Use of behavioural and physiological responses for scoring sound sensitivity in dogs
Use of behavioural and physiological responses for scoring sound sensitivity in dogs
Carla Caroline Franzini de Souza 0 1
Daniel Penteado Martins Dias 1
Raquel Nascimento de Souza 0 1
Magda Alves de Medeiros 0 1
0 Department of Physiological Sciences, Institute of Biological Sciences and Healthy, Federal Rural University of Rio de Janeiro , SeropeÂdica, RJ , Brazil , 2 Graduate Program in Veterinary Medicine, Federal Rural University of Rio de Janeiro , SeropeÂdica, RJ , Brazil , 3 Graduate Program in Physiological Sciences, Federal Rural University of Rio de Janeiro , SeropeÂdica, RJ , Brazil , 4 Barão de MauaÂ University Center , Ribeirão Preto, SP , Brazil
1 Editor: Carlos E. AmbroÂsio, Faculty of Animal Sciences and Food Engineering, University of São Paulo , BRAZIL
Sound sensitive dogs have exaggerated responses to sound stimuli that can negatively impact the welfare of the dog. Behavioural reactions combined with the response to sound involve a marked autonomic imbalance towards sympathetic predominance and release of cortisol. The purpose of the present study was to evaluate, in the laboratory, the cardiac autonomic modulation using heart rate variability (HRV) analysis, serum cortisol levels and behavioural parameters in response to sounds of fireworks in dogs with a history of sensitivity to fireworks. Based on these data, and combining qualitative measures and categorical measures, we propose one short and one full index of sound sensitivity in dogs. Six privately owned dogs with no history and another twelve dogs with a history of sound sensitivity to fireworks were used. The sound stimulus consisted of a standardised recording of fireworks (180-seconds long) with a peak intensity of 103±104 dB. The cardiac intervals were recorded using a frequency meter (Polar® RS800CX model) to evaluate the HRV, and the acquired data were processed using CardioSeries 2.4.1 software. Twenty-one behavioural parameters were analysed quantitatively by time, frequency or categorically by scores and were grouped in behavioural categories of arousal, fear, relaxation and ªotherº. Sound sensitive dogs had exacerbated autonomic responses to the sound stimulus in the laboratory compared to non-sensitive dogs, with higher LF/HF ratios suggesting autonomic imbalance towards sympathetic predominance, but the cortisol levels were similar between the sensitive and non-sensitive dogs. Sound sensitive dogs showed pronounced responses for the parameters: alert and attention, search sound, startle, trembling, hiding, run away and less intense responses for the parameters rest and wink/sleep. Furthermore, the behavioural categories of arousal, fear, relaxation (lack of) and LF/HF were correlated to the caregiver's perception of the sound sensitivity of the dogs. Not only the short index for sound sensitivity (behavioural categories arousal, fear and relaxation, and LF/HF ratio) but also the full index for sound sensitivity (all behavioural categories, LF/HF and cortisol levels) was highly
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: The present study was funded by Rio de
Janeiro Research Agency (FAPERJ), award
number: E-26/111.478/2014 and CCFS received
fellowship from National Counsel of Technological
and Scientific Development (CNPq). The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
Competing interests: The authors have declared
that no competing interests exist.
correlated to sound fear response at home. These indexes can contribute to the
development of strategies to treat sound sensitive dogs.
Loud and sudden sounds can induce a wide range of fearful behaviours in dogs, ranging from
evidence of minor anxiety to quite marked behaviours. Although the fear response is a natural
and self-protecting behaviour, in sound sensitive fearful dogs these responses are exaggerated
and inappropriate and negatively impact the welfare of the dog [
]. Sound sensitive dogs may
show a variety of signs such as restlessness, panting, increased startle response, trembling,
hiding, arched posture, salivation, destructiveness, defecation, vocalisation, self-mutilation,
among others [
]. Sound sensitivity in dogs is a widespread condition [
] and is frequently
associated to other behavioural problems and might cause extensive damage to property and
could be harmful to the dog itself, to people and other dogs [
Besides the behavioural reaction, sound stimuli may cause marked autonomic imbalance
towards sympathetic predominance and conspicuous cortisol release [
]. The exaggerate
autonomic activation, and cortisol release can ultimately lead to reduced immunity and increased
risk for conditions like hypertension, heart disease, fatigue and insomnia [
]. The diagnosis
of sound sensitivity in dogs can be quite inaccurate since it relies on owner perceptions
regarding the reactions of the dog when facing a noisy situation at home. Therefore, precise
measurements of physiological parameters should be considered to develop reliable methods of
diagnosis and evaluation of this condition (i.e. high sensitivity to sounds), and the assessment
of the efficacy of therapeutic approaches .
