Hygiene of housing conditions and proinflammatory signals alter gene expressions in porcine adipose tissues and blood cells.

Adipose tissue is an organ with metabolic, endocrine and immune functions. In this tissue, the expressions of genes associated with several metabolic pathways, including lipid metabolism, have been shown to be affected by genetic selection for feed efficiency, an important trait to consider in livestock. We hypothesized that the stimulation of immune system caused by poor hygiene conditions of housing impacts the molecular and cellular features of adipose tissue and that the impact may differ between pigs that diverge in feed efficiency. At the age of 12 weeks, Large White pigs from two genetic lines divergent for residual feed intake (RFI) were housed in two contrasting hygiene conditions (good vs poor). After six weeks of exposure, pigs were slaughtered (n = 36). Samples of blood, subcutaneous (SCAT) and perirenal (PRAT) adipose tissues were collected for cell response and gene expression investigations. The decrease in the relative weight of PRAT was associated with a decline in mRNA levels of FASN, ME, LCN2 and TLR4 (P < 0.05) in pigs housed in poor conditions compared with pigs housed in good conditions for both RFI lines. In SCAT, the expressions of only two key genes (PPARG and TLR4) were significantly affected by the hygiene of housing conditions. Besides, the mRNA levels of both LCN2 and GPX3 were influenced by the RFI line (P < 0.05). Because we suspected an effect of poor hygiene at the cellular levels, we investigated the differentiation of stromal vascular cells isolated from SCAT in vitro in the absence or presence of a pro-inflammatory cytokine, Tumor Necrosis Factor-α (TNF-α). The ability of these cells to differentiate in the absence or presence of TNF-α did not differ among the four groups of animals (P > 0.05). We also investigated the expressions of genes involved in the immune response and lipid metabolism in whole blood cells cultured in the absence and presence of LPS. The hygiene conditions had no effect but, the relative expression of the GPX3 gene was higher (P < 0.001) in high RFI than in low RFI pigs while the expressions of IL-10 (P = 0.027), TGFß1 (P = 0.023) and ADIPOR2 (P = 0.05) genes were lower in high RFI than in low RFI pigs. Overall, the current study indicates that the hygiene of housing had similar effects on both RFI lines on the expression of genes in adipose tissues and on the features of SCAT adipose cells and whole blood cells in response to TNF-α and LPS. It further demonstrates that the number of genes with expression impacted by housing conditions was higher in PRAT than in SCAT. It suggests a depot-specific response of adipose tissue to the current challenge.

Poor hygiene of housing conditions influences energy metabolism in a muscle type-dependent manner in growing pigs differing in feed efficiency.

The ability of pigs to cope with inflammatory challenges may by modified by selection for residual feed intake (RFI), a measure of feed efficiency. In the current study, we evaluated skeletal muscle metabolic responses to degraded hygiene conditions in pigs divergently selected for RFI. At 82 d of age, low RFI and high RFI pigs were housed in either poor or good hygiene conditions. After a 6-week challenge, the poor hygiene conditions induced a decrease in growth performance (P < 0.001) and in plasma IGF-I concentrations (P < 0.003) in both lines. In the slow-twitch oxidative semispinalis muscle, poor hygiene conditions induced a shift towards a more oxidative metabolism and an activation of the AMPK pathway in pigs of both RFI lines. In the fast-twitch glycolytic longississimus muscle, poor hygiene conditions were associated to a less glycolytic metabolism in the HRFI line only. Poor hygiene conditions also increased the protein level of lipidation of microtubule-associated protein 1 light-chain 3ß (LC3-II) in both RFI lines, suggesting an activation of the autophagy pathway. Altogether, the data revealed muscle-type specific metabolic adaptations to poor hygiene conditions, which may be related to different strategies to fuel the activated immune system.

A transcriptome multi-tissue analysis identifies biological pathways and genes associated with variations in feed efficiency of growing pigs.

