Molecular cytogenetic screening of sex chromosome abnormalities in young horse populations.

BACKGROUND: Chromosomal abnormalities occur in the equine population at a rate of approximately 2%. The use of molecular cytogenetic techniques allows a more accurate identification of chromosomal abnormalities, especially those with a low rate of abnormal metaphases, demonstrating that the actual incidence in equine populations is higher. OBJECTIVES: Estimation of the number of carriers of karyotypic abnormalities in a sample from a population of young horses of various breeds, using molecular cytogenetic techniques. STUDY DESIGN: Cross-sectional. METHODS: Venous blood samples were collected from 500 young horses representing 5 breeds (Purebred Arabian, Hucul, Polish primitive horse [Konik], Malopolska, Coldblood, Silesian). Chromosomes and DNA were obtained from blood lymphocytes and evaluated by fluorescence in situ hybridisation (FISH) and PCR, using probes and markers for the sex chromosomes and select autosomes. RESULTS: Nineteen horses, 18 mares and 1 stallion, were diagnosed with different chromosomal abnormalities: 17 cases of mosaic forms of sex chromosome aneuploidies with a very low incidence (0.6%-4.7%), one case of a SRY-negative 64,XY sex reversal mare, and one mare with X-autosome translocation. The percentage of sex chromosomal aberrations was established as 3.8% in the whole population, 6.08% in females and 0.49% in males. MAIN LIMITATIONS: Limited sample size, confined to horses from Poland. CONCLUSIONS: The rate of sex chromosomal abnormalities we identified was almost double that reported in previous population studies that used classical chromosome staining techniques. FISH allowed the detection of aneuploid cell lines which had a very low incidence. The FISH technique is a faster and more precise method for karyotype examination; however, it is usually focused on only one or two chromosomes while banding karyotyping includes the entire chromosome set.

Bioinformatic analysis of endometrial miRNA expression profile at day 26-28 of pregnancy in the mare.

The establishment of the fetomaternal interface depends on precisely regulated communication between the conceptus and the uterine environment. Recent evidence suggests that microRNAs (miRNAs) may play an important role in embryo-maternal dialogue. This study aimed to determine the expression profile of endometrial miRNAs during days 26-28 of equine pregnancy. Additionally, the study aimed to predict target genes for differentially expressed miRNAs (DEmiRs) and their potential role in embryo attachment, adhesion, and implantation. Using next-generation sequencing, we identified 81 DEmiRs between equine endometrium during the pre-attachment period of pregnancy (day 26-28) and endometrium during the mid-luteal phase of the estrous cycle (day 10-12). The identified DEmiRs appear to have a significant role in regulating the expression of genes that influence cell fate and properties, as well as endometrial receptivity formation. These miRNAs include eca-miR-21, eca-miR-126-3p, eca-miR-145, eca-miR-451, eca-miR-491-5p, members of the miR-200 family, and the miRNA-17-92 cluster. The target genes predicted for the identified DEmiRs are associated with ion channel activity and sphingolipid metabolism. Furthermore, it was noted that the expression of mucin 1 and leukemia inhibitory factor, genes potentially regulated by the identified DEmiRs, was up-regulated at day 26-28 of pregnancy. This suggests that miRNAs may play a role in regulating specific genes to create a favorable uterine environment that is necessary for proper attachment, adhesion, and implantation of the embryo in mares.

Changes in miRNA expression in the lungs of pigs supplemented with different levels and forms of vitamin D.

