A novel KRT71 variant in curly-coated dogs.

Curly fur is a common phenotype in many dog breeds, known to result from a missense variant (c.451C>T) in exon 2 of the keratin 71 (KRT71) gene. During screening for this variant across various breeds, we found that Curly Coated Retrievers (CCRs) fixed with the trait did not carry the known variant. By analysis of whole-genome sequencing data of one CCR we identified a novel genetic cause for curly fur. We found a novel structural variant in exon 7 of the KRT71 gene (c.1266_1273delinsACA) that was predicted to result in a frameshift and stop loss, therefore significantly affecting the structure of the protein, if translated. The variant was also found at lower frequencies in five other breeds, including Lagotto Romagnolo, Bichon Frise, Spanish Water Dog, Chesapeake Bay Retriever and Irish Terrier. One curly-coated Lagotto carried neither of the two KRT71 variants. These results identify a second variant for curly coat in KRT71 and suggest the existence of additional alleles. This study enables the development of an additional KRT71 gene test for breeders to understand and manage coat types.

Two MC1R loss-of-function alleles in cream-coloured Australian Cattle Dogs and white Huskies.

Loss-of-function variants in the MC1R gene cause recessive red or yellow coat-colour phenotypes in many species. The canine MC1R:c.916C>T (p.Arg306Ter) variant is widespread and found in a homozygous state in many uniformly yellow- or red-coloured dogs. We investigated cream-coloured Australian Cattle Dogs whose coat colour could not be explained by this variant. A genome-wide association study with 10 cream and 123 red Australian Cattle Dogs confirmed that the cream locus indeed maps to MC1R. Whole-genome sequencing of cream dogs revealed a single nucleotide variant within the MITF binding site of the canine MC1R promoter. We propose to designate the mutant alleles at MC1R:c.916C>T as e1 and at the new promoter variant as e2 . Both alleles segregate in the Australian Cattle Dog breed. When we considered both alleles in combination, we observed perfect association between the MC1R genotypes and the cream coat colour phenotype in a cohort of 10 cases and 324 control dogs. Analysis of the MC1R transcript levels in an e1 /e2 compound heterozygous dog confirmed that the transcript levels of the e2 allele were markedly reduced with respect to the e1 allele. We further report another MC1R loss-of-function allele in Alaskan and Siberian Huskies caused by a 2-bp deletion in the coding sequence, MC1R:c.816_817delCT. We propose to term this allele e3 . Huskies that carry two copies of MC1R loss-of-function alleles have a white coat colour.

Association study reveals novel risk loci for sporadic inclusion body myositis.

BACKGROUND AND PURPOSE: The aim was to identify potential genetic risk factors associated with sporadic inclusion body myositis (sIBM). METHODS: An association based case-control approach was utilized on whole exome sequencing data of 30 Finnish sIBM patients and a control cohort (n = 193). A separate Italian cohort of sIBM patients (n = 12) was used for evaluation of the results. RESULTS: Seven single nucleotide polymorphisms were identified in five genes that have a considerably higher observed frequency in Finnish sIBM patients compared to the control population, and the previous association of the genetic human leukocyte antigen region was confirmed. CONCLUSIONS: All seven identified variants could individually or in combination increase the susceptibility for sIBM.

Deep sequencing of a candidate region harboring the SOX9 gene for the canine XX disorder of sex development.

A disorder of sex development (DSD) in dogs with female sex chromosomes (78, XX), a lack of the SRY gene and the presence of testes or ovotestes is commonly diagnosed in numerous breeds. The molecular background of DSD is not fully recognized but has been linked to the copy number variation in the region harboring the SOX9 gene. We applied a genome-wide association study and targeted next-generation sequencing techniques to compare DSD and normal female dogs. The genome-wide association study did not indicate a significant chromosome region. Targeted next-generation sequencing of a 1.5-Mb region on canine chromosome 9 harboring the SOX9 gene revealed two putatively DSD-associated copy number variations 355 kb upstream and 691 kb downstream of SOX9, four blocks of low polymorphism and two blocks of an elevated heterozygosity. An initial next-generation sequencing analysis showed an association with two SNPs, but validation in larger cohorts did not confirm this result. We identified a large homologous fragment (over 243.8 kb), named hfMAGI2, located upstream of SOX9, that overlaps a known copy number variation region. It shows a high sequence similarity with the 5' flanking region of the MAGI2 gene located on canine chromosome 18 that encodes a protein involved in ovary formation during early embryonic development. Our study showed that the identified copy number variation region located upstream of the SOX9 gene contains potential regulatory sequences (long non-coding RNA and hfMAGI2) and led to the assumption that a multiplication of this element may alter expression of the SOX9 gene, triggering the DSD phenotype.

