual dog levels, must be consistently re‐evaluated based on the current population of dogs and can allow shelters to most usefully employ their resources and best serve the dogs in their care.
... The in uence of human gender on behaviour has been understudied in companion dogs [38,39] and, so far, has never been accounted as a potential confounding factor in eld tests aimed to assess age-related behavioural differences. Finally, cognitive tests designed to measure positive affective states have replicability issues and may not be reliable in ageing dogs, due to the learning required: for example, studies based on the cognitive bias test, a test for mood based on discrimination choices, showed that older dogs may struggle to learn the discrimination and therefore it may not be possible to test them [4,40]. Clinicians still need standardised testing for positive emotions in senior animals. ...
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The prolonged lifespan of companion dogs has resulted in an increased occurrence of behavioural and physical challenges linked to old age. The development of behavioural tests for identifying and monitoring age-related differences has begun. However, standardised testing requires validation. The present study aimed to assess external validity, interobserver reliability, and test-retest reliability of an indoor test battery for the rapid assessment of age-related behavioural differences in dogs. Two experimenters tested young and old dogs on a first occasion and after two weeks. Our results found external validity for two subtests out of six. On both test occasions, old dogs committed more errors than young dogs in a memory test and showed more object avoidance when encountering a novel object. Interobserver reliability and test-retest reliability was high. We conclude that the Memory and Novel object tests are valid and reliable for monitoring age-related memory performance and object neophobic differences in dogs.
... Similar findings have been reported in non-primate species. For example, Uccheddu et al. (2018) behaviors reflecting low-level acute stress or anxiety (Maestripieri et al., 1992) but FGCs reflecting high-level chronic stress (Sapolsky, 2002). Accordingly, self-directed behaviors have been found to increase in anxiety-inducing contexts, such as when animals are given anxiogenic drugs (Schino et al., 1996) or after aggression (Schino, 1998); FGC concentrations have been shown to increase when animals are exposed to high levels of stress, such as in the presence of tourists (Barja et al., 2007) or when exposed to the odor of a predator (Monclús et al., 2006). ...
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Captive breeding is vital for primate conservation, with modern zoos serving a crucial role in breeding populations of threatened species and educating the general public. However, captive populations can experience welfare issues that may also undermine their reproductive success. To enhance the wellbeing of endangered zoo primates, we conducted a study to assess the effects of a new scent enrichment program on captive red-ruffed lemurs (Varecia rubra), black howler monkeys (Alouatta caraya), siamangs (Symphalangus syndactylus), lar gibbons (Hylobates lar) and orangutans (Pongo pygmaeus pygmaeus). We combined behavioral observations and fecal endocrinology analyses to evaluate the effects of a series of essential oils (benzoin, lavender, lemongrass) on five captive troops (N = 19) housed at Dudley Zoo & Castle and Twycross Zoo (UK). We recorded observations of natural species-specific and abnormal stress-related behaviors for 480 h using instantaneous scan sampling. We collected 189 fecal samples and measured the fecal cortisol concentrations using radioimmunoassay. We found a significant effect of the scent enrichment on behaviors, with red-ruffed lemurs and black howler monkeys reducing their social interactions, as well as red-ruffed lemurs and lar gibbons decreasing their stress-related behaviors after they were exposed to the series of essential oils. We also found that red-ruffed lemurs displayed a significant increase in fecal glucocorticoids following exposure to essential oils. Our contradictory findings suggest that the effects of this series of essential oils may change depending on the species-specific social lives and olfactory repertoires of primates. In conclusion, we cannot recommend using these essential oils widely with zoo primates without additional evaluation.
... Methods used to generate a negative affective state are: unpredictable, aversive events [47,84,85], removal of environmental enrichment [19,51], warning cry of a conspecific and frightened eyes of a conspecific [86], shearing [87], social isolation [88][89][90], bright illumination [83], shaking [91], defeat against a rival [74] and neuromodulators that mimic stress [73]. Practices applied to induce a positive affective state are: environmental enrichment [48,50,92], successful foraging [93], olfactory enrichment with essential oils [94], administration of antidepressants [73] and the ending of an aversive event [85]. Following this manipulation, intermediate stimuli between the two learnt stimuli are presented, and it is evaluated whether the animals classify the ambiguous cues as a positive or negative stimulus. ...
