Porcine Genome Array Research Articles

Cryotop vitrification of large batches of pig embryos simultaneously provides excellent postwarming survival rates and minimal interference with gene expression

The most commonly used technique to vitrify pig embryos is the super open pulled straw (SOPS), where a maximum of 6 embryos can be vitrified simultaneously per device without compromising the minimum volume necessary for optimal preservation. Since optimal embryo transfer (ET) demands a transfer of 20–40 embryos per recipient, the customary use of SOPS complicates embryo warming and ET in field conditions. Such complications could be avoided when using the Cryotop® (OC) system, which has been proven to be an effective option for vitrifying at least 20 porcine embryos simultaneously. This study aimed to investigate the changes in the transcriptome of blastocysts caused by vitrification using both systems. In vivo-derived blastocysts were OC- (n = 60; 20 embryos/device) and SOPS- (n = 60; 4–6 embryos/device) vitrified and cultured for 24 h after warming. Nonvitrified blastocysts (n = 60) cultured for 24 h postcollection acted as controls. At the end of culture, 48 viable embryos from each group (6 pools of 8 embryos) were selected for microarray (GeneChip® Porcine Genome Array, P/N 900624, Affymetrix) analysis of differentially expressed genes (DEGs). The survival rate of embryos vitrified with the OC and SOPS systems (>97%) was similar to that of the control embryos (100%). Microarray analysis of each vitrification system compared to the control group showed 245 DEGs (89 downregulated and 156 upregulated) for the OC system and 210 (44 downregulated and 166 upregulated) for the SOPS system. Two pathways were enriched for the DEGs specifically altered in each vitrification system compared to the control (glycolysis/gluconeogenesis and carbon metabolism pathways for the OC system and amino sugar and nucleotide sugar metabolism and lysosome pathways in the SOPS group). The OC group showed 31 downregulated and 24 upregulated genes and two enriched pathways (mineral absorption and amino sugar and nucleotide sugar metabolism pathways) when compared to the SOPS group. In summary, vitrification with the OC system altered fewer genes related to apoptosis and activated genes related to cell proliferation. We conclude that vitrification with either the OC or SOPS system has a moderate to low effect on the transcriptome of in vivo-derived porcine blastocysts. Further investigation is needed to elucidate how the differences in the transcriptome of embryos vitrified with these systems affect their subsequent developmental ability after ET.

Altered mRNA Expression Due to Rectal Perforation in a Porcine Model - A Pilot Study.

Anastomotic leakage is the most serious and unwelcome complication in rectal surgery. It has a great impact on postoperative morbidity and mortality. In this pilot study, changes of mRNA expression in blood were analyzed in an animal model designed to imitate anastomotic leakage. Twelve pigs were randomized into two groups: A sham-operated control group and an experimental group in which iatrogenic rectal perforation was performed. The changes in the mRNA expression at 4 hours after creating the perforation were studied. Microarray analysis was performed using Gene Chip whole porcine genome array. mRNA expression of 19,124 genes was investigated. Significantly increased levels of genes with a fold change greater than 2 were found, including 276 coding for unknown proteins and 48 coding for known proteins. Eleven of those which coded for known proteins were up-regulated with a fold change >4. Eleven known genes were highly up-regulated after rectal perforation. These genes were mainly involved in inflammatory response, intracellular signaling and cell membrane regulation. Their corresponding proteins might potentially be clinical biomarkers of anastomotic leakage and should be evaluated in further clinical studies.

Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae.

Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70–75%), the pregnancy loss is 5–15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip® Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted <0.05 and a fold change cut-off of ±1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 ± 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.

Dose-Dependent Effect of Mesenchymal Stromal Cell Recruiting Chemokine CCL25 on Porcine Tissue-Engineered Healthy and Osteoarthritic Cartilage.