The canine era: the rise of a biomedical model.

Since the annotation of its genome a decade ago, the dog has proven to be an excellent model for the study of inherited diseases. A large variety of spontaneous simple and complex phenotypes occur in dogs, providing physiologically relevant models to corresponding human conditions. In addition, gene discovery is facilitated in clinically less heterogeneous purebred dogs with closed population structures because smaller study cohorts and fewer markers are often sufficient to expose causal variants. Here, we review the development of genomic resources from microsatellites to whole-genome sequencing and give examples of successful findings that have followed the technological progress. The increasing amount of whole-genome sequence data warrants better functional annotation of the canine genome to more effectively utilise this unique model to understand genetic contributions in morphological, behavioural and other complex traits.

Balancing selection and heterozygote advantage in major histocompatibility complex loci of the bottlenecked Finnish wolf population.

Maintaining effective immune response is an essential factor in the survival of small populations. One of the most important immune gene regions is the highly polymorphic major histocompatibility complex (MHC). We investigated how a population bottleneck and recovery have influenced the diversity and selection in three MHC class II loci, DLA-DRB1, DLA-DQA1 and DLA-DQB1, in the Finnish wolf population. We studied the larger Russian Karelian wolf population for comparison and used 17 microsatellite markers as reference loci. The Finnish and Karelian wolf populations did not differ substantially in their MHC diversities (GST″ = 0.047, P = 0.377), but differed in neutral microsatellite diversities (GST″ = 0.148, P = 0.008). MHC allele frequency distributions in the Finnish population were more even than expected under neutrality, implying balancing selection. In addition, an excess of nonsynonymous compared to synonymous polymorphisms indicated historical balancing selection. We also studied association between helminth (Trichinella spp. and Echinococcus canadensis) prevalence and MHC diversity at allele and SNP level. MHC-heterozygous wolves were less often infected by Trichinella spp. and carriers of specific MHC alleles, SNP haplotypes and SNP alleles had less helminth infections. The associated SNP haplotypes and alleles were shared by different MHC alleles, which emphasizes the necessity of single-nucleotide-level association studies also in MHC. Here, we show that strong balancing selection has had similar effect on MHC diversities in the Finnish and Russian Karelian wolf populations despite significant genetic differentiation at neutral markers and small population size in the Finnish population.

A large case-control study indicates a breed-specific predisposition to feline tooth resorption.

Tooth resorption (TR) is one of the most common dental diseases of cats. It is a painful condition leading to tooth loss. The etiology of TR remains unclear, but old age, breed, other oral and dental diseases, and environmental factors are suspected predisposing factors. In our study, we used part of the data from the extensive feline health online survey of 8115 Finnish cats. As TR is difficult to detect and as the feline health survey included diagnoses defined by both veterinarians and the owners, we limited our study to a subpopulation of cats diagnosed with oral or dental disease by a veterinarian and had dental examination or surgery under sedation (n=944). We utilized case-control study analysed by multivariable logistic regression to determine the risk factors and breed variation of feline TR. The 202 cats diagnosed with TR were defined as TR cases and the remaining 742 cats as controls. The frequency of veterinarian-diagnosed TR was 3.9% in the health survey data (316/8115) and 21% in the subpopulation (202/944). The risk of TR increased with age (14.7% in youngest and 25.3% in oldest age group). Our finding that TR was significantly associated with gingivitis or periodontitis in cats that had also calculus (OR: 2.49 and 3.70, respectively) suggests that inflammatory changes caused by calculus increase the risk of TR. We found that Cornish Rex, European, and Ragdoll are at higher risk for TR (OR: 2.44, 2.98 and 2.90, respectively). Exotic-Persians breed group had lower risk (OR: 0.28). TR was not observed in Turkish van or Devon Rex. The differences between breeds highlight a genetic contribution. In addition, female cats that had food available constantly had significantly less TR than female cats that had feeding times (OR: 0.44). The underlying reasons for this remain unexplained in our study.

