Behavioural and physiological responses of domestic ...

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Behavioural and physiological responses of domestic dogs (Canis familiaris) to agonistic growls from conspecifics Penney A. Wood a , Josine de Bie b , Jennifer A. Clarke a,∗ a b

Department of Biological Sciences, Macquarie University, Sydney 2109, NSW, Australia Australian School of Advanced Medicine, Macquarie University, Sydney 2109, NSW, Australia

a r t i c l e

i n f o

Article history: Accepted 14 October 2014 Available online xxx Keywords: Motivation–structural rules Aggression Coping strategies Cortisol Dogs Acoustic communication

a b s t r a c t Motivation–structural rule theory predicts that a sender producing harsh, low frequency sounds directed at a conspecific modifies the receiver’s behaviour, in part, by communicating its willingness to escalate to an attack. Motivation–structural (MS) rules generally assume that receivers respond to this signal by retreating because of the threat encoded in the acoustic characteristics of the vocalisation. This assumption does not consider if alternative behavioural responses exist or how internal and environmental contexts affect receivers. This becomes an area for potential development of the MS theory when acknowledging that physiological and behavioural reactions may be related to distinct antithetical responses, such as Passive and Proactive coping strategies. To test if aggressive sound stimuli elicit consistent retreat responses by receivers, 42 dogs from shelters and private dog breeders were graded on behaviour and measures of salivary cortisol (a stress related steroid hormone) in response to an agonistic growl from an unknown, similarly sized conspecific. Results revealed that 52.4% (22/42) of dogs displayed retreat behaviours (Passive response), 33.3% (14/42) actively approached the growl source (Proactive response) and 14.3% (6/42) neither approached nor retreated (Neutral response). Post-test cortisol levels differed significantly between dogs in the Proactive, Passive and Neutral categories. Passively responding dogs averaged an 80% change in cortisol levels, Proactive dogs exhibited a 16.5% average cortisol change and Neutral responders displayed only a 6.4% change in cortisol. Although shelter dogs exhibited greater changes in cortisol, they did not differ significantly from the privately-owned dogs. This study verified that overt, observable behavioural responses reflect the physiological stress responses as measured by cortisol changes exhibited by domestic dogs. Results also suggested that experiences may influence how dogs to threatening stimuli from conspecifics respond—behaviourally and physiologically. A protocol providing a simulated threatening interaction with a dog, which does not bring the animals into direct contact, would have important implications for shelter staff in terms of how dog–dog introductions are managed, housing arrangements, potential training, matching dogs to owners and what advice shelter staff could offer to new owners. © 2014 Elsevier B.V. All rights reserved.

1. Introduction

∗ Corresponding author. Tel.: +61 2 9850 8187; fax: +61 2 9850 9231. E-mail address: [email protected] (J.A. Clarke).

Natural selection has resulted in the distinct divergence of the acoustic structure of vocalisations used by mammals in short range “aggressive” vs. “appeasing” contexts (Collias, 1960) with wide bandwidth (harsh), relatively

http://dx.doi.org/10.1016/j.applanim.2014.10.004 0168-1591/© 2014 Elsevier B.V. All rights reserved.

Please cite this article in press as: Wood, P.A., et al., Behavioural and physiological responses of domestic dogs (Canis familiaris) to agonistic growls from conspecifics. Appl. Anim. Behav. Sci. (2014), http://dx.doi.org/10.1016/j.applanim.2014.10.004