BACKGROUND: Vitamin D is an immunomodulator, and its effects have been linked to many diseases, including the pathogenesis of cancer. However, the effect of vitamin D supplementation on the regulation of gene expression of the lungs is not fully understood. This study aims to determine the effect of the increased dose of cholecalciferol and a combination of cholecalciferol + calcidiol, as well as the replacement of cholecalciferol with calcidiol, on the miRNA profile of healthy swine lungs. METHODS AND RESULTS: The swine were long-term (88 days) supplemented with a standard dose (2000IU/kg) of cholecalciferol and calcidiol, the increased dose (3000 IU/kg) of cholecalciferol, and the cholecalciferol + calcidiol combination: grower: 3000 IU/Kg of vitamin D (67% of cholecalciferol and 33% of calcidiol), finisher 2500 IU/Kg of vitamin D (60% of cholecalciferol and 40% of calcidiol). Swine lung tissue was used for Next Generation Sequencing (NGS) of miRNA. Long-term supplementation with the cholecalciferol + calcidiol combination caused significant changes in the miRNA profile. They embraced altered levels of the expression of miR-150, miR-193, miR-145, miR-574, miR-340, miR-381, miR-148 and miR-96 (q-value < 0.05). In contrast, raising the dose of cholecalciferol only changed the expression of miR-215, and the total replacement of cholecalciferol with calcidiol did not significantly affect the miRNAome profile. CONCLUSIONS: The functional analysis of differentially expressed miRNAs suggests that the use of the increased dose of the cholecalciferol + calcidiol combination may affect tumorigenesis processes through, inter alia, modulation of gene regulation of the TGF- ß pathway and pathways related to metabolism and synthesis of glycan.

Benchmarking of bioinformatics tools for NGS-based microRNA profiling with RT-qPCR method.

MicroRNAs are vital gene expression regulators, extensively studied worldwide. The large-scale characterization of miRNAomes is possible using next-generation sequencing (NGS). This technology offers great opportunities, but these cannot be fully exploited without proper and comprehensive bioinformatics analysis. This may be achieved by the use of reliable dedicated software; however, different programs may generate divergent results, leading to additional discrepancies. Thus, the aim of this study was to compare three bioinformatic algorithms dedicated to NGS-based microRNA profiling and validate them using an alternative method, namely RT-qPCR. The comparison analysis revealed differences in the number and sets of identified miRNAs. The qPCR confirmed the expression of the investigated microRNAs. The correlation analysis of NGS and qPCR measurements showed strong and significant coefficients for a subset of the tested miRNAs, including those detected by all three algorithms. Single miRNA variants (isomiRs) showed different levels of correlation with the qPCR data. The obtained results revealed the good performance of all tested programs, despite the observed differences. Moreover, they implied that some specific miRNAs may be differentially estimated using NGS technology and the qPCR method, regardless of the used bioinformatics software. These discrepancies may stem from many factors, including the composition of the isomiR profile, their abundance, length, and investigated species. In conclusion, in this study, we shed light on the bioinformatics aspects of miRNAome profiling, elucidating its complexity and pinpointing potential features influencing validation. Thus, qPCR validation results should be open to interpretation when not fully concordant with NGS results until further, additional analyses are conducted.

The potential role of miRNAs and regulation of their expression in the development of mare endometrial fibrosis.

Mare endometrial fibrosis (endometrosis), is one of the main causes of equine infertility. Despite the high prevalence, both ethology, pathogenesis and the nature of its progression remain poorly understood. Recent studies have shown that microRNAs (miRNAs) are important regulators in multiple cellular processes and functions under physiological and pathological circumstances. In this article, we reported changes in miRNA expression at different stages of endometrosis and the effect of transforming growth factor (TGF)-ß1 on the expression of the most dysregulated miRNAs. We identified 1, 26, and 5 differentially expressed miRNAs (DEmiRs), in categories IIA (mild fibrosis), IIB (moderate fibrosis), and III (severe fibrosis) groups compared to category I (no fibrosis) endometria group, respectively (Padjusted < 0.05, log2FC ≥ 1.0/log2FC ≤ - 1.0). This study indicated the potential involvement of miRNAs in the regulation of the process associated to the development and progression of endometrosis. The functional enrichment analysis revealed, that DEmiRs target genes involved in the mitogen-activated protein kinases, Hippo, and phosphoinositide-3-kinase (PI3K)-Akt signalling pathways, focal adhesion, and extracellular matrix-receptor interaction. Moreover, we demonstrated that the most potent profibrotic cytokine-TGF-ß1-downregulated novel-eca-miR-42 (P < 0.05) expression in fibroblasts derived from endometria at early-stage endometrosis (category IIA).

