Abstracts of the 25th International Colloquium on Animal Cytogenetics and Genomics (25th ICACG), 26–29 June 2024, Naples, Italy

first_page Download PDF settings Order Article Reprints Font Type: Arial Georgia Verdana Font Size: Aa Aa Aa Line Spacing:    Column Width:    Background: Open AccessConference Report Abstracts of the 25th International Colloquium on Animal Cytogenetics and Genomics (25th ICACG), 26–29 June 2024, Naples, Italy by Leopoldo IannuzziLeopoldo Iannuzzi SciProfiles Scilit Preprints.org Google Scholar 1,*, Francesca CiotolaFrancesca Ciotola SciProfiles Scilit Preprints.org Google Scholar 2, Sara AlbarellaSara Albarella SciProfiles Scilit Preprints.org Google Scholar 2, Alessandra IannuzziAlessandra Iannuzzi SciProfiles Scilit Preprints.org Google Scholar 1, Angela PerucattiAngela Perucatti SciProfiles Scilit Preprints.org Google Scholar 1 and Vincenzo PerettiVincenzo Peretti SciProfiles Scilit Preprints.org Google Scholar 2 1 National Research Council (CNR), Institute of Animal Production System in Mediterranean Environment (ISPAAM), Portici, 80055 Naples, Italy 2 Department of Veterinary Medicine and Animal Production, University of Federico II, 80138 Naples, Italy * Author to whom correspondence should be addressed. Biol. Life Sci. Forum 2024, 33(1), 1; https://doi.org/10.3390/blsf2024033001 Published: 25 June 2024 Download keyboard_arrow_down Download PDF Download PDF with Cover Download XML Download Epub Versions Notes 1. IntroductionThe 25th International Colloquium on Animal Cytogenetics and Genomics is dedicated to the memory of Dr. James (Jim) Womack, a pioneer in gene mapping, especially in cattle. The meeting opened with an obituary presented by Prof. Penny Riggs, a former student at Texas A&M University (TAMU) and now a professor in the same department.The meeting was organized into 10 sessions, beginning with General Opening Session 1, which featured three main lectures highlighting the fields of animal cytogenetics and genomics. As expected, among the 83 accepted abstracts for publication, those related to animal genomics were more prevalent than those focused solely on cytogenetics. However, several abstracts combined the two disciplines (Cytogenomics) to provide a deeper understanding of animal genomes and to better identify latent chromosome abnormalities related to fertility. Various genomic approaches were reported in several abstracts, aimed at improving the selection of animals for productive traits, disease resistance, and animal biodiversity.Given the numerous abstracts on water buffalo (river type), a specific session was dedicated to this species, which is particularly important in Eastern, South American, and Mediterranean countries. Nonetheless, research on a wide range of animal species, including domestic and non-domestic animals, non-mammalian vertebrates, and invertebrates, was also presented. Special attention was given to the posters, which were displayed throughout the meeting. Additionally, 15 of the posters, selected by the chairpersons of the poster session, are presented and discussed on the final day. Five posters received awards. All abstracts underwent peer review, and only a few required corrections or modifications. In conclusion, the colloquium featured 13 lectures (L), 27 oral communications (O), and 43 posters (P). Each presentation was numbered according to the congress program. Special thanks to the editorial staff of the "Biology and Life Science Forum" journal for their assistance with the abstract's review and editing. 2. Dr. James (Jim) Womack Obituary O1—Gene Mapping Is Good for You!—Remembering Dr. James E. WomackPenny K Riggs and Womack Lab Former StudentsTexas A&M University, College Station, TX, USA; [email protected] (P.K.R.)A true pioneer in the field of comparative animal genomics, Prof. Jim Womack (30 March 1941–13 August 2023) is remembered for his remarkable career, scientific achievements, and mentorship of 50 doctoral students and countless additional graduate students, post-doctoral scientists, and visiting scholars. Jim completed a Bachelor of Science degree at Abilene Christian College (ACU), followed by a PhD in genetics at Oregon State University. Following positions at ACU and Jackson Laboratories, he joined the faculty at Texas A&M University in 1977, remaining until his retirement as a Distinguished Professor in 2018.Womack's extensive scientific contributions included the first comparative synteny map for cattle. This publication was key for connecting chromosome maps to linkage groups, launching advances in the field that led to the completion of the bovine genome sequence. Jim's research, as evidenced by more than 380 peer-reviewed publications, led to numerous recognitions and awards, including the CIBA Prize for Research in Animal Health (1993), election to the National Academy of Sciences (1999), and the Wolf Prize in Agriculture (2001). He was also recognized with awards for outstanding teaching, graduate mentorship, and service to the scientific community.Jim thoroughly enjoyed life outside the lab and cherished his family: his wife of 60 years, Raby Womack, his children Jimmy (deceased) and Wendy, and his grandsons Quaid Faltys and James Hamlin Hill. He is also survived by numerous colleagues, friends, and former students and advisees who will carry his legacy in genomics on to the next generation. 