BACKGROUND
with changes similar to those that occurred in the pig industry, the poultry market is now characterized by increasing diversity of processed products  <cit> .
as a consequence, poultry companies are now involved in food technology and product development, and improvement of meat processing ability has become a prevalent concern.
as in pigs, post-mortem ph is a key factor controlling chicken meat quality  <cit> .
variations in ultimate meat ph  are responsible for variations in several breast meat properties, including water-holding capacity, colour and firmness  <cit> .
low ultimate ph results in "acid meat", with a pale aspect and reduced water-holding capacity  <cit> , while high ultimate ph leads to dfd  meat, dark in colour, with reduced shelf-life  <cit> .
at the genetic level, there is a very strong negative correlation between the ultimate ph of breast meat and the level of muscle glycogen estimated by the glycolytic potential at the time of slaughter   <cit> .
the glycolytic potential has also been shown to be highly heritable   <cit> .
understanding the mechanisms and identifying the genes controlling muscle glycogen storage constitute a promising way to increase control of and improve chicken breast meat properties.
it would make it possible to develop useful breeding tools, such as molecular markers, to select birds with expected meat properties, and help optimize rearing practices, via the study of gene regulation.
glycogen is the main metabolic fuel for the anaerobic glycolysis which takes place post-mortem when muscles are no longer supplied with oxygen.
the genetic control of muscle glycogen, and therefore meat quality, was evidenced first in mammals and more recently in the chicken  <cit> .
in mammalian species, including the pig, mouse and human, a major gene, prkag <dig> which encodes the γ <dig> regulatory subunit of the amp-activated protein kinase , is responsible for variations in muscle glycogen content  <cit> .
in the chicken, there is no information available suggesting that a major gene could be involved in the control of glycogen content in muscle.
however, several studies have suggested that breast muscle glycogen content is related to growth and body composition: it decreases with growth rate and breast meat yield  <cit>  and increases with carcass fatness  <cit> .
the aim of the present study was to compare the expression profiles of muscles from chickens differing in muscle glycogen content and breast meat quality.
in the first experiment, birds originating from two experimental lines  and lean  lines) were compared.
the f and l lines were originally divergently selected for and against the amount of abdominal fat  <cit>  but they also exhibited differences in muscle glycogen content and in breast meat quality traits  <cit> .
despite a similar growth rate, the chickens from the f line were  <dig> times fattier than those from the l line.
moreover, due to high muscle glycogen content, the meat of the fat chickens exhibited a lower ultimate ph and higher drip loss and lightness than lean chickens.
the differences in muscle glycogen between the f and l lines have been related to variations in mrna encoding several enzymes regulating glycogen synthesis and degradation as well as in activation of ampk by phosphorylation  <cit> .
in the second experiment, the muscle transcriptomes were compared in individuals generated from the f <dig> population produced from these two lines  and exhibiting extremely high  or low  muscle glycogen content.
individuals for this analysis were chosen according to their levels of glycogen in muscle, while differences in body fatness were much less pronounced than in the first model.
the use of a  <dig> k oligo microarray provided the first description of genes differentially expressed between breast muscles exhibiting high or low glycogen content, correlated with poor or high meat quality traits, respectively.
this global approach was complemented by mrna analyses on previously studied candidate genes  <cit>  and on a subset of genes identified from array analysis.
for genes with a human ortholog, further interpretation was based on the use of ingenuity and gene ontology annotation databases highlighting several biological processes likely to be involved in muscle glycogen regulation.
RESULTS
carcass, muscle and meat quality traits of chickens used for gene expression analyses
the mean carcass and p. major muscle traits and sd are presented in table  <dig>  at  <dig> weeks of age, body weight was similar between f and l birds and between g+ and g- birds generated from the f2fl population.
breast meat yield was slightly higher in l than in f birds and similar between g+ and g-.
abdominal fat yield was 2-fold greater in f than in l birds while it was only 30% greater in g+ compared to g- birds.
however, the muscle glycogen reserves were 61% higher in g+ than in g- birds while they were only 34% higher in f than in l birds.
the ultimate ph of p. major muscle of f and g+, in relation to their greater glycogen content at the time of death, was higher than that of l and g- birds, respectively.
the breast meat was lighter  and less coloured , and exhibited more drip loss in f than in l birds.
only meat lightness  was higher in g+ than in g- birds generated from the f2fl population.
the g+ and g- chickens were generated from the f2fl population produced from the  <dig> generation intercross between the founder fat and lean lines.
the g- and g+ chickens correspond to the individual females exhibiting the lowest and the highest muscle glycogen content within the f2fl population.
ns = non-significant.
differential analysis on microarray
among the genes found to be expressed in chicken p. major muscle,  <dig> and  <dig> transcripts were differentially expressed between f and l and g+ and g-, respectively .
the gene expression fold-change ranged from  <dig>  to  <dig>  and  <dig>  to  <dig>  for the f vs. l and the g+ vs. g- comparisons, respectively.
a trend was observed for a higher percentage of genes down-regulated in muscle with high glycogen content .
full details of gene name, function, accession number, fold-change and p-value for all differentially expressed transcripts are listed in additional files  <dig> and  <dig>  only  <dig> transcripts were recorded as differential in both analyses, i.e.
f vs. l and g+ vs. g-, corresponding to  <dig> known genes .