Previous studies have analysed the behavioural response to fireworks or thunderstorm
stimuli in sound sensitivity dogs in a domestic environment and assigned scores to several
behaviours such as trembling, vocalisation, salivating, destruction, search for people and running
around [9±11]. On the other hand, others have analysed dogs that are non-sensitive to sounds,
regarding behavioural responses alone [
] or behavioural and physiological responses
combined [14±16]. However, to the best of our knowledge, no study has coupled behavioural,
autonomic and endocrine aspects together to propose a single index for sound sensitivity in dogs.
The purpose of the present study was to evaluate, in a laboratory environment, the cardiac
autonomic modulation using heart rate variability (HRV) analysis, serum cortisol levels and
several behavioural parameters in response to recorded sounds of fireworks in dogs with a
history of exaggerated fear of fireworks. Then by combining the qualitative measures of HRV and
cortisol data, with the qualitative and categorical measures of behavioural data, we propose
two sound sensitivity indexes (one short and one full) for dogs.
All procedures were assessed and approved by the ethics committee on animal use of the
Institute of Biological Healthy Sciences of the Federal Rural University of Rio de Janeiro (UFRRJ),
RJ, Brazil/ CEUA-ICBS, protocol nÊ 23083.4796/2014-14 and COMEP-UFRRJ nÊ 23083.
Dogs of both sexes, 2 to 6 years old, weighing from 10 to 30 kg and in good health were
recruited through an advertisement placed at the Veterinary Hospital for Small Animals and
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other facilities of the UFRRJ (SeropeÂdica, RJ, Brazil). The ad was also released onto social
media linked to the university community. Animals with signs or history of neurological
problems and aggression were excluded from the study. The recruitment period lasted from
October 2014 until February 2015.
The owners filled out a questionnaire regarding the daily routine of their dogs (S1 and S2
Appendices). and their reactions to sounds (adapted from [
]) (S3 and S4 Appendices).
The perception of the caregiver to responses such as panting, trembling, hiding, search for
caregiver, restlessness, vocalisation, destructive attitude, excessive salivation, inappropriate
elimination and self-trauma were recorded in scores ranging from 0 (absence of the behaviour)
to 5 (frequently observed or intense behaviour). Dogs were considered sensitive to sounds
when, during fireworks stimulus, scores 3 in at least three behavioural parameters were seen
or when one behavioural parameter alone reached the maximum score (5). Dogs were also
considered sensitive to sounds when their behaviours put their health and physical integrity at
risk or when the reaction to sound was very frequent (observed more than 70% of the
exposures to sound stimuli). The sum of the scores of each response (panting, trembling, hiding,
search for caregiver, restlessness, vocalisation, destruction, salivation, elimination,
auto-mutilation) resulted in a score which relies on the perception of the caregiver to the reactions of the
dogs to sounds. Considering the importance of dog-human interactions and the previous
history of the dog in the behavioural response, the caregivers also answered a questionnaire about
their home environment and the interaction of the dogs with humans and other dogs.
On the day of the sound test, all individual experimental procedures started at 0900h. First, with
the dogs (n = 6, non-sensitive dogs; n = 12, sound sensitive dogs) at their respective houses, a
non-invasive elastic band containing a contact electrode (Polar RS800cx, Polar1, Kempele,
Finland) was trapped to the chest of the dogs in order to have heart rate (HR) sampled on a
beat-by-beat basis. Then, the dogs were left undisturbed for 10 minutes for baseline
measurements of HR (Basal1) and were subjected to baseline collection of blood samples (SB1). Next,
the dogs were taken to the test room by car (a 10 to 15-minute-trip) in their transport boxes.
At the test room, the dogs were left to rest undisturbed for 30 minutes, and an additional
baseline measurement of HR was made (Basal2), followed by the second blood sample
collection (SB2). Animals were then placed 1 meter away from the sound source and exposed to a
180-second-long recording of fireworks acquired from the website http://www.sound-effect.
com. The sound level was adjusted to 103±104 dB and tested with a decibel meter (Sound
Level Meters, Model 732A, BK Precision1, Yorba Linda, CA, USA).
Other blood samples were collected 15 (S15), 30 (S30) and 60 (S60) minutes after the end of
the sound stimulus. To assess behavioural responses, dogs were videotaped during the whole
sound stimulus period and the 5 minutes following the sound stimulus. Finally, the HR
monitors were removed, and the dogs were returned to their houses (Fig 1).
