domain: sdv info shs

text:
A Dubost

D Micol

C Lethias

A Listrat
email: anne.listrat@clermont.inra.fr

New insight of some extracellular matrix molecules in beef muscles. Relationships with sensory qualities

Keywords: beef, muscle, connective tissue, extracellular matrix, sensory traits Implications

The aim of this study was to highlight the relationships between decorin, tenascin-X and type XIV collagen, three minor molecules of extracellular matrix (ECM), with some structural parameters of connective tissue and its content in total collagen, its cross-links (CLs) and its proteoglycans (PGs). In addition, we have evaluated impact of these minor molecules on beef quality traits. The relative abundance of these molecules was evaluated by western blot analysis in Longissimus thoracis (LT) and Biceps femoris (BF) muscles from Aberdeen Angus and Blond d'Aquitaine beef breeds. Decorin and tenascin-X were more abundant in BF than in LT (1.8 v. 0.5 arbitrary units (AU), respectively, P < 0.001, and 1.0 v. 0.6 AU, P < 0.05). There was no muscle effect for collagen XIV content. Decorin and tenascin-X relative abundance were positively correlated with perimysium and endomysium areas and with collagen characteristics (total, insoluble and CLs). Decorin was negatively correlated with total PG content and positively with tenascin-X. Collagen XIV was correlated with any of parameters measured. To assess the impact of decorin, tenascin-X and collagen XIV and of their ratios to total collagen and PGs on shear force and quality traits we realized, respectively, a multiple-linear regression analysis and a Pearson's correlation analysis. Decorin and tenascin-X relative abundance were, respectively, negatively and positively involved in juiciness. Decorin relative abundance was also negatively involved in abnormal flavour and positively in overall liking. The ratio of decorin to total collagen and PGs was negatively correlated to juiciness, together with collagen XIV ratio to total PGs. The ratios of decorin, tenascin-X and collagen XIV to total PGs were positively correlated to sensory tenderness, negatively to abnormal beef flavour and positively to overall liking. The ratio of decorin to total collagen was also negatively correlated to abnormal flavour and positively to overall liking while its ratio to total PGs was positively correlated to beef flavour and overall liking. Results of the present study highlighted for the first time the possible role of minor ECM molecules on beef quality traits. In addition, variations of meat texture and more generally of sensory qualities would depend not only to the quantity of total collagen and of its CLs, but also of components of ECM such as decorin, tenascin-X and collagen XIV and of their ratios to total collagen and PGs.

Introduction

Extracellular matrix (ECM) is the ground substance of connective tissue (CT), which is structured, by perimysium that bundles muscle fibres and by endomysium that surrounds individual skeletal muscle fibres. ECM is a dynamic network of molecules mainly composed of structural collagens but also of many other minor components. Among them, we can mention collagen XIV, a minor collagen, decorin, a proteoglycan (PG) and tenascin-X, a noncollagenic glycoprotein [START_REF] Velleman | Role of the extracellular matrix in muscle growth and development[END_REF]. They interact with structural collagens and each other (Font et al., 1993; Weber et al., 1996; Elefteriou et al., 2001; Lethias et al., 2006) and are co-localized in perimysium (Listrat). These interactions might have important biological implications on building of extracellular networks, alter flexibility of the ECM and, as a result, its mechanical properties [START_REF] Nishiyama | Type XII and type XIV collagens mediate interactions between banded collagen fibers in vitro and may modulate extracellular matrix deformability[END_REF]. This is confirmed by in vivo studies in mice showing that the inactivation of the decorin or tenascin-X genes leads to a weakness of CT [START_REF] Danielson | Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility[END_REF][START_REF] Mao | Tenascin-X deficiency mimics Ehlers-Danlos syndrome in mice through alteration of collagen deposition[END_REF] and by in vitro studies showing that addition of tenascin-X or collagen XIV to collagen gels modify the mechanical properties of these gels [START_REF] Nishiyama | Type XII and type XIV collagens mediate interactions between banded collagen fibers in vitro and may modulate extracellular matrix deformability[END_REF][START_REF] Margaron | Tenascin-X increases the stiffness of collagen gels without affecting fibrillogenesis[END_REF]. From this, we have hypothesized that decorin, tenascin-X and collagen XIV might, in association with structural collagens, play a role on mechanical properties of CT and thereby on meat beef quality traits.

Beef meat tenderness is generally attributed to structural collagen (generally referred to as total collagen), to its cross-links (CLs) and to its structural parameters. However, these parameters explain only a small part of changes in tenderness. This may be due to the fact that variations of texture would depend not only of the quantity of total collagen and of its CLs, but also of interactions between structural collagens and some minor components present in the ECM, such as those cited above.

In spite of hypothetical roles of ECM molecules in mechanical properties and in juiciness of poultry meat [START_REF] Velleman | Role of the extracellular matrix in muscle growth and development[END_REF], no direct relationship between ECM molecules and beef sensory quality has been reported.

The aim of this study was to analyze: (i) the muscle variations on decorin, tenascin-X and collagen XIV relative abundances, (ii) the relationships between these three molecules, perimysium and endomysium characteristics, collagen amount (total and insoluble contents) and its CLs and total PGs and (iii) to precise the effects of these ECM molecules on mechanical and sensory properties of beef meat.

Material and methods

The study was carried out in compliance with the French recommendations and those of the Animal Care and Use Committee of the National Institute for Agricultural Research (INRA, Institut National de la Recherche Agronomique) of Clermont-Ferrand/Theix, France for the use of experimental animals including animal welfare.

Beef production and muscle sampling

The experiment was performed on young bulls of Aberdeen Angus (n = 12) and Blond d'Aquitaine (n = 14) pure breeds. The former are early maturing breed [START_REF] Cuvelier | Performance, slaughter characteristics and meat quality of young bulls from Belgian Blue, Limousin and Aberdeen Angus breeds fattened with a sugar-beet pulp or a cereal-based diet[END_REF] whereas the latter are late maturing breed [START_REF] Listrat | Grass valorisation and muscular characteristics of blonde d'Aquitaine steers[END_REF]. Animals were assigned for a 100-day finishing period. The animals were housed in straw pens and individually fed. Diets consisted of concentrate (75%) and straw (25%). Animals were slaughtered, on average, at 17 months and at a final live weight of 665 kg. Longissimus thoracis (LT) muscle was taken from the 9th rib and Biceps femoris (BF) sample was taken from the middle of the muscle. Both were chosen due to their skeletal purpose. LT is a positional muscle and BF a locomotion muscle, LT is more oxidative than BF muscle [START_REF] Totland | Distribution patterns of muscle fibre types in major muscles of the bull (Bos taurus)[END_REF], LT has less total and insoluble collagen and CL contents than BF (Dubost et al., 2013a). These muscles and these breeds had been chosen to create a large variability within samples.

About structural (histology), PG and western blot analysis, muscle samples were taken 15 to 25 mn following exsanguination. For histological analysis, muscle samples of 1.5 × 1.5 × 1 cm were taken, fixed on a piece of cork and frozen in isopentane chilled in liquid nitrogen. About PG and western blot analyses, muscle samples (60 to 80 g) were cut up into small pieces, frozen and powdered in liquid nitrogen. The samples were stored at -80°C until analyses.

Carcasses were chilled in a cold dressing room (+2°C) and muscle samples of collagen and CL measurements (about 150 g) were taken up 24 h postmortem. They were cut into pieces of 1 cm cross-section, sealed under vacuum in plastic bags and frozen. Frozen muscle was homogenized in a household cutter, freeze-dried for 48 h, pulverized in a horizontal blade mill and finally stored at +4°C in stopper plastic flasks until analyses. About beef quality traits sensory evaluation, meat samples were aged in vacuum packs at +4°C for 14 days, packed under vacuum and stored at -20°C until Warner-Bratzler shear force measurement and sensory analysis.

Perimysium and endomysium characteristics Muscle blocks (1.5 × 1.5 × 1 cm) were fixed with Tissue-Tek ® OCT TM (Sakura Finetek, Villeneuve d'Ascq, France) in a cryostat (HM 500M; Microm Microtech, Francheville, France) maintained at -25°C and cross-sections of 10 µm thick were prepared. The cross-sections were stained according the Picro-Sirius red method previously described by [START_REF] Flint | Demonstration of collagen in meat-products by an improved picro-sirius red polarization method[END_REF].

