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The molecular background of disorders of sex development (DSD) in dogs is poorly understood. Several copies of the SRY genes have been reported in the dog genome. We used droplet digital PCR with the aim of determining variability in SRY copy number and its association with DSD in dogs. Altogether 19 DSD male dogs (XY DSD) of 10 breeds and 87 control dogs of eight breeds were analyzed. Moreover, we performed a comparative analysis of SRY copy number in other canids: wolves (3), red foxes (16), and Chinese raccoon dogs (10). We found that the modal number of SRY copies in dogs, wolves, red foxes, and Chinese raccoon dogs was 3, 3, 1, and 3 respectively. Variability of copy number was only observed in Yorkshire Terriers (two or three copies) and red foxes (one or two copies). An analysis of six DSD Yorkshire Terriers and 38 control males of this breed showed that 50% of the DSD dogs had two copies, while the incidence of this variant was significantly lower in the control dogs (10.5%). Searching for the copy number of the coding and 5'-flanking fragments revealed full concordance with the copy number. These fragments were also sequenced in DSD (19) and control (24) dogs, and no DNA variants were found. We conclude that, in the dog, two or three functional copies of the SRY gene are present, and a smaller number of copies showed an association with the risk of DSD phenotype in Yorkshire Terriers.

The equine graying with age causative mutation in the syntaxin-17 gene (STX17) has been known for over a decade, but proper genotyping of this variant remains challenging due to its molecular character (4.6-kb tandem duplication). Precise information on gray mutation status is important for horse breeders and veterinarians, since gray homozygous horses are more prone to developing aggressive melanoma tumors than heterozygotes. Since recent studies have confirmed that droplet digital PCR is a valuable technique for copy number analysis, we decided to investigate whether this method can be used for accurate genotyping of the horse graying-related variant and established the copy numbers of the 4.6-kb fragment in the available cohort (n = 75) of gray and nongray horses of various breeds. Surprisingly, we found that our STX17 genotype results varied from what has been previously published, suggesting that gray phenotype is associated with the presence of six (GG) or four (Gg) copies of studied region. All the examined nongray horses (gg) have the two copies of these fragments. This new pattern and its inheritance were also confirmed by an analysis conducted for the Polish Warmblood horse family. We noted no further copy number variation in the entire tested samples set. Our study confirmed the usefulness and accuracy of droplet digital PCR for genotyping STX17 gene variant. Further studies on a broader range of materials are needed to fully understand the origin and molecular structure of the graying causative mutation in the horse STX17.

Cryptorchidism is the most common disorder of sex development (DSD) in dogs. This malformation is associated with reduced fertility and with a higher risk of gonadal cancer. Testicular descent is a complex process, and the functions of many environmental and genetic factors are crucial for the proper migration of fetal gonads into the scrotum. Among these, the hormone INSL3 (insulin-like peptide 3) and its receptor RXFP2 (relaxin family peptide receptor 2) play crucial roles in the transabdominal migration of the testes. The genetic background of canine cryptorchidism is poorly elucidated. The aim of this study was to compare the transcript and methylation levels of INSL3 and RXFP2 genes in undescended and descended testes of isolated unilateral cryptorchids, and in gonads of control male dogs with scrotal testes. Next, we searched for polymorphic variants in the 5′-regulatory regions of both genes associated with predispositions to cryptorchidism. The INSL3 transcript level was significantly higher in the undescended testes than in the descended testes of both the affected and control dogs. On the other hand, the mRNA level of RXFP2 was significantly lower in the retained gonads of cryptorchids than in the scrotal testes. The methylation level of a single CpG site located 15 bp upstream of the translation start codon in INSL3 was significantly higher in the testes of the control dogs than in both gonads of cryptorchids. The methylation level of 14 CpG sites in the coding region of INSL3 was significantly higher in undescended testes than in the scrotal testes, which may be associated with the higher mRNA levels of INSL3 observed in these samples. The methylation pattern of two CpG sites in the 5′-flanking region of RXFP2 was similar in both descended and undescended testes. We detected three and seven single nucleotide polymorphisms (SNPs) in the 5′-regulatory regions of INSL3 and RXFP2, respectively. Among these, the frequency of A > C substitution (ss7093349755) located 495 bp upstream of the transcription start site of RXFP2 differed significantly between cryptorchids and control dogs. Our study showed two possible genetic biomarkers associated with canine cryptorchidism: a hypomethylation of a single CpG site in the 5′-flanking region of INSL3, and the ss7093349755 SNP in the 5′-flanking region of RXFP2.

Introduction A significant threat to public health is presented by antibiotic-resistant strains of bacteria, selective pressure on which results from antibiotic use. Colistin is an antibiotic commonly used in veterinary medicine, but also one of last resort in human medicine. Since the 2015 discovery in China of the mcr-1 gene encoding colistin resistance in Enterobacteriaceae, other countries have noted its presence. This study was to find the mcr-1 gene prevalence in E. coli isolated from poultry slaughtered in Poland. Material and Methods Cloacal swabs were taken from December 2017 to October 2018 from broiler chickens in three regions. The samples (n = 158) were grouped as flocks treated with colistin sulphate (n = 87) and those not treated (n = 71). Resistance to antimicrobials commonly used in poultry was evaluated by minimum inhibitory concentration. The presence of the mcr-1 gene was confirmed by PCR. Results Isolates containing the mcr-1 gene were yielded by 11.27% of the samples from not treated flocks and 19.54% of those from treated flocks, but no statistically significant difference in the prevalence of the gene was seen between the groups. Conclusion The results clearly preclude intensification of selective pressure for colistin resistance due to colistin sulphate treatment because they show that the avian gastrointestinal tract was already inhabited by colistin-resistant E. coli by the time the chickens came to the poultry house.

