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MicroRNAs (miRNAs) act as key gene expression regulators, influencing intracellular biological and pathological processes. They are of significant interest in animal genetics as potential biomarkers for animal selection and health. This study aimed to unravel the complex miRNA signature involved in mastitis in in vitro cell culture. For this purpose, we constructed a control and treatment model in ovarian mammary epithelial cells to analyze miRNA responses upon Staphylococcus aureus (S. aureus) stimulation. The high-throughput Illumina Small RNA protocol was employed, generating an average of 7.75 million single-end reads per sample, totaling 46.54 million reads. Standard bioinformatics analysis, including cleaning, filtering, miRNA quantification, and differential expression was performed using the miRbase database as a reference for ovine miRNAs. The results indicated differential expression of 63 miRNAs, including 33 up-regulated and 30 down-regulated compared to the control group. Notably, miR-10a, miR-10b, miR-21, and miR-99a displayed a significant differential expression (p ≤ 0.05) associated to signal transduction, transcriptional pathways, diseases of signal transduction by growth factor receptors and second messengers, MAPK signaling pathway, NF-κB pathway, TNFα, Toll Like Receptor 4 (TLR4) cascade, and breast cancer. This study contributes expanding miRNA databases, especially for sheep miRNAs, and identifies potential miRNA candidates for further study in biomarker identification for mastitis resistance and diagnosis. [ABSTRACT FROM AUTHOR]

This study examined the association, promoter methylation of Interferon alpha-inducible protein 6 (IFI6) gene, and their effects on mRNA expression and meat quality traits in pig. Muscle samples (n=300) were randomly collected from the Longissimus thoracis et lumborum (LTL) to determine meat quality traits including drip loss, thawing loss, cooking loss, meat pH [at 45 minutes (pH45min) and at 24 hours (pH24h) post-mortem], shear force (for tenderness), and lightness (L*) in pigs. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Muscle samples (5 per group) with divergent meat pH (low vs high) were selected for promoter methylation study using bisulfate and pyrosequencing. Muscle samples from animals (5 per group) with divergent phenotypes (low vs high meat pH and low vs high drip loss) were used for mRNA expression study by quantitative real-time polymerase chain reaction (qRT-PCR). Results showed that a polymorphism in IFI6 gene (g.370A>G) was significantly (P < 0.01) associated with meat pH24h. Furthermore, four CpG regions were identified in IFI6 promoter, of which DNA methylation in a CG site was higher in low meat pH24h group compared to high meat pH24h group (P < 0.05). The mRNA expression of IFI6 gene was significantly (P < 0.01) downregulated in low meat pH24h group than high meat pH24h group. This result postulated that high DNA methylation at CG site in the promoter region might downregulate the IFI6 gene expression, hence lower the muscle pH that might lead to higher drip loss and lower tenderness. Therefore, porcine IFI6 gene might be a potential candidate gene for meat quality trait in pig. Nevertheless, further research is needed to confirm the role of IFI6 gene in controlling meat quality traits in other commercial populations. This study analysed association of a SNP in IFI6 gene with meat quality traits, and promotor methylation and transcriptome expression of IFI6 gene in relation to meat pH. The result suggested that polymorphism affects meat pH. The high DNA methylation at a specific CG site in the IFI6 promoter is involved in IFI6 gene downregulation in low meat pH. [ABSTRACT FROM AUTHOR]

Land use and land cover (LULC) changes are one of the most important elements that shaped the global environmental setting. Day by day, the number of migrants from all over the country is increasing in Dhaka Metropolitan Area (DMA), thus forcing pressure on the surrounding environment. The objectives of the study are to identify the LULC changes along with urban growth and LULC prediction in the study area. ERDAS Imagine 2015 and ArcMap 10.4 were used for data management, analysis, and presentation. Urban growth and LULC change prediction are calculated by using Shannon's entropy and the CA-Markov model. The study observed that urbanization resulted in a considerable rise in the built-up area as well as a significant decline in agricultural land, vegetation, and water bodies in the study area. It is projected that the expansion of the built-up area will continue in the upcoming days while waterbody, agricultural land, and the vegetated cover area will decrease after 41 years. The study summarized that 59.27% area of DMA will be converted to built-up area by 2030, and it is expected to rise up to 70.86% by 2060. It is noted that less than 4% area will remain a waterbody in 2060. The study will serve as a guideline for government and non-government organizations, policymakers including urban planners, and scientific communities to assure sustainable urban land use planning and management. [ABSTRACT FROM AUTHOR]