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low-frequency sounds used in hostile contexts and narrow bandwidth (tonal), relatively high frequency sounds used in appeasement contexts. Motivation–structural rules predict that selective pressures favour the use of these acoustic cues in aggressive contexts due to the unambiguous relationship between body mass and the size of the vibrating membranes used to produce soundwaves in the mammalian larynx (Morton, 1977). The larger the vocal apparatus, the less tension is exerted on these membranes and harsher sounds can be produced (Morton, 1977). As Morton (1977) states, “Harsh, low-frequency sounds indicate that the sender is likely to attack if the receiver comes closer to the sender or remains at the same distance.” This relationship between acoustic structure of a vocalisation and an aggressive motivational context has been supported in numerous studies (e.g. Harrington, 1987; Gouzoules and Gouzoules, 2000; Compton et al., 2001; Feighny et al., 2006). However, an assumption behind this rule, that the receiver will avoid or retreat from the sender in response to the vocalisation’s acoustic structure (i.e. wide bandwidth and low frequency), has largely been unexplored. Additionally, how the physiological state of the receiver relates to its visible external response to aggressive and stress-inducing signals has rarely been considered or empirically tested. This becomes an area for development of the motivation–structural rules when considering that different behavioural responses can often be correlated to measurable fluctuations in the activation of the hypothalamic–pituitary–adrenal axis in response to socially stressful situations (Koolhaas et al., 1999). Measures of changes in salivary cortisol have been used as reliable indicators of an internal stress response for dogs in studies that have dealt with stressors such as loud noises, shocks, physical restriction and separation (Tuber et al., 1996; Beerda et al., 1998; Bergeron et al., 2002; Dreschel and Granger, 2005). However, these studies have not considered an individual’s overt behavioural tendencies to avoid or approach the stress source. Evidence from a variety of species indicates that behavioural and physiological reactions to physical threats are related to distinct, antithetical response styles or coping strategies (Wechsler, 1995; Koolhaas et al., 1999). This concept has been associated with ‘flight or fight’ personality dimensions in humans and non-human animals (Friedman and Silver, 2007). Animals that react to novel or potentially threatening situations with investigative or engaging behaviour and low cortical stress responses are often classified as having a the ‘fight’ or Proactive response style, while those responding with a reluctance to approach and fearful behaviours and high cortisol stress responses are classified as ‘flight’ or Passive responders (Koolhaas et al., 1999; Horváth et al., 1997). Additionally, these coping strategies may be influenced by an individual’s experiences and environment. In dogs (Canis familiaris), approach or avoidance tendencies are indicative of Proactive and Passive coping styles, but the physiological and behavioural measures related to coping strategies have been conducted only with dogs responding to stressful interactions with humans (Svartberg, 2002; Svartberg and Forkman, 2002; Horváth et al., 1997). No

study has investigated if couplings of behavioural and physiological responses form the basis of distinct coping strategies in dogs in relation to social stress from an aggressive conspecific. Because of the prevalence of dogs in human society and the number of important functions dogs perform, understanding their ability to cope with and interpret social stress is critical (Lucidi et al., 2005). This is particularly applicable to dogs sheltered in welfare facilities. Considering the large number of animals that circulate through animal shelters, being able to accurately and permanently re-home these animals is a paramount concern. Qualifying canine temperament for potential problem indicators is a difficult process. An individual’s tendency towards inappropriate behaviours during social interactions with conspecifics may be hazardous to test and cause problems post-adoption if not identified (Svartberg and Forkman, 2002). An assessment of a dog’s social tendencies with conspecifics would benefit from a protocol providing a simulated threatening interaction with a dog, which does not actually bring the animals into direct contact (Christensen et al., 2007). Such a protocol would have important implications for shelter staff in terms of how they manage introductions with other dogs, potential training and behaviour modification, housing arrangements in the kennels, how they would match dogs to owners and what advice they would give to these new owners. The goals of our study were to: (1) determine if, in the absence of visual cues, aggressive acoustic signals from a conspecific elicit avoidance behaviours, (2) determine how behavioural responses relate to changes in the stress hormone, cortisol, (3) investigate if a dog’s behavioural and physiological responses are related to its history/environment and (4) provide an additional tool for shelters to determine the least stressful housing conditions (e.g. with or without other dogs in a kennel) and to aid in the adoption process. To accomplish our goals, we exposed dogs from shelter environments and from private breeders to an aggressive “growl” stimulus from a similarly sized conspecific and to a “control” stimulus of a cicada insect chorus (similar to white noise), documented their behavioural responses and measured changes in salivary cortisol before and after exposure to the sounds. 2. Materials and methods 2.1. Subjects Forty-two healthy, domestic dogs were tested. Twenty dogs were residents (>2 wk) at the Animal Welfare League (AWL) re-homing shelter in Kemps Creek, Sydney, New South Wales, Australia. These were primarily crosses of Australian cattle dogs, Staffordshire bull terriers, border collies, Labrador retrievers and kelpies (17 were neutered). These dogs’ breeds were identified by the shelters’ veterinary staff during health checks. The backgrounds and histories of the mixed-breed shelter dogs were unknown. The 22 other dogs were privately-owned by eight registered breeders near Sydney, NSW. These were pure-bred dogs from three breeds: Australian cattle dogs,




