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INTRODUCTION: The Entlebucher Mountain Dog is predisposed to ureteral ectopia and associated diseases of the urinary tract as well as the kidneys, which can have severe to lethal consequences. Due to the clustered occurrence of clinical signs in 11 % of Entlebucher Mountain dogs in the absence of a genetic test for ureteral ectopia, screening was introduced in 2008 to allow phenotype-based breeding selection. The ureteral orifices of the dogs are visualized by ultrasound and existing urinary retention or urinary incontinence is documented. The diagnostic findings were evaluated centrally with assignment to one of five phenotypes depending on the localization of the ureteral orifices and the renal and ureteral shape. Breeding approval and mating restrictions are the responsibility of the respective breeding associations and predominantly Entlebucher Mountain Dogs with extravesical ectopic ureters and/or clinical signs were excluded from breeding. The effect of phenotype-based selective mating on the incidence of ureteral ectopia and its clinical signs, as well as possible factors influencing the expression of the phenotype, were determined in the birth cohorts after the introduction of screening. Analysis of the data set of 1456 phenotyped Entlebucher Mountain Dogs showed, that at 11 % versus 5 %, males were more frequently assigned to the extravesical phenotype than females. The effect of phenotype-based breeding selection was examined in a subpopulation consisting of phenotyped parents and their offspring (n = 876). The prevalence of the extravesical phenotype decreased from 24 % in the 2005 to 2007 birth cohorts to 1,4 % in the 2015 to 2017 birth cohorts. Since 2015 almost no Entlebucher Mountain Dogs with incontinence, hydroureter or hydronephrosis have been recorded. It was feared that the additional selection measures to control ureteral ectopia in the small Entlebucher Mountain Dog population would intensify the inbreeding increase. However, this has so far remained absent. Therefore, as long as no genetic test is available, it is recommended to continue phenotype-based breeding selection with exclusion of dogs with extravesical ureteral ectopia and/or hydroureter/hydronephrosis/urinary incontinence, while keeping an eye on the development of the inbreeding coefficient.

INTRODUCTION: Le Bouvier de l'Entlebuch est prédisposé à l'ectopie urétérale et aux maladies associées des voies urinaires ainsi que des reins, ce qui peut entraîner des conséquences fatales. En raison de l'apparition de signes cliniques chez 11 % des chiens et en l'absence d'un test génétique pour l'ectopie urétérale, un dépistage a été introduit en 2008 pour permettre une sélection d'élevage basée sur le phénotype. Les orifices urétraux des chiens ont été visualisés par échographie et la rétention ou l'incontinence urinaire existante documentée. Les résultats du diagnostic ont été évalués de manière centralisée avec attribution à l'un des cinq phénotypes en fonction de la localisation des orifices urétéraux ainsi que de la forme des reins et des uretères. L'approbation pour la reproduction et les restrictions d'accouplement relèvent de la responsabilité des associations d'élevage respectives et les bouviers de l'Entlebuch présentant des uretères ectopiques extravésicaux et/ou des signes cliniques ont majoritairement été exclus de la reproduction. L'effet de cet accouplement sélectif basé sur le phénotype sur l'incidence de l'ectopie urétérale et de ses signes cliniques ainsi que les facteurs possibles influençant l'expression du phénotype ont été déterminés dans les cohortes de naissance après l'introduction du dépistage. L'analyse de l'ensemble des données de 1456 Bouviers de l'Entlebuch phénotypés a montré que, à 11 % contre 5 %, les mâles étaient plus fréquemment affectés au phénotype extravésical que les femelles. L'effet de la sélection d'élevage basée sur le phénotype a été examiné dans une sous-population composée de parents phénotypés et de leur progéniture (n = 876). La prévalence du phénotype extravésical est passée de 24 % dans les cohortes de naissance de 2005 à 2007 à 1,4 % dans les cohortes de naissance de 2015 à 2017. Depuis 2015, presque aucun bouvier d'Entlebuch présentant une incontinence, un hydrouretère ou une hydronéphrose n'a été enregistré. Une possible augmentation de la consanguinité due aux mesures de sélection supplémentaires visant à contrôler l'ectopie urétérale ne s'est pas produite. Par conséquent, tant qu'aucun test génétique n'est disponible, il est recommandé de poursuivre la sélection d'élevage basée sur le phénotype avec exclusion des chiens présentant une ectopie urétérale extravésicale et/ou une hydrouretère/hydronéphrose/incontinence urinaire, tout en surveillant l'évolution du coefficient de consanguinité.