Stained cross-sections were then analyzed by image analysis with two home-made programs developed by our team using Visilog 6.7 Professional Software (Noesis, Gif-sur-Yvette, France): Perimysium Analyzer to study perimysium, and Endomysium Fibre Analyzer to study endomysium, previously described (Dubost et al., 2013a). About perimysium study, each stained muscle section (1.5 × 1.5 cm) was scanned entirely in transmission mode using an EPSON (Levallois Perret, France) Expression 10000XL PRO A3 scanner (resolution of 2400 dpi, corresponding to a spatial resolution of 10.58 µm/pixel). For endomysium, stained sections were visualized in bright field mode with an Olympus BX51 microscope (Olympus, Tokyo, Japan) using a 10 × objective (NA = 0.3). Five images were acquired randomly from the stained sections of 1.5 × 1.5 cm (the same as used for perimysium study) with a DP-72 colour camera and Cell-D image acquisition software (Olympus Soft Imaging Solutions, Münster, Germany). The resolution was 1360 × 1024 pixels, representing an 879 × 662 µm field of view. The resulting spatial resolution was 0.65 µm/pixel.

Perimysium, area (percentage of the total image) and number of branch points per cm² were determined. Only area of endomysium (percentage of the total image) was determined.

Collagen and CL measurements Total and insoluble collagen were determined according to the procedures of [START_REF] Woessner | The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid[END_REF] and [START_REF] Hill | The solubility of intramuscular collagen in meat animals of various ages[END_REF], previously described and updated by [START_REF] Listrat | Effect of the type of diet on muscle characteristics and meat palatability of growing Salers bulls[END_REF]Dubost, Micol, Lethias and Listrat Listrat and Hocquette, 2004). Both for total and insoluble collagen, each sample was weighed and measured in triplicate and data were expressed in milligrams of collagen per gram of dry matter (mg/g DM), using the factor 7.14 as ratio corresponding to the mean hydroxyproline content of the collagen present in intramuscular CT.

About CLs measurement, 1 ml of acid hydrolysate (in HCl 6 N) from total collagen was centrifuged at 16 000 × g for 5 min at +4°C. Then 600 µl of 6 N NaOH and 600 µl of 1 M Tris were added to 600 µl of acid supernatant. Final pH was adjusted between 7 and 8 with a few microliters of 6 N HCl or NaOH. Pyridinoline CLs were determined by the enzymelinked immunoassay Metra Pyd EIA kit (Quidel Corporation, San Diego, USA) according to the manufacturer. Previous assays had indicated no interfering effect of the 1/10 diluted muscle extract on the pyridinoline CL determination (data not shown). Results were expressed in nanomolar of pyridinoline per gram of dry matter (nM pyr/g DM) and in millimolar of pyridinoline per mole of collagen (mM pyr/M collagen) (assuming that collagen had a molecular weight of 300 000 Da) [START_REF] Etherington | Detection and estimation of collagen[END_REF].

Total PG content Muscle powder was incubated 24 h at +4°C with extraction buffer (6 M Urea, 1 M NaCl, 2% CHAPS and protease inhibitor cocktail (Complete; Roche Diagnostics GmbH, Meylan, France)). The solid-to-liquid ratio was 100 mg of muscle powder to 1 ml of extraction buffer. The next day, samples were centrifuged 40 min at +4°C, 15 000 g. Supernatant (muscle extract) was recovered and used to determine PG content according to a modified [START_REF] Barbosa | Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies[END_REF] Farndale's method [START_REF] Farndale | A direct spectrophotometric micro-assay for sulfated glycoaminoglycans in cartilage cultures[END_REF], based on the ability of sulphated glycosaminoglycans (GAGs) to bind the cationic dye 1,9-dimethylmethylene blue. Each sample was measured twice and data were expressed in µg of chondroitin-4-sulphate (C4S)-GAGs per gram of dry matter (µg C4S-GAGs/g DM) and in milligram of GAGs per gram of collagen (mg GAGs/g collagen).

Western blot analyses of decorin, tenascin-X and type XIV collagen Decorin, tenascin-X and collagen XIV were extracted with radio immunoprecipitation assay lysis buffer (150 mM NaCl, 10 mM Tris-HCl, 1 mM EGTA, 1 mM EDTA, adjusted at pH = 7.4 and completed with 100 mM sodium fluoride, 4 mM sodium pyrophosphate and 2 mM orthovanadate, 1% Triton 100 × , 0.5% Igepal CA-630 and protease inhibitor cocktail (Complete)). After extraction, protein concentration was determined by spectrophotometry (UVIKON 860, Serlabo Technologies, Entraigues, France) with Bradford assay [START_REF] Bradford | A rapid and sensitive method for quantification of microgram quantities of protein utilizing principle of protein-dye binding[END_REF].

Proteins were separated in denaturing conditions (10% SDS-polyacrylamide and β-mercaptoethanol). Samples were loaded on the gel (stacking gels of 4% and separation gels of 6%) at the rate of 30 µg of proteins for decorin and 50 µg for tenascin-X and collagen XIV. A mix of all samples was deposited (30 µg of proteins for decorin, 50 µg for collagen XIV and tenascin-X) on all the gels for normalization. Gels were run at 80 V for 20 min and then 120 V for 1 h, at +4°C.

Bands were transferred to a polyvinyl difluoride membrane (ref. IPVH00010; Millipore, Saint Quentin en Yvelines, France) at 120 mA for 5 h, at +4°C. Unspecific binding of antibodies to the membranes were blocked with 10% milk T-TBS 1 × blocking buffer at 37°C for 20 min. Membranes were washed 3 × for 5 min in T-TBS 1 × and then incubated overnight at +4°C with the primary antibodies. The three primary antibodies were monoclonal mouse anti-bovine antibodies. Anti-decorin (clone DS1) and anti-type XIV collagen (clone 15B8) were diluted to 1/50 and anti-tenascin-X (clone 8E7) was diluted to 1/25 in T-TBS 1 × . Anti-decorin was from Developmental Studies Hybridoma Bank, and anti-collagen XIV and anti-tenascin-X have been characterized by [START_REF] Lethias | Expression of type XIV collagen during the differenciation of fetal bovine skin: immunolabelling with monoclonal antibody is prominent in morphogenetic areas[END_REF] and [START_REF] Lethias | Identification and characterization of a conformational heparin-binding site involving two fibronectin type III modules of bovine tenascin-X[END_REF], respectively. Membranes were washed 2 × for 10 min in T-TBS 1 × and hybridized with the second antibody associated with horseradish peroxidase for chemiluminescence detection (IgG sheep anti-mouse, NA931; Amersham, GE Health Care, Vélizy Villacoublay, France) diluted to 1/5000.

Bands corresponding to the studied molecules were quantified under Image Quant software. Each band was normalized by the mix of the samples deposited on each gel. Quantities of decorin, tenascin-X and collagen XIV were expressed in arbitrary units (AU) or per milligram of total collagen or per microgram PGs in DM.

Meat quality evaluation About Warner-Bratzler shear force measurement, LT muscle cuts (3 cm thick pieces parallel as possible to the muscle fibres long axis) were grilled up to an internal temperature of 55°C and BF muscle cuts were cooked in a water bath (hermetically sealed in plastic bags) for 1 h to an internal temperature of 70°C

. Inside these cuts of 3 cm, 10 test pieces of 1 cm of crosssection shear forces were evaluated perpendicularly to muscle fibres using INSTRON 5944 (Elancourt, France). Forces at rupture were expressed in N/cm². About sensory analysis, LT steaks of 1.5 cm thickness were grilled up to an internal temperature of 55°C while BF muscle cuts (3 cm thick pieces parallel as possible to the muscle fibres long axis) were cooked in a water bath (hermetically sealed in plastic bags) during 1 h to an internal temperature of 70°C. At each sensory session, 12 panellists evaluated six different samples of the same muscle, randomly selected. Panellists evaluated six quality traits: total tenderness, juiciness, beef flavour (intensity of the beef flavour of the meat), abnormal flavour, residues after chewing and overall liking, on a continuous scale scored from 0 to 10 (0 = hard, dry, not tasty up to 10 = tender, juicy, tasty) [START_REF] Dransfield | Meat quality and composition of three muscles from French cull cows and young bulls[END_REF].