A detailed description of the experimental procedure and laboratory environment was
described elsewhere [
Cardiac interval variability analysis
The procedures for the cardiac interval variability (CIV) analysis in response to sound in dogs
was previously described [
]. Briefly, HR data were continuously acquired through the cardiac
monitor (RS 800cx, Polar, Kempele, Finland) and the data were transmitted from the HR
monitor to a custom computer software (Polar Pro Trainer v5, Polar, Kempele, Finland) via an
infrared interface. The time series of cardiac interval values from the moments Basal 1
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Fig 1. Experimental protocol.
(300-seconds-long; at the house), Basal 2 (300-seconds-long; at the test room), Sound
(180-seconds-long; during sound stimulus) and After-sound (300-seconds-long; 30 minutes after the
end of sound stimulus) were analysed.
CIV analysis in the time-frequency domain was performed using the freely-available
computer software (CardioSeries v2.4.1Ðhttp://www.danielpenteado.com). For the time domain
analysis, the square root of the mean of the sum of the squares of differences between adjacent
cardiac intervals (RMSSD) and the ratio between the standard deviation of cardiac interval
values (SDNN) and the RMSSD (SDNN/RMSSD) were calculated following processing of original
beat-by-beat series with cardiac interval values [
]. For further frequency domain analysis,
the beat-by-beat time series of cardiac interval values were converted to data points every 250
ms using cubic spline interpolation (4 Hz) and divided into half-overlapping sequential sets of
512 data points which were detrended by subtracting the linear trend (obtained by linear
regression calculation) from the data points. Next, a well-experienced researcher visually inspected
the data points (i.e. cardiac interval values) searching for transients that could affect the power
spectral density (PSD) calculation. To confirm that the visual inspection of the cardiac interval
time series was adequately performed, a Hanning window was used to attenuate the side effects,
and the spectrum of the segments was calculated using a direct fast Fourier Transform (FFT)
algorithm, followed by visual inspection for abnormal spectra. Noisy segments were not taken
into consideration for analysis. The spectra were integrated into the low-frequency band (LF,
0.04±0.15 Hz) and high-frequency band (HF, 0.15±0.40 Hz). The normalised values were
obtained by calculating the percentage of LF and HF power with regard to the total power of the
spectrum minus the very low-frequency band (VLF, <0.04Hz) power [
]. The LF/HF ratio
was calculated and considered as an index of the sympathovagal balance .
Blood samples, cortisol analysis
Serum cortisol concentrations were determined from blood serum by a double antibody
radioimmunoassay method using a commercial kit (RD Coated Tube Cortisol I125 RIA, Costa
Mesa, CA, USA), with an assay sensitivity of 0.17 μg/dL and an intraassay coefficient of
variation of 6.59%. The blood samples were collected from the cephalic vein using sterilised,
intravenous, disposable needles 22G and sample tubes. The tubes were centrifuged for 10 minutes
at 3200 rpm to obtain the serum which was stored in plastic containers at -20ÊC.
The behavioural responses of the dogs were analysed from videotape recordings (Digital
movie camera Sanyo C40, Moriguchi, Osaka, Japan) at the Baseline (2 min), Sound Stimulus
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(2.5 min) and After-Sound (2 min) moments. Video recordings of each test session were
analysed for behavioural signs without sound by a single trained observer (CCFZ) who was
blinded to the dog group. Twenty-one behaviours were measured according to the nature of
each response, by time (in seconds), frequency (number of occurrences) or by intensity (six
points scale: 0 = no signs, 1 = mild and occasional signs; 2 = occasional and moderate/some of
the time and mild signs; 3 = most of the times and mild/some of the time and modest signs;
4 = some of the time and severe/ most of the time and moderate signs; 5 = most of the time
and intense signs) [
]. Table 1 details the behavioural parameters assessed.
The behavioural parameters were assigned to four general categories of reaction frequently
associated with response to sound: a) behaviours of arousal, including Alert and Attention,
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Panting, Ambulation, Search sound, Startle; b) fear behaviours, including Trembling,
Whining, Tail between legs; Arched posture; Running around, Hiding and Freezing; c) behaviours
related to relaxation and "well-being" that is reduced during stress, including Rest, Wag tail,
and Blink sleep; d) other behaviours that are not actively associated with any of the previous
categories: Yawn, Bark, Growl, Elimination, Licking lips, Destruction. The grouping of
behavioural parameters into the categories was related to the primary neurobiological or
psychobiological meaning of each behavioural parameter.
Building the full and short indexes for sound sensitivity
To create a sound sensitivity index, the quantitative and qualitative measures were combined
]. Initially, the quantitative measures of LF/HF ratio (at the moment of the sound) and
cortisol levels (15 min after the sound stimulus) and the quantitative measures of each
behavioural reaction (at the moment of the sound) was transformed into a six-point scale
proportional to the magnitude of the response. The behavioural parameters measured as the duration
of the behaviour were converted into a percentage scale of time spent in that behaviour during
the period analysed (180 s). As the parameters measured by frequency had a more significant
variation, specific scales were attributed to each parameter. The parameters examined by
intensity were already in a six-point scale (see Table 2 for details).
