PBMC Transcription Profiles of Pigs with Divergent Humoral Immune Responses and Lean Growth Performance

licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2013.05.27; Accepted: 2013.08.20; Published: 2013.09.20 Background: The identification of key genes and regulatory networks in the transcriptomic responses of blood cells to antigen stimulation could facilitate the understanding of host defence and disease resistance. Moreover, genetic relationships between immunocompetence and the expression of other phenotypes, such as those of metabolic interest, are debated but incompletely understood in farm animals. Both positive and negative associations between immune responsiveness and performance traits such as weight gain or lean growth have been reported. We designed an in vivo microarray study of transcriptional changes in porcine peripheral blood mononuclear cells (PBMCs) during the immune response to tetanus toxoid (TT) as a model antigen for combined cellular (Th1) and humoral (Th2) responses. The aim of the study was to investigate the responsiveness of PBMCs against the background of divergent lean growth (LG) performance and anti-TT antibody (AB) titers and to compare lean growth and humoral immune performance phenotypes

A High Protein Diet during Pregnancy Affects Hepatic Gene Expression of Energy Sensing Pathways along Ontogenesis in a Porcine Model

In rodent models and in humans the impact of gestational diets on the offspring's phenotype was shown experimentally and epidemiologically. The underlying programming of fetal development was shown to be associated with an increased risk of degenerative diseases in adulthood, including the metabolic syndrome. There are clues that diet-dependent modifications of the metabolism during fetal life can persist until adulthood. This leads to the hypothesis that the offspring's transcriptomes show short-term and long-term changes depending on the maternal diet. To this end pregnant German landrace gilts were fed either a high protein diet (HP, 30% CP) or an adequate protein diet (AP, 12% CP) throughout pregnancy. Hepatic transcriptome profiles of the offspring were analyzed at prenatal (94 dpc) and postnatal stages (1, 28, 188 dpn). Depending on the gestational dietary exposure, mRNA expression levels of genes related to energy metabolism, N-metabolism, growth factor signaling pathways, lipid metabolism, nucleic acid metabolism and stress/immune response were affected either in a short-term or in a long-term manner. Gene expression profiles at fetal stage 94 dpc were almost unchanged between the diets. The gestational HP diet affected the hepatic expression profiles at prenatal and postnatal stages. The effects encompassed a modulation of the genome in terms of an altered responsiveness of energy and nutrient sensing pathways. Differential expression of genes related to energy production and nutrient utilization contribute to the maintenance of development and growth performance within physiological norms, however the modulation of these pathways may be accompanied by a predisposition for metabolic disturbances up to adult stages

A low protein diet during pregnancy provokes a lasting shift of hepatic expression of genes related to cell cycle throughout ontogenesis in a porcine model

<p>Abstract</p> <p>Background</p> <p>In rodent models and in humans the impact of gestational diets on the offspring's phenotype was shown experimentally and epidemiologically. Adverse environmental conditions during fetal development provoke an intrauterine adaptive response termed 'fetal programming', which may lead to both persistently biased responsiveness to extrinsic factors and permanent consequences for the organismal phenotype. This leads to the hypothesis that the offspring's transcriptome exhibits short-term and long-term changes, depending on the maternal diet. In order to contribute to a comprehensive inventory of genes and functional networks that are targets of nutritional programming initiated during fetal life, we applied whole-genome microarrays for expression profiling in a longitudinal experimental design covering prenatal, perinatal, juvenile, and adult ontogenetic stages in a porcine model. Pregnant sows were fed either a gestational low protein diet (LP, 6% CP) or an adequate protein diet (AP, 12% CP). All offspring was nursed by foster sows receiving standard diets. After weaning, all offspring was fed standard diets <it>ad libitum</it>.</p> <p>Results</p> <p>Analyses of the hepatic gene expression of the offspring at prenatal (94 <it>dies post conceptionem</it>, dpc) and postnatal stages (1, 28, 188 <it>dies post natum</it>, dpn) included comparisons between dietary groups within stages as well as comparisons between ontogenetic stages within diets to separate diet-specific transcriptional changes and maturation processes. We observed differential expression of genes related to lipid metabolism (e.g. Fatty acid metabolism, Biosynthesis of steroids, Synthesis and degradation of ketone bodies, FA elongation in mitochondria, Bile acid synthesis) and cell cycle regulation (e.g. Mitotic roles of PLK, G1/S checkpoint regulation, G2/M DNA damage checkpoint regulation). Notably, at stage 1 dpn no regulation of a distinct pathway was found in LP offspring.</p> <p>Conclusions</p> <p>The transcriptomic modulations point to persistent functional demand on the liver towards cell proliferation in the LP group but not in the AP group at identical nutritional conditions during postnatal life due to divergent 'programming' of the genome. Together with the observation that the offspring of both groups did not differ in body weight but in body composition and fat content, the data indicate that the activity of various genes led to diverse partitioning of nutrients among peripheral and visceral organs and tissues.</p