3. General Opening Session 1 3.1. L1—Cytogenetic Diagnostics—From Giemsa Staining to SNP Microarray and NGSMarek SwitonskiDepartment of Genetics and Animal Breeding, Poznan University of Life Sciences, Wolynska 33, 60-637 Poznan, Poland; [email protected]Implementation of cytogenetic diagnostics into veterinary medicine and animal breeding was launched in the late 1960s by Professor Ingemar Gustavsson, who, using Giemsa staining, showed a high incidence of 1/29 Robertsonian translocation in Swedish cattle. This simple approach was applied in studies on the worldwide distribution of this mutation in numerous cattle breeds. The development of banding techniques in the late 1960s and early 1970s, followed by the establishment of the first international G-banded standard karyotypes of several domestic animal species, was a mile stone in cytogenetic diagnostics. The use of G-banding revealed that reciprocal translocations are quite common in subfertile pigs, while C-banding was frequently applied in the diagnosis of X monosomy in sterile mares. Fluorescent in situ hybridization (FISH), developed in the middle 1980s, was another mile stone. Genome libraries and so-called flow karyotyping facilitated the use of locus specific BAC probes and whole chromosome painting probes for the identification of numerous chromosome aberrations. Recently, a multi-hybridization FISH approach, performed on microscopic slides with immobilized BAC probes specific for distal fragments of all chromosome arms, was successfully used for the rapid identification of reciprocal and Robertsonian translocations in AI bulls and AI boars. Classical and molecular cytogenetic techniques have a crucial limitation, which is a strict time regime regarding delivery of blood samples from a farm or clinic to the diagnostic laboratory; thus, molecular methods based on isolated DNA samples were highly demanded. The development of SNP microarrays and next generation sequencing (NGS) is the newest milestone in cytogenetic diagnostics. These methods were used in the diagnosis of autosomal or sex chromosome aneuploidies. Molecular detection of sex chromosome aneuploidies or XX/XY leukocyte chimerism associated with freemartinism can also be performed by digital droplet PCR (ddPCR). An interesting molecular approach recently developed is the use of copy number variation (CNV) in the pericentromeric region of bovine chromosome 29 for the detection of 1/29 centric fusion carriers. Concluding, the role of molecular techniques in cytogenetic diagnostics is increasing, and what is very important is that they allow for screening studies on archival DNA samples. Funding: This research was financed by statutory funding of 506.534.05.00 from the Faculty of Veterinary Medicine and Animal Science, Poznan University of Life Sciences, Poland. 3.2. L2—Bovine Research: From Genomics to EpigenomicsPaolo Ajmone Marsan 1,2,*, Licia Colli 1,3, Matilde Passamonti 1,4, John L. Williams 1,4 and Riccardo Negrini 1,51 Department of Animal Science, Food and Nutrition (DIANA), Università Cattolica del Sacro Cuore via Emilia Parmense, 84, 29122 Piacenza, Italy2 Romeo and Enrica Invernizzi Research Center on Sustainable Dairy Production (CREI), Università Cattolica del Sacro Cuore, via Emilia Parmense 84, 29122 Piacenza, Italy3 Biodiversity and Ancient DNA Research Center (BioDNA), Università Cattolica del Sacro Cuore, via Emilia Parmense 84, 29122 Piacenza, Italy4 Davies Livestock Research Centre, School of Animal and Veterinary Sciences, Faculty of Sciences, University of Adelaide, Roseworthy, SA 5371, Australia5 Nutrigenomics and Proteomics Research Center (PRONUTRIGEN) Università Cattolica del Sacro Cuore, via Emilia Parmense 84, 29122 Piacenza, Italy* Correspondence: [email protected]The field of bovine research is undergoing a significant transformation, driven by advances in long-read sequencing technologies. These developments have led to the construction of Telomere-to-Telomere (T2T) genomes, facilitating a thorough identification of genomic variations, including structural changes. This detailed mapping of all variants is instrumental in developing bovine pangenomes, which represent the whole genetic diversity of the species. Pangenomes consist of a core genome, present in all