Associations between canine temperament and salivary levels of cortisol and serotonin

Associations between canine temperament and salivary levels of cortisol and serotonin | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Associations between canine temperament and salivary levels of cortisol and serotonin View ORCID Profile Youngwook Jung , View ORCID Profile Yujin Song , Kayoung Yang , Kyongwon Yoo , Youngtae Heo , View ORCID Profile Minjung Yoon doi: https://doi.org/10.1101/2025.11.16.688729 Youngwook Jung a Department of Animal Science and Biotechnology, Kyungpook National University , Sangju, Republic of Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Youngwook Jung Yujin Song a Department of Animal Science and Biotechnology, Kyungpook National University , Sangju, Republic of Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yujin Song Kayoung Yang b Animal Welfare Division, National Institute of Animal Science , Wanju, Republic of Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site Kyongwon Yoo c Department of Companion Animal, Osan University , Osan, Republic of Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site Youngtae Heo c Department of Companion Animal, Osan University , Osan, Republic of Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site Minjung Yoon a Department of Animal Science and Biotechnology, Kyungpook National University , Sangju, Republic of Korea d Department of Horse, Companion and Wild Animal Science, Kyungpook National University , Sangju, Republic of Korea e Research Institute for Innovative Animal Science, Kyungpook National University , Sangju, Republic of Korea Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Minjung Yoon For correspondence: mjyoonemail{at}gmail.com Abstract Full Text Info/History Metrics Preview PDF Abstract Temperament influences canine behavior and helps determine a dog’s suitability as a companion or working animal. Although several temperament assessment tools exist, many rely on subjective evaluations, highlighting the need for scientifically validated methods. This study evaluated the validity of the Wesen temperament test by examining its association with physiological biomarkers—salivary cortisol and serotonin. Twenty-four dogs (11 females and 13 males) completed a modified Wesen test comprising seven subtests: Unconscious Confidence, Sociality, Noise Stability, Movement Stability, Desire for Play and Predation, Behavior in Stressful Situations, and Separation. Saliva samples were collected pre- and post-assessment, and cortisol and serotonin levels were measured using ELISA. Hormonal levels and temperament scores were analyzed using correlation, regression, and Kruskal-Wallis tests. Pre-assessment cortisol levels were negatively correlated with total average temperament score and several subtest scores. Post-assessment cortisol levels showed significant negative correlations with total average scores and all subtests. Changes in cortisol levels from pre- to post-assessment were negatively associated with temperament scores. Dogs with higher temperament scores exhibited significantly higher serotonin levels than those with lower scores. These findings support the utility of temperament assessments validated through physiological markers and provide novel evidence linking canine temperament with endocrine function. 1. Introduction The temperament of dogs is a key factor in determining their suitability as companion animals [ 1 ]. Additionally, temperament plays a critical role in working contexts, such as guiding [ 2 ], detection [ 3 ], and rescue operations [ 4 ]. Therefore, selecting dogs with appropriate temperaments for specific purposes is important. Temperament assessment can help predict a dog’s future performance and suitability [ 5 ]. Several tools have been developed for this purpose, including the Canine Behaviour Assessment and Research Questionnaire (C-BARQ) [ 6 ], the In-For Training test [ 7 ], and the Wesen test [ 8 ]. These assessment tools differ in their evaluation methods: Some rely on owners’ reports, while others require trained professionals. This variability raises concerns about potential subjectivity [ 7 ]. Thus, several studies have examined the validity of temperament assessments by comparing their scores with physiological biomarkers such as heart rate, respiratory rate, and hormone levels [ 9 – 11 ]. Among these biomarkers, hormones—particularly cortisol and serotonin—have emerged as key indicators owing to their close association with temperament and behavior. Cortisol is secreted in response to stress or negative emotional states [ 12 ]. It is commonly used as a biomarker of stress and overall well-being in dogs. Recent research has focused on the relationship between cortisol and temperament. Rosado and colleagues reported higher cortisol levels in aggressive dogs than in non-aggressive individuals [ 13 ]. Basal cortisol levels have also been linked to fear-related behaviors, such as panting and tongue protrusion [ 14 ]. Additionally, cortisol levels vary with the degree of sociality [ 15 ] and have been associated with touch sensitivity [ 16 ] and anxiety towards strangers [ 17 ]. Collectively, these findings indicate that cortisol is involved in several aspects of canine temperament, including aggression, fear responses, and social interaction. Therefore, cortisol serves as an indicator of temperament-related traits beyond its traditional role in stress response. Cortisol has been measured in several biological matrices, including plasma, urine, feces, hair, feathers, and