Gut-Brain Axis Impact on Canine Anxiety Disorders: New Challenges for Behavioral Veterinary Medicine

Anxiety disorders in dogs are ever-growing and represent an important concern in the veterinary behavior field. These disorders are often disregarded in veterinary clinical practice, negatively impacting the animal's and owner's quality of life. Moreover, these anxiety disorders can potentially result in the abandonment or euthanasia of dogs. Growing evidence shows that the gut microbiota is a central player in the gut-brain axis. A variety of microorganisms inhabit the intestines of dogs, which are essential in maintaining intestinal homeostasis. These microbes can impact mental health through several mechanisms, including metabolic, neural, endocrine, and immune-mediated pathways. The disruption of a balanced composition of resident commensal communities, or dysbiosis, is implicated in several pathological conditions, including mental disorders such as anxiety. Studies carried out in rodent models and humans demonstrate that the intestinal microbiota can influence mental health through these mechanisms, including anxiety disorders. Furthermore, novel therapeutic strategies using prebiotics and probiotics have been shown to ameliorate anxiety-related symptoms. However, regarding the canine veterinary behavior field, there is still a lack of insightful research on this topic. In this review, we explore the few but relevant studies performed on canine anxiety disorders. We agree that innovative bacterial therapeutical approaches for canine anxiety disorders will become a promising field of investigation and certainly pave the way for new approaches to these behavioral conditions.


Introduction
In modern societies, dogs assume important roles, mainly due to their companionship [1], as they are considered family members [2].Apart from dogs' intrinsic characteristics, their owners' lifestyles, socioeconomic conditions, work routines, and specifc personality traits can potentiate canine behavioral problems.Tese include anxiety disorders, which represent a serious concern for animal welfare, and can further impact every other aspect of human society, including the economy, public health, and others.Te combination of genetic factors, environment, and experiences in dogs' lives determines their behavioral development [1].
Anxiety is defned as a "preparatory response made in anticipation of threatening stimuli or scenarios" [3].Tis adaptative response is the root cause of many canine behavior problems [3] and can result in a chronically stressful life for dogs [4].An estimated 72.5% of dogs showed anxiety-like behaviors to a certain extent [4].Te Lincoln Canine Anxiety Scale has been validated as a reliable measurement to assess canine anxiety, which classifes this condition based on the severity of typical behaviors (Table 1) [3], which, from the most common to the least, include noise sensitivity (32%), fear (29%), excessive activity (15%), compulsive behavior (16%), aggression (14%), and separation-related behaviors (6%) [4].
Dogs with anxiety disorders are reported to be more vulnerable to other diseases and have shorter lifespans [4].Additionally, these traits, particularly aggressiveness, might be of public health importance [5].Anxiety disorders impact dogs' and owners' quality of life and afect their bond [6].Plus, it is likely to result in dogs being abandoned or even euthanized [7].Tis is a growing concern and requires further research [6].To this end, the role of the gut microbiome in the pathophysiology of these behavioral disorders has recently started to be investigated.
Although research has addressed the infuence of microbiota on anxiety disorders in humans and rodent models, there is a lack of insightful information on canine anxiety disorders.In this review, we intend to highlight recent data on the role of the microbiota in the gut-brain axis (GBA) and its impact on anxiety disorders in human and rodent models and, most importantly, to emphasize the exploratory yet relevant research applied to canine anxiety disorders.We also aim to highlight the positive outcomes that enhanced research in this area will provide for the future of behavioral veterinary medicine, the wellbeing of dogs, and the subsequent efect on society.

Materials and Methods
In this review, we provide a comprehensive overview of the gut-brain axis and its impact on anxiety disorders, summarizing the experimental and clinical evidence on the mechanisms involved and their infuence on mental health.Te revision was mostly based on scientifc articles published between 2015 and 2022, as well as a few earlier works.Te research was conducted on the PubMed and Google Scholar databases after removing duplicate references.Te Medical Subject Headings (MeSH) terms "anxiety," "gut-brain axis," "hormones," "immune cells," "microbiota," "neurotransmitters," "intestinal permeability," "zonulin," prebiotics," and "probiotics" were cross-referenced and used in the search platforms.Experimental or observational studies in humans, rodents, and dogs were identifed and compared.Relevant clinical and experimental articles written in the English language have been included.