Copy-number variation (CNV) is an important source of genetic variation, and one that played a role in the process of domestication. The adaptation to a new diet is a characteristic feature of dog domestication. We therefore sought genomic signatures of this process. The pancreatic alpha-amylase gene (AMY2B), expressed in the pancreas, exhibits a variable number of copies. It has been shown that the multiplication of this gene is associated with the adaptation of dogs to a starch-rich diet. To date, there has been no information made available on the copy-number variation of AMY2B in canid farm animals. The aim of the present study was to examine the AMY2B copy number in the red fox, the arctic fox, and in the Chinese raccoon dog. Droplet digital PCR (ddPCR) was used to count the gene copies in 152 animals (60 red foxes, 53 arctic foxes, and 39 Chinese raccoon dogs). We found that the majority (91%) of the animals had two copies of this gene. Of the red foxes and Chinese raccoon dogs, only 8% had three copies, while 32% of the arctic foxes had three copies. Our study showed that the multiplication of the AMY2B gene did not occur over several decades of breeding selection, which may reflect the low-starch feeding regime.

Background X monosomy is the most common disorder of sex development in horses. Although cytogenetic analysis is still the gold standard in the diagnosis of equine X monosomy, novel molecular techniques are being sought to quickly and reliably detect this chromosome abnormality. Objectives The goal of this study was to evaluate the usefulness of a novel variant of the PCR technique-namely, droplet digital PCR (ddPCR)-in the detection of X monosomy in mares. Study design A proof of concept of the usefulness of ddPCR in diagnosing an abnormal number of X chromosomes in mares. Methods We examined an infertile mare using cytogenetic (fluorescent in situ hybridisation-FISH) and molecular (droplet digital PCR-ddPCR) techniques. The X chromosome copy number in ddPCR was estimated via detection of the AMELX gene copy number. In addition, 70 mares homozygous for X-linked microsatellite marker (LEX3) were examined by ddPCR. For all mares, a PCR search for the Y-linked SRY gene was also performed. Results Cytogenetic analysis and ddPCR gave concordant results, indicating pure X monosomy in the studied mare. Of the 70 additional mares examined by ddPCR, a single copy of the X chromosome was found in two cases. All mares were SRY-negative and thus both freemartinism, manifested by leucocyte XX/XY chimerism, and sex reversal syndrome (XX, SRY-positive) could be excluded. Main limitations The ddPCR approach does not allow for unequivocal identification of mosaicism (63,X/64,XX or 65,XXX/64,XX), but may give an indication that further cytogenetic analysis is necessary. Conclusion The ddPCR approach appeared to be useful for diagnosing nonmosaic X monosomy in mares. If the number of X chromosome copies in a mare, as determined by ddPCR, differs from two (in our study, 2.2), additional cytogenetic investigation is recommended with the aim of detecting the mosaicism.

In humans, the dysfunction of the adenomatous polyposis coli (APC) gene causes hereditary familial adenomatous polyposis (FAP) and increased risk of colorectal cancer (CRC). The severity of polyposis varies between individuals, but genetic basis for this is in large part unknown. This variability also occurs in our porcine model of FAP, based on an APC1311 mutation (orthologous to human APC1309). Since loss of TAP1 function can lead to CRC in humans, we searched for germline polymorphisms in APC1311/+ pigs with low (LP) and high (HP) levels of polyposis, as well as in wild-type pigs representing six breeds and a commercial line. The distribution of 40 identified polymorphic variants was similar in the LP and HP pigs. In contrast, the TAP1 transcript level was significantly higher in normal colon mucosa of HP pigs than in LP pigs. Moreover, six SNPs showed significant effects on TAP1 promoter activity, but no correlation with severity of polyposis was observed. Analysis of DNA methylation in the promoter region showed that one CpG site differed significantly between LP and HP pigs. We conclude that TAP1 genotype may not itself be associated with polyposis, but our findings concerning its expression suggest a role in the development of polyps.

Obesity is a serious problem in numerous dog breeds, but knowledge of its hereditary background is scarce. On the contrary, numerous DNA polymorphisms associated with human obesity have been identified, with the strongest effect being demonstrated for FTO gene. We used targeted next-generation sequencing (tNGS) to search for polymorphisms in the region harboring FTO and IRX3 in 32 Labrador dogs. Moreover, we investigated the selected regions of FTO and IRX3, orthologous to the human regions associated with obesity, in 165 Labradors. For all dogs, the following information was available: age, sex, gonadal status, body weight, and body conformation score (BCS). The use of tNGS revealed 12,217 polymorphisms, but none of these obtained significance when lean and obese dogs were compared. Study of two SNPs in the 5’-flanking region of FTO in 165 dogs – creating two upstream reading frames (uORFs) – also showed no association with body weight and BCS but suggested the need for improvement in FTO annotation. No polymorphism was found in the 5’UTR of IRX3. Additionally, no differences of CpG islands methylation status between lean and obese dogs were found. Our study suggests that FTO and IRX3 are not useful markers of obesity in Labrador dogs.

Proper expression of the PPARG gene, which encodes a key transcription factor of adipogenesis, is indispensable in the formation of mature adipocytes. The positioning of a gene within the nuclear space has been implicated in gene regulation. We here report on the significance of the PPARG gene’s nuclear positioning for its activity during in vitro adipogenesis in the pig. We used an established system of differentiation of mesenchymal stem cells derived from bone marrow and adipose tissue into adipocytes. The differentiation process was carried out for 7 days, and the cells were examined using the 3D DNA/immuno-FISH and RNA/DNA-FISH approaches. PPARG transcript level was measured using real-time PCR, and PPARγ activity was detected with colorimetric assay. Changes in the nuclear location of the PPARG gene were observed when we compared undifferentiated mesenchymal stem cells with mature adipocytes. The gene moved from the nuclear periphery to the nuclear center as its transcriptional activity increased. The RNA/DNA-FISH approach shows that differences in primary transcript production correlated with the allele’s nuclear positioning. Transcriptionally active alleles preferentially occupy the central part of the nucleus, while inactive alleles are found on the nuclear periphery. We also show that transcription of PPARG begins with one allele, but that both alleles are active in later stages of differentiation. Our results provide evidence that functionally distinct alleles of the PPARG gene are positioned in different parts of the cell nucleus. This confirms the importance of nuclear architecture to the regulation of PPARG gene transcription, and thus to the fate of the adipose cell. Electronic supplementary material The online version of this article (10.1007/s10577-019-09604-2) contains supplementary material, which is available to authorized users.

