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Dodatek pieprzycy peruwiańskiej (Lepidium meyenii) w żywieniu świń i jej wpływ na gospodarkę lipidową - badanie nutrigenomiczne
Altered Transcript Levels of MMP13 and VIT Genes in the Muscle and Connective Tissue of Pigs with Umbilical Hernia
Umbilical hernia (UH) and inguinal hernia (IH) are among the most common defects in pigs, affecting their welfare and resulting in economic losses. In this study, we aimed to verify the association of previously reported differences in transcript levels of the ACAN, COL6A5, MMP13, and VIT genes with the occurrence of UH and IH. We examined mRNA levels in muscle and connective tissue from 68 animals—34 affected by UH and 34 controls. In a second cohort, we examined inguinal channel samples from 46 pigs (in four groups). We determined DNA methylation levels in muscle tissue for the UH and control animals. The transcript level of MMP13 changed in the UH cases, being upregulated and downregulated in muscle and connective tissue, respectively, and the VIT gene also showed an increased muscular mRNA level. The transcript of the ACAN gene significantly decreased in old pigs with IH. We further observed an increased DNA methylation level for one CpG site within the MMP13 gene in UH individuals. We conclude that these alterations in gene mRNA levels in the UH animals depend on the tissue and can sometimes be a consequence of, not a cause of, the affected phenotype.
Elevated serum concentration of anti‐Mullerian hormone and its association with SNP variants in the AMH gene in a tortoiseshell tomcat with a disorder of sex development (38,XX; SRY-negative)
AbstractTesticular disorders of sex development (DSD) in cats with XX sex chromosomes and the absence of the SRY gene are rare congenital abnormalities. A Maine Coon tomcat with a normal penis, gonads in the scrotum, low serum testosterone concentration, and an elevated level of anti‐Müllerian hormone (AMH) was subjected to genetic analyses due to an unusual tortoiseshell coat color for males. Primary studies revealed the presence of XX sex chromosomes, the lack of SRY and the presence of two copies of the candidate SOX9. The DSD tomcat and its parents were analyzed using whole genome sequencing. Candidate SNPs in AMH, ORC1, DOCK8, PRKAR1A, and TMEM186 genes, as well as a known intronic 5‐kb deletion in X‐linked ARHGAP36 gene, which is responsible for orange coat, were identified. Potentially pathogenic homozygous genotypes were observed in all candidate genes; however, only in AMH and ORC1 were these genotypes rare in a control cohort. Further studies were focused on two SNPs located in the 5′‐and 3′‐untranslated regions (UTRs) of AMH. It has been experimentally demonstrated that only a short AMH transcript is present in feline testes. In silico analysis revealed that the SNP located in the 3′UTR of AMH occurs within a sequence that partially matches the canonical binding site for human miR‐5571‐5p. This microRNA is expressed in mammalian testes, which we confirmed in feline testicular tissue. We concluded that SNP in the 3′UTR of AMH is associated with elevated expression of the encoded hormone; however, it is not the cause of the testicular DSD phenotype in the studied Maine Coon tomcat.
A confirmed association between DNA variants in CAPN9, OSM, and ITGAM candidate genes and the risk of umbilical hernia in pigs
AbstractUmbilical hernia (UH) is one of the most prevalent defects of swine, affecting their welfare and causing considerable economic loss. The molecular mechanisms behind UH in pigs remain poorly understood. The aim of this study was to verify the association between UH and previously reported DNA variants in theCAPN9,OSM,ITGAM, andNUGGCgenes. A case/control study design was applied in two different crossbred cohorts of commercial fatteners containing 412 and 171 pigs, respectively. SNPs withinCAPN9,OSM, andITGAMwere analyzed using Sanger sequencing, and 10 SNPs inCAPN9, five inOSM, and two inITGAMwere identified.A structural variant in theNUGGCgene was studied by droplet‐digital PCR, and an elevated copy number was detected in only a single individual. Significant differences in allele frequencies for four SNPs inCAPN9were detected. The haplotype analysis showed the effect on the risk of UH for two genes. The CAGGA haplotype withinOSMand AT haplotype inITGAMreduced the relative risk of UH by 52% and 45%, respectively, confirming that variants in those genes are associated with the risk of UH in pigs. Moreover, the interaction between theCAPN9haplotype and the sex of animals had also significant impact on UH risk.
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.
