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Cytogenetic and molecular insight into the genetic background of disorders of sex development in seventeen cats
AbstractThe genetic background of feline disorders of sex development (DSDs) is poorly understood. We performed comprehensive cytogenetic, molecular, and histological studies of 17 cats with abnormal external genitalia, unusual behavior, or tricolor coats (atypical in males). The DSD phenotype of three cats was associated with sex chromosome abnormalities: X/Y translocation (38,XXSRY+), 37,X/38,XY mosaicism, and XX/XY leukocyte chimerism. The remaining 14 affected cats were classified as XY DSD (SRY-positive). In this group and 38 normal males, we analyzed a priori selected candidate genes (SRY, TAC3, CYP11B1 and LHCGR). Only a previously reported nonpathogenic variant was found in SRY. Moreover, SRY gene copy number was determined, and three variants were observed: 6, 5 (modal), and 4 copies in a single DSD case. The known variants in TAC3 and CYP11B1, responsible for testicular hypoplasia, persistent primary dentition or congenital adrenal hyperplasia, were not found in the study group. Nine novel polymorphisms were identified in the LHCGR gene, one of which, a potentially regulatory indel variant in 5′UTR, was significantly associated (p = 0.0467) with XY DSD. Our report confirmed that abnormalities of sex chromosomes are important causes of feline DSDs. We also showed that the indel variant of LHCGR can be considered a promising marker associated with XY DSD phenotype.
Lack of causative mutation in the AMH and AMHR2 genes in a cat (38,XY) with persistent Mullerian duct syndrome (PMDS)
AbstractA 1‐year‐old European shorthair male cat with a normally developed penis was subjected to genetic, endocrinological and histological studies due to unilateral cryptorchidism. The blood testosterone level was typical for males, while the level of anti‐Mullerian hormone (AMH) was very low. Surgical removal of internal reproductive organs was followed by a histological study, which revealed inactive testicles with neoplastic changes and derivatives of Mullerian ducts. Cytogenetic analysis showed a normal XY sex chromosome complement and molecular analysis confirmed the presence of Y‐linked genes (SRY and ZFY). Although the level of AMH was low, two normal copies of the AMH gene were found using droplet digital PCR (ddPCR). Analysis of the coding sequences of two candidate genes (AMH and AMHR2) for persistent Mullerian duct syndrome (PMDS) in the affected cat and in control male cats (n = 24) was performed using the Sanger sequencing method. In the affected cat, homozygosity was found for three novel missense variants in Exon 1 (one SNP) and Exon 5 (two SNPs) of AMH, but the same homozygous genotypes were also observed in one and two control cats, respectively, whose sex development was not examined. Three known synonymous variants with homozygous status were found in AMHR2. We conclude that the DNA variants identified in AMH and AMHR2 are not responsible for PMDS in the affected cat.
First molecular characterisation of Sarcocystis miescheriana in a pig carcass condemned during routine meat inspection in Poland
Abstract Introduction This article presents the fourth detection of macroscopic cystic lesions due to sarcocystosis in domestic pigs during routine meat inspection worldwide, and the first molecular detection of Sarcocystis miescheriana in a domestic pig in Poland. Pigs can become intermediate hosts for S. miescheriana by accidental ingestion of oocysts or sporocysts present in food or water contaminated by the faeces of canids (definitive hosts). Material and Methods The affected swine showed no clinical symptoms such as weight loss, dermatitis or dyspnoea suggesting sarcocystosis. The presence of grossly visible cyst-like lesions was noticed by veterinary inspectors during post-mortem meat inspection of pig carcasses at a slaughterhouse located in central Poland. Ten rice-grain-shaped white lesions were isolated from the muscle tissue for molecular analysis, and four other macroscopic cyst-like lesions were also isolated for histopathological and microscopy analysis. The molecular characterisation included amplification and sequencing of the cytochrome C oxidase subunit 1 mitochondrial gene. Results The cyst-like structures were whitish, calcified, 1 cm long and 3 mm wide. The presence of S. miescheriana DNA was confirmed in all ten grossly visible cyst-like lesions. Conclusion This study shows that Sarcocystis spp. may be present in swine muscle tissue and cause lesions leading to carcass discard. Further analyses are needed to fully recognise the prevalence and impact of Sarcocystis spp. on animal and human health, especially taking into account the possible presence of the zoonotic S. suihominis.