Next, the correlations between the perception of the caregiver of the sound sensitivity of
his/her dog and the scores of the behavioural categories arousal, fear, relaxation and "other"
(average of the behavioural scores included in each the category), LF/HF and cortisol were
calculated. As all variables are directly proportional to the increase of the response intensity, the
category relaxation was transformed into a lack of relaxation, by subtracting five from the
average score of the relaxation category. Afterwards, two indexes of sound sensitivity were created:
the full index for sound sensitivity, considering the average score of all behavioural categories,
LF/HF and cortisol and the short index for sound sensitivity considering only scores strongly
correlated with the caregiver's perception of the dogs' sound sensitivity. Table 2 shows how the
quantitative data were transformed into categorical data to create the indexes of sound
Statistical analyses were performed with SPSS Version 21 (IBM SPSS Inc., Chicago, IL, USA)
using a two-way-random-model (confidence-interval 95%). Graphs were built using
GraphPad Prism 5.0 (GraphPad Software). Data are expressed as mean ± error.
To compare the response to sound stimulus between non-sensitive and sound sensitive
dogs, data of HRV, cortisol and behavioural parameters measured by time and frequency were
analysed by two-way ANOVA for the repeated-measures approach followed by Bonferroni's
multiple comparisons (factors time, group (non-sensitive and sound-sensitive), sex, age,
weight and neutered) and repeated measures for the factor time: Basal 1, Basal 2, Sound and
After Sound for HRV parameters; and Basal 1, Basal 2, S15 and S30 for cortisol, basal, sound
and after-sound for behaviours). Due to the significant difference between the groups in the
basal levels, for cortisol, HR, RMSSD and SDNN the data baseline values (Basal 1) were used
as a covariate. Behavioural parameters measured by intensity were analysed by non-parametric
The average score of the behavioural categories, LF/HF and cortisol, were correlated with
the general score of sound fear at home (owner's perception of the dog's reaction to fireworks
at home) by the Spearman test.
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LF/HF at sound
Alert and attention
Tail between legs
Score of LF/HF
Score of cortisol
Average Score of Arousal
Average Score of fear
Average score of relaxation = lack of relaxationa
An average score of other Behaviours
Parameters analysed quantitatively were converted to a six-point scale.
a Relaxation was transformed into a lack of relaxation, subtracting five from the average score of the relaxation category.
Thirty-six owners answered the advertisement, and 12 companion dogs with an exaggerated
fear of sounds and six dogs with no history of sensitivity to sound were included in the present
study. The non-sensitive dogs, 3.33±1.03 years old, consisted of three males and two
non-neutered females and one neutered female, weighing 20.67±7.42Kg. The sound sensitivity group
included five non-neutered and four neutered females and one non-neutered and two
neutered males, 3.33±1.37 years old and weighing 17.17±7.45Kg. No differences were found
between sound sensitive and non-sensitive dogs regarding sex, age, weight and castration. The
perception of the caregiver of the dog for sound sensitivity varied between 0 and 2, with an
average value of 0.8±0.9 in the non-sensitive dogs and 15 and 28, with an average value of
21.62±3.98 in the sound sensitive dogs (Supplementary data). All animals selected for the
study had reasonably similar management, feeding and family environment. The animals were
fed on commercial feed and lived in homes with unrestricted grounds and slept outdoors in
their own space with adequate shelter. All dogs included in the current study were left alone at
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home during specific periods of the day and most had the company of other animals (S5
The repeated measures ANOVA detected that the sound of thunder promoted an increase
in the power of the LF band of the cardiac interval spectrum and in the LF/HF ratio and a
decrease in the power of the HF band of the cardiac interval spectrum, (LF/HF: Wilks'
lambda = 0.173, F[
] = 22.234, p < 0.0001; LF: Wilks' lambda = 0.891, F[
] = 37.965,
p < 0.0001, HF: Wilks' lambda = 0.891, F[
] = 37.962, p < 0.0001). The sound stimulus did
not significantly change the HR, RMSSD, SDNN and SDNN/RMSSD values. Independent of
the time point (i.e. Basal 1, Basal 2, Sound, and After-Sound) sound sensitive dogs showed
higher HR (Group factor: F (1,15) = 6.594, p = 0.021). The fireworks sensitive dogs showed
higher LF/HF during the sound stimulus (Bonferroni test: p = 0.035) (Fig 2).