BACKGROUND: Animal's efficiency in converting feed into lean gain is a critical issue for the profitability of meat industries. This study aimed to describe shared and specific molecular responses in different tissues of pigs divergently selected over eight generations for residual feed intake (RFI). RESULTS: Pigs from the low RFI line had an improved gain-to-feed ratio during the test period and displayed higher leanness but similar adiposity when compared with pigs from the high RFI line at 132 days of age. Transcriptomics data were generated from longissimus muscle, liver and two adipose tissues using a porcine microarray and analyzed for the line effect (n = 24 pigs per line). The most apparent effect of the line was seen in muscle, whereas subcutaneous adipose tissue was the less affected tissue. Molecular data were analyzed by bioinformatics and subjected to multidimensional statistics to identify common biological processes across tissues and key genes participating to differences in the genetics of feed efficiency. Immune response, response to oxidative stress and protein metabolism were the main biological pathways shared by the four tissues that distinguished pigs from the low or high RFI lines. Many immune genes were under-expressed in the four tissues of the most efficient pigs. The main genes contributing to difference between pigs from the low vs high RFI lines were CD40, CTSC and NTN1. Different genes associated with energy use were modulated in a tissue-specific manner between the two lines. The gene expression program related to glycogen utilization was specifically up-regulated in muscle of pigs from the low RFI line (more efficient). Genes involved in fatty acid oxidation were down-regulated in muscle but were promoted in adipose tissues of the same pigs when compared with pigs from the high RFI line (less efficient). This underlined opposite line-associated strategies for energy use in skeletal muscle and adipose tissue. Genes related to cholesterol synthesis and efflux in liver and perirenal fat were also differentially regulated in pigs from the low vs high RFI lines. CONCLUSIONS: Non-productive functions such as immunity, defense against pathogens and oxidative stress contribute likely to inter-individual variations in feed efficiency.

Molecular alterations induced by a high-fat high-fiber diet in porcine adipose tissues: variations according to the anatomical fat location.

BACKGROUND: Changing the energy and nutrient source for growing animals may be an effective way of limiting adipose tissue expansion, a response which may depend on the genetic background of the animals. This study aims to describe the transcriptional modulations present in the adipose tissues of two pig lines divergently selected for residual feed intake which were either fed a high-fat high-fiber (HF) diet or an isocaloric low-fat high-starch diet (LF). RESULTS: Transcriptomic analysis using a porcine microarray was performed on 48 pigs (n = 12 per diet and per line) in both perirenal (PRAT) and subcutaneous (SCAT) adipose tissues. There was no interaction between diet and line on either adiposity or transcriptional profiles, so that the diet effect was inferred independently of the line. Irrespective of line, the relative weights of the two fat depots were lower in HF pigs than in LF pigs after 58 days on dietary treatment. In the two adipose tissues, the most apparent effect of the HF diet was the down-regulation of several genes associated with the ubiquitin-proteasome system, which therefore may be associated with dietary-induced modulations in genes acting in apoptotic and cell cycle regulatory pathways. Genes involved in glucose metabolic processes were also down-regulated by the HF diet, with no significant variation or decreased expression of important lipid-related genes such as the low-density lipoprotein receptor and leptin in the two fat pads. The master regulators of glucose and fatty acid homeostasis SREBF1 and MLXIPL, and peroxisome proliferator-activated receptor (PPAR)δ and its heterodimeric partner RXRA were down-regulated by the HF diet. PPARγ which has pleiotropic functions including lipid metabolism and adipocyte differentiation, was however up-regulated by this diet in PRAT and SCAT. Dietary-related modulations in the expression of genes associated with immunity and inflammation were mainly revealed in PRAT. CONCLUSION: A high-fat high-fiber diet depressed glucose and lipid anabolic molecular pathways, thus counteracting adipose tissue expansion. Interaction effects between dietary intake of fiber and lipids on gene expression may modulate innate immunity and inflammation, a response which is of interest with regard to chronic inflammation and its adverse effects on health and performance.

Whole Blood Transcriptomics Is Relevant to Identify Molecular Changes in Response to Genetic Selection for Feed Efficiency and Nutritional Status in the Pig.