saliva [ 14 , 18 – 20 ]. Blood cortisol is considered a reliable indicator of acute stress responses. However, because blood sampling is invasive, it has become less frequently used in recent studies. In contrast, saliva sampling has gained popularity as a noninvasive method that reduces stress in animals. Steroid hormones can diffuse rapidly into saliva from the bloodstream owing to their molecular properties [ 21 ]. Moreover, salivary cortisol reliably reflects plasma cortisol levels in dogs [ 22 ]. Therefore, we selected saliva sampling as the method for cortisol measurement. Serotonin plays a well-established role in temperament and behavior, including aggression, anxiety, and social interactions. Numerous studies have reported substantial differences in serotonin levels between aggressive and non-aggressive dogs [ 13 , 23 – 25 ]. Vermeire and colleagues further reported that serotonin receptors are expressed in cortical regions of the brain and are associated with anxiety disorders in dogs [ 26 ]. Associations between serotonin levels and dogs’ social behaviors toward humans have also been observed [ 27 ]. Collectively, these findings suggest that serotonin is a biomarker for temperament assessment in dogs. In dogs, serotonin has predominantly been measured in blood samples [ 25 , 28 – 30 ]. However, the invasiveness of blood sampling may limit its use in temperament monitoring. Therefore, noninvasive alternatives are needed. Saliva sampling has become increasingly popular because it minimizes stress in animals. In humans, although salivary serotonin is not directly correlated with serotonin levels in cerebrospinal fluid or platelets, it provides a reliable measure of peripheral serotonin levels [ 31 , 32 ]. Furthermore, salivary serotonin levels have been associated with indicators of happiness, mood, and depressive disorders [ 33 – 35 ]. On this basis, saliva sampling was adopted as the method for measuring serotonin levels in this study. This study aimed to validate a temperament assessment tool by measuring salivary cortisol and serotonin levels. Specifically, we (i) examined correlations between temperament scores and cortisol and serotonin levels and (ii) compared hormone levels between high- and low-scoring groups, as determined by the temperament assessment. We hypothesized that salivary hormone levels would correlate with temperament traits assessed by the Wesen test, thereby providing physiological evidence for the convergent validity of the test. The findings of this study may provide a scientific basis for the use of temperament assessments in dogs. 2. Materials and methods 2.1. Subjects Twenty-four dogs (eleven females and thirteen males) participated in the study, including seven mixedbreeds, six Beagles, four Border Collies, two Labrador Retrievers, two Malinois, one Pomeranian, one French Bulldog, and one Shepherd. The mean age was 5.13 ± 3.06 years. The Beagles were bred for experimental use, whereas the remaining dogs were owned by private individuals or professional trainers. All experimental procedures were conducted in accordance with institutional ethical guidelines and were approved by the Animal Experimentation Ethics Committee of Kyungpook National University (approval number: 2024-0531). 2.2. Temperament assessment To minimize stress associated with transportation, temperament assessments were conducted at three locations near the dogs’ original environments. The assessment was led by an experienced international dog show judge using a slightly modified version of the Wesen test. An overview of the temperament test process is presented in Fig 1 . The Wesen test (Wesensüberprüfung) was originally developed for German Shepherds aged 9–13 months [ 36 ], but its effectiveness and adaptability have led to its broad application across breeds and age groups [ 37 ]. The modified version used in this study consisted of seven subtests [ 5 , 8 , 36 ]: Unconscious Confidence: Attention, fear, confidence, interest, and relaxation were assessed during a physical examination that included inspection of the teeth, testes (for males), and a full-body check by the judge. Sociality: Responsiveness to the handler’s cues, confidence, cheerfulness, affinity, and energy were assessed during encounters with a group of strangers and an unfamiliar dog. Noise Stability: Calmness, relaxation, fear, and confidence were assessed in response to auditory stimuli produced by a motor, metal chain, and whip. Movement Stability: Dogs were guided by handlers to sit and walk on an unstable pallet and a series of connected tables. Relaxation, stability, fear, and activity were evaluated. Desire for Play and Predation: Dogs played with a toy or treat provided by both the handler and a stranger and were tasked with finding an object hidden under a box. Activity, persistence, affinity, and interest were evaluated. Behavior in Stressful Situations: Dogs played with a toy provided by the handler. Thereafter, the toy was hidden to prompt a search. During the search, a sudden loud noise was produced by dropping a metal chain to simulate a stress-inducing stimulus. Activity, confidence, persistence, interest, and reactions to the noise were evaluated. Separation: Dogs were tied to a fixed object and left alone for 5 min, followed by an encounter with a stranger. Anxiety, relaxation, and affinity were evaluated. Download figure Open in new tab Fig 1. Schematic representation of the temperament assessment test process (created using BioRender.com ) Each subtest comprised two to