The Canine Microbiome and the
Gut-Brain Axis 3.1.Microbiome.Te term microbiome is defned as a "characteristic microbial community occupying a reasonable welldefned habitat which has distinct physiochemical properties" [8].Tis term not only includes the microbiota, which are the microorganisms involved, but also encompasses the environmental conditions, microbial structural elements, and metabolites.Te microbiota encompasses groups of bacteria, fungi, algae, archaea, and protists inhabiting the gastrointestinal tract [8].Tis population of microorganisms is essential to support and promote the host's health [2,9], as they participate in a large variety of physiological processes, including energy demand, metabolic reactions, immunological responses, and neurobehavioral development [10].
In healthy dogs, the gut microbiota is dominated by members of the phyla Bacteroidetes, Fusobacteria, and Firmicutes and, to a lesser extent, of the phyla Actinobacteria and Proteobacteria [2,9,11].A high abundance of members of the genera Fusobacterium, Prevotella, and Bacteroides was detected in a metagenomic study that analyzed the feces of six wolves and 169 dogs from diferent breeds [9].Several environmental factors, such as diet [9], body weight [2], age [12], and others, can infuence the gut microbiota structure and composition.However, the impact of these factors is mild compared to the changes potentially induced by diseases [11].
Gut dysbiosis, defned as an altered composition of microbes [8], can lead to alterations in the microbial transcriptome, proteome, or metabolome [11].Imbalances in the gut microbiota population are linked to pathological states, such as infammation, obesity, metabolic alterations, and even mood disorders.Further, gut microbiota afects physiological, neuronal, and behavioral functions [13].Terefore, homeostasis in this ecosystem is fundamental for maintaining the overall health condition [10].

Te Gut-Brain Axis.
Te gut microbiome communicates bidirectionally with the host central nervous system through the GBA, a complex network capable of regulating cognitive functions and behavior through several diferent mechanisms, including neural, metabolic, endocrine, and immunemediated signaling pathways [14].Te mechanisms involved in this regulation are summarized in Figure 1.

Metabolic Pathways.
Te intestinal microbiome and the host have a complex symbiotic relationship [8].Gut bacteria produce metabolites, such as short-chain fatty acids (SCFAs), from undigested dietary fbers that reach the colon, obtaining energy for their metabolism.SCFAs, including acetate, propionate, and butyrate, are essential for intestinal homeostasis [17], as they represent the primary energy sources for luminal colon cells [18].In addition to the nourishment of intestinal epithelial cells, another important function is the maintenance of intestinal barrier functions [17].Of particular interest, butyrate is reported to have a major role in supporting intestinal barrier integrity through the modulation of the expression of tight junction proteins, which greatly infuence intestinal permeability [19].SCFAs also possess anti-infammatory and immunomodulatory efects [17], as they can modulate innate and adaptive immune responses [20].Furthermore, SCFAs are also capable of crossing the blood-brain barrier (BBB), infuencing endocrine responses [18,21], and signaling the brain through vagus aferent fbers [18].Propionate was found to protect the BBB from oxidative stress through nuclear factor erythroid 2-related factor 2 signaling and to inhibit pathways linked to microbial infections [21].Supplementation with butyrate and other SCFAs restored brain 2 Veterinary Medicine International function in rodents, with marked improvements in stress behaviors and no signifcant impact on microbial diversity [22,23].Diet and microbiota composition infuence the production of these organic acids [19].A decrease in microbiota diversity and reduced ratios of SCFA-producing bacteria was observed in subjects with generalized anxiety disorders, suggesting that lower SCFAs production could afect the intestinal barrier function [24].Furthermore, SCFAs have been shown to attenuate cortisol response to acute stress, suggesting their potential infuence on psychobiological processes [18].