Statistical analyses

Structural (perimysium and endomysium characteristics), biochemical (total collagen and its CLs, total PGs, decorin, tenascin-X and collagen XIV) data and the proportions of decorin, tenascin-X and collagen XIV to total collagen or PGs were submitted to ANOVA. We calculated these proportions Muscle ECM molecules and sensory beef qualities to determine if for the same quantity of collagen or PGs, the proportion of decorin, tenascin-X and collagen XIV was constant. All results were presented as LS means with appropriate SEM and the level of significance. Differences between the two muscles were claimed significant for P < 0.05. The model included also breed effect. The breed effect and the interactions between muscle and breed were not significant, then only muscle effect was presented in this paper. Pooled western blot data from the two muscles, for decorin, tenascin-X and collagen XIV expressed in AU were submitted to Pearson's correlation analysis, respectively, with structural and biochemical parameters of CT. It was possible to pool data from the two muscles, because both populations were evenly distributed homogeneously. ANOVA and Pearson's correlations were achieved using XLSTAT-Pro 2012.3.01. The REG procedure of SAS (1990, SAS Institute Inc.; Cary, NC, USA) was used to study the explained variance and regression coefficients of decorin, tenascin-X and collagen XIV relative abundance on beef quality traits from pooled data of the two muscles. In addition, to verify if the ratios of decorin, tenascin-X and collagen XIV to total collagen or PGs could explain quality parameters, Pearson's correlations were achieved on pooled data of the two muscles.

Results

Muscle effect on ECM characteristics BF and LT muscles were significantly different for all the measured parameters except for collagen XIV for which there was no difference. Parameters relative to perimysium, endomysium, collagen and its CLs, decorin and tenascin-X were significantly higher in BF than in LT muscle. Conversely there was significantly less PGs in BF than in LT muscle. Decorin ratio to total collagen was higher in the BF muscle than in the LT muscle while tenascin-X ratio to total collagen was not different between muscles. Proportions of decorin and tenascin-X to total PGs were higher in the BF muscle than in the LT (Table 1).

Relationships between CT structural and biochemical characteristics and ECM molecules

Table 2 summarized relationships between structural and biochemical characteristics of intramuscular CT and ECM molecules. Decorin and tenascin-X were positively correlated with perimysium area (r = 0.52 and 0.34, respectively), branch points of perimysium (r = 0.36 and 0.31, respectively) and endomysium area (r = 0.57 and 0.35, respectively). Decorin and tenascin-X were also positively correlated with CL content (r = 0.58 and 0.31, respectively), total collagen (r = 0.65 and 0.36) and with insoluble collagen (r = 0.60 and 0.37) content. There was no correlation between tenascin-X and total PGs, but there was a negative one between decorin and total PGs (r = -0.49). Decorin and tenascin-X (expressed in AU) were positively correlated together (r = 0.48). No correlation was found between type XIV collagen and the other parameters. Mean and SEM for all significant parameters are presented. For P < 0.05, 0.01 and 0.001, within a row, means differ significantly between the muscles. Dubost, Micol, Lethias and Listrat Relationships between beef quality traits and ECM characteristics Table 3 summarizes quality traits obtained after cooking of BF and LT muscles. BF and LT muscles have been cooked according two types of cooking in compliance with consumer habits. So we do not compare the results obtained for the two muscles. Table 4 summarized multi-regression analysis of meat quality traits with ECM molecules. In this model, neither tenderness, nor shear force variations were explained by the studied ECM molecules. Regression model of juiciness showed that 31% of variation (P < 0.01) was due for 19% to decorin and for 12% to tenascin-X (P < 0.05, decorin being negatively involved, and tenascin-X being positively involved). Abnormal flavour was explained at 38% by decorin (P < 0.001) and at 7% by collagen XIV (P > 0.05) (result not shown). A total of 21% of the overall liking variation were explained positively and mainly by decorin (P < 0.05).

Table 5 showed Pearson's correlations between decorin, tenascin-X or collagen XIV ratios to total collagen or PGs and data from meat quality evaluation. No ratio was correlated with shear force; tenascin-X ratio to total collagen was correlated with no meat quality parameter. Tenderness was positively correlated with decorin, tenascin-X and collagen XIV ratio to total PGs (+0.29; +0.31; +0.34, respectively). Juiciness was negatively correlated with decorin ratio to total collagen or PGs (-0.42 and -0.40, respectively). Beef flavour was positively correlated with decorin and collagen XIV ratio to total PGs (+0.40 and +0.38, respectively). Abnormal flavour and overall liking were, respectively, negatively and positively correlated with decorin ratio to total collagen or PGs, to tenascin-X ratio to total PGs and to collagen XIV ratio to total PGs.

Discussion

Endomysium and perimysium, the two subdivision of the meat intramuscular CT (IMCT) are composite networks of collagen embedded in a matrix of PGs. Major collagen types of muscle are fibrillar collagens. In the adult bovine muscle, the fibrillar collagens are linked between each other by different chemical CLs. Three reducible (DHLNL, HLNL and HHMD) and two non-reducible CLs have been highlighted (Ngapo et al., 2002). The reducible CLs concentrations increase from foetal life to about 18 months, after which an exponential decrease is observed. The two non-reducible CLs (pyridinoline and Ehrlich chromogen (EC)) are highly correlated [START_REF] Lepetit | A theoretical approach of the relationships between collagen content, collagen cross-links and meat tenderness[END_REF]. EC increases to a maximum concentration at about 1 year, and thereafter decreases exponentially. In contrast, pyridinoline concentration increases with animal age [START_REF] Ngapo | Perimysial collagen crosslinking in Belgian Blue double-muscled cattle[END_REF] and, consequently, is the major CLs of adult skeletal muscle.

Previously we shown [START_REF] Sifre | Influence of the spatial organization of the perimysium on beef tenderness[END_REF]Dubost et al., 2013b) that both IMCT organization, total collagen, pyridinoline and total PGs contents were involved in sensorial and mechanical meat properties but that they were far from explaining all variability measured. +0.57 0.12 <0.05 1 For decorin and tenascin-X (expressed in arbitrary units) we presented on the first line, the coefficient of the variable in the model, on the second line the partial R² of the variable, on the third line, the P-value of the variable. It is now accepted that major collagen types (type I and III), form large multi-protein complexes with on their surface some minor collagens, glycoproteins and some small PGs such as collagen XIV, tenascin-X and decorin, respectively. To our knowledge, any study has attempted to estimate the involvement of these molecules on meat beef qualities.

To do it, two beef muscles are been studied, LT and BF differing in collagen characteristics and meat quality traits. First, we have studied relationships between abundances of collagen XIV, tenascin-X and decorin, and of total and insoluble collagen, of CLs and total PGs and secondly the involvement of collagen XIV, tenascin-X and decorin in beef quality traits.

The results highlighted a muscle effect for decorin and tenascin-X. Decorin and tenascin-X were four and two times, respectively, higher in BF than in LT muscle. For decorin, this result was in accordance with the only study on the subject, that of [START_REF] Pedersen | Decorin and fibromodulin expression in two bovine muscles (M-semitendinosus and M-psoas major) differing in texture[END_REF], who had also found that Semitendinosus (ST) contained 1.5 times more of decorin that Psoas major (PM) muscle. The ST muscle as the BF muscle had more collagen and a lower PGs ratio to collagen than PM and LT muscles, respectively. To our knowledge, no result of this type is available in the literature regarding tenascin-X. In addition, we have shown a high positive correlation between total collagen and its CLs, decorin, tenascin-X and IMCT area. Tenascin-X mutation leads to CT disorders with primary skin and joint involvement. In skeletal muscle, its mutation results in weakness, atrophy [START_REF] Voermans | Reduced quantitative muscle function in tenascin-X deficient Ehlers-Danlos patients[END_REF][START_REF] Voermans | Neuromuscular involvement in various types of Ehlers-danlos syndrome[END_REF] and some features of myopathy described in several patients (Penisson-Besnier et al., 2013) and in tenascin-X deficient mice [START_REF] Voermans | Mild muscular features in tenascin-X knockout mice, a model of Ehlers-danlos syndrome[END_REF]. Mice with a decorin deficiency display abnormal fibril structure and organization, as well as fragile tissues with decreased strength and stiffness. These data associated to ours suggest that the locomotor muscles such as BF and ST would have a stiffer ECM than postural ones. This stiffness would be due to their higher content in collagen and CLs but also to their higher content in minor molecules. This difference of composition would impact the area occupied by the IMCT.