four categories, scored on a five-point scale (1 = lowest; 5 = highest). The judge evaluated all reactions according to standardized guidelines. During the Unconscious Confidence subtest, the judge held the leash; in all remaining subtests, the dog was handled either by the handler or a stranger. The entire temperament assessment was video-recorded by a designated observer. All subtests were conducted at an outdoor dog-training facility, except for the Behavior in Stressful Situations subtest, which took place in an indoor test room. 2.3. Sample collection To analyze hormonal levels, saliva samples were collected from each dog before and after temperament assessment by an experimenter not involved in judging. Samples were collected using a swab device and storage tube (Salimetrics, Carlsbad, CA, USA) by gently placing the swab under the tongue and in the cheek pouch. During transport to the laboratory, the samples were kept in an icebox at 4 ℃ to maintain integrity. Upon arrival, saliva was centrifuged at 1,000 × g for 2 min at 4 ℃ and stored at −80 ℃ until analysis. 2.4. Hormone analysis Hormone concentrations were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits. Samples were analyzed in duplicate without prior extraction or dilution. Optical density was measured using a microplate reader (Tecan, Männedorf, Switzerland), and concentrations were calculated based on a four-parameter logistic standard curve. 2.4.1. Cortisol ELISA Salivary cortisol levels before and after temperament assessment were measured using a Cortisol ELISA kit (1-3002; Salimetrics) with a sensitivity of 0.018 μg/dL. The intra- and inter-assay coefficients of variation (CVs) were 1.55% and 3.57%, respectively. Optical density was measured at 450 nm following the manufacturer’s instructions. 2.4.2. Serotonin ELISA Owing to insufficient saliva volume, sixteen samples that met the minimum quantity required for ELISA were included in the serotonin analysis. Salivary serotonin levels before temperament assessment were determined using a Serotonin ELISA kit (ADI-900-175; Enzo Life Sciences, Farmingdale, NY, USA) with a sensitivity of 0.293 ng/mL. The intra- and inter-assay CVs were 7.08% and 14.97%, respectively. Optical density was measured at 405 nm following the manufacturer’s protocol. 2.5. Statistical analysis Statistical analyses were performed using IBM SPSS Statistics (version 27; IBM, Armonk, NY, USA), and data visualization was conducted using GraphPad Prism (version 9; GraphPad Software, San Diego, CA, USA). Data normality was assessed using the Shapiro–Wilk test. Sex differences in hormone concentrations were evaluated using the Mann–Whitney U test. Variations in hormone concentrations among dogs bred for experimental use, privately owned dogs, and dogs raised by professional trainers were evaluated using the Kruskal–Wallis test. Spearman’s rank correlation coefficient (ρ) was used to examine relationships between hormone concentrations and temperament scores. Additionally, both Spearman’s ρ and linear regression analyses were used to evaluate associations between changes in cortisol concentrations and total temperament scores. The Kruskal–Wallis test was also used to compare hormone concentrations across groups categorized by temperament scores. Hormone concentrations and temperament scores are presented as mean ± standard error of the mean. Statistical significance was set at p < 0.05, and values between 0.05 and 0.1 were considered indicative of a trend. 3. Results 3.1. Correlation between cortisol levels and temperament assessment scores To examine potential sex effects, hormone concentrations were compared between females and males. No significant differences in cortisol or serotonin levels were observed between sexes or among dog populations. Therefore, data were pooled across sex and population for subsequent analyses. Correlations between salivary cortisol levels and temperament assessment scores are presented in Table 1. Pre-assessment cortisol levels were negatively correlated with total average scores ( p = 0.036). Negative correlations were also observed between pre-assessment cortisol levels and several subtests, including Unconscious Confidence ( p = 0.006), Sociality ( p = 0.035), Noise Stability ( p = 0.003), and Separation ( p = 0.006). No significant correlations were observed between pre-assessment cortisol levels and other subtests, including Movement Stability ( p = 0.291), Desire for Play and Predation ( p = 0.801), and Behavior in Stressful Situations ( p = 0.126). Post-assessment cortisol levels were negatively correlated with all subtest scores as well as with the total average scores ( p < 0.001). 3.2. Correlation between changes in cortisol levels and temperament assessment scores The correlation between changes in cortisol levels (from pre- to post-assessment) and total average scores is illustrated in Fig 2 . Cortisol levels were negatively correlated with total average scores ( p = 0.008). The coefficient of determination ( r ²) was 0.259 ( p = 0.011), and the adjusted r ² was 0.225. Download figure Open in new tab Fig 2. Linear regression between changes in cortisol levels (pre- to post-assessment) and total average temperament assessment scores. The analysis indicates a negative correlation (Spearman’s ρ = −0.526, p = 0.008) with an r ² of 0.259 ( p = 0.011) and an adjusted r ² of 0.225. 