MHC variability supports dog domestication from a large number of wolves: high diversity in Asia.

The process of dog domestication is still somewhat unresolved. Earlier studies indicate that domestic dogs from all over the world have a common origin in Asia. So far, major histocompatibility complex (MHC) diversity has not been studied in detail in Asian dogs, although high levels of genetic diversity are expected at the domestication locality. We sequenced the second exon of the canine MHC gene DLA-DRB1 from 128 Asian dogs and compared our data with a previously published large data set of MHC alleles, mostly from European dogs. Our results show that Asian dogs have a higher MHC diversity than European dogs. We also estimated that there is only a small probability that new alleles have arisen by mutation since domestication. Based on the assumption that all of the currently known 102 DLA-DRB1 alleles come from the founding wolf population, we simulated the number of founding wolf individuals. Our simulations indicate an effective population size of at least 500 founding wolves, suggesting that the founding wolf population was large or that backcrossing has taken place.

Association of Doberman hepatitis to canine major histocompatibility complex II.

Doberman hepatitis (DH) is a chronic and progressive inflammatory liver disease that mainly affects female dogs. The high incidence of chronic hepatitis in Dobermans is suggestive of a genetic predisposition. DH is characterized by mononuclear cell infiltration and copper accumulation in the liver and major histocompatibility complex (MHC) class II antigen expression in the hepatocytes. In dogs, the MHC is referred to as the dog leukocyte antigen (DLA) system. In this study, the potential role of DLA genes in DH was investigated by sequence-based typing in the exon 2 of DLA-DRB1, -DQA1 and -DQB1. The case group comprised 37 Dobermans with subclinical or clinical DH. The control group consisted of 37 healthy Dobermans, with normal liver enzyme values and without immunosuppressive medication. The control dogs were over 10 years old to include dogs with the lowest genetic risk of DH. Our results indicate that Dobermans with homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 [odds ratio (OR) = 14.9, confidence limit (CL) = 3.1-71.7, P < 0.00005], especially with homozygosity for DLA-DRB1*00601 (P < 0.0005), are susceptible to DH. The DQ heterodimer DLA-DQA1*00901/DQB1*00101 and the allele DLA-DRB1*01501 appear to confer protection against DH (P < 0.001). Allele and haplotype frequencies were compared using chi-squared statistics. The disease shows a complex pattern of inheritance, but the observed DLA class II association with DH suggests a role for the immune system in the development of the disease.

Evaluation of DLA promoters in Doberman hepatitis.

Doberman hepatitis (DH) is associated with homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 indicating a role for the immune system in the development of the disease. The dog leucocyte antigen (DLA) class II expression is controlled at the transcriptional level with proximal promoters. Differential expression of DLA class II molecules of antigen-presenting cells is reported to affect susceptibility to or protection from different immune-mediated diseases. The aim of this study was to evaluate, whether the variation in promoter areas of homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 Dobermans could explain why some dogs become afflicted with DH and others do not. Our findings suggest that promoter variants are not associated as risk modifiers in homozygous DLA-DRB1*00601/DQA1*00401/DQB1*01303 Dobermans, but additional factors are needed. Nevertheless, our study indicates that the whole DLA block is associated to the disease.

Regional occurrence, high frequency but low diversity of mitochondrial DNA haplogroup d1 suggests a recent dog-wolf hybridization in Scandinavia.

The domestic dog mitochondrial DNA (mtDNA)-gene pool consists of a homogenous mix of haplogroups shared among all populations worldwide, indicating that the dog originated at a single time and place. However, one small haplogroup, subclade d1, found among North Scandinavian/Finnish spitz breeds at frequencies above 30%, has a clearly separate origin. We studied the genetic and geographical diversity for this phylogenetic group to investigate where and when it originated and whether through independent domestication of wolf or dog-wolf crossbreeding. We analysed 582 bp of the mtDNA control region for 514 dogs of breeds earlier shown to harbour d1 and possibly related northern spitz breeds. Subclade d1 occurred almost exclusively among Swedish/Finnish Sami reindeer-herding spitzes and some Swedish/Norwegian hunting spitzes, at a frequency of mostly 60-100%. Genetic diversity was low, with only four haplotypes: a central, most frequent, one surrounded by two haplotypes differing by an indel and one differing by a substitution. The substitution was found in a single lineage, as a heteroplasmic mix with the central haplotype. The data indicate that subclade d1 originated in northern Scandinavia, at most 480-3000 years ago and through dog-wolf crossbreeding rather than a separate domestication event. The high frequency of d1 suggests that the dog-wolf hybrid phenotype had a selective advantage.

