BACKGROUND
aggressive behaviour in group-living animals is an important aspect of their daily lives, and this behaviour is partly used to establish social ranks in groups.
animals who rank highly in the social hierarchy have many advantages, such as better access to food and territories  <cit> .
in studies in chickens, highly ranked males mated more often and produced more offspring than low ranking males  <cit> .
likewise, dominant hens produced more offspring over their lifespan than sub-ordinate hens  <cit> .
the social hierarchy in chickens can be measured by the number of aggressive pecks, which are usually aimed at the head of a receiving bird  <cit> .
the onset of aggressive pecking differs between male and female chickens.
males initiate aggressive pecking behaviour in their second week after hatching, and the pecking reaches adult levels when the chicken are eight to nine weeks old.
females initiate aggressive pecks at approximately five weeks of age, and they reach adult levels at nine to  <dig> weeks of age  <cit> .
a stable hierarchy is established at approximately  <dig> weeks of age, and a number of different factors are involved in its formation.
kim and zuk  <cit>  demonstrated that previous social experience, parasite status, morphological characteristics and possibly age can be important factors in establishing a hen's rank in the group.
in the european union, poultry are commonly housed in free range housing systems .
aggression can be a problem in these flocks and result in increased social stress.
additionally, skin damage can trigger cannibalism.
the level of aggression has been shown to be lower in large groups of chickens than in small groups  <cit> .
in order to reduce aggressive encounters under practical settings, it is important to identify the genes involved in aggressive pecking behaviour to understand how the pecking order is established in chickens.
to date, little is known about the underlying genetic mechanisms behind aggressive pecking in chickens.
previous selection experiments showed that aggressive pecking was not related to feather pecking because while the propensity to peck feathers changed during selection, there was no effect on the aggressive pecking behaviour .
there are indications that 'group selection' experiments for high and low production and survivability can influence aggressive behaviour in laying hens  <cit> .
later studies on these selection lines demonstrated that there were changes in the dopaminergic and serotonergic systems  <cit> .
animals injected with dopamine d <dig> receptor blockers showed a reduced frequency of aggressive pecks on subordinates  <cit> .
administration of 5-ht1-a and 5ht1-b antagonists resulted in increased aggressive pecks depending on the selection line  <cit> .
both the dopaminergic and serotonergic systems have been shown to influence aggressive behaviours in both mammals and birds  <cit> .
the present study aimed to identify genes that regulate the aggressive pecking behaviour in chickens.
in order to identify these genes, we compared the genome-wide profiles of chicken brain samples from aggressive and receiver hens using a  <dig> k chicken microarray.
we tested the hypotheses that  differentially expressed  genes are associated with the number of aggressive pecks given or received and  genes are de among peckers, receivers and a mixed group of peckers and receivers.
RESULTS
phenotype
the number of pecks given and pecks received per hen is shown in figure  <dig>  the number of pecks given during a three hour period ranged from  <dig> to  <dig>  and the number of pecks received ranged from  <dig> to  <dig> .
there was no difference between the cages in terms of the number of aggressive pecks performed per bird  or in the number of pecks received per bird .
additionally, there was no difference in the animal weights between the cages .
number of pecks and gene expression
there was no relationship between the number of aggressive pecks given or received and the gene expression.
the spearman correlation ranged from - <dig>  to  <dig> , but the p value was between  <dig>  and  <dig>
grouping of animals according to the pecks performed and received
group  <dig> consisted of  <dig> animals , group  <dig> consisted of eight animals , group  <dig> consisted of five animals , and group  <dig> consisted of three animals.
group  <dig> was considered too small to perform a gene expression experiment on, so it was not used.
there was no difference in body weight between the groups .
gene expression analysis
in additional file  <dig>  the de genes  for the comparison between p&r and r are presented.
the logfc was in the range of  <dig>  to - <dig> .
of the  <dig> genes,  <dig> had a gene annotation, and  <dig> were mapped to the chicken genome.
however, none of these genes were significant at the false discovery rate  p <  <dig> .
in additional file  <dig>  the genes  for the comparison between p&r and p are presented.
the logfc was in the range of  <dig> to - <dig>  of the  <dig> genes,  <dig> had a gene annotation,  <dig> were mapped to the chicken genome, and  <dig> were significant at the fdr p <  <dig> .
in additional file  <dig>  the genes  of interest from the comparison between r and p are presented.