3.3. Comparison of cortisol levels among three groups Cortisol levels tended to increase from pre- to post-assessment (8.339 ± 1.352 pg/mL vs. 15.184 ± 3.682 pg/mL; p = 0.062). To further investigate these differences, dogs were categorized into three scoring groups—high (n = 8), medium (n = 8), and low (n = 8)—based on their total average temperament assessment scores ( Table 2 ). These groups differed significantly in their total average scores ( p < 0.01). Pre-assessment cortisol levels tended to be lower in the high-scoring group compared with the low-scoring group ( p = 0.058; Fig 3 ). Post-assessment cortisol levels were higher in the low-scoring group than in both the high-scoring ( p = 0.005) and medium-scoring ( p = 0.012) groups. Within the low-scoring group, post-assessment cortisol levels also tended to be higher than pre-assessment levels ( p = 0.059). Download figure Open in new tab Fig 3. Salivary cortisol levels in high-, medium-, and low-scoring groups. Cortisol levels were 5.996 ± 1.258 pg/mL (pre) and 6.295 ± 1.283 pg/mL (post) in the high-scoring group, 7.441 ± 2.379 pg/mL (pre) and 8.044 ± 1.296 pg/mL (post) in the medium-scoring group, and 11.581 ± 2.535 pg/mL (pre) and 31.213 ± 8.075 pg/mL (post) in the low-scoring group. Pre and Post indicate samples collected before and after temperament assessment, respectively. ** p < 0.01, * p < 0.05 3.4. Correlation between serotonin levels and temperament assessment scores Correlations between salivary serotonin levels and temperament assessment scores are summarized in Table 1 . A trend toward a positive correlation was observed between serotonin levels and the total average scores, although this result was not statistically significant ( p = 0.064). In contrast, a significant positive correlation was identified between serotonin levels and the Movement Stability subtest ( p = 0.009). No significant correlations were observed for the remaining subtests, including Unconscious Confidence ( p = 0.129), Sociality ( p = 0.118), Noise Stability ( p = 0.165), Desire for Play and Predation ( p = 0.108), Behavior in Stressful Situations ( p = 0.277), and Separation ( p = 0.159). View this table: View inline View popup Download powerpoint Table 1. Correlations between temperament assessment scores and salivary cortisol and serotonin levels 3.5. Comparison of serotonin levels among three groups To compare serotonin levels among groups, dogs were categorized into high- (n = 6), medium-(n = 5), and low-scoring (n = 5) groups based on their total average temperament assessment scores ( Table 2 ). Significant differences in the total average scores were observed among the groups ( p < 0.01). Serotonin levels in the high-scoring group were significantly higher than those in the low-scoring group ( p = 0.028; Fig 4 ). View this table: View inline View popup Download powerpoint Table 2. Average temperament assessment scores of each group for cortisol and serotonin analyses Download figure Open in new tab Fig 4. Salivary serotonin levels in high-, medium-, and low-scoring groups. Serotonin levels were 5.943 ± 1.958 ng/mL in the high-scoring group, 2.154 ± 0.666 ng/mL in the medium-scoring group, and 1.742 ± 0.348 ng/mL in the low-scoring group. * p < 0.05 4. Discussion In this study, we validated a temperament assessment based on the Wesen test by incorporating hormonal markers as physiological indicators. Establishing such physiological evidence strengthens the foundation for accurately evaluating canine temperament. A reliable assessment tool can help determine a dog’s suitability as a companion animal or for specific working purposes. Moreover, physiologically validated assessments may contribute to policies and legislation that promote the safe selection and responsible adoption of dogs with appropriate temperaments. We examined the associations between temperament assessment scores and salivary cortisol levels. Pre-assessment cortisol levels were negatively correlated with the total average score and several subtests. Post-assessment cortisol levels showed negative correlations with the total average score and all subtests. These findings provide physiological evidence supporting the convergent validity of the Wesen test. Lower cortisol levels, an established biomarker of acute stress and arousal in dogs, were associated with greater emotional stability. Our results align with previous studies reporting associations between cortisol levels and behavioral traits. Rayment and colleagues reported a slight positive correlation between cortisol levels and C-BARQ traits such as separation-related problems and touch sensitivity [ 16 ]. Similarly, McPeake and colleagues observed a negative correlation between post-assessment salivary cortisol levels and frustration-coping ability [ 10 ]. In a study investigating the relationship between long-term stress and temperament, Roth and colleagues discovered that hair cortisol levels were positively correlated with stranger-directed aggression and chasing traits, both assessed using the C-BARQ [ 9 ]. Rossi and colleagues also reported that lower cortisol levels were associated with more frequent play-soliciting and longer durations of exploratory behavior [ 38 ]. Collectively, these findings suggest that cortisol levels reflect behavioral traits related to stress responsiveness and emotional regulation across several temperament assessment tools. In this study, we identified a relationship between temperament scores and changes in salivary cortisol levels. Dogs with higher temperament scores exhibited more stable physiological responses. The negative correlation between cortisol changes and Wesen test scores suggests that behaviorally well-adjusted dogs may be more