Purpose: This study aims to examine sustainability reporting through the lens of global reporting initiative (GRI) standards in developing economies, particularly in Pakistan, from the perspective of stakeholder theory, legitimacy theory, and system theory. Design/methodology/approach: Qualitative and quantitative analyses on economic, social and environmental areas of sustainability reporting based on the GRI standards are applied across 57 organizations listed on the Pakistan stock exchange over the years 2016–2020. Findings: The results from the content analysis and descriptive statistics show that overall sustainability reporting increased persistently over time and limited organizations disclose economic, social and environmental sustainability based on GRI standards. Moreover, the result from the two-tailed correlation analysis shows positive relations between economic, social and environmental sustainability reporting. Research limitations/implications: Following the GRI standards, the regulators, government and policymakers need to assess the sustainability reporting based on GRI standards to improve corporate operations' transparency, stakeholder trust and legitimacy. The organizations should move beyond the compliance of regulatory norms and adopt the globally accepted sustainability GRI standards to improve sustainability reporting. The same kind of sustainability reporting is also advised for other countries with similar backgrounds and sustainability challenges. Social implications: The integrated sustainability reporting framework based on GRI standards enables the organizations to work as a system of interconnected economic, social and environmental sustainability to resolve the issue of sustainability reporting, ensure the trust of multiple stakeholders and legitimize their business operations in society. Originality/value: To the best of the authors' knowledge and thorough review of literature, this is the first study that examines the sustainability reporting based on GRI in the developing country of Pakistan to extend the findings of previous studies from conventional sustainability reporting to the globally accepted GRI based sustainability reporting. Using system theory, this study provides an additional contribution to the consideration concerning sustainability reporting based on GRI standards in the context of Pakistan. [ABSTRACT FROM AUTHOR]

Simple Summary: Tenderness influences repurchase decisions of sheep meat because it is a significant factor contributing to eating satisfaction and consumer acceptance. This study analyzed the transcriptome of five high- and five low-lamb tenderness samples. The result showed potential candidate hepatic genes and polymorphisms affecting lamb tenderness. These potential candidate genes and genetic markers could be used in lamb tenderness selection programs. Tenderness is a key meat quality trait that determines the public acceptance of lamb consumption, so genetic improvement toward lamb with higher tenderness is pivotal for a sustainable sheep industry. However, unravelling the genomics controlling the tenderness is the first step. Therefore, this study aimed to identify the transcriptome signatures and polymorphisms related to divergent lamb tenderness using RNA deep sequencing. Since the molecules and enzymes that control muscle growth and tenderness are metabolized and synthesized in the liver, hepatic tissues of ten sheep with divergent phenotypes: five high- and five low-lamb tenderness samples were applied for deep sequencing. Sequence analysis identified the number of reads ranged from 21.37 to 25.37 million bases with a mean value of 22.90 million bases. In total, 328 genes are detected as differentially expressed (DEGs) including 110 and 218 genes that were up- and down-regulated, respectively. Pathway analysis showed steroid hormone biosynthesis as the dominant pathway behind the lamb tenderness. Gene expression analysis identified the top high (such as TP53INP1, CYP2E1, HSD17B13, ADH1C, and LPIN1) and low (such as ANGPTL2, IGFBP7, FABP5, OLFML3, and THOC5) expressed candidate genes. Polymorphism and association analysis revealed that mutation in OLFML3, ANGPTL2, and THOC5 genes could be potential candidate markers for tenderness in sheep. The genes and pathways identified in this study cause variation in tenderness, thus could be potential genetic markers to improve meat quality in sheep. However, further validation is needed to confirm the effect of these markers in different sheep populations so that these could be used in a selection program for lamb with high tenderness. [ABSTRACT FROM AUTHOR]

Objective: The objective of this study was to identify polymorphism in olfactomedin like 3 (OLFML3) gene, and association analysis with meat quality, carcass characteristics, retail meat cut, and fatty acid composition in sheep, and expression quantification of OLFML3 gene in phenotypically divergent sheep. Methods: A total of 328 rams at the age of 10 to 12 months with an average body weight of 26.13 kg were used. A novel polymorphism was identified using high-throughput sequencing in sheep and genotyping of OLFML3 polymorphism was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Among 328 rams, 100 rams representing various sheep genotypes were used for association study and proc general linear model was used to analyse association between genotypes and phenotypic traits. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for the expression analysis of OLFML3 mRNA in phenotypically divergent sheep population. Results: The findings revealed a novel polymorphism in the OLFML3 gene (g.90317673 C>T). The OLFML3 gene revealed three genotypes: CC, CT, and TT. The single nucleotide polymorphism (SNP) was found to be significantly (p<0.05) associated with meat quality traits such as tenderness and cooking loss; carcass characteristics such as carcass length; retail meat cut such as pelvic fat in leg, intramuscular fat in loin and tenderloin, muscle in flank and shank; fatty acids composition such as tridecanoic acid (C13:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), ginkgolic acid (C17:1), linolenic acid (C18:3n3), arachidic acid (C20:0), eicosenoic acid (C20:1), arachidonic acid (C20:4n6), heneicosylic acid (C21:0), and nervonic acid (C24:1). The TT genotype was associated with higher level of meat quality, carcass characteristics, retail meat cut, and some fatty acids composition. However, the mRNA expression analysis was not different among genotypes. Conclusion: The OLFML3 gene could be a potential putative candidate for selecting higher quality sheep meat, carcass characteristics, retail meat cuts, and fatty acid composition in sheep. [ABSTRACT FROM AUTHOR]