the logfc was in the range of  <dig>  to - <dig>  of the  <dig> genes,  <dig> had a gene annotation,  <dig> were mapped to the chicken genome, and  <dig> were significant at the fdr p <  <dig> .
figure  <dig> shows the logfc distribution of the significant genes for the p&r vs. p and r vs. p comparisons.
figure  <dig> shows a venn diagram of the overlapping genes in the comparisons.
there were  <dig> genes in common between the p&r vs. p and r vs. p comparisons.
a heatmap of the  <dig> de genes in the r vs. p comparison is shown in figure  <dig>  clustering of the individuals based on their de genes  showed that three out of the five peckers clustered together.
the other two animals were assigned to two different clusters that contained receiver animals.
clustering of the individuals in the p&r vs. p comparison showed that the animals were mixed .
we tested for overrepresentation of gene sets representing biological processes , cellular components , and the molecular function  of gene ontology  in the p&r vs. p and r vs. p comparisons.
for the r vs. p comparison,  <dig> go identifiers were significant .
of these  <dig> go identifiers,  <dig> belonged to the bp set,  <dig> belonged to the cc set, and six belonged to the mf gene set .
the go identifiers that could potentially be involved in behavioural processes were related to synaptosomes , the regulation of excitatory postsynaptic membrane potential , the regulation of membrane potential , and glutamate receptor binding .
the go: <dig>  go: <dig>  go: <dig> genes were the glutamate receptors .
the go: <dig> genes were mainly acetylcholine receptors .
go identifiers that were involved in muscle development and lipid biosynthesis were also identified.
the go identifiers detected from the p&r vs. p comparison are shown in additional file 5: figure s <dig>
discussion
this study identified genes that are involved in aggressive pecking behaviour in chickens, which is a behaviour related to social dominance.
this research will help gain a better understanding of the underlying genetics of this behaviour.
our results showed that there was no association between number of pecks given or received and the gene expression level.
however, comparison between the pecker and receiver animals showed that some genes were differentially expressed in the two groups.
grouping of animals
the aim of this study was to detect differences between pecker and receiver animals, and we assumed that the peckers were the aggressive animals and the receivers were the submissive animals or victims.
in our study, we clearly divided groups of pecker  and receiver  animals.
we are aware that there are animals in the pecker and receiver  group that have phenotypic profiles that may match either the pecker group or receiver group .
however, it is difficult to assign a clear cut-off point to define an animal as a pecker or receiver when it both performs and receives aggressive pecks.
some researchers have used aggressive encounters  to rank animals in a social dominance hierarchy  <cit> .
these approaches have varied, with some researchers counting only the number of pecks and receiving pecks  <cit> , while others have taken the interactions  between animals into account  <cit> .
nevertheless, there are no major differences between the animal rankings in groups when the different ranking methods are used.
in most cases, the animals defined as 'pure' peckers or receivers in our study were also among the top ranking or bottom ranking animals using the previously proposed formulas to rank animals in groups  <cit> .
these similarities indicate that the groupings used in this study represent aggressive and submissive animals.
genes and behaviour
the p&r group had great variability in terms of the number of pecks given and received and is large compared to both the r and the p groups .
this may explain why there were no significant genes at the fdr p <  <dig>  level for the p&r vs. r comparison .
the p&r vs. p comparison detected  <dig> significant genes, and of these  <dig> overlapped with significant genes from the r vs. p comparison.
this suggests that in both of these cases, the gene expression differences were caused by the p group.
interpreting the de genes in the r vs. p comparison is a first step towards understanding the underlying genetic mechanism behind aggressive behaviour in chickens.
the r vs. p comparison identified  <dig> significant genes.
it was not fully clear how the annotated genes were involved in regulating aggressive behaviour; however, as the annotation of the chicken genome improves, this may provide a clearer picture of how the genes are related to aggressive behaviour.
none of the most obvious candidate genes for aggressive behaviour  were identified in our comparisons.
for example, it was previously shown that serotonergic receptors play a role in aggression in chickens  <cit> .
it is possible that these genes were up- or down-regulated in a specific part of the brain, but could not be detected in our study of whole brain gene expression because the effect was diluted.
another explanation is that these genes influence aggressive behaviour via different allelic forms.
the gene set enrichment analysis, which involved all of the genes on the array, demonstrated that genes involved with muscle development were among the significant go identifiers.
in drosophila tested for aggressive behaviour, genes involved in muscle contraction were among the significant go identifiers  <cit> .
