Breath characteristics and adventitious lung sounds in healthy and asthmatic horses.

BACKGROUND: Standard thoracic auscultation suffers from limitations, and no systematic analysis of breath sounds in asthmatic horses exists. OBJECTIVES: First, characterize breath sounds in horses recorded using a novel digital auscultation device (DAD). Second, use DAD to compare breath variables and occurrence of adventitious sounds in healthy and asthmatic horses. ANIMALS: Twelve healthy control horses (ctl), 12 horses with mild to moderate asthma (mEA), 10 horses with severe asthma (sEA) (5 in remission [sEA-], and 5 in exacerbation [sEA+]). METHODS: Prospective multicenter case-control study. Horses were categorized based on the horse owner-assessed respiratory signs index. Each horse was digitally auscultated in 11 locations simultaneously for 1 hour. One-hundred breaths per recording were randomly selected, blindly categorized, and statistically analyzed. RESULTS: Digital auscultation allowed breath sound characterization and scoring in horses. Wheezes, crackles, rattles, and breath intensity were significantly more frequent, higher (P < .001, P < .01, P = .01, P < .01, respectively) in sEA+ (68.6%, 66.1%, 17.7%, 97.9%, respectively), but not in sEA- (0%, 0.7%, 1.3%, 5.6%) or mEA (0%, 1.0%, 2.4%, 1.7%) horses, compared to ctl (0%, 0.6%, 1.8%, -9.4%, respectively). Regression analysis suggested breath duration and intensity as explanatory variables for groups, wheezes for tracheal mucus score, and breath intensity and wheezes for the 23-point weighted clinical score (WCS23). CONCLUSIONS AND CLINICAL IMPORTANCE: The DAD permitted characterization and quantification of breath variables, which demonstrated increased adventitious sounds in sEA+. Analysis of a larger sample is needed to determine differences among ctl, mEA, and sEA- horses.

Quantitative Gait Analysis Before and After a Cross-country Test in a Population of Elite Eventing Horses.

Early recognition of lameness is crucial for injury prevention. Quantitative gait analysis systems can detect low-grade asymmetries better than the human eye and may be useful in early lameness recognition. The aims of this study were (1) to investigate the frequency of gait asymmetries based on head and pelvic movement in elite eventing horses using inertial mounted measurement units and (2) to assess the association between asymmetries and muscle enzymes and blood lactate (LA) levelspost-exercise. Movement asymmetry of the head, wither, and pelvis were quantified in 33 elite eventing horses prior to and one day after the cross-country test of three Concours Complet International (CCI3* and CCI4*) events held three weeks apart. The effects of LA concentration immediately after completion of the cross-country course and of serum levels of creatine kinase (CK) and aspartate amino-transferase (AST) four hours post-exercise on gait asymmetry parameters were tested with linear models. A total of 58% and 77% of the 33 horses exhibited gait asymmetries that exceeded published threshold values before and after the cross-country course, respectively. The magnitude of pre-existing gait asymmetries was not significantly increased after the cross-country test and no associations with post-exercise levels of CK, AST, or LA were detected. The stride duration was significantly shorter the day following the cross-country test and was associated with LA, the age and the weight of the horses. In conclusion, a majority of the horses studied presented gait asymmetries and strenuous exercise resulted in decreased stride duration but did not worsen gait asymmetries.

Atopic dermatitis in a cohort of West Highland white terriers in Switzerland. Part II: estimates of early life factors and heritability.

BACKGROUND: There is accumulating evidence in studies of allergic diseases in humans and dogs that environmental experiences during the first months of life can influence the development of allergic disease. No prospective study has evaluated this in veterinary medicine. HYPOTHESIS/OBJECTIVES: To assess early-life risk factors for canine atopic dermatitis (cAD) and estimate its heritability. ANIMALS: A West Highland white terrier birth cohort (n = 107) followed up to three years of age recording the development of cAD. METHODS AND MATERIALS: The effect of environmental factors [house dust mites (HDM), hygiene, feeding, lifestyle] and early-life determinants [breeder, mode of delivery, birth season, sex, litter size, early-life immunoglobulin E (IgE) levels] were assessed, using Stata SE 15.1 statistical analysis. Heritabilities were estimated using the R program packages MCMCglmm and QGglmm. RESULTS: Maternal allergic status [P = 0.013, odds ratio (OR 3.3)], male sex (P = 0.06), mode of delivery (P = 0.12), breeder (P = 0.06), presence of HDM (P = 0.11) and environmental hygiene level (P = 0.15) were identified as possible influence factors by bivariate analyses. In the multivariate analysis the male sex was significantly associated with the development of cAD in the offspring (P = 0.03, OR 2.4). The heritabilities on the observed scale were 0.31 (direct), 0.04 (maternal genetic effects) and 0.03 (maternal permanent environmental effects). CONCLUSION AND CLINICAL IMPORTANCE: These results suggest that several environmental factors could influence the development of cAD but clearly demonstrate the genetic influence of the individual and the dam. Further studies are needed to identify specific environmental factors, which could be potential targets for primary disease intervention.