functional annotation
among the genes that were reported to be differentially expressed between muscles with high or low glycogen content,  <dig> with a human ortholog were submitted to annotation analyses.
when compiling the lists of genes expressed differentially between g+ and g- and between f and l chickens, the software ingenuity analysis pathways  <dig>   highlighted several biological functions , including lipid and carbohydrate metabolism.
the genes associated with these two functions represent about 15% of the  <dig> genes considered for annotation analysis.
when considering findings separately, ipa highlighted lipid metabolism and molecular transport as common pathways in the two models  and also specific biological functions for each of them: cell morphology, cell cycle, and cell to cell signalling and interaction for f/l, and small molecular biochemistry, cell death, and cellular development for g+/g-.
gene ontology  terms are also widely used for global interpretation of the functions of genes revealed by differential microarray analysis.
according to gene ontology, genes differentially expressed between f and l participated in several biological processes that can be grouped in  <dig> main biological functions: molecule transport and localization, and lipid, energy, and amino acid metabolism.
genes differentially expressed between g+ and g- belonged to biological processes especially related to lipid and energy metabolism, as well as developmental processes including cell growth, proliferation, differentiation and organization.
the biological interpretation of expressional data was performed using ingenuity pathway analysis  <dig>  .
the genes included in the analysis were shown to be differential between f and l and/or between g+ and g- by either microarray or real-time rt-pcr.
genes are presented in alphabetical order for each category.
the genes over expressed in muscles with high  and low glycogen content  are in bold and normal characters, respectively.
genes in italic were differentially regulated between models .
validation by real-time rt-pcr of a subset of genes revealed by differential microarray analysis
the mrna levels of  <dig> genes involved in skeletal and muscular system development and function, lipid metabolism or carbohydrate metabolism, and found to be differential on microarray were further quantified by real-time rt-pcr in both models  .
the level of 18s rrna was chosen as reference and confirmed to be invariable.
the expression levels  of genes were compared between g+ and g- and between f and l  for each of the  <dig> genes selected.
ratios of gene expression determined by real-time rt-pcr were compared to ratios obtained using microarray analysis .
over expression in g+ compared to g- was clearly confirmed for cebpb and rgs <dig> , and suggested for foxo <dig> as similar fold-changes reached significance in the microarray analysis but not with real-time rt-pcr.
under expression was confirmed for lpar <dig>  pdk <dig>  rps <dig> and srf , and suggested for ppp1r2b and ucp <dig>  as similar fold-changes were observed in microarray and rt-pcr studies.
none of the differences suggested between f and l chickens by the microarray study could be statistically confirmed by rt-pcr.
slightly higher expression of ugdh  and lower expression of srf  were suggested in f compared to l chickens.
it is of note that the genes showed completely different variations between f and l and between g+ and g-.
none of the  <dig> genes over expressed in g+ compared to g-  differed between f and l chickens.
similarly, lpar <dig> and rps <dig>  which were under expressed in g+ compared to g-, did not differ between f and l. by contrast to what was observed when comparing g+ to g-, pdk <dig> was over expressed in f compared to l, suggesting an inverse relationship between muscle glycogen and pdk <dig> expression in the two models.
the observation of higher expression of abhd <dig> in g+ compared to g- on microarray prompted real-time rt-pcr measurements in f and l. while the differential expression between g+ and g- was not confirmed, significantly higher expression was observed in f compared to l chickens .
the biological interpretation of the real-time rt-pcr findings highlighted a gene network involved in several molecular and cellular functions, including lipid and carbohydrate metabolism, molecular transport, small molecule biochemistry, and cell morphology .
for both microarray and real-time rt-pcr analyses, results are expressed as fat to lean and g+ to g- ratios of the expression gene.
ns = non-significant; nd = not determined.
statistical significance is indicated as follows: †p value <  <dig> , *p value <  <dig> , **p value <  <dig> .
§ only genes identified as differentially expressed in at least one model are presented in the table.
differential analysis of candidate genes
the transcript expression of  <dig> genes  directly involved in muscle glycogen turnover was quantified by real-time rt-pcr.
only  <dig> of them were significantly differentially expressed between g+ and g- and their expression ratios are presented in table  <dig>  none of the genes assayed was significantly differentially expressed between f and l chickens.
the transcript levels of prkab <dig> and prkag <dig>  which encode the amp-activated protein kinase  regulatory β <dig> and γ <dig> subunits, respectively was lower in g+ than in g- muscles: the ratio of g+/g- expression was  <dig>   for prkab <dig> and  <dig>  for prkag <dig> .
the phkd gene, which encodes the δ subunit of phosphorylase kinase , was also significantly down-regulated  in g+ compared to g- muscles, with a g+/g- expression ratio of  <dig> .
transcription factor analysis
transcription factor analysis highlighted several interrelations between genes whose expression differential was confirmed between g+ and g- animals by real-time rt-pcr .
binding sites for the transcription factor cebpb were thus reported in the promoters of rgs <dig>  ucp <dig>  srf, foxo <dig>  similarly, promoters of cebpb, prkab <dig> and pdk <dig> possess the foxo <dig> binding site, and rps <dig> and lpar <dig> possess binding sites for srf.
ugdh is under the control of ppara  that also activates ucp <dig> and pdk <dig>
discussion
few studies have reported global gene expression surveys in the chicken to date.