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Abnormalities of chromosomes are an important and well documented cause of disorders of sexual development, fertility problems and congenital anomalies in mammals. Detection of low-level 63,X/64,XX mosaicism during routine cytogenetic evaluation is a challenge because its clinical significance is not yet fully clear. This study describes the prevalence and levels of 63,X mosaicism for a cohort of fertile mares and compares the results with eight problem mares for which no clinical cause of sub-fertility was found. The study design allowed for the analysis of micronuclei which are biomarkers of genomic instability and can disturb cell divisions, drive cancer development or cause congenital diseases. Although 27% of the fertile mares were identified to be 63,X mosaics, the results showed that the rates of abnormal cells were very low (1-3%). Levels of abnormal cells in problem mares with 63,X mosaicism were similar or higher. The average rate of micronuclei in the blood of the fertile mares was ∼1%, well below the baseline (5%) which was proposed for peripheral blood of normal healthy humans. We found weak to modest, but not significant, correlations between the age of fertile mares and 63,X cells (Kendall's tau b = 0.2905; p > 0.05) as well as the rate of micronuclei (Kendall's tau b = 0.1896; p > 0.05). Likewise, the correlation between presence of a 63,X cell line and micronuclei rate was not significant (Kendall's tau b = 0.3201; p > 0.05). The presence of 63,X cells in rates greater than 3% may indeed indicate a higher risk for sub-fertility and eventually for associated health problems in such mares. Detection and elimination of mares with high level of X aneuploidies from breeding may have a positive effect on the fertility within the general horse population. This data may support the evaluation of problem mares with mosaic karyotypes involving the X chromosome.

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In domestic goats, the polled intersex syndrome (PIS) refers to XX female-to-male sex reversal associated with the absence of horn growth (polled). The causal variant was previously reported as a 11.7 kb deletion at approximately 129 Mb on chromosome 1 that affects the transcription of both FOXL2 and several long non-coding RNAs. In the meantime the presence of different versions of the PIS deletion was postulated and trials to establish genetic testing with the existing molecular genetic information failed. Therefore, we revisited this variant by long-read whole-genome sequencing of two genetically female (XX) goats, a PIS-affected and a horned control. This revealed the presence of a more complex structural variant consisting of a deletion with a total length of 10 159 bp and an inversely inserted approximately 480 kb-sized duplicated segment of a region located approximately 21 Mb further downstream on chromosome 1 containing two genes, KCNJ15 and ERG. Publicly available short-read whole-genome sequencing data, Sanger sequencing of the breakpoints and FISH using BAC clones corresponding to both involved genome regions confirmed this structural variant. A diagnostic PCR was developed for simultaneous genotyping of carriers for this variant and determination of their genetic sex. We showed that the variant allele was present in all 334 genotyped polled goats of diverse breeds and that all analyzed 15 PIS-affected XX goats were homozygous. Our findings enable for the first time a precise genetic diagnosis for polledness and PIS in goats and add a further genomic feature to the complexity of the PIS phenomenon.