Heritabilities for the puppy weight at birth in Labrador retrievers.

BACKGROUND: Weight at birth is an important predictor of neonatal mortality and morbidity in dogs. In addition, the birthweight of the puppies in a litter influences the decision to perform a cesarean section. The goal of the present study was to estimate heritabilities for the puppy birth weight in Labrador retrievers. RESULTS: Of the 1138 Labrador retriever litters whelped at the Guiding Eye for the Blind between September 2001 and February 2018, 1013 were included in the analyses after data editing. Puppy weight at birth was the target trait, measured on a continuous scale in pounds, and converted to grams. Linear mixed models were used to identify factors influencing puppy weight at birth. The analyses showed that the sex of the puppy, litter size, length of gestation, adult weight of the dam, parity, year of birth and inbreeding coefficient of the puppies and dams contributed to the variance of the puppy birth weight. Dam and litter effects were included as random effects. A multiple trait derivative free restricted maximum likelihood approach was used to estimate variance components and genetic parameters with two animal models, one without covariates (Model 1) and one with covariates (Model 2). Sex of the puppy and litter size had moderate effects, whereas gestation length, adult weight of the dam, parity, year of birth and inbreeding coefficients of the dam and the puppies had minor effects. Estimates for Model 1 and Model 2 were 0.21 and 0.17 for the direct heritabilities, 0.22 and 0.22 for the maternal additive genetic heritabilities, 0.07 and 0.07 for the maternal permanent environmental proportions, and 0.14 and 0.08 for the environmental proportion of the litter. CONCLUSIONS: In order to estimate reliable breeding values for puppy weight at birth, sex of puppy, litter size, length of gestation and the adult weight of the dam should be included. Estimates could benefit from weighing the dams prior to each mating.

Factors contributing to the decision to perform a cesarean section in Labrador retrievers.

BACKGROUND: In the past 10 years, the frequency of unplanned cesarean sections in the Labrador Retriever breeding colony at Guiding Eyes for the Blind stayed around 10% (range 5% to 28%). To reduce the number of cesarean sections, factors influencing the occurrence of a cesarean section need to be known. The goal of this study was to identify factors that contribute to the decision to perform a cesarean section. RESULTS: Of the 688 Labrador Retriever litters whelped between 2003 and 2016, 667 litters had sufficient data and remained in the analysis. The target trait was ordinal with the three levels "normal whelping", "assisted whelping" and "cesarean section". A general ordinal logistic regression approach was used to analyze the data. Model selection with possible predictors resulted in a final model including weight of the dam, the weight of the heaviest puppy of a litter, the number of fetuses malpositioned and the quality of uterine contractions. Weight and size of a litter, parity, maternal inbreeding coefficient, whelping season, dam and sire were dropped from the model because they were not significant. The risk of a cesarean section was influenced by the combination of the weight of the dam and the weight of the heaviest puppy in the litter, as well as by the number of malpositioned fetuses and the quality of the contractions. Larger puppies increased the risk of cesarean section especially when the dam had a lighter weight. For dams weighing 23.6 kg and 32.8 kg the predicted probability of a cesarean section was low, with 0.06 and 0.02, respectively, when the heaviest puppy in a litter was light (0.42 kg), contractions were normal and no fetus was malpositioned. However, the probability of a cesarean section was much higher, ranging from 0.24 to 0.08, when the heaviest puppy in a litter was heavy (0.66 kg). CONCLUSIONS: Means to reduce the cesarean section frequency in this Labrador Retriever breeding colony should include genetic selection for ideal puppy weight. In addition, dams with an adult body weight substantially below average should not be selected as breeders in this colony.