individuals, and accessory genomes, which are unique to certain populations, breeds, or individuals. Pangenomes will enhance detailed investigations of intraspecific diversity and facilitate the identification of variants responsible for specific traits within genomic regions. Long-read sequencing technologies are also enhancing our ability to study gene expression and DNA methylation. In addition to being at the basis of tissue differentiation, DNA methylation is one of the epigenetic mechanisms involved in environmental adaptation, enabling organisms to adjust to local conditions and stress responses. This adaptability is vital for cattle, which face diverse environmental challenges, including changes in maternal diet, heat stress, and disease. These challenges can lead to epigenetic modifications, some of which may be heritable, thereby affecting subsequent generations. The forthcoming release of the first bovine pangenome represents a pivotal achievement, capturing the species' collective variation and signifying a new chapter in cattle genetic research. Together with new findings on the heritability of epigenetic alterations triggered by environmental stressors, the pangenome opens new perspectives in the identification of causal genetic and epigenetic variants, to understand biology, and for assisted selection. The current challenge is to incorporate epigenetic insights into traditional genetic improvement programs. This presentation will shed light on the latest progress in bovine genomics and epigenomics, focusing on the development of pangenomes and the effects of epigenetic modifications on the resilience and adaptability of cattle, and discussing the potential for integrating these innovative discoveries into genetic improvement strategies to improve cattle performance and welfare. 3.3. L3—Clinical Veterinary Genetics in the Age of Whole Genome SequencingCord Drögemüller Institute of Genetics, Vetsuisse Faculty, University of Bern, Switzerland; [email protected]Domesticated animals' genomes are a goldmine for medical research because they often have rare diseases that are very similar to those in humans, and their genomes contain selection signatures that explain, for example, breed-specific traits such as coat color and horn status. The large SNP genotyping data available from farm animal genomic selection programmes could be readily used to map potentially associated regions in the genomes and could also help to detect chromosomal abnormalities in affected animals. Over the past decade, short-read whole genome sequencing of individual cases compared to thousands of control genomes has been used to identify causal variants for Mendelian disorders, including protein-altering single nucleotide variants and indels, as well as larger structural variants such as copy number variations, translocations, or deletions predicted to cause haploinsufficiency. In the case of chromosomal abnormalities, depth of read coverage analysis allows the WGS-based detection of reciprocal translocations and other structural constitutional rearrangements beyond the resolution limit of classical cytogenetic techniques, including precise mapping of breakpoints in the genome. Although only recently introduced, long-read sequencing technologies have already demonstrated their potential in veterinary genetic research, with the potential to greatly improve diagnostic yield, particularly for the identification of structural variants and repeat expansions. Using recent examples, this presentation will highlight the emerging potential of precision genomic diagnostics in livestock and show how these methods can contribute to the sustainable improvement of the reproductive success of livestock populations by increasing the diagnostic rate of potentially genetic losses during pregnancy, birth, and rearing. 4. Session 2—Cytogenetics and Genomics in Animal Diagnostics 4.1. L4—Detection and Characterization of Cytogenetic Defects in Cattle Using Large Genotypic and Phenotypic Data Sets Generated for Genomic EvaluationJeanlin Jourdain 1,2, Harmonie Barasc 3, Sébastien Fritz 1,2, Clémentine Escouflaire 1,2, Anne Barbat 2, Cécile Grohs 2, Chris Hozé 1,2, Thomas Faraut 3, Didier Boichard 2, Alain Pinton 3 and Aurélien Capitan 1,2,*1 Eliance, 75012 Paris, France2 Université Paris-Saclay, INRAE, AgroParisTech, GABI, G2B, 78350 Jouy-en-Josas, France3 GenPhySE, Université de Toulouse, INRAE, ENVT, 31320 Castanet-Tolosan, France* Correspondence: [email protected]Over the past two decades, the French National Cattle Database (FNCD) has accumulated information on the life, performance, and pedigree of more than one hundred million animals, as well as the SNP array genotypes of 2 million individuals from various breeds generated as part of genomic selection. The exploitation of this vast amount of data