Deregulation of oxidative phosphorylation pathways in embryos derived in vitro from prepubertal and pubertal heifers based on whole-transcriptome sequencing.

BACKGROUND: Although, oocytes from prepubertal donors are known to be less developmentally competent than those from adult donors it does not restrain their ability to produce full-term pregnancies. The transcriptomic profile of embryos could be used as a predictor for embryo's individual developmental competence. The aim of the study was to compare transcriptomic profile of blastocysts derived from prepubertal and pubertal heifers oocytes. Bovine cumulus-oocyte complexes (COCs) were obtained by ovum pick- up method from prepubertal and pubertal heifers. After in vitro maturation COCs were fertilized and cultured to the blastocyst stage. Total RNA was isolated from both groups of blastocysts and RNA-seq was performed. Gene ontology analysis was performed by DAVID (Database for Annotation, Visualization and Integrated Discovery). RESULTS: A higher average blastocyst rate was obtained in the pubertal than in the pre-pubertal group. There were no differences in the quality of blastocysts between the examined groups. We identified 436 differentially expressed genes (DEGs) between blastocysts derived from researched groups, of which 247 DEGs were downregulated in blastocysts derived from pubertal compared to prepubertal heifers oocytes, and 189 DEGs were upregulated. The genes involved in mitochondrial function, including oxidative phosphorylation (OXPHOS) were found to be different in studied groups using Kyoto Encyclopedia of Genes (KEGG) pathway analysis and 8 of those DEGs were upregulated and 1 was downregulated in blastocysts derived from pubertal compared to prepubertal heifers oocytes. DEGs associated with mitochondrial function were found: ATP synthases (ATP5MF-ATP synthase membrane subunit f, ATP5PD- ATP synthase peripheral stalk subunit d, ATP12A- ATPase H+/K + transporting non-gastric alpha2 subunit), NADH dehydrogenases (NDUFS3- NADH: ubiquinone oxidoreductase subunit core subunit S3, NDUFA13- NADH: ubiquinone oxidoreductase subunit A13, NDUFA3- NADH: ubiquinone oxidoreductase subunit A3), cytochrome c oxidase (COX17), cytochrome c somatic (CYCS) and ubiquinol cytochrome c reductase core protein 1 (UQCRC1). We found lower number of apoptotic cells in blastocysts derived from oocytes collected from prepubertal than those obtained from pubertal donors. CONCLUSIONS: Despite decreased expression of genes associated with OXPHOS pathway in blastocysts from prepubertal heifers oocytes, the increased level of ATP12A together with the lower number of apoptotic cells in these blastocysts might support their survival after transfer.

Unveiling the population genetic structure of Iranian horses breeds by whole-genome resequencing analysis.

Preserving genetic diversity is pivotal for enhancing genetic improvement and facilitating adaptive responses to selection. This study focuses on identifying key genetic variants, including single nucleotide polymorphisms (SNPs), insertion/deletion polymorphisms (INDELs), and copy number variants (CNVs), while exploring the genomic evolutionary connectedness among seven Iranian horses representing five indigenous breeds: Caspian, Turkemen, DareShuri, Kurdish, and Asil. Using whole-genome resequencing, we generated 2.7 Gb of sequence data, with raw reads ranging from 1.2 Gb for Caspian horses to 0.38 Gb for Turkoman horses. Post-filtering, approximately 1.9 Gb of reads remained, with ~ 1.5 Gb successfully mapped to the horse reference genome (EquCab3.0), achieving mapping rates between 76.4% (Caspian) and 98.35% (Turkoman). We identified 2,909,816 SNPs in Caspian horses, constituting around 0.1% of the genome. Notably, 71% of these SNPs were situated in intergenic regions, while 8.5 and 6.8% were located upstream and downstream, respectively. A comparative analysis of SNPs between Iranian and non-Iranian horse breeds showed that Caspian horses had the lowest number of shared SNPs with Turkoman horses. Instead, they showed a closer genetic relationship with DareShuri, Quarter, Arabian, Standardbred, and Asil breeds. Hierarchical clustering highlighted Caspian horses as a distinct cluster, underscoring their distinctive genomic signature. Caspian horses exhibit a unique genetic profile marked by an enrichment of private mutations in neurological genes, influencing sensory perception and awareness. This distinct genetic makeup shapes mating preferences and signifies a separate evolutionary trajectory. Additionally, significant non-synonymous single nucleotide polymorphisms (nsSNPs) in reproductive genes offer intervention opportunities for managing Caspian horses. These findings reveal the population genetic structure of Iranian horse breeds, contributing to the advancement of knowledge in areas such as conservation, performance traits, climate adaptation, reproduction, and resistance to diseases in equine science.

