BACKGROUND
for decades intensive genetic selection has increased the growth rate of broiler chickens by more than 300% , improving feed efficiency, increasing breast muscle size, and increasing weight concomitantly .
these selection-induced body changes were accompanied by a significant increase in metabolic rate mainly attributable to major metabolic organs including liver and muscle, and increased environmental temperature sensitivity .
furthermore, a higher death rate due to sudden death syndrome is observed in these broiler chickens during the early growth phase than in slower growing breeds  <cit> .
the internal organs of broiler chickens develop slower, remain smaller in size, and have reduced oxygen supplies than non-broiler chickens, resulting in several metabolic disturbances  <cit> .
however, the biological mechanisms underlying the relationship between increased growth rates and delayed and reduced organ development remain largely unknown.
detailed studies comparing chicken breed naturally differentiating for growth rate could elucidate these biological mechanisms, resulting in development of biomarkers to control both growth rate and organ development simultaneously.
to enable the study of biological mechanisms underlying a trait, a good animal model should have the following features:  the animal model should display high variation for the trait of interest, preferably without an extreme selection background for the trait, as selection may induce unwanted and probably unnoticed side effects, and  the animal model should display variation in traits related to the trait under investigation.
we studied the biological mechanisms underlying growth rate in  chickens.
as a result of increased growth rate, body weight is usually also increased, while organ weights may be reduced.
indigenous chinese chicken breeds comprise a wide variety of phenotypes, including growth rate and body weight.
most indigenous chinese breeds have not undergone artificial selection and are used both as layers and broilers.
the wuding chicken breed is a fast growing large size chicken breed, and the daweishan mini chicken breed is a slow growing small size chicken breed.
the average daily gain  of the wuding chicken breed is more than five times the adg of the daweishan mini chicken.
in addition, the adult wuding chicken body size and weight are double that of the daweishan mini chicken.
furthermore, the internal organs of the daweishan mini chicken are relatively larger than the internal organs of the wuding chicken  <cit> .
thus, these two breeds may be an ideal model system to study the effects of broiler chicken growth rates in a background without artificial selection.
transcriptomics offers the possibility to study genome activation, highlighting the biological mechanisms underlying differences in phenotypes .
studying the transcriptome of breast muscle and liver will highlight differences in tissue-specific expression profiles in these two breeds.
the objective of this study was to investigate differences in biological mechanisms between these two breeds related to differences in growth rate and body weight phenotypes.
RESULTS
body composition and growth pattern differences in wuding and daweishan chicken breeds
a
bw body weight; borgan weight percentage of body weight
fig.
<dig> the bursa of fabricius of wuding chicken  and daweishan chicken .
despite the difference in body size the bursa of fabricius were of similar size, so the relative size is larger in the daweishan compared to the wuding chicken
muscle and liver transcriptome profile differences between wuding and daweishan chicken breeds
the  microarray hybridization data for muscle and liver are provided in additional files  <dig> and  <dig>  respectively.
table  <dig> shows the number of differentially expressed genes in the breast muscle and liver of wuding and daweishan mini chicken.
the number of differentially expressed genes with higher expression in daweishan than in wuding chicken was always higher than the number of differentially expressed genes showing the opposite expression ratio.table  <dig> number of genes differentially expressed between the wuding and the daweishan chicken breeds, and number of genes with annotation to be analyzed
wuding > minib
mini > wudingb
wuding > minib
mini > wudingb
an is number of genes b>: higher expressed
the annotation of the microarray showed many probes as either expressed sequence tag  numbers or unknowns.
the probes were converted into gene names, and the number of genes with sufficient identification to be included in the functional analyses is given in the “n analysis” column.
unannotated genes were not used for the functional analyses.
athe direction of the differential expression:>: higher expressed
genes displaying higher expression in the wuding than in the daweishan chicken breed
breast muscle : cytoscape-reactomefi analysis identified one large network and four small networks based on the differentially expressed genes.
three additional analyses methods  indicated that the large network consisted of muscle-specific differentially expressed genes, including muscle developmental genes.
biological mechanisms enriched for differentially expressed genes included both striated skeletal muscle and cardiac muscle development and functioning, as well as mechanisms associated with muscle development and function, including energy metabolism, extra cellular matrix development and composition, protein metabolism, and the scavenger receptor pathway.
another biological mechanism enriched for differentially expressed genes included a network of diverse immune-related functions.
genes involved in the hydroxy lysine and proline metabolisms were also enriched.
liver tissue : cytoscape-reactomefi could not create a network based on the differentially expressed genes.
however, a network was identified when allowing for the addition of linker genes .
string was unable to create a network, just a few connections between protein could be formed.
functional analysis using david did not identify any enriched biological mechanisms.
cytoscape-cluego identified enrichment of genes displaying increased expression involved in catecholamine mechanisms, including dopamine, monoamine, and ammonium ion metabolism.fig.