offers numerous prospects in applied and basic research.In a recent study, we developed a highly sensitive approach to detect interchromosomal rearrangements (IR) by searching for abnormal linkage disequilibrium patterns between markers from non-homologous chromosomes in large paternal half-sib families (Jourdain et al., Genome Res. 2023 Jun; 33(6)). After validation by cytogenetic analyses, we reported one Robertsonian fusion, 10 reciprocal translocations, and the first case of insertional translocation reported in cattle among 5571 normozoospermic bulls (prevalence = 2.15/1000). By combining multiple sources of information, we demonstrated that most of these IRs are the result of recent de novo mutations due to abnormal male meiosis and that they have dramatic negative effects on several fitness-related traits in the carrier sires and their carrier daughters. Finally, we performed long-read sequencing to better characterize the exact nature of seven of these IRs and to identify candidate genes whose haploinsufficiency may be health threatening.In this talk, we will detail the main results of this study, which, is to our knowledge, the most comprehensive and thorough screen for interchromosomal rearrangements compatible with normal spermatogenesis in livestock species. In addition, we will also present the possibilities offered by the analysis of signal intensity and genotypes of markers located on chromosomes X and Y to identify other cytogenetic defects, with a special emphasis on heterochromosome aneuploidy and free martinism. 4.2. L5—The Impact of Bioinformatics in Animal CytogeneticsPietro ParmaDipartimento di Scienze Agrarie e Ambientali, Università di Milano, Via Celoria 2, 20133, Milano, Italia; [email protected]The number of chromosomes of the bovine species was defined as 2n = 60 in 1927 and this observation was later confirmed in several papers published from 1944 to 1957.There is no doubt that the impetus for the development of the cytogenetics of cattle, and thus of animals of zoo-economic interest, began in 1964 when Gustavsson published the identification of the first cases of Rob1;29.However, even though 60 years have now passed, cytogenetic approaches have not changed much. Technological advances that can be recalled are the application of banding techniques that have enabled the unambiguous recognition of chromosomes, especially for those species where most chromosomes are acrocentric, or the development of FISH that initiated the mapping of genetic factors in the pre-genomic era.In recent decades, an evolving discipline has emerged as a result of the need to analyze the multitude of data produced by high-throughput sequencing technologies: bio-informatics.In this presentation, the relationships between cytogenetics and bio-informatics and how the latter can be of great help to cytogenetics are presented and discussed. 4.3. O2—Three New 65,XXY Horses Detected Using Medium-Density Genomic Screening in the Pura Raza Español Breed Miguel Moreno-Millán 1, Ana Encina 2, Monika Bugno Poniewierzka 3, Mercedes Valera 2 and Sebastián Demyda Peyrás 1,*1 Facultad de Veterinaria, Universidad de Córdoba, España2 Escuela Técnica Superior De Ingeniería Agronómica, Universidad de Sevilla, España3 Uniwersytet Rolniczy im. Hugona Kołłątaja w Krakowie, Polska* Correspondence: [email protected]Chromosomal abnormalities are the most common cause of genetic infertility in the domestic horse. Nowadays, the age of genomics allows us to not only improve the accuracy of diagnostics but also perform large-scale screening programs. This is the case of the Pura Raza Español horse, in which all the individuals enrolled in the studbook are analyzed for chromosomal abnormalities by a genomic procedure. Hereby, we present three new cases of 65,XXY horses detected during the last 16 months. The individuals were flagged due to abnormal results in the initial STR parentage testing and then submitted to MD SNP genotyping (GGP Equine, Neogen, UK). Copy number aberration (CNA) was made per chromosome using an in-house pipeline based on the analyses of B-allele frequencies (BAF) and Log R Ratio (LRR). In addition, two of the individuals were karyotyped, determining the sex-pair complements by FISH. The three cases showed ~172 ECAY-linked SNP-makers with positive amplification. In ECAX, the non-par region (SNP located > 1.8 Mb) showed an average BAF close to 0, depicting heterozygosity. However, in the PAR region (SNP located in the first 1.8 Mb of ECAX), the three individuals showed four different peaks in BAF values, commonly associated with mosaicism. This CNA in the PAR region was produced by the presence of two ECAX and one ECAY in the same individual. In addition, no differences were observed between the results obtained from analyzing DNA obtained from blood and hair follicles, ruling out the presence of hematopoietic chimerism. Two of the three cases were confirmed as 65, XXY, by FISH analysis. We demonstrated the validity of genomic screening to allow the early and accurate detection of CNA in domestic horses. 