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In the past two decades, average litter size (ALS) in Entlebucher Mountain dogs decreased by approximately 0.8 puppies. We conducted a GWAS for ALS using the single-step methodology to take advantage of 1632 pedigree records, 892 phenotypes and 372 genotypes (173 662 markers) for which only 12% of the dogs had both phenotypes and genotypes available. Our analysis revealed associations towards the growth differentiation factor 9 gene (GDF9), which is known to regulate oocyte maturation. The trait heritability was estimated at 43.1%, from which approximately 15% was accountable by the GDF9 locus alone. Therefore, markers flanking GDF9 explained approximately 6.5% of the variance in ALS. Analysis of WGSs revealed two missense substitutions in GDF9, one of which (g.11:21147009G>A) affected a highly conserved nucleotide in vertebrates. The derived allele A was validated in 111 dogs and shown to be associated with decreased ALS (-0.75 ± 0.22 puppies per litter). The variant was further predicted to cause a proline to serine substitution. The affected residue was immediately followed by a six-residue deletion that is fixed in the canine species but absent in non-canids. We further confirmed that the deletion is prevalent in the Canidae family by sequencing three species of wild canids. Since canids uniquely ovulate oocytes at the prophase stage of the first meiotic division, requiring maturation in the oviduct, we conjecture that the amino acid substitution and the six-residue deletion of GDF9 may serve as a model for insights into the dynamics of oocyte maturation in canids.

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Black and tan animals have tan-coloured ventral body surfaces separated by sharp boundaries from black-coloured dorsal body surfaces. In the at mouse mutant, a retroviral 6 kb insertion located in the hair cycle-specific promoter of the murine Asip gene encoding agouti signalling protein causes the black and tan phenotype. In rabbits, three ASIP alleles are thought to exist, including an at allele causing a black and tan coat colour that closely resembles the mouse black and tan phenotype. The goal of our study was to identify the functional genetic variant causing the rabbit at allele. We performed a WGS-based comparative analysis of the ASIP gene in one black and tan and three wt agouti-coloured rabbits. The analysis identified 75 at -associated variants including an 11 kb deletion. The deletion is located in the region of the hair cycle-specific ASIP promoter and thus in a region homologous to the site of the retroviral insertion causing the at allele in mice. We observed perfect association of the genotypes at this deletion with the coat colour phenotype in 49 rabbits. The comparative analysis and the previous knowledge about the regulation of ASIP expression suggest that the 11 kb deletion is the most likely causative variant for the black and tan phenotype in rabbits.

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An ectopic ureter is a congenital anomaly which may lead to urinary incontinence and without a surgical intervention even to end-stage kidney disease. A genetic component contributes to the development of this anomaly in Entlebucher mountain dogs (EMD); however, its nature remains unclear. Using the Illumina CanineHD bead chip, a case-control genome-wide association study was performed to identify SNPs associated with the trait. Six loci on canine chromosomes 3, 17, 27 and 30 were identified with 16 significantly associated SNPs. There was no single outstanding SNP associated with the phenotype, and the association signals were not close to known genes involved in human congenital anomalies of the kidney or lower urinary tract. Additional research will be necessary to elucidate the potential role of the associated genes in the development of ectopic ureters in the EMD breed.

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INTRODUCTION: The present case report describes a female cotton-top tamarin which was taken over by the Zoo Basel to form a new breeding pair. The animal demonstrated increased marking behavior without any obvious medical reasons, dominant behavior, failure to become pregnant and its external genitals were ambiguous. A disorder of the sexual development (DSD) was suspected by the zoo veterinarians and in a first step, the chromosomes of the monkey were analyzed. Six cell lines with different karyotypes were observed. The two most frequent cell lines had a 46,XX or a 46,XY karyotype which are normal chromosome complements of female and male cotton-top tamarins, respectively. The other much less frequent cell lines showed numerical aberrations with and without a marker chromosome. Specific biological features of the Callitrichidae, such as natural twinning and genetic chimerism impeded the clarification of the pathogenesis and prevented a reliable prognosis on the fertility of the cotton-top tamarin.