Thymus-expressed chemokine (CCL25) is a potent cell attractant for mesenchymal stromal cells, and therefore it is a candidate for in situ cartilage repair approaches focusing on the recruitment of endogenous repair cells. However, the influence of CCL25 on cartilage is unknown. Accordingly, in this study, we investigated the effect of CCL25 on tissue-engineered healthy and osteoarthritic cartilage. Porcine chondrocytes were cultured in a three-dimensional (3D) micromass model that has been proven to mimic key-aspects of human cartilage and osteoarthritic alterations upon stimulation with tumor necrosis factor-α (TNF-α). Micromass cultures were stimulated with CCL25 (0, 0.05, 0.5, 5, 50, 500 nmol/L) alone or in combination with 0.6 nmol/L TNF-α for seven days. Effects were evaluated by life/dead staining, safranin O staining, histomorphometrical analysis of glycosaminoglycans (GAGs), collagen type II (COL2A1) real-time RT-PCR and Porcine Genome Array analysis. 500 nmol/L CCL25 led to a significant reduction of GAGs and COL2A1 expression and induced the expression of matrix metallopeptidases (MMP) 1, MMP3, early growth response protein 1 (EGR1), and superoxide dismutase 2 (SOD2). In concentrations lower than 500 nmol/L, CCL25 seems to be a candidate for in situ cartilage repair therapy approaches.

Body composition, serum lipid levels, and transcriptomic characterization in the adipose tissue of male pigs in response to sex hormone deficiency

It is known that the male hypogonadism plays an important role in regulating adipose metabolism. In the present study, fifteen pairs of full male sibs were divided into a castrated group and an intact group with a paired experiment design. The pigs were slaughtered at an age of 175days. The carcass characteristics and fat deposit of the studied animal were measured, and the hormone and serum lipid levels of the peripheral blood samples were determined, and the differentially expressed genes of the back fat between the two groups were screened with porcine genome array. Our results showed that the absence of male gonadal steroids attributed to castration significantly raised the serum lipid levels and increased fat accumulation in the pigs. A total of 225 differentially expressed genes were identified between the boars and barrows and 135 of them were upregulated. The analysis of Gene Ontology categories and KEGG pathway indicated that these differentially expressed genes were mainly involved in metabolism of lipid, carbohydrate, amino acid, xenobiotics biodegradation, and immune diseases pathways. Our results indicated that there were higher capacity of fatty acid of synthesis, enhanced uptaking capacity of fatty acids and cholesterol, inhibited lipolysis, and enhanced carbohydrate metabolism in the adipose tissue of barrows compared to boars. The findings of the present study provide new insight into the mechanisms of adipose metabolism induced by hormone deficiency.

Abstract 11685: Early Myocardial Gene Expression Profiling After Deep Hypothermic Circulatory Arrest Reveals Ontogenetic Adaptive Mechanisms Underlying Ischemia-Reperfusion Tolerance in the Swine

Previous studies suggest that immature hearts are more tolerant to ischemia than adult myocardium; whether differences exist during ontogenic development in severity of global myocardial ischemia/reperfusion (I/R) injury associated with cardiac surgery remains unknown. We employed swine models of deep hypothermic circulatory arrest (DHCA) to test the hypothesis that early differential regulation of cardioprotective genes is associated with increased tolerance of neonatal myocardium to surgical I/R. Male neonatal and adult pigs were randomized to DHCA or sham (N=5-14/group). DHCA animals underwent cardiopulmonary bypass (18°C), DHCA 60 min, and were recovered for 3h. Severity of perioperative myocardial injury was determined biochemically (Troponin I) and echocardiographically. Transcript levels were quantified in left ventricular myocardium using Affymetrix Porcine Genome arrays, followed by differential expression and pathway analysis. A 2-fold increase in severity of perioperative myocardial injury was found in adult pigs (Fig.A). Of 596 transcripts differentially expressed in neonates (≥1.5-fold change, DHCA vs. sham, FDR&lt;0.05), 53 were shared with adults (Fig.B). Among them, divergent expression patterns were found for oxysterol binding protein-like 6 (down-regulated in adults up-regulated in neonates) and bromodomain adjacent to zinc finger domain 2B (up-regulated in adults - down-regulated in neonates). Top transcripts uniquely upregulated in neonates but unchanged in adults were the apoptosis inhibitor baculoviral IAP repeat containing 2, and signal transducer and activator of transcription 3. mTOR signaling and p53 signaling were the top canonical pathways deregulated in adults and neonates, respectively. A high resistance to surgical I/R injury in neonatal vs adult swine heart is correlated with robust differences in early transcriptional programs, suggesting the involvement of fundamental adaptive ontogenetic processes.