Neural Mechanisms.
Communication through the GBA is ensured by the autonomic nervous system.Vagus nerve neurons can perceive microbiota metabolites at the mucosa level, such as SCFAs, through its aferent fbers, which are distributed in the intestinal wall.Te gut information is transmitted to the central nervous system, and consequently, a response can be generated.Moreover, vagus nerve fbers integrate a cholinergic anti-infammatory system, which can decrease peripheral infammation and reduce intestinal permeability.Tus, stress stimuli might inhibit a vagal tone and afect gut microbiota composition and intestinal health [25].
Te gut microbiota can interfere with neural mechanisms by regulating neurotransmitter levels, including serotonin (5-HT), dopamine, norepinephrine, and gamma-aminobutyric acid (GABA).Microbiota can produce and contribute to regulate their metabolic pathways in intestinal cells, thus infuencing nervous system signaling and, ultimately, mental health [26].
GABA is an inhibitory neurotransmitter in the central nervous system and enteric nervous system [36].Important microbial sources of GABA are lactic acid bacteria, namely, the Lactobacillus, Enterococcus, Leuconostoc, Pediococcus, Propionibacterium, and Weissella genera [37].GABA-ergic neurotransmission inhibits the amygdala and prevents inappropriate emotional and behavioral responses [38].Changes in GABA regulation are linked with stress and anxiety [38], and daily GABA administration [39] and modulation of GABA-A receptor signaling [40] has been found to reduce anxiety symptoms in stressed rodents.Veterinary Medicine International All these data strongly suggest the interplay between gut microbiota, neural mechanisms, and behavior; however, further studies are required to evaluate this link accurately.

Endocrine Routes.
In addition to the abovementioned pathways, gut microbiota controls endocrine mechanisms that integrate the GBA by regulating the hypothalamic-pituitary-adrenal (HPA) axis.Tis system is fundamental for basal homeostasis and controls several body processes in response to stress factors [41].In stress situations, corticotropin-releasing factor (CRF) is secreted from the hypothalamus and induces adrenocorticotropic hormone (ACTH) discharge in blood circulation.In turn, glucocorticoids are secreted from the cortex of the adrenal gland.Stress events can activate the HPA axis, leading to an increase in cortisol levels [41].Te In an infammatory environment, characterized by gut dysbiosis and increased intestinal permeability, microbiota can translocate through the intestinal barrier.Moreover, immune cells may produce proinfammatory cytokines, while the gut microbiota itself may produce metabolites such as SCFAs and neurotransmitters that can directly impact mental health.Original illustration based on [15,16] captioned as shown above: 1-hypothalamus, 2-pituitary gland, 3-adrenal gland, ACTH-adrenocorticotropic hormone, CRF-corticotropin-releasing factor, and SCFAs-short chain fatty acids.Veterinary Medicine International hyperactivation of the HPA axis has been associated with, anxiety traits in children with low socioeconomic environments [42], and stressful circumstances [43].Te complex feedback mechanism behind the HPA axis might be partially regulated by the gut microbiota since some bacteria might induce its overactivity, thus infuencing behavior patterns [44].On the other hand, stressful situations and further activation of the HPA axis can result in changes in gut microbiota composition [41].Consequently, the intestinal barrier integrity might be afected, thus infuencing intestinal permeability, which might induce an infammatory response wherein mediators such as cytokines and prostaglandins can activate the HPA axis [45].Besides the negative impact on immunologic states, a persistent hyperactivation of the HPA axis might alter the hippocampal structure, potentially resulting in altered neurogenesis, the morphology of neurons, or even cellular death [44].In this complex interplay, a role for gut microbiota in these immune-neuroendocrine mechanisms is suggested [41].
In the brain, cytokines might infuence the metabolism of neurotransmitters, such as 5-HT, dopamine, and glutamate; interfere with the function of the HPA axis, thus afecting the production of the involved hormones; activate the nuclear factor kappa-light-chain-enhancer of activated B cells, consequently afecting the development of neural tissues; or target brain neurocircuits that control motivation, reward, anxiety, arousal, and alarm states [47].Hence, infammatory cytokines can have essential roles in neurodegenerative mood disorders' onset and development stages [48].

Intestinal Permeability
Te intestinal barrier is a complex functional structure that protects the organism from pathogen invasion and toxins, simultaneously allowing the absorption of nutrients and electrolyte changes [19].Tight junctions (Tj) play an important role in this regulation as they support the barrier's integrity.Tese multiprotein complexes are located at the IECs, specifcally at the apical ends of the lateral membranes.Tj proteins, including claudin and occludin, support its structure as they form a selectively permeable barrier in the paracellular pathways [53].Several other elements are essential to intestinal barrier homeostasis, such as goblet cells that secrete mucin, essential to forming the outside mucus layer, which protects the epithelial surface [19].Moreover, Paneth cells produce important antimicrobial peptides in the small intestine's crypts.Also, humoral elements, such as defensins, have a role in this regulation [19].
Infammatory states emerging from diseases might induce metabolic, immunologic, and neuroendocrine alterations that infuence the integrity of this barrier, afecting gut permeability [19,54].On the other hand, dysbiosis may lead to the loss of intestinal barrier integrity, resulting in the movement of luminal content, namely, pathogens, toxins, and antigens, to the bloodstream [19], which might induce an imbalanced mucosal immune system as well as an infammatory state, with the production of proinfammatory cytokines [53,55].Alterations in intestinal permeability can either precede or appear secondary to an infammatory status involved in diseases [15].Chronic stress situations and sleep deprivation can lead to alterations in microbiota composition, suggesting the interplay between both factors [56].

