INTRODUCTION:Monorchidism is a rarely described condition in the horse and is not to be confused with cryptorchidism. The diagnosis is challenging and confirmed by surgery and histology in combination with hormonal assays. This report describes, to the best of the author's knowledge, the first case of monorchidism and abdominal cryptorchidism of the developed testicle in a horse. METHODS:An Irish Cob underwent laparoscopic castration for removal of bilateral cryptorchid testicles. At surgery, the horse was diagnosed as a monorchid with the testicle retained intra-abdominally. Histopathological, hormonal, molecular and cytogenetic analysis was performed. This included measuring testosterone and anti-Mullerian hormone (AMH) in serum blood, isolating genomic DNA from EDTA- and heparin-treated blood, PCR amplification of the SRY gene, metaphase chromosome preparation, and DAPI banding before metaphase analysis with fluorescence in situ hybridisation (FISH) analysis. RESULTS:The horse was positive for the SRY gene and had a mosaic 63,X/64,XY karyotype with the aneuploid cells being present in only 2% of metaphases. FISH showed that the missing sex chromosome of the aneuploid cell line was the Y chromosome embedded in micronuclei. An abnormal high rate of micronuclei (6.6%) was observed indicating genotoxic events and/or genome instability. Hormonal assay results confirmed that AMH was not significantly increased, suggesting that no further testicular tissue was present. Histopathology was consistent with testicular tissue displaying a Sertoli cell-only pattern with bipolar ductal structures. CONCLUSION:The exact causes of monorchidism and cryptorchidism are unclear, but the elevated rate of micronuclei is clear evidence for genome instability which might have been involved in the failure of normal testicular development and descent. Future cases could further clarify the disease mechanism based on this report.

Muscular dystrophies represent a group of disorders characterized by progressive muscle degeneration and weakness. An important subgroup are the dystrophin-related muscular dystrophies caused by variants in the DMD gene. They can be divided into the more severe Duchenne muscular dystrophy and the milder Becker muscular dystrophy. Here, we characterize the clinical, histopathological and molecular genetic aspects of two male Entlebucher Mountain Dogs with clinical signs of muscular dystrophy. The two dogs presented with marked dysphagia starting at the age of several weeks and in the later course recognizable exercise intolerance with highly increased serum creatine kinase levels. Histopathological signs of a dystrophic myopathy represented by degeneration of muscle fibers and signs of regeneration were present. Whole genome sequencing of one affected dog identified an intragenic 8.6 kb duplication in the X-chromosomal DMD gene, c.7528-4048_7645 + 4450dup. No other protein-changing variants in candidate genes for muscular dystrophy were identified. The duplication includes exon 52 of DMD and is predicted to lead to a frameshift and truncation of 30% of the wild-type open reading frame. Genotyping of the whole family confirmed the presence of the mutant allele in both affected dogs and the unaffected dam. The correct co-segregation of the mutant allele in the affected family as well as knowledge from humans and other species suggest the identified DMD variant as the most likely candidate variant for the muscular dystrophy phenotype in the two investigated dogs.

Background: Between 2006 and 2014, 10 cases of urolithiasis in yaks with calcium carbonate uroliths were confirmed in Switzerland, and at the same time, a sixfold calcium overhang in roughage in an affected farm was evident. The purpose of this study was the question of whether urolithiasis in yaks in the alpine regions of Switzerland poses a husbandry risk. The hypothesis was that elevated calcium levels in roughage led to hypercalcemia and, thus, the formation of calcium carbonate stones. Methods: Blood samples from 99 animals from 10 farms were examined (n = 6 problem farms; n = 4 control farms). Several metabolites were analyzed in the blood and urine. In addition, roughage, soil, and water samples were analyzed. The farms were distributed within different regions of Switzerland. Results: This study revealed different Ca:P ratios, from 1.56 to 7.74:1, in the forages and mild hypercalcemia in the animals of the problem farms. In a univariate analysis of the problem versus control farms, about 20 other significant factors emerged. Multivariate analysis showed that altitude, CP (crude protein), P, Mg, NDF, and ADF in the diet are important factors. Based on analysis of the pedigree, there was no evidence of an obvious genetic background of urolithiasis. Limitations: A limitation of this study is the small number of yaks in Switzerland. Conclusions: The question of whether urolithiasis in yaks in alpine Switzerland poses a husbandry risk can be answered affirmatively. Calcium-rich forages play a crucial role in this multifactorial process.