QTL for microstructural and biophysical muscle properties and body composition in pigs

BACKGROUND: The proportion of muscle fibre types and their size affect muscularity as well as functional properties of the musculature and meat quality. We aimed to identify QTL for microstructural muscle properties including muscle fibre size, their numbers and fibre type proportions as well as biophysical parameters of meat quality and traits related to body composition, i.e. pH, conductivity, area of M. longissimus dorsi and lean meat content. A QTL scan was conducted in a porcine experimental population that is based on Duroc and Berlin Miniature Pig. RESULTS: Least square regression interval mapping revealed five significant and 42 suggestive QTL for traits related to muscle fibre composition under the line-cross model as well as eight significant and 40 suggestive QTL under the half-sib model. For traits related to body composition and biophysical parameters of meat quality five and twelve significant plus nine and 22 suggestive QTL were found under the line-cross and half-sib model, respectively. Regions with either significant QTL for muscle fibre traits or significant QTL for meat quality and muscularity or both were detected on SSC1, 2, 3, 4, 5, 13, 14, 15, and 16. QTL for microstructural properties explained a larger proportion of variance than did QTL for meat quality and body composition. CONCLUSION: Microstructural properties of pig muscle and meat quality are governed by genetic variation at many loci distributed throughout the genome. QTL analysis under both, the line-cross and half-sib model, allows detecting QTL in case of fixation or segregation of the QTL alleles among the founder populations and thus provide comprehensive insight into the genetic variation of the traits under investigation. Genomic regions affecting complex traits of muscularity and meat quality as well as microstructural properties might point to QTL that in first instance affect muscle fibre traits and by this in second instance meat quality. Disentangling complex traits in their constituent phenotypes might facilitate the identification of QTL and the elucidation of the pleiotropic nature of QTL effects

Trait correlated expression combined with expression QTL analysis reveals biological pathways and candidate genes affecting water holding capacity of muscle

<p>Abstract</p> <p>Background</p> <p>Leakage of water and ions and soluble proteins from muscle cells occurs during prolonged exercise due to ischemia causing muscle damage. Also <it>post mortem </it>anoxia during conversion of muscle to meat is marked by loss of water and soluble components from the muscle cell. There is considerable variation in the water holding capacity of meat affecting economy of meat production. Water holding capacity depends on numerous genetic and environmental factors relevant to structural and biochemical muscle fibre properties a well as <it>ante </it>and <it>post </it>slaughter metabolic processes.</p> <p>Results</p> <p>Expression microarray analysis of M. <it>longissimus dorsi </it>RNAs of 74 F2 animals of a resource population showed 1,279 transcripts with trait correlated expression to water holding capacity. Negatively correlated transcripts were enriched in functional categories and pathways like extracellular matrix receptor interaction and calcium signalling. Transcripts with positive correlation dominantly represented biochemical processes including oxidative phosphorylation, mitochondrial pathways, as well as transporter activity. A linkage analysis of abundance of trait correlated transcripts revealed 897 expression QTL (eQTL) with 104 eQTL coinciding with QTL regions for water holding capacity; 96 transcripts had <it>trans </it>acting and 8 had <it>cis </it>acting regulation.</p> <p>Conclusion</p> <p>The complex relationships between biological processes taking place in live skeletal muscle and meat quality are driven on the one hand by the energy reserves and their utilisation in the muscle and on the other hand by the muscle structure itself and calcium signalling. Holistic expression profiling was integrated with QTL analysis for the trait of interest and for gene expression levels for creation of a priority list of genes out of the orchestra of genes of biological networks relevant to the liability to develop elevated drip loss.</p

Genetic Regulation of Liver Metabolites and Transcripts Linking to Biochemical-Clinical Parameters