Disorders of sex development (DSD) are a well-known cause of sterility and reduced fertility in dogs. Here, we report on 6 new cases of DSD in French bulldogs diagnosed by us within the last year. The animals were phenotypical females who underwent detailed diagnosis due to the presence of an enlarged clitoris or a penis-like structure with atypically located urethral orifice (hypospadias). A female karyotype (78,XX) and lack of Y-linked genes (SRY and ZFY) were observed in 5 cases, which were classified as XX (SRY-negative) DSD. Gonads histology could be performed in 4 XX DSD cases: testicular tissue was observed in 3 cases and ovotesticular tissue in 1 case. In the last case, leukocyte chimerism (XX/XY) was detected alongside an ovotestis with epididymis and fallopian tubes. This case was classified as freemartinism. Our study and a literature review showed that heritable XX (SRY-negative) DSD is quite common in French bulldogs, but spontaneously developed freemartinism can be also diagnosed. In conclusion, we postulate that the description of the sexual phenotype of DSD females (enlarged clitoris and presence of testicular tissue) does not distinguish between hereditary XX DSD and nonhereditary freemartinism. Cytogenetic and molecular analyses are thus necessary to correctly diagnose such cases. This is especially important for the French bulldog breed, as a rapid increase in its population could spread hereditary DSD.

Freemartinism is a disorder of sex development (DSD) caused by the formation of placental anastomoses between fetuses of different sex. It leads to development of an abnormal reproductive tract in the female fetus, including the presence of testes or ovotestes. In pig production, such conditions can affect both reproductive performance and meat quality, through undesired boar taint. A common approach to diagnosing freemartinism is to detect leukocyte chimerism (XX/XY). In the study described here, we performed a complex evaluation of 28 DSD pigs with ambiguous external genitalia (enlarged clitoris, presence of scrotum, etc.). Sex chromosomes were cytogenetically identified by Giemsa staining, as well as with fluorescence in situ hybridization (FISH), while Y-linked (ZFY) and X-linked (ZFX) genes were detected by PCR-RFLP. Histological analysis of the gonads was performed on eosin–hematoxylin-stained microscope slides. Chromosome studies revealed that, of 28 DSD animals, 20 were freemartins (38,XX/38,XY), 2 had a female karyotype (38,XX), and 6 had a male karyotype (38,XY). Analysis of breeding data showed that the mean litter size in which DSD animals occurred was about 17. Histology of the gonads could be performed for 8 DSD pigs, including 6 freemartins and 2 XX DSD. In all these cases, testicular tissue without signs of spermatogenesis was seen. In XX DSD pigs, a copy number variation (CNV) polymorphism downstream from SOX9, tentatively associated with this type of DSD, was detected by FISH. On the other hand, the background of the XY DSD remains unknown. Since cytogenetic analysis is labor-intensive and time-consuming, we evaluated the usefulness of the droplet digital PCR (ddPCR) approach to diagnosing XX/XY leukocyte chimerism. Estimation of X and Y chromosome copy numbers was based on the quantitative detection of AMELX and AMELY genes. Different sex chromosome sets (XX, XY and XX/XY) were unequivocally identified by ddPCR. In conclusion, we suggest that freemartinism, beside testicular or ovotesticular XX DSD, is an emerging problem in highly prolific pigs and that detection of XX/XY leukocyte chimerism can be performed using a fast and reliable ddPCR approach.

Disorders of sex development (DSDs) are congenital malformations defined as discrepancies between sex chromosomes and phenotypical sex. Testicular or ovotesticular XX DSDs are frequently observed in female dogs, while monogenic XY DSDs are less frequent. Here, we applied whole genome sequencing (WGS) to search for causative mutations in XX DSD females in French Bulldogs (FB) and American Staffordshire Terries (AST) and in XY DSD Yorkshire Terries (YT). The WGS results were validated by Sanger sequencing and ddPCR. It was shown that a missense SNP of the PADI6 gene, is significantly associated with the XX DSD (SRY-negative) phenotype in AST (P = 0.0051) and FB (P = 0.0306). On the contrary, we did not find any associated variant with XY DSD in YTs. Our study suggests that the genetic background of the XX DSD may be more complex and breed-specific.

