Metabolomic Characterization of Canine Behavioural Disorders : Fearfulness and Hyperactivity/Impulsivity

Psychiatric disorders in humans and their counterparts, behavioural disorders in dogs, are the major welfare problems in both species affecting the wellbeing of millions of people and dogs worldwide. Despite extensive research elucidating the pathobiological events leading to the onset of psychiatric/behavioural disorders, the exact etiology remains unattainable. Moreover, the complexity and multicausality of these mental disorders hampers research, addressing the need for more comprehensive approaches to provide insights into the molecular mechanisms of these disorders and to identify disorder-specific biomarkers. The aim of this thesis was to pilot the use of a non-targeted metabolomics approach in canine behavioural research, and to provide novel molecular information concerning two specific behavioural disorders in pet dogs, fearfulness and hyperactivity/impulsivity. Fearfulness is the most common behavioural disorder in dogs, characterized by excessive fear response when confronted with a threatening stimulus. During this thesis work, two separate non-targeted metabolomics characterization studies of canine fearfulness were conducted. The first study aimed to pilot the feasibility and potential of metabolomics technology in canine behavioural research, whereas the second metabolic characterization of canine fear was conducted with a larger sample size to optimize theoretical and analytical limitations observed in the first pilot study. The results showed clear differences in the blood metabolic profiles of fearful and non-fearful control dogs, including increased plasma glutamine abundance in fearful dogs. These alterations potentially originate from the systemic effects of chronic psychological stress. Hyperactive/impulsive dogs manifest inappropriate levels of activity, impulsivity and inattention, corresponding to attention deficit hyperactive disorder (ADHD) in humans. The non-targeted plasma metabolomics showed lower levels of plasma phospholipids in addition to altered tryptophan metabolism in hyperactive and impulsive dogs. These changes may reflect disturbances in the gut microbiota composition in the affected dogs. Collectively, this thesis has demonstrated the feasibility of metabolomics in canine behavioural research and provided novel molecular correlates and potential biomarkers for canine fearfulness and hyperactivity/impulsivity. Additionally, this study identifies changes which also had been reported in other species. This suggests that dogs could be used as a model to aid in gaining better understanding of human psychiatric disorders.Mielenterveyden häiriöt ihmisillä sekä koirien vastaavat tautitilat, käytöshäiriöt, ovat yleisimpiä elämänlaatua heikentäviä hyvinvointiongelmia molemmilla lajeilla. Laajamittaisista tutkimuksista huolimatta kyseisten tautitilojen etiologia on edelleen suurilta osin tuntematon. Sekä mielenterveyden häiriöt että käytöshäiriöt ovat taustaltaan monisyisiä sairauksia, mikä vaikeuttaa tutkimuksia. Sen vuoksi tarvitaan kokonaisvaltaisempia lähestymistapoja, jotka voivat tarjota lisätietoa tautitilojen molekulaarisista mekanismeista sekä tunnistaa tautikohtaisia merkkiaineita, biomarkkereita. Tässä väitöskirjassa pilotoitiin kohdentamattoman metabolomiikan eli aineenvaihdunnan tutkimuksen käyttöä koirien käyttäytymistutkimuksessa. Tavoitteena oli tuottaa uutta molekyylitason tietoa kahdesta käytöshäiriöstä, arkuudesta sekä yliaktiivisuudesta ja impulsiivisuudesta. Arkuus on yleisin koirilla esiintyvä käytöshäiriö, ja se ilmenee liiallisina pelkoreaktioina uhkaavan ärsykkeen, kuten toisen koiran tai vieraan ihmisen, läsnä ollessa. Tässä väitöskirjatyössä arkojen koirien aineenvaihduntaa tutkittiin kahdessa erillisessä tutkimuksessa. Ensimmäisen tutkimuksen tarkoitus oli testata metabolomiikan soveltuvuutta koirien käyttäytymistutkimuksessa, kun taas toisessa tutkimuksessa kehitettiin pilottitutkimuksessa havaittuja teoreettisia ja analyyttisiä puutteita suuremmassa aineistossa. Tulokset osoittivat, että arkojen ja ei-arkojen verrokkikoirien aineenvaihdunnassa oli selkeitä eroja, kuten kohonneet plasman glutamiinipitoisuudet aroilla koirilla. Nämä löydökset saattavat johtua arkojen koirien kärsimän kroonisen psykologisen stressin kokonaisvaltaisista seurauksista. Yliaktiiviset ja impulsiiviset koirat kärsivät jatkuvasta ja sopimattomasta yliaktiivisuudesta, impulsiivisuudesta ja tarkkaamattomuudesta. Kyseinen tautitila vastaa ihmisillä esiintyvää tarkkaavuus- ja yliaktiivisuushäiriötä (ADHD). Metabolomiikka-analyysi paljasti madaltuneen fosfolipidien määrän sekä aminohappo tryptofaanin aineenvaihdunnan häiriöiden olevan yhteydessä koirien yliaktiiviseen ja impulsiiviseen käytökseen. Havaitut muutokset voivat heijastua häiriintyneestä suoliston mikrobitasapainosta. Kaiken kaikkiaan tämä väitöskirjatyö on luonut pohjaa metabolomiikan laajemmalle käytölle koirien käyttäytymistutkimuksessa ja tarjonnut uutta molekyylitason tietoa koirien arkuudesta sekä hyperaktiivisuudesta ja impulsiivisuudesta. Lisäksi tutkimus on paljastanut molekyylitason yhtäläisyyksiä eri lajien välillä, mikä voi tulevaisuudessa hyödyttää eläinlääketieteen lisäksi myös ihmisen mielenterveyden häiriöiden tutkimusta