Assessment of the functionality of genome-wide canine SNP arrays and implications for canine disease association studies.

Domestic dogs share a wide range of important disease conditions with humans, including cancers, diabetes and epilepsy. Many of these conditions have similar or identical underlying pathologies to their human counterparts and thus dogs represent physiologically relevant natural models of human disorders. Comparative genomic approaches whereby disease genes can be identified in dog diseases and then mapped onto the human genome are now recognized as a valid method and are increasing in popularity. The majority of dog breeds have been created over the past few hundred years and, as a consequence, the dog genome is characterized by extensive linkage disequilibrium (LD), extending usually from hundreds of kilobases to several megabases within a breed, rather than tens of kilobases observed in the human genome. Genome-wide canine SNP arrays have been developed, and increasing success of using these arrays to map disease loci in dogs is emerging. No equivalent of the human HapMap currently exists for different canine breeds, and the LD structure for such breeds is far less understood than for humans. This study is a dedicated large-scale assessment of the functionalities (LD and SNP tagging performance) of canine genome-wide SNP arrays in multiple domestic dog breeds. We have used genotype data from 18 breeds as well as wolves and coyotes genotyped by the Illumina 22K canine SNP array and Affymetrix 50K canine SNP array. As expected, high tagging performance was observed with most of the breeds using both Illumina and Affymetrix arrays when multi-marker tagging was applied. In contrast, however, large differences in population structure, LD coverage and pairwise tagging performance were found between breeds, suggesting that study designs should be carefully assessed for individual breeds before undertaking genome-wide association studies (GWAS).

Association of a dog leukocyte antigen class II haplotype with hypoadrenocorticism in Nova Scotia Duck Tolling Retrievers.

Canine hypoadrenocorticism (Addison's disease) is due to a deficiency of corticosteroids and mineralocorticoids produced by the adrenals. Although this is a relatively uncommon disease in the general dog population, some breeds, including the Nova Scotia Duck Tolling Retriever (NSDTR), are at increased risk for developing hypoadrenocorticism. A prior study has shown that the increased risk is due to a heritable component. This potentially lethal disorder is hypothesized to have an autoimmune etiology, thus the aim of this study was to determine whether genetic susceptibility to hypoadrenocorticism in NSDTRs is associated with genes of the canine major histocompatibility complex [MHC; dog leukocyte antigen system (DLA)]. Samples were collected from NSDTRs diagnosed with hypoadrenocorticism and healthy siblings or country-matched controls. The DLA class II alleles and haplotypes were determined and compared between cases and controls. We found seven different haplotypes of which the haplotype DLA-DRB1*01502/DQA*00601/DQB1*02301 was significantly more prevalent in the diseased dogs (P = 0.044). In addition, these affected dogs also were more likely to be homozygous across the DLA class II region than the control dogs (OR = 6.7, CI = 1.5-29.3, P = 0.011). We also found that homozygous dogs, regardless of their haplotype, tended to have earlier disease onset compared with heterozygous dogs. These data indicate a limited MHC diversity in North American NSDTRs and suggest that the MHC may play a role in the development of hypoadrenocorticism in the NSDTR, supporting the autoimmune origin of the disease.

Ventricular arrhythmia and sudden cardiac death in young Leonbergers.