Given the central metabolic role of the liver, hepatic metabolites and transcripts reflect the organismal physiological state. Biochemical-clinical plasma biomarkers, hepatic metabolites, transcripts, and single nucleotide polymorphism (SNP) genotypes of some 300 pigs were integrated by weighted correlation networks and genome-wide association analyses. Network-based approaches of transcriptomic and metabolomics data revealed linked of transcripts and metabolites of the pentose phosphate pathway (PPP). This finding was evidenced by using a NADP/NADPH assay and HDAC4 and G6PD transcript quantification with the latter coding for first limiting enzyme of this pathway and by RNAi knockdown experiments of HDAC4. Other transcripts including ARG2 and SLC22A7 showed link to amino acids and biomarkers. The amino acid metabolites were linked with transcripts of immune or acute phase response signaling, whereas the carbohydrate metabolites were highly enrich in cholesterol biosynthesis transcripts. Genome-wide association analyses revealed 180 metabolic quantitative trait loci (mQTL) (p &lt; 10-4). Trans-4-hydroxy-L-proline (p = 6 × 10-9), being strongly correlated with plasma creatinine (CREA), showed strongest association with SNPs on chromosome 6 that had pleiotropic effects on PRODH2 expression as revealed by multivariate analysis. Consideration of shared marker association with biomarkers, metabolites, and transcripts revealed 144 SNPs associated with 44 metabolites and 69 transcripts that are correlated with each other, representing 176 mQTL and expression quantitative trait loci (eQTL). This is the first work to report genetic variants associated with liver metabolite and transcript levels as well as blood biochemical-clinical parameters in a healthy porcine model. The identified associations provide links between variation at the genome, transcriptome, and metabolome level molecules with clinically relevant phenotypes. This approach has the potential to detect novel biomarkers displaying individual variation and promoting predictive biology in medicine and animal breeding

The three-way relationship of polymorphisms of porcine genes encoding terminal complement components, their differential expression, and health-related phenotypes

<p>Abstract</p> <p>Background</p> <p>The complement system is an evolutionary ancient mechanism that plays an essential role in innate immunity and contributes to the acquired immune response. Three modes of activation, known as classical, alternative and lectin pathway, lead to the initiation of a common terminal lytic pathway. The terminal complement components (TCCs: C6, C7, C8A, C8B, and C9) are encoded by the genes <it>C6</it>, <it>C7</it>, <it>C8A</it>, <it>C8B</it>, <it>C8G</it>, and <it>C9</it>. We aimed at experimentally testing the porcine genes encoding TCCs as candidate genes for immune competence and disease resistance by addressing the three-way relationship of genotype, health related phenotype, and mRNA expression.</p> <p>Results</p> <p>Comparative sequencing of cDNAs of animals of the breeds German Landrace, Piétrain, Hampshire, Duroc, Vietnamese Potbelly Pig, and Berlin Miniature Pig (BMP) revealed 30 SNPs (21 in protein domains, 12 with AA exchange). The promoter regions (each ~1.5 kb upstream the transcription start sites) of <it>C6</it>, <it>C7</it>, <it>C8A</it>, <it>C8G</it>, and <it>C9</it> exhibited 29 SNPs. Significant effects of the TCC encoding genes on hemolytic complement activity were shown in a cross of Duroc and BMP after vaccination against Mycoplasma hyopneumoniae, Aujeszky disease virus and PRRSV by analysis of variance using repeated measures mixed models. Family based association tests (FBAT) confirmed the associations. The promoter SNPs were associated with the relative abundance of TCC transcripts obtained by real time RT-PCR of 311 liver samples of commercial slaughter pigs. Complement gene expression showed significant relationship with the prevalence of acute and chronic lung lesions.</p> <p>Conclusions</p> <p>The analyses point to considerable variation of the porcine TCC genes and promote the genes as candidate genes for disease resistance.</p

Breed, Diet, and Interaction Effects on Adipose Tissue Transcriptome in Iberian and Duroc Pigs Fed Different Energy Sources

In this study, we analyzed the effects of breed, diet energy source, and their interaction on adipose tissue transcriptome in growing Iberian and Duroc pigs. The study comprised 29 Iberian and 19 Duroc males, which were kept under identical management conditions except the nutritional treatment. Two isoenergetic diets were used with 6% high oleic sunflower oil (HO) or carbohydrates (CH) as energy sources. All animals were slaughtered after 47 days of treatment at an average live weight of 51.2 kg. Twelve animals from each breed (six fed each diet) were employed for ham subcutaneous adipose tissue RNA-Seq analysis. The data analysis was performed using two different bioinformatic pipelines. We detected 837 and 1456 differentially expressed genes (DEGs) according to breed, depending on the pipeline. Due to the strong effect of breed on transcriptome, the effect of the diet was separately evaluated in the two breeds. We identified 207 and 57 DEGs depending on diet in Iberian and Duroc pigs, respectively. A joint analysis of both effects allowed the detection of some breed–diet interactions on transcriptome, which were inferred from RNA-Seq and quantitative PCR data. The functional analysis showed the enrichment of functions related to growth and tissue development, inflammatory response, immune cell trafficking, and carbohydrate and lipid metabolism, and allowed the identification of potential regulators. The results indicate different effects of diet on adipose tissue gene expression between breeds, affecting relevant biological pathways