Pelger-Huët anomaly (PHA) in humans is an autosomal dominant hematological phenotype without major clinical consequences. PHA involves a characteristic hyposegmentation of granulocytes (HG). Human PHA is caused by heterozygous loss of function variants in the LBR gene encoding lamin receptor B. Bi-allelic variants and complete deficiency of LBR cause the much more severe Greenberg skeletal dysplasia which is lethal in utero and characterized by massive skeletal malformation and gross fetal hydrops. HG phenotypes have also been described in domestic animals and homology to human PHA has been claimed in the literature. We studied a litter of Australian Shepherd Dogs with four stillborn puppies in which both parents had an HG phenotype. Linkage analysis excluded LBR as responsible gene for the stillborn puppies. We then investigated the HG phenotype in Australian Shepherd Dogs independently of the prenatal lethality. Genome-wide association mapped the HG locus to chromosome 27 and established an autosomal recessive mode of inheritance. Whole genome sequencing identified a splice site variant in LMBR1L, c.191+1G>A, as most likely causal variant for the HG phenotype. The mutant allele abrogates the expression of the longer X2 isoform but does not affect transcripts encoding the shorter X1 isoform of the LMBR1L protein. The homozygous mutant LMBR1L genotype associated with HG is common in Australian Shepherd Dogs and was found in 39 of 300 genotyped dogs (13%). Our results point to a previously unsuspected function of LMBR1L in the myeloid lineage of leukocytes.

BackgroundWeight 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.ResultsOf 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.ConclusionsIn 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.

Chromosomal abnormalities are notable causes of infertility in horses. Mares show various degrees of estrous behavior, and ultrasound examination often reveals an underdeveloped genital tract. This article reports investigations on fertility in a Haflinger sibship with a healthy, normally developed, fertile mare with at least three healthy offspring. Chromosomal analysis performed incidentally and blinded for this mare revealed 63,X/64,XX/65,XXX mosaicism. Two closely related mares were also mosaics (63,X/64,XX), and one of them was a carrier of a marker chromosome. Repeated examinations of the mare and seven relatives (four mares and three stallions) did not provide evidence for sub- or in-fertility. They had no developmental abnormalities or conspicuous body conditions. Peripheral blood samples were collected for analysis of the karyotype and molecular analyses. Chromosomes were Giemsa stained and 4',6-diamidino-2-phenylindole banded to identify numerical or structural aberrations of chromosomes and identification of sex chromosomes, respectively. Fluorescence in situ hybridization was performed with an equine Y-chromosome painting probe to identify and count the sex chromosomes, and polymerase chain reaction analysis was used to test for the presence of the SRY gene and investigating chimerism. The present article demonstrates the necessity of further studies analyzing chromosomal X0 mosaics to improve the predictive value of chromosomal aberrations on fertility.

The present report describes a 4-year-old Trakehner mare which was referred to the clinic for a breeding soundness evaluation. Clinical, histological, and postmortem examination revealed an underdeveloped genital tract, the absence of a cervix uteri, and small inactive ovaries without male gonadal tissue. Blood lymphocyte analysis revealed an unusual mosaic karyotype consisting of 2 cell lines. For the majority of cells (70%), monosomy X (63,X) was observed. The remaining cells (30%) contained 64 chromosomes including one X chromosome and a small rudimentary Y chromosome consisting mostly of heterochromatin. The centromere was retained, but its full functionality was questionable. PCR analysis revealed that the entire male-specific region of Y (Yq14), including the SRY gene, was deleted. It remained unclear if the pseudoautosomal region (Yq15) and parts of the heterochromatic region (Yq13) were affected by this deletion. The phenotype of the mare with this disorder of sex development associated with sex chromosome abnormalities is genetically comparable to 63,X monosomy which fully explains the clinical findings.

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.