<dig> cytoscape-reactomefi network of genes displaying higher expression in wuding than in daweishan liver.
the network was created using the linker gene option.
this software option adds genes to the network through which two or more differentially expressed genes are connected, thereby creating a network.
circles denote differentially expressed genes, diamonds indicate linker genes.
without the use of linker genes no network was created
genes displaying higher expressed in the daweishan than in the wuding breed
breast muscle : cytoscape-reactomefi analysis was unable to create a network based on the differentially expressed genes.
allowing the inclusion of linker genes resulted in formation of a network .
as shown in the figure, no direct gene connections between differently expressed genes were observed.
david analysis indicated over representation of genes involved in biological mechanisms of the endoplasmic reticulum.
cytoscape-cluego identified enrichment of genes involved in metabolism of glycerolipids/neutral lipids and the scavenger receptor mechanism.
string identified networks of differentially expressed molecular chaperones, protein synthesis, t-cell activation, adherence junction, and mitochondrial genes.fig.
<dig> cytoscape-reactomefi network of genes displaying higher expression in daweishan than in wuding breast muscle.
the network was created using the linker gene option.
this software option adds genes to the network through which two or more differentially expressed genes are connected, thereby creating a network.
circles denote differentially expressed genes, diamonds indicate linker genes.
without the use of linker genes no network was created
liver tissue : cytoscape-reactomefi analysis was unable to create a network based on the differentially expressed genes.
allowing for the use of linker genes resulted in formation of a network.
cytoscape-cluego indicated overrepresentation of abc receptors.
david and string indicated that genes involved in defense/immune processes – especially the il- <dig> cytokine and acute phase mechanisms – and protein metabolism and signal transduction were higher expressed in the daweishan than in the wuding breed.
biological mechanisms enriched for up- and down-regulated differentially expressed genes
breast muscle
the results were highly complementary.
only a few additional enriched metabolic mechanisms were identified: the statin pathway and the selenium-folate-vitb <dig>  pathway.
liver tissue
the results identified enriched biological mechanisms associated with the same functions as the individual analysis, namely immune processes and cellular metabolism.
genes involved in immune processes were more commonly differentially expressed than those involved in functional metabolic processes.
enriched functional metabolic processes were primarily involved in the regulation of energy metabolism.
discussion
the model system: breeds, growth rate, body size, and organ development
breeding in broiler chickens is primarily focused on selection to increase growth rate and muscularity .
we used two indigenous chinese chicken breeds to investigate the biological mechanisms underlying differences in body growth rate.
high phenotypic variation is observed between these two breeds, with the wuding breed displaying an almost five times higher growth rate and more than two times higher adult body weight than the daweishan breed.
the high contrast in our model system is ideal for investigating biological mechanisms underlying these phenotypes.
our results indicated that the wuding chicken internal organs are relatively smaller than the daweishan chicken.
increased growth rate in broiler chickens is associated with the development of ascites  <cit> .
reduced organ development is also observed in chickens with ascites compared to healthy chickens  <cit> .
ascites has also been described as a metabolic disorder  <cit> , and the liver is the major organ involved in metabolism.
we therefore conclude that our model system is ideal for the study of the biological mechanisms underlying phenotypic differences in growth rate, and proceeded to investigate the transcriptome profiles of both breast muscle and liver.
indeed, it may be argued that our model system did not show signs of ascites.
the underlying reason may be that the breeds of our model system did not undergo high intensity artificial selection like commercial broiler lines.
this resulted in more balanced metabolic changes as we have shown.
this may also be a lesson for breeding companies.
validation of microarray results using real time pcr
the breast muscle and liver tissue microarrays identified a high number of differentially expressed genes between the wuding and the daweishan mini chicken breeds.
genes displaying both higher and lower expression levels were identified between breeds.
microarray results were verified using real time pcr, confirmed both the magnitude and the directional changes reported between the two breeds.
only minor differences in the amplitude of the expression changes were observed between the microarray and real time pcr results.
these differences may originate from experimental conditions such as differences in hybridization efficiency between the microarray probes and real time pcr primers, or taq-polymerase efficiencies.
morey et al.
<cit>  showed that the correlation between microarrays and real time pcr varies due to experimental factors including expression level, fold change, etc.
therefore, we concluded that the microarray data used in our bioinformatics analysis was accurate.
biological mechanisms: growth rate and breast muscle development
in both tissues the number of genes displaying increased expression in the daweishan chicken was higher than in the wuding chicken breed.
this may indicate a higher general metabolic rate in the daweishan breed than in the wuding breed.
wuding breed metabolism seems to be focused on specific metabolic pathways, e.g.
muscle development , while the general metabolism of the wuding breed was depressed compared to the daweishan breed.
this may also help explained the relationship between the above-mentioned metabolic effects and ascites.
the wuding chicken breed displayed increased expression of genes involved in several muscle growth, development, and functional biological mechanisms compared to the daweishan chicken breed.
this result indicates that muscle growth is a major biological factor affecting body growth rates.
this is in agreement with previous results from broiler chicken lines selected for increased body growth rate  <cit> , indicating that our model system is a good model for studying the mechanisms underlying differences in growth rates in broiler chickens.
several of our analyses indicated that striated muscle activity and heart functioning pathways were enriched for differentially expressed genes, although we only analyzed breast muscle tissue.