Effect of vitamin D3 supplementation on blood parameters and liver gene expression in female rats.

BACKGROUND: To better understand the molecular mechanism responsible for the therapeutic potential of vitamin D, we conducted an analysis of the liver transcriptomes of adult female rats. METHODS: Adult female rats (n = 18) were divided into three groups, receiving different doses of vitamin D: group I, 0; group II, 1000 U/kg; and group III, 5000 U/kg. Growth, body weight, the weight of main organs, blood haematological and biochemical parameters were evaluated. Gene expression in the liver were analyzed using RNA-seq and qPCR techniques. RESULTS: We observed a lower platelet count (p < 0,008) and a significantly greater (p < 0.02) number of WBCs in rats supplemented with 1000 U/kg than in rats from group III (5000 U/kg). Moreover, we noted a trend (p < 0.06) in total cholesterol concentration, suggesting a linear decrease with increasing doses of vitamin D. RNA-seq analysis did not reveal any differentially expressed genes with FDR < 0.05. However, GSEA revealed significant activation of a number of processes and pathways, including: "metallothionein, and TspO/MBR family", and "negative regulation of tumor necrosis factor production". qPCR analysis revealed significant upregulation of the Mt1, Mt2 and Orm1 genes in animals receiving high doses of vitamin D (p < 0.025, p < 0.025, and p < 0009, respectively). Moreover, Srebp2 and Insig2 were significantly lower in both experimental groups than in the control group (p < 0.003 and p < 0.036, respectively). CONCLUSIONS: Our results support the anti-inflammatory, anitioxidant and anticholesterologenic potential of vitamin D but suggest that high doses of vitamin D are needed to obtain significant results in this regard.

First transcriptomic insight into the working muscles of racing pigeons during a competition flight.

BACKGROUND: The currently known homing pigeon is a result of a sharp one-sided selection for flight characteristics focused on speed, endurance, and spatial orientation. This has led to extremely well-adapted athletic phenotypes in racing birds. METHODS: Here, we identify genes and pathways contributing to exercise adaptation in sport pigeons by applying next-generation transcriptome sequencing of m.pectoralis muscle samples, collected before and after a 300 km competition flight. RESULTS: The analysis of differentially expressed genes pictured the central role of pathways involved in fuel selection and muscle maintenance during flight, with a set of genes, in which variations may therefore be exploited for genetic improvement of the racing pigeon population towards specific categories of competition flights. CONCLUSIONS: The presented results are a background to understanding the genetic processes in the muscles of birds during flight and also are the starting point of further selection of genetic markers associated with racing performance in carrier pigeons.

Correction: Gabrys et al. Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome. Int. J. Mol. Sci. 2024, 25, 3262.

In the original publication [...].

Follicular Fluid-Derived Extracellular Vesicles Influence on In Vitro Maturation of Equine Oocyte: Impact on Cumulus Cell Viability, Expansion and Transcriptome.

Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus-oocyte complexes (COCs) that underwent in vitro maturation (IVM) in the presence (200 µg protein/mL) or absence (control) of ffEVs were assessed for cumulus expansion and viability. CCs were isolated after 12 h of IVM, followed by RNA extraction, cDNA library generation, and subsequent transcriptome analysis using next-generation sequencing. Confocal microscopy images illustrated the internalization of labeled ffEVs by CCs. Supplementation with ffEVs significantly enhanced cumulus expansion in both compacted (Cp, p < 0.0001) and expanded (Ex, p < 0.05) COCs, while viability increased in Cp groups (p < 0.01), but decreased in Ex groups (p < 0.05), compared to the controls. Although transcriptome analysis revealed a subtle effect on CC RNA profiles, differentially expressed genes encompassed processes (e.g., MAPK and Wnt signaling) potentially crucial for cumulus properties and, consequently, oocyte maturation.

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.

Transcriptome and methylome sequencing reveals altered long non-coding RNA genes expression and their aberrant DNA methylation in equine sarcoids.

Recent publications confirmed that long non-coding RNAs (lncRNAs) perform an essential function in gene-specific transcription regulation. Nevertheless, despite its important role, lncRNA has not yet been described in equine sarcoids, the skin neoplasia of horses. Therefore, the aim of this study is to deepen the knowledge about lncRNA expression in the pathogenesis of equine sarcoids and provide new insight into the regulatory function of lncRNA in the bovine papillomavirus-dependent neoplasia of horse dermal tissues. RNA sequencing (RNA-seq) data from 12 equine sarcoid samples and the corresponding controls were reanalyzed in this study. A total of 3396 differentially expressed (DE) lncRNAs and 128 DElncRNA-DE genes (DEGs) pairs were identified. Differentially expressed lncRNAs predicted target genes were enriched in pathways associated with inter alia the extracellular matrix disassembly and cancer pathways. Furthermore, methylation data from the same samples were integrated into the analysis, and 12 DElncRNAs were described as potentially disturbed by aberrant methylation. In conclusion, this study presents novel data about lncRNA's role in the pathogenesis of equine sarcoids.

Knockout of TRDMT1 methyltransferase affects DNA methylome in glioblastoma cells.

PURPOSE: We have previously shown that TRDMT1 methyltransferase is a regulator of chemotherapy-associated responses in glioblastoma cells. Despite the fact that glioblastoma, a common and malignant brain tumor, is widely characterized in terms of genetic and epigenetic markers, there are no data on TRDMT1-related changes in 5-methylcytosine pools in the genome. In the present study, the effect of TRDMT1 gene knockout (KO) on DNA methylome was analyzed. METHODS: CRISPR-based approach was used to obtain TRDMT1 KO glioblastoma cells. Total 5-methylcytosine levels in DNA, DNMT1 pools and DNMT activity were studied using ELISA. Reduced representation bisulfite sequencing (RRBS) was considered to comprehensively evaluate DNA methylome in glioblastoma cells with TRDMT1 KO. RESULTS: TRDMT1 KO cells were characterized by decreased levels of total 5-methylcytosine in DNA and DNMT1, and DNMT activity. RRBS-based methylome analysis revealed statistically significant differences in methylation-relevant DMS-linked genes in control cells compared to TRDMT1 KO cells. TRDMT1 KO-associated changes in DNA methylome may affect the activity of several processes and pathways such as telomere maintenance, cell cycle and longevity regulating pathway, proteostasis, DNA and RNA biology. CONCLUSIONS: TRDMT1 may be suggested as a novel modulator of gene expression by changes in DNA methylome that may affect cancer cell fates during chemotherapy. We postulate that the levels and mutation status of TRDMT1 should be considered as a prognostic marker and carefully monitored during glioblastoma progression.

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.

Changes in DNA Methylation and mRNA Expression in Lung Tissue after Long-Term Supplementation with an Increased Dose of Cholecalciferol.