Methylome and transcriptome data integration reveals aberrantly regulated genes in equine sarcoids.

DNA methylation is a key mechanism in transcription regulation, and aberrant methylation is a common and important mechanism in tumor initiation, maintenance, and progression. To find genes that are aberrantly regulated by altered methylation in horse sarcoids, we used reduced representation bisulfite sequencing (RRBS) accompanied by RNA sequencing (RNA-Seq) for methylome (whole genome DNA methylation sequencing) and transcriptome profiling, respectively. We found that the DNA methylation level was generally lower in lesion samples than in controls. In the analyzed samples, a total of 14,692 differentially methylated sites (DMSs) in the context of CpG (where cytosine and guanine are separated by a phosphate), and 11,712 differentially expressed genes (DEGs) were identified. The integration of the methylome and transcriptome data suggests that aberrant DNA methylation may be involved in the deregulation of expression of the 493 genes in equine sarcoid. Furthermore, enrichment analysis of the genes demonstrated the activation of multiple molecular pathways related to extracellular matrix (ECM), oxidative phosphorylation (OXPHOS), immune response, and disease processes that can be related to tumor progression. The results provide further insight into the epigenetic alterations in equine sarcoids and provide a valuable resource for follow-up studies to identify biomarkers for predicting susceptibility to this common condition in horses.

Methylation Status of Gene Bodies of Selected microRNA Genes Associated with Neoplastic Transformation in Equine Sarcoids.

Horses are of great importance in recreation, livestock production, as working animals in poorly developed countries, and for equine-assisted therapy. Equine sarcoids belong to the most commonly diagnosed tumors in this species. They may cause discomfort, pain, and can lead to the permanent impairment of motor function. The molecular bases of their formation are still under investigation. Our previous studies revealed altered microRNA (miRNA) expression and DNA methylation levels in sarcoid tumors. Abnormal patterns of methylation may be responsible for changes in gene expression levels, including microRNAs. Recently, the DNA methylation of gene bodies has also been shown to have an impact on gene expression. Thus, the aim of the study was to investigate the methylation pattern of gene bodies of chosen miRNAs identified in sarcoid tissue (miR-101, miR-10b, miR-200a, and miR-338-3p), which have also been established to play roles in neoplastic transformation. To this end, we applied qRT-PCR, Bisulfite Sequencing PCR (BSP), and Mquant methods. As a result, we identified the statistically significant downregulation of pri-mir-101-1, pri-mir-10b, and pri-mir-200a in the sarcoid samples in comparison to the control. The DNA methylation analysis revealed their hypermethylation. This suggests that DNA methylation may be one mechanism responsible for the downregulation of theses miRNAs. However, the identified differences in the methylation levels are not very high, which implies that other mechanisms may also underlie the downregulation of the expression of these miRNAs in equine sarcoids. For the first time, the results obtained shed light on microRNA expression regulation by gene body methylation in equine sarcoids and provide bases for further deeper studies on other mechanisms influencing the miRNA repertoire.

Tracking the Molecular Scenarios for Tumorigenic Remodeling of Extracellular Matrix Based on Gene Expression Profiling in Equine Skin Neoplasia Models.