this may be due to the involvement of similar genes in striated muscle activity and heart functioning pathways.
furthermore, networks regulating collagen, extra cellular matrix, and energy metabolism accompanied the network.
these networks represent parts of breast muscle tissue and necessary muscle functions.
this suggests that both muscle growth rate and function are regulated in a balanced way.
the breast muscle results also showed increased expression of genes involved in immune function in the wuding compared to the daweishan chicken breed.
the results indicated that several immune mechanisms involved in the acute inflammatory reaction were activated .
this may suggest that the wuding breast muscle tissue was less healthy than the daweishan chicken.
so, despite the balanced biological mechanisms underlying the growth rate of the wuding breast muscle tissue, the increased inflammatory status may indicate an imbalance compared to the daweishan chicken.
next, we investigated the hub genes of the networks in more detail combining the network information with the information from david and genecards .
the hub genes were defined as the genes with the most connections to other genes in the networks.
this analysis identified several hub genes active in more than one process, indicating a possible relationship between these biological processes.
the fhl <dig> gene was upregulated in the breast muscle of the wuding compared to the daweishan breed.
this gene encodes a member of the four-and-a-half-lim-only protein family, thought to have a role in the assembly of extracellular membranes.
this gene is down-regulated during transformation of normal myoblasts to rhabdomyosarcoma cells.
its upregulation in wuding breast muscle suggests involvement in the regulation of muscle development.
the tnnt <dig> gene was also upregulated in the breast muscle of the wuding breed.
the tnnt <dig> gene is fast skeletal muscle specific, suggesting that the muscle mass of wuding chickens is comprised mostly of the fast  muscle type.
it was previously reported that the muscle fiber composition of high muscle mass chickens consists mostly of fast muscle fibers  <cit> .
however, the upregulation of another hub gene, myh <dig>  which is mainly involved in heart function, suggests that slow  muscle fibers are also responsible for the high muscularity of the wuding chicken.
the c1qa, c1qb, and c1qc genes are hub genes involved in immune processes  that are also active in the hydroxylysine and hydroxyproline collagen synthesis pathways.
col6a <dig>  another hub gene, is a collagen associated with the c1qa, c1qb, and c1qc genes, the hydroxylysine/hydroxyproline pathways, and muscle organ development.
these results suggest that the upregulation of these hub genes is associated with increased growth rates.
the biological mechanisms enriched for genes displaying increased expression in the muscle tissue of the daweishan compared to wuding chicken breed were related to more general metabolism.
the metabolism of proteins – especially those that need to be exported from the cell – was affected, as was lipid metabolism and molecular chaperones.
the hub genes in the network created after allowing linker genes  were either linker genes or differentially expressed genes mainly connected to linker genes.
therefore, we focused on genes central to diverse functions rather than hub genes.
important genes involved in diverse mechanisms include hsp90b <dig>  hspa <dig>  pdia <dig>  and hyou <dig>  hsp90b <dig>  hspa <dig>  and hyou <dig> are chaperone proteins involved in regulating protein folding.
hsp90b <dig> and hspa <dig> are related to endoplasmic reticulum stress.
hsp90b <dig> binds calcium and is atp metabolizing, while hspa <dig> is regulated by glucose levels.
furthermore, hspa <dig> is involved in monitoring the transport of proteins through the cell.
hyou <dig> is also glucose regulated and is also a chaperone protein.
upregulation of hyou <dig> is suggested to be involved in reduction of apoptosis.
the pdia <dig> protein is an electron carrier.
cytoscape cluego analysis related these genes, and especially the hsp90b <dig> gene, directly to the regulation of the subthalamus development, which regulates movement – i.e.
skeletal muscle activity.
together these  genes may be involved in regulating increased cellular protein metabolism and breast muscle functioning in daweishan chickens.
finally, hsp90b <dig> activity is also associated with immune chaperone activity regulating innate and adaptive immunity  <cit> .
these immune mechanisms differ between the breast muscle of daweishan and wuding chicken breeds.
since these genes are involved in regulating the immune response rather than the proinflammatory immune response, these expression changes may not result in increased tissue damage.
high  growth rate and ascites in chickens
although we did not observe ascites in these chicken, health problems related to increased muscle growth rate and body weight of broiler chickens have been reported previously.
specifically, these health problems were related to ascites development and sudden death syndrome  <cit> .
the development of ascites and sudden death syndrome results from an imbalance in the development of the internal organs and skeletal muscle  <cit> .
indeed we observed differences between the relative organ weights of the wuding and daweishan chicken breeds.
so, despite the balanced biological mechanisms of the musculature of the wuding chicken breed, the differential organ development may cause problems leading to increased expression of acute inflammatory genes.
although speculative, this may indicate that the wuding chicken is less balanced than the daweishan chicken, and have activated biological mechanisms that could lead to ascites.
it should be noted that the growth rate of wuding chicken is still less than selected broiler lines.
we also observed increased expression of the scavenger receptors in the wuding compared to the daweishan chicken breed.
scavenger receptors are involved in the removal of foreign substances and waste from tissues  <cit> .
