Elisabeth Laville

A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep

Texel sheep are renowned for their exceptional meatiness. To identify the genes underlying this economically important feature, we performed a whole-genome scan in a Romanov × Texel F2 population. We mapped a quantitative trait locus with a major effect on muscle mass to chromosome 2 and subsequently fine-mapped it to a chromosome interval encompassing the myostatin (GDF8) gene. We herein demonstrate that the GDF8 allele of Texel sheep is characterized by a G to A transition in the 3′ UTR that creates a target site for mir1 and mir206, microRNAs (miRNAs) that are highly expressed in skeletal muscle. This causes translational inhibition of the myostatin gene and hence contributes to the muscular hypertrophy of Texel sheep. Analysis of SNP databases for humans and mice demonstrates that mutations creating or destroying putative miRNA target sites are abundant and might be important effectors of phenotypic variation.

Chemical oxidation decreases proteolytic susceptibility of skeletal muscle myofibrillar proteins

The objective of this study was to investigate the effect of chemical oxidation on proteolysis susceptibility of myofibrillar proteins. Myofibrils were prepared from pig M. longissimus dorsi and oxidised by a hydroxyl radical generating system. Protein oxidation level was measured by the carbonyl content, free thiol group content and bityrosine formation. Oxidised or non-oxidised myofibrillar proteins were exposed to papain and proteolysis was estimated by fluorescence using fluorescamine. Oxidation of myofibrillar proteins was dependent upon the oxidising agent concentration. Disulfide bridge and bityrosine formation indicated that oxidation by OH° can induce protein polymerization. Electrophoretic study showed that myosin was the protein most sensitive to oxidation. Results showed a direct and quantitative relationship between protein damages by hydroxyl radical and decreased proteolytic susceptibility. Electrophoretic observations suggest that polymerization and aggregation may explain in part decreased susceptibility of myofibrillar proteins to proteolysis.

Functional Metagenomics Reveals Novel Pathways of Prebiotic Breakdown by Human Gut Bacteria

The human intestine hosts a complex bacterial community that plays a major role in nutrition and in maintaining human health. A functional metagenomic approach was used to explore the prebiotic breakdown potential of human gut bacteria, including non-cultivated ones. Two metagenomic libraries, constructed from ileum mucosa and fecal microbiota, were screened for hydrolytic activities on the prebiotic carbohydrates inulin, fructo-oligosaccharides, xylo-oligosaccharides, galacto-oligosaccharides and lactulose. The DNA inserts of 17 clones, selected from the 167 hits that were identified, were pyrosequenced in-depth, yielding in total 407, 420 bp of metagenomic DNA. From these sequences, we discovered novel prebiotic degradation pathways containing carbohydrate transporters and hydrolysing enzymes, for which we provided the first experimental proof of function. Twenty of these proteins are encoded by genes that are also present in the gut metagenome of at least 100 subjects, whatever are their ages or their geographical origin. The sequence taxonomic assignment indicated that still unknown bacteria, for which neither culture conditions nor genome sequence are available, possess the enzymatic machinery to hydrolyse the prebiotic carbohydrates tested. The results expand the vision on how prebiotics are metabolized along the intestine, and open new perspectives for the design of functional foods.

Characterisation of PSE zones in semimembranosus pig muscle.

Pig semimembranosus muscles, sampled from normal hams or from PSE-zones of defective hams, were analysed by histochemistry and electrophoretic techniques. PSE zones were characterised by a disorganisation of fibre alignment and a significant increase of inter fibre spacing (26.2% vs. 16.9%, p<0.05). Protein solubility was significantly lower in defective muscle (55.4 vs. 91.5mg/g, p<0.001). SDS-PAGE evidenced in such samples a lower abundance of the 97, 40 and 26kDa bands in the sarcoplasmic fraction and a higher abundance of the 97, 58, 34, 31, 15 and 11kDa bands in the myofibrillar fraction. Intensity of the MHC band (200kDa) was lower in PSE zone samples. By 2-D electrophoresis, it was shown that troponin T, MLC 1 and alpha-crystallin were less proteolysed in defective muscles, while creatine kinase fragments were more represented. One form of HSP 27 was absent from PSE zone samples. Overall, meat from PSE-zones and fast pH fall-PSE meat show numerous histological and biochemical similarities, particularly in their protein characteristics.