4.4. O3—Sperm-FISH Analysis for Validating Sexed Italian Mediterranean River Buffalo (Bubalus bubalis) SemenRamona Pistucci 1, Pietro Parma 2, Roberta Cimmino 3, Gianluca Neglia 4 and Alessandra Iannuzzi 1,*1 Institute of Animal Production System in Mediterranean Environment (ISPAAM), National Research Council (CNR), Portici, Italy2 Department of Agricultural and Enviromental Sciences University of Milan, 20133 Milan, Italy3 Italian Buffalo Species Breeders Association (ANASB), V. Petrarca 42/44, 81100 Caserta, Italy4 Department of Veterinary Medicine and Animal Production, Federico II University, V. F. Delpino 1, 80137 Naples, Italy* Correspondence: [email protected]In both cattle and Italian Mediterranean River Buffalo breeding, sexed semen plays a vital role in economic and reproductive management. The most recent technology employed for semen sexing is laser ablation, where female sperm, containing more DNA chromosomes, are typically larger than male sperm. This technique involves passing the semen through a laser that can identify stained X chromosomes and use ablation to remove the Y chromosomes, resulting in female-sexed semen.This study aims to assess the accuracy of this sexed semen technology through Fluorescence in Situ Hybridization of Spermatozoa (Sperm FISH). For the analysis, we utilized two different probes containing sequences complementary to those of the buffalo X and Y chromosomes from CHORI BAC libraries 240 (cattle) and validated on river buffalo metaphases. We counted 1000 spermatozoa in six bulls under the fluorescence microscope, both in the total and sexed semen fractions of each animal. The total sperm fraction showed an X signal percentage close to 50%, while sexed semen ranged between 70% and 80%. Only one sexed semen sample showed an X signal percentage identical to the total fraction, highlighting the importance of FISH analysis for sexed semen validation. Moreover, it emphasizes the need for cytogenetic methods to assess X chromosome-carrying sperm concentration, a fundamental aspect of dairy farming. Additionally, validating sexed semen is crucial as it can cost up to 10 times more than unsorted semen, underscoring the economic significance of accurate validation procedures.This study highlights practical implications for the dairy industry and buffalo breeding, stressing the need for further research to optimize semen sexed validation methods.Funding: This study has been supported by project"PON01_486 GENOBU", Ministry of Education, Universities, and Research. 4.5. P1—Monozygotic Origin of Three Cases of Female Dicephalic Buffalo and Bovine Calves Sara Albarella 1,*, Joanna Nowacka-Woszuk 2, Marek Switonski 2, Marita Giorgia Riccardi 3, Emanuele D'Anza 1, Giovanna Bifulco 1, Dario Costanza 1, Mariagiulia Pugliano 1, Francesca Ciotola 1 and Vincenzo Peretti 11 Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy2 Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Poznan, Poland3 Department of Animal Health, Experimental Zooprophylactic Institute of Southern Italy, Portici, Italy* Correspondence: [email protected]Dicephaly is a rare congenital malformation observed in different mammalian species. In humans, its frequency is estimated between 1:50,000 and 1:100,000 in newborns, and the majority of the cases were female. In livestock breeding, dicephalic newborns cause economic losses due to the negative consequences of dystocia for a cow delivering such an animal. There are several reports concerning the anatomy of dicephalic bovine or buffalo calves; however, there are no reports on their molecular characteristics. The aim of the present study was DNA analysis of tissue samples isolated from both heads (ear, hair follicles, or brain) of stillborn dicephalic calves (two bovine and one buffalo) to reveal their origin (mono- or dizygotic) and chromosomal sex. A panel of 12 microsatellites (BM1818, BM1824, BM2113, ETH3, ETH10, ETH225, INRA23, SPS115, TGLA227, TGLA122, TGLA126, and TGLA45), accepted by ISAG for parentage testing in cattle, was used to study both bovine cases. Moreover, PCR searching for Y-linked (SRY and AMELY) and X-linked (AMELX) was also performed. The genotype of the buffalo calf was analyzed at 14 microsatellite sites specific for this species (CSSM60, BMC1013, CSSM47, INRA026, CSSM19, BMO922, RM4, INRA006, CSSM42, MAF65, D5S2, CSSM38, BM1706, and CYP21), along with searching for the presence of Y-linked and X-linked genes. These analyses revealed the same genotype for all microsatellites and a lack of Y-linked sequences. In conclusion, our study showed that the studied calves were female and had a monozygotic origin. 