L-Carnitine sustainably affects bioenergetic profile of bovine blastocysts and transcriptome profile of elongation-stage embryos.

L-Carnitine (LC) is known to play key roles in lipid metabolism and antioxidative activity, implicating enhanced cryotolerance of bovine blastocysts. However, sustainability of LC supplementation during culture period on preimplantation development beyond the blastocyst stage has not been investigated so far. Therefore, all embryos were cultured under fatty acid free conditions, one group with LC (LC-Embryos) and the control group without LC (Control) supplementation. Transfer to recipients was conducted on day 6. Elongation-stage embryos were recovered on day 14; metrics of embryo recollection, developmental rates to early elongation-stage as well as mean embryo length did not differ between the groups. Gene expression analysis via NGS revealed 341 genes to be differentially regulated between elongation-stage embryos derived from LC supplementation compared to controls. These played mainly a role in molecular functions and biological processes like oxidoreductase activity, ATP dependent activity, cellular stress and respiration. Pathways like oxidative phosphorylation and thermogenesis, ECM receptor signaling, PI3K-Akt and focal adhesion were affected by differentially regulated genes. Moreover, all DEGs located on the mitochondria were significantly downregulated in the LC-Embryos, being in line with lower mitochondrial copy number and mtDNA integrity compared to the control group. Finally, we uncovered alterations of the bioenergetic profile on day 7 as a consequence of LC supplementation for the first time, revealing significantly higher oxygen consumption rates, ATP linked respiration and spare capacity for LC-embryos. Conclusively, we uncovered direct effects of LC supplementation during the culture period on the bioenergetic profile going along with sustainable effects on mtDNA copy numbers and transcriptome profile of bovine day 14 embryos.

Sexual dimorphic miRNA-mediated response of bovine elongated embryos to the maternal microenvironment.

A skewed male-to-female ratio in cattle is believed to be due to the biased embryo losses during pregnancy. The changes in biochemical secretion such as miRNAs by the embryo due to altered maternal environment could cause a sex biased selective implantation resulting in a skewed male to female ratio at birth. Nevertheless, it is still not clear whether the male and female embryos could modify their miRNA expression patterns differently in response to altered physiological developmental conditions. Therefore, this study was focused on identifying sex specific miRNA expression patterns induced in the embryo during the elongation period in response to the maternal environment. For this, in vitro produced day female and male embryos were transferred to Holsteins Frisian cows and heifers. The elongated female and male embryos were then recovered at day 13 of the gestation period. Total RNA including the miRNAs was isolated from each group of elongated embryo samples were subjected to the next generation miRNA sequencing. Sequence alignment, identification and quantification of miRNAs were done using the miRDeep2 software package and differential miRNA expression analyses were performed using the edgeR bioconductor package. The recovery rate of viable elongating embryos at day 13 of the gestation period was 26.6%. In cows, 2.8 more viable elongating male embryos were recovered than female embryos, while in heifers the sex ratio of the recovered elongating embryos was close to one (1.05). The miRNA analysis showed that 254 miRNAs were detected in both male and female elongated embryos developed either in cows or heifers, of which 14 miRNAs including bta-miR-10b, bta-miR-148a, bta-miR-26a, and bta-miR-30d were highly expressed. Moreover, the expression level of 32 miRNAs including bta-let-7c, bta-let-7b, bta-let-7g, bta-let-7d and bta-let-7e was significantly different between the male and female embryos developed in cows, but the expression level of only 4 miRNAs (bta-miR-10, bta-mR-100, bta-miR-155 and bta-miR-6119-5p) was different between the male and female embryos that were developed in heifers. Furthermore, 19 miRNAs including those involved in cellular energy homeostasis pathways were differentially expressed between the male embryos developed in cows and heifers, but no significantly differentially expressed miRNAs were detected between the female embryos of cows and heifers. Thus, this study revealed that the sex ratio skewed towards males in embryos developed in cows was accompanied by increased embryonic sexual dimorphic miRNA expression divergence in embryos developed in cows compared to those developed in heifers. Moreover, male embryos are more sensitive to respond to the maternal reproductive microenvironment by modulating their miRNA expression.