Inheritance of equine sarcoid disease in Franches-Montagnes horses.

The mode of inheritance for susceptibility to equine sarcoid disease (ES) remains unknown. The objectives of this study were to analyse a large sample of the Franches-Montagnes (FM) horse population and investigate the heritability and mode of inheritance for susceptibility to ES. Horses were clinically examined for the presence of sarcoid tumours. A standardized examination protocol and client questionnaire were used and a pedigree- and subsequent segregation-analysis for the ES trait performed. To investigate the mode of inheritance, five models were evaluated and compared in a hierarchical way. The analyses reveal that variation in susceptibility to ES is best explained by a model incorporating polygenic variation. The possible effect of a major gene, such as specific equine leukocyte antigen alleles, is unlikely, but cannot be ruled-out entirely. The heritability of the phenotype on the observation scale for the trait 'affected with ES' was estimated to be 8%. A corrected value for the heritability on a liability scale was estimated at 21% and it is therefore possible to estimate breeding values for ES. The arguments against the practical implementation of an estimated breeding value in a multifactorial condition like ES are discussed.

A COL11A2 mutation in Labrador retrievers with mild disproportionate dwarfism.

We describe a mild form of disproportionate dwarfism in Labrador Retrievers, which is not associated with any obvious health problems such as secondary arthrosis. We designate this phenotype as skeletal dysplasia 2 (SD2). It is inherited as a monogenic autosomal recessive trait with incomplete penetrance primarily in working lines of the Labrador Retriever breed. Using 23 cases and 37 controls we mapped the causative mutation by genome-wide association and homozygosity mapping to a 4.44 Mb interval on chromosome 12. We re-sequenced the genome of one affected dog at 30x coverage and detected 92 non-synonymous variants in the critical interval. Only two of these variants, located in the lymphotoxin A (LTA) and collagen alpha-2(XI) chain gene (COL11A2), respectively, were perfectly associated with the trait. Previously described COL11A2 variants in humans or mice lead to skeletal dysplasias and/or deafness. The dog variant associated with disproportionate dwarfism, COL11A2:c.143G>C or p.R48P, probably has only a minor effect on collagen XI function, which might explain the comparatively mild phenotype seen in our study. The identification of this candidate causative mutation thus widens the known phenotypic spectrum of COL11A2 mutations. We speculate that non-pathogenic COL11A2 variants might even contribute to the heritable variation in height.

Temporal genetic variation of the red fox, Vulpes vulpes, across western Europe and the British Isles.

Quaternary climatic fluctuations have had profound effects on the phylogeographic structure of many species. Classically, species were thought to have become isolated in peninsular refugia, but there is limited evidence that large, non-polar species survived outside traditional refugial areas. We examined the phylogeographic structure of the red fox (Vulpes vulpes), a species that shows high ecological adaptability in the western Palaearctic region. We compared mitochondrial DNA sequences (cytochrome b and control region) from 399 modern and 31 ancient individuals from across Europe. Our objective was to test whether red foxes colonised the British Isles from mainland Europe in the late Pleistocene, or whether there is evidence that they persisted in the region through the Last Glacial Maximum. We found red foxes to show a high degree of phylogeographic structuring across Europe and, consistent with palaeontological and ancient DNA evidence, confirmed via phylogenetic indicators that red foxes were persistent in areas outside peninsular refugia during the last ice age. Bayesian analyses and tests of neutrality indicated population expansion. We conclude that there is evidence that red foxes from the British Isles derived from central European populations that became isolated after the closure of the landbridge with Europe.

Segregation analysis of overweight body condition in an experimental cat population.

The goal of this study was to analyze the mode of inheritance of an overweight body condition in an experimental cat population. The cat population consisted of 95 cats of which 81 cats could be clearly classified into lean or overweight using the body condition scoring system according to Laflamme. The lean or overweight classification was then used for segregation analyses. Complex segregation analyses were employed to test for the significance of one environmental and 4 genetic models (general, mixed inheritance, major gene, and polygene). The general genetic model fit the data significantly better than the environmental model (P ≤ 0.0013). Among all other models employed, the major gene model explained the segregation of the overweight phenotype best. This is the first study in which a genetic component could be shown to be responsible for the development of overweight in cats.

A nonsense mutation in the optic atrophy 3 gene (OPA3) causes dilated cardiomyopathy in Red Holstein cattle.