Fatty acids (FA) in ruminants, especially unsaturated FA (USFA) have important impact in meat quality, nutritional value, and flavour quality of meat, and on consumer's health. Identification of the genetic factors controlling the FA composition and metabolism is pivotal to select sheep that produce higher USFA and lower saturated (SFA) for the benefit of sheep industry and consumers. Therefore, this study was aimed to investigate the transcriptome profiling in the liver tissues collected from sheep with divergent USFA content in longissimus muscle using RNA deep-sequencing. From sheep (n = 100) population, liver tissues with higher (n = 3) and lower (n = 3) USFA content were analysed using Illumina HiSeq 2500. The total number of reads produced for each liver sample were ranged from 21.28 to 28.51 million with a median of 23.90 million. Approximately, 198 genes were differentially regulated with significance level of p-adjusted value <0.05. Among them, 100 genes were up-regulated, and 98 were down-regulated (p<0.01, FC>1.5) in the higher USFA group. A large proportion of key genes involved in FA biosynthesis, adipogenesis, fat deposition, and lipid metabolism were identified, such as APOA5, SLC25A30, GFPT1, LEPR, TGFBR2, FABP7, GSTCD, and CYP17A. Pathway analysis revealed that glycosaminoglycan biosynthesis- keratan sulfate, adipokine signaling, galactose metabolism, endocrine and other factors-regulating calcium metabolism, mineral metabolism, and PPAR signaling pathway were playing important regulatory roles in FA metabolism. Importantly, polymorphism and association analyses showed that mutation in APOA5, CFHR5, TGFBR2 and LEPR genes could be potential markers for the FA composition in sheep. These polymorphisms and transcriptome networks controlling the FA variation could be used as genetic markers for FA composition-related traits improvement. However, functional validation is required to confirm the effect of these SNPs in other sheep population in order to incorporate them in the sheep breeding program. [ABSTRACT FROM AUTHOR]

This study was carried out to assess the levels of trace metals (Fe, Zn, Mn and Cu) in the macroalgae and sediment of the Sundarban mangrove estuary in Bangladesh. In this connection, macroalgae, water and sediment samples were collected seasonally (pre-monsoon, monsoon and post-monsoon) for a year from the study area. The surface water of the estuary was found to be quite rich in NO3, PO4 and NH4, which is more than that of the sediment pore water. Trace elements were determined using an atomic absorption spectrophotometer. The amount of trace metals in the samples were found in the order of Fe > Mn > Zn > Cu. Enrichment factor was done for trace metals in sediment and showed non-crustal origin of Zn and Cu in the monsoon period. Analysis of variance revealed the seasonal variation of trace metal concentration in sediment (except Mn; p > 0.05) and macroalgae (except Zn; p > 0.05). Iron in sediment showed significant positive correlation both with Fe (r = 0.84, p < 0.05) and Mn (r = 0.87, p < 0.01) in macroalgae. Similarly, Cu in sediment demonstrated a significant positive correlation with Cu in macroalgae (r = 0.81, p < 0.01). The findings reveal a significant communication of metals in sediment and macroalgae. [ABSTRACT FROM AUTHOR]

Porcine reproductive and respiratory syndrome (PRRS) is a devastating viral disease affecting the swine industry worldwide. Genetic variation in host immunity has been considered as one of the potential determinants to improve the immunocompetence, thereby resistance to PRRS. Therefore, the present study aimed to investigate the breed difference in innate immune response to PRRSV vaccination between German Landrace (DL) and Pietrain (Pi) pigs. We analyzed microarray-based transcriptome profiles of peripheral blood mononuclear cells (PBMCs) collected before (0 h) and 24 h after PRRSV vaccination from purebred DL and Pi pigs with three biological replicates. In total 4,269 transcripts were identified to be differentially expressed in PBMCs in at least any of four tested contrast pairs (i.e. DL-24h vs. DL-0h, Pi-24h vs. Pi-0h, DL-0h vs. Pi-0h and DL-24h vs. Pi-24h). The number of vaccine-induced differentially expressed genes (DEGs) was much higher (2,459) in DL pigs than that of Pi pigs (291). After 24 h of PRRSV vaccination, 1,046 genes were differentially expressed in PMBCs of DL pigs compared to that of Pi (DL-24h vs. Pi-24h), indicating the breed differences in vaccine responsiveness. The top biological pathways significantly affected by DEGs of both breeds were linked to immune response functions. The network enrichment analysis identified ADAM17, STAT1, MMS19, RPA2, BAD, UCHL5 and APC as potential regulatory genes for the functional network of PRRSV vaccine response specific for DL; while FOXO3, IRF2, ADRBK1, FHL3, PPP2CB and NCOA6 were found to be the most potential hubs of Pi specific transcriptome network. In conclusion, our data provided insights of breed-specific host transcriptome responses to PRRSV vaccination which might contribute in better understanding of PPRS resistance in pigs. [ABSTRACT FROM AUTHOR]