The mitochondrial respiration signature of the bovine blastocyst reflects both environmental conditions of development as well as embryo quality.

The major limitation of the widespread use of IVP derived embryos is their consistent deficiencies in vitality when compared with their ex vivo derived counterparts. Although embryo metabolism is considered a useful metric of embryo quality, research connecting mitochondrial function with the developmental capacity of embryos is still lacking. Therefore, the aim of the present study was to analyse bovine embryo respiration signatures in relation to developmental capacity. This was achieved by taking advantage of two generally accepted metrics for developmental capacity: (I) environmental conditions during development (vivo vs. vitro) and (II) developmental kinetics (day 7 vs. day 8 blastocysts). Our study showed that the developmental environment affected total embryo oxygen consumption while different morphokinetics illustrating the embryo qualities correlate with maximal mitochondrial respiration, mitochondrial spare capacity, ATP-linked respiration as well as efficiency of ATP generation. This respiration fingerprint for high embryo quality is reflected by relatively lower lipid contents and relatively higher ROS contents. In summary, the results of the present study extend the existing knowledge on the relationship between bovine embryo quality and the signature of mitochondrial respiration by considering contrasting developmental environments as well as different embryo morphokinetics.

Multivariate genome-wide associations for immune traits in two maternal pig lines.

BACKGROUND: Immune traits are considered to serve as potential biomarkers for pig's health. Medium to high heritabilities have been observed for some of the immune traits suggesting genetic variability of these phenotypes. Consideration of previously established genetic correlations between immune traits can be used to identify pleiotropic genetic markers. Therefore, genome-wide association study (GWAS) approaches are required to explore the joint genetic foundation for health biomarkers. Usually, GWAS explores phenotypes in a univariate (uv), trait-by-trait manner. Besides two uv GWAS methods, four multivariate (mv) GWAS approaches were applied on combinations out of 22 immune traits for Landrace (LR) and Large White (LW) pig lines. RESULTS: In total 433 (LR: 351, LW: 82) associations were identified with the uv approach implemented in PLINK and a Bayesian linear regression uv approach (BIMBAM) software. Single Nucleotide Polymorphisms (SNPs) that were identified with both uv approaches (n = 32) were mostly associated with immune traits such as haptoglobin, red blood cell characteristics and cytokines, and were located in protein-coding genes. Mv GWAS approaches detected 647 associations for different mv immune trait combinations which were summarized to 133 Quantitative Trait Loci (QTL). SNPs for different trait combinations (n = 66) were detected with more than one mv method. Most of these SNPs are associated with red blood cell related immune trait combinations. Functional annotation of these QTL revealed 453 immune-relevant protein-coding genes. With uv methods shared markers were not observed between the breeds, whereas mv approaches were able to detect two conjoint SNPs for LR and LW. Due to unmapped positions for these markers, their functional annotation was not clarified. CONCLUSIONS: This study evaluated the joint genetic background of immune traits in LR and LW piglets through the application of various uv and mv GWAS approaches. In comparison to uv methods, mv methodologies identified more significant associations, which might reflect the pleiotropic background of the immune system more accurately. In genetic research of complex traits, the SNP effects are generally small. Furthermore, one genetic variant can affect several correlated immune traits at the same time, termed pleiotropy. As mv GWAS methods consider strong dependencies among traits, the power to detect SNPs can be boosted. Both methods revealed immune-relevant potential candidate genes. Our results indicate that one single test is not able to detect all the different types of genetic effects in the most powerful manner and therefore, the methods should be applied complementary.

Genetic parameters of immune traits for Landrace and Large White pig breeds.