It has been shown in vitro that decorin and tenascin-X interact with the fibrillar collagens (particularly type I) (Svensson et al., 1995; Lethias et al., 2006), and that type XIV collagen interacts with decorin [START_REF] Font | Characterization of the interactions of type XII collagen with two small proteoglycans from fetal bovine tendon, decorin and fibromodulin[END_REF][START_REF] Ehnis | Localization of a binding site for the proteoglycan decorin on collagen XIV (undulin)[END_REF][START_REF] Elefteriou | Binding of tenascin-X to decorin[END_REF]. Tenascin-X and collagen XIV could modulate the flexibility of the ECM and, as a result, its mechanical properties. Their addition to collagen gels increases compressive resistance of these gels [START_REF] Nishiyama | Type XII and type XIV collagens mediate interactions between banded collagen fibers in vitro and may modulate extracellular matrix deformability[END_REF][START_REF] Margaron | Tenascin-X increases the stiffness of collagen gels without affecting fibrillogenesis[END_REF]. Decorin could also play a role in mechanical properties of tissues, however, its role has not been elucidated [START_REF] Lewis | Effect of decorin and dermatan sulfate on the mechanical properties of a neocartilage[END_REF][START_REF] Dourte | Influence of decorin on the mechanical, compositional, and structural properties of the mouse patellar tendon[END_REF].

Our study shows a high muscle effect on the decorin ratio to collagen or PGs and tenascin-X to PGs. As a matter of fact the decorin ratios to collagen and PGs were 2.4 and two times, respectively, higher in BF than in LT muscle and the tenascin-X ratio to PGs twice higher in BF than in LT muscle. So for each muscle there might be a balance between the different ECM molecules.

In the present study, we failed to show a role of decorin, tenascin-X or collagen XIV in mechanical properties of meat beef; however, their ratios to PGs were positively correlated to tenderness and overall liking in the sensory evaluation. These molecules could have an additive role on mechanical properties and tenderness of meat by increasing the complexity of ECM by the interactions that they develop between them and with the fibrillary collagens and PGs. It would be more the respective proportion of different ECM molecules that would play a role on meat quality than their absolute quantities. They could participate to increase the area of IMCT and then the hardness of meat, since the muscles the richest in collagen are also the hardest [START_REF] Lepetit | A theoretical approach of the relationships between collagen content, collagen cross-links and meat tenderness[END_REF]. As suggested by [START_REF] Nishimura | The role of intramuscular connective tissue in meat texture[END_REF] for PGs, during postmortem aging and cooking, they could be degraded, which would facilitate the denaturation of fibrillary collagen.

Negative relationship between decorin and total PG content was pointed out. PGs are divided in two families, aggregating and non-aggregating PGs that have distinct properties. The first have a high molecular weight (core protein >200 kDa) and would participate to maintain tissue hydration through their high anionic charge which generates an osmotic gradient and promotes the retention of water [START_REF] Velleman | Role of the extracellular matrix in muscle growth and development[END_REF]. Decorin belongs to the family of non-aggregating PGs. The role of this PG is to maintain space between collagen fibrils, hence tissue hydration, and movement of water soluble components [START_REF] Scott | Supramolecular organization of extracellular matrix glycosaminoglycans, in vitro and in the tissues[END_REF]. From their properties, [START_REF] Velleman | Role of the extracellular matrix in muscle growth and development[END_REF] has deduced that aggregating and non-aggregating PGs could play a role in juiciness. Dubost et al. (2013b) had shown that total PG content influenced negatively juiciness of cooked meat according to Dubost, Micol, Lethias and Listrat muscle and the cooking mode. In this paper our results showed also a negative involvement of decorin in beef cooked meat juiciness. The multi-regression analysis showed that tenascin-X would be involved positively in juiciness. Juiciness depends on the amount of water retained in the cooked meat namely water-holding capacity of muscle [START_REF] Pearce | Water distribution and mobility in meat during the conversion of muscle to meat and ageing and the impacts on fresh meat quality attributesa review[END_REF]. In our knowledge, previously, no role of tenascin-X on waterholding capacity has been shown.

These preliminary results give new insights on the role of IMCT and its ECM on beef meat. Further investigations would be essential to validate them, both on raw and cooked meat, with a greater number of observations and an experimental design with a higher variability.

Conclusion

Results of this study highlighted a muscle effect on decorin and tenascin-X abundances and their ratio to total collagen or PGs. The originality of this study was to have shown the positive relationship between the relative abundance of these two molecules and some structural parameters of perimysium and endomysium of CT. This supports the role of these molecules on CT organization. In addition, impact of relative abundance of these minor ECM molecules and of their proportions to total collagen or PGs on beef quality traits was highlighted for the first time. These results allow assuming that to understand the role of CT on the meat quality we do not have to consider the molecules that compose it one by one but as a whole.

2

  Beef quality traits were evaluated by sensory panel on a continuous scale scored from 0 to 10.

Table 1

 1 Effect of muscle on structural parameters of bovine intramuscular connective tissue and composition of its extracellular matrix

		Biceps femoris	Longissimus thoracis	SEM	P-value
	Perimysium
	Area (%)	9.44	6.03	0.56	<0.001
	Number of branch points cm -2	87.64	73.83	5.88	<0.05
	Endomysium area (%)	6.57	4.71	0.29	<0.001
	Collagen
	Total (mg/g DM)	7.45	4.40	0.24	<0.001
	Insoluble (mg/g DM)	4.77	3.19	0.18	<0.001
	Cross-links (CLs)
	CLs (nM pyr/g DM)	38.37	19.52	2.36	<0.001
	CLs (mM pyr/M collagen)	0.22	0.19	0.01	<0.01
	Proteoglycans (PGs) (µg C4S-GAGs\g DM)	439.28	639.56	34.26	<0.001
	Decorin (Dcn)
	Dcn (AU)	1.80	0.46	0.16	<0.001
	Dcn/col (AU/mg per g DM)	0.24	0.10	0.02	<0.001
	Dcn/PGs (AU/µg C4S-GAGs per g DM)	0.0040	0.0008	0.0002	<0.001
	Tenascin-X (Ten-X)
	Ten-X (AU)	1.01	0.58	0.16	<0.05
	Ten-X/col (AU/mg per g DM)	0.13	0.13	0.02	>0.05
	Ten-X/PGs (AU/µg C4S-GAGs per g DM)	0.0020	0.0010	0.0004	<0.01
	Collagen XIV (Col XIV)
	Col XIV (AU)	1.58	1.31	0.23	>0.05
	Col XIV/col (AU/mg per g DM)	0.21	0.31	0.06	>0.05
	Col XIV/PGs (AU/µg C4S-GAGs per g DM)	0.0020	0.0022	0.0004	>0.05

DM = dry matter; AU = arbitrary units; C4S-GAGs = chondroitin-4-sulphate-glycosaminoglycans.

Table 2

 2 Pearson's correlation coefficients between decorin, tenascin-X and collagen XIV and structural and biochemical characteristics of bovine intramuscular connective tissue

		P area	Branch points	E area	Total col Insoluble col Total CLs	Total PGs	Decorin Tenascin-X Col XIV
	Decorin	0.52***	0.36***	0.57*** 0.65***	0.60***	0.58***	-0.49***	1	0.48***	0.03
	Tenascin-X 0.34**	0.31*	0.35**	0.36**	0.37**	0.31*	-0.29*	0.48**	1	0.07
	Col XIV	0.10	0.01	0.13	0.13	0.15	0.14	-0.09	0.03	0.07	1

P = perimysium; E = endomysium; Col = collagen; CLs = cross-links; PGs = proteoglycans. Correlations were significant for *P < 0.05, **P < 0.01), ***P < 0.001.

Table 4

 4 Multiple-linear regression analysis of beef quality traits with extracellular matrix moleculesJuiciness 2 Abnormal beef flavour 2 Overall liking 2

	Model R²	0.31	0.45	0.21
	Model P-value	<0.01	<0.001	<0.05
	Decorin 1	-0.67	-0.51	+0.46
		0.19	0.38	0.17
		<0.05	<0.001	<0.05
	Tenascin-X 1

Table 3

 3 Mean of quality traits of bovine Biceps femoris (BF) and Longissimus thoracis (LT) muscles obtained after cooking for all animals

	BF 1	LT 1

Table 5

 5 Pearson's correlation coefficients between ratios of decorin, tenascin-X, collagen XIV to total collagen or PGs and quality traits in beef meat Dcn = decorin; Col = collagen; PGs = proteoglycans; Ten = tenascin. Correlations were significant for *P < 0.05, **P < 0.01, ***P < 0.001.

		Shear force	Tenderness	Juiciness	Beef flavour	Abnormal flavour	Overall liking
	Dcn/total Col	+0.20	+0.16	-0.42**	+0.14	-0.50**	+0.30*
	Dcn/total PGs	+0.20	+0.29 *	-0.40**	+0.40**	-0.64**	+0.55**
	Ten-X/total Col	+0.05	+0.25	+0.19	-0.06	-0.04	+0.01
	Ten-X/total PGs	+0.004	+0.31 *	+0.03	+0.28	-0.42**	+0.48**
	Col XIV/total Col	-0.08	+0.13	+0.09	-0.20	+0.14	-0.20
	Col XIV/total PGs	+0.07	+0.34 *	-0.25	+0.38 *	-0.50**	+0.45**

Sensory traits were evaluated by sensory panel on a continuous scale scored from 0 to 10.

