Annie Vincent

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.

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.

Characterisation of oxidative phosphorylation in skeletal muscle mitochondria subpopulations in pig: a study using top-down elasticity analysis

In skeletal muscle, two mitochondrial populations are present which, on the basis of their localisation, are termed intermyofibrillar and subsarcolemmal mitochondria (IMF and SS, respectively). These two populations have different biochemical characteristics and show different responses to physiological stimuli. In this paper, we characterise the oxidative phosphorylation of SS and IMF using ‘top‐down’ elasticity analysis. We excluded the possibility that their different characteristics can be attributed to a different degree of breakage of the two types of mitochondria due to the different isolation procedures used in their preparation. The higher respiration rate and higher respiratory control ratio shown by IMF compared with those shown by SS are principally due to the higher activities of the reactions involved in substrate oxidation as confirmed by the measurement of cytochrome oxidase activity. There is no difference in the leak of protons across the inner mitochondrial membrane between IMF and SS; a faster rate of ATP synthesis and turnover is driven by the lower membrane potential in SS compared with in IMF.

First evidence of uncoupling protein-2 (UCP-2) and -3 (UCP-3) gene expression in piglet skeletal muscle and adipose tissue.

Uncoupling proteins (UCPs) facilitate proton transport inside the mitochondria and decrease the proton gradient, leading to heat production. Until now, the presence of UCP1 or other UCP homologs had not been detected in tissues of pig, a species where evidence for the presence of brown adipose tissue has only been provided in 2-3 month old animals. In the light of the improving knowledge on the UCPs family, we decided to examine both UCP2 and UCP3 mRNA expression in piglet skeletal muscle and adipose tissue. Using RT-PCR we have successfully cloned a partial UCP2 sequence and a complete UCP3 cDNA. UCP3s open reading frame (936bp) shares 90, 89 and 85% similarity with bovine, human and rat UCP3 nucleotide sequences, respectively. In 3-5 day old piglets, these genes are expressed in adipose tissue and in both longissimus thoracis (LT) and rhomboïdeus (RH) muscles, without any effect of muscle metabolic type. This is in good agreement with the measurement of the same membrane potential in mitochondria isolated from both types of muscles. In triiodothyronine-treated piglets, UCP3 mRNA is more expressed in LT than in RH muscle. These genes may be involved in the control of the energy metabolism of the piglet.

Lipid and protein oxidation in microsomal fraction from turkeys: influence of dietary fat and vitamin E supplementation.

The objectives of this study were to investigate the effects of dietary fats (6% soya oil or rapeseed oil or tallow) and α-tocopheryl acetate supplementation (30 ppm for control and 200 ppm for supplemented animals), on lipid and protein oxidation, induced by Fe(3+)/ascorbate, of microsomal fraction in turkey muscles (M. pectoralis major and M. sartorius). Supplementation of turkeys with α-tocopheryl acetate increased the vitamin E content of microsomal membranes. Vitamin E supplementation strongly decreased lipid oxidation in membranal fractions when animals were fed rapeseed oil or tallow; this effect was less pronounced in animals fed soya oil. Vitamin E supplementation induced a slight decrease in protein carbonyl content, especially in animals fed soya oil. Level of protein free thiols was considerably enhanced in diet enriched with soya oil. Vitamin E supplementation had a stabilizing effect on glucose-6-phosphatase activity of microsomes when oxidized by Fe(3+)/ascorbate. No muscle effect was detected on the level of lipid and protein oxidation in membranal fractions even if M. sartorius is known to be more oxidative than M. pectoralis major.

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.

Review: divergent selection for residual feed intake in the growing pig.