Improving the immunocompetence towards pathogens represents a desirable objective of breeding strategies to increase resilience. However, the immune system is complex and the genetic foundation of the underlying components is not yet clarified. In the present study, we focused on 22 blood parameters of 1,144 Landrace (LR) and Large White (LW) piglets at the age of 6-7 weeks. The immune profiles covered immune cells, red blood cell characteristics and cytokines. Genetic parameters based on pedigree information along with possible environmental effects were estimated. Litter effects play an important role in the expression of immune parameters of their young progenies. Hence, litter impacts on the piglet's immune profile including the immune parameters of the dam itself were investigated by different models. To incorporate the complexity of the immune network, the data were further investigated with a principal component analysis. Immune traits showed low to high breed-specific heritabilities (h2 ). Strong positive rg were estimated among red blood cell characteristics (0.77-0.99) and among cytokines (0.48-0.99). Neutrophils and lymphocytes illustrated a high negative rg (-0.96 to -0.98). The litter impact on piglet's immunity was examined and strengthened already observed breed differences. In LR, h2 (0.22-0.15) and litter effect (c2 ) (0.52-0.44) for IFN-γ decreased after statistical consideration of maternal impact. In LW, a decrease in h2 (0.32-0.18) for IFN-γ and an increase in c2 (0.54-0.56) were observed. Here, sufficient correlations were detected within various immune traits and functional biological networks of principal components. Most immune traits are heritable and are promising to cover global breed-specific immunocompetence in pigs. The analysis of immune traits has to be extended in order to find an optimal range and to characterize relationships between immunity and performance to gain an improved immune system without accidental losses in productivity.

The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events.

At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions between varying technical settings as well as the time point of electroporation in a bovine zygote's cell cycle on developmental metrics and the frequency and type of editing events are largely unknown. The present study uncovers that increasing pulse lengths result in higher Full Edit rates, with Mosaicism in Full-Edit embryos being significantly affected by adjusting RNP-electroporation relative to zygote cell cycle. A considerable proportion of Full Edit embryos demonstrated loss-of-heterozygosity after RNP-electroporation prior to S-phase. Some of these loss-of-heterozygosity events are a consequence of chromosomal disruptions along large sections of the target chromosomes making it necessary to check for their presence prior use of this technique in animal breeding. One out of 2 of these loss-of-heterozygosity events, however, was not associated with loss of an entire chromosome or chromosomal sections. Whether analysed loss-of-heterozygosity in these cases, however, was a false negative result due to loss of PCR primer sequences after INDEL formation at the target side or indeed due to interhomolog recombination needs to be clarified in follow up studies since the latter would for sure offer attractive options for future breeding schedules.

Comparison of the choice of animals for re-sequencing in two maternal pig lines.

Next-generation sequencing is a promising approach for the detection of causal variants within previously identified quantitative trait loci. Because of the costs of re-sequencing experiments, this application is currently mainly restricted to subsets of animals from already genotyped populations. Imputation from a lower to a higher marker density could represent a useful complementary approach. An analysis of the literature shows that several strategies are available to select animals for re-sequencing. This study demonstrates an animal selection workflow under practical conditions. Our approach considers different data sources and limited resources such as budget and availability of sampling material. The workflow combines previously described approaches and makes use of genotype and pedigree information from a Landrace and Large White population. Genotypes were phased and haplotypes were accurately estimated with AlphaPhase. Then, AlphaSeqOpt was used to optimize selection of animals for re-sequencing, reflecting the existing diversity of haplotypes. AlphaSeqOpt and ENDOG were used to select individuals based on pedigree information and by taking into account key animals that represent the genetic diversity of the populations. After the best selection criteria were determined, a subset of 57 animals was selected for subsequent re-sequencing. In order to evaluate and assess the advantage of this procedure, imputation accuracy was assessed by setting a set of single nucleotide polymorphism (SNP) chip genotypes to missing. Accuracy values were compared to those of alternative selection scenarios and the results showed the clear benefits of a targeted selection within this practical-driven approach. Especially imputation of low-frequency markers benefits from the combined approach described here. Accuracy was increased by up to 12% compared to a randomized or exclusively haplotype-based selection of sequencing candidates.

Oregano essential oil showed limited effects on pigs' carcass quality and haematology whereas a transcriptome analysis revealed significant modulations in the jejunum and the ileum.