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A good reproductive performance is a central element of animal breeding. The breeders of Entlebucher Mountain dogs observed a decrease of the mean litter size and an increase of the number of unsuccessful matings in the past years. The aim of the present study was to identify factors with an influence on fertility in this breed. In total, 915 litters from 202 sires and 348 dams from 1986 to 2013 entered the analyses. The total puppy losses (7.4%) reduced the mean litter size at birth of 5.49 ± 2.13 to a mean litter size at registration of 5.08 ± 2.05. There was no deviation from the expected equal sex distribution for puppies at birth and at registration, as well as for puppy losses consisting of stillborn puppies and puppies which died or had to be euthanized before registration. The mean annual litter inbreeding coefficient increased from 0.37 in 1986 to 0.40 in 2013 and was correlated with the year of birth of the litter (Kendall's tau b = 0.46). The age of the dam and parental inbreeding were identified as significant predictors with a negative effect on litter size at birth. For the litter size at registration the age and inbreeding of the dam had a significant negative effect and a 1% increase of dam inbreeding is expected to decrease the litter size at birth and registration by 0.1 and 0.09 puppies, respectively. The occurrence of total puppy losses decreased during the years and was more frequent in larger litters. In addition, in litters of older parents the occurrence of puppy losses was more frequent than in litters from younger parents. The final generalized linear mixed-effects models for litter size at birth, litter size at registration and for total puppy losses explained 36%, 33% and 22% of the total variance, respectively. The impact of inbreeding and parental age on fertility of the Entlebucher Mountain dog was small and the influence of the dam was much bigger than the one of the sire. Other factors must be responsible for the variability of litter sizes not explained by the models. Without changes of breeding circumstances, a further increase of inbreeding must be expected. Therefore, a close monitoring and minimizing of inbreeding must be followed up by the breeding community.

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INTRODUCTION: The present case report describes a 6-year old subfertile pony mare, which became pregnant after the eleventh artificial insemination. The examination of the ovaries and the uterus did not reveal any abnormal clinical findings and the mare showed a regular oestrous cycle. Based on cytogenetic and molecular genetic analyses it became possible to elucidate the observed subfertility. The mosaic karyotype of the mare consisted of 63,X (20%) and 64,XX (80%) cells. A PCR analysis failed to amplify sequences from the equine SRY gene. The observed classic 63,X/64,XX mosaicism is a plausible explanation for the subfertility of the mare.

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The present case report describes a stray cat with a female appearance. The new owners requested to neuter the animal. During surgery the veterinarian could not find any gonadal tissue. After puberty the cat showed more and more male behaviour. The owners of the cat were interested to know the cause of the abnormal behaviour, but forbid any further clinical tests or surgery. Based upon cytogenetic and molecular genetic experiments a diagnosis became possible. The uniform karyotype (38,XY) was in accordance with the karyotype of a male cat and it was possible to amplify the SR Y gene by PCR. The cat represents a case of SRYpositive sex reversal.

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Numerical and structural aberrations of chromosomes may cause malformations, embryonal losses and reduced or missing fertility. In male horses the fertility is rather well controlled through their semen evaluation. For mares there are no mandatory regulations which specify their use in a breeding programme. Therefore, mares with chromosomal aberrations, which exclude reproduction success may be chosen for breeding. The present case describes a horse, offically registered as a female, which was presented for a breeding exam. On this occasion, small and inactive ovaries were diagnosed. Although the cytogenetical analysis excluded a numerical or gross structural chromosome aberration, the karyotype (64,XY) corresponded to the male sex. The diagnosis male to female sex-reversal (SRY-negative) excludes reproductive success for this horse. This case underlines the importance of a cytogenetic analysis for female horses, before they enter a breeding programme.