resilient to stress. These findings support the convergent validity of the Wesen test in assessing emotional stability and stress reactivity in dogs. Similar findings have been reported in other studies. McPeake and colleagues reported significant correlations between scores on the Canine Frustration Questionnaire and changes in salivary cortisol levels [ 10 ]. Similarly, the startling test has been validated through its association with physiological indicators such as cortisol levels and heart rate [ 11 ]. Another study found that high sociability, as measured by behavioral assessment tests, was associated with smaller variations in cortisol levels [ 39 ]. Overall, these findings underscore the consistent relationship between canine temperament traits—across several temperament assessment tools—and physiological responses related to stress. Additionally, we compared cortisol levels among three groups classified by temperament assessment scores. Pre-assessment cortisol levels tended to be lower in the high-scoring group than in the low-scoring group, consistent with reports that shelter dogs with more sociable temperaments exhibit lower baseline cortisol levels [ 40 ]. Likewise, dogs engaging in more positive interactions generally exhibit lower cortisol levels [ 41 ]. Post-assessment cortisol levels were significantly higher in the low-scoring group than in the medium- and high-scoring groups, indicating greater stress reactivity among dogs with lower temperament scores. Consistent with this finding, Shin and Shin reported that more sociable dogs exhibited lower cortisol levels following behavioral assessment than less sociable dogs [ 39 ]. These findings suggest that dogs with more stable or socially adaptive temperaments are less reactive to testing situations and experience lower stress levels. In the low-scoring group, cortisol levels tended to increase after the assessment, reflecting the overall trend observed in this study. Collectively, these results indicate that both basal and reactive cortisol levels reflect underlying temperament traits, with socially adaptive dogs exhibiting lower physiological stress responses in routine and test-related situations. Interestingly, Lensen and colleagues reported that salivary cortisol and chromogranin A (CgA) levels measured 10 min after a behavioral test were positively associated with high trainability and negatively associated with stranger-directed fear, respectively, in adult dogs [ 42 ]. However, these associations varied depending on the timing of sample collection. Cortisol levels measured 40 min after the test were associated with high energy, whereas CgA levels were related to low excitability, both of which may be considered undesirable traits. These findings highlight the importance of sampling time when interpreting stress-related biomarkers. We also analyzed the relationship between temperament assessment scores and salivary serotonin levels. Although overall temperament scores did not show a statistically significant correlation with serotonin levels, a positive trend was observed. This finding suggests that behavioral indicators of emotional stability are partly associated with serotonergic activity. The Movement Stability subtest, which evaluates traits such as relaxation, stability, and activeness, showed a significant positive correlation with serotonin levels. This finding supports the convergent validity of the subtest, indicating that higher serotonin levels are associated with calmer and more regulated behaviors. These results are consistent with previous findings on serotonin–behavior relationships in dogs. Rayment and colleagues reported a negative trend between serotonin levels and C-BARQ traits related to separation-related problems and touch sensitivity [ 16 ]. Similarly, another study identified a weak linear correlation between social behavioral scores and serotonin levels in shelter dogs [ 27 ]. Wright and colleagues also found that higher impulsivity scores on the Dog Impulsivity Assessment Scale (DIAS) were associated with lower serotonin levels [ 43 ]. Taken together, these studies and our findings suggest that serotonin levels are associated with several positive behavioral characteristics across diverse behavioral assessment tools. This highlights the potential of serotonin as a physiological marker of emotional and behavioral regulation in dogs. We also compared serotonin levels among three groups classified by temperament scores. Dogs in the high-scoring group exhibited significantly higher serotonin levels than those in the low-scoring group. This finding aligns with numerous studies reporting that higher serotonin levels are associated with lower aggression and anxiety in dogs [ 27 , 30 , 44 – 46 ]. Collectively, these findings provide physiological evidence supporting the reliability of the temperament assessment used in this study. However, one study reported no significant differences in serotonin levels between groups classified by factors assessed through the C-BARQ or DIAS [ 16 ]. This inconsistency may stem from methodological differences. Unlike the temperament assessment in our study, which involved direct behavioral observation, both the C-BARQ and DIAS rely on owner-reported data. Although these tools are validated, their outcomes can be influenced by individual variation in owner perception and experience. Moreover, previous studies suggest that factors such as learning history, environmental conditions, and breed characteristics also influence test outcomes [ 16 ]. Therefore, these