Pig production depends on a health and performance balance. An approach to improve intestinal health is the oregano essential oil (OEO) supplementation within a conventional diet. Intestinal integrity regulating effects, for example gene expression, of some feed ingredients are important key factors for that balance. We hypothesized that OEO affects the expression of genes associated with pigs' intestinal integrity. In four trials, a total of 86 pigs have been used. From weaning, the 'treated' group (n = 42) was additionally fed an oregano flavour additive [1500 mg/kg (7.5% pure OEO)] within the basal diet. The 'control' group (n = 44) was kept under identical environmental conditions, except the OEO. At age of 6 months, pigs were slaughtered with an average weight of 111.1 ± 10.9 kg. In addition to automatically generated 'Fat-o-Meter' (AutoFOM) data, carcass quality factors have been measured manually. Valuable cuts of meat, such as ham and belly, were significantly reduced in the OEO group. Effects of OEO on pigs' haematologic parameters were very limited. For transcriptome analysis, the most interesting microarray expression results have been listed in a table (topTable). Selected genes were technically validated by qPCR. As a result, few significant differences in animal development and meat quality have been found between the OEO treated and the control group. Depending on OEO supplementation, we found 93 differently regulated genes in the jejunal tissue (70 up, 23 down) and 60 in the ileal tissue (48 up, 12 down). Just three genes (GRIN3B [glutamate ionotropic receptor NMDA type subunit 3B], TJP1/ZO-1 [tight junction protein ZO-1] and one uncharacterized gene) were affected by OEO both in jejunum and ileum. qPCR validation revealed AKT serine/threonine kinase 3 (AKT3), Interferon (IFN) -ε, -ω, tight junction protein (TJP1)/ZO-1 (ZO-1) to be upregulated in the jejunum and C-C motif chemokine ligand 21 (CCL21) was upregulated in the ileum of pigs that were supplemented with OEO. OEO supplementation had limited effects on pigs' performance traits. However, we were able to demonstrate that OEO alters the expression of genes associated with adaptive immune response in pigs' small intestine. These findings help to explain OEOs' beneficial impact on pigs' intestinal integrity.

Genome-wide associations for immune traits in two maternal pig lines.

BACKGROUND: In recent years, animal welfare and health has become more and more important in pig breeding. So far, numerous parameters have been considered as important biomarkers, especially in the immune reaction and inflammation. Previous studies have shown moderate to high heritabilities in most of these traits. However, the genetic background of health and robustness of pigs needs to be extensively clarified. The objective of this study was to identify genomic regions with a biological relevance for the immunocompetence of piglets. Genome-wide Association Studies (GWAS) in 535 Landrace (LR) and 461 Large White (LW) piglets were performed, investigating 20 immune relevant traits. Besides the health indicators of the complete and differential blood count, eight different cytokines and haptoglobin were recorded in all piglets and their biological dams to capture mediating processes and acute phase reactions. Additionally, all animals were genotyped using the Illumina PorcineSNP60v2 BeadChip. RESULTS: In summary, GWAS detected 25 genome-wide and 452 chromosome-wide significant SNPs associated with 17 immune relevant traits in the two maternal pig lines LR and LW. Only small differences were observed considering the maternal immune records as covariate within the statistical model. Furthermore, the study identified across- and within-breed differences as well as relevant candidate genes. In LR more significant associations and related candidate genes were detected, compared with LW. The results detected in LR and LW are partly in accordance with previously identified quantitative trait loci (QTL) regions. In addition, promising novel genomic regions were identified which might be of interest for further detailed analysis. Especially putative pleiotropic regions on SSC5, SSC12, SSC15, SSC16 and SSC17 are of major interest with regard to the interacting structure of the immune system. The comparison with already identified QTL gives indications on interactions with traits affecting piglet survival and also production traits. CONCLUSION: In conclusion, results suggest a polygenic and breed-specific background of immune relevant traits. The current study provides knowledge about regions with biological relevance for health and immune traits. Identified markers and putative pleiotropic regions provide first indications in the context of balancing a breeding-based modification of the porcine immune system.