Maintaining an appropriate concentration of vitamin D is essential for the proper functioning of the body, regardless of age. Nowadays, there are more and more indications that vitamin D supplementation at higher than standard doses may show protective and therapeutic effects. Our study identified differences in the body's response to long-term supplementation with cholecalciferol at an increased dose. Two groups of pigs were used in the experiment. The first group received a standard dose of cholecalciferol (grower, 2000 IU/kg feed, and finisher, 1500 IU/kg feed), and the second group received an increased dose (grower, 3000 IU/kg feed, and finisher, 2500 IU/kg feed). After slaughter, lung samples were collected and used for RRBS and mRNA sequencing. Analysis of the methylation results showed that 2349 CpG sites had significantly altered methylation patterns and 1116 (47.51%) identified DMSs (Differentially Methylated Sites) were related to genes and their regulatory sites. The mRNA sequencing results showed a significant change in the expression of 195 genes. The integrated analysis identified eleven genes with DNA methylation and mRNA expression differences between the analyzed groups. The results of this study suggested that an increased vitamin D intake may be helpful for the prevention of lung cancer and pulmonary fibrosis. These actions may stem from the influence of vitamin D on the expression of genes associated with collagen production, such as SHMT1, UGT1A6, and ITIH2.The anti-cancer properties of vitamin D are also supported by changes in KLHL3 and TTPA gene expression.

Interaction networks and pathway analysis of genetic resistance to gastrointestinal nematodes in sheep.

Gastrointestinal nematode (GINs) infections are one of the causative agents of health and economic issues in sheep production systems worldwide. Considerable genetic variations in resistance or susceptibility in different sheep breeds are documented, but published results are conflicting. Recent advances obtained by high-throughput technologies such as commercial SNP chips, whole-genome sequencing, or whole transcriptome profiling provide new insights into breeding for host resistance or nematode control at the genetic levels. This study aimed to identify potential biomarkers associated with the resistance to ovine GINs through a network analysis approach. Comprehensive gene and protein interaction networks were reconstructed for candidate genes involved in the most related immune pathways associated with resistance to ovine GINs using data mining from literature. Generally, 30 genes including CD53, CHIA, RELN, HRH1, EPS15, LRP8, ATP2B1, IL4, IL5, IL13, IL2RA, IL23R, TNFα, IFNγ, TBX21, SH3RF1, HERC2, PTPN1, BIN1, HERC5, C3AR1, NOS2, STAT5B, STAT4, CCL1, CCL8, VIL1, CXCR1, CXCR2, and CXCR4 located on chromosomes 1, 2, 3, 4, 5, 6, 11, 13, 19, and 20 have been found as containing effective regions with the most related pathways to nematode infections. The results obtained by network analysis showed two functional modules, belonging to the interleukins family (IL4, IL5, IL13, IL23R, and IL2RA) and chemokine receptors or ligands family (CXCR1, CXCR2, CXCR4, CCL1, and CCL8). Interleukins are a group of cytokines that are expressed by white blood cells with a major role in the immune system. Chemokines are also a family of chemoattractant cytokines which play a vital role in cell migration that influence the immune system by a process known as chemotaxis. The results provide useful information for the functional annotation of candidate genes related to parasite resistance and add new information towards a consensus on quantitative trait loci (QTLs) related to the incidence of nematode infections.

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.

Trichostatin A-Mediated Epigenetic Modulation Predominantly Triggers Transcriptomic Alterations in the Ex Vivo Expanded Equine Chondrocytes.

Epigenetic mechanisms of gene regulation are important for the proper differentiation of cells used for therapeutic and regenerative purposes. The primary goal of the present study was to investigate the impacts of 5-aza-2' deoxycytidine (5-AZA-dc)- and/or trichostatin A (TSA)-mediated approaches applied to epigenomically modulate the ex vivo expanded equine chondrocytes maintained in monolayer culture on the status of chondrogenic cytodifferentiation at the transcriptome level. The results of next-generation sequencing of 3' mRNA-seq libraries on stimulated and unstimulated chondrocytes of the third passage showed no significant influence of 5-AZA-dc treatment. Chondrocytes stimulated with TSA or with a combination of 5-AZA-dc+TSA revealed significant expressional decline, mainly for genes encoding histone and DNA methyltransferases, but also for other genes, many of which are enriched in canonical pathways that are important for chondrocyte biology. The TSA- or 5-AZA-dc+TSA-induced upregulation of expanded chondrocytes included genes that are involved in histone hyperacetylation and also genes relevant to rheumatoid arthritis and inflammation. Chondrocyte stimulation experiments including a TSA modifier also led to the unexpected expression incrementation of genes encoding HDAC3, SIRT2, and SIRT5 histone deacetylases and the MBD1 CpG-binding domain protein, pointing to another function of the TSA agent besides its epigenetic-like properties. Based on the transcriptomic data, TSA stimulation seems to be undesirable for chondrogenic differentiation of passaged cartilaginous cells in a monolayer culture. Nonetheless, obtained transcriptomic results of TSA-dependent epigenomic modification of the ex vivo expanded equine chondrocytes provide a new source of data important for the potential application of epigenetically altered cells for transplantation purposes in tissue engineering of the equine skeletal system.