Acknowledgements

The authors thank the staff of Experimental Unit (UERT) of INRA (Theix) for animal management, slaughtering and Warner-Bratzler shear force measurements, the staff of Amuvi (Animal, Muscle, Viande) team for muscle sampling and Arnaud Delavaud for western blot contribution. The authors also thank the staff and panellists of Experimental Unit of INRA (Le Magneraud) for sensory evaluation.


Ahmed Sami
email: assami70@yahoo.com

Edward Mills

Jean-François Hocquette

Relationships between DNAJA1 Expression and Beef Tenderness: Effects of Electrical Stimulation and Post-mortem Aging in two Muscles

Keywords: DNAJA1, Tenderness, Beef muscles, Electrical stimulation, Ageing

This study aimed to check if if relationships between DNAJA1 expression and beef tenderness is valid in two muscles, which differ in toughness from Angus steers with or without electric stimulation at different times of post-mortem ageing. Electric stimulation did not significantly affect DNAJA1 expression at the time of slaughter. But, RNA degradation soon after slaughter was a major factor, which affected the assessment of DNAJA1 expression, even at 3 h post-mortem. Correlation of DNAJA1 expression at slaughter with shear force values varied according to ageing time (24 h, 3, 7, 14 or 21 days post-mortem) or according to the use of electric stimulation and was not significantly correlated with any of these shear force peak values despite a few high correlation values (-0.60 or +0.34). In conclusion, DNAJA1 is not an omnipotent marker of beef toughness with this particular data set since the correlation of its expression level with shear force values varies according to slaughtering conditions and ageing time.

Introduction

Tenderness, juiciness and flavor affect beef palatability, with tenderness being the most economically important trait [START_REF] Boleman | Consumer evaluation of beef of known categories of tenderness[END_REF]. Due to advances in genomics, researchers have identified previously unrecognized genes associated with meat quality. Gene expression controls the biological characteristics of muscles. Knowledge of gene expression profiles has identified the gene DNAJA1, which is strongly and negatively related to tenderness, explaining up to 63% of variation in tenderness [START_REF] Bernard | New indicators of beef sensory quality revealed by expression of specific genes[END_REF]. Study of gene expression in cattle may lead to improved understanding of post-mortem effects on beef quality [START_REF] Eggen | Genomic approaches to economic trait loci and tissue expression profiling: application to muscle biochemistry and beef quality[END_REF][START_REF] Mullen | Understanding meat quality through the application of genomic and proteomic approaches[END_REF].

The DNAJA1 gene encodes a member of the 40 kDa heat shock protein family (Hsp40), which is a co-chaperone of the 70 kDa heat shock protein family (Hsp70) which affects protein folding and mitochondrial protein import. The DNAJA1/Hsp70 complex inhibits ordered cellular death, or apoptosis [START_REF] Bernard | New indicators of beef sensory quality revealed by expression of specific genes[END_REF]. After animal bleeding, cells enter anoxia and receive no more nutrients, thus initiating apoptosis. It has been hypothesized that apoptosis is the first stage of meat aging [START_REF] Ouali | Biomarkers of meat tenderness: Present knowledge and perspectives in regards to our current understanding of the mechanisms involved[END_REF], and a delay or inhibition of apoptosis by the DNAJA1/Hsp 70 complex could reduce meat tenderization.

To achieve desirable organoleptic properties, meat is typically aged before consumption. Post-mortem tenderization of meat is a complicated process consisting of a series of biochemical pathways [START_REF] Herrera-Mendez | Meat Aging: Reconsideration of the current concept[END_REF]. However, high quality RNA is essential for gene expression studies and aging of meat may cause concern about RNA quality or integrity [START_REF] Bojlul | Long-term stability of RNA in post-mortem bovine skeletal muscle, liver and subcutaneous adipose tissues[END_REF]. Some studies have shown that a delay in tissue processing may not decrease RNA integrity as reflected by the abundance of messenger RNA (mRNA) [START_REF] Marchuk | Postmortem stability of total RNA isolated from rabbit ligament, tendon and cartilage[END_REF][START_REF] Seyboldt | Reverse transcription-polymerase chain reaction assay for speciesspecific detection of bovine central nervous system tissue in meat and meat products[END_REF].

A common post-mortem treatment at large commercial beef abattoirs is electrical stimulation (ES) of carcasses, in which an electrical current passed through the carcass to accelerate anaerobic glycolysis and subsequent pH decline such that carcasses can be chilled more quickly without risk of cold-shortening [START_REF] Bendall | The electrical stimulation of beef carcasses[END_REF]. Electrical stimulation has been shown to reduce Warner-Bratzler Shear Force (WBSF) values, improve tenderness, in beef, lamb and goat [START_REF] Savell | Effect of electrical stimulation of palatability of beef, lamb and goat meat[END_REF]. As well, ES of beef carcasses has been shown to accelerate post-mortem tenderization [START_REF] George | The tenderizing effect of electrical stimulation of beef carcasses[END_REF].

The objective of this study was to relate DNAJA1 expression to beef toughness in muscles that may differ in tenderness and to evaluate RNA integrity and expression of the DNAJA1 gene during meat ageing in non-electrically and electrically stimulated muscles.

Materials and Methods

Animals and Harvesting Procedure

Five Angus steers were fed total mixed ration of corn silage, cracked corn, roasted soybeans, and feedlot vitamin and mineral mix for 258 days to a final average body weight 615±27 kg and approximately 15 months of age. Animals were slaughtered at the meat laboratory facility of the Department of Animal Science, College of Agriculture Sciences, Penn State University, PA, USA, in accordance of the USDA approved humane slaughter guidelines. After carcass dressing (ca. 20 min postmortem), the right side of the carcass was electrically stimulated (ES, 350 V, 3 cycles, 30s per each cycle), the left side was non-stimulated (NES). Carcasses were weighed and average hot carcass weight was 369±15 kg, and then chilled directly at 4˚C for 24 h.

Muscles Samples and Storage

After 25 min post-mortem (zero time, 0 h) 300 mg muscle samples were collected from Longissimus thoracis (LT) and Bicepes femoris (BF) from ES and NES sides then homogenized in 3 mL TRIzol reagent using power homogenizer. Samples were directly kept at -80˚C until the next steps of RNA extraction. Further samples of the LD and BF muscles from ES and NES sides were taken at 1, 3, 6, 9 and 24 h post-mortem. After 24 h LD and BF muscles were dissected from each side and cut into steaks (2.5 cm thickness) that were vacuum packed, stored at 4˚C and sampled for RNA extraction following the same procedure at 3, 7, 14 and 21 d post-mortem. Steaks were kept frozen at -20˚C after completing their respective aging period for further analysis.

Extraction, Quantification and Purity of RNA

Total RNA was extracted from muscles samples (n=200) with TRIzol Reagent® (Molecular Research Centre, Inc. USA) according to the manufacturer's instructions. Reextraction of RNA was carried out using chloroformisopropryl alcohol. Extracted RNA pellet was washed once with 75% ethanol. The RNA pellet was finally dissolved in 50 µL of nuclease-free water. RNA sample concentration and purity were determined using a NanoDrop spectrophotometer. RNA concentrations were adjusted to be within the quantitative range of an Agilent RNA 6000 Nano Chip. Each RNA sample was analyzed on an Agilent Bioanalyzer with an RNA 6000 Nano Chip according to the manufacturer's instructions (Agilent Technologies, Santa Clara, CA).

Relative Level of DNAJA1 Gene Expression

Primers and probes for DNAJA1 gene (the genetic marker for beef tenderness) were designed by the Nucleic Acid Facility of the Pennsylvania State University using sequence data from Genbank and the real-time PCR probe/primer design software Primer Express (v2.0, Applied Biosystems). For real-time qPCR analysis performed by the Nucleic Acid Facility of the Pennsylvania State University, DNAsetreated RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City CA) and the protocol provided with the kit. Quantitation by real-time PCR was done by adding 10 or 20 ng of cDNA in a reaction with 2X TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City CA) in a volume of 20 µL. Primer was added at a concentration of 400 nM and a probe at 200 nM which was labeled with a 5' FAM and a 3' Black Hole Quencher (Biosearch Tech, Novato, CA). The amplification protocol consisted of 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and one min at 60°C in the 7300 Real-Time PCR System (Foster City CA). Ct values of the DNAJA1 gene and the reference gene 18S were used with the delta Ct method [START_REF] Livak | Analysis of Relative Gene Expression Data Using Real Time Quantitative PCR and the 2 -∆∆C T method[END_REF] to determine relative levels of DNAJA1 gene expression.