Funder: Swedish Insitute, Funder: Polish Ministry of Science and Higher Education post-doctoral fellowship (Mobility Plus Programme); Grant(s): Polish Ministry of Science and Higher Education post-doctoral fellowship (Mobility Plus Programme, no:609/MOB/2011/0), BackgroundReproductive biology methods rely on in vitro follicle cultures from mature follicles obtained by hormonal stimulation for generating metaphase II oocytes to be fertilised and developed into a healthy embryo. Such techniques are used routinely in both rodent and human species. DNA methylation is a dynamic process that plays a role in epigenetic regulation of gametogenesis and development. In mammalian oocytes, DNA methylation establishment regulates gene expression in the embryos. This regulation is particularly important for a class of genes, imprinted genes, whose expression patterns are crucial for the next generation. The aim of this work was to establish an in vitro culture system for immature mouse oocytes that will allow manipulation of specific factors for a deeper analysis of regulatory mechanisms for establishing transcription regulation-associated methylation patterns.ResultsAn in vitro culture system was developed from immature mouse oocytes that were grown to germinal vesicles (GV) under two different conditions: normoxia (20% oxygen, 20% O2) and hypoxia (5% oxygen, 5% O2). The cultured oocytes were sorted based on their sizes. Reduced representative bisulphite sequencing (RRBS) and RNA-seq libraries were generated from cultured and compared to in vivo-grown oocytes. In the in vitro cultured oocytes, global and CpG-island (CGI) methylation increased gradually along with oocyte growth, and methylation of the imprinted genes was similar to in vivo-grown oocytes. Transcriptomes of the oocytes grown in normoxia revealed chromatin reorganisation and enriched expression of female reproductive genes, whereas in the 5% O2 condition, transcripts were biased towards cellular stress responses. To further confirm the results, we developed a functional assay based on our model for characterising oocyte methylation using drugs that reduce methylation and transcription. When histone methylation and transcription processes were reduced, DNA methylation at CGIs from gene bodies of grown oocytes presented a lower methylation profile.ConclusionsOur observations reveal changes in DNA methylation and transcripts between oocytes cultured in vitro with different oxygen concentrations and in vivo-grown murine oocytes. Oocytes grown under 20% O2 had a higher correlation with in vivo oocytes for DNA methylation and transcription demonstrating that higher oxygen concentration is beneficial for the oocyte maturation in ex vivo culture condition. Our results shed light on epigenetic mechanisms for the development of oocytes from an immature to GV oocyte in an in vitro culture model.

Simple Summary Congenital malformations, defined as defects of morphogenesis present at birth, are an important problem in livestock production, if they are caused by hereditary mutations. They can lead to animal death, reduce their productive ability and influence animal welfare. Thus, the identification of the causes of congenital abnormalities are greatly needed. In the present report, we described a Holstein-Fresian calf with multiple congenital malformations including head asymmetry, the relocation of the frontal sinus and eye orbits, hypoplastic thymus, ductus Botalli, unfinished obliteration in umbilical arteries, and a bilateral series of tooth germs in the temporal region. Cytogenetic examination revealed a unique mosaic karyotype with a small marker chromosome, which could not be identified by standard banding techniques. It can be assumed that the presence the marker chromosome may be associated with observed congenital malformations in the studied calf. Abstract A Holstein-Fresian calf with multiple congenital malformations was subjected postmortem to anatomical and genetic investigation. The calf was small (20 kg), had shortened limbs and was unable to stand up. It lived only 44 days. Detailed anatomical investigation revealed the following features: head asymmetry, the relocation of the frontal sinus and eye orbits, hypoplastic thymus without neck part, ductus Botalli, unfinished obliteration in umbilical arteries, and a bilateral series of tooth germs in the temporal region. Cytogenetic examination, performed on in vitro cultured fibroblasts, showed a unique mosaic karyotype with a marker chromosome—60,XX[9 2%]/60,XX,+mar[8%], which was for the first time described in cattle. No other chromosome abnormalities indicating chromosome instabilities, like chromatid breaks or gaps were identified, thus teratogenic agent exposure during pregnancy was excluded. The marker chromosome (mar) was small and it was not possible to identify its origin, however, sequential DAPI/C (4’,6-diamidino-2-phenylindole) band staining revealed a large block of constitutive heterochromatin, which is characteristic for centromeric regions of bovine autosomes. We suppose that the identified marker chromosome was a result of somatic deletion in an autosome and its presence could be responsible for the observed developmental malformations. In spite of the topographic distance among the affected organs, we expected a relationship between anatomical abnormalities. To the of our best knowledge, this is the first case of a mosaic karyotype with a cell line carrying a small marker chromosome described in a malformed calf.

Disorders of sex development (DSD) are important causes of infertility and sterility, and are risk factors for gonadal carcinogenesis. Many DSDs are caused by genetic factors, mainly sex chromosome abnormalities or mutations of genes involved in sexual development, as well as structural variants (SVs) - large deletions, duplications, and insertions, if these overlap genes involved in sex development. The aim of this study was to determine if there were SVs in four candidate genes - NR0B1 (DAX1), NR5A1, RSPO1, and SOX3 - using droplet digital PCR (ddPCR). There was study of two cohorts of dogs with DSD, including 55 animals with XX DSD and 15 with XY DSD. In addition, 40 control females and 10 control males were included in the study. Among cases, for which there were evaluations, a large deletion consisting of four exons of the NR5A1 gene was identified in a Yorkshire Terrier with a rudimentary penis, hypospadias, bilateral cryptorchidism, and spermatogenesis inactive testes. This is the first mutation in the NR5A1 gene leading to XY DSD phenotype to be reported in domestic animals. There were no SVs in the genes evaluated in the present study in the cohort of dogs with XX DSD. The results from this study provide evidence that the large structural variants of these genes are rarely associated with the DSD phenotype in dogs.

Purpose Short stature in children is a significant medical problem which, without proper diagnosis and treatment, can lead to long-term consequences for physical and psychological health in adult life. Since human height is a polygenic and highly heritable trait, numerous variants in the genes involved in growth—including the growth hormone (GH1) gene—have been identified as causes of short stature. Methods In this study, we performed for the first time molecular analysis of the GH1 gene in a cohort (n = 186) of Polish children and adolescents with short stature, suffering from growth hormone deficiency (GHD) or idiopathic short stature (ISS), and a control cohort (n = 178). Results Thirteen SNP variants were identified, including four missense variants, six in 5′UTR, and three in introns. The frequency of minor missense variants was low ( Conclusions Mutations of GH1 causing short stature are very rare in the Polish population, but two potentially causative variants need further studies in a larger cohort of GHD patients.