The primary objective of the present study was to assess the effects of vitamin and mineral premix (VMP) withdrawal from the diets 30 and 60 days ahead of slaughter on carcass and meat quality of Holstein Friesian steers. A total of 45 animals at 16 to 17 months of age were used and the selected animals were divided into three experimental groups: control group (fed with a diet with VMP), VMP withdrawal 30 days ahead of slaughter (VMP30 group), and VMP withdrawal 60 days ahead of slaughter (VMP60 group). Meat samples were taken at 24 h postmortem from the 13th rib section and meat quality was evaluated on the Longissimus dorsi thoracis (LT) muscle. After slaughter, carcass yield and meat drip loss, cooking loss, thawing loss, and shear force traits were determined. Meat pH and color parameters were measured at 24, 48, 72, 96, 120, and 144 h of postmortem. The fatty acid composition in 13th rib section' adipose tissue was determined. The hot and cold carcass weights, carcass yield and chilling loss were not affected by the withdrawal of VMP from the diet. Withdrawal of VMP from the diets 30 and 60 days ahead of slaughter did not have any significant effects on ultimate pH, drip loss, cooking loss, thawing loss, shear force, and meat color. Additionally, dry matter, crude protein, ash, fat contents, moisture-protein ratio of the meat samples, and fatty acid profiles were not affected by VMP30 and VMP60 treatments. It was concluded based on present finding VMP could be withdrawn safely from the diets 30 and 60 days ahead of slaughter without any negative effects on carcass and meat quality traits of feedlot steers. Withdrawal of VMP may reduce feeding costs and environmental damages generated by animal breeding systems. [ABSTRACT FROM AUTHOR]

The porcine reproductive and respiratory syndrome (PRRS) is an infectious disease that leads to high financial and production losses in the global swine industry. The pathogenesis of this disease is dependent on a multitude of factors, and its control remains problematic. The immune system generally defends against infectious diseases, especially dendritic cells (DCs), which play a crucial role in the activation of the immune response after viral infections. However, the understanding of the immune response and the genetic impact on the immune response to PRRS virus (PRRSV) remains incomplete. In light of this, we investigated the regulation of the host immune response to PRRSV in porcine lung DCs using RNA-sequencing (RNA-Seq). Lung DCs from two different pig breeds (Pietrain and Duroc) were collected before (0 hours) and during various periods of infection (3, 6, 9, 12, and 24 hours post infection (hpi)). RNA-Seq analysis revealed a total of 20,396 predicted porcine genes, which included breed-specific differentially expressed immune genes. Pietrain and Duroc infected lung DCs showed opposite gene expression courses during the first time points post infection. Duroc lung DCs reacted more strongly and distinctly than Pietrain lung DCs during these periods (3, 6, 9, 12 hpi). Additionally, cluster analysis revealed time-dependent co-expressed groups of genes that were involved in immune-relevant pathways. Key clusters and pathways were identified, which help to explain the biological and functional background of lung DCs post PRRSV infection and suggest IL-1β1 as an important candidate gene. RNA-Seq was also used to characterize the viral replication of PRRSV for each breed. PRRSV was able to infect and to replicate differently in lung DCs between the two mentioned breeds. These results could be useful in investigations on immunity traits in pig breeding and enhancing the health of pigs. [ABSTRACT FROM AUTHOR]