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he evaluation of an animal’s emotional state and its well-being poses a challenge to current research. Direct measurement of emotion is inaccessible, so assessment relies on measures of affective states that are predominantly influenced by emotions and important indicators. Studies that used physiological, cognitive or behavioural measurements have shown the assessment of cognitive and judgement bias to be a suitable method to gain insight into the affective state and welfare of animals. Although these method does not always allow a clear interpretation of the results with respect to the affective state, animals in discrimination tasks generally show a pessimistic judgement bias when they are in a negative affective state. This tendency agrees with cognitive theories stating that an individual’s assessment of a situation gives information about the involved emotions. Therefore, the analysis of cognitive bias may allow assessing the influence of various factors on an animal’s affective state and, if appropriate, creating conditions that result in a positive affective state for animals in husbandry. As it is our aim to highlight useful methods for determining the welfare of animals in husbandry, we here review underlying theories of cognition and affect, evaluate their relevancy to animal behaviour and give an overview of applicable methods and their outcomes.
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The prolonged lifespan of companion dogs has resulted in increased behavioural and physical challenges linked to old age. The development of behavioural tests to identify and monitor age‑related differences has begun. However, standardised testing requires validation. The present study aimed to assess external validity, interobserver reliability, and test–retest reliability of an indoor test battery for the rapid assessment of age‑related behavioural differences in dogs. Two experimenters tested young dogs (N = 20, mean age ± SD = 2.7 ± 0.4 years) and old dogs (N = 18, mean age ± SD = 11.8 ± 1.3 years) in the test battery once and then again after two weeks. Our results found external validity for two subtests out of six. On both test occasions, old dogs committed more errors than young dogs in a memory subtest and showed more object avoidance when encountering a novel object. Interobserver reliability and test–retest reliability was high. We conclude that the Memory and Novel object subtests are valid and reliable for monitoring age‑related memory performance and object neophobic differences in dogs.
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Laboratory animals are raised in a fixed space during the study period and are environmentally bound. Laboratory animal may be under stress on the constrained environment, which changes physiological indicators, affecting the reproducibility and accuracy of animal study. Therefore, reducing animal stress by providing proper breeding environment and environmental enrichment can be the basis for animal study. In this study, the stress level was assessed according to the mouse breeding environment. According to the results of the experiment, it was determined that the individual ventilation cage had less cortisol concentration in serum and body weight increased in the individual ventilation cage than individual isolated cage, when providing environmental enrichment rather than group breeding or not providing environmental enrichment. The results will provide appropriate guidelines for laboratory animal welfare.
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Previous research on humans and animals suggests that the analysis of sleep patterns may reliably inform us about welfare status, but little research of this kind has been carried out for non-human animals in an applied context. This study explored the use of sleep and resting behaviour as indicators of welfare by describing the activity patterns of dogs (Canis lupus familiaris) housed in rescue shelters, and comparing their sleep patterns to other behavioural and cognitive measures of welfare. Sleep and activity patterns were observed over five non-consecutive days in a population of 15 dogs. Subsequently, the characteristics of sleep and resting behaviour were described and the impact of activity on patterns of sleep and resting behaviour analysed. Shelter dogs slept for 2.8% of the day, 14.3% less than previously reported and experienced less sleep fragmentation at night (32 sleep bouts). There were no statistically significant relationships between behaviours exhibited during the day and sleep behaviour. A higher proportion of daytime resting behaviour was significantly associated with a positive judgement bias, less repetitive behaviour and increased time spent coded as ‘relaxed’ across days by shelter staff. These results suggest that, in the context of a busy shelter environment, the ability to rest more during the day could be a sign of improved welfare. Considering the non-linear relationship between sleep and welfare in humans, the relationship between sleep and behavioural indicators of welfare, including judgement bias, in shelter dogs may be more complex than this study could detect.
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Domestic dogs can be housed in a variety of confined conditions, including kennels, shelters and laboratories. Concern over the well-being of dogs housed in human care has prompted much research in recent years into the enrichment of environments for kennelled dogs. This paper highlights the findings and recommendations arising from this work. Two types of general enrichment method are discussed, namely animate (i.e. enrichment through the provision of social contacts with conspecifics and humans) and inanimate (i.e. enrichment through the provision of toys, cage furniture, auditory and olfactory stimulation). The benefits and, where relevant, possible disadvantages, to these various types of enrichment method are highlighted throughout.