Differences in Pig Muscle Proteome According to HAL Genotype: Implications for Meat Quality Defects

Bidimensional electrophoresis was used to compare sarcoplasmic protein profiles of early post-mortem pig semimembranosus muscles, sampled from pigs of different HAL genotypes (RYR1 mutation 1841T/C): 6 NN, 6 Nn, 6 nn. ANOVA showed that 55 (18%) of the total of 300 matched protein spots were influenced by genotype, and hierarchical clustering analysis identified 31 (10% of the matched proteins) additional proteins coregulated with these proteins. Fold-changes of differentially expressed proteins were between 1.3 and 21.8. Peptide mass fingerprinting identification of 78 of these 86 proteins indicates that faster pH decline of nn pigs was not explained by higher abundance of glycolytic enzymes. Results indicate further that nn muscles contained fewer proteins of the oxidative metabolic pathway, fewer antioxidants, and more protein fragments. Lower abundance of small heat shock proteins and myofibrillar proteins in nn muscles may at least partly be explained by the effect of pH on their extractability. Possible consequences of lower levels of antioxidants and repair capacities, increased protein fragmentation, and lower extractability of certain proteins in nn muscles on meat quality are discussed.

Early post-mortem sarcoplasmic proteome of porcine muscle related to protein oxidation

Oxidative deterioration or modifications of proteins which appear during meat storage and processes can result in the impairment of technological, sensorial and nutritional qualities. Improving the quality involves a better understanding of the biochemical mechanisms responsible for protein oxidation in meat. For that purpose, an analysis was conducted to investigate the relationships between the early post-mortem sarcoplasmic proteome, which contains the majority of enzymes involved in the oxidative process, and protein oxidation generated during meat storage and cooking. This study was performed in Longissimus lumborum pig muscle. In order to have sufficient variability in the proteome and in the meat oxidation level, five groups of 10 animals issued from two different breeds and raised in three different rearing systems were analysed. Protein oxidation was estimated by the measurement of carbonyl groups after 1 and 4days of refrigerated storage, and after 100°C experimental cooking of the 4days aged meat. Significant correlations (p<0.05) were observed between the level of carbonyl groups and the intensities of 104 spots of the 2D electrophoresis, out of which 52 were clearly identified. The possible involvement of some proteins in the muscle oxidative stress leading to protein oxidation is discussed.

Estimation of genetic trends in French Large White pigs from 1977 to 1998 for growth and carcass traits using frozen semen.

Genetic trends for growth, feed efficiency, composition, and morphometry of carcasses were estimated in a French Large White (LW) pig population using frozen semen. Two groups of pigs were produced by inseminating LW sows with either stored, frozen semen from 17 LW boars born in 1977 or with semen from 23 LW boars born in 1998. In each group, 15 males and 90 females were randomly chosen and mated to produce approximately 1,000 pigs/group. These pigs were performance tested with individual ADFI and serial BW and backfat thickness measurements, slaughtered at 105 kg of BW, and measured for carcass traits. The data were analyzed using mixed linear animal models, including the fixed effect of the experimental group (offspring of 1977 or 1998 boars), the random effect of the additive genetic value of each animal, and, when significant, the fixed effects of sex, fattening batch, and slaughterhouse, the linear regression on BW, and the random effect of the common environment of birth litter. For each trait, the genetic trend was estimated as twice the difference between the 2 experimental groups. Results showed moderately favorable trends for on-test ADG (3.7 +/- 1.3 g/d per year) and feed conversion ratio (-0.014 +/- 0.005 kg/kg per year) in spite of a tendency toward an increase in ADFI (7.6 +/- 4.7 g/yr). A strong reduction in carcass fatness (-0.35 +/- 0.07 mm/yr for carcass average backfat thickness) and a large improvement in carcass leanness (0.31 +/- 0.10 mm(2)/yr and 0.41 +/- 0.08%/yr for loin eye area and carcass muscle content, respectively) were observed. Carcass shape measurements (back and leg length, back width, muscle thickness of hind limbs) were not affected by selection. Serial measurements of BW and backfat thickness showed that the major part of the genetic gains occurred during late growth and that the reduction in the backfat layer was more pronounced in the rear than in the front part of the carcass. The use of frozen semen appears to be a powerful practice to thoroughly investigate changes attributable to selection.