Prebiotics and Probiotics
Considering the mechanisms involved in regulating the GBA, microbiome modulation stands out as a promising therapeutic solution for anxiety disorders [16].
According to the International Scientifc Association for Probiotics and Prebiotics consensus statement (ISAPP), the term probiotic was revised to "live microorganisms that, when administered in adequate amounts, confer a health beneft on the host" [57].Probiotics can impact the regulation of the central and enteric nervous systems through diferent mechanisms that include attenuation of the HPA axis, restraining cytokine production, and infuence the neuroendocrine system.Te use of probiotics, such as L. plantarum P-8 [44], various Streptococcus, Bifdobacterium, Lactobacillus, and Lactococcus strains [48,58], and Lactobacillus rhamnosus HN001 [59], has been shown to improve stress, anxiety [16,44,48,58], memory, and cognitive potential in humans [16,44,48,59].Additionally, the use of probiotics induced benefcial changes in gut microbiota, increasing diversity and infuencing specifc bacterial species [16].Alongside, probiotic supplementation also led to reduced proinfammatory cytokines such as IFN-c and TNF-α [44], and potentially lowered plasma cortisol [44].Probiotics might also infuence metabolite production, such as SCFAs, and neurotransmitters, including 5-HT and GABA.Tis strongly suggests their multidimensional action in the GBA [60].
More recently, the ISAPP has also updated the defnition of prebiotics to "a substrate that is selectively utilized by host microorganisms conferring a health beneft" [61].Tese Veterinary Medicine International nondigestible compounds act as nutrient sources, promoting the growth of benefcial bacteria (probiotics) in the colon [60].Prebiotics more widely accepted for their clinical evidence are fructooligosaccharides (FOS) and galactooligosaccharides (GOS).Others include mannan oligosaccharides (MOS), xylooligosaccharides (XOS), human milk oligosaccharides, inulin, conjugated linoleic acid, polyunsaturated fatty acids, phenolics, and phytochemicals [62].Prebiotic administration, including lactoferrin [63], FOS [63][64][65], GOS [63,64,66,67], and polydextrose [63], has been found to improve anxiety symptoms in both humans [65][66][67] and rodents [63,64].It has also resulted in changes in the gut microbiota, specifcally increases in Lactobacillus spp.[63] and Bifdobacteria [65].Counterintuitively, some results have shown a decrease in Bifdobacterium and Lactobacillus spp., which could be explained by the enhancement of indigenous commensals through prebiotic administration, reducing the relative abundance of these added probiotics [64].Prebiotic supplementation also showed a reduction in proinfammatory cytokines, such as TNF-α [64], increased acetate and propionate, but decreased isobutyrate levels [64].Additionally, it appeared to reduce hyperactivation of the HPA [64,67] and lower tryptophan levels, possibly due to increased bacterial uptake or the production of 5-HT [64].Additionally, the combined administration of Lactobacillus casei and inulin signifcantly improved anxiety symptoms in rodents more efectively than either treatment alone, impacting their endocrine and neurochemical systems [62].Te term "psychobiotic" has been attributed to probiotics that ofer health benefts to individuals with psychiatric conditions.Tese microorganisms produce neuroactive substances that infuence the GBA and hold potential in alleviating symptoms of disorders such as depression and anxiety [68].Tis defnition also encompasses prebiotics, which contribute to the proliferation of benefcial gut bacteria [69].
Tis novel concept is well known for its anti-infammatory, antidepressant, and antianxiety efects [60].Te strength of these formulations is that they lack cognitive side efects, nor are they addictive, like other drugs usually prescribed to treat anxiety disorders [70].Tus, psychobiotics appear as a novel alternative for the treatment of anxiety disturbances [16,60,70].