An important component of tissues is the extracellular matrix (ECM), which not only forms a tissue scaffold, but also provides the environment for numerous biochemical reactions. Its composition is strictly regulated, and any irregularities can result in the development of many diseases, including cancer. Sarcoid is the most common skin cancer in equids. Its formation results from the presence of the genetic material of the bovine papillomavirus (BPV). In addition, it is assumed that sarcoid-dependent oncogenic transformation arises from a disturbed wound healing process, which may be due to the incorrect functioning of the ECM. Moreover, sarcoid is characterized by a failure to metastasize. Therefore, in this study we decided to investigate the differences in the expression profiles of genes related not only to ECM remodeling, but also to the cell adhesion pathway, in order to estimate the influence of disturbances within the ECM on the sarcoid formation process. Furthermore, we conducted comparative research not only between equine sarcoid tissue bioptates and healthy skin-derived explants, but also between dermal fibroblast cell lines transfected and non-transfected with a construct encoding the E4 protein of the BP virus, in order to determine its effect on ECM disorders. The obtained results strongly support the hypothesis that ECM-related genes are correlated with sarcoid formation. The deregulated expression of selected genes was shown in both equine sarcoid tissue bioptates and adult cutaneous fibroblast cell (ACFC) lines neoplastically transformed by nucleofection with gene constructs encoding BPV1-E1^E4 protein. The identified genes (CD99, ITGB1, JAM3 and CADM1) were up- or down-regulated, which pinpointed the phenotypic differences from the backgrounds noticed for adequate expression profiles in other cancerous or noncancerous tumors as reported in the available literature data. Unravelling the molecular pathways of ECM remodeling and cell adhesion in the in vivo and ex vivo models of epidermal/dermal sarcoid-related cancerogenesis might provide powerful tools for further investigations of genetic and epigenetic biomarkers for both silencing and re-initiating the processes of sarcoid-dependent neoplasia. Recognizing those biomarkers might insightfully explain the relatively high capacity of sarcoid-descended cancerous cell derivatives to epigenomically reprogram their nonmalignant neoplastic status in domestic horse cloned embryos produced by somatic cell nuclear transfer (SCNT).

Assessment of BPV-1 Mediated Matrix Metalloproteinase Genes Deregulation in the In Vivo and In Vitro Models Designed to Explore Molecular Nature of Equine Sarcoids.

Matrix metalloproteinases (MMPs) represent a family of enzymes capable of biocatalytically breaking down the structural and functional proteins responsible for extracellular matrix (ECM) integrity. This capability is widely used in physiological processes; however, imbalanced MMP activity can trigger the onset and progression of various pathological changes, including the neoplasmic transformation of different cell types. We sought to uncover molecular mechanisms underlying alterations in transcriptional profiles of genes coding for MMPs, which were comprehensively identified in equine adult dermal tissue bioptates, sarcoid-derived explants, and ex vivo expanded adult cutaneous fibroblast cell (ACFC) lines subjected to inducible oncogenic transformation into sarcoid-like cells. The results strongly support the hypothesis that the transcriptional activity of MMP genes correlates with molecular modifications arising in equine dermal cells during their conversion into sarcoid cells. The alterations in MMP transcription signatures occurs in both sarcoid tissues and experimentally transformed equine ACFC lines expressing BPV1-E4^E1 transgene, which were characterized by gene up- and down-regulation patterns.

Feeding pigs with coconut oil affects their adipose miRNA profile.

BACKGROUND: Circulating miRNA molecules are intensively studied for their usefulness as biomarkers of civilization diseases. Meanwhile, it is known that diet can influence the level of miRNA expression in tissues. METHODS: Our research aimed to determine how a diet containing various sources of fat (rapeseed oil, beef tallow, coconut oil) and different amounts of cDDGS (corn Dried Distilled Grains with Solubles) affects the miRNA profile in pig fat-the main source of circulating miRNAs. For this purpose, we used Next Generation Sequencing of miRNA libraries. RESULTS: We observed the highest number of differentially expressed miRNAs in the samples from animals that were fed with coconut oil in the diet compared to all other treatments. In contrary, cDDGS appeared to have little effect on miRNA expression. CONCLUSIONS: We propose a subset of diet-related, adipose-specific, conservative miRNAs among mammals, namely: ssc-miR-99b, ssc-miR-4334-3p, ssc-miR-146b, ssc-miR-23a. Moreover, we observed that several miRNAs regulated by dietary fats are considered as biomarkers in human and animal diseases.