factors should be considered when interpreting associations between behavioral assessments and physiological markers. Although this study yielded several noteworthy findings, certain limitations should be considered when interpreting the results. Owing to constraints in salivary sampling, fewer samples were available for serotonin analysis, resulting in a smaller dataset compared with that of cortisol. To improve reliability and generalizability, future studies should include a larger number of dogs. Previous research has shown that salivary stimulants do not significantly affect hormonal measurements [ 47 ]. Thus, their use may be considered in future studies to increase saliva yield. In this study, no significant differences in hormonal levels were observed between groups classified by sex or living environment. Nevertheless, these factors may influence hormonal responses under different conditions or study designs and should be considered in future investigations. Cobb and colleagues reported that variables such as breed and body weight have minimal effects on salivary cortisol levels [ 47 ]; however, they also found that intact females exhibited significantly higher cortisol levels than intact males and neutered dogs. Additionally, living environment influenced cortisol levels, and puppies under 6 months of age exhibited significantly lower cortisol levels than older dogs. Similarly, Sandri and colleagues found that cortisol levels were associated with body size and housing conditions [ 48 ]. Collectively, these findings suggest that biological and environmental factors, including body size, sex, reproductive status, age, and housing conditions, influence basal cortisol levels. Future research should explore how these factors interact with temperament assessment outcomes, such as those derived from the Wesen test, to clarify their combined effects on physiological stress responses in dogs. Although cortisol is a well-established biomarker of stress, future studies should incorporate other physiological indicators to improve the robustness of findings [ 49 ]. Heart rate is one such parameter. For example, Robinson and colleagues reported that heart rate correlated with several behavioral test batteries [ 50 ]. Moreover, King and colleagues found a strong correlation between heart rate and startling test scores [ 11 ]. Incorporating validated physiological markers and examining their interrelationships may enhance the reliability and robustness of future findings. In conclusion, this study validated a canine temperament assessment tool by examining its associations with cortisol and serotonin. Significant correlations were observed between temperament assessment scores and cortisol levels, and a potential association was observed with serotonin levels. Furthermore, both hormonal markers differed among groups categorized by temperament scores. These results support the utility of temperament assessments validated through physiological measures and contribute to a better understanding of the relationships among canine temperament, behavior, and endocrine function. Author contributions Conceptualization : Youngwook Jung, Kyongwon Yoo, Youngtae Heo, Minjung Yoon Data curation : Youngwook Jung Formal analysis : Youngwook Jung Funding acquisition : Kayoung Yang, Kyongwon Yoo, Youngtae Heo, Minjung Yoon Investigation : Youngwook Jung, Yujin Song, Kayoung Yang, Kyongwon Yoo, Youngtae Heo, Minjung Yoon Methodology : Youngwook Jung, Yujin Song, Kayoung Yang, Kyongwon Yoo, Youngtae Heo, Minjung Yoon Project administration : Minjung Yoon Resources : Kayoung Yang, Kyongwon Yoo, Youngtae Heo, Minjung Yoon Software : Youngwook Jung Supervision : Kayoung Yang, Kyongwon Yoo, Youngtae Heo, Minjung Yoon Validation : Youngwook Jung Visualization : Youngwook Jung Writing – original draft : Youngwook Jung Writing - review & editing : Youngwook Jung, Minjung Yoon Acknowledgements The authors would like to thank Dr. Yeonju Choi (Malgill Healing and Leadership, Republic of Korea) and Yubin Song, Taelang Kim, Jaewoo Choi, and Junseo Ko (Kyungpook National University, Republic of Korea) for their assistance with the temperament assessment. We also extend our gratitude to Dr. Heejun Jung (Korea Polytechnics, Republic of Korea) and Junyoung Kim, Gaeun Jeong, and Suhyun Kim (Kyungpook National University, Republic of Korea) for their support. Reference 1. ↵ Taylor KD , Mills DS . The development and assessment of temperament tests for adult companion dogs . Journal of Veterinary Behavior . 2006 ; 1 ( 3 ): 94 – 108 . OpenUrl 2. ↵ Bray EE , Sammel MD , Cheney DL , Serpell JA , Seyfarth RM . Effects of maternal investment, temperament, and cognition on guide dog success . Proceedings of the National Academy of Sciences . 2017 ; 114 ( 34 ): 9128 – 33 . OpenUrl Abstract / FREE Full Text 3. ↵ Maejima M , Inoue-Murayama M , Tonosaki K , Matsuura N , Kato S , Saito Y , et al. Traits and genotypes may predict the successful training of drug detection dogs . Applied Animal Behaviour Science . 2007 ; 107 ( 3-4 ): 287 – 98 . OpenUrl 4. ↵ Hare E , Kelsey KM , Serpell JA , Otto CM . Behavior differences between search-and-rescue and pet dogs . Frontiers in veterinary science . 2018 ; 5 : 118 . OpenUrl PubMed 5. ↵ Ruefenacht S , Gebhardt-Henrich S , Miyake T , Gaillard C . A behaviour test on German Shepherd dogs: heritability of seven different traits . Applied Animal Behaviour Science . 2002 ; 79 ( 2 ): 113 – 32 . OpenUrl CrossRef 6. ↵ Duffy DL , Serpell JA . Predictive validity of a method for evaluating temperament in young guide and service dogs . Applied Animal Behaviour Science . 