Endocrine Fertility Parameters-Genomic Background and their Genetic Relationship to Boar Taint in German Landrace and Large White.

The surgical castration of young male piglets without anesthesia is no longer allowed in Germany from 2021. One alternative is breeding against boar taint, but shared synthesis pathways of androstenone (AND) and several endocrine fertility parameters (EFP) indicate a risk of decreasing fertility. The objective of this study was to investigate the genetic background between AND, skatole (SKA), and six EFP in purebred Landrace (LR) and Large White (LW) populations. The animals were clustered according to their genetic relatedness because of their different origins. Estimated heritabilities (h2) of AND and SKA ranged between 0.52 and 0.34 in LR and LW. For EFP, h2 differed between the breeds except for follicle-stimulating hormone (FSH) (h2: 0.28-0.37). Both of the breeds showed unfavorable relationships between AND and testosterone, 17-ß estradiol, and FSH. The genetic relationships (rg) between SKA and EFP differed between the breeds. A genome-wide association analysis revealed 48 significant associations and confirmed a region for SKA on S us S crofa chromosome (SSC) 14. For EFP, the results differed between the clusters. In conclusion, rg partly confirmed physiologically expected antagonisms between AND and EFP. Particular attention should be spent on fertility traits that are based on EFP when breeding against boar taint to balance the genetic progress in both of the trait complexes.

Genomic background and genetic relationships between boar taint and fertility traits in German Landrace and Large White.

BACKGROUND: Due to ethical reasons, surgical castration of young male piglets in their first week of life without anesthesia will be banned in Germany from 2021. Breeding against boar taint is already implemented in sire breeds of breeding organizations but in recent years a low demand made this trait economically less important. The objective of this study was to estimate heritabilities and genetic relationships between boar taint compounds androstenone and skatole and maternal/paternal reproduction traits in 4'924 Landrace (LR) and 4'299 Large White (LW) animals from nucleus populations. Additionally, genome wide association analysis (GWAS) was performed per trait and breed to detect SNP marker with possible pleiotropic effects that are associated with boar taint and fertility. RESULTS: Estimated heritabilities (h2) were 0.48 (±0.08) for LR (0.39 ± 0.07 for LW) for androstenone and 0.52 (±0.08) for LR (0.32 ± 0.07 for LW) for skatole. Heritabilities for reproduction did not differ between breeds except age at first insemination (LR: h2 = 0.27 (±0.05), LW: h2 = 0.34 (±0.05)). Estimates of genetic correlation (rg) between boar taint and fertility were different in LR and LW breeds. In LR an unfavorable rg of 0.31 (±0.15) was observed between androstenone and number of piglets born alive, whereas this rg in LW (- 0.15 (±0.16)) had an opposite sign. A similar breed-specific difference is observed between skatole and sperm count. Within LR, the rg of 0.08 (±0.13) indicates no relationship between the traits, whereas the rg of - 0.37 (±0.14) in LW points to an unfavorable relationship. In LR GWAS identified QTL regions on SSC5 (21.1-22.3 Mb) for androstenone and on SSC6 (5.5-7.5 Mb) and SSC14 (141.1-141.6 Mb) for skatole. For LW, one marker was found on SSC17 at 48.1 Mb for androstenone and one QTL on SSC14 between 140.5 Mb and 141.6 Mb for skatole. CONCLUSION: Knowledge about such genetic correlations could help to balance conventional breeding programs with boar taint in maternal breeds. QTL regions with unfavorable pleiotropic effects on boar taint and fertility could have deleterious consequences in genomic selection programs. Constraining the weighting of these QTL in the genomic selection formulae may be a useful strategy to avoid physiological imbalances.

PBMCs transcriptome profiles identified breed-specific transcriptome signatures for PRRSV vaccination in German Landrace and Pietrain pigs.

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.

MicroRNAs: tiny molecules with a significant role in mammalian follicular and oocyte development.