Maternal cafeteria diet influences kisspeptin (Kiss1), kisspeptin receptor(Gpr54), and sirtuin (Sirt1) genes, hormonal and metabolic profiles, and reproductive functions in rat offspring in a sex-specific manner
Abstract Kisspeptin (KP, encoded by Kiss1, binding to the Gpr54 receptor) is a neuropeptide conveying information on the metabolic status to the hypothalamic–pituitary–gonadal axis. KP acts together with dynorphin A (encoded by Pdyn) and neurokinin B (encoded by Tac2) to regulate reproduction. KP is crucial for the onset of puberty and is under the control of sirtuin (encoded by Sirt1). We hypothesize that the maternal cafeteria (CAF) diet has adverse effects on the offspring’s hormonal, metabolic, and reproductive functions due to sex-specific alterations in the expression of Kiss1, Gpr54, Pdyn, Tac2, and Sirt1 in the hypothalamus, and Kiss1, Gpr54, and Sirt1 in the liver. Rats were fed a CAF diet before pregnancy, during pregnancy, and during lactation. The vaginal opening was monitored. Offspring were sacrificed in three age points: PND 30, PND 35, and PND 60 (females) and PND 40, PND 45, and PND 60 (males). Their metabolic and hormonal status was assessed. mRNA for Kiss1, Gpr54, Pdyn, Tac2, and Sirt1 were measured by real-time PCR in the hypothalamus and/or livers. We found that CAF offspring had lower weight and altered body composition; increased cholesterol and triglyceride levels, sex-specific changes in glucose and insulin levels; sex-dependent changes in Sirt1/Kiss1 mRNA ratio in the hypothalamus; sex-specific alterations in Kiss1 and Sirt1 mRNA in the liver with more diversity in males; and a delayed puberty onset in females. We concluded that the mother’s CAF diet leads to sex-specific alterations in metabolic and reproductive outcomes via Kiss1/Gpr54 and Sirt1 systems in offspring.
From cytogenetics to cytogenomics: a new era in the diagnosis of chromosomal abnormalities in domestic animals
The Effects of Peruvian maca (Lepidium meyenii) Root Extract on In Vitro Cultured Porcine Fibroblasts and Adipocytes
Peruvian maca (Lepidium meyenii) is a plant known for its nutritional and medicinal properties whose use as a supplement in animal diets has attracted much interest. We studied the effects of powdered maca root extract on the growth potential of in vitro cultured porcine cells prior to its use as an additive in animal nutrition. Fibroblast cell viability (MTT), cell proliferation (BrdU), and apoptosis level (TUNEL) were measured for a range of extract doses (0, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, and 10 mg/mL). Transcript levels of CCND1, MCM2, and PCNA genes as molecular markers of cell proliferation were also determined. Next, the effects of maca extract at 2 and 5 mg/mL on in vitro induced adipogenesis were evaluated over eight days of differentiation. The transcript levels of three adipocyte marker genes (CEBPA, PPARG, and FABPB4) were measured at days 0, 4, and 8 of adipose differentiation, and lipid droplet accumulation (BODIPY staining) was also noted. No cytotoxic effect was detected on fibroblast cell viability, and the inhibitory concentration (IC50) value was determined to be IC50 > 10 mg/mL. Doses of maca extract above 3 mg/mL decreased cell proliferation. The transcript level decreased in concentrations above 5 for the MCM2 and PCNA genes. For the CCND1 gene, the transcript level decreased when the greatest maca dose was used. In the in vitro adipogenesis experiment, it was found that the rate of lipid droplet formation increased on day 4 of differentiation for both doses, while decreased lipid droplet formation was observed on day 8 for 5 mg/mL of maca extract. Significant changes were seen in the mRNA level for CEBPA and PPARG on days 4 and 8, while the transcript of FABP4 increased only on day 8 at 2 mg/mL dose. It can be concluded that the addition of Peruvian maca in small doses (<3 mg/mL) has no negative effect on porcine fibroblast growth or proliferation, while 2 mg/mL of maca extract enhances adipocyte differentiation.