One-carbon metabolism is critical to pregnancy outcomes, because it determines the availability of nutrients involved in cell divisions and DNA methylation. The aim of this study was to analyze how 50% prenatal calorie restriction affected one-carbon metabolism in pregnant Wistar rats of the F0 to F2 generations. Mean choline (p 0.001), betaine (p 0.001), and S-adenosylmethionine (SAM) (p 0.05) concentrations were respectively about 40%, 45%, and 20% lower in the F0_R (R - restricted diet) than in the F0_C (C - control diet). Homocysteine, S-adenosylhomocysteine (SAH), and trimethylamine oxide concentrations were unaffected. In the F1_R, the SAM-to-SAH ratio was 25% higher (p 0.05) than in the F1_C. No differences between the C and R groups were observed in the F2 generation. The SAM concentrations in the F1_R were higher than in the F0_R and the F2_R (p 0.01). The relative transcript levels of Mat1a, Bhmt, Cbs, Pemt, and Mthfr were only slightly affected by the diet, with changes of less than a factor of 2.0. Cbs activity in the F2_R was significantly higher than in the F2_C (p 0.001). Food deprivation may affect one-carbon metabolism in pregnant rats, but it does not stimulate persistent metabolic changes that can be observed during the pregnancy of their progeny of the F1 or F2 generations.

Hypospadias, the abnormal position of the urethral orifice, is considered a rare congenital malformation of the reproductive organs in male dogs. We present 5 new cases of hypospadias - 2 of the penile type in German Shepherd Dogs and 3 perineal types in a Bavarian Mountain Hound, a French Bulldog, and an American Staffordshire Terrier. Other abnormalities (rudimentary or underdeveloped penis, incompletely formed preputial sheath, and bilateral cryptorchidism) were also observed. Molecular analysis of all cases revealed the presence of Y-linked genes (SRY and ZFY). Cytogenetic and histological analysis could be performed for only 2 cases: a normal male sex chromosome complement (78,XY) and spermatogenetically inactive testicles were observed. A retrospective search for hypospadias in 19,950 medical records of male dogs from a single veterinary clinic in Poland (2006-2017) was also performed. Altogether, 10 reports of penile hypospadias were found (0.05%). The majority of the reports concerned German Shepherd Dogs (8 cases among 1,511 male dogs of this breed), and thus, the estimated incidence of hypospadias in this breed was 0.5%. Moreover, we performed a review of 26 cases of canine hypospadias reported in the years 2004-2017. Our study and the review of the literature suggest that hypospadias is not rare in dogs and that some breeds (such as German Shepherd Dog and Boston Terrier) may be prone to this disorder.

Testicular XX disorder of sex development (XX DSD, SRY -negative), causing sterility, is quite frequently diagnosed in pig populations, however, its molecular background is still unknown. This disorder may affect carcass quality (boar taint) due to the presence of testicular tissue in animals with ambiguous female external genitalia. A chromosome fragment encompassing the SOX9 gene was previously considered as a candidate region in functional and association studies. We analyzed distribution of polymorphic variants in 5’UTR and in 3’-flanking regions of the SOX9 gene in a cohort of 12 XX DSD pigs and a panel of 116 normal females. Altogether, 8 previously known polymorphic sites were analyzed and no significant association under Bonferroni correction (P>0.0063) between DSD phenotype and identified SNPs was found. The region harboring the SOX9 gene was also searched for the presence of copy number variation (CNV) by fluorescence in situ hybridization technique (FISH), using a set of 7 BAC probes. A potential CNV region, manifested by size variation (large and small) of fluorescence signals, produced by a single BAC probe hybridizing downstream (approx. 500 kb) of the SOX9 gene, was observed in 4 DSD cases. We conclude, that a new CNV polymorphism in the region harboring SOX9 gene can be considered as a promising marker for the DSD phenotype.

Background Undernutrition is an increasingly common problem. Insufficient calorie intake and nutrient deficiencies during pregnancy may have an impact not only on the mother, but may also alter metabolism in the infant. In this study, we have applied a calorie-restricted diet during gestation and examined its effect on hepatic Fasn mRNA and DNA methylation profiles in rats and their female progeny. The body composition and blood lipid profiles were also evaluated in both generations. Results The results showed that the investigated diet regimen exerted a greater effect on the dams than on the offspring. We found that, in the calorie-restricted group, the transcript level of the Fasn gene in the liver increased in the mothers, while in the progeny it was only slightly enhanced. The implemented diet altered lipid profile in the dams by decreasing total cholesterol, HDL, and TG levels. An increase in LDL was noted in the offspring. No change in DNA methylation profile was observed in response to the calorie-restricted diet. Conclusions Calorie restriction during pregnancy modified the hepatic Fasn mRNA transcript level and altered the blood cholesterol concentrations in dams, but there were no such effects in their four-week-old offspring. The examined dietary regimen had no effect on DNA methylation of the Fasn 5′-flanking region in the rat liver. Electronic supplementary material The online version of this article (doi:10.1186/s12863-017-0544-0) contains supplementary material, which is available to authorized users.