this may further indicate that unhealthy or unbalanced processes exist in wuding chicken breast.
such processes may potentially be related with inflammation, as described above.
they may also be partly responsible for the impaired health of the wuding chicken.
biological mechanisms: growth rate and liver metabolism
major differences in liver gene expression levels between the two chicken breeds were not observed, suggesting general liver metabolism is not significantly different between the two breeds.
with the use of linker genes a single network was created based on the genes displaying higher expression in the wuding than in the daweishan breed, including genes involved in catecholamine related pathways.
as all hub genes in the network were linker genes, we will not discuss them further.
the catecholamine pathway is upregulated during high stress levels and also affects the immune response .
this may help explain the altered expression of genes involved in the immune response  in breast muscle tissue.
the dopamine and monoamine genes are neurotransmitters that stimulate the reward-motivation behavior and emotion/arousal behavior, respectively.
upregulation of these genes in the wuding chicken breed may suggest that these chicken are more nervous/agitated compared to the daweishan breed.
in the body dopamine is also involved in gastrointestinal motility and  in regulating vasodilatation.
these mechanisms may be necessary to regulate uptake and distribution of feed components required for the higher growth rate of the wuding chicken breed than for the growth rate of the daweishan chicken.
vasodilatation may also be a mechanism to overcome the adverse effects of early ascites enabling increased oxygen supply to the cells.
similarly, in the liver of the daweishan mini chicken the hub genes in the network created allowing linker genes were either linker genes or differentially expressed genes mainly connected with linker genes.
a number of upregulated genes included genes involved in the atp binding cassette, the abc receptors, signal transduction, the il- <dig> pathway, and a number of transcription factors.
the diversity of the identified biological mechanisms indicates higher expression of several metabolic aspects in the daweishan than in the wuding chicken breed.
as indicated above, these results also indicate that increased growth rates come at the expense of general metabolic activity.
genes of major importance in this study were the abc receptor  genes abcb <dig>  abcb <dig>  and abcc <dig>  all three are involved in multi drug resistance, including xenobiotic compounds, bile salt export, and cyclic nucleotide export.
there main function is to lower the cellular concentration of these substances via translocation.
this increases cellular survival.
similarly, the il <dig> cytokine functions on cellular proliferation and differentiation to increased cellular survival via the lif gene and its receptor, all of which showed increased expression in the liver of the daweishan mini chicken.
il <dig> is a myokine discharged in the bloodstream after muscle contraction and acts to increase the breakdown of fats and improve insulin resistance.
this mechanism represents a direct link between muscle and liver functioning: in daweishan mini chicken increased muscle cell metabolic activity leads to increased liver activity.
other important genes include the psma <dig> and the hspd <dig> genes.
psma <dig> is an atp/ubiquitin proteasome protein  that uses atp to degrade proteins.
this reduces inflammatory immune responses in the liver of daweishan mini chicken, adding to the increased cellular survival.
hspd <dig> is a mitochondrial chaperonin protein essential for protein import in the mitochondrion and for correct protein folding.
all these functions use atp, or are involved in atp synthesis.
this points again to the increased metabolic activity in the daweishan compared to the wuding chicken.
CONCLUSIONS
our results identified increased expression of genes involved in muscle development and acute inflammatory responses associated with selection for fast body and muscle growth rates, while the daweishan breed displayed higher expression of genes involved in general metabolism than the wuding breed.
the general conclusion of this study is that selection for increased body growth rates focuses on muscle growth leading to a declined and imbalanced general metabolism.
this may be the underlying cause of ascites development in broiler chickens.
furthermore, the expression changes in breast muscle seem to be more related to these growth-rate changes than those observed in liver tissue.