Repeated measures ANCOVA also compared the effect `animal group’ on cortisol levels at
different time points after the sound stimulus, using Basal 1 as a covariate to account for any
potential initial differences. The sound stimulus increased the cortisol levels (Wilks' lambda =
0.803, F (4,10) = 10.167, p = 0.002), and although the cortisol values showed an apparent
difference between fireworks sensitive and non-sensitive dogs, no statistical difference between
sensitive and non-sensitive dogs (no effect of group and group x time) was observed (Fig 3).
Table 3 shows the behavioural parameters in response to fireworks of the sound sensitive
and non-sensitive dogs. The sound stimulus increased, regardless of the group, in the
behaviours: Alert and Attention (Wilks' lambda = 0.726, F(2,15) = 19.862, p = 0.001); Panting
(Wilks' lambda = 0.349, F(2,14) = 3.751, p = 0.049); Search sound (Wilks' lambda = 0.549,
F(2,14) = 9.145, p = 0.003); Startle (Kruskal Wallis test, p< 0.0001); Trembling (Wilks'
lambda = 0.549, F(2,14) = 6.161, p = 0.011); Hiding (Wilks' lambda = 0.663, F(2,14) = 3.816,
p = 0.046) and run away (Wilks' lambda = 0.640, F(2,14) = 4.211, p = 0.035). The behavioural
analysis shows that the sound sensitive dogs had more intense responses to sound in the
parameters Alert and attention (p = 0.001), Search sound (p = 0.036), Trembling (p = 0.009),
Hiding (p = 0.040) and run away (p = 0.039) and less intense response in the parameters Rest
(p = 0.0001) and Wink/sleep (p = 0.0001). Other behavioural parameters were not statistically
different between sound sensitive and non-sensitive dogs.
The Spearman's test shows a significant correlation between the perception of the caregiver
of the dog's sound sensitivity and the behavioural categories arousal (r2 = 0.57, p = 0.002), fear
(r2 = 0.771, p = 0.002), lack of relaxation (r2 = 0.716, p = 0.002) and LF/HF (r2 = 0.523,
p = 0.03). Both full index for sound sensitivity (when considering all behavioural categories,
LF/HF and cortisol scores) and short index for sound sensitivity (considering only correlated
categories: arousal, fear, lack of relaxation and LF/HF score) were correlated to sound fear
responses at home (r2 = 0.750, p = 0.001 and r2 = 0.78, p<0.0001; respectively) (Fig 4).
Our findings indicate that sound sensitive dogs had exacerbated autonomic and behavioural
responses to the sound stimulus in the laboratory compared to non-sensitive dogs. Sound
sensitive dogs had a more pronounced increase of LF/HF ratios that suggests autonomic
imbalance towards sympathetic predominance and more intense response in the behavioural
parameters related to arousal and fear and less intense response in behaviours related to
relaxation. Although the sound-induced cortisol levels increased in both groups, there were no
significant differences between sensitive and non-sensitive dogs.
The present study analysed changes in behavioural responses, serum cortisol levels and
HRV parameters in dogs subjected to sound stimulus in a laboratory setting. Although there
are differences between playing a recorded sound and real noisy situation at home, this
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Fig 2. Effect of fireworks sounds on the cardiac interval variability, examined through frequency domain analysis, and heart rate in sound sensitive dogs
and non-sensitive dogs. The ratio between the power of the low and high frequency bands of the cardiac interval spectrum (LF/HF, Panel A), power of the LF
9 / 18
(Panel B) and HF (HF, Panel C) bands, heart rate (HR, Panel D), the square root of the mean of the sum of the squares of differences between adjacent cardiac
intervals (RMSSD, Panel E), standard deviation of cardiac interval values (SDNN, Panel F) and the ratio between the RMSSD and SDNN (SDNN/RMSSD, Panel
G). Data obtained at Basal 1 (dogs at the house), Basal 2 (dogs in the test room), Sound (during the acoustic stimulus) and After-Sound (30 min after the end of
the sound). P<0.05 compared to non-sensitive dogs. Data are shown as the mean ± standard error of the mean.
approach has been used in several studies to categorize the response of dogs to these sounds
since it allows a more reliable analysis of the response of individual dogs without the
confounding factors of home context [6, 10±12, 24±28]. Laboratory testing can be advantageous to
analyse physiological and ethological measures to study the progression and nature of the
sound sensitivity in dogs and to test treatment strategies.
Besides the behavioural evaluation, the present study carried out an HRV analysis which
suggested that sound sensitive dogs had an exacerbated autonomic response. Changes in
several different HRV parameters have been related to physical, pathological or emotional stress
]. In healthy dogs, HRV analysis is useful to estimate the emotional state of the animal.