The Induced Expression of BPV E4 Gene in Equine Adult Dermal Fibroblast Cells as a Potential Model of Skin Sarcoid-like Neoplasia.

The equine sarcoid is one of the most common neoplasias in the Equidae family. Despite the association of this tumor with the presence of bovine papillomavirus (BPV), the molecular mechanism of this lesion has not been fully understood. The transgenization of equine adult cutaneous fibroblast cells (ACFCs) was accomplished by nucleofection, followed by detection of molecular modifications using high-throughput NGS transcriptome sequencing. The results of the present study confirm that BPV-E4- and BPV-E1^E4-mediated nucleofection strategy significantly affected the transcriptomic alterations, leading to sarcoid-like neoplastic transformation of equine ACFCs. Furthermore, the results of the current investigation might contribute to the creation of in vitro biomedical models suitable for estimating the fates of molecular dedifferentiability and the epigenomic reprogrammability of BPV-E4 and BPV-E4^E1 transgenic equine ACFC-derived sarcoid-like cell nuclei in equine somatic cell-cloned embryos. Additionally, these in vitro models seem to be reliable for thoroughly recognizing molecular mechanisms that underlie not only oncogenic alterations in transcriptomic signatures, but also the etiopathogenesis of epidermal and dermal sarcoid-dependent neoplastic transformations in horses and other equids. For those reasons, the aforementioned transgenic models might be useful for devising clinical treatments in horses afflicted with sarcoid-related neoplasia of cutaneous and subcutaneous tissues.

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).

Pet ownership in pregnancy and methylation pattern in cord blood.

Having pets in the house during the first years of life has been shown to protect against allergies. However, the result of different studies is heterogeneous. The aim of this study was to evaluate the methylation pattern in cord blood in relation to pet ownership during pregnancy.We investigated the methylation patterns of 96 cord blood samples, participants of the Epigenetic Hallmark of Maternal Atopy and Diet-ELMA project, born to mothers who either owned pets (n = 32) or did not own pets (n = 64) during their pregnancy. DNA from cord blood was analysed using the Infinium methylation EPIC. For statistical analysis, RnBeads software was applied.We found 113 differentially methylated sites (DMs) in the covariate-adjusted analysis (FDR p < 0.05), with small methylation differences. The top DMs were associated with genes: UBA7, THRAP3, GTDC1, PDE8A and SBK2. In the regional analysis, two promoter regions presented with significance: RN7SL621P and RNU6-211P. Cis-regulatory element analysis revealed significant associations with several immune-related pathways, such as regulation of IL18, Toll signalling, IL6 and complement.We conclude that pet exposure during pregnancy causes subtle but significant changes in methylation patterns in cord blood, which are reflected in the biological processes governing both innate and adaptive immune responses.

Genotyping-by-sequencing performance in selected livestock species.