Silver birch pollen-derived microRNAs promote NF-κB-mediated inflammation in human lung cells.

The pollen of Betula pendula Roth (silver birch) is considered to be the main cause of allergy-related rhinitis in Europe and its protein-based allergens such as Bet v 1 are well characterized. However, little is known about non-protein components of birch pollen, e.g., small RNAs and their proinflammatory activity. In the present study, next-generation sequencing (NGS) and bioinformatic approaches were used for silver birch pollen (SBP)-derived microRNA profiling and evaluation of microRNA target genes and pathways in human. Human lung cells, namely WI-38 fibroblasts and A549 alveolar epithelial cells were then stimulated with SBP microRNA in vitro and imaging cytometry-based analysis of the levels of proinflammatory cytokines, autophagy parameters and small RNA processing regulators was conducted. Bioinformatic analysis revealed that SBP microRNA may interfere with autophagy, inflammation and allergy pathways in human. SBP and SBP-derived microRNA induced NF-κB-mediated proinflammatory response in human lung cells as judged by increased levels of NF-κB p65, IL-8 and TNFα. NSUN2 and NSUN5 were involved in pollen-derived microRNA processing. Pollen-derived microRNA also modulated autophagic pathway by changes in the pools of LC3B and p62 that may affect autophagy-based adaptive responses during allergic lung inflammation. We postulate that SBP-derived microRNAs can be considered as novel proinflammatory environmental agents.

Application of the targeted sequencing approach reveals the single nucleotide polymorphism (SNP) repertoire in microRNA genes in the pig genome.

MicroRNAs (miRNAs) are recognized as gene expression regulators, indirectly orchestrating a plethora of biological processes. Single nucleotide polymorphism (SNP), one of the most common genetic variations in the genome, is established to affect miRNA functioning and influence complex traits and diseases. SNPs in miRNAs have also been associated with important production traits in livestock. Thus, the aim of our study was to reveal the SNP variability of miRNA genes in the genome of the pig, which is a significant farm animal and large-mammal human model. To this end, we applied the targeted sequencing approach, enabling deep sequencing of specified genomic regions. As a result, 73 SNPs localized in 50 distinct pre-miRNAs were identified. In silico analysis revealed that many of the identified SNPs influenced the structure and energy of the hairpin precursors. Moreover, SNPs localized in the seed regions were shown to alter targeted genes and, as a result, enrich different biological pathways. The obtained results corroborate a significant impact of SNPs on the miRNA processing and broaden the state of knowledge in the field of animal genomics. We also report the targeted sequencing approach to be a promising alternative for the whole genome sequencing in miRNA genes focused studies.

MicroRNA expression profile in bovine mammary gland parenchyma infected by coagulase-positive or coagulase-negative staphylococci.

MicroRNAs (miRNAs) are short, non-coding RNAs, 21-23 nucleotides in length which are known to regulate biological processes that greatly impact immune system activity. The aim of the study was to compare the miRNA expression in non-infected (H) mammary gland parenchyma samples with that of glands infected with coagulase-positive staphylococci (CoPS) or coagulase-negative staphylococci (CoNS) using next-generation sequencing. The miRNA profile of the parenchyma was found to change during mastitis, with its profile depending on the type of pathogen. Comparing the CoPS and H groups, 256 known and 260 potentially new miRNAs were identified, including 32 that were differentially expressed (p ≤ 0.05), of which 27 were upregulated and 5 downregulated. Comparing the CoNS and H groups, 242 known and 171 new unique miRNAs were identified: 10 were upregulated (p ≤ 0.05), and 2 downregulated (p ≤ 0.05). In addition, comparing CoPS with H and CoNS with H, 5 Kyoto Encyclopedia of Genes and Genomes pathways were identified; in both comparisons, differentially-expressed miRNAs were associated with the bacterial invasion of epithelial cells and focal adhesion pathways. Four gene ontology terms were identified in each comparison, with 2 being common to both immune system processes and signal transduction. Our results indicate that miRNAs, especially miR-99 and miR-182, play an essential role in the epigenetic regulation of a range of cellular processes, including immunological systems bacterial growth in dendritic cells and disease pathogenesis (miR-99), DNA repair and tumor progression (miR-182).