An increase in HR and LF/HF ratio are associated with emotional arousal [
] with positive
or negative valence. The measures of HRV have also been used to address the effect of different
types of music on stress levels of sheltered dogs and to evaluate the impact of human-dog
]. The analysis of HRV has been used to measure the response to sound
stimulation in non-sensitive dogs. A previous study by our group showed that thunder sound
increases the power of the LF band of the cardiac interval spectrum, the LF/HF ratio and the
HR, and decreases the power of the HF band of the cardiac interval spectrum [
]. In the
present study, among all HRV parameters analysed, only the ratio LF/HF was different between
sound sensitive and non-sensitive dogs at the moment of the sound (Fig 2, panel A). Wormald
and colleagues showed that dogs affected by anxiety-related behavioural problems had reduced
HRV during manual restraint when compared to unaffected dogs: with a lower standard
deviation of RR intervals (SDNN), high frequency (HF) spectrum and low frequency (LF) spectrum
power. Unfortunately, there is no discussion about the LF/HF ratio in Wormald's paper . In
recent years, the LF/HF ratio has received censure as a measure of cognitive and physical
aspects of stress, mainly because some authors believe that the sympathovagal balance cannot
be quantified by a single number and the LF/HF ªassumes a simplistic linear relationship
between the activity of the nervous systems and the frequency bands" [
]. Although we
Fig 3. Changes in serum cortisol in response to the fireworks sounds in fireworks sensitive dogs and non-sensitive
dogs. Data obtained from Basal 1 (dogs at the house), Basal 2 (dogs in the test room), 15, 30 and 60 minutes after the
end of the fireworks sound. Data are shown as media ± error.
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indicates a difference between sound sensitive and non-sensitive dogs at the moment of sound
# indicates the parameters with significant effect of sound (effect time independently of the group).
agree with this criticism, in the present study, the ratio LF/HF was used combined with
behavioural and endocrine measures, which provides a more comprehensive view of the stress
response. In other HRV parameters, sound sensitive dogs presented, independently of the
sound stimulus (and in basal condition) higher HR than non-sensitive dogs and no statistical
difference in SDNN and RMSSD parameters. However, there are controversies about the
meaning of the RMSSD and SDNN during stress and in the basal situation, since some studies
reported a decrease in RMSSD and HF for a more favourable valence of the emotional state
], while another study related a reduction in RMSSD in a negative situation and a
decrease in SDNN associated with a positive condition . Furthermore, although sound
sensitive dogs showed a tendency to present higher SDNN/RMSSD during sound than
non-sensitive, the difference was not statistically significant. Thus, the ratio LF/HF was the most
consistent HRV parameter to show discrepancies between sound sensitive and non-sensitive
dogs during the sound stimulus applied here.
Differently, from HRV, cortisol analyses have been used mainly as a tool to validate stress
models. The analysis of cortisol has not been used as a sensible tool for comparing the
magnitudes of different types of stress or analysing the effect of anti-stress strategies. While some
studies have shown an apparent increase of cortisol levels in response to sound stress [
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Fig 4. Indexes for sound sensitivity in dogs. Correlation between the perception of the caregiver of the dog's sound sensitivity and the
full index for sound sensitivity (when considering all behavioural categories, LF/HF, cortisol scores) and short index for sound
sensitivity (considering behavioural categories of arousal, fear, lack of relaxation and LF/HF score). Data of all animals see S1 Dataset.
other studies have failed to show any increase  or this elevation was not observed in all the
]. In the present study, although the sound increased the cortisol
levels, there was no statistical difference between sound sensitive and non-sensitive dogs. The
lack of difference between the groups of dogs, at least in part, was due to the substantial
individual variation at basal levels, since the basal levels were used as a covariate in the statistical
analysis. Furthermore, the limited number of animals also contributed to this result. We
expected that the sound sensitive dogs would have a higher cortisol response. Dreschel and
colleagues tested the effect of thunder sound in thunder-anxious dogs with their caregivers at
home. The sound stimulus significantly increased the cortisol levels when compared to a
situation of when a non-sound stimulus was applied . Another important point about cortisol
levels is that the results do not indicate a positive or negative valence of a reaction to the
environmental stimulus, and a range of other measures are necessary to evaluate emotional distress
Another aspect that should be addressed is the stressor effect of blood collection. It is well
known that venous blood collection can produce changes in the cortisol levels, in behavioural
and HRV parameters. In the present study, the first blood collection was performed with the
animals in their respective houses. As the cortisol peak in dogs is 15 to 30 min after the
stimulus, this first collection (Basal 1) was not influenced by venipuncture. The chest strip for RR
interval data acquisition was placed before venipuncture. Consequently, HRV data assessed at
basal 1 moment was not affected by blood collection. The second blood collection was
performed with the animals in the experimental room (Basal 2). The cortisol levels at basal 2
moment had a slight increase in relation to basal 1, but this increase, in addition to not being
statistically significant, cannot be solely associated with venipuncture, but also with other
stimuli such as transport. The LF/HF and other HRV parameters at basal 2 moment, were not
different from basal one 1 either. Thus, even recognising the blood collection as a stressor, the
venipuncture did not significantly influence the parameters studied.