Warner-Bratzler Shear Force (WSF)

The 1, 3, 7, 14 and 21 day steaks were evaluated for WBSF. Steaks were thawed at 4ºC for 24 h, packages were opened, and steaks were lightly blotted and then weighed. Steaks were wrapped in aluminum foil and cooked to 70ºC in a standard conventional oven set at 177ºC. Internal temperature at the approximate geometric center of each steak was monitored intermittently with a digital thermocouple thermometer (Electro-therm, Model HT 680A, Geraberg, Germany). Steaks were cooled to room temperature (24ºC) over a 4 h period. Three 1.3 cm diameter steak cores parallel to muscle fiber direction were removed from each steak by hand. Cores were sheared perpendicularly to muscle fiber direction using a Warner-Bratzler shear cell attached to a shear force instrument (Model TMS-90, Meat Shear Cell Model CW-2, Food Technology Corporation, Virginia, USA) with a crosshead speed of 250 mm/min. Peak shear force (kg force) and shear work (joules) were recorded then averaged by steak.

Statistical analyses

Comparisons between muscles with and without electric stimulation or between muscles from the same animals were achieved by the paired Student's t test. Pearson correlation coefficients between gene expression levels and shear force values were calculated for the combined data from both muscles using the statistical software (StatSoft Inc., Tulsa, OK, USA).

Results

Toughness of Beef

Shear force (either peak force or work) did not significantly differ between the two muscle types without electric stimulation. However, with electrical stimulation, a significant difference not only was observed in shear force (peak force and work) after 21 days of ageing, but also after 14 days of ageing for shear work values only (Table 1). Thus, for electrically stimulated beef, BF muscle was tougher than LT muscle at 14 and 21 days of ageing. The effect of electric stimulation was significant for peak shear force at 21 days of ageing for the LT muscle only (Table 1).

DNAJA1 Expression

DNAJA1 expression relative to that of 18S RNA did not significantly differ between muscle type and treatment (with or without electrical stimulation) at the time of slaughter. It is also highly variable except for Longissimus thoracis muscle without electrical stimulation (Table 2). Correlation between DNA expression values across muscles was 0.794 between the two treatments (with or without electrical stimulation) (Table 3). From 2 h onwards, total RNA from at least one sample was degraded and all of them were degraded at 21 days post-mortem (Fig. 1). DNAJA1 expression was very low in samples with a high RNA degradation (data not shown). We therefore considered that the assessment of DNAJA1 expression was not reliable except at the time of slaughter.

Correlations between DNAJA1 Expression and Shear Force Value

On average, shear force peak values were poorly correlated across ageing times with or without electric stimulation (Table 3) except a few of them (PF24 h with PF14d, PF3d with PF7d, PF3d with PF3dES, PF7d with PF14dES). Correlations between shear force peak values and DNAJA1 expression in the two muscles (n=10 values) varied according to time of ageing or to the use of electric stimulation (Table 3). Furthermore, DNAJA1 expression values were not significantly correlated with shear force peak value at any time. However, because it was much higher than any other correlation, the correlation with PF14d (r = -0.60) with no electric stimulation is worth being mentioned even if it was not significant, as well the correlation of 0.34 between DNAJA1 expression values with shear force values at 7 days of ageing after electric stimulation (Table 3).

Discussion

The biological mechanisms which determine meat tenderness have been studied for decades in order to find good biomarkers of tenderness which can be used as predictors in order to optimize livestock practices and slaughtering conditions with the ultimate goal to produce beef of the best palatability. Nevertheless, this research strategy was not productive despite some important discoveries: (i) meat ageing is a very important process for the ultimate beef tenderness, (ii) this process involves several proteolytic systems involved in the degradation of muscle proteins during ageing (calpain/calpastatin, cathepsin, proteasome and more recently described caspases and serpins) and (iii) more recently, it was discovered that the onset of apoptosis is among the first steps involved in the conversion of muscle into meat just after slaughtering (reviewed by [START_REF] Ouali | Biomarkers of meat tenderness: Present knowledge and perspectives in regards to our current understanding of the mechanisms involved[END_REF]. Therefore, looking for biological markers involved in the process of apoptosis became a recent active area of research. Furthermore, this approach became easier thanks to the development of genomic tools (transcriptomics, proteomics) through the examination of associated molecular signatures of all genes or proteins, which means that no working hypothesis regarding the underlying mechanisms is required (reviewed by [START_REF] Cassar-Malek | Application of gene expression studies in livestock production systems: a European perspective[END_REF].

So far, different research teams have identified markers of beef tenderness involved in the energy metabolism pathway, in cell detoxification, or which belong to the heat shock protein family or to the annexin family (reviewed by [START_REF] Ouali | Biomarkers of meat tenderness: Present knowledge and perspectives in regards to our current understanding of the mechanisms involved[END_REF]. Within the heat shock family, several markers were identified so far among, which we can cite the Hsp27 protein [START_REF] Morzel | Muscle proteome and meat eating qualities of Longissimus thoracis of "Blonde d'Aquitaine" young bulls: a central role of HSP27 isoforms[END_REF] or the [START_REF] Bernard | New indicators of beef sensory quality revealed by expression of specific genes[END_REF]. The DNAJ proteins (also known as heat shock protein 40) act as ascochaperones to the molecular chaperone DnaK (Hsp70), which is responsible for several cellular processes such as rescuing misfolded proteins, folding polypeptide chains, transport of polypeptides through membranes, assembly and disassembly of protein complexes and control of regulatory proteins [START_REF] Qui | The diversity of the DNAJ/Hsp40 family, the crucial partners for Hsp70 chaperones[END_REF][START_REF] Jiang | Structural basis of J cochaperone binding and regulation of Hsp70[END_REF]. In general, Jdomain proteins modulate protein assembly, disassembly, and translocation [START_REF] Walsh | The Jprotein family: Modulating protein assembly, disassembly and translocation[END_REF]. DNAJ/Hsp40 proteins are indeed important for protein translation, folding, unfolding, translocation and degradation, primarily by stimulating the ATPase activity of chaperone proteins, Hsp70 [START_REF] Qui | The diversity of the DNAJ/Hsp40 family, the crucial partners for Hsp70 chaperones[END_REF]. DNAJA1 has been studied in biomedicine and has been shown to be a biomarker for pancreatic cancer [START_REF] Stark | Structure and function of human DnaJ homologue subfamily a member 1 (DNAJA1) and its relationship to pancreatic cancer[END_REF].

In cattle, initial work has shown a strong negative relationship between DNAJA1 expression in Longissimus thoracis muscle and tenderness score assessed by a sensory panel [START_REF] Bernard | New indicators of beef sensory quality revealed by expression of specific genes[END_REF]. However, further work showed that DNAJA1 was not the only gene belonging to the heat shock protein family related to tenderness and that the genes individually correlated with tenderness are not consistent across genders and slaughtering years in Longissimus thoracis muscle from the Charolais breed indicating the strong influence of rearing conditions on the relationships between DNAJA1 expression and beef palatability [START_REF] Hocquette | The GENOTEND chip: a new tool to analyses gene expression in muscles of beef cattle for beef quality prediction[END_REF]. This may be explained by the fact that developmental age and management factors influence DNAJA1 expression at the mRNA [START_REF] Cassar-Malek | Expression of DNAJA1 in bovine muscles according to developmental age and management factors[END_REF] or protein levels [START_REF] Guillemin | Variations in the abundance of 24 proteins biomarkers of beef tenderness according to muscle and animal type[END_REF]. DNAJA1 expression also differs between muscle types [START_REF] Cassar-Malek | Expression of DNAJA1 in bovine muscles according to developmental age and management factors[END_REF]. All together, the absence of consistency of relationship between DNAJA1 expression with tenderness (across cattle population, age and muscle type) are likely to explain the absence of any association in the current study with Angus animals.

According to [START_REF] Fontanesi | Microarray gene expression analysis of porcine skeletal muscle sampled at several post mortem time points[END_REF], transcriptomic studies can be performed in porcine skeletal muscle up to 24 h post-mortem. Therefore, we could conclude that microarray data obtained from specimens collected in the processing plant, over a relatively long period, have the potential to assess mRNA biomarkers of beef quality, with no potential bias from RNA degradation. In one way, our study confirmed this observation since most of the RNA degradation occurs after 1 or 3 days of ageing. However, careful examination of individual samples indicated that at least one sample RNA was degraded from two hours postmortem onwards with a concomitant fall in DNAJA1 expression. When performing correlation studies between two sets of data, any change in one point may affect the correlation dramatically, especially with a low number of samples. Therefore, the quality of RNA samples is surely a crucial factor determining the association between any potential biomarkers of tenderness and the real tenderness score.