The dog is considered to be a useful biomedical model for human diseases and disorders, including obesity. One of the numerous genes associated with human polygenic obesity is MC4R, encoding the melanocortin 4 receptor. The aim of our study was to analyze polymorphisms and methylation of the canine MC4R in relation to adiposity. Altogether 270 dogs representing four breeds predisposed to obesity: Labrador Retriever (n = 187), Golden Retriever (n = 38), Beagle (n = 28) and Cocker Spaniel (n = 17), were studied. The dogs were classified into three groups: lean, overweight and obese, according to the 5-point Body Condition Score (BCS) scale. In the cohort of Labradors a complete phenotypic data (age, sex, neutering status, body weight and BCS) were collected for 127 dogs. The entire coding sequence as well as 5′ and 3′-flanking regions of the studied gene were sequenced and six polymorphic sites were reported. Genotype frequencies differed considerably between breeds and Labrador Retrievers appeared to be the less polymorphic. Moreover, distribution of some polymorphic variants differed significantly (P C, c.868C>T and c.*33C>G) and Beagles (c.-435T>C and c.637G>T). On the contrary, in Labradors no association between the studied polymorphisms and BCS or body weight was observed. Methylation analysis, using bisulfite DNA conversion followed by Sanger sequencing, was carried out for 12 dogs with BCS = 3 and 12 dogs with BCS = 5. Two intragenic CpG islands, containing 19 cytosines, were analyzed and the methylation profile did not differ significantly between lean and obese animals. We conclude that an association of the MC4R gene polymorphism with dog obesity or body weight is unlikely, in spite of the fact that some associations were found in small cohorts of Beagles and Golden Retrievers. Also methylation level of this gene is not related with dog adiposity. Electronic supplementary material The online version of this article (doi:10.1007/s11033-017-4114-3) contains supplementary material, which is available to authorized users.

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

The genetic background of disorders of sex development (DSD) in dogs with a normal male sex chromosome set (78,XY) is poorly described. In this study, we present for the first time, an analysis of six genes of the testosterone pathway, encoding enzymes (CYP17A1, HSD3B2, HSD17B3, SRD5A2) and transcription factors (NR5A1, AR). The entire coding sequence and flanking regions of the introns, 5'-UTR and 3'-UTR were analyzed in five DSD dogs (78,XY, SRY-positive) with ambiguous external genitalia and in 15 control dogs. A homozygous deletion of 2 bp in exon 2 of HSD17B3 (hydroxysteroid 17-beta dehydrogenase 3) was found in a Dachshund dog with enlarged clitoris, vulva and abdominal gonads and decreased serum testosterone level. In silico analysis revealed that this deleterious variant causes truncation of the encoded polypeptide (from 306 to 65 amino acids) and deprivation of the active site of the encoded enzyme. Genotyping of 23 control Dachshund dogs showed a normal homozygous genotype. Thus, we assumed that the 2-bp deletion is the causative variant. Moreover, 24 SNPs (four in CYP17A1, three in HSD3B2, six in HSD17B3, five in SRD5A2, one in AR and five in NR5A1), two intronic indels (one in HSD3B2 and one in SRD5A2) and two microsatellite polymorphisms in exon 1 of AR were found. Six SNPs appeared to be novel. No association with DSD phenotype was observed. Identification of the first case of DSD in domestic animals caused by a deleterious variant of a gene involved in testosterone synthesis showed that these genes are important candidates in such studies.

In humans, the dysfunction of the adenomatous polyposis coli (

Freemartinism is the most common type of disorder of sex development in cattle. It leads to sterility in the female co-twin in heterosexual twin pregnancy, and is thus a serious problem in cattle production. The incidence of freemartin syndrome is directly dependent on the prevalence of twinning, which has increased in dairy cattle populations in recent years. Thus, early and rapid identification of freemartins is needed to reduce economic loss. Of the various methods used to diagnose this condition, identifying the XX and XY cell lines in blood samples using cytogenetic techniques is the gold standard; however, this technique is time consuming. Faster and more reliable techniques are thus being sought. Droplet digital PCR (ddPCR) is a third-generation PCR method and it has not previously been used to detect XX/XY leukocyte chimerism in cattle. The aim of the present study was to verify the usefulness of ddPCR to detect and quantify leukocyte chimerism in this species. The X and Y copy numbers were estimated by identifying the copy numbers of 2 genes located on the sex chromosomes: amelogenin X-linked (AMELX) on the X chromosome and amelogenin Y-linked (AMELY) on the Y chromosome. In the first step, we performed ddPCR on samples prepared from female DNA mixed with male DNA in serially diluted proportions. We determined that the sensitivity of this method was sufficient to detect a low-frequency (

Hernia is a condition in which internal organs, most commonly the intestines, protrude through an opening in the layers of muscle and connective tissue. There are two major types of hernia—umbilical and inguinal. This pathology is a serious problem in pig production, leading to economic loss through the cost of surgical intervention, increased mortality rate, and reduced carcass value. The frequency of hernia in pig populations has been estimated from 1.5% to over 6%, and it heritability is around 0.3. Different approaches, including candidate gene sequencing, linkage analysis, genome-wide association studies, and next-generation sequencing, have been used to search for the causative mutations responsible for this pathology. In this way, different genomic regions have been suggested as associated with both types of hernia, but no strong candidate gene has been proposed to play a major role in its pathogenesis. In this review, current knowledge of the genetic background of porcine hernia is presented.

Testicular or ovotesticular disorder of sex development (DSD) in genetic females (78,XX; SRY-negative) has been reported quite frequently in numerous dog breeds and is usually diagnosed due to the presence of female external genitalia with an enlarged clitoris. The molecular background of this disorder, diagnosed also in human and other mammals, is not fully understood. However, it has recently been proposed that a copy number variation (CNV) in the region upstream of the SOX9 gene is associated with it. We described a rare case of this disorder in a French Bulldog with abdominal testes and male external genitalia (a slightly malformed penis). FISH studies showed a female karyotype, lack of a translocation involving the Y chromosome, and a distinct size variation in the CNV region (CNVR) upstream of the SOX9 gene, located on chromosome 9 (CFA9). A large FISH variant on a single CFA9 and a lack of the variant on its homologue was observed. Surprisingly, in the mother of this DSD dog, 2 normal-sized variants were identified which means that the CNV in the DSD dog was de novo. Our observations are in agreement with earlier suggestions that a high number of copies at the CNVR upstream of SOX9 may be associated with this type of DSD.