The porcine reproductive and respiratory syndrome (PRRS) is a devastating viral disease affecting swine production, health and welfare throughout the world. A synergistic action of the innate and the adaptive immune system of the host is essential for mounting a durable protective immunity through vaccination. Therefore, the current study aimed to investigate the transcriptome profiles of peripheral blood mononuclear cells (PBMCs) to characterize the innate and the adaptive immune response to PRRS Virus (PRRSV) vaccination in Pietrain pigs. The Affymetrix gene chip porcine gene 1.0 ST array was used for the transcriptome profiling of PBMCs collected at immediately before (D0), at one (D1) and 28 days (D28) post PRRSV vaccination with three biological replications. With FDR <0.05 and log2 fold change ±1.5 as cutoff criteria, 295 and 115 transcripts were found to be differentially expressed in PBMCs during the stage of innate and adaptive response, respectively. The microarray expression results were technically validated by qRT-PCR. The gene ontology terms such as viral life cycle, regulation of lymphocyte activation, cytokine activity and inflammatory response were enriched during the innate immunity; cytolysis, T cell mediated cytotoxicity, immunoglobulin production were enriched during adaptive immunity to PRRSV vaccination. Significant enrichment of cytokine-cytokine receptor interaction, signaling by interleukins, signaling by the B cell receptor (BCR), viral mRNA translation, IFN-gamma pathway and AP-1 transcription factor network pathways were indicating the involvement of altered genes in the antiviral defense. Network analysis revealed that four network modules were functionally involved with the transcriptional network of innate immunity, and five modules were linked to adaptive immunity in PBMCs. The innate immune transcriptional network was found to be regulated by LCK, STAT3, ATP5B, UBB and RSP17. While TGFß1, IL7R, RAD21, SP1 and GZMB are likely to be predictive for the adaptive immune transcriptional response to PRRSV vaccine in PBMCs. Results of the current immunogenomics study advances our understanding of PRRS in term of host-vaccine interaction, and thereby contribute to design a rationale for disease control strategy. [ABSTRACT FROM AUTHOR]

West Nile virus (WNV) is one of the most common causes of epidemic viral encephalitis in horses worldwide. Peripheral blood mononuclear cells (PBMCs) are amongst the first to encounter the virus following a mosquito bite. This study aimed to elucidate the transcription kinetics of cytokine, Toll-like receptor (TLRs) and TLRs-associated genes following WNV challenge of equine PBMCs. PBMCs were challenged with an Australian strain of WNV (WNVNSW2011) and transcriptomes were quantified at 2, 6, 12 and 24 h post-infection (pi) using qRT-PCR. Type I and II interferons (IFNα, β and γ) mRNA transcription increased following WNV exposure, as did the transcripts for IL1α, IL1β, IL6, IL8, and IL22, but with slightly varying kinetics. TLR1, 3, 5, 7-9 transcripts were also upregulated in equine PBMCsin response to WNV challenge, as were those for MyD88, NF-κB, TRAF3, STAT1 and 2, IRF3 and 7, ISG15, as well as SOCS1 and 3 compared to the control cells. Expression of selected genes in the draining lymph node, spleen and brain (medulla oblongata) of experimentally infected horses was also assessed and transcription of most of these genes was also upregulated here. Although qRT-PCR detected higher viral RNA at 24 h pi compared to 6 h pi, the virus did not replicate productively in equine PBMCs. The up-regulation of gene-transcription for selected cytokines, IFNs, TLRs and TLRs-associated molecules suggests their involvement in virus recognition and control of WNV infection in the horse. [ABSTRACT FROM AUTHOR]

Histone acetylation, regulated by histone deacetylases (HDACs) is a key epigenetic mechanism controlling gene expressions. Although dendritic cells (DCs) are playing pivotal roles in host immune responses, the effect of epigenetic modulation of DCs immune responses remains unknown. Sulforaphane (SFN) as a HDAC inhibitor has anti-inflammatory properties, which is used to investigate the epigenetic regulation of LPS-induced immune gene and HDAC family gene expressions in porcine monocyte-derived dendritic cells (moDCs). SFN was found to inhibit the lipopolysaccharide LPS induced HDAC6, HDAC10 and DNA methyltransferase (DNMT3a) gene expression, whereas up-regulated the expression of DNMT1 gene. Additionally, SFN was observed to inhibit the global HDAC activity, and suppressed moDCs differentiation from immature to mature DCs through down-regulating the CD40, CD80 and CD86 expression and led further to enhanced phagocytosis of moDCs. The SFN pre-treated of moDCs directly altered the LPS-induced TLR4 and MD2 gene expression and dynamically regulated the TLR4-induced activity of transcription factor NF-κB and TBP. SFN showed a protective role in LPS induced cell apoptosis through suppressing the IRF6 and TGF-ß1 production. SFN impaired the pro-inflammatory cytokine TNF-α and IL-1ß secretion into the cell culture supernatants that were induced in moDCs by LPS stimulation, whereas SFN increased the cellular-resident TNF-α accumulation. This study demonstrates that through the epigenetic mechanism the HDAC inhibitor SFN could modulate the LPS induced innate immune responses of porcine moDCs. [ABSTRACT FROM AUTHOR]