Belted cattle have a circular belt of unpigmented hair and skin around their midsection. The belt is inherited as a monogenic autosomal dominant trait. We mapped the causative variant to a 37 kb segment on bovine chromosome 3. Whole genome sequence data of 2 belted and 130 control cattle yielded only one private genetic variant in the critical interval in the two belted animals. The belt-associated variant was a copy number variant (CNV) involving the quadruplication of a 6 kb non-coding sequence located approximately 16 kb upstream of the TWIST2 gene. Increased copy numbers at this CNV were strongly associated with the belt phenotype in a cohort of 333 cases and 1322 controls. We hypothesized that the CNV causes aberrant expression of TWIST2 during neural crest development, which might negatively affect melanoblasts. Functional studies showed that ectopic expression of bovine TWIST2 in neural crest in transgenic zebrafish led to a decrease in melanocyte numbers. Our results thus implicate an unsuspected involvement of TWIST2 in regulating pigmentation and reveal a non-coding CNV underlying a captivating Mendelian character.

A 23-month-old tomcat was referred to our clinic because of male behavioral problems, cryptorchidism, and an undefined intra-abdominal organ resembling a uterus. Ultrasonography and computed tomography showed 2 fluid-filled tubular structures dorsolaterally to the bladder and connected to the pelvic urethra. The cat was castrated, and the tubular structures were surgically removed. Histology identified them as Müllerian duct remnants. The testes were hypoplastic, the epididymes and deferent ducts were normal. Cytogenetic analyses revealed the presence of a mosaic 37,X/38,XY karyotype which explains the clinical findings.

Hereditary footpad hyperkeratosis (HFH) represents a palmoplantar hyperkeratosis, which is inherited as a monogenic autosomal recessive trait in several dog breeds, such as e.g. Kromfohrländer and Irish Terriers. We performed genome-wide association studies (GWAS) in both breeds. In Kromfohrländer we obtained a single strong association signal on chromosome 5 (p(raw) = 1.0×10(-13)) using 13 HFH cases and 29 controls. The association signal replicated in an independent cohort of Irish Terriers with 10 cases and 21 controls (p(raw) = 6.9×10(-10)). The analysis of shared haplotypes among the combined Kromfohrländer and Irish Terrier cases defined a critical interval of 611 kb with 13 predicted genes. We re-sequenced the genome of one affected Kromfohrländer at 23.5× coverage. The comparison of the sequence data with 46 genomes of non-affected dogs from other breeds revealed a single private non-synonymous variant in the critical interval with respect to the reference genome assembly. The variant is a missense variant (c.155G>C) in the FAM83G gene encoding a protein with largely unknown function. It is predicted to change an evolutionary conserved arginine into a proline residue (p.R52P). We genotyped this variant in a larger cohort of dogs and found perfect association with the HFH phenotype. We further studied the clinical and histopathological alterations in the epidermis in vivo. Affected dogs show a moderate to severe orthokeratotic hyperplasia of the palmoplantar epidermis. Thus, our data provide the first evidence that FAM83G has an essential role for maintaining the integrity of the palmoplantar epidermis.

The dog was the first domesticated animal but it remains uncertain when the domestication process began and whether it occurred just once or multiple times across the Northern Hemisphere. To ascertain the value of modern genetic data to elucidate the origins of dog domestication, we analyzed 49,024 autosomal SNPs in 1,375 dogs (representing 35 breeds) and 19 wolves. After combining our data with previously published data, we contrasted the genetic signatures of 121 breeds with a worldwide archeological assessment of the earliest dog remains. Correlating the earliest archeological dogs with the geographic locations of 14 so-called "ancient" breeds (defined by their genetic differentiation) resulted in a counterintuitive pattern. First, none of the ancient breeds derive from regions where the oldest archeological remains have been found. Second, three of the ancient breeds (Basenjis, Dingoes, and New Guinea Singing Dogs) come from regions outside the natural range of Canis lupus (the dog's wild ancestor) and where dogs were introduced more than 10,000 y after domestication. These results demonstrate that the unifying characteristic among all genetically distinct so-called ancient breeds is a lack of recent admixture with other breeds likely facilitated by geographic and cultural isolation. Furthermore, these genetically distinct ancient breeds only appear so because of their relative isolation, suggesting that studies of modern breeds have yet to shed light on dog origins. We conclude by assessing the limitations of past studies and how next-generation sequencing of modern and ancient individuals may unravel the history of dog domestication.