Cardiomyopathies are severe degenerative disorders of the myocardium that lead to heart failure. During the last three decades bovine dilated cardiomyopathy (BDCMP) was observed worldwide in cattle of Holstein-Friesian origin. In the Swiss cattle population BDCMP affects Fleckvieh and Red Holstein breeds. The heart of affected animals is enlarged due to dilation of both ventricles. Clinical signs are caused by systolic dysfunction and affected individuals die as a result of severe heart insufficiency. BDCMP follows an autosomal recessive pattern of inheritance and the disease-causing locus was mapped to bovine chromosome 18 (BTA18). In the present study we describe the successful identification of the causative mutation in the OPA3 gene located on BTA18 that was previously reported to cause 3-methylglutaconic aciduria type III in Iraqi-Jewish patients. We demonstrated conclusive genetic and functional evidence that the nonsense mutation c.343C>T in the bovine OPA3 gene causes the late-onset dilated cardiomyopathy in Red Holstein cattle.

Haematological parameters are normal in dominant white Franches-Montagnes horses carrying a KIT mutation.

The KIT receptor protein-tyrosine kinase plays an important role during embryonic development. Activation of KIT is crucial for the development of various cell lineages such as melanoblasts, stem cells of the haematopoietic system, spermatogonia and intestinal cells of Cajal. In mice, many mutations in the Kit gene cause pigmentation disorders accompanied by pleiotropic effects on blood cells and male fertility. Previous work has demonstrated that dominant white Franches-Montagnes horses carry one copy of the KIT gene with the p.Y717X mutation. The targeted breeding of white horses would be ethically questionable if white horses were known to suffer from anaemia or leukopenia. The present study demonstrates that no statistically significant differences in peripheral blood parameters are detectable between dominant white and solid-coloured Franches-Montagnes horses. The data indicate that KIT mutations may have different effects in mice, pigs, and horses. The KIT p.Y717X mutation does not have a major negative effect on the haematopoietic system of dominant white horses.

A whole-genome scan for recurrent airway obstruction in Warmblood sport horses indicates two positional candidate regions.

Recurrent airway obstruction (RAO), or heaves, is a naturally occurring asthma-like disease that is related to sensitisation and exposure to mouldy hay and has a familial basis with a complex mode of inheritance. A genome-wide scanning approach using two half-sibling families was taken in order to locate the chromosome regions that contribute to the inherited component of this condition in these families. Initially, a panel of 250 microsatellite markers, which were chosen as a well-spaced, polymorphic selection covering the 31 equine autosomes, was used to genotype the two half-sibling families, which comprised in total 239 Warmblood horses. Subsequently, supplementary markers were added for a total of 315 genotyped markers. Each half-sibling family is focused around a severely RAO-affected stallion, and the phenotype of each individual was assessed for RAO and related signs, namely, breathing effort at rest, breathing effort at work, coughing, and nasal discharge, using an owner-based questionnaire. Analysis using a regression method for half-sibling family structures was performed using RAO and each of the composite clinical signs separately; two chromosome regions (on ECA13 and ECA15) showed a genome-wide significant association with RAO at P < 0.05. An additional 11 chromosome regions showed a more modest association. This is the first publication that describes the mapping of genetic loci involved in RAO. Several candidate genes are located in these regions, a number of which are interleukins. These are important signalling molecules that are intricately involved in the control of the immune response and are therefore good positional candidates.

The bovine dilated cardiomyopathy locus maps to a 1.0-Mb interval on chromosome 18.

Cardiomyopathies are myocardial diseases that lead to cardiac dysfunction, heart failure, arrhythmia, and sudden death. In human medicine, cardiomyopathies frequently warrant heart transplantation in children and adults. Bovine dilated cardiomyopathy (BDCMP) is a heart muscle disorder that has been observed during the last 30 years in cattle of Holstein-Friesian origin. In Switzerland BDCMP affects Swiss Fleckvieh and Red Holstein breeds. BDCMP is characterized by a cardiac enlargement with ventricular remodeling and chamber dilatation. The common symptoms in affected animals are subacute subcutaneous edema, congestion of the jugular veins, and tachycardia with gallop rhythm. A cardiomegaly with dilatation and hypertrophy of all heart chambers, myocardial degeneration, and fibrosis are typical postmortem findings. It was shown that all BDCMP cases reported worldwide traced back to a red factor-carrying Holstein-Friesian bull, ABC Reflection Sovereign. An autosomal recessive mode of inheritance was proposed for BDCMP. Recently, the disease locus was mapped to a 6.7-Mb interval MSBDCMP06-BMS2785 on bovine Chr 18 (BTA18). In the present study the BDCMP locus was fine mapped by using a combined strategy of homozygosity mapping and association study. A BAC contig of 2.9 Mb encompassing the crucial interval was constructed to establish the correct marker order on BTA18. We show that the disease locus is located in a gene-rich interval of 1.0 Mb and is flanked by the microsatellite markers DIK3006 and MSBDCMP51.