One of the primary factors contributing to boar taint is the level of androstenone in porcine adipose tissues. A majority of the studies performed to identify candidate biomarkers for the synthesis of androstenone in testis tissues follow a reductionist approach, identifying and studying the effect of biomarkers individually. Although these studies provide detailed information on individual biomarkers, a global picture of changes in metabolic pathways that lead to the difference in androstenone synthesis is still missing. The aim of this work was to identify major pathways and interactions influencing steroid hormone synthesis and androstenone biosynthesis using an integrative approach to provide a bird’s eye view of the factors causing difference in steroidogenesis and androstenone biosynthesis. For this purpose, we followed an analysis procedure merging together gene expression data from boars with divergent levels of androstenone and pathway mapping and interaction network retrieved from KEGG database. The interaction networks were weighted with Pearson correlation coefficients calculated from gene expression data and significant interactions and enriched pathways were identified based on these networks. The results show that 1,023 interactions were significant for high and low androstenone animals and that a total of 92 pathways were enriched for significant interactions. Although published articles show that a number of these enriched pathways were activated as a result of downstream signaling of steroid hormones, we speculate that the significant interactions in pathways such as glutathione metabolism, sphingolipid metabolism, fatty acid metabolism and significant interactions in cAMP-PKA/PKC signaling might be the key factors determining the difference in steroidogenesis and androstenone biosynthesis between boars with divergent androstenone levels in our study. The results and assumptions presented in this study are from an in-silico analysis done at the gene expression level and further laboratory experiments at genomic, proteomic or metabolomic level are necessary to validate these findings. [ABSTRACT FROM AUTHOR]

Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB) staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down) in exosomes and 30 miRNAs differentially expressed (21 up and 9 down) in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment. [ABSTRACT FROM AUTHOR]

Boar taint is the unpleasant odour of meat derived from non-castrated male pigs, caused by the accumulation of androstenone and skatole in fat. Skatole is a tryptophan metabolite produced by intestinal bacteria in gut and catabolised in liver. Since boar taint affects consumer’s preference, the aim of this study was to perform transcriptome profiling in liver of boars with divergent skatole levels in backfat by using RNA-Seq. The total number of reads produced for each liver sample ranged from 11.8 to 39.0 million. Approximately 448 genes were differentially regulated (p-adjusted <0.05). Among them, 383 genes were up-regulated in higher skatole group and 65 were down-regulated (p<0.01, FC>1.5). Differentially regulated genes in the high skatole liver samples were enriched in metabolic processes such as small molecule biochemistry, protein synthesis, lipid and amino acid metabolism. Pathway analysis identified the remodeling of epithelial adherens junction and TCA cycle as the most dominant pathways which may play important roles in skatole metabolism. Differential gene expression analysis identified candidate genes in ATP synthesis, cytochrome P450, keratin, phosphoglucomutase, isocitrate dehydrogenase and solute carrier family. Additionally, polymorphism and association analysis revealed that mutations in ATP5B, KRT8, PGM1, SLC22A7 and IDH1 genes could be potential markers for skatole levels in boars. Furthermore, expression analysis of exon usage of three genes (ATP5B, KRT8 and PGM1) revealed significant differential expression of exons of these genes in different skatole levels. These polymorphisms and exon expression differences may have impacts on the gene activity ultimately leading to skatole variation and could be used as genetic marker for boar taint related traits. However, further validation is required to confirm the effect of these genetic markers in other pig populations in order to be used in genomic selection against boar taint in pig breeding programs. [ABSTRACT FROM AUTHOR]

Boar taint is an unpleasant smell and taste of pork meat derived from some entire male pigs. The main causes of boar taint are the two compounds androstenone (5α-androst-16-en-3-one) and skatole (3-methylindole). It is crucial to understand the genetic mechanism of boar taint to select pigs for lower androstenone levels and thus reduce boar taint. The aim of the present study was to investigate transcriptome differences in boar testis and liver tissues with divergent androstenone levels using RNA deep sequencing (RNA-Seq). The total number of reads produced for each testis and liver sample ranged from 13,221,550 to 33,206,723 and 12,755,487 to 46,050,468, respectively. In testis samples 46 genes were differentially regulated whereas 25 genes showed differential expression in the liver. The fold change values ranged from −4.68 to 2.90 in testis samples and −2.86 to 3.89 in liver samples. Differentially regulated genes in high androstenone testis and liver samples were enriched in metabolic processes such as lipid metabolism, small molecule biochemistry and molecular transport. This study provides evidence for transcriptome profile and gene polymorphisms of boars with divergent androstenone level using RNA-Seq technology. Digital gene expression analysis identified candidate genes in flavin monooxygenease family, cytochrome P450 family and hydroxysteroid dehydrogenase family. Moreover, polymorphism and association analysis revealed mutation in IRG6, MX1, IFIT2, CYP7A1, FMO5 and KRT18 genes could be potential candidate markers for androstenone levels in boars. Further studies are required for proving the role of candidate genes to be used in genomic selection against boar taint in pig breeding programs. [ABSTRACT FROM AUTHOR]