The molecular mechanisms underlying feed efficiency need to be better understood to improve animal efficiency, a research priority to support a competitive and sustainable livestock production. This study was undertaken to determine whether pig blood transcriptome was affected by differences in feed efficiency and by ingested nutrients. Growing pigs from two lines divergently selected for residual feed intake (RFI) and fed isoproteic and isocaloric diets contrasted in energy source and nutrients were considered. Between 74 and 132 days of age, pigs (n = 12 by diet and by line) received a regular diet rich in cereals and low in fat (LF) or a diet where cereals where partially substituted by lipids and fibers (HF). At the end of the feeding trial, the total number of white blood cells was not affected by the line or by the diet, whereas the red blood cell number was higher (P<0.001) in low RFI than in high RFI pigs. Analysis of the whole blood transcriptome using a porcine microarray reveals a higher number of probes differentially expressed (DE) between RFI lines than between diets (2,154 versus 92 probes DE, P<0.01). This corresponds to 528 overexpressed genes and 477 underexpressed genes in low RFI pigs compared with high RFI pigs, respectively. Overexpressed genes were predominantly associated with translational elongation. Underexpressed genes were mainly involved in the immune response, regulation of inflammatory response, anti-apoptosis process, and cell organization. These findings suggest that selection for RFI has affected the immune status and defense mechanisms of pigs. Genes DE between diets were mainly related to the immune system and lipid metabolism. Altogether, this study demonstrates the usefulness of the blood transcriptome to identify the main biological processes affected by genetic selection and feeding strategies.

The Longissimus and Semimembranosus muscles display marked differences in their gene expression profiles in pig.

BACKGROUND: Meat quality depends on skeletal muscle structure and metabolic properties. While most studies carried on pigs focus on the Longissimus muscle (LM) for fresh meat consumption, Semimembranosus (SM) is also of interest because of its importance for cooked ham production. Even if both muscles are classified as glycolytic muscles, they exhibit dissimilar myofiber composition and metabolic characteristics. The comparison of LM and SM transcriptome profiles undertaken in this study may thus clarify the biological events underlying their phenotypic differences which might influence several meat quality traits. METHODOLOGY/PRINCIPAL FINDINGS: Muscular transcriptome analyses were performed using a custom pig muscle microarray: the 15 K Genmascqchip. A total of 3823 genes were differentially expressed between the two muscles (Benjamini-Hochberg adjusted P value ≤0.05), out of which 1690 and 2133 were overrepresented in LM and SM respectively. The microarray data were validated using the expression level of seven differentially expressed genes quantified by real-time RT-PCR. A set of 1047 differentially expressed genes with a muscle fold change ratio above 1.5 was used for functional characterization. Functional annotation emphasized five main clusters associated to transcriptome muscle differences. These five clusters were related to energy metabolism, cell cycle, gene expression, anatomical structure development and signal transduction/immune response. CONCLUSIONS/SIGNIFICANCE: This study revealed strong transcriptome differences between LM and SM. These results suggest that skeletal muscle discrepancies might arise essentially from different post-natal myogenic activities.

Associations between muscle gene expression pattern and technological and sensory meat traits highlight new biomarkers for pork quality assessment.

Meat quality (MQ) results from complex phenomenon and despite improved knowledge on MQ development, its variability remains high. The identification of biomarkers and the further development of rapid tests would thus be helpful to evaluate MQ in pork industries. Using transcriptomics, the present study aimed at identifying biomarkers of eight pork quality traits: ultimate pH, drip loss, lightness, redness, hue angle, intramuscular fat, shear force and tenderness, based on an experimental design inducing a high variability in MQ. Associations between microarray gene expression and pork traits (n=50 pigs) highlighted numerous potential biomarkers of MQ. Using quantitative RT-PCR, 113 transcript-trait correlations including 40 of these genes were confirmed (P<0.05, |r|≤0.73), out of which 60 were validated (P<0.05, |r|≤0.68) on complementary experimental data (n=50). Multiple regression models including 3 to 5 genes explained up to 59% of MQ trait variability. Moreover, functional analysis of correlated-trait genes provided information on the biological phenomena underlying MQ.