Staffordshire bull terriers and border collies (two were neutered). All had been born and raised on the breeder’s property. Six of these dogs had show-ring experience (a fact we discovered after testing the dogs). Dogs were subjected to a single testing session consisting of a control sound (cicada, Cicadidae, chorus) and an agonistic growl vocalisation, both at ∼70 dbA. The cicada chorus was structurally dissimilar from the dog growl and higher in frequency (4392 Hz) than the growl and but well within the range of canine hearing (Sales et al., 1997). The cicada chorus sounds were downloaded from www.sounddogs.com and played back using a SMSAFS amplified field speaker (Saul Mineroff Electronics, Inc., Elmont, NY USA) connected to a laptop computer. Growl stimuli were elicited from four unfamiliar dogs at another shelter that were similar to the test dogs in age, weight and breed. These dogs exhibited growling while ‘kennel guarding,’ which is a form of aggressive behaviour associated with territorial resource defence (Yeon, 2007; Handelman, 2008). While standing adjacent to the dog’s kennel for 5 min, we recorded (real-time) a minimum of 3 min of growling using a Roland RO9 portable 24 bit Digital Audio Recorder (Roland Corporation Australia, NSW, Australia) and a Sennheiser K3U Microphone (Sennheiser Australia Pty Ltd, Chatswood, NSW, Australia). The peak (dominant or loudest) frequency of the growl vocalisations ranged between 344 and 430 Hz. Growls used in the test stimulus were elicited from: (1) male, 2 years old, Australian cattle dog X Staffordshire bull terrier, 18.7 kg, (2) female, 1 year old Staffordshire bull terrier X unknown, 19.7 kg, (3) male, >3 year old, Staffordshire cross X unknown, 19.5 kg and (4) male, >3 year, Staffordshire bull terrier X unknown, 17.3 kg. Playback material was constructed by editing each of these four dogs’ growl series to play for 3 min at ∼70 dbA. The shelter dogs and privately-owned dogs were then tested with one of these four dogs’ 3 min growl series—randomly assigned. Stimulus order was also randomised. All dogs were tested in a portable, enclosed outdoor arena constructed of cloth and wooden posts (Fig. 1) on their ‘home site’ to reduce effects of transport stress and novelty (Bergeron et al., 2002). For the breeder’s dogs, this was on the grounds of the breeder’s facility. For the shelter dogs, this was on the grounds of the shelter facility at which they were maintained. Black cloth, 1.6 m high, was attached to posts in the ground at 1 m intervals. Two sides were each 3.2 m in length and the far end was 2 m long. The speaker producing the sounds was placed behind a screen and the camera filming the dogs’ responses was placed behind the dog. This arena was mobile and provided a relative consistent setting for the tests as it screened the dogs from distracting visual stimuli. Dogs were accompanied by a familiar handler and allowed to move freely around the test area for 10 min (during this time they trotted around the test area, sniffing, tail wagging). Each dog was then put on a lead and led in into the arena with a SMS-AFS amplified field speaker (Saul Mineroff Electronics, Inc., Elmont, NY USA) connected to a laptop computer placed behind a screen at one end and a Sony HDR CX130 Handicam camcorder (Sony Australia,

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Fig. 1. Diagrammatic illustration of the test arena in which each dog was positioned during sound stimulus testing. The arena measured 320 cm × 200 cm with 160 cm high walls (see Section 2 for details).