 for decades intensive genetic selection has increased the growth rate of broiler chickens by more than 300%, improving feed efficiency, increasing breast muscle size, and increasing weight concomitantly. these selection-induced body changes were accompanied by a significant increase in metabolic rate mainly attributable to major metabolic organs including liver and muscle, and increased environmental temperature sensitivity. furthermore, a higher death rate due to sudden death syndrome is observed in these broiler chickens during the early growth phase than in slower growing breeds. the internal organs of broiler chickens develop slower, remain smaller in size, and have reduced oxygen supplies than non-broiler chickens, resulting in several metabolic disturbances. however, the biological mechanisms underlying the relationship between increased growth rates and delayed and reduced organ development remain largely unknown. detailed studies comparing chicken breed naturally differentiating for growth rate could elucidate these biological mechanisms, resulting in development of biomarkers to control both growth rate and organ development simultaneously. to enable the study of biological mechanisms underlying a trait, a good animal model should have the following features: the animal model should display high variation for the trait of interest, preferably without an extreme selection background for the trait, as selection may induce unwanted and probably unnoticed side effects, and the animal model should display variation in traits related to the trait under investigation. we studied the biological mechanisms underlying growth rate in chickens. as a result of increased growth rate, body weight is usually also increased, while organ weights may be reduced. indigenous chinese chicken breeds comprise a wide variety of phenotypes, including growth rate and body weight. most indigenous chinese breeds have not undergone artificial selection and are used both as layers and broilers. the wuding chicken breed is a fast growing large size chicken breed, and the daweishan mini chicken breed is a slow growing small size chicken breed. the average daily gain of the wuding chicken breed is more than five times the adg of the daweishan mini chicken. in addition, the adult wuding chicken body size and weight are double that of the daweishan mini chicken. furthermore, the internal organs of the daweishan mini chicken are relatively larger than the internal organs of the wuding chicken. thus, these two breeds may be an ideal model system to study the effects of broiler chicken growth rates in a background without artificial selection. transcriptomics offers the possibility to study genome activation, highlighting the biological mechanisms underlying differences in phenotypes. studying the transcriptome of breast muscle and liver will highlight differences in tissue-specific expression profiles in these two breeds. the objective of this study was to investigate differences in biological mechanisms between these two breeds related to differences in growth rate and body weight phenotypes.  body composition and growth pattern differences in wuding and daweishan chicken breeds a bw body weight; borgan weight percentage of body weight fig. the bursa of fabricius of wuding chicken and daweishan chicken. despite the difference in body size the bursa of fabricius were of similar size, so the relative size is larger in the daweishan compared to the wuding chicken muscle and liver transcriptome profile differences between wuding and daweishan chicken breeds the microarray hybridization data for muscle and liver are provided in additional files and respectively. table shows the number of differentially expressed genes in the breast muscle and liver of wuding and daweishan mini chicken. the number of differentially expressed genes with higher expression in daweishan than in wuding chicken was always higher than the number of differentially expressed genes showing the opposite expression ratio.table number of genes differentially expressed between the wuding and the daweishan chicken breeds, and number of genes with annotation to be analyzed  wuding > minib  mini > wudingb  wuding > minib  mini > wudingb an is number of genes b>: higher expressed the annotation of the microarray showed many probes as either expressed sequence tag numbers or unknowns. the probes were converted into gene names, and the number of genes with sufficient identification to be included in the functional analyses is given in the “n analysis” column. unannotated genes were not used for the functional analyses. athe direction of the differential expression:>: higher expressed genes displaying higher expression in the wuding than in the daweishan chicken breed breast muscle: cytoscape-reactomefi analysis identified one large network and four small networks based on the differentially expressed genes. three additional analyses methods indicated that the large network consisted of muscle-specific differentially expressed genes, including muscle developmental genes. biological mechanisms enriched for differentially expressed genes included both striated skeletal muscle and cardiac muscle development and functioning, as well as mechanisms associated with muscle development and function, including energy metabolism, extra cellular matrix development and composition, protein metabolism, and the scavenger receptor pathway. another biological mechanism enriched for differentially expressed genes included a network of diverse immune-related functions. genes involved in the hydroxy lysine and proline metabolisms were also enriched. liver tissue: cytoscape-reactomefi could not create a network based on the differentially expressed genes. however, a network was identified when allowing for the addition of linker genes. string was unable to create a network, just a few connections between protein could be formed. functional analysis using david did not identify any enriched biological mechanisms. cytoscape-cluego identified enrichment of genes displaying increased expression involved in catecholamine mechanisms, including dopamine, monoamine, and ammonium ion metabolism.fig. cytoscape-reactomefi network of genes displaying higher expression in wuding than in daweishan liver. the network was created using the linker gene option. this software option adds genes to the network through which two or more differentially expressed genes are connected, thereby creating a network. circles denote differentially expressed genes, diamonds indicate linker genes. without the use of linker genes no network was created genes displaying higher expressed in the daweishan than in the wuding breed breast muscle: cytoscape-reactomefi analysis was unable to create a network based on the differentially expressed genes. allowing the inclusion of linker genes resulted in formation of a network. as shown in the figure, no direct gene connections between differently expressed genes were observed. david analysis indicated over representation of genes involved in biological mechanisms of the endoplasmic reticulum. cytoscape-cluego identified enrichment of genes involved in metabolism of glycerolipids/neutral lipids and the scavenger receptor mechanism. string identified networks of differentially expressed molecular chaperones, protein synthesis, t-cell activation, adherence junction, and mitochondrial genes.fig. cytoscape-reactomefi network of genes displaying higher expression in daweishan than in wuding breast muscle. the network was created using the linker gene option. this software option adds genes to the network through which two or more differentially expressed genes are connected, thereby creating a network. circles denote differentially expressed genes, diamonds indicate linker genes. without the use of linker genes