Visualizing non infectious and infectious Anopheles gambiae blood feedings in naive and saliva-immunized mice.

BACKGROUND: Anopheles gambiae is a major vector of malaria and lymphatic filariasis. The arthropod-host interactions occurring at the skin interface are complex and dynamic. We used a global approach to describe the interaction between the mosquito (infected or uninfected) and the skin of mammals during blood feeding. METHODS: Intravital video microscopy was used to characterize several features during blood feeding. The deposition and movement of Plasmodium berghei sporozoites in the dermis were also observed. We also used histological techniques to analyze the impact of infected and uninfected feedings on the skin cell response in naive mice. RESULTS: The mouthparts were highly mobile within the skin during the probing phase. Probing time increased with mosquito age, with possible effects on pathogen transmission. Repletion was achieved by capillary feeding. The presence of sporozoites in the salivary glands modified the behavior of the mosquitoes, with infected females tending to probe more than uninfected females (86% versus 44%). A white area around the tip of the proboscis was observed when the mosquitoes fed on blood from the vessels of mice immunized with saliva. Mosquito feedings elicited an acute inflammatory response in naive mice that peaked three hours after the bite. Polynuclear and mast cells were associated with saliva deposits. We describe the first visualization of saliva in the skin by immunohistochemistry (IHC) with antibodies directed against saliva. Both saliva deposits and sporozoites were detected in the skin for up to 18 h after the bite. CONCLUSION: This study, in which we visualized the probing and engorgement phases of Anopheles gambiae blood meals, provides precise information about the behavior of the insect as a function of its infection status and the presence or absence of anti-saliva antibodies. It also provides insight into the possible consequences of the inflammatory reaction for blood feeding and pathogen transmission.

Mitochondrial function, fatty acid metabolism, and immune system are relevant features of pig adipose tissue development.

The molecular mechanisms underlying the genetic control of fat development in humans and livestock species still require characterization. To gain insights on gene expression patterns associated with genetic propensity for adiposity, we compared subcutaneous adipose tissue (SCAT) transcriptomics profiles from two contrasted pig breeds for body fatness. Samples were obtained from Large White (LW; lean phenotype) and Basque pigs (B; low growth and high fat content) at 35 kg (n = 5 per breed) or 145 kg body weight (n = 10 per breed). Using a custom adipose tissue microarray, we found 271 genes to be differentially expressed between the two breeds at both stages, out of which 123 were highly expressed in LW pigs and 148 genes were highly expressed in B pigs. Functional enrichment analysis based on gene ontology (GO) terms highlighted gene groups corresponding to the mitochondrial energy metabolism in LW pigs, whereas immune response was found significantly enriched in B pigs. Genes associated with lipid metabolism, such as ELOVL6, a gene involved in fatty acid elongation, had a lower expression in B compared with LW pigs. Furthermore, despite enlarged adipocyte diameters and higher plasma leptin concentration, B pigs displayed reduced lipogenic enzyme activities compared with LW pigs at 145 kg. Altogether, our results suggest that the development of adiposity was associated with a progressive worsening of the metabolic status, leading to a low-grade inflammatory state, and may thus be of significant interest for both livestock production and human health.

Comparison of muscle transcriptome between pigs with divergent meat quality phenotypes identifies genes related to muscle metabolism and structure.