Identification of Molecular Mechanisms Related to Pig Fatness at the Transcriptome and miRNAome Levels.

Fat deposition and growth rate are closely related to pork quality and fattening efficiency. The next-generation sequencing (NGS) approach for transcriptome and miRNAome massive parallel sequencing of adipocyte tissue was applied to search for a molecular network related to fat deposition in pigs. Pigs were represented by three breeds (Large White, Pietrain, and Hampshire) that varied in fat content within each breed. The obtained results allowed for the detection of significant enrichment of Gene Ontology (GO) terms and pathways associated directly and indirectly with fat deposition via regulation of fatty acid metabolism, fat cell differentiation, inflammatory response, and extracellular matrix (ECM) organization and disassembly. Moreover, the results showed that adipocyte tissue content strongly affected the expression of leptin and other genes related to a response to excessive feed intake. The findings indicated that modification of genes and miRNAs involved in ECM rearrangements can be essential during fat tissue growth and development in pigs. The identified molecular network within genes and miRNAs that were deregulated depending on the subcutaneous fat level are proposed as candidate factors determining adipogenesis, fatness, and selected fattening characteristics in pigs.

Genetic Differentiation of the Two Types of Polish Cold-blooded Horses Included in the National Conservation Program.

The current role of the horse as a companion animal resulted in a decrease of interest in breeding and usage of draft horses. This meant that the population of cold-blooded horses in Poland has been dramatically reduced during the last decades. To avoid impoverishment of the gene pool of the local horse population, a conservation program was established which involves draft horses and other local horse breeds. The draft horses bred in Poland can be subdivided in a few horse types of which the most widespread and consolidated are Sztumski and Sokólski horses. These two subpopulations are phenotypically diversified, however, the overall level of their genetic differentiation seems to be relatively low and not precisely determined, especially with the use of molecular markers. In reference to this, in this study we used Illumina genotyping arrays to describe in detail the genetic differentiation of these two cold-blooded horse populations. We describe the genetic distance between them, as well as within-population variation, admixture patterns, and level of relatedness within populations. We also made an attempt to detect genome regions divergently selected between those horses by the detection of diversifying selection signals. The results of this study provide initial evidence supporting breeding decisions that were made during conservation breeding program design and answer questions raised by the breeders of Sokólski and Sztumski horses concerning the level of their genetic variation and differentiation.

MicroRNA profiling of the pig periaqueductal grey (PAG) region reveals candidates potentially related to sex-dependent differences.

BACKGROUND: MicroRNAs indirectly orchestrate myriads of essential biological processes. A wide diversity of miRNAs of the neurodevelopmental importance characterizes the brain tissue, which, however, exhibits region-specific miRNA profile differences. One of the most conservative regions of the brain is periaqueductal grey (PAG) playing vital roles in significant functions of this organ, also those observed to be sex-influenced. The domestic pig is an important livestock species but is also believed to be an excellent human model. This is of particular importance for neurological research because of the similarity of pig and human brains as well as difficult access to human samples. However, the pig PAG profile has not been characterized so far. Moreover, molecular bases of sex differences connected with brain functioning, including miRNA expression profiles, have not been fully deciphered yet. METHODS: Thus, in this study, we applied next-generation sequencing to characterize pig PAG expressed microRNAs. Furthermore, we performed differential expression analysis between females and males to identify changes of the miRNA profile and reveal candidates underlying sex-related differences. RESULTS: As a result, known brain-enriched, and new miRNAs which will expand the available profile, were identified. The downstream analysis revealed 38 miRNAs being differentially expressed (DE) between female and male samples. Subsequent pathway analysis showed that they enrich processes vital for neuron growth and functioning, such as long-term depression and axon guidance. Among the identified sex-influenced miRNAs were also those associated with the PAG physiology and diseases related to this region. CONCLUSIONS: The obtained results broaden the knowledge on the porcine PAG miRNAome, along with its dynamism reflected in different isomiR signatures. Moreover, they indicate possible mechanisms associated with sex-influenced differences mediated via miRNAs in the PAG functioning. They also provide candidate miRNAs for further research concerning, i.e., sex-related bases of physiological and pathological processes occurring in the nervous system.