moreover, our study is the first to relate global gene expression profiles to muscle glycogen content and variations in meat quality.
in mammals, especially in cattle and the pig, several microarray studies have investigated global muscle gene expression in relation to sensorial meat attributes such as tenderness, juiciness, flavour and marbling, which are not directly related to muscle glycogen content  <cit> .
recent analyses in the pig helped to link gene expression profiles to variations in meat water loss, this characteristic being strongly related to variations in muscle ph, but without considering glycogen variations in muscle  <cit> .
the transcripts being up-regulated with high drip loss in the pig belong to groups of genes functionally categorized as genes of membrane proteins, signal transduction, cell communication, response to stimulus, and the cytoskeleton.
among genes down-regulated with high drip loss, functional groups of oxidoreductase activity, electron transport, and lipid metabolism were identified.
the originality of our study lies in the models chosen for microarray analyses.
as already shown, there is a positive relationship between body fatness and glycogen content in breast muscle in the chicken  <cit> .
comparing divergently selected fat and lean chickens, which also differ in muscle glycogen content  <cit> , is therefore relevant to identify mechanisms underlying variations in muscle glycogen directly related to variations in body fatness.
it may however not be optimal for distinguishing the mechanisms involved in the control of adiposity and muscle glycogen metabolism.
we therefore used chickens generated from a f <dig> cross between the fat and lean lines.
in this population, phenotypically extreme individuals with high and low muscle glycogen content  displayed only limited differences in abdominal fat content .
being able to dissociate carcass fatness and muscle glycogen content highlighted that, although muscle glycogen metabolism and carcass adiposity are under the control of shared regulation in chicken, they also involve specific pathways.
working on f <dig> birds also allowed comparison of birds with a more homogeneous genetic background  while specifically differing in muscle glycogen content.
among the genes found to be expressed in chicken p. major muscle,  <dig> and  <dig> transcripts were differentially expressed between f and l and g+ and g-, respectively.
notably, only  <dig> transcripts, corresponding to  <dig> known genes, were recorded as differential in both models.
with the hypothesis that the g+/g- model was the most powerful to identify genes controlling glycogen metabolism and to rule out the possibility that some of them could have been missed in the f/l comparison, qrtpcr comparisons were conducted on both models and focused on genes linked to glycogen and lipid metabolism and differential between g+ and g-.
the results confirm that the differences are indeed specific of the g+/g- model for most genes  and even for the genes showing a strong differential expression  no difference was observed between f and l. moreover, the results also pointed out that pdk <dig> and ugdh were inversely regulated in relation to glycogen content in g+/g- compared to f/l.
similarly, we observed significant differences between g+ and g- and not between f and l for three candidate genes .
altogether, the data further supported that the comparison of extreme animals with high or low glycogen in the f <dig> population was the most adapted to identify the mechanisms controlling glycogen metabolism in chicken muscle.
for this reason we decided to further analyse  <dig> genes confirmed as differential between g+ and g-.
a bioinformatic analysis of the promoter sequences of these genes indicated a transcriptional link between  <dig> of them and suggested a key role for the three transcription factors, cebpb, foxo <dig>  srf as potential regulators of several functional candidates affecting glycogen turnover in the muscle .
figure  <dig> attempts to summarize how the differences observed at transcript level could impact on glycogen metabolism.
the lower expression of ugdh  in the g+ muscle is consistent with reduced conversion of udp-glucose into udp-glucuronate, and therefore higher use of glycogen synthesis.
phkd, that encodes the δ subunit of the phosphorylase kinase complex , was also expressed at lower levels in g+ muscle.
this regulatory subunit  contains the ca2+-binding site which allows the activation of the phosphorylase kinase complex that both activates glycogenolysis and inhibits glycogen synthesis  <cit> .
its up-regulation in the g- muscle is therefore consistent with increased activity of the phk complex and a reduced amount of glycogen in muscle.
the activity of the phk complex is under the control of both camp-dependent protein kinase  and amp-activated protein kinase .
the nuclear transcription factor ccaat/enhancer-binding protein beta  and the regulator of g protein signalling  <dig>  are involved in the control of the cellular camp level and therefore in the activation of camp-dependent protein kinase   <cit> .
pka is known to suppress inhibition of the gamma subunit of phosphorylase kinase  and thereby activate the phosphorylase kinase enzyme complex, which in turn activates glycogen phosphorylase and inhibits glycogen synthase  <cit> .
our observations are therefore consistent with a potential role of cebpb and rgs <dig> in the regulation of glycogen levels in chicken skeletal muscle, through the camp-dependent pka pathway.
however, the lpar <dig> gene , which exerts a similar effect on camp levels, showed different regulation, being down-regulated in g+.
in the muscle of g+ chickens, we observed lower expression of the gene encoding the regulatory γ <dig> subunit of ampk .
the amp-dependence of the ampk complex is markedly affected by the identity of the γ isoform present, with γ2-containing complexes having a greater response to amp than those containing γ <dig> or γ <dig>  <cit> .
lower prkag <dig> mrna levels in g+ muscles could imply a lower response of the ampk complex to amp, which in turn would be consistent with greater amounts of glycogen in muscle.
although the levels of ampk activation were not measured in the present study, a reduced level of ampk activation was previously reported in f compared to l chickens in conditions where prkag <dig> mrna levels were lower in f than in l  <cit> .