BACKGROUND: Meat quality depends on physiological processes taking place in muscle tissue, which could involve a large pattern of genes associated with both muscle structural and metabolic features. Understanding the biological phenomena underlying muscle phenotype at slaughter is necessary to uncover meat quality development. Therefore, a muscle transcriptome analysis was undertaken to compare gene expression profiles between two highly contrasted pig breeds, Large White (LW) and Basque (B), reared in two different housing systems themselves influencing meat quality. LW is the most predominant breed used in pig industry, which exhibits standard meat quality attributes. B is an indigenous breed with low lean meat and high fat contents, high meat quality characteristics, and is genetically distant from other European pig breeds. METHODOLOGY/PRINCIPAL FINDINGS: Transcriptome analysis undertaken using a custom 15 K microarray, highlighted 1233 genes differentially expressed between breeds (multiple-test adjusted P-value<0.05), out of which 635 were highly expressed in the B and 598 highly expressed in the LW pigs. No difference in gene expression was found between housing systems. Besides, expression level of 12 differentially expressed genes quantified by real-time RT-PCR validated microarray data. Functional annotation clustering emphasized four main clusters associated to transcriptome breed differences: metabolic processes, skeletal muscle structure and organization, extracellular matrix, lysosome, and proteolysis, thereby highlighting many genes involved in muscle physiology and meat quality development. CONCLUSIONS/SIGNIFICANCE: Altogether, these results will contribute to a better understanding of muscle physiology and of the biological and molecular processes underlying meat quality. Besides, this study is a first step towards the identification of molecular markers of pork quality and the subsequent development of control tools.

A comparison of subcutaneous adipose tissue proteomes in juvenile piglets with a contrasted adiposity underscored similarities with human obesity.

Subcutaneous fat tissues from an indigenous fat-type breed and an intensively-lean selected breed were studied in juvenile pigs. Combining DIGE with bioinformatics and target analyses of key genes, enzymes or terminal routes, this study identifies metabolic and homeostatic processes, response to organic substances, and acute-phase responses as the main pathways whose proteins were regulated in association with adiposity. Breed-related differences in abundance and activities of malic enzyme and glucose-6-phosphate dehydrogenase NADPH-supplying enzymes suggested up-regulation of the lipogenic pathway to dispose for a greater adiposity. Over-abundance in the lipolytic protein carboxylesterase-1 was revealed in fat-type piglets. A panel of pro- and anti-inflammatory proteins such as serpins, had an altered abundance in the fat-type piglets, suggesting adverse consequences of fat accumulation even in early post-weaning stages. Propensity to low-grade inflammation in fat pigs was reinforced by the up-regulation of genes encoding pro-inflammatory cytokines IL6 and TNF-α in these piglets. Differential abundance in annexin-A5 and pericentrin suggested a positive regulation of cell apoptosis in lean piglets. Our results are relevant in the context of data linking the accretion of body lipids to the physiology and pathology of adipose tissue in models other than rodents for a better control of human health and nutrition.

Differentially-expressed genes in pig Longissimus muscles with contrasting levels of fat, as identified by combined transcriptomic, reverse transcription PCR, and proteomic analyses.

Intramuscular fat content is important for many meat quality parameters. This work is aimed at identifying functional categories of genes associated with natural variation among individuals in intramuscular fat content to help the design of genetic schemes for high marbling potential. Taking advantage of the global nature of transcriptomic and proteomic technologies, 40 genes were identified as differently expressed between high fat and low fat pig Longissimus muscles at slaughter weight. They are involved in metabolic processes, cell communication, binding, and response to stimulus. Using real-time PCR in muscle biopsies taken earlier in the fattening period, the group with a high intramuscular fat content was also characterized by the down-expression of genes playing a negative role in adipogenesis, such as architectural transcription factor high-motility hook A1, mitogen activated protein-kinase14, and cyclin D1. These results suggest that interindividual variability in intramuscular fat content might arise essentially from differences in early adipogenesis.

Phospholipase A2 diversity and polymorphism in European viper venoms: paradoxical molecular evolution in Viperinae.