no network was created liver tissue: cytoscape-reactomefi analysis was unable to create a network based on the differentially expressed genes. allowing for the use of linker genes resulted in formation of a network. cytoscape-cluego indicated overrepresentation of abc receptors. david and string indicated that genes involved in defense/immune processes – especially the il- cytokine and acute phase mechanisms – and protein metabolism and signal transduction were higher expressed in the daweishan than in the wuding breed. biological mechanisms enriched for up- and down-regulated differentially expressed genes breast muscle the results were highly complementary. only a few additional enriched metabolic mechanisms were identified: the statin pathway and the selenium-folate-vitb pathway. liver tissue the results identified enriched biological mechanisms associated with the same functions as the individual analysis, namely immune processes and cellular metabolism. genes involved in immune processes were more commonly differentially expressed than those involved in functional metabolic processes. enriched functional metabolic processes were primarily involved in the regulation of energy metabolism. discussion the model system: breeds, growth rate, body size, and organ development breeding in broiler chickens is primarily focused on selection to increase growth rate and muscularity. we used two indigenous chinese chicken breeds to investigate the biological mechanisms underlying differences in body growth rate. high phenotypic variation is observed between these two breeds, with the wuding breed displaying an almost five times higher growth rate and more than two times higher adult body weight than the daweishan breed. the high contrast in our model system is ideal for investigating biological mechanisms underlying these phenotypes. our results indicated that the wuding chicken internal organs are relatively smaller than the daweishan chicken. increased growth rate in broiler chickens is associated with the development of ascites. reduced organ development is also observed in chickens with ascites compared to healthy chickens. ascites has also been described as a metabolic disorder, and the liver is the major organ involved in metabolism. we therefore conclude that our model system is ideal for the study of the biological mechanisms underlying phenotypic differences in growth rate, and proceeded to investigate the transcriptome profiles of both breast muscle and liver. indeed, it may be argued that our model system did not show signs of ascites. the underlying reason may be that the breeds of our model system did not undergo high intensity artificial selection like commercial broiler lines. this resulted in more balanced metabolic changes as we have shown. this may also be a lesson for breeding companies. validation of microarray results using real time pcr the breast muscle and liver tissue microarrays identified a high number of differentially expressed genes between the wuding and the daweishan mini chicken breeds. genes displaying both higher and lower expression levels were identified between breeds. microarray results were verified using real time pcr, confirmed both the magnitude and the directional changes reported between the two breeds. only minor differences in the amplitude of the expression changes were observed between the microarray and real time pcr results. these differences may originate from experimental conditions such as differences in hybridization efficiency between the microarray probes and real time pcr primers, or taq-polymerase efficiencies. morey et al. showed that the correlation between microarrays and real time pcr varies due to experimental factors including expression level, fold change, etc. therefore, we concluded that the microarray data used in our bioinformatics analysis was accurate. biological mechanisms: growth rate and breast muscle development in both tissues the number of genes displaying increased expression in the daweishan chicken was higher than in the wuding chicken breed. this may indicate a higher general metabolic rate in the daweishan breed than in the wuding breed. wuding breed metabolism seems to be focused on specific metabolic pathways, e.g. muscle development, while the general metabolism of the wuding breed was depressed compared to the daweishan breed. this may also help explained the relationship between the above-mentioned metabolic effects and ascites. the wuding chicken breed displayed increased expression of genes involved in several muscle growth, development, and functional biological mechanisms compared to the daweishan chicken breed. this result indicates that muscle growth is a major biological factor affecting body growth rates. this is in agreement with previous results from broiler chicken lines selected for increased body growth rate, indicating that our model system is a good model for studying the mechanisms underlying differences in growth rates in broiler chickens. several of our analyses indicated that striated muscle activity and heart functioning pathways were enriched for differentially expressed genes, although we only analyzed breast muscle tissue. this may be due to the involvement of similar genes in striated muscle activity and heart functioning pathways. furthermore, networks regulating collagen, extra cellular matrix, and energy metabolism accompanied the network. these networks represent parts of breast muscle tissue and necessary muscle functions. this suggests that both muscle growth rate and function are regulated in a balanced way. the breast muscle results also showed increased expression of genes involved in immune function in the wuding compared to the daweishan chicken breed. the results indicated that several immune mechanisms involved in the acute inflammatory reaction were activated. this may suggest that the wuding breast muscle tissue was less healthy than the daweishan chicken. so, despite the balanced biological mechanisms underlying the growth rate of the wuding breast muscle tissue, the increased inflammatory status may indicate an imbalance compared to the daweishan chicken. next, we investigated the hub genes of the networks in more detail combining the network information with the information from david and genecards. the hub genes were defined as the genes with the most connections to other genes in the networks. this analysis identified several hub genes active in more than one process, indicating a possible relationship between these biological processes. the fhl gene was upregulated in the breast muscle of the wuding compared to the daweishan breed. this gene encodes a member of the four-and-a-half-lim-only protein family, thought to have a role in the assembly of extracellular membranes. this gene is down-regulated during transformation of normal myoblasts to rhabdomyosarcoma cells. its upregulation in wuding breast muscle suggests involvement in the regulation of muscle development. the tnnt gene was also upregulated in the breast muscle of the wuding breed. the tnnt gene is fast skeletal muscle specific, suggesting that the muscle mass of wuding chickens is comprised mostly of the fast muscle type. it was previously reported that the muscle fiber composition of high muscle mass chickens consists mostly of fast muscle fibers. however, the upregulation of another hub gene, myh which is mainly involved in heart function, suggests that slow muscle fibers are also responsible for the high muscularity of the wuding chicken. the c1qa, c1qb, and c1qc genes are hub genes involved in immune processes that are also active in the hydroxylysine and hydroxyproline collagen synthesis pathways. col6a another hub gene, is a collagen associated with the c1qa, c1qb, and c1qc genes, the hydroxylysine/hydroxyproline pathways, and muscle