A mutation in hairless dogs implicates FOXI3 in ectodermal development.

Mexican and Peruvian hairless dogs and Chinese crested dogs are characterized by missing hair and teeth, a phenotype termed canine ectodermal dysplasia (CED). CED is inherited as a monogenic autosomal semidominant trait. With genomewide association analysis we mapped the CED mutation to a 102-kilo-base pair interval on chromosome 17. The associated interval contains a previously uncharacterized member of the forkhead box transcription factor family (FOXI3), which is specifically expressed in developing hair and teeth. Mutation analysis revealed a frameshift mutation within the FOXI3 coding sequence in hairless dogs. Thus, we have identified FOXI3 as a regulator of ectodermal development.

Genomic characterisation, chromosomal assignment and in vivo localisation of the canine high mobility group A1 (HMGA1) gene.

BACKGROUND: The high mobility group A1 proteins (HMGA1a/HMGA1b) are highly conserved between mammalian species and widely described as participating in various cellular processes. By inducing DNA conformation changes the HMGA1 proteins indirectly influence the binding of various transcription factors and therefore effect the transcription regulation. In humans chromosomal aberrations affecting the HMGA1 gene locus on HSA 6p21 were described to be the cause for various benign mesenchymal tumours while high titres of HMGA1 proteins were shown to be associated with the neoplastic potential of various types of cancer. Interestingly, the absence of HMGA1 proteins was shown to cause insulin resistance and diabetes in humans and mice. Due to the various similarities in biology and presentation of human and canine cancers the dog has joined the common rodent animal model for therapeutic and preclinical studies. Accordingly, the canine genome was sequenced completely twice but unfortunately this could not solve the structure of canine HMGA1 gene. RESULTS: Herein we report the characterisation of the genomic structure of the canine HMGA1 gene consisting of 7 exons and 6 introns spanning in total 9524 bp, the in vivo localisation of the HMGA1 protein to the nucleus, and a chromosomal assignment of the gene by FISH to CFA12q11. Additionally, we evaluated a described canine HMGA1 exon 6 SNP in 55 Dachshunds. CONCLUSION: The performed characterisations will make comparative analyses of aberrations affecting the human and canine gene and proteins possible, thereby providing a basis for revealing mechanisms involved in HMGA1 related pathogenesis in both species.

Chromosomal assignment of canine THADA gene to CFA 10q25.

BACKGROUND: Chromosomal translocations affecting the chromosome 2p21 cluster in a 450 kb breakpoint region are frequently observed in human benign thyroid adenomas. THADA (thyroid adenoma associated) was identified as the affected gene within this breakpoint region. In contrast to man tumours of the thyroid gland of dogs (Canis lupus familiaris) constitute mainly as follicular cell carcinomas, with malignant thyroid tumours being more frequent than benign thyroid adenomas. In order to elucidate if the THADA gene is also a target of chromosomal rearrangements in thyroid adenomas of the dog we have physically mapped the canine THADA gene to canine chromosome 10.A PCR was established to screen a canine genome library for a BAC clone containing the gene sequence of canine THADA. Further PCR reactions were done using the identified BAC clone as a template in order to verify the corresponding PCR product by sequencing.Canine whole blood was incubated with colcemid in order to arrest the cultured cells in metaphases. The verified BAC DNA was digoxigenin labeled and used as a probe in fluorescence in situ hybridization (FISH). Ten well spread metaphases were examined indicating a signal on canine chromosome 10 on both chromatids. A detailed fine mapping was performed indicating the canine THADA gene locus on the q-arm of chromosome 10. RESULTS: The canine THADA gene locus was mapped on chromosome 10q25. Our mapping results obtained in this study following the previously described nomenclature for the canine karyotype. CONCLUSION: We analysed whether the THADA gene locus is a hotspot of canine chromosomal rearrangements in canine neoplastic lesions of the thyroid and in addition might play a role as a candidate gene for a possible malignant transformation of canine thyroid adenomas. Although the available cytogenetic data of canine thyroid adenomas are still insufficient the chromosomal region to which the canine THADA has been mapped seems to be no hotspot of chromosomal aberrations seen in canine thyroid adenomas.