The miR-30 gene family includes potential regulators of adipogenesis. We searched for polymorphism in this gene family in 4 pig breeds: Duroc, Pietrain, Polish Landrace (PL), Polish Large White (PLW) and a synthetic line (L990). Altogether 5 single nucleotide polymorphisms (SNPs) in 3 genes were found, including one already known SNP rs340704946 within the pre-miR-30d genomic sequence. An association of the rs340704946 with intramuscular fat (IMF) content, abdominal fat accumulation and daily body mass gain in L990 was found. Moreover, the AG genotype was associated with an increased content of monounsaturated fatty acids (MUFA) in subcutaneous (P=0.027) and visceral fat (P=0.007) tissues when compared to the GG genotype. Analysis of PLW pigs revealed an association of the rs340704946 with transcript level of the ME1 gene in longissimus dorsi (LD) muscle (P=0.002), as well as the ME1 protein product in the LD muscle (P

The genetic background of disorders of sex development (DSDs) in cats is poorly understood, due to a relatively low number of such studies in this species. Here we present three new DSD cases with different complements of sex chromosomes. The first, an Oriental Shorthair cat with a rudimentary penis, abdominal atrophic testicles and lack of uterus appeared to be a freemartin, since leucocyte chimerism XX/XY and a lack of Y-linked genes (SRY and ZFY) were observed in DNA isolated from hair follicles. XXY trisomy was identified in the second case, a tortoiseshell Devon Rex male cat with atrophic scrotal testicles and a normal penis. Finally, a European Shorthair cat with atrophic testicles in a bifid scrotum, rudimentary penis and a lack of uterus had XY complement, including Y chromosome of normal size and morphology. Also presence of eight Y-linked genes, detected by PCR, was confirmed. Due to the low testosterone level in this last patient, we searched for a causative mutation in two candidate genes (HSD3B2 and HSD17B3) involved in the metabolism of this steroid hormone. Altogether, five polymorphic sites in HSD3B2 and two in HSD17B3 were found, but none of them showed associations with DSD phenotype. We thus excluded a possibility that the causative mutation is present in these genes. In conclusion, we confirmed that analysis of the sex chromosome complement is a crucial step in diagnosis of DSDs. However, extensive molecular studies of the genes involved in sex development are needed to elucidate the causes of DSDs in cats with normal complements of sex chromosomes.

Disorders of sex development (DSD) are a serious health problem in dogs. Different types of DSD have been described, including persistent Mullerian duct syndrome (PMDS), for which the molecular background has been identified in miniature schnauzers. Human patients with PMDS are at increased risk for cancers of the gonads (predominantly) or the Mullerian duct structures (rarely). This report describes two miniature schnauzer dogs with PMDS caused by a known nonsense mutation in the AMHR2 gene, with concurrent development of genital neoplasia. The first case (78,XY and SRY-positive) had unilateral cryptorchidism and a Sertoli cell tumour in the abdominal testicle. The second case (mosaic karyotype 77,XY,rob/78,XY and SRY-positive) had both gonads descended in the scrotum and developed an abdominal mass derived from the uterine wall, which showed histological features typical of leiomyoma.

78,XX testicular or ovotesticular disorder of sex development (DSD) is the most common sex anomaly in dogs, but its molecular background remains unknown. It was hypothesized that the causative mutation may reside in canine chromosome 23 (CFA23), where two genes playing a pivotal role in ovarian development (CTNNB1 and FOXL2) are located. The aim of our study was to search for polymorphism in both candidate genes in 15 DSD dogs (78,XX and a lack of the SRYgene) and 29 normal females. Altogether, 7 novel polymorphic variants were identified: 5 SNPs in CTNNB1 and 2 indels in the FOXL2 gene. The distribution of the identified variants was similar in the DSD and control dogs. Therefore, we concluded that the conducted research did not prove an association between these polymorphisms and canine testicular or ovotesticular XX DSD.

An 18-month-old European shorthair cat was subjected to genetic studies due to ambiguous external genitalia (underdeveloped both penis and scrotum). Further anatomic and histopathological studies revealed the presence of abdominal, atrophic testes and uterus. Cytogenetic analysis showed two cell lines, one with X monosomy-37,X [90% of the analysed metaphase spreads], and other line had 38 chromosomes with normal X chromosome and abnormally small Y-derived chromosome-38,X,der(Y) [10%]. Further fluorescence in situ hybridization study with telomeric probe revealed a ring structure of the der(Y). Eight Y chromosome-specific genes, SRY, TETY1, TETY2, CUL4BY, CYORF15, HSFY, FLJ36031Y and ZFY, were detected. We conclude that the described abnormality of the reproductive system, leading to sterility, was caused by a very rare type of chromosomal mosaicism-37,X/38,X,r(Y).

Freemartinism, a primary disorder of sex development (DSD) in cattle, is associated with leu - kocyte chimerism (60,XX/60,XY). The diagnosis of DSD is easy if it is known that a heifer with abnormally developed reproductive tracts originates from a heterosexual twin birth, but it is not so obvious in the case of single born calves. In the present study twelve DSD heifers which were single born (singletons) and culled due to the abnormal development of internal genitalia were studied using cytogenetic and molecular tech - niques. Among the heifers 7 appeared to be chimeric (60,XX/60,XY and the presence of the genes residing in the Y chromosome: SRY and AMELY) and 5 had a normal female karyotype (60,XX and a lack of the Y-linked genes). In addition, milk productivity was analyzed in relation to the incidence of twinning at a local Dairy Cattle Breeding Centre, from which 8 studied singletons (6 chimeric and 2 with a normal female karyotype) originated. It was found that in the years 2005-2013 an upward trend for average milk yield (from 9700 kg in 2005 to 11 500 kg in 2013) was associated with the increase of twin births (from 1.5% in 2005 to 5.9% in 2013). Our study showed that approximately 60% of single born heifers with abnormally developed internal genitalia were freemartins (a male co-twin died during pregnancy), while DSD etiology of the other cases (60,XX and a lack of the Y-linked genes) remains unknown. It cannot be excluded that some of these heifers represent a testicular/ovotesticular DSD (60,XX and SRY-negative). In conclusion, our study suggests that the occurrence of freemartins and other DSD in single born heifers seems to be an underestimated problem in cattle breeding.