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To test the hypothesis of a heritable base of ectopic ureters (EU) in Entlebucher Mountain Dogs (EMD) and to elucidate associated risk factors and mode of inheritance of the disease, 565 EMD were clinically investigated and population genetic analyses performed. Based on the location of the most caudal termination of the ureteral openings, 552 EMD were classified into three phenotype groups trigone, intravesically and extravesically ectopic based on results of abdominal sonography, urethra-cystoscopy and/or contrast-enhanced computed tomography. One-third (32.9%) of the phenotyped animals had normal terminations of both ureters in the bladder trigone, 47.3% had at least one intravesicular ectopic termination and 19.8% had at least one extravesicular ectopic termination. Multivariate mixed logistic regression revealed gender as a risk factor associated with EU as males were more often affected than females. Complex segregation analysis indicated a hereditary basis for EU in EMD and the involvement of a major gene in the occurrence of the extravesicular EU phenotype.

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The aim of our study was to diagnose aneuploidy in equine spermatozoa by multicolour fluorescence in situ hybridization (FISH) technique using specific molecular probes for equine sex chromosomes and autosome pair four (EGFR probe) labeled by different fluorochromes. These were applied on decondensed spermatozoa of four stallions. In total, more than 8800 sperm cells were examined. The total frequency of aberrant cells was 0.496%: aneuploidy of XX (0.135%), YY (0.023%), XY (0.102%), diploidy (0.057%), lack of sex chromosome (0.18%). In one stallion the ratio of normal X- and Y-bearing cells was different from the expected 1:1 ratio (p = 0.0002), in all three other stallions this ratio was close to 1:1. The present study demonstrated that the FISH technique is a powerful method to identify sex chromosome aberrations in equine spermatozoa and allows for the determination of the ratio between X-Y-spermatozoa.

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The objective of this study was to investigate whether the occurrence of cryptorchidism in a litter is associated with the sex ratio in this litter. The analysis included for dog 1,339 litters of 4 different breeds, of which 12.8% had 1 and 3.1% had 2 cryptorchids, and for pig 119,920 litters of 14 different populations, of which 2.2% had 1 and 0.2% had 2 cryptorchids. The data were analyzed using the GLIMMIX procedure of SAS. In dog, the presence of cryptorchids in a litter does not affect litter size or the stillborn rate. In pig, litter size increases significantly with an increasing number of cryptorchids in a litter. The stillborn rate in pig is larger in litters with cryptorchids than in litters without cryptorchids. To characterize the imbalance of the sexes in a litter, the sex ratio, defined as the number of live males divided by the number of live offspring, and the sex difference, defined as the number of live males minus the number of live females, were used as dependent variables in the analyses. In both dog and pig, the presence of 1 or 2 cryptorchids in a litter clearly goes together with a shift in both the sex ratio and the sex difference in a litter, in favor of male offspring. Our results in dog and pig suggest a common underlying mechanism affecting both cryptorchidism and sex ratio, which may hold true in other mammals as well.

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We have analysed the chromosomes of three wild and endangered canid species: the maned wolf (Chrysocyon brachyurus), the bat-eared fox (Otocyon megalotis) and the fennec fox (Fennecuszerda) using classical and molecular cytogenetic methods. For the first time detailed and encompassing descriptions of the chromosomes are presented including the chromosomal assignment of nucleolar organizer regions and the 5S rRNA gene cluster. We propose a karyotype nomenclature with ideograms including more than 300 bands per haploid set for each of these three species which will form the basis for further research. In addition, we propose four basic different patterns of karyotype organization in the family Canidae. A comparison of these patterns with the most recent molecular phylogeny of Canidae revealed that the karyotype evolution of a species is not always strongly connected with its phylogenetic position. Our findings underline the need and justification for basic cytogenetic work in rare and exotic species.

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Cytogenetic and molecular genetic studies of an intersex horse have been carried out. The investigated animal had overall male body conformation; however, its external genitalia consisted of incompletely developed vulva and penis. The X and Y chromosome painting probes detected three cell lines in the examined horse: 63,X, 64,XX and 65,XX with a fragment of a Y chromosome (del Y). The DNA analysis with the PCR and PCR/RFLP methods showed absence of SRY,AMELY and ZFY genes as well as of six Y microsatellite markers (YM2, YP9, YJ10, YE1, YH12, and YA16). These results suggest that the Y chromosome fragment detected in the investigated animal was the result of a deletion of a euchromatic fragment comprising the above-mentioned markers.