Recent Advances in the Veterinary Behavioral Medicine
In veterinary practice, there have been remarkable advances in neurodevelopment research associated with the canine gut microbiome.Canine aging and cognitive performance have been associated with dysbiosis.In a study involving 29 mixed breed dogs, behavioral tests were conducted to assess cognitive performance.Te results showed a lower proportion of Fusobacteria in older dogs and fewer Actinobacteria in dogs with higher memory faculties [71].Canine aggressiveness, a type of anxiety-like behavior, was evaluated in a group of 31 American Pit Bull Terriers [72].Whereas, aggressive dogs showed a higher proportion of Firmicutes, particularly members of the Lactobacillus genus; dogs lacking this behavior demonstrated a higher abundance of Proteobacteria and Fusobacteria strains.It is relevant to highlight that dogs exhibiting non-aggressive behavior showed a higher abundance of Bacteroides and Dorea strains from the Bacteroidetes and Firmicutes phyla, respectively, compared to aggressive dogs.Tese results led to the following question: are the detected alterations in microbiota composition a predisposing factor or a consequence of canine aggressiveness?A third option also considered the possibility of unexplored variants intrinsic to dogs with aggressive behavior that could infuence their microbiota [72].A preliminary study established a correlation between behavior phenotypes and specifc gut microbiota structures in a sample of 42 dogs of various breeds [73].Te authors suggested that a chronic stress scenario might infuence the gut's internal environment by interacting with neuroactive metabolites secreted by commensal bacteria, thereby infuencing host behavior.A behavioral specialist grouped dogs as aggressive, phobic, and healthy, and those with aggressive behavior had a lower abundance of Bacteroidetes.Tis group of dogs exhibited a lower abundance of members of the Oscillospira, Peptostreptococcus, Bacteroides, Sutterella, and Coprobacillus genera.Furthermore, an increase of typically low-prevalence bacteria was detected in aggressive dogs, including Dorea, Blautia, Collinsella, and Slackia, with an even higher prevalence of strains of Catenicabacterium and Megamonas [73].Concerning dogs with phobic disorders, no substantial alterations were detected at the phylum level.Surprisingly, canine phobic behavioral disorders were associated with a higher proportion of Lactobacillus, a genus usually found predominantly in probiotic formulations.Te authors also analyzed fecal cortisol and testosterone levels, showing no signifcant correlation between these and aggressive or phobic disorders.Although the population cohort in this study was not sufcient to draw signifcant conclusions, this investigation undoubtedly paved the way for further large-scale studies [73].
Regarding neuroendocrine mechanisms, a recent study investigated the relationship between 5-HT and tryptophan and dogs' behavioral response to stress stimuli [74].A mixed-breed group of 39 healthy shelter dogs was classifed according to their behavioral response to medical examination and blood collection procedures.Serum 5-HT and tryptophan concentrations were analyzed, and no correlation was established.Tis could be because none of the dogs in this study displayed intense behavioral disorders.Another possible explanation suggested by the authors is the opposite way of circulation of 5-HTand tryptophan in the BBB, which regulates the passage between central and peripheral circulation [74].Likewise, a 107 mixed-breed group of dogs with diferent levels of aggressive, fearful, and impulsive behaviors, defned accordingly to the owner-answered questionnaires, did not show signifcant alterations in serum 5-HT and cortisol levels after being restrained when exposed to a novel environment [75].It is possible that the exposition to a new environment could not be sufciently distressful to cause alterations in these markers' concentrations or that the methods of behavioral evaluation performed were not accurate enough.Another possible explanation was the lack of 6 Veterinary Medicine International a relationship between these markers and canine behavioral tendencies [75].
Regarding probiotic therapeutic use, a fourteen-day supplementation of Lactiplantibacillus plantarum PS128 seemed to stabilize aggression and separation anxiety behaviors in dogs.Moreover, plasma 5-HT turnover ratio decreased after supplementation, specifcally in dogs with separation anxiety.Tis implicates 5-HT as a potential factor in the GBA, indicating a slower breakdown of 5-HT into its metabolites, and consequently a higher availability of this neurotransmitter in the system [76].
Furthermore, Purina ® researchers have highlighted the potential benefts of Bifdobacterium longum (BL999) in dogs with anxiety behaviors in a study supporting a new product (https://www.purinaproplanvets.com/media/521317/086602_vet1900-0918cc_abstract.pdf).Involving 24 anxious Labrador Retrievers, the study assessed the efects of BL999 on behavior and physiological markers like heart rate and salivary cortisol.Over a 12-week period, with a midway washout, dogs alternated between BL999 and a placebo.Signifcant improvements were noted in anxious behaviors and physiological markers in the BL999 group.Tese preliminary fndings are promising, and releasing comprehensive study details for peer review would greatly beneft the scientifc community, enabling further evaluation and expansion upon these results.A double-blind, placebo-controlled clinical trial probed the efects of a novel nutraceutical supplement in canine stress-related behaviors and its relationship with the fecal microbiome [77].Relaxigen Pet dog ® contains a mixture of prebiotics (FOS), probiotics (Lactobacillus reuteri), postbiotics (butyric acid), 5-hydroxytryptophan (5-HTP), a 5-TH precursor, L-theanine, derived from glutamine, a precursor of GABA, and other natural infammatory compounds, including conjugated linoleic acid, a neuroprotective supplement with anti-infammatory properties, and Krill, an Omega-3 rich oil.Forty dogs enrolled in this study were classifed by a veterinary behaviorist according to their stress behavior level.Anxious dogs treated with Relaxigen Pet dog ® had lower levels of Bacteroides, Prevotella, Porphyromonas, Bifdobacterium, Lactobacillus, and Enterobacteriaceae strains.Tis group also demonstrated less anxiety-like behaviors [77].Tis innovative study showed promising results, which will bring new insights into veterinary behavioral medicine and pave the way for future research.To our knowledge, no other research has been conducted on the utilization of probiotics for the treatment of canine anxiety disorders.Despite recent signifcant progress in veterinary behavioral medicine, the link between canine anxiety disorders and the gut microbiome, as well as the potential benefts of probiotics in this condition, remains to be further elucidated and tested on a larger scale.