Chatswood, NSW, Australia) filming the dogs’ responses at the other end of the arena for 3 min (in real-time). Recordings of the control and test sounds were played for 3 min with the dog 2–3 m from the speaker. Dogs’ responses were scored 1 or 0 for the presence or absence of specific behaviours. Nominal behavioural states were grouped according to their contribution to a larger behavioural category (Table 1; after Beerda et al., 1998; Vas et al., 2005; Horváth et al., 1997). Neutral postures were the body posture specific to that breed under relaxed conditions (Handelman, 2008) or determined using observations of post-test postures for the mixed-breed dogs. Behaviours and postures associated with fearful or acute stress responses or competing motivational states were scored as present or absent (after Horváth et al., 1997). Pacing was considered as a deliberate and repetitive movement directed towards the sound source. A retreat response was characterised by a sustained withdrawal from the sound source, and an ambivalent response was a partial approach followed by a sustained retreat. Salivary cortisol sampling took place 1 min before and 20 min after the stimulus playbacks (Fig. 2). Pre-stimulus collections of cortisol levels established a baseline for each test. This timing took into account the 20 min lag that may occur in the change in salivary cortisol following a stressful stimulus. Samples were collected using hydrocellulose sponges swabbed the cheeks and jowls of each dog. Swabs were placed into labelled centrifuge tubes and stored at approximately −20 ◦ C (Kalman and Grahn, 2004). During transport from sampling sites, samples were kept in an insulated container for ≤2 h before refrigeration (Kalman and Grahn, 2004). Tubes were warmed to room temperature and 25 ␮m of saliva was extracted via centrifugation at 3000 rpm for 15 min (Horváth et al., 1997). Saliva samples with sufficient volume were analysed in duplicate at ASLAN Medical labs at Macquarie University Hospital using a concentration sensitive (0.003 to 3.0 ␮g/dL) 1-3002—cortisol salivary immunoassay kit ELISA/EIA (Salimetrics, Sydney, NSW, Australia).




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Fig. 2. Diagrammatic illustration of the testing protocol for filming overt, observable behavioural responses of the dogs to playbacks of agonistic growls (from a similarly sized dog) or a control sound (a cicada chorus) and for taking salivary cortisol samples for measuring concurrent physiological stress responses. Table 1 Behavioural categories exhibited by domestic dogs in response to playback of growl and control sound stimulus (after Beerda et al., 1998; Vas et al., 2005; Horváth et al., 1997; Handelman, 2008). Behavioural category

Description of states

Movement response

No approach attempt Retreat: run or trotting withdrawal from the sound source within the first 30 s of the playback, subject maintains this distance Ambivalent approach: an individual begins to approach the sound then withdraws as in ‘retreat’ and/or has a latent approach (that takes place 1 min after the stimulus sound commences) Fast approach: individual approaches the sound source within the first 30 s of the test without retreat Passive: After commencement of playback the tail position is lowered and ears are flattened or drawn back and/or legs are bent Neutral: After commencement of stimulus sound, the subject maintains post-test posture or a posture that is specific to the individual’s breed and displayed under relaxed conditions Alert: After commencement of the stimulus sound, the tail is position is elevated, and/or the head position is higher and accompanied with elevated forward pointing ears, and or the subject is standing extremely erect Tongue flicking: repetitive movement of the tongue along the upper lip. Paw lifting: a fore paw is lifted at an approximate angle of 45◦ Gaze aversion/looking away hiding, handler orientation Repetitive movement directed at the sound source. Legs on one side of the dog’s body move in the same direction in parallel fashion Moving tail from side to side Barking, whining or howling