A disorder of sexual development (DSD) of dogs with a female karyotype, missing SRY gene, and presence of testicles or ovotestes is quite commonly diagnosed. It is suggested that this disorder is caused by an autosomal recessive mutation; however, other models of inheritance have not been definitely ruled out. In an earlier study it was hypothesized that the mutation may reside in a pericentromeric region of canine chromosome 23 (CFA23). Three positional candidate genes (CLASP2, UBP1, and FBXL2) were selected in silico in the search for polymorphisms in 7 testicular or ovotesticular XX DSD dogs, 8 XX DSD dogs of unknown cause (SRY-negative, with enlarged clitoris and unknown histology of gonads), and 29 normal female dogs as a control group. Among the 15 molecularly studied dogs with enlarged clitoris there were 3 new cases of testicular or ovotesticular XX DSD and 4 new cases of XX DSD with unknown cause (histology of the gonads unknown). Altogether, 11 (including 10 novel) polymorphisms in 5′- and 3′-flanking regions of the studied genes were found. The distribution analysis of these polymorphisms showed no association with the DSD phenotypes. Thus, it was concluded that the presence of the causative mutation for testicular or ovotesticular XX DSD in the pericentromeric region of CFA23 is unlikely.

The nutritional factors acting during early life can affect the development of the organism. It has been hypothesized that such programmed traits can be inherited by later generations. In this work, we present for the first time the effect of food deprivation in pregnant dams and its consequences for the transcription and DNA methylation profiles in the offspring of the next three generations. We used a 50% reduction in dietary intake during pregnancy in the rat and determined whether this altered the hepatic DNA methylation and transcription levels in female fetuses over three generations. Targeted next-generation sequencing (tNGS) was used in the first generation for 1748 genes associated with six selected biological processes. The selected cytosines were then studied by pyrosequencing in F1-F3. The transcript level of the selected genes was determined by the real-time PCR. The tNGS approach indicated 394 cytosines, in close proximity to 374 genes, with a statistically significant difference in methylation levels between the control and restricted groups. A gene clustering analysis revealed 23 molecular pathways to which the studied genes were assigned. Only seven cytosines were differently methylated to more than 10%, and so these sites were studied next using pyrosequencing. The observation from NGS was confirmed for only one cytosine located near the St6galnac5 gene, though this was with the opposite effect. A difference was also observed for the Usp30 gene, though in proximity to the cytosine selected from NGS. In F3, the differences were observed for the Oxct2b gene. We also found differences in methylation levels between generations for the Grb10 and St6galnac5 genes, but independently of the diet used. The transcript levels of selected genes (Usp30, Grb10, Pld1, St6galnac5, Oxct2b, Khk, and Acsl4) were not altered in F1, while changes were detected for Pld1 and Oxct2b in F2 and F3, respectively. Prenatal food deprivation did not induce broad changes in hepatic DNA methylation of the genes involved in lipid or carbohydrate metabolism, and did not result in alterations in their transcription. Thus, the hypothesis that transgenerational inheritance is induced by dietary restriction was not confirmed.

Among functional and positional candidate genes for pig fatness traits, there are genes encoding acetyl-CoA carboxylase alpha (ACACA) and sterol regulatory element binding transcription factor 1 (SREBF1). In our study, 5 known and 2 novel SNPs in the ACACA gene and 2 known and 6 novel polymorphisms in the SREBF1 were found. The SNPs segregated as 6 and 10 haplotypes in ACACA and SREBF1, respectively. Two SNPs in ACACA were associated with fatness and performance traits in Polish breeds. In the SREBF1 gene, two novel SNPs were associated with daily gain and one, within a potential intronic micro-RNA gene, showed an association with fatness traits. Breed-specific differences in the transcript level were observed for ACACA only. No correlation between the transcript levels and fatty acid compositions was found in muscle and fat tissues. We concluded that polymorphisms of the ACACA and SREBF1 genes are promising markers for pig carcass and performance traits.

The fat mass and obesity associated (Fto) and iroquois homeobox 3 (Irx3) genes have been recognised as important obesity-related genes. Studies on the expression of these genes in the fat tissue of human and mouse have produced inconsistent results, while similar data on rat are limited. Environmental factors such as diet, should be considered as potential modulators of gene transcript levels through epigenetic mechanisms including DNA methylation. The aim of this study was to evaluate transcription levels and DNA methylation profiles of rat Fto and Irx3 genes in two white adipose tissue depots in response to high-fat and high-protein diets. The relative transcript levels of Fto and Irx3 were shown to be tissue-specific with higher levels detected in subcutaneous fat tissue than in abdominal fat tissue. Moreover, negative correlations between the transcripts of both genes were observed for subcutaneous fat tissue. The identified interactions (e.g. diet×duration of diet regimen) indicated that the diet had an impact on the transcript level; however, this effect was dependent on the duration of the diet regimen. The high-fat diet led to upregulation of Fto and Irx3 linearly with time across the two tissues. DNA methylation of the regulatory regions of the studied genes was very low and not related with the tissue, diet, or duration of diet regimen. Our study revealed that diet was an important factor modulating transcription of Fto and Irx3, but its effect is time-dependent. In contrast, the DNA methylation profiles of Fto and Irx3 were not altered by nutrition, which may indicate that the feeding type, when applied postnatally, did not affect DNA methylation of these genes.