Gene Expression Profiling of Valvular Interstitial Cells in Rapacz Familial Hypercholesterolemic Swine.

Rapacz familial hypercholesterolemic (RFH) swine is a well-established model of human FH, a highly prevalent hereditary disease associated with increased risk of coronary artery disease and calcific aortic valve disease (CAVD). However, while these animals have been used extensively for the study of atherosclerosis, the heart valves from RFH swine have not previously been examined. We report the analysis of valvular interstitial cell gene expression in adult (two year old) and juvenile (three months old) RFH and WT swine by microarray analysis via the Affymetrix Porcine Genome Array (GEO #: GSE53997). Principal component and hierarchical clustering analysis revealed grouping and almost no variability between the RFH juvenile and WT juvenile groups. Additionally, only 21 genes were found differentially expressed between these two experimental groups whereas over 900 genes were differentially expressed when comparing either RFH or WT juvenile swine to RFH adults.

Hepatic expression patterns in psychosocially high-stressed pigs suggest mechanisms following allostatic principles

Psychosocial challenges are known to introduce cellular and humoral adaptations in various tissues and organs, including parts of the sympatho-adrenal-medullary system and hypothalamic–pituitary–adrenal axis as well as other peripheral tissue being responsive to cortisol and catecholamines. The liver is of particular interest given its vital roles in maintaining homeostasis and health as well as regulating nutrient utilization and overall metabolism. We aimed to evaluate whether and how response to psychosocial stress is reflected by physiological molecular pathways in liver tissue. A pig mixing experiment was conducted to induce psychosocial stress culminating in skin lesions which reflect the involvement in aggressive behavior and fighting. At 27weeks of age, animals prone to psychosocially low- and high-stress were assigned to mixing groups. Skin lesions were counted before mixing and after slaughter on the carcass. Individual liver samples (n=12) were taken. The isolated RNA was hybridized on Affymetrix GeneChip porcine Genome Arrays. Relative changes of mRNA abundances were estimated via variance analyses. Molecular routes related to tRNA charging, urea cycle, acute phase response, galactose utilization, and steroid receptor signaling were found to be increased in psychosocially high-stressed animals, whereas catecholamine degradation and cholesterol biosynthesis were found to be decreased. In particular, psychosocially high-stressed animals show decreased expression of catechol-O-methyltransferase (COMT) which has been linked to molecular mechanisms regulating aggressiveness and stress response. The expression patterns of high-stressed animals revealed metabolic alterations of key genes related to energy-mobilizing processes at the expense of energy consuming processes. Thus, the coping following psychosocial challenges involves transcriptional alterations in liver tissue which may be summarized with reference to the concept of allostasis, a strategy which is critical for survival.

Chronic ammonia exposure does not influence hepatic gene expression in growing pigs

Housed pigs are often exposed to elevated concentrations of atmospheric ammonia. This aerial pollutant is widely considered to be an environmental stressor that also predisposes to reduced growth rates and poor health, although evidence to support this view is limited. Hepatic gene expression is very responsive to stress and metabolic effects. Two batches of growing pigs were therefore exposed to a nominal concentration of atmospheric ammonia of either 5 ppm (low) or 20 ppm (high) from 4 weeks of age for 15 weeks. Growth rates were monitored. Samples of liver were taken after slaughter (at ∼19 weeks of age). Samples from the second batch were analysed for global gene expression using 23 K Affymetrix GeneChip porcine genome arrays. Samples from both batches were subsequently tested for five candidate genes using quantitative real-time PCR (qPCR). The array analysis failed to detect any significant changes in hepatic gene expression following chronic exposure to atmospheric ammonia. Animals clustered into two main groups but this was not related to the experimental treatment. There was also no difference in growth rates between groups. The qPCR analyses validated the array results by showing similar fold changes in gene expression to the arrays. They revealed a significant batch effect in expression of lipin 1 (LPIN1), Chemokine (C-X-C motif) ligand 14 (CXCL14), serine dehydratase (SDS) and hepcidin antimicrobial peptide (HAMP). Only CXCL14, a chemotactic cytokine for monocytes, was significantly down-regulated in response to ammonia. As chronic exposure to atmospheric ammonia did not have a clear influence on hepatic gene expression, this finding implies that 20 ppm of atmospheric ammonia did not pose a significant material risk to the health or metabolism of housed pigs.