Posture

Stress indicators

Fear-typical indicators Pacing

Tail wagging Vocalising

2.2. Statistical analysis A paired group cluster analysis for Manhattan variance was performed (PAST version 2.16) in order to group subjects based on similarities in behaviours. Clusters were formed on both weak and strong similarity relationships and a non-parametric multivariate analysis of variance (NPMANOVER) was performed to test the strength of these groupings. Optical densities for cortisol levels were converted to concentrations and analysed for normality using a Shapiro–Wilks test and log transformed (using SPSS). Cortisol concentrations were expressed as percent change between pre-test and post-test situations, allowing a comparative analysis of changes in the stress response at an individual subject level. An ANOVA comparison was made of the range and mean of salivary cortisol concentrations from each of the subjects before and after the growl and control stimulus exposure. Independent sample two-tailed t-tests and one-way ANOVA for repeated measurements were used to determine if differences existed between percent changes in cortisol concentrations from each cluster and: (a) stimulus conditions (growl vs. control), (b) subject characteristics (sex, breed, size), and (c) environmental variables (time of day). A Pearson correlation test was performed to determine if a relationship existed between size and the percent change in cortisol. Independent sample two-tailed t-tests and one-way ANOVA for repeated measurements were used to determine if differences existed between percent change in cortisol concentrations and the presence and absence of each of the behavioural categories. Significance was determined at P ≤ 0.05 or less using FDR control to decrease type I errors (Verhoeven et al., 2005).

3. Results There were no relationships between breed, sex or reproductive status (spayed/neutered or intact) and the average and range of cortisol level changes for the four


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dogs). The ratios of neutered to intact dogs in the Proactive and Passive response groups differed very little. The ratio of dogs responding proactively to the growl stimulus was 36% intact to 64% neutered, which is similar to the ratio of dogs responding passively which was 41% intact to 59% neutered (Fig. 5). In contrast, the Neutral group was 100% intact dogs from private breeders. 3.2. Changes in salivary cortisol concentrations and relationships to behavioural clustering

Fig. 3. Percent of dogs responding (n = 42) with passive (i.e. avoidance), proactive (i.e. approach) and neutral (i.e. no change in position) behaviours to the control (cicada) sound and the test (agonistic growl) sound.

saliva samples pre and post control and pre and post growl stimuli (P > 0.05, all cases, n = 42). Because source group (shelter and privately-owned) was significantly related to the average and range of salivary cortisol level changes (F1,41 = 6.34, P = 0.02, n = 42; F1,41 = 5.43, P = 0.03, n = 42), relationships between source group and behavioural and physiological responses were analysed further. There were no relationships between stimuli order or test stimuli type and response (P > 0.05, all cases). 3.1. Grouping of behavioural responses In response to the control stimulus (cicada chorus), 90.5% (38/42) of the dogs maintained a Neutral posture and displayed none of the indicators related to fear, stress or excitement (Fig. 3). Four dogs (9.5%) approached the sound. These responses to the control stimulus were clustered (non-significantly) into two asymmetrical groups (F1,41 = 0.27, P = 0.57, n = 42). In contrast, the analysis for responses to the growl stimulus revealed a distinctly different arrangement of subjects into three clusters (F1,41 = 118.91, P < 0.0001, n = 42; Fig. 4): Neutral (14.3%, 6/42 dogs), Proactive (33.3%, 14/42 dogs), and Passive (52.4%, 22/42 dogs). Dogs in the Neutral response group made no attempt to approach or retreat from the stimulus. These six dogs were all members of the privately-owned group. Dogs in the Proactive response group displayed an alert posture, fast approach and no acute stress, fear or withdrawal behaviours. The majority of Proactive dogs (64%; 9/14) were shelter dogs. Dogs in the Passive response group displayed avoidance/fearful behavioural indicators with 73% (16/22) exhibiting ambivalent (alternating between towards and away) movements and 27% (6/22) retreating from the growl stimuli. The Passive response group was composed of equal numbers of shelter dogs and privately-owned dogs (11 dogs from each source). Stress behaviours were observed only in this Passive response group (59%, 13/22