To aid in the rapid diagnosis of myxomatosis in rabbits, a real-time polymerase chain reaction (PCR) for the specific detection of Myxoma virus is described. Primers and probe were designed to amplify a 147-bp fragment within the Serp2 gene. The assay was able to detect 23 copies of a synthesized oligo indicating a reliable sensitivity. In addition, the real-time PCR did not detect the Rabbit fibroma virus used in myxomatosis vaccines. The novel PCR was shown to be able to detect Myxoma virus in fresh and paraffin-embedded rabbit tissues originating from myxomatosis cases from various regions in Switzerland.

Melanoma is a disease with high incidence in gray horses and has limited therapeutic options in metastatic disease. Gene therapy has shown some success in animal models and human patients. A randomized double-blind, placebo-controlled study was conducted to investigate 2 treatment options using cytokine-encoding plasmid DNA in horses with metastatic melanoma to induce immunologic antitumor effects. Adult gray horses with spontaneously occurring metastatic melanoma (n=26) were included in the study. Treatment of 26 gray horses with metastatic melanoma consisted of interleukin-18-encoding plasmid DNA, interleukin-12-encoding plasmid DNA, or empty plasmid DNA (control group), injected intratumorally, respectively. Tumor response was assessed using ultrasound and caliper measurements and histologic assessment of tumor biopsies. Significant tumor regression could be shown in both the treatment groups receiving IL-18 and IL-12-encoding plasmid DNA whereas placebo-treated control patients showed tumor growth over the course of the treatment. In addition, 7 of 10 tumors from horses treated with IL-18 or IL-12 showed peritumoral and/or intratumoral inflammatory infiltrates after treatment compared with 1 of the 6 in the control group. The treatment as assessed by serial blood draws and clinical investigation, was safe and well tolerated. These data suggest that the intratumoral treatment with IL-18 and IL-12-encoding plasmid DNA has antitumor effects, which is well tolerated and thus holds promise for the treatment of patients with metastatic melanoma.

Tracing maternal and paternal lineages independently to explore breeding systems and dispersal strategies in natural populations has been high on the wish-list of evolutionary biologists. As males are the heterogametic sex in mammals, such sex-specific patterns can be indirectly observed when Y chromosome polymorphism is combined with mitochondrial sequence information. Over the past decade, Y-chromosomal markers applied to human populations have revealed remarkable differences in the demographic history and behaviour between the sexes. However, with a few exceptions, genetic data tracing the paternal line are lacking in most other mammalian species. This deficit can be attributed to the difficulty of developing Y-specific genetic markers in non-model organisms and the general low levels of polymorphisms observed on the Y chromosome. Here, we present an overview of the currently employed strategies for developing paternal markers in mammals. Moreover, we review the practical feasibility and requirements of various methodological strategies and highlight their future prospects when combined with new molecular techniques such as next generation sequencing.