A genetical genomics approach reveals new candidates and confirms known candidate genes for drip loss in a porcine resource population

In this study lean meat water-holding capacity (WHC) of a Duroc×Pietrain (DuPi) resource population with corresponding genotypes and transcriptomes was investigated using genetical genomics. WHC was characterized by drip loss measured in M. longissimus dorsi. The 60K Illumina SNP chips identified genotypes of 169 F2 DuPi animals. Whole-genome transcriptomes of muscle samples were available for 132 F2 animals using the Affymetrix 24K GeneChip® Porcine Genome Array. Performing genome-wide association studies of transcriptional profiles, which are correlated with phenotypes, allows elucidation of cis- and trans-regulation. Expression levels of 1,228 genes were significantly correlated with drip loss and were further analyzed for enrichment of functional annotation groups as defined by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathways. A hypergeometric gene set enrichment test was performed and revealed glycolysis/glyconeogenesis, pentose phosphate pathway, and pyruvate metabolism as the most promising pathways. For 267 selected transcripts, expression quantitative trait loci (eQTL) analysis was performed and revealed a total of 1,541 significant associations. Because of positional accordance of the gene underlying transcript and the eQTL location, it was possible to identify eight eQTL that can be assumed to be cis-regulated. Comparing the results of gene set enrichment and the eQTL detection tests, molecular networks and potential candidate genes, which seemed to play key roles in the expression of WHC, were detected. The α-1-microglobulin/bikunin precursor (AMBP) gene was assumed to be cis-regulated and was part of the glycolysis pathway. This approach supports the identification of trait-associated SNPs and the further biological understanding of complex traits.

Early inflammatory response to the saponin adjuvant Matrix-M in the pig

The early inflammatory response to Matrix-M was evaluated in pigs. Adverse reactions measured as body temperature, appetite, activity level and reaction at the site of injection were not observed after s.c. injection with three doses of the adjuvant (75, 100 or 150μg) into one week old piglets. Analyses of the immediate cytokine response of PBMC after in vitro exposure to Matrix-M (AbISCO-100®) revealed only a low expression of mRNA for tumour necrosis factor-α (p<0.05) after 6h incubation. Histological examination revealed an infiltration of leukocytes, haemorrhage and necrosis in muscle 24h after i.m. injection of 150μg Matrix-M in pigs aged eleven weeks. At this time, different grades of reactive lymphoid hyperplasia were recorded in the draining lymph node that was enlarged in three of these six pigs injected with Matrix-M. The global transcriptional response at the site of injection and in the draining lymph node was analyzed using Affymetrix GeneChip Porcine Genome Array. A significant enrichment of gene signatures for the cell types described as “myeloid cells” and “plasmacytoid dendritic cells” was observed at the site of injection in Matrix-M injected pigs compared with pigs injected with saline. A number of genes encoding cytokines/chemokines or their receptors were upregulated at the injection site as well as in the draining lymph node. In the draining lymph node, a majority of the upregulated genes were interferon-regulated genes (IRGs). The expression of IFN-β, but not IFN-α, was increased in the draining lymph nodes of a majority of the pigs exposed to Matrix-M. These IFN-β expressing pigs also expressed increased levels of osteopontin (OPN) or stimulator of interferon genes (STING), two factors known to facilitate the expression of type I IFNs in response to viral infection. Thus, Matrix-M does not appear to induce any harmful inflammatory response in piglets whilst contributing to the innate immunity by activating the type I IFN system, possibly through several alternative signalling pathways.