Expression of adropin in the reproductive organs of healthy female dogs and those with cystic endometrial hyperplasia, pyometra, and ovarian cysts
Effect of zinc oxide nanoparticle supplementation on parasite infection and rumen environment of grazing lambs
This study investigated the effect of zinc nanoparticles (ZnO-NPs) on the growth parameters, parasitological status, ruminal fermentation, and histopathology of lambs that were experimentally infected with Haemonchus contortus larvae. The infected lambs were divided into two groups ( n = 10/group) and grazed on pasture while being fed one of two diets: a control diet consisting of 350 g/d of a dietary concentrate (CONTROL), and a diet consisting of 350 g/d of concentrate enriched with ZnO-NPs (ZINC). Pasture aboveground plant coverage and plant taxa from phytosociological relevés were used as descriptors to investigate differences in vegetation based on plant medicinal properties and the nutritional value. Communities dominated by plants with medicinal properties were mainly found in the CONTROL group's pasture, while the pasture of the ZINC group contained most plants with outstanding nutritional value. The number of eggs per gram of feces was quantified on days D14, D20, D28, D42, D56, D70, D84, D98, and D107 post-infection. There was a significant decrease in egg shedding from D42 onwards in the ZINC group, and from D56 and D70 onwards in the CONTROL group. The ruminal concentration of ammonia nitrogen ( p = 0.018), n -butyrate ( p = 0.025), n-valerate ( p = 0.002), total protozoa count ( p < 0.001), and the enzymatic activities of α-amylase ( p < 0.001) and xylanase ( p = 0.006) were significantly higher in the ZINC group than in the CONTROL group. The molar proportion of acetate was lower ( p = 0.011) in the ZINC group than in the CONTROL group. Morphological observations of the rumen indicated that the homogeneity of the ruminal papillae was slightly impaired, the lamina propria was inflamed, or lymphocytes had infiltrated. In conclusion, the dynamics of gastrointestinal nematode infection were significantly reduced, probably due to the medicinal and nutritional properties of the pasture plants. This effect was also enhanced by the supplementation with ZnO nanoparticles, which possess strong anthelmintic potential.
XX/XY Chimerism in Internal Genitalia of a Virilized Heifer
Five DSD heifers underwent genetic analysis in the present study. We cytogenetically analyzed in vitro cultured leukocytes and searched for SRY, AMELX/AMELY and ZFX/ZFY genes in leukocytes and hair follicles, finding that four of the studied heifers were freemartins (XX/XY leukocyte chimerism). The fifth case had an underdeveloped vulva localized ventrally and cranially to the mammary gland, a normal female sex chromosome complement (60,XX) in the leukocytes, and a lack of Y-chromosome-derived genes in the leukocytes and hair follicles. Postmortem anatomical examination of this heifer revealed the presence of normal ovaries with follicles, uterus, and oviducts, but molecular detection of the SRY, ZFX, ZFY,AMELX, and AMELY genes in these organs indicated the presence of a cell line carrying the Y chromosome. Further analysis of twelve microsatellite markers revealed the presence of additional variants at six loci in DNA samples derived from the reproductive organs; XX/XY chimerism was thus suspected in these samples. On the basis of the detection of AMELY (Y-linked) versus AMELX (X-linked) and SOX9 (autosomal) versus AMELY genes by droplet digital PCR (ddPCR), the Y/X and Y/autosome ratios were evaluated; they indicated the presence of XX and XY cell lines in the reproductive tissues. Our study showed that XX/XY chimerism can be present in the internal reproductive organs of the virilized heifers with a normal female set of sex chromosomes (60,XX) and a lack of Y-chromosome-derived genes in the leukocytes. The etiology of this phenomenon remains unknown.