INTRODUCTION: To describe unexpected sudden cardiac death (SCD) in young Leonbergers (<3 years) and to review the circumstances before death and necropsy findings; to prospectively evaluate the presence of possible arrhythmias in young Leonbergers; and to examine pedigrees for determining potential modes of inheritance. ANIMALS: Postmortem evaluations included 21 Leonbergers. Clinical evaluation consisted of 46 apparently healthy Leonbergers with and without a close family history of SCD. MATERIALS AND METHODS: Necropsy reports were reviewed retrospectively. Prospective clinical evaluation included physical examination, 5-min electrocardiogram, 24-h Holter, echocardiography, and laboratory tests. Pedigree data were examined for mode of inheritance. RESULTS: Based on necropsy reports, SCD occurred at a median age of 12 months (range, 2.0-32.0 months) without any previous clinical signs and usually in rest. No evidence of structural cardiac disease was found; arrhythmia-related death was suspected. Clinical evaluation and 24-h Holter showed ventricular arrhythmia (VA) in 14 apparently healthy Leonbergers (median age, 18 months; range, 12-42 months). Severity of VA varied from infrequent couplets/triplets to frequent complexity (couplets, triplets, nonsustained ventricular tachycardias,VTs) characterized by polymorphology. During follow-up, two dogs with polymorphic VT died. Although breed specificity and high prevalence indicate a heritable disease, based on available pedigree data, the mode of inheritance could not be determined. CONCLUSIONS: Sudden cardiac death in young Leonbergers is associated with malignant VA characterized by complexity and polymorphic nature. Diagnosis is based on 24-h Holter monitoring. Pedigree analysis suggests that the arrhythmia is familial.

A novel genomic region on chromosome 11 associated with fearfulness in dogs.

The complex phenotypic and genetic nature of anxieties hampers progress in unravelling their molecular etiologies. Dogs present extensive natural variation in fear and anxiety behaviour and could advance the understanding of the molecular background of behaviour due to their unique breeding history and genetic architecture. As dogs live as part of human families under constant care and monitoring, information from their behaviour and experiences are easily available. Here we have studied the genetic background of fearfulness in the Great Dane breed. Dogs were scored and categorised into cases and controls based on the results of the validated owner-completed behavioural survey. A genome-wide association study in a cohort of 124 dogs with and without socialisation as a covariate revealed a genome-wide significant locus on chromosome 11. Whole exome sequencing and whole genome sequencing revealed extensive regions of opposite homozygosity in the same locus on chromosome 11 between the cases and controls with interesting neuronal candidate genes such as MAPK9/JNK2, a known hippocampal regulator of anxiety. Further characterisation of the identified locus will pave the way for molecular understanding of fear in dogs and may provide a natural animal model for human anxieties.

Breed-specific reference ranges for standard echocardiographic measurements in salukis.

OBJECTIVES: To establish normal reference ranges for standard echocardiographic measurements in Salukis and to examine whether bodyweight, age, gender and heart rate had an influence on the echocardiographic variables. MATERIALS AND METHODS: Seventy-five privately owned healthy purebred Salukis, aged 2 to 10 years were included. Case history was obtained and dogs were examined by physical examination, complete blood cell count, serum biochemistry and echocardiography. Associations between bodyweight, gender, sex and heart rate and echocardiographic variables were examined using multiple linear regression analysis and allometric scaling. Reference values with 95% prediction intervals were calculated from regression equations. RESULTS: Bodyweight was a significant predictor of left ventricular diameters, left ventricular volumes and E-point-to-septal-separation. Associations between bodyweight and the echocardiographic variables were best described by multiple linear regression models, providing bodyweight-based reference values. Age, gender and heart rate had significant effect on some of the echocardiographic variables and were included in the final models. When the equation included heart rate or age, reference values were calculated using mean heart rate value (80 bpm) and median age value (73 months). CLINICAL SIGNIFICANCE: This study provides detailed bodyweight-based echocardiographic values in normal Salukis which can be used as reference values.

Two novel genomic regions associated with fearfulness in dogs overlap human neuropsychiatric loci.

Anxiety disorders are among the leading health issues in human medicine. The complex phenotypic and allelic nature of these traits as well as the challenge of establishing reliable measures of the heritable component of behaviour from the associated environmental factors hampers progress in their molecular aetiology. Dogs exhibit large natural variation in fearful and anxious behaviour and could facilitate progress in the molecular aetiology due to their unique genetic architecture. We have performed a genome-wide association study with a canine high-density SNP array in a cohort of 330 German Shepherds for two phenotypes, fear of loud noises (noise sensitivity) and fear of strangers or in novel situations. Genome-widely significant loci were discovered for the traits on chromosomes 20 and 7, respectively. The regions overlap human neuropsychiatric loci, including 18p11.2, with physiologically relevant candidate genes that contribute to glutamatergic and dopaminergic neurotransmission in the brain. In addition, the noise-sensitivity locus includes hearing-related candidate genes. These results indicate a genetic contribution for canine fear and suggest a shared molecular aetiology of anxiety across species. Further characterisation of the identified loci will pave the way to molecular understanding of the conditions as a prerequisite for improved therapy.

