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Transcriptome analysis of muscle in order to identify genes which determine muscle characteristics and sensory quality traits of beef
K. Sudre1, I. Cassar-Malek1, C. Leroux1, A. Listrat1, Y. Ueda1, C. Jurie1, G. Renand2, P. Martin3, J.F. Hocquette1
INRA
INTRODUCTION
Beef meat consumption has decreased about 20% since 1985. This can be explained, at least in part, by the severe competition with white meats, by the bad image of beef (BSE crisis, etc) and by the dissatisfaction of consumers with the considerable and uncontrolled variability in beef sensory quality traits. The latter depend not only on slaughter conditions and transformation processes but also on muscle characteristics at slaughter (fibres, intramuscular fat, con- nective tissue, etc). Rearing conditions (growth rate, nutrition, age) and genetic type of animals (breed, etc) are of paramount importance to control muscle characteristics and hence to ensure consumers are offered high and consistent quality meat (GEAY et al., 2002).
However, the determinism of beef meat quality traits depends on many fac- tors. The muscle characteristics that have been analysed so far do not explain the major part of variability in sensorial quality traits (RENAND et al., 2001). The- refore, our major objective is to discover new muscle characteristics that may explain variability in meat quality. Besides specific cases (double-muscled cattle for instance), muscle characteristics generally depend on the expression of a great number of genes. Knowledge of these genes, of their expression profile and of the factors that regulate their expression is thus necessary to improve animal selection and to optimise husbandry conditions. It is also important to acquire a better understanding of the biological links between muscle growth and qualitative muscle characteristics. The ultimate objective is indeed to find the optimum compromise in terms of selection between muscle growth poten- tial and meat quality. Furthermore, as the media focuses on natural feeding of
1. Unité de Recherches sur les Herbivores, Theix, 63122 Saint-Genès Champanelle, 2. Station de Génétique Quantitative et Appliquée, 78352 Jouy-en-Josas, 3. Unité de Génomique et Physiologie de la Lactation, 78352 Jouy-en-Josas.
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ruminants, it is of great importance to valorise grass feeding, especially in Mas- sif Central where extensive animal husbandry and pasture have a high social and economic impact.
Thus, our objectives are:
1) to identify and to analyse genes that determine muscular characteristics and beef meat sensory quality,
2) to relate those data with divergent phenotypes for muscle growth poten- tial, for feeding conditions and for meat quality traits,
3) to deduce practical strategies to improve selection schema and rearing conditions of cattle.
METHODS
Experimental design:
Two muscles (one oxidative, Rectus abdominis [RA] and one glycolytic Semitendinosus [ST]) of divergent groups of animals were studied in two experi- ments:
• Sixty four young Charolais bulls, progeny of 25 Charolais sires divergently selected for their muscle growth capacity index (a synthetic breeding value index for high muscle weight and low carcass fat percentage) were slaugh- tered at 15 or 19 months of age. Carcass composition was estimated from dissection of the 6th rib. Animals were ranked according to their own mus- cle growth capacity index and two groups of six bulls from each extremity of the index distribution were used in this study. Bulls were fed a complete pelleted diet distributed ad libitum until slaughter.
• Two groups of eight 30-month-old steers were fed similar net energy intake from weaning onwards either with grass at pasture or with maize silage indoors.
In both experiments, biochemical and metabolic muscle characteristics of fibres and connective tissue were determined as previously described by LISTRAT et al. (2001).
Transcriptome analysis:
Total RNAs were extracted and were pooled in order to provide one sample for each muscle and each genetic type.
Arrays (high density filters) containing 1339 printed cDNA from a human muscle library were first hybridised with radiolabelled complex cDNA from bovine mRNA as previously described (SUDRE et al., 2000).
Then, a bovine muscle cDNA library was prepared using a pool of messenger RNA extracted from oxidative or glycolytic muscles sampled from fœtus, grow- ing or adult cattle of various genotypes. Furthermore, 77 cDNAs from genes studied by biochemical and metabolic approaches were prepared by RT-PCR
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using specific primers. Glass slide arrays with a total of 480 bovine muscle cDNA probes were hybridised with fluorescently labelled total RNAs.
RESULTS
Animal and muscle characteristics:
Young bulls with a high muscle growth potential were characterised by a mus- cle mass 27% higher (316.4 vs 249.2 kg) and a proportion of fat in the carcass 28% lower (12.6 vs 17.4%) than bulls with a low growth potential (P < 0.01).