Spatial distribution of myosin heavy chain isoforms and lactate dehydrogenase M4 in the limb musculature of two crossbred lambs

Sixteen different skeletal muscle samples were distributed in the cross-section of eight hip and thigh muscles. Contractile characteristics were assessed by measuring myosin heavy chain (MHCI, MHCIIa, MHCIIb) composition by electrophoresis. Glycolytic capacity was estimated by immunochemical quantitation of the LDH-M4. Histochemistry was used as a reference. The MHC isoform composition of most of the muscles in this study was heterogeneous. When an intramuscular transversal regionality was observed (semitendinosus, vastus lateralis, vastus medialis and rectus femoris muscles), MHCI percentage increased toward deeper layers, while MHCIIb and LDH-M4 decreased. The pattern of MHCIIa isoform distribution was less evident. Within semimembranosus and gluteus medius muscles, proportions of MHC isoforms were constant. Gradients of variation of MHCI and MHCIIb isoforms across rectus femoris and vastus medialis muscles were sharper than those of semitendinosus and vastus lateralis muscles. For the vastus lateralis muscle, these gradients may also be modified according to the breed. Breed effect was mainly shown by MHCIIb and MHCI isoforms and was not observed at all the sampling points of the muscles. These observations show that breed effect on muscle contractile and metabolic characteristics is not uniformly expressed throughout the muscle. Results of a comparison may differ according to the muscle and sampling location.

Early post-mortem sarcoplasmic proteome of porcine muscle related to lipid oxidation in aged and cooked meat

In order to identify specific markers of lipid oxidation generated in meat during refrigerated storage and cooking an analysis was conducted to investigate the relationships between the early post-mortem sarcoplasmic proteome, which contains the majority of enzymes involved in the oxidative process, and the level of lipid oxidation. This study was performed in Longissimus lumborum pig muscle. Proteome was analysed by 2-D electrophoresis in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and lipid oxidation was estimated by the TBA reactive substances (TBA-RS) measurement. Many markers of lipid oxidation were identified, but no single marker covered the oxidative process in its entirety. The role of five protein groups (albumin, redoxins, annexins, lipid transporters and enzymes of aerobic respiration), from which a link with lipid oxidation can be established, is discussed. This study, which completes a precedent work focused on protein oxidation, clearly demonstrates that a combination of several markers is needed to assess the sensitivity of meat to oxidation during both ageing and cooking.

Estimation of the muscle to bone ratio of the bovine pelvic limb using a morphometric method

Conformation is an indicator of carcass composition. The aim of this study was to derive an equation for estimating the composition of the bovine pelvic limb using morphometric variates. From a mixed group of 38 French bred bovines, the sample was chosen to have a wide range of conformation. The muscle to bone ratio (M B ) was used as an index of composition. The carcasses were weighed and the other variates of the equation were measured on carcass photographs: M B = 6.2 + 0.005W - 0.62EF AB + 12GH with W: carcass weight (kg); GH: medio-lateral diameter of the distal part of the leg (cm); EF AB : medio-lateral diameter of thigh (cm)/leg length(cm); (R(2) = 0.91 and rsd = 0.27).