Comprehensive diagnosis of Rett's syndrome relying on genetic, epigenetic and expression evidence of deficiency of the methyl-CpG-binding protein 2 gene: study of a cohort of Israeli patients.

BACKGROUND: Despite advances in the characterisation of mutations in the MECP2-coding region, a small proportion of classic RTT cases remain without recognisable mutations. OBJECTIVE AND METHODS: To identify previously unknown mutations, a quantitative assay was established, providing estimates of MECP2_e1 and MECP2_e2 expression levels in peripheral blood. A systematic analysis of an Israeli cohort of 82 patients with classic and atypical RTT is presented, including sequence analysis of the MECP2-coding region, MLPA, XCI and quantitative expression assays. RESULTS AND CONCLUSION: A novel mis-sense mutation at ca 453C-->T (pD151E), resulting in a change of a conserved residue at the methyl-binding domain, and a rare GT deletion of intron 1 donor splice site are reported. It is shown that various MECP2 mutations had distinct effects on MECP2 expression levels in peripheral blood. The most significant (p<0.001) reduction in the expression of both MECP2 isoforms was related to the presence of the intron 1 donor splice-site mutation. Using quantitative expression assays, it was shown that several patients with classic and atypical RTT with no mutation findings had significantly lower MECP2 expression levels. Further research on these patients may disclose still elusive non-coding regulatory MECP2 mutations.

Absence Seizures as a Feature of Juvenile Myoclonic Epilepsy in Rhodesian Ridgeback Dogs.

Myoclonic epilepsy in Rhodesian Ridgeback (RR) dogs is characterized by myoclonic seizures occurring mainly during relaxation periods, a juvenile age of onset and generalized tonic-clonic seizures in one-third of patients. An 8-month-old female intact RR was presented for myoclonic seizures and staring episodes that both started at 10 weeks of age. Testing for the DIRAS1 variant indicated a homozygous mutant genotype. Unsedated wireless video-electroencephalography (EEG) identified frequent, bilaterally synchronous, generalized 4 Hz spike-and-wave complexes (SWC) during the staring episodes in addition to the characteristic myoclonic seizures with generalized 4-5 Hz SWC or 4-5 Hz slowing. Photic stimulation did not evoke a photoparoxysmal response. Repeat video-EEG 2 months after initiation of levetiracetam treatment disclosed a >95% decrease in frequency of myoclonic seizures, and absence seizures were no longer evident. Absence seizures represent another seizure type in juvenile myoclonic epilepsy (JME) in RR dogs, which reinforces its parallels to JME in humans.

Interbreed variation in serum serotonin (5-hydroxytryptamine) concentration in healthy dogs.

INTRODUCTION: Serotonin (5-hydroxytryptamine [5-HT]) has several biological functions. In different species, excessive 5-HT has been linked to valvular lesions, similar to those seen in dogs with myxomatous mitral valve disease. Previous studies suggest higher 5-HT in healthy Cavalier King Charles Spaniels (CKCSs), a breed highly affected by myxomatous mitral valve disease, compared to other breeds. OBJECTIVE: To investigate potential interbreed variation in serum 5-HT in healthy dogs. ANIMALS: 483 healthy dogs of nine breeds aged 1-7 years. METHODS: Dogs were examined at five European centers. Absence of cardiovascular, organ-related, or systemic diseases was ensured by thorough clinical investigations including echocardiography. Serum was frozen and later analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Median 5-HT concentration was 252.5 (interquartile range = 145.5-390.6) ng/mL. Overall breed difference was found (p<0.0001), and 42% of pairwise breed comparisons were significant. Univariate regression analysis showed association between serum 5-HT concentration and breed, center of examination, storage time, and sex, with higher 5-HT in females. In multiple regression analysis, the final model had an adjusted R2 of 0.27 with breed (p<0.0001), center (p<0.0001), and storage time (p=0.014) remaining significant. Within centers, overall breed differences were found at 3/5 centers (p≤0.028), and pairwise comparisons within those centers showed breed differences in 42% of comparisons. Among the included breeds, Newfoundlands, Belgian Shepherds and CKCSs had highest 5-HT concentrations. CONCLUSIONS: Interbreed variation in serum 5-HT concentration was found in healthy dogs aged 1-7 years. These differences should be taken into account when designing clinical studies.