Transcriptional analysis of intramuscular fatty acid composition in the longissimus thoracis muscle of Iberian × Landrace back‐crossed pigs

This study aimed at identifying differential gene expression conditional on the fatty acid profile of the longissimus thoracis (Lt) muscle, a prime cut of economic relevance for fresh and cured pork production. A population of 110 Iberian (25%)×Landrace (75%) back-crossed pigs was used, because these two breeds exhibit extreme profiles of intramuscular saturated fatty acid, monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA) contents. Total RNA from Lt muscle was individually hybridized to GeneChip Porcine Genome arrays (Affymetrix). A principal component analysis was performed with data from the 110 animals to select 40 extreme animals based on the total fatty acid profile and the MUFA composition (MAP). Comparison of global transcription levels between extreme fatty acid profile pigs (n=40) resulted in 219 differentially expressed probes (false discovery rate <0.10). Gene ontology, pathway and network analysis indicated that animals with higher percentages of PUFA exhibit a shift toward a more oxidative muscular metabolism state, with a raise in mitochondria function (PPARGC1A, ATF2), fatty acid uptake and oxidation (FABP5, MGLL). On the other hand, 87 probes were differentially expressed between MUFA composition groups (n=40; false discovery rate <0.10). In particular, muscles rich in n-7 MUFA expressed higher levels of genes involved in lipid metabolism (GLUL, CRAT, PLA2G15) and lower levels of fatty acid elongation genes (ELOVL5). Moreover, the chromosomal position of FABP5, PAQR3, MGLL, PPARGC1A, GLUL and ELOVL5 co-localized with very relevant QTL for fat deposition and composition described in the same resource population. This study represents a complementary approach to identifying genes underlying these QTL effects.

Identification of temporal and region-specific myocardial gene expression patterns in response to infarction in swine.

Molecular mechanisms associated with pathophysiological changes in ventricular remodelling due to myocardial infarction (MI) remain poorly understood. We analyzed changes in gene expression by microarray technology in porcine myocardial tissue at 1, 4, and 6 weeks post-MI.MI was induced by coronary artery ligation in 9 female pigs (30–40 kg). Animals were randomly sacrificed at 1, 4, or 6 weeks post-MI (n = 3 per group) and 3 healthy animals were also included as control group. Total RNA from myocardial samples was hybridized to GeneChip® Porcine Genome Arrays. Functional analysis was obtained with the Ingenuity Pathway Analysis (IPA) online tool. Validation of microarray data was performed by quantitative real-time PCR (qRT-PCR).More than 8,000 different probe sets showed altered expression in the remodelling myocardium at 1, 4, or 6 weeks post-MI. Ninety-seven percent of altered transcripts were detected in the infarct core and 255 probe sets were differentially expressed in the remote myocardium. Functional analysis revealed 28 genes de-regulated in the remote myocardial region in at least one of the three temporal analyzed stages, including genes associated with heart failure (HF), systemic sclerosis and coronary artery disease. In the infarct core tissue, eight major time-dependent gene expression patterns were recognized among 4,221 probe sets commonly altered over time. Altered gene expression of ACVR2B, BID, BMP2, BMPR1A, LMNA, NFKBIA, SMAD1, TGFB3, TNFRSF1A, and TP53 were further validated.The clustering of similar expression patterns for gene products with related function revealed molecular footprints, some of them described for the first time, which elucidate changes in biological processes at different stages after MI.