Non-mosaic X monosomy (77,X) in a female dog with signs of virilization
AbstractA 14-month-old female Miniature Poodle dog with an enlarged clitoris and asymmetry in the placement of the teats was subjected to clinical, histopathological, and genetic studies. Macroscopically, the uterus and fallopian tubes appeared normal, while both ovaries were diffusely altered. At histology, the ovarian parenchyma was almost completely effaced by a diffuse hyperplasia of theca cells with atretic primary follicles. Chromosome analysis showed pure (non-mosaic) X monosomy (77,X). This finding was confirmed by the highly sensitive droplet digital PCR (ddPCR) approach. Despite the observed virilization, molecular analysis did not show the presence of Y-linked genes (SRY,ZFY, andTSPY1) in the blood cells or ovary tissue. To the best of our knowledge, this is the first case of X monosomy in a dog associated with virilization.
Insights into the role of bioactive plants for lambs infected with Haemonchus contortus parasite
Bioactive plants provide therapeutic and prophylactic effects to ruminants. We determined the effect of grazing on natural meadow grassland enriched with experimentally sown chicory (Cichorium intybus) on parasitological status, pasture larval infectivity, antioxidant parameters, and the histology of abomasal tissue in lambs experimentally infected with the parasitic gastrointestinal nematode (GIN) Haemonchus contortus. We also qualitatively identified the main polyphenols in the meadow grassland and phenolic metabolites in the feces of the lambs. Sixteen lambs were orally infected with approximately 5,000 infective larvae (L3) of H. contortus. The lambs were divided into two groups: lambs grazing on a plot consisting exclusively of meadow pasture which serves as control group and lambs grazing on a plot where approximately 25% of a meadow grassland was reclaimed with chicory. The experimental period was 144 days. The number of eggs per gram (EPG) of feces was quantified on D21, D34, D48, D62, D76, D89, D103, D118, D131, and D144 post-infection. Pasture contamination with H. contortus L3 was examined. EPG in both groups of lambs was highest at D34. Egg shedding was significantly lower in both groups from D48 onwards, with a reduction of >95% from D103 onwards. Pasture contamination with L3 was highest at D41 but was then significantly lower in both groups. The total antioxidant capacity, the activity of glutathione peroxidase and the concentration of malondialdehyde in the serum changed significantly during the experiment (p < 0.003, < 0.001, and < 0.016, respectively). At least 54 species of meadow plants were identified on both pasture plots; plant bioactive compounds identified were mainly phenolic acids, flavonoids, and glucosides. Phenolic metabolites (e.g., coumaric acid, chicory acid, salvigenin, and esters of gallic acid) were identified in the feces of the lambs. In some lambs, the morphological observation identified small histopathological changes in the abomasal tissues typical of hemonchosis. Both the natural meadow pasture and the pasture enriched with experimentally sown chicory slowed the dynamics of GIN infection and pasture contamination with L3 by mobilizing the antioxidant defensive system and gradually increasing the resistance of the infected lambs, probably due to the beneficial effects of plant bioactive substances.
Nesfatin-1 expression and blood plasma concentration in female dogs suffering from cystic endometrial hyperplasia and pyometra and its possible interaction with phoenixin-14
Abstract Background Nesfatin-1 is a neuropeptide that regulates the hypothalamic-pituitary-gonadal axis and may play a role in uterus function. It is co-expressed with other peptides, such as phoenixin, which can influence sex hormone secretion. Our previous research has confirmed that phoenixin-14 is involved in the development of cystic endometrial hyperplasia (CEH) and pyometra in dogs. Therefore, based on the similarities and interactions between these neuropeptides, we hypothesized that nesfatin-1 might also regulate the reproductive system in dogs. This study aimed to determine the expression of nesfatin-1 and its interaction with phoenixin-14 in dogs with CEH or pyometra compared to healthy females, and concerning animals’ body condition score (BCS 4–5/9 vs. BCS > 5/9). Results The analysis of nesfatin-1 in the uterus of bitches consisted of qPCR, western blot and immunofluorescence assays, and ELISAs. The results showed significantly higher nesfatin-1 encoding gene, nucleobindin-2 mRNA (Nucb2) and nesfatin-1 protein expression in overweight females and those suffering from CEH or pyometra compared to healthy animals. The immunoreactivity of nesfatin-1 was elevated in the uteri of bitches with higher BCS > 5/9. Moreover, nesfatin-1 blood concentrations increased in all examined overweight bitches. In the case of phoenixin signals, we found opposite results, regardless of the female body condition score. Conclusion The etiology of CEH and pyometra are not fully known, although we have expanded the level of knowledge with respect to the possible interaction of nesfatin-1 and phoenixin in female dogs’ uteri. They interact oppositely. With increasing female body weight, the expression of nesfatin-1 in the uterus and its peripheral blood concentration increased. However, for female dogs affected by CEH and pyometra, a decreased level of phoenixin-14, irrespective of their body condition score is characteristic. This knowledge could be crucial in the development of biomarkers for these conditions, which may lead to earlier recognition.