Background: As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs. Results: The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study. Conclusion: There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes. [ABSTRACT FROM AUTHOR]

Background: Leg weakness issues are a great concern for the pig breeding industry, especially with regard to animal welfare. Traits associated with leg weakness are partly influenced by the genetic background of the animals but the genetic basis of these traits is not yet fully understood. The aim of this study was to identify quantitative trait loci (QTL) affecting leg weakness in pigs. Methods: Three hundred and ten F2 pigs from a Duroc × Pietrain resource population were genotyped using 82 genetic markers. Front and rear legs and feet scores were based on the standard scoring system. Osteochondrosis lesions were examined histologically at the head and the condylus medialis of the left femur and humerus. Bone mineral density, bone mineral content and bone mineral area were measured in the whole ulna and radius bones using dual energy X-ray absorptiometry. A line-cross model was applied to determine QTL regions associated with leg weakness using the QTL Express software. Results: Eleven QTL affecting leg weakness were identified on eight autosomes. All QTL reached the 5% chromosome-wide significance level. Three QTL were associated with osteochondrosis on the humerus end, two with the fore feet score and two with the rear leg score. QTL on SSC2 and SSC3 influencing bone mineral content and bone mineral density, respectively, reached the 5% genome-wide significance level. Conclusions: Our results confirm previous studies and provide information on new QTL associated with leg weakness in pigs. These results contribute towards a better understanding of the genetic background of leg weakness in pigs. [ABSTRACT FROM AUTHOR]

Background: Serum lipids are associated with many serious cardiovascular diseases and obesity problems. Many quantitative trait loci (QTL) have been reported in the pig mostly for performance traits but very few for the serum lipid traits. In contrast, remarkable numbers of QTL are mapped for serum lipids in humans and mice. Therefore, the objective of this research was to investigate the chromosomal regions influencing the serum level of the total cholesterol (CT), triglyceride (TG), high density protein cholesterol (HDL) and low density protein cholesterol (LDL) in pigs. For this purpose, a total of 330 animals from a Duroc × Pietrain F2 resource population were phenotyped for serum lipids using ELISA and were genotyped by using 122 microsatellite markers covering all porcine autosomes for QTL study in QTL Express. Blood sampling was performed at approximately 175 days before slaughter of the pig. Results: Most of the traits were correlated with each other and were influenced by average daily gain, slaughter date and age. A total of 18 QTL including three QTL with imprinting effect were identified on 11 different porcine autosomes. Most of the QTL reached to 5% chromosome-wide (CW) level significance including a QTL at 5% experiment-wide (GW) and a QTL at 1% GW level significance. Of these QTL four were identified for both the CT and LDL and two QTL were identified for both the TG and LDL. Moreover, three chromosomal regions were detected for the HDL/LDL ratio in this study. One QTL for HDL on SSC2 and two QTL for TG on SSC11 and 17 were detected with imprinting effect. The highly significant QTL (1% GW) was detected for LDL at 82 cM on SSC1, whereas significant QTL (5% GW) was identified for HDL/LDL on SSC1 at 87 cM. Chromosomal regions with pleiotropic effects were detected for correlated traits on SSC1, 7 and 12. Most of the QTL identified for serum lipid traits correspond with the previously reported QTL for similar traits in other mammals. Two novel QTL on SSC16 for HDL and HDL/LDL ratio and an imprinted QTL on SSS17 for TG were detected in the pig for the first time. Conclusion: The newly identified QTL are potentially involved in lipid metabolism. The results of this work shed new light on the genetic background of serum lipid concentrations and these findings will be helpful to identify candidate genes in these QTL regions related to lipid metabolism and serum lipid concentrations in pigs. [ABSTRACT FROM AUTHOR]

The aim of the present study was to detect quantitative trait loci (QTL) for innate and adaptive immunity in pigs. For this purpose, a Duroc × Pietrain F resource population (DUPI) with 319 offspring was used to map QTL for the immune traits blood antibodies and interferon-gamma using 122 microsatellites covering all autosomes. Antibodies response to Mycoplasma hyopneumoniae and tetanus toxoid vaccine and the interferon-gamma (IFNG) serum concentration were measured at three different time points and were used as phenotypes. The differences of antibodies and interferon concentration between different time points were also used for the linkage mapping. Line-cross and imprinting QTL analysis, including two-QTL, were performed using QTL Express. A total of 30 QTL (12, 6, and 12 for mycoplasma, tetanus antibody, and IFNG, respectively) were identified at the 5% chromosome-wide-level significant, of which 28 were detected by line-cross and 2 by imprinting model. In addition, two QTL were identified on chromosome 5 using the two-QTL approach where both loci were in repulsion phase. Most QTL were detected on pig chromosomes 2, 5, 11, and 18. Antibodies were increased over time and immune traits were found to be affected by sex, litter size, parity, and month of birth. The results demonstrated that antibody and IFNG concentration are influenced by multiple chromosomal areas. The flanking markers of the QTL identified for IFNG on SSC5 did incorporate the position of the porcine IFNG gene. The detected QTL will allow further research in these QTL regions for candidate genes and their utilization in selection to improve the immune response and disease resistance in pig. [ABSTRACT FROM AUTHOR]