the prkab <dig> gene encoding the regulatory β <dig> subunit of ampk was also expressed at lower levels in g+ muscle, as previously described in  <cit>  when comparing f and l chickens.
the ampk β subunit contains a glycogen-binding site which allows the kinase to act as a glycogen sensor, ampk activation being inhibited by glycogen  <cit> .
how the changed expression of the β <dig> regulatory subunit is related to muscle glycogen content remains to be elucidated.
while in the former study  <cit> , prkab <dig>  prkag <dig> and phkd were relatively over expressed in the muscles of l compared to f chickens, none of them were found to be differentially expressed between f and l chickens in the present study.
one explanation could be that the divergence in abdominal fatness was much more marked  between the fat and lean birds used in our previous study  <cit>  than in those used in the present study , possibly related to the composition of the diet.
in fact, the birds used in the first experiment received a diet with a higher crude protein level than those used in the present study  which might explain the lower adiposity differential between fat and lean lines reported here.
it can be expected that more marked phenotypic differences could be related to more marked differences in gene expression.
a recent study  <cit>  showed that ampk activation combined with fatty acid administration synergistically induced pyruvate dehydrogenase kinase  <dig>  expression, and in turn decreased cellular glucose oxidation.
it can be expected that the preferential expression of the γ <dig> ampk subunit  in the g- muscles is consistent with the increased expression of pdk <dig> reported in our study.
ampk activation also increases uncoupling protein  <dig>  expression in muscles in mammals  <cit> .
in chicken muscle, stimulation of ampk is also associated with significant over expression of the avian ucp, the ortholog gene of mammalian ucp <dig>  <cit> .
the relative down-regulation of the ampk γ <dig> subunit was thus consistent with that of ucp <dig>  reported concomitantly in g+ muscle.
as already mentioned, pdk <dig> was not down-regulated by higher glycogen content in the muscle of the f line, but in contrast, up-regulated, suggesting a strong interaction with lipid metabolism, which could also result from the absence of regulation of ampk subunits between f and l in the present study which contrasted with earlier results  <cit> .
several microarray studies conducted in genetically modified mice reported very distinct global gene expression profiles between animals exhibiting high or low glycogen content in muscle  <cit> .
there are only a few common genes between those highlighted in our study and those demonstrated to be regulated in such models.
this could arise from the fact that our birds were much less divergent in terms of glycogen content than the transgenic mice used previously, and also from the fact that different mechanisms may be involved.
in mice, glycogen content was altered by invalidation or overexpression of genes directly controlling glycogen content such as gys and prkag <dig>  the study of parker et al.
<cit>  comparing mice lacking  <cit>  or accumulating  <cit>  glycogen in muscle, as a result of glycogen synthase  inactivation or overexpression, revealed marked differences in expression for a number of enzymes involved in the regulation of glycogen metabolism.
comparison of mutant and knockout mice for prkag <dig> showed that changes in the activity of the γ <dig> subunit of ampk were accompanied by coordinated and reciprocal regulation of carbohydrate and lipid metabolism  <cit> .
indeed, mutation 225q , which causes accumulation of glycogen in muscle, was associated with a gene expression profile suggesting increased glucose and lipid uptake, oxidative capacity and glycogen synthesis, and resistance of muscle to fatigue.
only slight disturbances in gene expression were observed between g+ and g- chickens, suggesting that the corresponding phenotypes resulted from the additive effects of several genes on the muscle glycogen in the chicken rather than a major effect of a single gene such as the rn gene in the pig  <cit> .
the present study highlighted changes in several candidate genes directly involved in the control of glycogen metabolism such as phkd, prkab <dig> & g <dig> and pdk <dig> and also in genes involved in camp signalling such as cebpb, rgs <dig> and lpar <dig>  these transcriptional candidate genes warrant further study in larger populations to correlate their expression with muscle glycogen levels, and to investigate the underlying mechanisms.
CONCLUSIONS
the aim of the study presented here was to identify candidate genes involved in the control of glycogen content in muscle.
studying phenotypically extreme chickens for muscle glycogen content generated from a f <dig> cross population helped to distinguish mechanisms involved in lipid and carbohydrate metabolism, which are highly related in the chicken.
several genes, related to carbohydrate metabolism or not, were suggested as potentially active in the regulation of muscle glycogen content and hence meat quality in the chicken.
a qtl search is in progress on the f <dig> cross population used in the present study that aims to identify chromosomal regions involved in the control of phenotypes related to chicken meat quality, including muscle glycogen content.
this should help to identify both functional and positional genes that could be subsequently included in large scale expression studies to validate the relationship between variations in gene expression and meat quality.
these studies should together allow the identification of molecular markers that could be used to select birds with expected muscle and meat properties, and to optimize rearing practices via the study of gene regulation.