Application of next generation sequencing for large scale genotyping in livestock is limited by high costs and challenging data analysis process. However, available restriction enzyme-based enrichment techniques like e.g. genotyping-by-sequencing (GBS) are promising tools allowing reduction of financial outlies by a high sample multiplexing and narrowing down the sequenced genome areas to the randomly distributed read tags. In this study, we tested the performance of standard, PstI endonuclease-adapted GBS protocol for population genetics in cattle, horse and sheep with application of different, including low-depth sequencing setups. It was found that the detected SNPs display desirable polymorphism parameters and are evenly scattered across the whole genome including gene coding regions. It was also shown that the SNPs can be successfully applied in population genetics, revealing the genetic differentiation of the studied breeds. The GBS approach represents a cost-effective alternative to existing genotyping methods which may find adoption in various research applications.

Detection of runs of homozygosity in conserved and commercial pig breeds in Poland.

Runs of homozygosity (ROH) are continuous segments of the genome that arose as a result of inbreeding, resulting in the inheritance of identical haplotypes from both parents who shared a common ancestor. In the present study, we performed a detailed characterization and comparison of ROH in four pig breeds, including intensively selected Polish Landrace as well as native unselected animals of Pulawska and two Zlotnicka breeds (White and Spotted). We used a medium-density PorcineSNP60 BeadChip assay (Illumina) and cgaTOH software to detect ROH covering a minimum of 30 adjacent SNPs and maintaining a size over 1 Mb. By analysing ROH distribution and frequency across the genome, we also identified genomic regions with high ROH frequency (so-called "ROH hotspots"). The obtained results showed that the analysed conserved breeds were characterized by a higher ROH span and higher ROH-based inbreeding coefficients (FROH ), which likely result from past population bottlenecks, increasing the overall inbreeding level within these populations. The analysis of ROH distribution across the genomes revealed the presence of both shared and breed-specific ROH hotspots. These hotspots, presumably representing genome regions under selection, overlapped with a variety of genes associated with processes connected with immune system functioning, reproduction, glucose homeostasis and metabolism. The genome regions with ROH hotspots overlapping in all analysed populations, located on SSC4 (51.9-55.9 Mb) and 13 (92.6-97.8 Mb), covered thirty-one different genes, including MMP16, SLC7A13, ATP6V0D2, CNGB3, WWiP1, RiMDN1 and CPNE3. These genes are primarily associated with biological regulation and metabolism, processes that could be responsible for the variety of the selected production and functional features.

The use of the SLC16A1 gene as a potential marker to predict race performance in Arabian horses.

BACKGROUND: Arabian horses are commonly believed to be one of the oldest and the most popular horse breeds in the world, characterized by favourable stamina traits and exercise phenotypes. During intensive training, the rates of lactate production and utilization are critical to avoid muscle fatigue and a decrease in exercise performance. The key factor determining transmembrane lactate transport is the monocarboxylate transporter 1 protein coded for by the SLC16A1 gene. The aim of the present research was to identify polymorphisms in the coding sequence and UTRs in the equine SLC16A1 gene and to evaluate their potential association with race performance traits in Arabian horses. Based on RNA-seq data, SNPs were identified and genotyped using PCR-RFLP or PCR-HRM methods in 254 Arabian horses that competed in flat races. An association analysis between polymorphisms and racing results was performed. RESULTS: Novel polymorphisms in the equine SLC16A1 locus have been identified (missense and 5'UTR variants: g.55601543C > T and g.55589063 T > G). Analysis showed a significant association between the 5'UTR polymorphism and several racing results as follows: the possibility of winning first or second place, the number of races in which horses started and total financial benefits. The analysis also showed differences in genotype distribution depending on race distance. In the studied population, the shorter distance races were only won by TT horses. The GG and TG horses took first and second places in middle- and long-distance races, and the percentage of winning heterozygotes increased from 19.5 to 27% at the middle and long distances, respectively. The p.Val432Ile (g.55601543C > T) polymorphism was not significantly related to the analysed racing results. CONCLUSION: Our results showed that g.55589063 T > G polymorphism affected the possibility of winning first or second place and of competing in more races. The different distribution of genotypes depending on race distance indicated the possibility of using a SNP in the SLC16A1 gene as a marker to predict the best race distance for a horse. The presented results provide a basis for further research to validate the use of the SLC16A1 gene as a potential marker associated with racing performance.