Simple Summary: Epigenetic modifications of the genes regulate the inflammation process that includes the DNA methylation and histone acetylation. Sulforaphane is well known for its immunomodulatory properties. Notably, the mechanism of its anti-inflammatory functions involving epigenetic modifications is unclear. This study highlighted the regulatory mechanism of sulforaphane in the innate immunity responses in an acute inflammatory state employ in vivo cell culture model. Porcine monocyte-derived dendritic cells were exposed to LPS with or without sulforaphane pre-treatment for these purposes. Epigenetics modulations of the important genes and regulatory factors were studies as well as the immune responses of the cells were vigorously studied over the period of time. This study deciphers the mechanism of SFN in restricting the excessive inflammatory reactions, thereby, exerting its protective and anti-inflammatory function though epigenetic mechanism. Inflammation is regulated by epigenetic modifications, including DNA methylation and histone acetylation. Sulforaphane (SFN), a histone deacetylase (HDAC) inhibitor, is also a potent immunomodulatory agent, but its anti-inflammatory functions through epigenetic modifications remain unclear. Therefore, this study aimed to investigate the epigenetic effects of SFN in maintaining the immunomodulatory homeostasis of innate immunity during acute inflammation. For this purpose, SFN-induced epigenetic changes and expression levels of immune-related genes in response to lipopolysaccharide (LPS) stimulation of monocyte-derived dendritic cells (moDCs) were analyzed. These results demonstrated that SFN inhibited HDAC activity and caused histone H3 and H4 acetylation. SFN treatment also induced DNA demethylation in the promoter region of the MHC-SLA1 gene, resulting in the upregulation of Toll-like receptor 4 (TLR4), MHC-SLA1, and inflammatory cytokines' expression at 6 h of LPS stimulation. Moreover, the protein levels of cytokines in the cell culture supernatants were significantly inhibited by SFN pre-treatment followed by LPS stimulation in a time-dependent manner, suggesting that inhibition of HDAC activity and DNA methylation by SFN may restrict the excessive inflammatory cytokine availability in the extracellular environment. We postulate that SFN may exert a protective and anti-inflammatory function by epigenetically influencing signaling pathways in experimental conditions employing porcine moDCs. [ABSTRACT FROM AUTHOR]

Simple Summary: Disease resistance is the ability of animals to inhibit the growth of invading pathogens within the body, which is influenced by the interaction of the host immune system, host genetics, and the pathogens. Resistant animals can be produced by molecular breeding by introducing the genomic marker responsible for disease resistance or immunocompetence. Immunogenomics is an information science that enables the genome-scale investigation of host immune response to pathogenic infection thereby identification of the genomic marker for disease resistance. Once the genomic marker is determined, it could be implemented in producing disease resistance animals by applying the advanced reproductive biotechnology like genome editing. The technical ease and decreasing cost over time might enhance the application of genome editing techniques for producing disease resistance livestock. Disease occurrence adversely affects livestock production and animal welfare, and have an impact on both human health and public perception of food–animals production. Combined efforts from farmers, animal scientists, and veterinarians have been continuing to explore the effective disease control approaches for the production of safe animal-originated food. Implementing the immunogenomics, along with genome editing technology, has been considering as the key approach for safe food–animal production through the improvement of the host genetic resistance. Next-generation sequencing, as a cutting-edge technique, enables the production of high throughput transcriptomic and genomic profiles resulted from host-pathogen interactions. Immunogenomics combine the transcriptomic and genomic data that links to host resistance to disease, and predict the potential candidate genes and their genomic locations. Genome editing, which involves insertion, deletion, or modification of one or more genes in the DNA sequence, is advancing rapidly and may be poised to become a commercial reality faster than it has thought. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) [CRISPR/Cas9] system has recently emerged as a powerful tool for genome editing in agricultural food production including livestock disease management. CRISPR/Cas9 mediated insertion of NRAMP1 gene for producing tuberculosis resistant cattle, and deletion of CD163 gene for producing porcine reproductive and respiratory syndrome (PRRS) resistant pigs are two groundbreaking applications of genome editing in livestock. In this review, we have highlighted the technological advances of livestock immunogenomics and the principles and scopes of application of CRISPR/Cas9-mediated targeted genome editing in animal breeding for disease resistance. [ABSTRACT FROM AUTHOR]