 with changes similar to those that occurred in the pig industry, the poultry market is now characterized by increasing diversity of processed products. as a consequence, poultry companies are now involved in food technology and product development, and improvement of meat processing ability has become a prevalent concern. as in pigs, post-mortem ph is a key factor controlling chicken meat quality. variations in ultimate meat ph are responsible for variations in several breast meat properties, including water-holding capacity, colour and firmness. low ultimate ph results in "acid meat", with a pale aspect and reduced water-holding capacity, while high ultimate ph leads to dfd meat, dark in colour, with reduced shelf-life. at the genetic level, there is a very strong negative correlation between the ultimate ph of breast meat and the level of muscle glycogen estimated by the glycolytic potential at the time of slaughter. the glycolytic potential has also been shown to be highly heritable. understanding the mechanisms and identifying the genes controlling muscle glycogen storage constitute a promising way to increase control of and improve chicken breast meat properties. it would make it possible to develop useful breeding tools, such as molecular markers, to select birds with expected meat properties, and help optimize rearing practices, via the study of gene regulation. glycogen is the main metabolic fuel for the anaerobic glycolysis which takes place post-mortem when muscles are no longer supplied with oxygen. the genetic control of muscle glycogen, and therefore meat quality, was evidenced first in mammals and more recently in the chicken. in mammalian species, including the pig, mouse and human, a major gene, prkag which encodes the γ regulatory subunit of the amp-activated protein kinase, is responsible for variations in muscle glycogen content. in the chicken, there is no information available suggesting that a major gene could be involved in the control of glycogen content in muscle. however, several studies have suggested that breast muscle glycogen content is related to growth and body composition: it decreases with growth rate and breast meat yield and increases with carcass fatness. the aim of the present study was to compare the expression profiles of muscles from chickens differing in muscle glycogen content and breast meat quality. in the first experiment, birds originating from two experimental lines and lean lines) were compared. the f and l lines were originally divergently selected for and against the amount of abdominal fat but they also exhibited differences in muscle glycogen content and in breast meat quality traits. despite a similar growth rate, the chickens from the f line were times fattier than those from the l line. moreover, due to high muscle glycogen content, the meat of the fat chickens exhibited a lower ultimate ph and higher drip loss and lightness than lean chickens. the differences in muscle glycogen between the f and l lines have been related to variations in mrna encoding several enzymes regulating glycogen synthesis and degradation as well as in activation of ampk by phosphorylation. in the second experiment, the muscle transcriptomes were compared in individuals generated from the f population produced from these two lines and exhibiting extremely high or low muscle glycogen content. individuals for this analysis were chosen according to their levels of glycogen in muscle, while differences in body fatness were much less pronounced than in the first model. the use of a k oligo microarray provided the first description of genes differentially expressed between breast muscles exhibiting high or low glycogen content, correlated with poor or high meat quality traits, respectively. this global approach was complemented by mrna analyses on previously studied candidate genes and on a subset of genes identified from array analysis. for genes with a human ortholog, further interpretation was based on the use of ingenuity and gene ontology annotation databases highlighting several biological processes likely to be involved in muscle glycogen regulation.  carcass, muscle and meat quality traits of chickens used for gene expression analyses the mean carcass and p. major muscle traits and sd are presented in table at weeks of age, body weight was similar between f and l birds and between g+ and g- birds generated from the f2fl population. breast meat yield was slightly higher in l than in f birds and similar between g+ and g-. abdominal fat yield was 2-fold greater in f than in l birds while it was only 30% greater in g+ compared to g- birds. however, the muscle glycogen reserves were 61% higher in g+ than in g- birds while they were only 34% higher in f than in l birds. the ultimate ph of p. major muscle of f and g+, in relation to their greater glycogen content at the time of death, was higher than that of l and g- birds, respectively. the breast meat was lighter and less coloured, and exhibited more drip loss in f than in l birds. only meat lightness was higher in g+ than in g- birds generated from the f2fl population. the g+ and g- chickens were generated from the f2fl population produced from the generation intercross between the founder fat and lean lines. the g- and g+ chickens correspond to the individual females exhibiting the lowest and the highest muscle glycogen content within the f2fl population. ns = non-significant. differential analysis on microarray among the genes found to be expressed in chicken p. major muscle, and transcripts were differentially expressed between f and l and g+ and g-, respectively. the gene expression fold-change ranged from to and to for the f vs. l and the g+ vs. g- comparisons, respectively. a trend was observed for a higher percentage of genes down-regulated in muscle with high glycogen content. full details of gene name, function, accession number, fold-change and p-value for all differentially expressed transcripts are listed in additional files and only transcripts were recorded as differential in both analyses, i.e. f vs. l and g+ vs. g-, corresponding to known genes. functional annotation among the genes that were reported to be differentially expressed between muscles with high or low glycogen content, with a human ortholog were submitted to annotation analyses. when compiling the lists of genes expressed differentially between g+ and g- and between f and l chickens, the software ingenuity analysis pathways highlighted several biological functions, including lipid and carbohydrate metabolism. the genes associated with these two functions represent about 15% of the genes considered for annotation analysis. when considering findings separately, ipa highlighted lipid metabolism and molecular transport as common pathways in the two models and also specific biological functions for each of them: cell morphology, cell cycle, and cell to cell signalling and interaction for f/l, and small molecular biochemistry, cell death, and cellular development for