243 IMPACT OF MATERNAL LOW-DOSE ESTRADIOL-17β EXPOSURE DURING PREGNANCY ON THE REPRODUCTIVE ORGANS OF MALE PIG PROGENY

In male developing piglets, low endogenous oestrogenic hormone levels prevail. Sensitive tissues expressing high levels of oestrogen receptors, such as the epididymis, might thus be of specific relevance for early disturbances by estrogenic endocrine disruptors. We have previously shown that distinct concentrations of the natural oestrogen oestradiol-17β (E2) orally applied during the whole period of pregnancy in sows lead to a distinct shift in body composition. Therefore, we aimed at investigating whether prenatal E2 exposure had the potential to program male reproductive organs toward altered semen quality. The E2 treatment groups (n = 4 sows/group) were designed to represent doses of pharmacological relevance (PR; 1000 µg kg–1 of body weight per day), oral no-effect level (10 µg kg–1 of body weight per day), and acceptable daily intake level (0.05 µg kg–1 of body weight per day). Total RNA was extracted from the epididymis of 8-week-old piglet twins (n = 2 per sow). The RNA of littermates was pooled and subjected to microarray analysis (MA) using Agilent Porcine Genome Arrays (Agilent Technologies Inc., Santa Clara, CA, USA). Ejaculates of adult male pigs from the control group and the pharmacological group (n = 4) were collected and analysed for quantitative and qualitative parameters by using computer-assisted sperm analysis and flow cytometry. In total, 105 transcripts exhibited a significant difference of more than 1.5-fold for at least one of the treatment groups compared with the control group (P &lt; 0.05; FDR 5%). Several transcripts revealed gradually decreasing amounts subject to the dose of E2 exposure. Eight transcripts showed significant differences in all 3 treatment groups compared with the control group. Thirty-two transcripts had reduced abundance in the PR group and the oral no-effect level group, whereas 20 transcripts were downregulated in the PR group only. Semen parameters of adult males did not differ regarding volume and semen concentration, morphology, and membrane integrity. Computer-assisted sperm analysis evaluations did not show differences in motility and other kinematic parameters. Eight weeks after E2 exposure, male prepubertal piglets exhibited significant differences in epididymal messenger RNA transcript abundance subject to an in utero E2 treatment. This points toward possible epigenetic programming of the epididymis. Although the differential transcript abundance indicates a perturbation, normal semen quality was found after adolescence. Either these transcriptional changes are of minor functional importance or flexible mechanisms account for the developmental adaptations. Considering that identical transcripts were regulated in offspring exposed to both pharmacological and very low doses, further investigations of set points obviating long-term adverse consequences are needed.

Abstract 213: Physiogenomic Response to Prolonged Cardiac Arrest and Resuscitation: Hyporesponders Fail to Upregulate Certain Genes

Background: In our porcine laboratory studies of prolonged cardiac arrest and resuscitation we have observed that approximately one out of every four animals appears to be hyporesponsive to vasopressor drug therapy during CPR. These hyporesponsive animals fail to generate an increase in coronary perfusion pressure (CPP) sufficient to attain a return of spontaneous circulation (ROSC) and cannot be resuscitated. Some humans unexplainably fail resuscitation despite promising conditions (age, witnessed arrest, bystander CPR, rapid EMS response). We conducted this natural experiment in order to compare the physiogenomic responses of animals that were hyporesponsive, to those that responded as expected to vasopressor therapy. Hypothesis: We hypothesized that we would identify genes that were differentially expressed between the two groups (hyporesponders and normal responders). Methods: We sedated, anesthetized, and instrumented 14 female swine (mean mass 28.6 kgs). Micro-manometer tipped catheters were placed to measure CPP for determination of response status. Ventricular fibrillation (VF) was induced and untreated for 10 minutes, then CPR was begun. Two minutes later pressors were given (epinephrine 0.1 mg/kg and vasopressin 0.6 U/kg). At minute 15 the first rescue shock was delivered. Twenty-eight minutes after the induction of VF, femoral artery samples were harvested and snap-frozen. Gene expression was assessed on the Affymetrix Porcine Genome Array, and affected pathways were ordered with Pathway Express (with Impact Analysis of p&lt;0.05 considered significant). Results: We compared 4 hyporesponsive pigs to a sample of 5 normal responders. There were 213 differentially expressed genes affecting 10 pathways shown in the Table (with the apoptosis pathway at p=0.056). Conclusion: Hyporesponders differentially express genes that affect vasoregulation, myocardial contractility, calcium regulation and 7 other pathways.

The effect of enterotoxigenic Escherichia coli F4ab,ac on early-weaned piglets: A gene expression study