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The recent developments in liquid chromatography (LC) are mainly dedicated to both system miniaturization (micro-, capillary-, and nano-LC) and analysis time decrease (fast-, and ultra-fast-LC). For the latter, several strategies can be used, and high temperature liquid chromatography (HTLC) seems very promising and easy to implement, especially in miniaturized system. In LC, the evaporative light scattering detector (ELSD) is considered an attractive alternative to conventional detector such as UV-vis due to its versatility and quasi-universality. Therefore, the compatibility of ELSD with micro-LC and micro-HTLC was investigated for several pharmaceutical compounds of interest. The nebulization process appeared to be the most critical parameter for performing the coupling and maintaining an efficient separation. Therefore, appropriate modifications in the nebulization cell geometry were brought to make ELSD fully compatible with micro-LC. The impact of optimized nebulization cell on chromatographic performance was evaluated in terms of efficiency and sensitivity. Finally, highly efficient, sensitive and fast separations of pharmaceutical drugs were performed with both techniques and the customized nebulization cell design.

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Cytogenetic investigations of the nucleolar-organizing regions (NORs) show that there is variation in the transcriptional activity of rDNA in many organisms. As a consequence, genetic polymorphism of these regions has been detected. The aim of the present study was to evaluate the hypothetic genetic mechanisms determining the NORs polymorphism of the domestic horse chromosomes. Molecular cytogenetic analyses were carried out on Hucul horses and the following techniques were used: fluorescence in situ hybridization (FISH), telomere primed in situ synthesis (PRINS), in situ nick-translation with HpaII, silver staining (AgNOR) and C-banding technique (CBG). The obtained results suggest that variation in the number and size of silver deposits is related to the number of rDNA copies, DNA methylation and the localization of ribosomal DNA loci in telomeric regions. Moreover, we have found that chromosome pairs 28 and 31 are characterized by higher variation in the NORs number.

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An infertile mare with hypoplastic ovaries was subjected to cytogenetic analysis. Fluorescence in situ hybridisation (FISH) using the equine X whole chromosome painting probe (WCPP) was carried out on a chromosome preparation obtained from blood lymphocyte culture. The number of analysed spreads was high (235) and in the X chromosome aneuploidy in mosaic form was diagnosed. The karyotype formula was 63,X / 64,XX / 65,XXX. The ratio of the three lines was 15%, 82% and 3%, respectively. The application of the FISH technique with WCPP is discussed.

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Bovine dilated cardiomyopathy (BDCMP) is a severe and terminal disease of the heart muscle observed in Holstein-Friesian cattle over the last 30 years. There is strong evidence for an autosomal recessive mode of inheritance for BDCMP. The objective of this study was to genetically map BDCMP, with the ultimate goal of identifying the causative mutation. A whole-genome scan using 199 microsatellite markers and one SNP revealed an assignment of BDCMP to BTA18. Fine-mapping on BTA18 refined the candidate region to the MSBDCMP06-BMS2785 interval. The interval containing the BDCMP locus was confirmed by multipoint linkage analysis using the software loki. The interval is about 6.7 Mb on the bovine genome sequence (Btau 3.1). The corresponding region of HSA19 is very gene-rich and contains roughly 200 genes. Although telomeric of the marker interval, TNNI3 is a possible positional and a functional candidate for BDCMP given its involvement in a human form of dilated cardiomyopathy. Sequence analysis of TNNI3 in cattle revealed no mutation in the coding sequence, but there was a G-to-A transition in intron 6 (AJ842179:c.378+315G>A). The analysis of this SNP using the study's BDCMP pedigree did not conclusively exclude TNNI3 as a candidate gene for BDCMP. Considering the high density of genes on the homologous region of HSA19, further refinement of the interval on BTA18 containing the BDCMP locus is needed.

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