g+/g-. gene ontology terms are also widely used for global interpretation of the functions of genes revealed by differential microarray analysis. according to gene ontology, genes differentially expressed between f and l participated in several biological processes that can be grouped in main biological functions: molecule transport and localization, and lipid, energy, and amino acid metabolism. genes differentially expressed between g+ and g- belonged to biological processes especially related to lipid and energy metabolism, as well as developmental processes including cell growth, proliferation, differentiation and organization. the biological interpretation of expressional data was performed using ingenuity pathway analysis. the genes included in the analysis were shown to be differential between f and l and/or between g+ and g- by either microarray or real-time rt-pcr. genes are presented in alphabetical order for each category. the genes over expressed in muscles with high and low glycogen content are in bold and normal characters, respectively. genes in italic were differentially regulated between models. validation by real-time rt-pcr of a subset of genes revealed by differential microarray analysis the mrna levels of genes involved in skeletal and muscular system development and function, lipid metabolism or carbohydrate metabolism, and found to be differential on microarray were further quantified by real-time rt-pcr in both models. the level of 18s rrna was chosen as reference and confirmed to be invariable. the expression levels of genes were compared between g+ and g- and between f and l for each of the genes selected. ratios of gene expression determined by real-time rt-pcr were compared to ratios obtained using microarray analysis. over expression in g+ compared to g- was clearly confirmed for cebpb and rgs, and suggested for foxo as similar fold-changes reached significance in the microarray analysis but not with real-time rt-pcr. under expression was confirmed for lpar pdk rps and srf, and suggested for ppp1r2b and ucp as similar fold-changes were observed in microarray and rt-pcr studies. none of the differences suggested between f and l chickens by the microarray study could be statistically confirmed by rt-pcr. slightly higher expression of ugdh and lower expression of srf were suggested in f compared to l chickens. it is of note that the genes showed completely different variations between f and l and between g+ and g-. none of the genes over expressed in g+ compared to g- differed between f and l chickens. similarly, lpar and rps which were under expressed in g+ compared to g-, did not differ between f and l. by contrast to what was observed when comparing g+ to g-, pdk was over expressed in f compared to l, suggesting an inverse relationship between muscle glycogen and pdk expression in the two models. the observation of higher expression of abhd in g+ compared to g- on microarray prompted real-time rt-pcr measurements in f and l. while the differential expression between g+ and g- was not confirmed, significantly higher expression was observed in f compared to l chickens. the biological interpretation of the real-time rt-pcr findings highlighted a gene network involved in several molecular and cellular functions, including lipid and carbohydrate metabolism, molecular transport, small molecule biochemistry, and cell morphology. for both microarray and real-time rt-pcr analyses, results are expressed as fat to lean and g+ to g- ratios of the expression gene. ns = non-significant; nd = not determined. statistical significance is indicated as follows: †p value <, *p value <, **p value <. § only genes identified as differentially expressed in at least one model are presented in the table. differential analysis of candidate genes the transcript expression of genes directly involved in muscle glycogen turnover was quantified by real-time rt-pcr. only of them were significantly differentially expressed between g+ and g- and their expression ratios are presented in table none of the genes assayed was significantly differentially expressed between f and l chickens. the transcript levels of prkab and prkag which encode the amp-activated protein kinase regulatory β and γ subunits, respectively was lower in g+ than in g- muscles: the ratio of g+/g- expression was for prkab and for prkag. the phkd gene, which encodes the δ subunit of phosphorylase kinase, was also significantly down-regulated in g+ compared to g- muscles, with a g+/g- expression ratio of. transcription factor analysis transcription factor analysis highlighted several interrelations between genes whose expression differential was confirmed between g+ and g- animals by real-time rt-pcr. binding sites for the transcription factor cebpb were thus reported in the promoters of rgs ucp srf, foxo similarly, promoters of cebpb, prkab and pdk possess the foxo binding site, and rps and lpar possess binding sites for srf. ugdh is under the control of ppara that also activates ucp and pdk discussion few studies have reported global gene expression surveys in the chicken to date. moreover, our study is the first to relate global gene expression profiles to muscle glycogen content and variations in meat quality. in mammals, especially in cattle and the pig, several microarray studies have investigated global muscle gene expression in relation to sensorial meat attributes such as tenderness, juiciness, flavour and marbling, which are not directly related to muscle glycogen content. recent analyses in the pig helped to link gene expression profiles to variations in meat water loss, this characteristic being strongly related to variations in muscle ph, but without considering glycogen variations in muscle. the transcripts being up-regulated with high drip loss in the pig belong to groups of genes functionally categorized as genes of membrane proteins, signal transduction, cell communication, response to stimulus, and the cytoskeleton. among genes down-regulated with high drip loss, functional groups of oxidoreductase activity, electron transport, and lipid metabolism were identified. the originality of our study lies in the models chosen for microarray analyses. as already shown, there is a positive relationship between body fatness and glycogen content in breast muscle in the chicken. comparing divergently selected fat and lean chickens, which also differ in muscle glycogen content, is therefore relevant to identify mechanisms underlying variations in muscle glycogen directly related to variations in body fatness. it may however not be optimal for distinguishing the mechanisms involved in the control of adiposity and muscle glycogen metabolism. we therefore used chickens generated from a f cross between the fat and lean lines. in this population, phenotypically extreme individuals with high and low muscle glycogen content displayed only limited differences in abdominal fat content. being able to dissociate carcass fatness and muscle glycogen content highlighted