The genetic regulation of female fertility (follicular development, oocyte maturation and early preimplantation embryo development) involves the spatio-temporal regulation of those genes that play key roles in various stages of the female reproductive axis. MicroRNAs (miRNAs), a class of small non-coding RNAs, are known to regulate the expression of a large proportion of such genes. In recent decades, multiple studies have aimed to determine the roles of these non-coding RNAs in mammalian follicular development, oocyte growth and embryo development. These studies have applied a variety of approaches, including conditional knockout of miRNA biogenesis genes, high-throughput sequencing technologies for pattern recognition in miRNA expression and loss- and gain-of-function of miRNAs in various animal models. In addition to the cellular miRNAs, a large variety of RNAs are found in circulation, being coupled with extracellular vesicles, proteins and lipids. Because of their potential as diagnostic markers for abnormal physiologies, there is increasing interest in the identification of extracellular miRNAs in various biological fluids and spent in vitro culture media. This review focuses on studies addressing the expression and potential role of cellular and extracellular miRNAs in mammalian follicular cell physiology and subsequent ovarian functionality and oocyte maturation.

Transcriptome profile of lung dendritic cells after in vitro porcine reproductive and respiratory syndrome virus (PRRSV) infection.

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-1ß1 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.

PBMC transcriptome profiles identifies potential candidate genes and functional networks controlling the innate and the adaptive immune response to PRRSV vaccine in Pietrain pig.

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.

LPS-induced expression of CD14 in the TRIF pathway is epigenetically regulated by sulforaphane in porcine pulmonary alveolar macrophages.

Pulmonary alveolar macrophages (AMs) are important in defense against bacterial lung inflammation. Cluster of differentiation 14 (CD14) is involved in recognizing bacterial lipopolysaccharide (LPS) through MyD88-dependent and TRIF pathways of innate immunity. Sulforaphane (SFN) shows anti-inflammatory activity and suppresses DNA methylation. To identify CD14 epigenetic changes by SFN in the LPS-induced TRIF pathway, an AMs model was investigated in vitro. CD14 gene expression was induced by 5 µg/ml LPS at the time point of 12 h and suppressed by 5 µM SFN. After 12 h of LPS stimulation, gene expression was significantly up-regulated, including TRIF, TRAF6, NF-κB, TRAF3, IRF7, TNF-α, IL-1ß, IL-6, and IFN-ß. LPS-induced TRAM, TRIF, RIPK1, TRAF3, TNF-α, IL-1ß and IFN-ß were suppressed by 5 µM SFN. Similarly, DNMT3a expression was increased by LPS but significantly down-regulated by 5 µM SFN. It showed positive correlation of CD14 gene body methylation with in LPS-stimulated AMs, and this methylation status was inhibited by SFN. This study suggests that SFN suppresses CD14 activation in bacterial inflammation through epigenetic regulation of CD14 gene body methylation associated with DNMT3a. The results provide insights into SFN-mediated epigenetic down-regulation of CD14 in LPS-induced TRIF pathway inflammation and may lead to new methods for controlling LPS-induced inflammation in pigs.

Integrative Analysis of Metabolomic, Proteomic and Genomic Data to Reveal Functional Pathways and Candidate Genes for Drip Loss in Pigs.

The aim of this study was to integrate multi omics data to characterize underlying functional pathways and candidate genes for drip loss in pigs. The consideration of different omics levels allows elucidating the black box of phenotype expression. Metabolite and protein profiling was applied in Musculus longissimus dorsi samples of 97 Duroc × Pietrain pigs. In total, 126 and 35 annotated metabolites and proteins were quantified, respectively. In addition, all animals were genotyped with the porcine 60 k Illumina beadchip. An enrichment analysis resulted in 10 pathways, amongst others, sphingolipid metabolism and glycolysis/gluconeogenesis, with significant influence on drip loss. Drip loss and 22 metabolic components were analyzed as intermediate phenotypes within a genome-wide association study (GWAS). We detected significantly associated genetic markers and candidate genes for drip loss and for most of the metabolic components. On chromosome 18, a region with promising candidate genes was identified based on SNPs associated with drip loss, the protein "phosphoglycerate mutase 2" and the metabolite glycine. We hypothesize that association studies based on intermediate phenotypes are able to provide comprehensive insights in the genetic variation of genes directly involved in the metabolism of performance traits. In this way, the analyses contribute to identify reliable candidate genes.