Purpose: Mutations in bestrophin 1 (BEST1) are associated with a group of retinal disorders known as bestrophinopathies in man and canine multifocal retinopathies (cmr) in the dog. To date, the dog is the only large animal model suitable for the complex characterization and in-depth studies of Best-related disorders. In the first report of cmr, the disease was described in a group of mastiff-related breeds (cmr1) and the Coton de Tulear (cmr2). Additional breeds, e. g., the Lapponian herder (LH) and others, subsequently were recognized with similar phenotypes, but linked loci are unknown. Analysis of the BEST1 gene aimed to identify mutations in these additional populations and extend our understanding of genotype-phenotype associations.Methods: Animals were subjected to routine eye exams, phenotypically characterized, and samples were collected for molecular studies. Known BEST1 mutations were assessed, and the canine BEST1 coding exons were amplified and sequenced in selected individuals that exhibited a cmr compatible phenotype but that did not carry known mutations. Resulting sequence changes were genotyped in several different breeds and evaluated in the context of the phenotype.Results: Seven novel coding variants were identified in exon 10 of cBEST1. Two linked mutations were associated with cmr exclusive to the LH breed (cmr3). Two individuals of Jamthund and Norfolk terrier breeds were heterozygous for two conservative changes, but these were unlikely to have disease-causing potential. Another three substitutions were found in the Bernese mountain dog that were predicted to have a deleterious effect on protein function. Previously reported mutations were excluded from segregation in these populations, but cmr1 was confirmed in another mastiff-related breed, the Italian cane corso.Conclusions: A third independent canine model for human bestrophinopathies has been established in the LH breed. While exhibiting a phenotype comparable to cmr1 and cmr2, the novel cmr3 mutation is predicted to be based on a distinctly different molecular mechanism. So far cmr2 and cmr3 are exclusive to a single dog breed each. In contrast, cmr1 is found in multiple related breeds. Additional sequence alterations identified in exon 10 of cBEST1 in other breeds exhibit potential disease-causing features. The inherent genetic and phenotypic variation observed with retinal disorders in canines is complicated further by cmr3 being one of four distinct genetic retinal traits found to segregate in LH. Thus, a combination of phenotypic, molecular, and population analysis is required to establish a strong phenotype-genotype association. These results indicate that cmr has a larger impact on the general dog population than was initially suspected. The complexity of these models further confirms the similarity to human bestrophinopathies. Moreover, analyses of multiple canine models will provide additional insight into the molecular basis underlying diseases caused by mutations in BEST1.

The cardiac troponin complex, which is an important component of the contractile apparatus, is composed of the three subunits troponin I (TnI), troponin C (TnC) and troponin T (TnT). Troponin I is the inhibitory subunit and consists of three isoforms encoded by TNNI1, TNNI2 and TNNI3 genes, respectively. Due to the different types of cardiomyopathies caused by mutations in the TNNI3 gene and its fluorescence in situ hybridization (FISH) mapping on bovine chromosome 18q26, which was shown to be linked to the recessively inherited bovine dilated cardiomyopathy (BDCMP), bovine TNNI3 was considered as candidate gene for BDCMP. Real-time polymerase chain reaction (PCR) TNNI3 expression analysis resulted in a significant difference between BDCMP affected and unaffected animals when normalized to ACTB gene expression, but there was no significant difference in expression when normalized to GAPDH. Northen blotting experiment was in agreement with the expression analysis and did not reveal a significant difference between the group of BDCMP affected and unaffected animals. Sequencing of the bovine TNNI3 gene revealed a single nucleotide polymorphism in intron 6 (c.378+315G>A), but this single nucleotide polymorphism (SNP)was present regardless of the BDCMP status. In summary our data provide evidence to exclude the bovine TNNI3 gene as a candidate for BDCMP.

Fluorescent in situ hybridisation (FISH) using a panel of molecular probes for all chromosome pairs obtained by chromosome microdissection of the domestic horse ( Equus caballus ) was used to diagnose karyotype abnormalities in 35 horses (32 mares, 2 stallions and 1 intersex), which were selected for the study due to infertility (23 horses), reduced fertility (10 horses) and developmental anomalies (2 horses). The use of the FISH technique with probes for each horse chromosome pair enabled the diagnosis of many different chromosome aberrations in this population. Among the horses analysed, 21 animals had normal karyotype - 64,XX (19 mares) and 64,XY (2 stallions). Fourteen animals, constituting 40% of the population studied, showed the following chromosome abnormalities: 63,X (1 mare); 63,X/64,XX (6 mares); 63,X/64,XX/65,XXX (3 mares); 63,X/65,XXX (1 mare); 64,XX/65,XX+Xp (1 mare); 63,X/64,XX/65,XX+Xq (1 mare), and 63,X/64,XX/65,XX+delY (1 intersex). When only the mares studied because of complete infertility were taken into consideration, this proportion exceeded 56%. Due to the increased frequency of the above-mentioned aberrations in the mosaic form of two or more lines, it was necessary to analyse a large number (100-300) of metaphase spreads. The use of specific molecular probes obtained by chromosome microdissection made these diagnoses much easier.