This review summarizes the results from the INRA (Institut National de la Recherche Agronomique) divergent selection experiment on residual feed intake (RFI) in growing Large White pigs during nine generations of selection. It discusses the remaining challenges and perspectives for the improvement of feed efficiency in growing pigs. The impacts on growing pigs raised under standard conditions and in alternative situations such as heat stress, inflammatory challenges or lactation have been studied. After nine generations of selection, the divergent selection for RFI led to highly significant (P<0.001) line differences for RFI (−165 g/day in the low RFI (LRFI) line compared with high RFI line) and daily feed intake (−270 g/day). Low responses were observed on growth rate (−12.8 g/day, P<0.05) and body composition (+0.9 mm backfat thickness, P=0.57; −2.64% lean meat content, P<0.001) with a marked response on feed conversion ratio (−0.32 kg feed/kg gain, P<0.001). Reduced ultimate pH and increased lightness of the meat (P<0.001) were observed in LRFI pigs with minor impact on the sensory quality of the meat. These changes in meat quality were associated with changes of the muscular energy metabolism. Reduced maintenance energy requirements (−10% after five generations of selection) and activity (−21% of time standing after six generations of selection) of LRFI pigs greatly contributed to the gain in energy efficiency. However, the impact of selection for RFI on the protein metabolism of the pig remains unclear. Digestibility of energy and nutrients was not affected by selection, neither for pigs fed conventional diets nor for pigs fed high-fibre diets. A significant improvement of digestive efficiency could likely be achieved by selecting pigs on fibre diets. No convincing genetic or blood biomarker has been identified for explaining the differences in RFI, suggesting that pigs have various ways to achieve an efficient use of feed. No deleterious impact of the selection on the sow reproduction performance was observed. The resource allocation theory states that low RFI may reduce the ability to cope with stressors, via the reduction of a buffer compartment dedicated to responses to stress. None of the experiments focussed on the response of pigs to stress or challenges could confirm this theory. Understanding the relationships between RFI and responses to stress and energy demanding processes, as such immunity and lactation, remains a major challenge for a better understanding of the underlying biological mechanisms of the trait and to reconcile the experimental results with the resource allocation theory.

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.

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.

Genetic variability of transcript abundance in pig skeletal muscle at slaughter: Relationships with meat quality traits

A family structured population of 325 pigs (females and barrows) was produced as an intercross between 2 commercial sire lines and was subjected to a systematic transcriptome analysis of LM samples obtained shortly after slaughter. Additionally, measurements of meat quality traits of fresh and cooked loin were gathered from the same animals. The transcriptome analysis was achieved by microarray hybridization, using a custom repertoire of 15,000 6mer DNA probes targeting transcripts expressed in growing pig skeletal muscle. These data allowed us to estimate the heritability of expression abundance for each of the quantified RNA species. The abundance of 9,765 RNA was estimated as heritable with a false discovery rate of 5%, from which 1,174 were deemed as highly heritable (h(2) > 0.50). We also observed a large number of transcripts whose LM expression abundance is genetically correlated with 4 meat quality traits: the loin pH measured at 45 min postmortem (pH45), 253 transcripts; the loin cooking loss (CL), 134 transcripts; the cooked loin shear force (SFc), 184 transcripts; and the loin color redness (a*) value, 190 transcripts. Heritable and meat quality genetically correlated transcripts showed an over-representation of biological processes involved in the induction of apoptosis (genetically correlated with CL), complement activation (genetically correlated with SFc), glucose metabolism (genetically correlated with a*), and cation channel activity (genetically correlated with pH45). Overall, the biological functions highlighted in the highly heritable transcripts and the lack of transcript that would be genetically correlated with LM glycolytic potential suggest that the genetic variability of the LM postmortem transcriptome is focused on muscle tissue response to postmortem ischemia and reflects more distantly the antemortem muscle physiology. Because of the contrasting distributions of the genetic correlations between LM RNA concentrations and the different meat quality traits studied, indirect selection strategies of meat quality traits based on measurements of selected LM RNA species could be only proposed for a subset of the analyzed meat characteristics (pH45, SFc, a*, CL). A substantial improvement in the efficiency of selection for these meat quality traits could result from measuring muscle RNA concentrations on selection candidates, if the same genetic parameters can be verified using in vivo-sampled muscles.