Salivary cortisol responses were significantly greater in response to the growl stimulus compared to the control stimulus (t82 = 2.43, P = 0.01, n = 42; growl: 48.31 ± 51.33%, control: 25.37 ± 33.35%). Dogs in the Neutral response group exhibited lower average change in cortisol concentrations of 6.19 ± 5.60% post-test to the growl stimuli, compared to dogs in the Passive and Proactive response groups (t18 = −2.85, P = 0.01, n = 20; t26 = −3.60, P < 0.01, n = 28; respectively; Fig. 6). In response to the growl stimulus, the percent change in cortisol concentrations for the Proactive response group (16.52 ± 8.02%) was significantly less than the Passive response group (t34 = −4.39, P ≤ 0.01, n = 34). Dogs in the Passive response group exhibited the highest average cortisol change of 80 ± 53.50% post-test to the growl stimuli. 3.3. Relationships between dogs’ sources, breed, sex, time and cortisol responses Only privately-owned dogs displayed Neutral behaviours and these dogs also exhibited a significantly lower average percent change in cortisol (6.19 ± 5.56%) compared to privately-owned dogs displaying Proactive or Passive responses (t20 = 2.88, P < 0.01, n = 22). Average cortisol change associated with the growl stimulus did not differ between shelter dogs and privately-owned dogs in the Proactive group (t12 = 0.56, P > 0.02, n = 14) or in the Passive response group, (t20 = 2.08, P > 0.008; n = 22; shelter: 102.1 ± 63.98%, privately-owned 58.00 ± 29.09%). Breed, sex and time of day of testing were not related to changes in cortisol concentrations or behavioural responses to either stimulus (P > 0.05, all cases, n = 42). Also, no relationship existed between dog size and change in cortisol concentration for either stimulus (growl: r = −0.2201, P = 0.16, n = 42; control: r = 0.0504, P = 0.75, n = 42) or for stimulus order (t40 = −0.46, P = 0.64, n = 42). 4. Discussion The first goal of our study was to determine if, in the absence of visual cues, aggressive acoustic signals from a similarly-sized conspecific elicit avoidance behaviours, and the results confirm that this is the case but other responses are also elicited. Our results revealed that 52.4% of the dogs tested did indeed exhibit retreat behaviours in response to harsh, low frequency growls, but 47.6% remained in position or actively approached the sound. These distinct and antithetical responses in response to an aggressive vocalisation indicate that a harsh, low frequency sound alone may not consistently elicit the assumed retreat



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Fig. 4. Behavioural variables in response to playback ‘growl’ stimulus clustered according to Manhattan variance performed using PAST (version ver. 2.16) with grouping distance of 0.45. Subjects marked with an asterisk indicate the ‘privately-owned’ dogs and those and without an asterisk are from ‘shelter’ sources.

behaviour. That said, in reality, growls by dogs are produced in close proximity to the perceived threatening conspecific and are typically accompanied by visual and chemosensory signals that may elicit different responses on the part of the receiver through multimodal signalling. However, the effects of personality, coping styles, experiences and training are also important considerations when predicting dogs’ responses to threatening stimuli. Our second goal was to determine how behavioural responses relate to changes in the stress hormone, cortisol and the results revealed that behavioural responses

Fig. 5. Percent of intact and neutered dogs (from shelters and breeders, n = 42) responding proactively, passively and neutrally to the growl stimulus. All dogs responding neutrally were privately-owned dogs.

are reliably reflective of physiological changes in the stress hormone, cortisol. The behavioural and physiological responses characteristic of Passive responses include cautious approach, displays of fear-related behaviour and high cortisol stress reactions, while Proactive responses include rapid approach and low cortisol changes (Koolhaas et al.,

Fig. 6. Percent change in salivary cortisol of dogs (n = 42) responding with passive, proactive and neutral behaviours to the control sound and the growl sound. Box plots display 10th, 25th, 50th, 75th, 90th percentiles as lines on the bar centred about the mean, and the 5th and 95th percentiles are presented as error bars.