Conclusions
Canine anxiety disorders present a signifcant issue in veterinary behavior today, posing medical challenges and affecting both pet and owner quality of life.
Te infuence of microbiota on mental and behavioral disorders, including anxiety, is well-documented in human and rodent models, considering the metabolic, neuroendocrine and immune-mediated mechanisms involved.However, there is a signifcant gap in understanding these relationships in the context of canine veterinary behavior.
In this study, several conclusions have been drawn: (1) Te limited number of studies included on canine anxiety disorders and GBA refects the scarcity of research on this topic.Current studies are broad and focus on a few specifc markers only.In addition, most clinical trials relied solely on behavioral patterns to assess anxiety.Future studies should incorporate more biomarkers such as specifc cytokines, metabolites, hormones, neurotransmitters, and others to accurately evaluate canine anxiety levels.(2) Extensive research in human and rodent models has elucidated the role of microbiota in behavioral disorders.Te authors propose that leveraging this knowledge could be pivotal in advancing canine research and enhancing veterinary practices.(3) Despite microbiota diferences among humans, rodents, and dogs, one further step in the investigation on this topic could be the assessment of specifc microbiota in dogs, which have been previously associated with anxiety symptoms in humans and rodents.(4) Regarding prebiotic and probiotic therapies, to our knowledge, there have only been two peer-reviewed studies conducted on dogs [76,77].Although promising, it is not enough to draw frm conclusions, indicating the need for further research.
Continuous eforts in this area are likely to shed light on the impact of GBA on canine anxiety disorders, as well as on its treatment, potentially improving both dog's and owner's quality of life.Additionally, this would represent a signifcant milestone in the feld of veterinary behavioral medicine.

Figure 1 :
Figure1: Mechanisms involved in the GBA.Anxiety disorders may lead to the hyperactivation of the HPA axis, causing the release of cortisol into the systemic circulation.In an infammatory environment, characterized by gut dysbiosis and increased intestinal permeability, microbiota can translocate through the intestinal barrier.Moreover, immune cells may produce proinfammatory cytokines, while the gut microbiota itself may produce metabolites such as SCFAs and neurotransmitters that can directly impact mental health.Original illustration based on[15,16] captioned as shown above: 1-hypothalamus, 2-pituitary gland, 3-adrenal gland, ACTH-adrenocorticotropic hormone, CRF-corticotropin-releasing factor, and SCFAs-short chain fatty acids.

Table 1 :
[3]t of the behaviors evaluated in the Lincoln Canine Anxiety Scale (adapted from[3]).