The sound stimulus can produce a clear behavioural response in dogs, even in non-sensitive
dogs as alert and attention, panting, search sound, startle, trembling and hiding. Sound
sensitive dogs have shown a more pronounced response to the parameters alert and attention,
search sound, trembling, hiding and less intense response in the parameters rest and wink/
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sleep. Some studies have assessed several behavioural parameters in beagles or in
non-soundsensitive dogs submitted to a sound-stress model in the laboratory [6, 12±16]. Our previous
study showed that the thunderstorm stimulus could induce reactions of vigilance (alert and
attention), trembling, hiding and restlessness (ambulation) in laboratory dogs and domestic
dogs with no history of sensitivity to sounds [
]. Few studies have addressed the effect of noise
recordings in sound sensitivity dogs. Dreschel and Granger (2005) compared the impact of
thunder sound with no stimulation in thunder-anxious dogs at their home environment and
observed a higher occurrence of the parameters salivation, vocalisation, hiding, panting,
trembling and interaction with the owner [
]. Therefore, behavioural analysis can be considered
very complex in both experimental and clinical settings since there is a vast individual
difference in the behavioural responses. Even in sound sensitive dogs, the behavioural response can
be variable, with some dogs showing signs of arousal and little signs of fear, while others
respond with noticeable signs of fear and scarce signs of arousal. Therefore, the behavioural
analysis still involves the measurement of a large number of behavioural variables.
Scoring autonomic, endocrine and behavioural parameters to build an index of sound sensitivity in dogs
Previous studies have used scores to classify the behaviour in response to a sound stimulus. In
the study of Dreschel and colleagues (2005) with sound sensitive dogs, reactions such as
salivation, vocalization, hiding, pacing, panting, remaining close to the owner, and trembling were
graded on a scale of 1±5 based on the severity or amount observed during playing a
thunderstorm recording at home (1, small amount / not severe, to 5, extensive amount, very serious).
The dog's behavioural score was calculated from the sum of the scores for whining, hiding,
pacing, panting, remaining close to the owner [
]. Landsberg and colleagues (2015), playing
thunderstorm recordings for beagles created a global score, based on the classification of the
behavioural response in: positive score, with behaviours that increase in response to sound
(startle, scan (orient), bolt; aimless, repetitive or stereotypic pacing, running, or circling;
digging, climbing, jumping, barking), and negative score with behaviours associated with
suppression of activity and triggering of an autonomic response (freeze-against wall-at door;
crouch (cower), tail between legs, ears back and pant, shake (tremble), alert/tense/vigilant,
salivate, yawn, lick lip, lift foreleg, whine) [
]. Other studies have also grouped the behaviour in
active and inactive anxiety signs and created a global score, where active anxiety-associated
behaviours included startling; bolting; vigilance; scanning; and active responses, such as
pacing, aimless activity, stereotypic circling etc.; and inactive anxiety-associated behaviours
included decreased activity, such as freezing; positioning in corners, against the wall, or at
door; lowered body postures, such as crouching, tail tucking, and ears back; and autonomic/
conflict behaviours, such as panting, shaking, salivating, yawning, licking lips, or elimination
In the present study, twenty-one behaviours were grouped into four categories (arousal,
fear and lack of relaxation), trying to relate each behaviour to its
neurobiological/psychobiological meaning. The arousal category included active behaviours frequently associated to an
aversive, novel or suddenly situation, without being specifically related to fear, like the
behaviours alert and attention, panting, ambulation, search sound, startle. The fear behaviour
category included classical behaviours associated to fear and were grouped as passive behaviours
such as trembling, tail between legs, arched posture and freezing and as active behaviours such
as running around, hiding and whining. In the relaxation category, passive behaviours were
related to sleepiness and repose, such as rest and blink sleep and behaviours related to
happiness in dogs as wagtail. These behaviours are typically reduced in a stress situation. Other
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behaviours that are frequently described in sound sensitive dogs such as yawn, bark, growl,
elimination, licking lips and destruction were grouped as "others" since they are not strongly
associated with any of the previous categories. The classification of the behaviours into
categories has a limitation since dogs have large behavioural repertories and can learn to express
specific behaviour to obtain attention from their caregivers. One example of this is the wagging
tail, typically associated with happiness can also be shown during an aversive situation.