A novel canine nuclear magnetic resonance spectroscopy-based metabolomics platform : Validation and sample handling

Background Metabolomics has been proven to be an invaluable research tool by providing comprehensive insight into systemic metabolism. However, the lack of scalable and quantitative methods with known reference intervals (RIs) and documented reproducibility has prevented the use of metabolomics in the clinical setting. Objective The objective of this study was to validate the developed quantitative nuclear magnetic resonance (NMR) spectroscopy-based metabolomics platform for canine serum and plasma samples and determine optimal sample handling conditions for its use. Methods Altogether, 8247 canine samples were analyzed using a Bruker's 500 MHz NMR spectrometer. Using statistical approaches derived from international guidelines, we studied method precision, measurand stability in various long- and short-term storage conditions, as well as the effect of prolonged contact with red blood cells (RBCs), and differences among blood collection tubes. We also screened interferences with lipemia, hemolysis, and bilirubinemia. The results were compared against routine clinical chemistry methods, and RIs were defined for all measurands. Results We determined RIs for 123 measurands, most of which were previously unpublished. The reproducibility of the results of the NMR platform appeared generally outstanding, and the integrity of the results can be ensured by following standard blood drawing and processing guidelines. Conclusions Owing to the advantages of quantitative results, high reproducibility, and scalability, this canine metabolomics platform holds great potential for numerous clinical and research applications to improve canine health and well-being.Peer reviewe

Age, breed, sex and diet influence serum metabolite profiles of 2000 pet dogs

As an individual's metabolism reflects health and disease states well, metabolomics holds a vast potential in biomedical applications. However, normal physiological factors, such as age, can also influence metabolism, challenging the establishment of disease-specific metabolic aberrations. Here, we examined how physiological and diet-related factors drive variance in the metabolism of healthy pet dogs. We analysed 2068 serum samples using a canine nuclear magnetic resonance (NMR) spectroscopy-based metabolomics platform. With generalized linear models, we discovered that age, breed, sex, sterilization, diet type and fasting time significantly affected the canine metabolite profiles. Especially, breed and age caused considerable variation in the metabolite concentrations, and breeds with very different body conformations systematically differed in several lipid measurands. Our results enhance the understanding how normal physiological factors influence canine metabolism, aid accurate interpretation of the NMR results, and suggest the NMR platform might be applied in identifying aberrations in nutrient absorption and metabolism.Peer reviewe

Personality traits associate with behavioral problems in pet dogs

Personality traits, especially neuroticism, strongly predict psychopathology. The domestic dog (Canis lupus familiaris Linnaeus, 1758) is used as a natural model for psychiatric disorders, but the similarity between dog and human personality and the association between dog personality and unwanted behavioral traits, such as fearfulness, aggressiveness, and impulsivity/inattention, remain unknown. This study utilized structural equation modeling (SEM) with survey data of 11,360 dogs to examine the associations and correlations between seven personality and ten unwanted behavioral traits. Personality traits included insecurity, energy, training focus, aggressiveness/dominance, human sociability, dog sociability, and perseverance. Unwanted behavioral traits included fearfulness, noise sensitivity, fear of surfaces/heights, separation anxiety, barking, stranger-directed aggression, owner-directed aggression, dog-directed aggression, hyperactivity/impulsivity, and inattention. We first fitted confirmatory factor models for the unwanted behavioral traits and the best model grouped unwanted behaviors into four latent traits: fear-related behavior, fear-aggression, aggression, and impulsivity/inattention and used this structure in the subsequent SEM model. Especially, insecurity, which resembles the human neuroticism trait, was strongly associated with unwanted behavior, paralleling the association between neuroticism and psychopathology. Similarly, training focus, resembling conscientiousness, was negatively related to impulsivity/inattention, and aggressiveness/dominance was associated with aggressive behaviors, resembling associations of conscientiousness and agreeableness with attention deficit hyperactivity disorder and aggression-related psychopathology, respectively. These results indicate that dog personality traits resemble human personality traits, suggesting that their neurological and genetic basis may also be similar and making the dog a suitable animal model for human behavior and psychiatric disorders.Peer reviewe