Diarrhoea in neonatal and early-weaned piglets due to enterotoxigenic Escherichia coli-F4 (ETEC-F4) is an important problem in the pig farming industry. There is substantial evidence for a genetic basis for susceptibility to ETEC-F4 since not all pigs suffer from diarrhoea after an ETEC-F4 infection. A region on SSC13 has been found to be in close linkage to the susceptibility of piglets for ETEC-F4ab,ac. Potential candidate genes on SSC13 have been examined and although some polymorphisms were found to be in linkage disequilibrium with the phenotype, the causative mutation has not yet been found. In this study we are looking at the expression of porcine genes in relation to ETEC-F4ab,ac. With the aid of the Affymetrix GeneChip Porcine Genome Array we were able to find differentially expressed genes between ETEC-F4ab,ac receptor positive (Fab,acR+) piglets without diarrhoea and F4ab,acR+ piglets with diarrhoea or F4ab,acR− animals. Since the susceptibility to ETEC-F4ab,ac was described as a Mendelian trait, it is not so surprisingly that only two differentially expressed genes, transferrin receptor (TFRC) and trefoil factor 1 (TFF1), came out of the analysis. Although both genes could pass for functional candidate genes only TFRC also mapped to the region on SSC13 associated with susceptibility for ETEC-F4, which makes TFRC a positional functional candidate gene. Validation by qRT-PCR confirmed the differential expression of TFRC and TFF1. In piglets without diarrhoea, the expression of both genes was higher in F4ab,acR+ than in F4ab,acR− piglets. Similarly, TFRC and TFF1 expression in F4ab,acR+ piglets without diarrhoea was also higher than in F4ab,acR+ piglets with diarrhoea. Consequently, although both genes might not play a role as receptor for F4 fimbriae, they could be of great importance during an ETEC-F4 outbreak. An upregulation of TFRC can be a consequence of the piglets ability to raise an effective immune response. An elevation of TFF1, a protein involved in mucin formation, may also affect the piglet's capability to cope with ETEC bacteria, rather than being a receptor for its fimbriae.

Paradigm of Time-sequence Development of the Intestine of Suckling Piglets with Microarray.

The interaction of the genes involved in intestinal development is the molecular basis of the regulatory mechanisms of intestinal development. The objective of this study was to identify the significant pathways and key genes that regulate intestinal development in Landrace piglets, and elucidate their rules of operation. The differential expression of genes related to intestinal development during suckling time was investigated using a porcine genome array. Time sequence profiles were analyzed for the differentially expressed genes to obtain significant expression profiles. Subsequently, the most significant profiles were assayed using Gene Ontology categories, pathway analysis, network analysis, and analysis of gene co-expression to unveil the main biological processes, the significant pathways, and the effective genes, respectively. In addition, quantitative real-time PCR was carried out to verify the reliability of the results of the analysis of the array. The results showed that more than 8000 differential expression transcripts were identified using microarray technology. Among the 30 significant obtained model profiles, profiles 66 and 13 were the most significant. Analysis of profiles 66 and 13 indicated that they were mainly involved in immunity, metabolism, and cell division or proliferation. Among the most effective genes in these two profiles, CN161469, which is similar to methylcrotonoyl-Coenzyme A carboxylase 2 (beta), and U89949.1, which encodes a folate binding protein, had a crucial influence on the co-expression network.

Supplementary tryptophan downregulates the expression of genes induced by the gut microbiota in healthy weaned pigs susceptible to enterotoxigenic Escherichia coli F4

Supplementary l-tryptophan (l-Trp) limits the decrease of feed intake and growth in Enterotoxigenic Escherichia coli F4 (ETEC) susceptible pigs upon oral challenge with this pathogen. Susceptibility to ETEC is genetically controlled and related to the presence of receptors for the F4 fimbriae (F4R). We aimed to assess if dietary Trp affects genes involved in the intestinal barrier of healthy pigs carrying or not the F4R. Thirty-six littermate weaning pigs were selected to have potentially eighteen ETEC-susceptible and eighteen ETEC-non-susceptible subjects, based on a Mucin 4 gene polymorphism. For 21 days they were fed a diet with 0.17 or 0.22 ileal digestible Trp:Lys ratio. Using the test of ETEC adhesion to the intestinal villi, the pigs were divided into F4R negative (no bacteria adhering, F4R−), F4R positive (F4R+), and F4R mildly positive (F4Rm+). A preliminary test (GeneChip® Porcine Genome Array) highlighted the differentially expressed genes in the jejunum of 3 F4R− and 3 F4R+ pigs. The expression of the most interesting genes was assessed on the whole sample. In F4R+ pigs, Trp reduced the mRNA of four genes involved in the intestinal barrier and/or induced by several bacteria-associated molecular patterns, like lipopolysaccharide (LPS) (REG3G, Regenerating islet-derived 3 gamma; SFTPD, Surfactant pulmonary-associated protein D; CFB, Complement factor B; LBP, LPS-binding protein) (P<0.05). In pigs fed the low-Trp diet, REG3G, SFTPD and LPB mRNA increased with F4R presence (P<0.05). Interleukine-8 tended to be less expressed with the higher Trp level whatever the F4R presence (P=0.09). No DNA from ETEC was detected in the jejunum contents. Trp favorably interacts to reduce the bacterial induction of some genes involved in the intestinal barrier in ETEC susceptible pigs, but the causative mechanism is yet to be established.