Activities of metabolic enzymes significantly differed between RA and ST muscles confirming the metabolic differences between these muscles: for ins- tance, lactate dehydrogenase activity (characteristic of glycolytic metabolism) was 23% higher in ST than in RA (P < 0.001). Conversely, isocitrate dehydroge- nase activity (characteristic of oxidative metabolism) was 29% higher in RA than in ST (P < 0.03).
A low muscle growth potential was associated with a more oxidative muscle metabolism as indicated by higher citrate synthase activity compared to that in bulls with a high growth potential (+62%, P < 0.004). Muscle collagen content and solubility did not significantly differ between the two groups of animals.
Grass-fed steers on pasture were characterised by a lower insoluble colla- gen content in ST muscle (-15%, P < 0.05) and a higher oxidative metabolic activity in RA muscle compared to maize silage-fed steers. The activity of two mitochondrial enymes (citrate synthase and isocitrate dehydrogenase) were indeed 19-46 et 25-30% higher in RA of grass-fed steers (P < 0.05) (LISTRAT et al., 2001).
Transcriptome studies:
Hybridisation on human muscle arrays allowed the detection of 1232 hybri- disation signals. More than 500 could be exploited and 375 were considered to compare the different samples. Thirty four genes were differentially expressed in RA and/or ST between the two groups of bulls with high or low muscle growth potential. Many of them are involved in muscle structure (e.g. sarcosin, titin) or in cellular regulation (e.g. thyroid hormone receptor interacting protein 10, LIM protein, a tumour suppressor involved in B-cell chronic lymphocytic leukaemia (LEU5), heat shock protein 90a) which were more expressed in muscles from low muscle growth potential bulls. Conversely, three genes were more expressed in muscles from high growth potential bulls (e.g. NADH dehydroge- nase Fe-S protein 3). This experiment also allowed genes differentially expressed between RA and ST to be detected. For instance, LEU5, carnitine acetyltransferase, and the non-ATPasic proteasome 26S subunit 4 were more expressed in ST than in RA from low muscle growth potential bulls and the doli- chol-phosphate-mannose synthase gene was more expressed in ST than in RA from high muscle growth potential bulls. The two latter differences were confir- med by Northern blot analysis.
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Among the 1440 cDNAs from the bovine library which were sequenced, 1019 from several gene families could be exploited: mitochondrial genes (133), genes encoding ribosomal proteins (100), contractile proteins, metabolic enzy- mes, etc. Finally, 353 cDNAs from this library and 77 cDNAs individually prepa- red by RT-PCR with specific primers were considered as a first bovine muscle cDNA repertoire. This was printed on glass slides and hybridised with RNA extracted from muscles of high or low muscle growth potential bulls. On ave- rage, 30 to 60% of the hybridisation signals could be exploited. This indicates that this cDNA repertoire may be used for transcriptome studies in bovine mus- cles. Less than four genes appeared to be differentially expressed between both genotypes and less than 10 between the two muscle types. Northern blot analyses are in progress to check these differences.
Transcriptome analysis of muscles from the two groups of steers with diver- gent types of diets is in progress.
CONCLUSION AND PERSPECTIVES
In this study, we established powerful experimental designs, we used availa- ble human arrays and we constructed bovine molecular tools. They are the first steps for carrying out transcriptome analysis of bovine muscles.
This strategy forms part of an integrative approach to many molecular and biochemical data from the gene to the muscle with the ultimate objective of dis- covering new muscle characteristics that determine beef meat quality. These new characteristics will be further analysed by classic biochemical studies. This research will complete other studies on gene single nucleotide polymorphisms (European Project GEMQUAL) and proteomic analyses in progress at INRA (EGGEN and HOCQUETTE, 2003).
ACKNOWLEDGEMENTS
This work was supported by an INRA grant for functional genomics devoted to the analysis of genome from farm animals (AGENAE). The authors wish to gratefully acknowledge the staff of the Experimental Domain of "Pin au Haras", the team of the slaughterhouse, the staff of different INRA research laboratories (URH, Theix, LGBC, Jouy-en-Josas), the laboratory of C. AUFFRAY (CNRS, Vil- lejuif) and the private Company Diagnogène (Clermont-Ferrand).
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