organ development. these results suggest that the upregulation of these hub genes is associated with increased growth rates. the biological mechanisms enriched for genes displaying increased expression in the muscle tissue of the daweishan compared to wuding chicken breed were related to more general metabolism. the metabolism of proteins – especially those that need to be exported from the cell – was affected, as was lipid metabolism and molecular chaperones. the hub genes in the network created after allowing linker genes were either linker genes or differentially expressed genes mainly connected to linker genes. therefore, we focused on genes central to diverse functions rather than hub genes. important genes involved in diverse mechanisms include hsp90b hspa pdia and hyou hsp90b hspa and hyou are chaperone proteins involved in regulating protein folding. hsp90b and hspa are related to endoplasmic reticulum stress. hsp90b binds calcium and is atp metabolizing, while hspa is regulated by glucose levels. furthermore, hspa is involved in monitoring the transport of proteins through the cell. hyou is also glucose regulated and is also a chaperone protein. upregulation of hyou is suggested to be involved in reduction of apoptosis. the pdia protein is an electron carrier. cytoscape cluego analysis related these genes, and especially the hsp90b gene, directly to the regulation of the subthalamus development, which regulates movement – i.e. skeletal muscle activity. together these genes may be involved in regulating increased cellular protein metabolism and breast muscle functioning in daweishan chickens. finally, hsp90b activity is also associated with immune chaperone activity regulating innate and adaptive immunity. these immune mechanisms differ between the breast muscle of daweishan and wuding chicken breeds. since these genes are involved in regulating the immune response rather than the proinflammatory immune response, these expression changes may not result in increased tissue damage. high growth rate and ascites in chickens although we did not observe ascites in these chicken, health problems related to increased muscle growth rate and body weight of broiler chickens have been reported previously. specifically, these health problems were related to ascites development and sudden death syndrome. the development of ascites and sudden death syndrome results from an imbalance in the development of the internal organs and skeletal muscle. indeed we observed differences between the relative organ weights of the wuding and daweishan chicken breeds. so, despite the balanced biological mechanisms of the musculature of the wuding chicken breed, the differential organ development may cause problems leading to increased expression of acute inflammatory genes. although speculative, this may indicate that the wuding chicken is less balanced than the daweishan chicken, and have activated biological mechanisms that could lead to ascites. it should be noted that the growth rate of wuding chicken is still less than selected broiler lines. we also observed increased expression of the scavenger receptors in the wuding compared to the daweishan chicken breed. scavenger receptors are involved in the removal of foreign substances and waste from tissues. this may further indicate that unhealthy or unbalanced processes exist in wuding chicken breast. such processes may potentially be related with inflammation, as described above. they may also be partly responsible for the impaired health of the wuding chicken. biological mechanisms: growth rate and liver metabolism major differences in liver gene expression levels between the two chicken breeds were not observed, suggesting general liver metabolism is not significantly different between the two breeds. with the use of linker genes a single network was created based on the genes displaying higher expression in the wuding than in the daweishan breed, including genes involved in catecholamine related pathways. as all hub genes in the network were linker genes, we will not discuss them further. the catecholamine pathway is upregulated during high stress levels and also affects the immune response. this may help explain the altered expression of genes involved in the immune response in breast muscle tissue. the dopamine and monoamine genes are neurotransmitters that stimulate the reward-motivation behavior and emotion/arousal behavior, respectively. upregulation of these genes in the wuding chicken breed may suggest that these chicken are more nervous/agitated compared to the daweishan breed. in the body dopamine is also involved in gastrointestinal motility and in regulating vasodilatation. these mechanisms may be necessary to regulate uptake and distribution of feed components required for the higher growth rate of the wuding chicken breed than for the growth rate of the daweishan chicken. vasodilatation may also be a mechanism to overcome the adverse effects of early ascites enabling increased oxygen supply to the cells. similarly, in the liver of the daweishan mini chicken the hub genes in the network created allowing linker genes were either linker genes or differentially expressed genes mainly connected with linker genes. a number of upregulated genes included genes involved in the atp binding cassette, the abc receptors, signal transduction, the il- pathway, and a number of transcription factors. the diversity of the identified biological mechanisms indicates higher expression of several metabolic aspects in the daweishan than in the wuding chicken breed. as indicated above, these results also indicate that increased growth rates come at the expense of general metabolic activity. genes of major importance in this study were the abc receptor genes abcb abcb and abcc all three are involved in multi drug resistance, including xenobiotic compounds, bile salt export, and cyclic nucleotide export. there main function is to lower the cellular concentration of these substances via translocation. this increases cellular survival. similarly, the il cytokine functions on cellular proliferation and differentiation to increased cellular survival via the lif gene and its receptor, all of which showed increased expression in the liver of the daweishan mini chicken. il is a myokine discharged in the bloodstream after muscle contraction and acts to increase the breakdown of fats and improve insulin resistance. this mechanism represents a direct link between muscle and liver functioning: in daweishan mini chicken increased muscle cell metabolic activity leads to increased liver activity. other important genes include the psma and the hspd genes. psma is an atp/ubiquitin proteasome protein that uses atp to degrade proteins. this reduces inflammatory immune responses in the liver of daweishan mini chicken, adding to the increased cellular survival. hspd is a mitochondrial chaperonin protein essential for protein import in the mitochondrion and for correct protein folding. all these functions use atp, or are involved in atp synthesis. this points again to the increased metabolic activity in the daweishan compared to the wuding chicken.  our results identified increased expression of genes involved in muscle development and acute inflammatory responses associated with selection for fast body and muscle growth rates, while the daweishan breed displayed higher expression of genes involved in general metabolism than the wuding breed. the general conclusion of this study is that selection for increased body growth rates focuses on muscle growth leading to a declined and imbalanced general metabolism. this may be the underlying cause of ascites development in broiler chickens. furthermore, the expression changes in breast muscle seem to be more related to these growth-rate changes than those observed in liver tissue. 