A Gestational High Protein Diet Affects the Abundance of Muscle Transcripts Related to Cell Cycle Regulation throughout Development in Porcine Progeny

BACKGROUND: In various animal models pregnancy diets have been shown to affect offspring phenotype. Indeed, the underlying programming of development is associated with modulations in birth weight, body composition, and continual diet-dependent modifications of offspring metabolism until adulthood, producing the hypothesis that the offspring's transcriptome is permanently altered depending on maternal diet. METHODOLOGY/PRINCIPAL FINDINGS: To assess alterations of the offspring's transcriptome due to gestational protein supply, German Landrace sows were fed isoenergetic diets containing protein levels of either 30% (high protein--HP) or 12% (adequate protein--AP) throughout their pregnancy. Offspring muscle tissue (M. longissimus dorsi) was collected at 94 days post conception (dpc), and 1, 28, and 188 days post natum (dpn) for use with Affymetrix GeneChip Porcine Genome Arrays and subsequent statistical and Ingenuity pathway analyses. Numerous transcripts were found to have altered abundance at 94 dpc and 1 dpn; at 28 dpn no transcripts were altered, and at 188 dpn only a few transcripts showed a different abundance between diet groups. However, when assessing transcriptional changes across developmental time points, marked differences were obvious among the dietary groups. Depending on the gestational dietary exposure, short- and long-term effects were observed for mRNA expression of genes related to cell cycle regulation, energy metabolism, growth factor signaling pathways, and nucleic acid metabolism. In particular, the abundance of transcripts related to cell cycle remained divergent among the groups during development. CONCLUSION: Expression analysis indicates that maternal protein supply induced programming of the offspring's genome; early postnatal compensation of the slight growth retardation obvious at birth in HP piglets resulted, as did a permanently different developmental alteration and responsiveness to the common environment of the transcriptome. The transcriptome modulations are interpreted as the molecular equivalent of developmental plasticity of the offspring that necessitates adaptation and maintenance of the organismal phenotype

Identification of expression QTL (eQTL) of genes expressed in porcine M. longissimus dorsi and associated with meat quality traits

<p>Abstract</p> <p>Background</p> <p>Genetic analysis of transcriptional profiles is a promising approach for identifying and dissecting the genetics of complex traits like meat performance. Accordingly, expression levels obtained by microarray analysis were taken as phenotypes in a linkage analysis to map eQTL. Moreover, expression levels were correlated with traits related to meat quality and principle components with high loadings of these traits. By using an up-to-date annotation and localization of the respective probe-sets, the integration of eQTL mapping data and information of trait correlated expression finally served to point to candidate genes for meat quality traits.</p> <p>Results</p> <p>Genome-wide transcriptional profiles of <it>M. longissimus dorsi </it>RNAs samples of 74 F2 animals of a pig resource population revealed 11,457 probe-sets representing genes expressed in the muscle. Linkage analysis of expression levels of these probe-sets provided 9,180 eQTL at the suggestive significance threshold of LOD > 2. We mapped 653 eQTL on the same chromosome as the corresponding gene and these were designated as 'putative <it>cis-</it>eQTL'. In order to link eQTL to the traits of interest, probe-sets were addressed with relative transcript abundances that showed correlation with meat quality traits at p ≤ 0.05. Out of the 653 'putative <it>cis-</it>eQTL', 262 transcripts were correlated with at least one meat quality trait. Furthermore, association of expression levels with composite traits with high loadings for meat quality traits generated by principle component analysis were taken into account leading to a list of 85 genes exhibiting <it>cis-</it>eQTL and trait dependent expression.</p> <p>Conclusion</p> <p>Holistic expression profiling was integrated with QTL analysis for meat quality traits. Correlations between transcript abundance and meat quality traits, combined with genetic positional information of eQTL allowed us to prioritise candidate genes for further study.</p