2012 ; 138 ( 1-2 ): 99 – 109 . OpenUrl CrossRef 7. ↵ Bray EE , Levy KM , Kennedy BS , Duffy DL , Serpell JA , MacLean EL . Predictive models of assistance dog training outcomes using the canine behavioral assessment and research questionnaire and a standardized temperament evaluation . Frontiers in veterinary science . 2019 ; 6 : 49 . OpenUrl PubMed 8. ↵ Fuchs T , Gaillard C , Gebhardt-Henrich S , Ruefenacht S , Steiger A . External factors and reproducibility of the behaviour test in German shepherd dogs in Switzerland . Applied Animal Behaviour Science . 2005 ; 94 ( 3-4 ): 287 – 301 . OpenUrl 9. ↵ Roth LS , Faresjö Å , Theodorsson E , Jensen P . Hair cortisol varies with season and lifestyle and relates to human interactions in German shepherd dogs . Scientific Reports . 2016 ; 6 ( 1 ): 19631 . OpenUrl PubMed 10. ↵ McPeake KJ , Collins LM , Zulch H , Mills DS . Behavioural and physiological correlates of the Canine Frustration Questionnaire . Animals . 2021 ; 11 ( 12 ): 3346 . OpenUrl PubMed 11. ↵ King T , Hemsworth PH , Coleman GJ . Fear of novel and startling stimuli in domestic dogs . Applied Animal Behaviour Science . 2003 ; 82 ( 1 ): 45 – 64 . OpenUrl CrossRef 12. ↵ Dickerson SS , Kemeny ME . Acute stressors and cortisol responses: a theoretical integration and synthesis of laboratory research . Psychological bulletin . 2004 ; 130 ( 3 ): 355 . OpenUrl CrossRef PubMed Web of Science 13. ↵ Rosado B , Garcia-Belenguer S , Leon M , Chacon G , Villegas A , Palacio J . Effect of fluoxetine on blood concentrations of serotonin, cortisol and dehydroepiandrosterone in canine aggression . J Vet Pharmacol Ther . 2011 ; 34 ( 5 ): 430 – 6 . OpenUrl PubMed 14. ↵ Shiverdecker MD , Schiml PA , Hennessy MB . Human interaction moderates plasma cortisol and behavioral responses of dogs to shelter housing . Physiology & behavior . 2013 ; 109 : 75 – 9 . OpenUrl CrossRef PubMed 15. ↵ Shin YJ , Shin NS . Relationship between sociability toward humans and physiological stress in dogs . J Vet Med Sci . 2017 ; 79 ( 7 ): 1278 – 83 . OpenUrl PubMed 16. ↵ Rayment DJ , Peters RA , Marston LC , De Groef B . Relationships between serum serotonin, plasma cortisol, and behavioral factors in a mixed-breed,-sex, and-age group of pet dogs . Journal of Veterinary Behavior . 2020 ; 38 : 96 – 102 . OpenUrl 17. ↵ Packer RM , Davies AM , Volk HA , Puckett HL , Hobbs SL , Fowkes RC . What can we learn from the hair of the dog? Complex effects of endogenous and exogenous stressors on canine hair cortisol . PLoS One . 2019 ; 14 ( 5 ): e0216000 . OpenUrl PubMed 18. ↵ Sheriff MJ , Dantzer B , Delehanty B , Palme R , Boonstra R . Measuring stress in wildlife: techniques for quantifying glucocorticoids . Oecologia . 2011 ; 166 ( 4 ): 869 – 87 . OpenUrl CrossRef PubMed Web of Science 19. Buttner AP . Neurobiological underpinnings of dogs’ human-like social competence: How interactions between stress response systems and oxytocin mediate dogs’ social skills . Neuroscience & Biobehavioral Reviews . 2016 ; 71 : 198 – 214 . OpenUrl PubMed 20. ↵ Gold A , Langlois D , Refsal K . Evaluation of basal serum or plasma cortisol concentrations for the diagnosis of hypoadrenocorticism in dogs . Journal of veterinary internal medicine . 2016 ; 30 ( 6 ): 1798 – 805 . OpenUrl PubMed 21. ↵ Groschl M . Current status of salivary hormone analysis . Clinical chemistry . 2008 ; 54 ( 11 ): 1759 – 69 . OpenUrl Abstract / FREE Full Text 22. ↵ Jung Y , Yang K , Yoon M . Exploring the relationship between plasma and salivary levels of oxytocin, vasopressin, and cortisol in beagles: A preliminary study . Domestic Animal Endocrinology . 2025 ; 92 : 106937 . OpenUrl PubMed 23. ↵ Popova NK . From genes to aggressive behavior: the role of serotonergic system . Bioessays . 2006 ; 28 ( 5 ): 495 – 503 . OpenUrl CrossRef PubMed Web of Science 24. Amat M , Le Brech S , Camps T , Torrente C , Mariotti VM , Ruiz JL , et al. Differences in serotonin serum concentration between aggressive English cocker spaniels and aggressive dogs of other breeds . Journal of veterinary behavior . 2013 ; 8 ( 1 ): 19 – 25 . OpenUrl 25. ↵ León M , Rosado B , García-Belenguer S , Chacón G , Villegas A , Palacio J . Assessment of serotonin in serum, plasma, and platelets of aggressive dogs . Journal of Veterinary Behavior . 2012 ; 7 ( 6 ): 348 – 52 . OpenUrl 26. ↵ Vermeire ST , Audenaert KR , Dobbeleir AA , De Meester RH , De Vos FJ , Peremans KY . Evaluation of the brain 5-HT2A receptor binding index in dogs with anxiety disorders, measured with 123I-5I-R91150 and SPECT . Journal of Nuclear Medicine . 2009 ; 50 ( 2 ): 284 – 9 . OpenUrl Abstract / FREE Full Text 27. ↵ Alberghina D , Rizzo M , Piccione G , Giannetto C , Panzera M . An exploratory study about the association between serum serotonin concentrations and canine-human social interactions in shelter dogs (Canis familiaris) . Journal of Veterinary Behavior . 2017 ; 18 : 96 – 101 . OpenUrl 28. ↵ Humphrey J , Toh C . Absorption of serotonin (5-hydroxytryptamine) and histamine by dog platelets . The Journal of Physiology . 1954 ; 124 ( 2 ): 300 . OpenUrl PubMed Web of Science 29. González-Martínez Á , Muñiz de Miguel S , Graña N , Costas X , Diéguez FJ . Serotonin and dopamine blood levels in ADHD-like dogs . Animals . 2023 ; 13 ( 6 ): 1037 . OpenUrl PubMed 30. ↵ Rosado B , García-Belenguer S , León M , Chacón G , Villegas A , Palacio J . Blood concentrations of serotonin, cortisol and dehydroepiandrosterone in aggressive dogs . Applied Animal Behaviour Science . 