4.6. P2—Assessment of the Influence of Methylation and the Position Effect on the Inactivation of Nucleolar Organizer Regions in Mares with Karyotype 64,xx,t(x;1)(xp;1p)(xq;1q)Monika Bugno-Poniewierska *, Kornelia Gala, Natalia Ciesla and Barbara Kij-MitkaDepartment of Animal Reproduction, Anatomy and Genomics; University of Agriculture in Krakow* Correspondence: [email protected]The nucleolar organizer regions (NORs) in the equine karyotype are located on three pairs of autosomal chromosomes 1, 28, and 31. The activity of NORs may be influenced by many factors, including age or breed, but also by mechanisms such as methylation, inactivation of one of the X chromosomes, and the position effect. The above-mentioned mechanisms led to the formulation of a hypothesis assuming that the translocation involving the p arm of chromosome pair 1 (where the NORs are located) and the p arm of the X chromosome will affect the activity of the NORs in the newly formed 1p:Xp structure. Therefore, the aim of the conducted research was to assess the influence of methylation and the position effect on the inactivation of NORs in mares with karyotype 64,XXt(X:1)(Xp:1p)(Xq:1q).Cytogenetic analyses, such as lymphocyte culture, AgNOR staining, and immunofluorescence with anti-5-methylcytosine antibody techniques, were performed on the metaphase chromosomes of a 2-year-old Hutsul mare, carrier of a reciprocal translocation between the arms of chromosomes no.1 and the X chromosome—karyotype 64,XXt(X:1)(Xp:1p)(Xq:1q). The control consisted of preparations from 3 Hutsul mares, 2 years old, with a normal karyotype.The immunofluorescence technique showed that this mutation influenced the methylation pattern of p arm chromosomes no. 1, silencing the signal within the NOR, but did not affect the transcriptional activity of this region. The AgNOR staining technique used allowed for the determination of the number of active NORs, which ranged from one to six. The observed variability in the number of silvered NORs was primarily caused by the different frequency of these regions appearing on chromosomes 28 and 31. However, no differences were found in the number of active NORs on chromosome pair 1 in mares with translocation 64,XXt(X:1)(Xp:1p)(Xq:1q), which indicates the lack of influence of the effect of inactivation of one of the X chromosomes and methylation on a translocated chromosome consisting of the p arm of chromosome no.1 and the p arm of the X chromosome. 4.7. P3—A Case of 78,XX/78,XY Leukocyte Chimerism in a Great Dane Dog with Disorder of Sex DevelopmentEmanuele D'Anza 1, Pietro Parma 2,*, Fausto Cremonesi 3, Patrizio Donati 4, Izabela Szczerbal 5, Marek Switonski 5, Sara Albarella 1 and Francesca Ciotola 11 Dipartimento di Medicina Veterinaria e Produzioni Animali, Università degli Studi di Napoli Federico II, via Delpino 1, 80137 Napoli, Italy2 Dipartimento di Scienze Agrarie e Ambientali, Università di Milano, Via Celoria 2, 20133 Milano, Italy3 Dipartimento di Medicina Veterinaria e Scienze Animali, Università di Milano, Lodi—Via dell'Università 6, 26900 Lodi, Italy4 Ambulatorio Veterinario Donati, 20023 Cerro Maggiore (MI)5 Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Wolynska 33, 60-637 Poznan, Poland* Correspondence: [email protected]Disorders of sex development (DSDs) in dogs are diagnosed more and more frequently, and affected animals are often of pure breed. Canine DSDs caused by sex chromosome aneuploidies (X monosomy, X trisomy, and XXY complement) are rather rarely diagnosed. The most common type of DSD in dogs with a normal set of XY sex chromosomes is polygenic cryptorchidism, however, monogenic XY DSDs caused by mutations of the AMHR2, NR5A1, and HSD17B3 genes were also reported. On the other hand, in chromosomal female dogs (XX) with the presence of testes or ovotestes, the following DNA variants (causative or candidate) were observed: duplication of SOX9, CNV in the 5′flanking region of SOX9, and SNP in PADI6. The aim of this study was the genetic characterization of a Great Dane dog with a malformed uro-genital system. The animal presented a female phenotype with a normal vulva, hypertrophy of the clitoris, a urethral outlet at the clitoral tip, and the presence, laterally to the vulvar lips, of two skin folds resembling the scrotum. During the surgery performed to correct the malformations, two small and rounded gonads were found. Cytogenetic analyses of in vitro cultured leukocytes by fluorescent in situ hybridization (FISH) with the use of X- and Y-specific probes showed the presence of two cell lines: 78,XX (approx. 95%) and 78,XY (approx. 5%). PCR searching for the SRY gene in DNA isolated from blood cells confirmed its presence and a normal sequence. The observed abnormalities of the uro-genit