the muscle tissue of the daweishan mini chicken breed showed higher expression of genes involved in several metabolic mechanisms including endoplasmic reticulum, protein and lipid metabolism, energy metabolism, as well as specific immune traits than in the wuding chicken. intensive selection has resulted in increased growth rates and muscularity in broiler chickens, in addition to adverse effects, including delayed organ development, sudden death syndrome, and altered metabolic rates. the wuding chicken breed is a fast growing large chicken breed, and the daweishan mini chicken breed is a slow growing small chicken breed. genes highly expressed in the daweishan mini chicken breed compared to the wuding breed were enriched for protein metabolism, abc receptors, signal transduction, and il6-related mechanisms. non-artificially-selected indigenous chinese chicken breeds display a wide variety of phenotypes, including differential growth rate, body weight, and muscularity. the muscle tissue in the wuding breed showed higher expression of muscle development genes than muscle tissue in the daweishan chicken breed. the biological mechanisms underlying selection responses remain largely unknown. together they form an ideal model system to study the biological mechanisms underlying broiler chicken selection responses in a natural system. the objective of this study was to study the biological mechanisms underlying differential phenotypes between the two breeds in muscle and liver tissues, and relate these to the growth rate and body development phenotypes of the two breeds. growth ratechicken breedsbreast musclelivermicroarraymetabolic differencesbiological mechanismsnational high technology research and program, no. we conclude that faster growth rates and larger body size are related to increased expression of genes involved in muscle development and immune response in muscle, while slower growth rates and smaller body size are related to increased general cellular metabolism. the liver tissue showed fewer differences between the two breeds. genes displaying higher expression in the wuding breed than in the daweishan breed were not associated with a specific gene network or biological mechanism. the liver of the daweishan breed displayed increased expression of metabolic genes. this expression was accompanied by higher expression of acute inflammatory response genes in wuding chicken than in daweishan chicken.
 intensive selection has resulted in increased growth rates and muscularity in broiler chickens, in addition to adverse effects, including delayed organ development, sudden death syndrome, and altered metabolic rates. the biological mechanisms underlying selection responses remain largely unknown. non-artificially-selected indigenous chinese chicken breeds display a wide variety of phenotypes, including differential growth rate, body weight, and muscularity. the wuding chicken breed is a fast growing large chicken breed, and the daweishan mini chicken breed is a slow growing small chicken breed. together they form an ideal model system to study the biological mechanisms underlying broiler chicken selection responses in a natural system. the objective of this study was to study the biological mechanisms underlying differential phenotypes between the two breeds in muscle and liver tissues, and relate these to the growth rate and body development phenotypes of the two breeds.  the muscle tissue in the wuding breed showed higher expression of muscle development genes than muscle tissue in the daweishan chicken breed. this expression was accompanied by higher expression of acute inflammatory response genes in wuding chicken than in daweishan chicken. the muscle tissue of the daweishan mini chicken breed showed higher expression of genes involved in several metabolic mechanisms including endoplasmic reticulum, protein and lipid metabolism, energy metabolism, as well as specific immune traits than in the wuding chicken. the liver tissue showed fewer differences between the two breeds. genes displaying higher expression in the wuding breed than in the daweishan breed were not associated with a specific gene network or biological mechanism. genes highly expressed in the daweishan mini chicken breed compared to the wuding breed were enriched for protein metabolism, abc receptors, signal transduction, and il6-related mechanisms.  we conclude that faster growth rates and larger body size are related to increased expression of genes involved in muscle development and immune response in muscle, while slower growth rates and smaller body size are related to increased general cellular metabolism. the liver of the daweishan breed displayed increased expression of metabolic genes. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords growth ratechicken breedsbreast musclelivermicroarraymetabolic differencesbiological mechanismsnational high technology research and program, no. 2011aa100305jia junjing http://dx.doi.org/ /501100001809national natural science foundation of china31260532ge changrong yunnan provincial natural science foundation research projects2009cd and 2011fa015zhao sumei issue-copyright-statement© the author 2017 