Induced androgenetic development in rainbow trout and transcriptome analysis of irradiated eggs.

Ionizing radiation is administered to damage nuclear genome in fish eggs during induced androgenesis. In this study, we examined whether 350 Gy of X-ray applied to damage chromosomes in the rainbow trout eggs affects maternal RNA. Shortly after irradiation, we did not find any symptoms of RNA degradation in the treated eggs. Significant (p < 0.01) differences between non-irradiated and irradiated eggs concerned only a few transcripts including increased expression of immediate early response 2 (IER2) and early growth response 1 (EGR1) genes observed in the irradiated eggs. Both genes belong to the group of "immediate early genes" that respond quickly to the diverse extracellular stimuli. Elevated expression of these genes was accompanied by decreased level of ssa-miR-10b-5p and ssa-miR-21b-5p (p < 0.05), for which IER2 and EGR1 are target genes. The level of RNA in the fertilized irradiated eggs was highly significantly lower than in the non-irradiated eggs (p < 0.001) and in the unfertilized irradiated eggs (p < 0.0001). However, transcriptome profiles of fertilized non-irradiated eggs and fertilized irradiated eggs did not differ significantly. Thus, we assume that reduced abundance of mRNA in the fertilized irradiated eggs was associated with post-translational degradation and clearance of the maternal transcripts rather than from the irradiation of eggs.

A genome-wide scan for diversifying selection signatures in selected horse breeds.

The genetic differentiation of the current horse population was evolutionarily created by natural or artificial selection which shaped the genomes of individual breeds in several unique ways. The availability of high throughput genotyping methods created the opportunity to study this genetic variation on a genome-wide level allowing detection of genome regions divergently selected between separate breeds as well as among different horse types sharing similar phenotypic features. In this study, we used the population differentiation index (FST) that is generally used for measuring locus-specific allele frequencies variation between populations, to detect selection signatures among six horse breeds maintained in Poland. These breeds can be classified into three major categories, including light, draft and primitive horses, selected mainly in terms of type (utility), exterior, performance, size, coat color and appearance. The analysis of the most pronounced selection signals found in this study allowed us to detect several genomic regions and genes connected with processes potentially important for breed phenotypic differentiation and associated with energy homeostasis during physical effort, heart functioning, fertility, disease resistance and motor coordination. Our results also confirmed previously described association of loci on ECA3 (spanning LCORL and NCAPG genes) and ECA11 (spanning LASP1 gene) with the regulation of body size in our draft and primitive (small size) horses. The efficiency of the applied FST-based approach was also confirmed by the identification of a robust selection signal in the blue dun colored Polish Konik horses at the locus of TBX3 gene, which was previously shown to be responsible for dun coat color dilution in other horse breeds. FST-based method showed to be efficient in detection of diversifying selection signatures in the analyzed horse breeds. Especially pronounced signals were observed at the loci responsible for fixed breed-specific features. Several candidate genes under selection were proposed in this study for traits selected in separate breeds and horse types, however, further functional and comparative studies are needed to confirm and explain their effect on the observed genetic diversity of the horse breeds.