Furthermore, behavioural responses to stress are interlinked and share neural pathways.
Therefore, the categories cannot be related to specific neuroanatomical pathways.
To create a score of global reaction and homogenise the magnitude of the autonomic,
endocrine and behavioural response, each behavioural parameter, cortisol levels and LF/HF analysis
were transformed in scores (Table 2). This method is commonly used to create sustainable
indicators by aggregating a set of sustainable development indicators in thematic and
dimensional indices [
]. Thus, this method allows the analysis of each parameter separately, in
categories and with all the parameters together. In this way, each behavioural type (arousal,
fear, relaxation and other), LF/HF and cortisol levels were correlated to the caregiver's
perception of the fear of the dogs at home. A moderate correlation was found between the caregiver's
perception of the dogs' sound sensitivity and the behavioural categories arousal and fear and a
high correlation between the caregiver's perception of the dogs' sound sensitivity and the
behavioural category lack of relaxation. These results reinforce the idea that testing in a controlled
environment, the sound sensitivity dogs will have more intense responses than non-sensitive
dogs in the parameters autonomic (LF/HF ratio) and behavioural categories of arousal, fear,
lack of relaxation. Unfortunately, despite the evidence and because of the limited number of
animals, we could not run the linear regression to show that high scores in the behavioural
categories arousal, fear and lack of relaxation and in the HRV parameter ratio LF/HF are
predictors of sound sensitivity in dogs. After that, not only the short index for sound sensitivity (only
behavioural categories arousal, fear and relaxation and LF/HF ratio were included) but also
the full index for sound sensitivity (all behavioural categories, LF/HF and cortisol were
included) were highly correlated to sound fear at home. These indexes can be used in future
research to help diagnose sound sensitivity and can be used to evaluate treatment strategies.
Moreover, the short index has an advantage as compared to the full index since it does not
include the cortisol measures and therefore does not require blood collection and thus
simplifies the analysis. Another important characteristic of these analyses evaluates the dog's
response to a sound stimulus in the laboratory, and it is based exclusively on the dog's
response, excluding the caregiver evaluation or the dog-caregiver relationship. In a clinical
condition, the evaluation of the owner is fundamental for the diagnosis of behavioural
disorders in pets. However, it is well known that this assessment is not based solely on the behaviour
of the dog itself, but also on the owner's expectation regarding animal behaviour, which can
often be unrealistic.
The combined analysis of behavioural, endocrine and autonomic data allows a global and
integrated view of the fear response of sound sensitive dogs. Although the diagnosis of sound
sensitivity in dogs is based on behavioural responses and physiological data, it can be extremely
useful in understanding the neurophysiological mechanisms of fear responses and their
possible consequences for the health and well-being of the individual.
Therefore, despite the limited number of animals used, this article presents indexes of
sound sensitivity that merge behavioural, endocrine and autonomic responses into a single
score value. This association can be very advantageous because it represents a quantitative and
14 / 18
less subjective analysis than the behavioural evaluation alone. Both the short and full indexes
for sound sensibility can be valuable tools in future studies concerning neurophysiological
mechanisms and treatment strategies of sound sensitivity in dogs.
S1 Appendix. General behavioural form in English.
S2 Appendix. General behavioural form in Portuguese.
S3 Appendix. Sound sensitive form. Owner's Perception of dogs fear in English.
S4 Appendix. Sound sensitive form. Owner's Perception of dogs fear in Portuguese.
S5 Appendix. Dogs information.
S1 Dataset. Data of all animals.
The authors would like to thank Dr Newton Canteras for the valuable discussion about the
possible pathways involved in different behavioural categories and Dr Celso G. Barbosa for his
important help in the statistic procedure of blending qualitative and quantitative data.
Conceptualization: Carla Caroline Franzini de Souza, Magda Alves de Medeiros.
Data curation: Carla Caroline Franzini de Souza, Raquel Nascimento de Souza.
Formal analysis: Carla Caroline Franzini de Souza, Magda Alves de Medeiros.
Investigation: Carla Caroline Franzini de Souza, Magda Alves de Medeiros.
Methodology: Daniel Penteado Martins Dias, Raquel Nascimento de Souza, Magda Alves de
Project administration: Magda Alves de Medeiros.
Software: Daniel Penteado Martins Dias.
Supervision: Magda Alves de Medeiros.
Writing ± original draft: Carla Caroline Franzini de Souza, Magda Alves de Medeiros.
Writing ± review & editing: Daniel Penteado Martins Dias, Magda Alves de Medeiros.
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