PRNP promoter polymorphisms are associated with BSE susceptibility in Swiss and German cattle.

BACKGROUND: Non-synonymous polymorphisms within the prion protein gene (PRNP) influence the susceptibility and incubation time for transmissible spongiform encephalopathies (TSE) in some species such as sheep and humans. In cattle, none of the known polymorphisms within the PRNP coding region has a major influence on susceptibility to bovine spongiform encephalopathy (BSE). Recently, however, we demonstrated an association between susceptibility to BSE and a 23 bp insertion/deletion (indel) polymorphism and a 12 bp indel polymorphism within the putative PRNP promoter region using 43 German BSE cases and 48 German control cattle. The objective of this study was to extend this work by including a larger number of BSE cases and control cattle of German and Swiss origin. RESULTS: Allele, genotype and haplotype frequencies of the two indel polymorphisms were determined in 449 BSE cattle and 431 unaffected cattle from Switzerland and Germany including all 43 German BSE and 16 German control animals from the original study. When breeds with similar allele and genotype distributions were compared, the 23 bp indel polymorphism again showed a significant association with susceptibility to BSE. However, some additional breed-specific allele and genotype distributions were identified, mainly related to the Brown breeds. CONCLUSION: Our study corroborated earlier findings that polymorphisms in the PRNP promoter region have an influence on susceptibility to BSE. However, breed-specific differences exist that need to be accounted for when analyzing such data.

Polymorphism and chromosomal location of the MC4R (melanocortin-4 receptor) gene in the dog and red fox.

The melanocortin-4 receptor (MC4R) is expressed in the hypothalamus and regulates energy intake and body weight. In silico screening of the canine chromosome 1 sequence and a comparison with the porcine MC4R sequence by BLAST were performed. The nucleotide sequence of the whole coding region and 3'- and 5'-flanking regions of the dog (1214 bp) and red fox (1177 bp) MC4R gene was established and high conservation of the nucleotide sequences was revealed (99%). Five sets of PCR primers were designed and a search for polymorphism was performed by the SSCP technique in a group of 31 dogs representing nineteen breeds and 35 farm red foxes. Sequencing of DNA fragments, representing the identified SSCP patterns, revealed three single nucleotide polymorphisms (including a missense one) in dogs and four silent SNPs in red foxes. An average SNP frequency was approx. 1/400 bp in the dog and 1/300 bp in the red fox. We mapped the MC4R gene by FISH to the canine chromosome 1 (CFA1q1.1) and to the red fox chromosome 5 (VVU5p1.2).

Isolation and expression analysis of the canine insulin-like factor 3 gene.

The insulin-like factor 3 (INSL3 or relaxin-like factor) is a hormone produced mainly in gonadal tissues in males and females. Deletion of INSL3 or its receptor in male mice leads to the undescended testes, or cryptorchidism. Here we describe an isolation and analysis of full-length canine INSL3 gene. The INSL3 gene is composed of two exons within a small genomic region. Putative translation of the isolated cDNA yields 132 amino acid preproINSL3 that has the domain structure characteristic for the insulin-relaxin peptide superfamily with a well-conserved receptor-binding domain. Northern blot hybridization showed stronger expression of INSL3 in testis than in ovary. Reverse transcription-polymerase chain reaction analysis of the INSL3 expression revealed a minor splice variant of INSL3 potentially encoding 105 amino acids peptide. We established that the medium, conditioned with recombinant canine INSL3, produced from the full-length cDNA, but not from the minor splice variant, activated human GREAT/LGR8 receptor in vitro. In addition to the functional allele of INSL3, genomic DNA of one of the analyzed dogs contained an intronless nonexpressed pseudogene of INSL3. We isolated canine INSL3 promoter and showed that its activity was strongly mediated by steroidogenic factor-1 in vitro. Using site-specific mutagenesis, we identified a well-conserved steroidogenic factor-1 binding site within canine INSL3 promoter.