Corn dried distillers grains with solubles (cDDGS) in the diet of pigs change the expression of adipose genes that are potential therapeutic targets in metabolic and cardiovascular diseases.

BACKGROUND: Corn dried distillers grains with solubles (cDDGS) are a byproduct of biofuel and alcohol production. cDDGS have been used in pig feed for many years, because they are readily available and rich in protein, fiber, unsaturated fatty acids and phytosterols. However, feed mixtures too high in cDDGS result in the worsening of backfat quality. We performed RNA-sequencing analysis of backfat from crossbred pigs fed different diets. The diets were isoenergetic but contained different amounts of cDDGS and various sources of fats. The animals were divided into four dietary groups during the two months of experimentation: group I (control (-cDDGS+rapeseed oil)), group II (+cDDGS+rapeseed oil), group III (+cDDGS+beef tallow), and group IV (+cDDGS+coconut oil). The aim of the present experiment was to evaluate changes in the backfat transcriptome of pigs fed isoenergetic diets that differed in cDDGS presence. RESULTS: Via DESeq2 software, we identified 93 differentially expressed genes (DEGs) between groups I and II, 13 between groups I and III, and 125 between groups I and IV. DEGs identified between group I (-cDDGS+rapeseed oil) and group II (+cDDGS+rapeseed oil) were highly overrepresented in several KEGG pathways: metabolic pathways (FDR < 1.21e-06), oxidative phosphorylation (FDR < 0.00189), fatty acid biosynthesis (FDR < 0.00577), Huntington's disease (FDR < 0.00577), fatty acid metabolism (FDR < 0.0112), Parkinson's disease (FDR < 0.0151), non-alcoholic fatty liver disease (NAFLD) (FDR < 0.016), Alzheimer's disease (FDR < 0.0211) and complement and coagulation cascades (FDR < 0.02). CONCLUSIONS: We observed that the addition of cDDGS positively affects the expression of several genes that have been recently proposed as potential targets for the treatment of obesity, diabetes, cardiovascular disease, and Alzheimer's disease (e.g., FASN, AACS, ALAS1, HMGCS1, and VSIG4). Thus, our results support the idea of including cDDGS into the diets of companion animals and humans and encourage research into the bioactive ingredients of cDDGS.

A genome-wide detection of selection signatures in conserved and commercial pig breeds maintained in Poland.

BACKGROUND: Identification of selection signatures can provide a direct insight into the mechanism of artificial selection and allow further disclosure of the candidate genes related to the animals' phenotypic variation. Domestication and subsequent long-time selection have resulted in extensive phenotypic changes in domestic pigs, involving a number of traits, like behavior, body composition, disease resistance, reproduction and coat color. In this study, based on genotypes obtained from PorcineSNP60 Illumina assay we attempt to detect both diversifying and within-breed selection signatures in 530 pigs belonging to four breeds: Polish Landrace, Pulawska, Zlotnicka White and Zlotnicka Spotted, of which the last three are a subject of conservative breeding and substantially represent the native populations. RESULTS: A two largely complementary statistical methods were used for signatures detection, including: pairwise FST and relative extended haplotype homozygosity (REHH) test. Breed-specific diversifying selection signals included several genes involved in processes connected with fertility, growth and metabolism which are potentially responsible for different phenotypes of the studied breeds. The diversifying selection signals also comprised PPARD gene that was previously found to have a large effect on the shape of the external ear in pigs or two genes encoding neuropeptide Y receptors (Y2 and Y5) involved in fat deposition and stress response which are important features differentiating the studied breeds. REHH statistics allowed detecting several within-breed selection signatures overlapping with genes connected with a range of functions including, among others: metabolic pathways, immune system response or implantation and development of the embryo. CONCLUSIONS: The study provides many potential candidate genes with implication for traits selected in the individual breeds and gives strong basis for further studies aiming at identification of sources of variation among the studied pig breeds.