Deciphering transcriptome profiles of peripheral blood mononuclear cells in response to PRRSV vaccination in pigs.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important viral diseases affecting swine industry worldwide. Despite routine farm vaccination, effective control strategies for PRRS remained elusive which underscores the need for in-depth studies to gain insight into the host immune response to vaccines. The current study aimed to investigate transcriptional responses to PRRS Virus (PRRSV) vaccine in the peripheral blood mononuclear cells (PBMCs) within 3 days following vaccination in German Landrace pigs. RESULTS: Transcriptome profiling of PBMCs from PRRSV vaccinated and age-matched unvaccinated pigs at right before (0 h), and at 6, 24 and 72 h after PRRSV vaccination was performed using the Affymetrix gene chip porcine gene 1.0 st array. Comparison of PBMCs transcriptome profiles between vaccinated and unvaccinated pigs revealed a distinct host innate immune transcriptional response to PRRSV vaccine. There was a significant temporal variation in transcriptional responses of PRRSV vaccine in PBMCs accounting 542, 2,263 and 357 differentially expressed genes (DEGs) at 6, 24 and 72 h post vaccination, respectively compared to the time point before vaccination (controls). Gene ontology analysis revealed the involvement of these DEGs in various biological process including innate immune response, signal transduction, positive regulation of MAP kinase activity, TRIF-dependent toll-like receptor signaling pathway, T cell differentiation and apoptosis. Immune response specific pathways such as cytokine-cytokine receptor interaction, chemokine signaling pathway, signal transduction, JAK-STAT pathway and regulation, TRAF6 mediated induction of NF-kB and MAPK, the NLRP3 inflammasome, endocytosis and interferon signaling were under regulation during the early stage of PRRSV vaccination. Network enrichment analysis revealed APP, TRAF6, PIN1, FOS, CTNNB1, TNFAIP3, TIP1, CDKN1, SIRT1, ESR1 and HDAC5 as the highly interconnected hubs of the functional network of PRRSV vaccine induced transcriptome changes in PBMCs. CONCLUSIONS: This study showed that a massive gene expression change occurred in PBMCs following PRRSV vaccination in German Landrace pigs. Within first 3 days of vaccine exposure, the highest transcript abundance was observed at 24 h after vaccination compared to that of control. Results of this study suggest that APP, TRAF6, PIN1, FOS, CDKN1A and TNFAIP3 could be considered as potential candidate genes for PRRSV vaccine responsiveness.

Different Statistical Approaches to Investigate Porcine Muscle Metabolome Profiles to Highlight New Biomarkers for Pork Quality Assessment.

The aim of this study was to elucidate the underlying biochemical processes to identify potential key molecules of meat quality traits drip loss, pH of meat 1 h post-mortem (pH1), pH in meat 24 h post-mortem (pH24) and meat color. An untargeted metabolomics approach detected the profiles of 393 annotated and 1,600 unknown metabolites in 97 Duroc × Pietrain pigs. Despite obvious differences regarding the statistical approaches, the four applied methods, namely correlation analysis, principal component analysis, weighted network analysis (WNA) and random forest regression (RFR), revealed mainly concordant results. Our findings lead to the conclusion that meat quality traits pH1, pH24 and color are strongly influenced by processes of post-mortem energy metabolism like glycolysis and pentose phosphate pathway, whereas drip loss is significantly associated with metabolites of lipid metabolism. In case of drip loss, RFR was the most suitable method to identify reliable biomarkers and to predict the phenotype based on metabolites. On the other hand, WNA provides the best parameters to investigate the metabolite interactions and to clarify the complex molecular background of meat quality traits. In summary, it was possible to attain findings on the interaction of meat quality traits and their underlying biochemical processes. The detected key metabolites might be better indicators of meat quality especially of drip loss than the measured phenotype itself and potentially might be used as bio indicators.