2010 ; 123 ( 3-4 ): 124 – 30 . OpenUrl CrossRef PubMed 31. ↵ Egri C , Dunbar M , Horvath GA . Correlation between salivary, platelet and central serotonin levels in children . Canadian Journal of Neurological Sciences . 2020 ; 47 ( 2 ): 214 – 8 . OpenUrl PubMed 32. ↵ Leung J , Selvage C , Bosdet T , Branov J , Rosen-Heath A , Bishop C , et al. Salivary serotonin does not correlate with central serotonin turnover in adult phenylketonuria (PKU) patients . Molecular Genetics and Metabolism Reports . 2018 ; 15 : 100 – 5 . OpenUrl 33. ↵ Matsunaga M , Ishii K , Ohtsubo Y , Noguchi Y , Ochi M , Yamasue H . Association between salivary serotonin and the social sharing of happiness . PloS one . 2017 ; 12 ( 7 ): e0180391 . OpenUrl CrossRef PubMed 34. Karbownik MS , Hicks SD . The Association of Salivary Serotonin With Mood and Cardio-Autonomic Function: A Preliminary Report . Frontiers in Psychiatry . 2022 ; 13 : 788153 . OpenUrl PubMed 35. ↵ Tan Z-L , Bao A-M , Tao M , Liu Y-J , Zhou J-N . Circadian rhythm of salivary serotonin in patients with major depressive disorder . Neuroendocrinology Letters . 2007 ; 28 ( 4 ): 395 – 400 . OpenUrl PubMed 36. ↵ Dogs A-TGS . Wesensüberprüfung – Wesen Test n.d. [Available from: https://24kgsd.com/wesen-test/ . 37. ↵ Mittmann A. Untersuchung des Verhaltens von 5 Hunderassen und einem Hundetypus im Wesenstest nach den Richtlinien der Niedersächsischen Gefahrtierverordnung vom 05.07. 2000 2002 . 38. ↵ Rossi A , Parada FJ , Stewart R , Barwell C , Demas G , Allen C . Hormonal correlates of exploratory and play-soliciting behavior in domestic dogs . Frontiers in psychology . 2018 ; 9 : 1559 . OpenUrl PubMed 39. ↵ Shin Y-J , Shin N-S . Relationship between sociability toward humans and physiological stress in dogs . J Vet Med Sci . 2017 ; 79 ( 7 ): 1278 – 83 . OpenUrl PubMed 40. ↵ De Palma C , Viggiano E , Barillari E , Palme R , Dufour AB , Fantini C , et al. Evaluating the temperament in shelter dogs . Behaviour . 2005 : 1307 – 28 . 41. ↵ Mârza SM , Munteanu C , Papuc I , Radu L , Diana P , Purdoiu RC. Behavioral, Physiological, and Pathological Approaches of Cortisol in Dogs . Animals . 2024 ; 14 ( 23 ): 3536 . OpenUrl PubMed 42. ↵ Lensen RC , Moons CP , Diederich C . Physiological stress reactivity and recovery related to behavioral traits in dogs (Canis familiaris) . PloS one . 2019 ; 14 ( 9 ): e0222581 . OpenUrl PubMed 43. ↵ Wright HF , Mills DS , Pollux PM . Behavioural and physiological correlates of impulsivity in the domestic dog (Canis familiaris) . Physiology & behavior . 2012 ; 105 ( 3 ): 676 – 82 . OpenUrl CrossRef PubMed 44. ↵ Çakiroǧlu D , Meral Y , Sancak A , Cifti G . Relationship between the serum concentrations of serotonin and lipids and aggression in dogs . Veterinary record . 2007 ; 161 ( 2 ): 59 – 61 . OpenUrl Abstract / FREE Full Text 45. Rosado B , García-Belenguer S , Palacio J , Chacón G , Villegas A , Alcalde AI . Serotonin transporter activity in platelets and canine aggression . The Veterinary Journal . 2010 ; 186 ( 1 ): 104 – 5 . OpenUrl PubMed 46. ↵ Bochiș TA , Imre K , Marc S , Vaduva C , Florea T , Dégi J , et al. The variation of serotonin values in dogs in different environmental conditions . Veterinary Sciences . 2022 ; 9 ( 10 ): 523 . OpenUrl PubMed 47. ↵ Cobb M , Iskandarani K , Chinchilli V , Dreschel N . A systematic review and meta-analysis of salivary cortisol measurement in domestic canines . Domestic Animal Endocrinology . 2016 ; 57 : 31 – 42 . OpenUrl CrossRef PubMed 48. ↵ Sandri M , Colussi A , Perrotta MG , Stefanon B . Salivary cortisol concentration in healthy dogs is affected by size, sex, and housing context . Journal of Veterinary Behavior . 2015 ; 10 ( 4 ): 302 – 6 . OpenUrl 49. ↵ Diederich C , Giffroy J-M . Behavioural testing in dogs: A review of methodology in search for standardisation . Applied Animal Behaviour Science . 2006 ; 97 ( 1 ): 51 – 72 . OpenUrl CrossRef Web of Science 50. ↵ Robinson LM , Skiver Thompson R , Ha JC . Puppy temperament assessments predict breed and American Kennel Club group but not adult temperament . Journal of Applied Animal Welfare Science . 2016 ; 19 ( 2 ): 101 – 14 . OpenUrl PubMed View the discussion thread. Back to top Previous Next Posted November 17, 2025. Download PDF Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Share Associations between canine temperament and salivary levels of cortisol and serotonin Youngwook Jung , Yujin Song , Kayoung Yang , Kyongwon Yoo , Youngtae Heo , Minjung Yoon bioRxiv 2025.11.16.688729; doi: https://doi.org/10.1101/2025.11.16.688729 Share This Article: Copy Citation Tools Associations between canine temperament and salivary levels of cortisol and serotonin Youngwook Jung , Yujin Song , Kayoung Yang , Kyongwon Yoo , Youngtae Heo , Minjung Yoon bioRxiv 2025.11.16.688729; doi: https://doi.org/10.1101/2025.11.16.688729 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Physiology Subject Areas All Articles Animal Behavior and Cognition (7635) Biochemistry (17697) Bioengineering (13895) Bioinformatics (41951) Biophysics (21456) Cancer Biology (18594) Cell Biology (25520) Clinical Trials (138) Developmental Biology (13381) Ecology (19903) Epidemiology (2067) Evolutionary Biology (24323) Genetics (15612) Genomics (22510) Immunology (17738) Microbiology (40401) Molecular Biology (17184) Neuroscience (88622) Paleontology (667) Pathology (2833) Pharmacology and Toxicology (4825) Physiology (7644) Plant Biology (15158) Scientific Communication and Education (2046) Synthetic Biology (4296) Systems Biology (9825) Zoology (2271)