that, although muscle glycogen metabolism and carcass adiposity are under the control of shared regulation in chicken, they also involve specific pathways. working on f birds also allowed comparison of birds with a more homogeneous genetic background while specifically differing in muscle glycogen content. among the genes found to be expressed in chicken p. major muscle, and transcripts were differentially expressed between f and l and g+ and g-, respectively. notably, only transcripts, corresponding to known genes, were recorded as differential in both models. with the hypothesis that the g+/g- model was the most powerful to identify genes controlling glycogen metabolism and to rule out the possibility that some of them could have been missed in the f/l comparison, qrtpcr comparisons were conducted on both models and focused on genes linked to glycogen and lipid metabolism and differential between g+ and g-. the results confirm that the differences are indeed specific of the g+/g- model for most genes and even for the genes showing a strong differential expression no difference was observed between f and l. moreover, the results also pointed out that pdk and ugdh were inversely regulated in relation to glycogen content in g+/g- compared to f/l. similarly, we observed significant differences between g+ and g- and not between f and l for three candidate genes. altogether, the data further supported that the comparison of extreme animals with high or low glycogen in the f population was the most adapted to identify the mechanisms controlling glycogen metabolism in chicken muscle. for this reason we decided to further analyse genes confirmed as differential between g+ and g-. a bioinformatic analysis of the promoter sequences of these genes indicated a transcriptional link between of them and suggested a key role for the three transcription factors, cebpb, foxo srf as potential regulators of several functional candidates affecting glycogen turnover in the muscle. figure attempts to summarize how the differences observed at transcript level could impact on glycogen metabolism. the lower expression of ugdh in the g+ muscle is consistent with reduced conversion of udp-glucose into udp-glucuronate, and therefore higher use of glycogen synthesis. phkd, that encodes the δ subunit of the phosphorylase kinase complex, was also expressed at lower levels in g+ muscle. this regulatory subunit contains the ca2+-binding site which allows the activation of the phosphorylase kinase complex that both activates glycogenolysis and inhibits glycogen synthesis. its up-regulation in the g- muscle is therefore consistent with increased activity of the phk complex and a reduced amount of glycogen in muscle. the activity of the phk complex is under the control of both camp-dependent protein kinase and amp-activated protein kinase. the nuclear transcription factor ccaat/enhancer-binding protein beta and the regulator of g protein signalling are involved in the control of the cellular camp level and therefore in the activation of camp-dependent protein kinase. pka is known to suppress inhibition of the gamma subunit of phosphorylase kinase and thereby activate the phosphorylase kinase enzyme complex, which in turn activates glycogen phosphorylase and inhibits glycogen synthase. our observations are therefore consistent with a potential role of cebpb and rgs in the regulation of glycogen levels in chicken skeletal muscle, through the camp-dependent pka pathway. however, the lpar gene, which exerts a similar effect on camp levels, showed different regulation, being down-regulated in g+. in the muscle of g+ chickens, we observed lower expression of the gene encoding the regulatory γ subunit of ampk. the amp-dependence of the ampk complex is markedly affected by the identity of the γ isoform present, with γ2-containing complexes having a greater response to amp than those containing γ or γ. lower prkag mrna levels in g+ muscles could imply a lower response of the ampk complex to amp, which in turn would be consistent with greater amounts of glycogen in muscle. although the levels of ampk activation were not measured in the present study, a reduced level of ampk activation was previously reported in f compared to l chickens in conditions where prkag mrna levels were lower in f than in l. the prkab gene encoding the regulatory β subunit of ampk was also expressed at lower levels in g+ muscle, as previously described in when comparing f and l chickens. the ampk β subunit contains a glycogen-binding site which allows the kinase to act as a glycogen sensor, ampk activation being inhibited by glycogen. how the changed expression of the β regulatory subunit is related to muscle glycogen content remains to be elucidated. while in the former study, prkab prkag and phkd were relatively over expressed in the muscles of l compared to f chickens, none of them were found to be differentially expressed between f and l chickens in the present study. one explanation could be that the divergence in abdominal fatness was much more marked between the fat and lean birds used in our previous study than in those used in the present study, possibly related to the composition of the diet. in fact, the birds used in the first experiment received a diet with a higher crude protein level than those used in the present study which might explain the lower adiposity differential between fat and lean lines reported here. it can be expected that more marked phenotypic differences could be related to more marked differences in gene expression. a recent study showed that ampk activation combined with fatty acid administration synergistically induced pyruvate dehydrogenase kinase expression, and in turn decreased cellular glucose oxidation. it can be expected that the preferential expression of the γ ampk subunit in the g- muscles is consistent with the increased expression of pdk reported in our study. ampk activation also increases uncoupling protein expression in muscles in mammals. in chicken muscle, stimulation of ampk is also associated with significant over expression of the avian ucp, the ortholog gene of mammalian ucp. the relative down-regulation of the ampk γ subunit was thus consistent with that of ucp reported concomitantly in g+ muscle. as already mentioned, pdk was not down-regulated by higher glycogen content in the muscle of the f line, but in contrast, up-regulated, suggesting a strong interaction with lipid metabolism, which could also result from the absence of regulation of ampk subunits between f and l in the present study which contrasted with earlier results. several microarray studies conducted in genetically modified mice reported very distinct global gene expression profiles between animals exhibiting high or low glycogen content in muscle. there are only a few common genes between those highlighted in our study and those demonstrated to be regulated in such models. this could arise from the fact that our birds were much less divergent in terms of glycogen content than