Prevalence of the SOD1, PRCD and SLC2A9 gene mutations responsible for degenerative myelopathy, progressive rod-cone degeneration, and hyperuricosuria in Polish population of Labrador Retriever dogs
Wykorzystanie analizy transkryptomicznej do identyfikacji genów związanych z występowaniem przepuklin pępkowych świń
A massive alteration of gene expression in undescended testicles of dogs and the association of KAT6A variants with cryptorchidism
Cryptorchidism is the most common form of disorder of sex development in male dogs, but its hereditary predisposition is poorly elucidated. The gonadal transcriptome of nine unilaterally cryptorchid dogs and seven control dogs was analyzed using RNA-seq. Comparison between the scrotal and inguinal gonads of unilateral cryptorchid dogs revealed 8,028 differentially expressed genes (DEGs) (3,377 up-regulated and 4,651 down-regulated). A similar number of DEGs (7,619) was found by comparing the undescended testicles with the descended testicles of the control dogs. The methylation status of the selected DEGs was also analyzed, with three out of nine studied DEGs showing altered patterns. Bioinformatic analysis of the cDNA sequences revealed 20,366 SNP variants, six of which showed significant differences in allelic counts between cryptorchid and control dogs. Validation studies in larger cohorts of cryptorchid ( n = 122) and control ( n = 173) dogs showed that the TT genotype (rs850666472, p.Ala1230Val) and the AA genotype in 3′UTR (16:23716202G>A) in KATA6 , responsible for acetylation of lysine 9 in histone H3, are associated with cryptorchidism ( P = 0.0383). Both the transcript level of KAT6A and H3K9 acetylation were lower in undescended testes, and additionally, the acetylation depended on the genotypes in exon 17 and the 3′UTR. Our study showed that the massive alteration of the transcriptome in undescended testicles is not caused by germinal DNA variants in DEG regulatory sequences but is partly associated with an aberrant DNA methylation and H3K9 acetylation patterns. Moreover, variants of KAT6A can be considered markers associated with the risk of this disorder.
Cleft Lip and Palate in Four Full-Sib Puppies from a Single Litter of Staffordshire Bull Terrier Dogs: An Anatomical and Genetic Study
Cleft lip and palate (CLP) is a well-known congenital defect in dogs, characterized by abnormal communication between the oral and nasal cavities. Its incidence rate is high and affects all dog breeds. The etiology of CLP is thought to be multifactorial, caused by both genetic and environmental factors. In this study, four puppies out of seven from a single litter of Staffordshire Bull Terrier dogs with craniofacial abnormalities were anatomically and genetically examined. Classical anatomical preparation, dyed-latex-injection of the arterial vessels, and cone-beam computed tomography were used. The puppies showed variations in their observable abnormalities: three of them had a complete cleft of the palate on both sides, while one puppy had a cleft on the right side only. Cytogenetic analysis showed a normal diploid chromosome number (2n = 78,XX or 78,XY) in the studied animals. Known genomic variants of CLP were examined in the ADAMTS20, DLX6, and MYH3 genes, but no mutations were identified. Further studies are needed to identify the breed-specific genetic variants associated with canine CLP.
First report on the presence of a common pathogenic variant in the PKD1 gene, causing polycystic kidney disease, in a series of related Sphynx cats
Testicular Disorder of Sex Development in a Mare With a Low Incidence of SRY‐Positive Cell Line in the Gonads
ABSTRACTAn adult mare with ambiguous external genitalia, observed at a slaughterhouse, was subjected to detailed examination. The mare exhibited fused labia and an enlarged clitoris located at the ventral commissure of the vulva. Anatomical and histopathological studies revealed the presence of two testicles, vas deferens, fallopian tubes, a small uterus with blindly ending horns and a normally developed cervix. Cytogenetic analysis, using fluorescent in situ hybridisation (FISH) of in vitro cultured lymphocytes, showed a female karyotype—64,XX. Molecular detection of X‐ and Y‐linked genes (SRY and ZFX/ZFY) in blood cells confirmed the presence of X‐linked genes only. In contrast, in hair follicles and gonadal tissue, the presence of genes originating from the Y chromosome was also detected. The use of digital droplet PCR (ddPCR) revealed the presence of a SRY‐positive cell line; however, at a very low level (< 5%). Analysis of polymorphic short tandem repeats (STRs), recommended for parentage testing, did not detect chimerism, which would be indicated by the presence of three or four variants at some STR loci. In conclusion, the studied case was classified as a sex chromosome disorder of sex development (DSD) due to gonadal XX/XY mosaicism. To the best of our knowledge, this is the first reported case of such an abnormality in a DSD horse.