We report the diversity and polymorphism of phospholipase A(2) (PLA(2)) transcripts from snakes belonging to nine European viper subspecies. This diversity results in the expression of a combination of six PLA(2) species--ammodytin I1, ammodytin I2, ammodytin L, ammodytoxin, vaspin A and vaspin B--with 19 known isoforms of the first five of these species. Most of the European viper venoms studied contained either a myotoxin or a neurotoxin, and all contained ammodytin I1 and ammodytin I2. There is no evidence that a given pattern of PLA(2) species constitutes a taxonomic criterion, and isoform analysis would be required for such discrimination. Analysis of the phylogenetic relationships between PLA(2) species from European vipers and those of other members of the Viperinae revealed a strong correlation between the geographical source of the viper and the clustering seen for the different isoforms, for each PLA(2) species. The K(a)/K(s) values calculated for the mature protein-coding region of paralogous genes showed that ratios for pairs including vaspin B or one ammodytoxin isoform were greater than 1.09, whereas those for most of the remaining pairs were less than 1. Different patterns of mutation were observed in comparisons of the different PLA(2) isoforms. The mechanisms directing a mutation toward a precise exon remain unresolved.

Epidemiological data, clinical admission gradation and biological quantification by ELISA of scorpion envenomations in Algeria: effect of immunotherapy.

An epidemiological and biological survey of scorpion envenomation was conducted in Algeria. Analysis of 182 medical files showed that 70% of the patients were stung by Androctonus australis. Most accidents occurred during the morning (40%) or the evening (30%). Two-thirds of the patients reached a hospital 1 hour after being stung. Their clinical symptoms classified 78% of them as Grade I (mild envenomation) and 17% of them as Grade II (moderate envenomation) on admission to hospital. No severe envenomation (Grade III) was reported. Most patients were treated with antivenom by the intramuscular route. Blood samples were collected before and after antivenom immunotherapy. A good correlation was observed between the grade of envenomation on admission and the blood venom concentrations measured by ELISA. The venom concentration decreased as function of the interval between the sting and blood collection (t1/2 = 2 h). Intramuscular injection of 10 ml of antivenom did not efficiently neutralize scorpion venom. Inflammation was followed by measuring IL6 concentration. IL6 peaked 1 h after scorpion envenomation. This study shows that optimization of the administration of antivenom is required to achieve clinical efficiency. In particular, intravenous injection of a larger dose of a more potent antivenom should be considered.

Mitochondrial and peroxisomal fatty acid oxidation capacities increase in the skeletal muscles of young pigs during early postnatal development but are not affected by cold stress.

In pigs, the optimal utilization of energy substrates within muscle fibers is a prerequisite of the utmost importance for successful adaptation to extra-uterine life. In the present work we demonstrate that fatty acid (FA) oxidative capacities increased within the first five days of life in piglet skeletal muscle. Mitochondrial FA oxidation capacities increased more in the rhomboideus oxidative than in the longissimus lumborum glycolytic muscle (+114% vs. +62%, P < 0.001). The apparent rate of fatty acid degradation by peroxisomes represents 30 to 40% of total FA oxidation capacities and increased by about 170% (P < 0.001) with age in both muscles. The postnatal enhancement of skeletal muscle oxidative capacities was further supported by a rise in acid-soluble and long-chain acylcamitine tissue levels (+67%, P < 0.01), and plasma levels of albumin (+160%, P < 0.001). Cold stress had no effect on mitochondrial and peroxisomal FA oxidation but greatly enhanced (+61%, P < 0.05) the circulating levels of non-esterified fatty acids at five days of life.

Toxicity evolution of Vipera aspis aspis venom: identification and molecular modeling of a novel phospholipase A(2) heterodimer neurotoxin.

We report the simultaneous presence of two phospholipase A(2) (PLA(2)) neurotoxins in the venom of Vipera aspis aspis, the first such observation. One is monomeric and identical to ammodytoxin B of Vipera ammodytes ammodytes. Its presence may result from gene flux after interbreeding between V. aspis aspis and V. ammodytes ammodytes. The second, a novel heterodimer named vaspin, is very similar to vipoxin of Vipera ammodytes meridionalis and to PLA(2)-I of Vipera aspis zinnikeri. It may result from expression of preexisting genes, the acidic subunit evolving from an ancestor common to ammodytin I2 from V. ammodytes ammodytes, which we also found in V. aspis aspis.