Toughness (the opposite of tenderness) is routinely assessed by shear force on cooked meat. A first crucial parameter is cooking temperature which may be to an internal temperature of 55°C (rare cooking) as in France [START_REF] Allais | Effects of polymorphisms in the calpastatin and µ-calpain genes on meat tenderness in three French beef breeds[END_REF] or at 74°C in UK [START_REF] Farag | A comparison of conventional and radio frequency tempering of beef meats: Effects on product temperature distribution[END_REF] or China [START_REF] Hou | Effect of suspension method and aging time on meat quality of Chinese fattened cattle M. Longissimus dorsi[END_REF]. Shear force can also be measured after different days of ageing with poor correlations between shear force values depending on ageing time [START_REF] Hou | Effect of suspension method and aging time on meat quality of Chinese fattened cattle M. Longissimus dorsi[END_REF] as in this study (where correlations varies up to 0.69 maximum). Therefore, depending on the set of shear force values used to be correlated with potential biomarkers of beef quality, different results will be obtained. For instance, [START_REF] Tizioto | Identification of KCNJ11 as a functional candidate gene for bovine meat tenderness[END_REF] reported that KCNJ11 SNPs were markers for tenderness using shear force values at 0 and 7 days post-mortem whereas KCNJ11 expression level was a negative marker of tenderness at 7 days of ageing only, but not at 0 or 14 days of ageing.

In our study, we found no significant relationships between DNAJA1 expression levels and shear force values despite the highest negative correlation of -0.60 at 14 days of ageing. However, the major observation of this study is the high variability of this relationship between DNAJA1 expression levels and shear force values which ranged from -0.60 to 0.34.

Many factors previously discussed are likely to explain at least in part this variability. Some technical factors related to the experiment itself are important such as any potential RNA degradation in at least one sample. The methodology used to generate reference values of toughness (in this case shear force) is also crucial since shear values vary with or without electric stimulation (this study), according to ageing time (this study, [START_REF] Tizioto | Identification of KCNJ11 as a functional candidate gene for bovine meat tenderness[END_REF][START_REF] Hou | Effect of suspension method and aging time on meat quality of Chinese fattened cattle M. Longissimus dorsi[END_REF] as well as according to other factors not studied here such as, for instance, suspension method of carcasses [START_REF] Hou | Effect of suspension method and aging time on meat quality of Chinese fattened cattle M. Longissimus dorsi[END_REF]. In the case of ageing, biomarkers of tenderness (in this case protein expression levels because mRNA levels are not considered as reliable) vary according to ageing time [START_REF] Laville | Proteome changes during meat ageing in tough and tender beef suggest the importance of apoptosis and chaperone proteins for beef ageing and tenderisation[END_REF].

Other authors argue that the reference values should not be shear force but sensory scores from expert panels. Indeed, phenotypic correlations between tenderness scores and shear force values were approximately -0.35 or -0.40 for French young bulls indicating that shear force is a poor indicator of the real tenderness assessed by real people eating beef [START_REF] Van Wezemael | Relationships between sensory evaluations of beef tenderness, shear force measurements and consumer characteristics[END_REF]. This problem, however, is not so important for genetic studies because the genetic correlations are much higher between these two parameters (around -0.90) [START_REF] Allais | Effects of polymorphisms in the calpastatin and µ-calpain genes on meat tenderness in three French beef breeds[END_REF]. For both approaches, cooking method and cooking temperature in addition to muscle type affect shear force and tenderness values [START_REF] Modzelewska-Kapituła | The effect of muscle, cooking method and final internal temperature on quality parameters of beef roast[END_REF]. Generally speaking, consumers prefer the appearance, aroma and flavor of beef strip loin samples cooked at the highest temperatures and the tenderness and juiciness of samples cooked at the lowest temperatures [START_REF] Gomes | Sensory descriptive profiling and consumer preferences of beef strip loin steaks[END_REF].

Other authors even think that the best descriptor of tenderness is the assessment by untrained consumers and not expert panels. Trained panels have generally a smaller variance due to a controlled methodology thanks to training, but the training procedure can lead to biased results. By comparison, a consumer panel is unbiased, but has a larger variance. The decision in Australia was to use untrained consumer taste panels and this decision was based on the need to have a reliable, transparent, system of testing samples that would engender confidence with both the beef industry and consumer sectors. In this country, the final assessment of palatability of beef is often done by the target consumer from the street eating beef [START_REF] Thompson | Managing meat tenderness[END_REF]. Experiments relating biomarkers of beef quality to sensory scores determined by untrained consumers are still lacking, but results are likely to differ from those obtained so far with trained expert panels or with shear force as in this study. In contrast to previous results with one muscle type from young Charolais bulls, we observed that DNAJA1 expression level was not a marker of beef tenderness with this particular data set made from two muscle types sampled from Angus steers. Besides animal differences (breed, gender, age, rearing practices), other technical factors related to the methodology (RNA degradation and storage), to the overall experimental design (muscle type, slaughtering conditions with or without electric stimulation) or to the assessment of tenderness (shear force vs score from expert panels) as well as temperature and cooking method of beef are likely to explain discrepancies between studies.

Fig. 1 :

 1 Fig. 1: Stability of total RNA in (A) Non-ES Longissimus thoracis, (B) ES Longissimus thoracis, (C) Non-ES Biceps femoris and (D) ES Biceps femoris muscles during postmortem storage by Agilent Bio-analyzer assay

Table 1 :

 1 Shear force AA of LT and BF muscles at 24 hours, 3, 7, 14 or 21 days of ageing Shear Force measured as Peak Force (PF, newtons) and Shear Work (SW, N cm) A, B indicate differences of shear force values between LD and BF muscles for electrically stimulated muscles a, b indicate differences of shear force values due to electrical stimulation for LT muscle Means with different letters are significantly different (P<0.05) LT: Longissimus thoracis, BF: Biceps femoris NES: non-electrical stimulation. ES: electrical stimulation

	Variables		PF 24 h	SW 24 h	PF 3 d	SW 3 d	PF 7 d	SW 7 d	PF 14 d	SW 14 d	PF 21 d	SW 21 d
	LT NES Mean	43.10	46.30	41.90	44.70	37.10	41.90	38.30	43.80	32.90a	38.40
		SD	13.04	9.28	8.52	8.62	8.17	7.64	6.34	5.61	3.41	3.69
	BF NES Mean	37.90	46.70	41.90	48.40	48.40	50.10	38.30	48.70	32.90	41.90
		SD	14.55	17.82	12.32	17.30	9.90	17.68	6.98	9.52	6.59	8.99
	LT ES	Mean	39.90	42.00	40.80	43.30	39.60	42.90	34.10	37.10A	28.6Ab	34.6 A
		SD	8.95	7.31	4.41	5.63	4.38	4.85	6.49	5.96	1.82	2.47
	BF ES	Mean	39.40	46.80	40.80	46.80	35.40	46.80	39.50	43.00B	37.30B	45.8 B
		SD	4.10	4.58	6.02	8.03	4.22	7.43	3.71	4.87	3.62	5.20

AA

Table 2 :

 2 DNAJA1 expression at the time of slaughter

	Variables	Mean	SD	CV (%)
	LT NES	1.02	0.01	1.40
	BF NES	0.90	0.85	94.20
	LT ES	0.54	0.43	79.40
	BF ES	0.92	0.88	96.50
	LT:Longissimus thoracis, BF: Biceps femoris
	NES: non-electrical stimulation. ES: electrical stimulation
	DNAJA1 expression levels are expressed in arbitrary units


Paolo Annicchiarico

Bernadette Julier

Gaëtan Louarn

Amel Maamouri

Alfalfa intercropping and competitive ability

Keywords: interspecific interference, legumegrass mixtures, organic agriculture, plant competition, weed competition

Alfalfa is subjected to interspecific competition in two situations: (i) intercropping with a forage grass, where the required longlasting equilibrium between species is frequently threatened by excessive alfalfa competitive ability; (ii) competition against weeds, which can be severe in monoculture and requires high competitive ability to avoid any chemical weed control. In both cases, competition for light interception is the main mechanism that drives the species proportion. Vegetative vigour and plant architectural traits can affect the alfalfa competition dynamics across successive harvests. Once completely unveiled, these traits could be selected for new varieties with improved adaptation to competition.