The Role of a High-Fat, High-Fructose Diet on Letrozole-Induced Polycystic Ovarian Syndrome in Prepubertal Mice
This study aims to investigate the effects of a high-fat, high-fructose (HF/HFr) diet on metabolic/endocrine dysregulations associated with letrozole (LET)-induced Polycystic Ovarian Syndrome (PCOS) in prepubertal female mice. Thirty-two prepubertal C57BL/6 mice were randomly divided into four groups of eight and implanted with LET or a placebo, with simultaneous administration of an HF/HFr/standard diet for five weeks. After sacrifice, the liver and blood were collected for selected biochemical analyses. The ovaries were taken for histopathological examination. The LET+HF/HFr group gained significantly more weight than the LET-treated mice. Both the LET+HF/HFr and the placebo-treated mice on the HF/HFr diet developed polycystic ovaries. Moreover the LET+HF/HFr group had significantly elevated testosterone levels, worsened lipid profile and indices of insulin sensitivity. In turn, the HF/HFr diet alone led to similar changes in the LET-treated group, except for the indices of insulin sensitivity. Hepatic steatosis also occurred in both HF/HFr groups. The LET-treated group did not develop endocrine or metabolic abnormalities, but polycystic ovaries were seen. Since the HF/HFr diet can cause substantial metabolic and reproductive dysregulation in both LET-treated and placebo mice, food items rich in simple sugar—particularly fructose—and saturated fat, which have the potential to lead to PCOS progression, should be eliminated from the diet of young females.
Expression and localization of the neuropeptide phoenixin-14 and its receptor GRP173 in the canine reproductive organs and periovarian adipose tissue
Whole genome sequencing identifies a missense polymorphism in PADI6 associated with testicular/ovotesticular XX disorder of sex development in dogs
Effect of Sainfoin (Onobrychis viciifolia) Pellets on Rumen Microbiome and Histopathology in Lambs Exposed to Gastrointestinal Nematodes
Our study analyzed the ruminal fermentation and microbiome, hematological profile, and abomasal histopathology of lambs experimentally infected with a gastrointestinal nematode (GIN) and fed sainfoin pellets (SFPs; 600 g DM/d/animal) for 14 d. Twenty-four lambs infected with Haemonchus contortus were divided into two separated groups: animals fed meadow hay (control) and animals fed SFPs. The ruminal contents, fermentation parameters, and microbiome in vitro and in vivo were determined using molecular and microscopic techniques. Ruminal contents in the SFP group indicated smaller populations of Archaea (p < 0.001), Methanomicrobiales (p = 0.009), and lower methane concentrations in vitro (p = 0.046) and in vivo (p = 0.030) than the control group. The relative abundance of Butyrivibrio fibrisolvens quantified by real-time PCR was higher in the lambs with the SFP diet (p = 0.05). Haemonchosis affected the number of red blood cells of the lambs (p < 0.001). The lambs in the SFP group had a higher percentage of damaged abomasa glands than did the control group (p = 0.004). The consumption of SFPs by GIN-infected lambs may affect ruminal methanogens and subsequently decrease methane emission without undesirable changes in the ruminal microbiome or the health of the animals.