the transgenic mice used previously, and also from the fact that different mechanisms may be involved. in mice, glycogen content was altered by invalidation or overexpression of genes directly controlling glycogen content such as gys and prkag the study of parker et al. comparing mice lacking or accumulating glycogen in muscle, as a result of glycogen synthase inactivation or overexpression, revealed marked differences in expression for a number of enzymes involved in the regulation of glycogen metabolism. comparison of mutant and knockout mice for prkag showed that changes in the activity of the γ subunit of ampk were accompanied by coordinated and reciprocal regulation of carbohydrate and lipid metabolism. indeed, mutation 225q, which causes accumulation of glycogen in muscle, was associated with a gene expression profile suggesting increased glucose and lipid uptake, oxidative capacity and glycogen synthesis, and resistance of muscle to fatigue. only slight disturbances in gene expression were observed between g+ and g- chickens, suggesting that the corresponding phenotypes resulted from the additive effects of several genes on the muscle glycogen in the chicken rather than a major effect of a single gene such as the rn gene in the pig. the present study highlighted changes in several candidate genes directly involved in the control of glycogen metabolism such as phkd, prkab & g and pdk and also in genes involved in camp signalling such as cebpb, rgs and lpar these transcriptional candidate genes warrant further study in larger populations to correlate their expression with muscle glycogen levels, and to investigate the underlying mechanisms.  the aim of the study presented here was to identify candidate genes involved in the control of glycogen content in muscle. studying phenotypically extreme chickens for muscle glycogen content generated from a f cross population helped to distinguish mechanisms involved in lipid and carbohydrate metabolism, which are highly related in the chicken. several genes, related to carbohydrate metabolism or not, were suggested as potentially active in the regulation of muscle glycogen content and hence meat quality in the chicken. a qtl search is in progress on the f cross population used in the present study that aims to identify chromosomal regions involved in the control of phenotypes related to chicken meat quality, including muscle glycogen content. this should help to identify both functional and positional genes that could be subsequently included in large scale expression studies to validate the relationship between variations in gene expression and meat quality. these studies should together allow the identification of molecular markers that could be used to select birds with expected muscle and meat properties, and to optimize rearing practices via the study of gene regulation. 
several other genes were suggested to have roles in the regulation of glycogen storage in chicken muscle. real-time rt-pcr was subsequently performed to validate the differential expression of genes either selected from the microarray analysis or whose function in regulating glycogen metabolism was well known. pdk may act as a glycogen sensor in muscle, ugdh may compete for glycogen synthesis by using udp-glucose for glucoronidation, and prkab prkag and phkd may impact on glycogen turnover in muscle, through amp-activated signalling pathways. the genetic determinism of glycogen and related meat quality traits has been established in the chicken but the molecular mechanisms involved in variations in these traits remain to be fully described. among the genes found to be expressed in chicken p. major muscle, and transcripts appeared to be differentially expressed on microarrays for the f vs. l and the g+ vs. g- comparisons, respectively. we confirmed that, as in mammals, the down-regulation of cebpb and rgs coincides with a decrease in peripheral adiposity in the chicken, but these genes are also suggested to affect muscle glycogen turnover through their role in the camp-dependent signalling pathway. some involved particularly in lipid and carbohydrate metabolism were selected for further validation studies by real-time rt-pcr. large scale analyses are now required to validate the role of the genes identified and ultimately to find molecular markers that can be used for selection or to optimize rearing practices. in this study, chicken genome arrays were used to compare muscle gene expression profiles of chickens from fat and lean lines that exhibited high and low muscle glycogen content, respectively, and of individuals exhibiting extremely high or low muscle glycogen content originating from the f cross between the fat and lean lines. the processing ability of poultry meat is highly related to its ultimate ph, the latter being mainly determined by the amount of glycogen in the muscle at death.
 the processing ability of poultry meat is highly related to its ultimate ph, the latter being mainly determined by the amount of glycogen in the muscle at death. the genetic determinism of glycogen and related meat quality traits has been established in the chicken but the molecular mechanisms involved in variations in these traits remain to be fully described. in this study, chicken genome arrays were used to compare muscle gene expression profiles of chickens from fat and lean lines that exhibited high and low muscle glycogen content, respectively, and of individuals exhibiting extremely high or low muscle glycogen content originating from the f cross between the fat and lean lines. real-time rt-pcr was subsequently performed to validate the differential expression of genes either selected from the microarray analysis or whose function in regulating glycogen metabolism was well known.  among the genes found to be expressed in chicken p. major muscle, and transcripts appeared to be differentially expressed on microarrays for the f vs. l and the g+ vs. g- comparisons, respectively. some involved particularly in lipid and carbohydrate metabolism were selected for further validation studies by real-time rt-pcr. we confirmed that, as in mammals, the down-regulation of cebpb and rgs coincides with a decrease in peripheral adiposity in the chicken, but these genes are also suggested to affect muscle glycogen turnover through their role in the camp-dependent signalling pathway. several other genes were suggested to have roles in the regulation of glycogen storage in chicken muscle. pdk may act as a glycogen sensor in muscle, ugdh may compete for glycogen synthesis by using udp-glucose for glucoronidation, and prkab prkag and phkd may impact on glycogen turnover in muscle, through amp-activated signalling pathways.  this study is the first stage in the understanding of molecular mechanisms underlying variations in poultry meat quality. large scale analyses are now required to validate the role of the genes identified and ultimately to find molecular markers that can be used for selection or to optimize rearing practices. 