Fearful dogs have increased plasma glutamine and gamma-glutamyl glutamine

Anxiety-related disorders, including fearfulness are common and leading welfare problems among the worldwide dog population. The etiology of anxieties is complex and affected by genetic and environmental factors. Thus, there is a need for more comprehensive approaches, such as metabolomics, to understand the causes of anxiety and to identify anxiety-related biomarkers for more efficient diagnostic and treatment options. To study metabolic alterations related to canine fearfulness, a non-targeted plasma metabolite profiling was performed in a cohort of 20 fearful and 21 non-fearful dogs. The results showed that nine metabolic features were significantly associated with fearfulness. The most prominent change included increased plasma glutamine and gamma-glutamyl glutamine (gamma-Glu Gln) in fearful dogs across breeds. Alterations in glutamine metabolism have previously been associated with several psychiatric disorders, indicating the relevance of this finding also in dogs. In addition, we describe a novel breed-specific association between renal biomarker symmetric dimethylarginine (SDMA) and canine fearfulness. These observed metabolic alterations may result from high levels of prolonged psychological stress in fearful dogs.Peer reviewe

Metabolic changes associated with two endocrine abnormalities in dogs : elevated fructosamine and low thyroxine

Introduction Metabolomics studies in canine endocrine abnormalities are sparse and basic information on these abnormalities must be generated. Objectives To characterize the metabolic changes associated with elevated fructosamine, reflecting poor glycemic control, and low thyroxine, a thyroid hormone controlling metabolism. Methods Leftovers of clinical serum samples; 25 controls, 79 high fructosamine, and 47 low thyroxine, were analyzed using H-1 NMR and differences were evaluated using Firth logistic regression. Results Both high fructosamine and low thyroxine were associated with changes in concentrations of multiple metabolites, including glycoprotein acetyls and lipids. Conclusion These findings suggest promising makers for further research and clinical validation.Peer reviewe

Aggressiveness, ADHD-like behaviour, and environment influence repetitive behaviour in dogs

Repetitive behaviour ranges from variants of normal repetitive behaviours to abnormal repetitive behaviours. The domestic dog spontaneously performs different repetitive behaviours, which can be severe and impair the quality of life and the dog-owner relationship. We collected comprehensive behavioural questionnaire data from almost 4500 Finnish pet dogs and studied the effect of several demographic, environmental, and behavioural factors on canine repetitive behaviour with logistic regression. We replicated findings from previous studies by revealing comorbidity between repetitive behaviour and behavioural factors aggressiveness, hyperactivity/impulsivity, and inattention. We also found a novel association between repetitive behaviour and the owner's dog experience. In addition, we showed that repetitive behaviour is more common in dogs that live without conspecifics, dogs that were given a low amount of exercise, dogs that lived in larger families, young dogs and elderly dogs, and neutered dogs. Finally, we identified breed differences in repetitive behaviour, suggesting that some breeds are more vulnerable to repetitive behaviour and indicate a genetic susceptibility. As abnormal repetitive behaviour can considerably worsen the well-being of dogs and impair the dog-owner relationship, a better understanding of the environmental, lifestyle, and molecular factors affecting canine repetitive behaviour can benefit both dogs and humans.Peer reviewe

Active and social life is associated with lower non-social fearfulness in pet dogs

Behavioural problems are leading welfare issues in domestic dogs. In particular, anxiety-related behavioural problems, such as fearfulness and noise sensitivity are highly prevalent conditions that cause distress to fearful dogs. To better understand the environmental factors associated with non-social fear, including noise sensitivity, fear of novel situations, and fear of surfaces and heights, a large online survey including data on 13,700 Finnish pet dogs was performed by the dog owners. After fulfilling inclusion criteria, this data consisted of 9,613 dogs with fear of fireworks, 9,513 dogs with fear of thunder, 6,945 dogs with fear of novel situations, and 2,932 dogs with fear of surfaces and heights. Logistic regression analyses revealed that dogs with frequent non-social fear had experienced less socialisation during puppyhood, were more often neutered, had inexperienced owners, lived without conspecifics, participated less frequently in activities or training, and lived in more urban environments. In addition, we identified several breed differences, and a tendency of more common non-social fear in small dog breeds, which suggests a genetic background. Non-social fearfulness has a negative effect on well-being of the dogs. Our findings suggest that the socialisation and the living environment and the value of other dogs' company and owner interaction via activities and training may improve the well-being of the dogs.Peer reviewe

Inadequate socialisation, inactivity, and urban living environment are associated with social fearfulness in pet dogs

Problematic behaviours are severe welfare issues for one of the world's most popular pets, the domestic dog. One of the most prevalent behavioural problem that causes distress to dogs is social fearfulness, meaning fear of conspecifics or unfamiliar people. To identify demographic and environmental factors associated with fear of dogs and strangers, logistic regression was utilised with a large dataset of 6,000 pet dogs collected through an owner-filled behavioural survey. Social fearfulness was associated with several factors, including urban environment, poor socialisation during puppyhood, infrequent participation in training and other activities, small body size, female sex, and neutering. In addition, we identified several breed differences, suggesting a genetic contribution to social fearfulness. These findings highlight the role of inadequate socialisation, inactivity, and urban living environmental in fear-related behavioural problems in dogs. Improvements in the management and breeding practices of dogs could, therefore, enhance the welfare of man's best friend.Peer reviewe