1999; Svartberg, 2002; Svartberg and Forkman, 2002). In our study, dogs that responded to the growl stimuli with avoidance and fear clearly met ‘Passive’ categorisation criteria and they exhibited high changes in cortisol concentrations. Dogs categorised as behaviourally ‘Proactive’ responded with high levels of activity, a rapid approach towards the growl stimulus and low changes in cortisol. These behavioural and physiological responses of Passive and Proactive dogs also corresponded with the findings Horváth et al. (1997), who compared the behavioural and cortisol stress responses of working dogs to the threatening social stimulus of an intimidating human aggressor. The exceptions to the Passive and Proactive responses concerned the dogs in the Neutral response category, who neither retreated nor approached and who showed no change in cortisol levels. The Neutral response group distinctly reveals the importance of our third line of investigation, which was to determine if and how a dog’s behavioural and physiological responses to potentially threatening stimuli are related to its experiences and/or environment. All the Neutral response dogs were the privately-owned dogs that had show-ring experiences, as we discovered post-sampling. These dogs may have had repeated exposure to aggressive vocalisations from unfamiliar dogs in the controlled conditions of dog shows, where dogs are prevented from following vocal threats with attacks. It is possible that these experiences decreased these dogs’ assessment of and reaction to the threat, which suggests an area needing further research. As noted in the methods, the backgrounds/histories of the shelter dogs were unknown, but it is unlikely that any of these mixed-breed dogs had showring experience. Although shelter dogs showed the highest cortisol responses of all the 42 subjects (i.e. 128%, 168%, 170% and 234% changes in cortisol levels), these levels were not significantly different from privately-owned dogs’ cortisol measures. These slight differences in the physiological stress response may be a reflection of the typical histories of dogs surrendered to welfare facilities and the potentially stressful nature of the shelter environment. Studies suggest that shelter environments contain intense and consistently present social stressors that may cause dogs with limited social exposure to experience a physiological sensitisation of the HPA axis in stressful situations (Tuber et al., 1996; Hennessy et al., 1997; Coppola et al., 2006; Rooney et al., 2007). However, we did not find that significantly more shelter dogs responded passively nor were their cortisol levels were significantly higher compared to privately-owned dogs. Thus, we cannot conclude that these dogs’ responses to the growl stimulus to which we subjected them are specifically related to their shelter vs. privately-owned history/environments. However, unlike the Proactive and Passive responders, Neutral responders maintained a relaxed posture, made no approach attempts and exhibited the lowest changes in cortisol in response to the growl stimuli. We discounted hearing loss as a factor contributing to this response because dogs in this study had full hearing capabilities and all six subjects in this group briefly turned their heads towards the sound source in the initial minute of

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the growl playback. An alternative explanation for these Neutral responses relates to the effects of experience and training, which can influence a subject’s assessment of threat and/or cause an extinction of the threat response (Stankowich and Blumstein, 2005). Dogs in the Neutral response cluster were the only dogs known to have been regular participants in competitive dog shows. The nature of competitive dog shows requires subjects to be unresponsive and appear calm during official assessment while simultaneously being exposed to a distracting social environment—often composed of vocalising conspecifics. Repeated exposure to a threatening stimulus may habituate a subject’s behavioural response and reduce displays of threatening and threatened behaviour (Clayton and Hinde, 1968; Gallagher et al., 1972). This study demonstrates that a dog’s response to an aggressive vocal stimulus from an unfamiliar conspecific can reveal physiological and behavioural coping styles that reflect the individual’s interpretation of social threats. Our results confirm that behavioural observations can be used as a proxy for stress hormone level measurements in dogs subjected to an auditory threat from a conspecific. This procedure can provide shelters with a tool to predict how an individual dog assesses a conspecific threat and, consequently, used to assess the level of stress a dog may experience when placed in a situation with multiple conspecifics, such as being adopted into a home with other resident dogs. In addition to adoption assessments, this procedure could be used in kennel placement procedures in shelters when determining whether to house a dog singly or in a multiple dog kennel, and thus improve welfare practices and management of shelter dogs. 5. Conclusions Our findings confirmed that behavioural responses are reliable indicators of internal, physiological stress responses as measured by changes in salivary cortisol. Our findings reveal that a dog’s behavioural and physiological responses may be related to its training. We propose that a modified version of the methods employed in this study may provide an additional tool by which shelter dogs can be assessed for adoption into appropriate homes. Conflict of interest statement There are no conflicts of interest associated with the publication of this article. Acknowledgements We thank T. Pearson for guidance regarding recording techniques and equipment, D. Nipperess and D. Allen for statistical advice and S. Giuliano for field research assistance. We also thank the supportive dog-shelter staff and dog-owners and who made this study possible: R. Wicks and K. Parfrey from the Animal Welfare League and the owners of the privately-owned dogs from Avadale, CodeBonnie, Dalheath, Dreamwood, Knobel, Moonstream, Staffplay, and Tiamostaff breeders—and of course all the




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