Introduction

Our focus on alfalfa competitive relationships is two-fold here, i.e.: (i) intercropping with a grass companion, to obtain a long-lasting, balanced mixture of the two components of the mixture; (ii) competition against weeds, to avoid any chemical weed control. Legume-grass mixtures are gaining new interest in Europe and elsewhere, owing to the energy and environmental costs associated with the synthesis and use of nitrogen fertilizer required for grass forage production and the quest for greatest self-sufficiency in feed proteins at the farm and the country levels. Legume-grass mixtures are currently grown over 70% of the sown grassland in France, exploited as hay, silage or grazed forage. Alfalfa monoculture is still prevalent in other countries, e.g. Italy, where it is the backbone of organic croplivestock systems and contributes significantly to conventionally managed systems. Alfalfa competitive ability against weeds is crucial for organic systems and increasingly important for conventional ones, especially for the pure stand. We briefly review alfalfa competitive relationships mainly with respect to their relevance to breeders. One major issue in this context is whether alfalfa varieties targeted to mixed cropping with grasses, or targeted to cropping under severe weed competition (as in most organic systems), could be successfully selected or recommended from testing trials carried out in the simpler condition of absence of competition. The response, which depends on the degree of genetic correlation between selection and target environments, has farreaching implications, e.g. for the choice of phenotyping conditions in marker-assisted selection studies, or procedures for assessing the variety value for cultivation and use.

Competition

Alfalfa-grass intercropping and weed-invaded alfalfa crops are situations of interspecific plant competition which imply different targets. The target is maintaining a desirable legume-grass balance in the former case, and excluding the weeds in the latter. Competition for resources is one of the major processes controlling plant growth and explaining the dynamics of plant mixtures. Competition for light is of primary importance, because it determines the energy available for all physiological processes and partially drives the acquisition of other resources. Furthermore, as light availability declines exponentially with the distance from the top of the canopy, minor differences in plant size can have major effects on the relative yield of the intercropped species. The amount of captured light defines the potential growth rate and the nitrogen demand of the crops. Besides energy capture and photosynthesis, competition for water and nutrients is also critical in explaining the dynamics of plant mixtures.

Competitive ability and compatibility with grasses

The companion grass tends to be at competitive disadvantage and less persistent than alfalfa when associated with highly productive alfalfa varieties under favourable cropping conditions, while the reverse may occur when alfalfa is grown in shallow soils or suffers from poor establishment.

In alfalfa, traits related to growth habit, shoot development and branching (leaf area expansion), and internode length (vertical leaf area distribution) showed a prominent role in light interception, and mainly contribute to its advantage compared to grasses (4). For legumes

RESEARCH

in mixture, intercepted light amount also determines the maximal amount of nitrogen that can be fixed by nodules [START_REF] Corre-Hellou | Interspecific competition for soil N and its interaction with N 2 fixation, leaf expansion and crop growth in pea-barley intercrops[END_REF], which can reach around 180 kg N ha -1 year -1 in alfalfa pure stands (9).

The root systems of grasses and alfalfa differ strongly in terms of architecture (fasciculate vs. taproot), root length density (high vs. low) and rooting depth (short vs. long) [START_REF] Haynes | Competitive ability of the grass-legume association[END_REF]. Grasses are better competitors for water and most soil nutrients (mineral N, P and K in particular) than alfalfa, especially within the topsoil layers, but alfalfa explores deeper soil horizons.

However, alfalfa is little affected by soil N competition under conditions favourable to N 2 fixation, owing to its ability to fix atmospheric nitrogen (N 2 ). The proportion of N derived from fixation is even higher in mixtures than in pure stands as a consequence of the specialization of each species on a particular N resource (atmospheric N for alfalfa; soil N for grasses). N 2 fixation is intensively affected by water limitation, because this physiological process is one of the most sensitive to water stress. A drought event can thus quickly reduce the nitrogen autotrophy of alfalfa, changing drastically its ability to compete with grasses.

The relationships between species in a mixture are not only competitive: they may produce beneficial effects by modifying the environment of the other species. This is frequent is grass-legume mixtures, because a portion of the nitrogen fixed by legumes can be transferred to the associated grasses. Such effects tend to be more limited in alfalfa-grass than in clover-grass mixtures (10).

Maintaining a desirable balance between grass and alfalfa may be achieved by strategic choices at sowing (choice of alfalfa cultivars, grass species, date of seeding; density, seeding pattern) and by tactical management options (fertilisation, cutting). The selection of alfalfa varieties which are specifically adapted to mixed cropping because of greater compatibility with (hence, lower competitiveness against) grasses is also worth being investigated. A set of early studies in Lodi indicated the consistent ranking of alfalfa cultivars for biomass yield across purestand and mixed-stand conditions (11). However, preliminary results in Lusignan suggest at least some degree of genotype inconsistency between these conditions (Fig. 1). This experiment, which includes the observation of a number of morphological traits, will be used to test whether other alfalfa traits besides lower vigour may be associated with greater compatibility with associated grasses. We hypothesize that an alfalfa cultivar with lower stem height, more branching and good regrowth dynamics after cutting would have a reasonable high yielding ability in pure stand and would be a less aggressive companion crop for the grass in mixed stand, because of less competition for light interception. Breeding for all other traits that contributes to alfalfa persistency and quality would still be required for these new varieties. Choosing highly competitive grass companions can also be important to ensure more balanced associations.

Competitive ability with weeds

Weed density tends to be higher in alfalfa monocultures than alfalfa-grass mixtures. The false seeding technique, which is frequently adopted in organic agriculture, can be very useful to limit weed density and aggressiveness in the early, crucial stage of crop establishment. Weed competition after the first mowing is limited to species which are able to regrow.

The genetic variation for ability to compete with weeds has been poorly studied in alfalfa. One study whose results are partly summarized in Fig. 2 revealed substantially consistent responses of eight cultivars across conditions of high or almost nil weed competition (reproducing organic and conventional systems, respectively) which were established in one experiment in Lombardy (northern Italy). On average, landrace and variety germplasms showed comparable competitive ability against weeds. Competitive ability of the cultivars was strictly related to their forage yielding ability in the absence of weeds [START_REF] Annicchiarico | Forage and seed yield response of lucerne cultivars to chemically-weeded and non-weeded managements and implications for germplasm choice in organic farming[END_REF], with a semi-prostrate cultivar showing lowest values of both traits (Fig. 2). This relationship agrees with the crucial importance attributed to high relative growth rate for competitive success of genotypes belonging to erect, vigorous forage species grown in relatively favourable environments [START_REF] Campbell | A trade-off between scale and precision in resource foraging[END_REF], when considering that high relative growth rate also implies high forage yield in the absence of competition. This relationship may be much looser in less vigorous, subordinate species such as white clover, whose genotype competitive ability mainly relies on the ability to make finescale exploitation of light and nutrients from undepleted zones through morphological plasticity [START_REF] Annicchiarico | Breeding white clover for increased ability to compete with associated grasses[END_REF]. The high consistency of cultivar yield responses across high vs. negligible weed competiton conditions contrasts with the low consistency across two Italian cropping regions (northern vs. central Italy) (3; Fig. 2), suggesting that distinct material for each of these regions be bred with no specific selection for organic systems and/or higher competitive ability.

Compatibility with grass companions, and ability to outcompete weeds, likely inquire different optima of alfalfa competitive ability. They are associated with vegetative vigour and plant architectural traits yet to be discovered, which could eventually be selected for to produce varieties with improved adaptation to conditions of competition. ■

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 1 Consiglio per la Ricerca e la Sperimentazione in Agricoltura, Centro di Ricerca per le Produzioni Foraggere e Lattiero-Casearie, Lodi, Italy 2 INRA, UR4, Unité de Recherche Pluridisciplinaire Prairies et Plantes Fourragères, Lusignan, France (bernadette.julier@lusignan.inra.fr)

Figure 1 .

 1 Figure 1. Biomass yield in the first harvest of 2011 of alfalfa genotypes grown in mixtures (one target alfalfa plant surrounded by 2 alfalfa and 4 tall fescue plants) and in pure stands (one target alfalfa plant surrounded by 6 alfalfa plants from the tall, erect cultivar Orca); the line is for equal biomass production in both stands

Figure 2 .

 2 Figure 2. Forage yield of eight alfalfa cultivars across conditions reproducing organic or conventional cropping systems in two geographic areas (Lombardy, northern Italy; Umbria, central Italy), and genotype × environment (GE) interaction and phenotypic correlation for cultivar yield across environments; the graph pools results from the studies (2) and (3)