Gene expression analysis of mammary tissue during fetal bud formation and growth in two pig breeds – indications of prenatal initiation of postnatal phenotypic differences

BackgroundThe mammary gland is key to all mammal species; in particular in multiparous species like pigs the number and the shape of functional mammary gland complexes are major determinants of fitness. Accordingly, we aimed to catalog the genes relevant to mammogenesis in pigs. Moreover, we aimed to address the hypothesis that the extent and timing of proliferation, differentiation, and maturation proccesses during prenatal development contribute to postnatal numerical, morphological and functional properties of the mammary gland. Thus we focused on differentially expressed genes and networks relevant to mammary complex development in two breeds that are subject to different selection pressure on number, shape and function of teats and show largely different prevalence of non-functional inverted teats. The expression patterns of fetal mammary complexes obtained at 63 and 91 days post conception (dpc) from German Landrace (GL) and Pietrain (PI) were analyzed by Affymetrix GeneChip Porcine Genome Arrays.ResultsThe expression of 11,731 probe sets was analysed between the two stages within and among breeds. The analysis showed the largest distinction of samples of the breed GL at 63 dpc from all other samples. According to Ingenuity Pathways Analysis transcripts with abundance at the four comparisons made (GL63-GL91, PI63-PI93, GL63-PI63 and GL91-PI91) were predominantly assigned to biofunctions relevant to `cell maintenance, proliferation, differentiation and replacement´, `organismal, organ and tissue development´ and `genetic information and nucleic acid processing´. Moreover, these transcripts almost exclusively belong to canonical pathways related to signaling rather than metabolic pathways. The accumulation of transcripts that are up-regulated in GL compared to PI indicate a higher proliferating activity in GL, whereas processes related to differentiation, maturation and maintenance of cells are more prominent in PI. Differential expression was validated by quantitative RT-PCR of five genes (GAB1, MAPK9, PIK3C2B, PIK3C3 and PRKCH) that are involved in several relevant signaling pathways.ConclusionsThe results indicate that mammary complex development in PI precedes GL. The differential expression between the two breeds at fetal stages likely reflects the prenatal initiation of postnatal phenotypes concerning the number and shape as well as functionality of teats.

Toxicogenomic analysis of chlorine vapor-induced porcine skin injury

Chlorine is an industrial chemical that can cause cutaneous burns. Understanding the molecular mechanisms of tissue damage and wound healing is important for the selection and development of an effective post-exposure treatment. This study investigated the effect of cutaneous chlorine vapor exposure using a weanling swine burn model and microarray analysis. Ventral abdominal sites were exposed to a mean calculated chlorine vapor concentration of 2.9 g/L for 30 min. Skin samples were harvested at 1.5 h, 3 h, 6 h, and 24 h post-exposure and stored in RNAlater® until processing. Total RNA was isolated, processed, and hybridized to Affymetrix GeneChip® Porcine Genome Arrays. Differences in gene expression were observed with respect to sampling time. Ingenuity Pathways Analysis revealed seven common biological functions among the top ten functions of each time point, while canonical pathway analysis revealed 3 genes (IL-6, IL1A, and IL1B) were commonly shared among three significantly altered signaling pathways. The transcripts encoding all three genes were identified as common potential therapeutic targets for Phase II/III clinical trial, or FDA-approved drugs. The present study shows transcriptional profiling of cutaneous wounds induced by chlorine exposure identified potential targets for developing therapeutics against chlorine-induced skin injury.