it is not clear whether genes related to muscle development have the same function in the brain; however, in chickens, the largest bird usually ranks the highest  <cit> .
in spite of this, we observed no difference in body weight between the pecking and receiver animals in our study.
other go identifiers detected in this study were involved in lipid metabolism, lipid synthesis and low density lipo-protein binding .
fatty acid binding proteins have been shown to play a role in the differentiation of neurons and glial cells in rats  <cit> .
as a consequence of improved neural development, subsequent changes in the brain and behaviour could occur between low and high ranking animals, such as differences in the development of memory functions.
in our study, some of the significant go identifiers coded for synaptosomes, glutamate receptor binding, and the regulation of excitatory postsynaptic membrane potentials.
glutamate receptors, which were detected in the go identifiers, play an important role in the development of memory formation following passive avoidance training in young chickens  <cit> .
therefore, memory may play a role in remembering the social hierarchy of the group.
CONCLUSIONS
in conclusion, our study provides a first insight into which genes are involved in aggressive behaviour in chickens.
the results of our study showed that the level of expression is not dependent on the number of pecks given or received.
it was not fully clear how the de genes were involved in regulating aggressive behaviour.
however, the gene set enrichment test showed that the de genes coded for synaptosomes, and genes involved with lipid metabolism and memory formation.
when confirmed in future studies, the de genes may help scientists understand how the pecking order forms in laying hens at a molecular level.

 aggressive behaviour in group-living animals is an important aspect of their daily lives, and this behaviour is partly used to establish social ranks in groups. animals who rank highly in the social hierarchy have many advantages, such as better access to food and territories. in studies in chickens, highly ranked males mated more often and produced more offspring than low ranking males. likewise, dominant hens produced more offspring over their lifespan than sub-ordinate hens. the social hierarchy in chickens can be measured by the number of aggressive pecks, which are usually aimed at the head of a receiving bird. the onset of aggressive pecking differs between male and female chickens. males initiate aggressive pecking behaviour in their second week after hatching, and the pecking reaches adult levels when the chicken are eight to nine weeks old. females initiate aggressive pecks at approximately five weeks of age, and they reach adult levels at nine to weeks of age. a stable hierarchy is established at approximately weeks of age, and a number of different factors are involved in its formation. kim and zuk demonstrated that previous social experience, parasite status, morphological characteristics and possibly age can be important factors in establishing a hen's rank in the group. in the european union, poultry are commonly housed in free range housing systems. aggression can be a problem in these flocks and result in increased social stress. additionally, skin damage can trigger cannibalism. the level of aggression has been shown to be lower in large groups of chickens than in small groups. in order to reduce aggressive encounters under practical settings, it is important to identify the genes involved in aggressive pecking behaviour to understand how the pecking order is established in chickens. to date, little is known about the underlying genetic mechanisms behind aggressive pecking in chickens. previous selection experiments showed that aggressive pecking was not related to feather pecking because while the propensity to peck feathers changed during selection, there was no effect on the aggressive pecking behaviour. there are indications that'group selection' experiments for high and low production and survivability can influence aggressive behaviour in laying hens. later studies on these selection lines demonstrated that there were changes in the dopaminergic and serotonergic systems. animals injected with dopamine d receptor blockers showed a reduced frequency of aggressive pecks on subordinates. administration of 5-ht1-a and 5ht1-b antagonists resulted in increased aggressive pecks depending on the selection line. both the dopaminergic and serotonergic systems have been shown to influence aggressive behaviours in both mammals and birds. the present study aimed to identify genes that regulate the aggressive pecking behaviour in chickens. in order to identify these genes, we compared the genome-wide profiles of chicken brain samples from aggressive and receiver hens using a k chicken microarray. we tested the hypotheses that differentially expressed genes are associated with the number of aggressive pecks given or received and genes are de among peckers, receivers and a mixed group of peckers and receivers.  phenotype the number of pecks given and pecks received per hen is shown in figure the number of pecks given during a three hour period ranged from to and the number of pecks received ranged from to. there was no difference between the cages in terms of the number of aggressive pecks performed per bird or in the number of pecks received per bird. additionally, there was no difference in the animal weights between the cages. number of pecks and gene expression there was no relationship between the number of aggressive pecks given or received and the gene expression. the spearman correlation ranged from - to, but the p value was between and grouping of animals according to the pecks performed and received group consisted of animals, group consisted of eight animals, group consisted of five animals, and group consisted of three animals. group was considered too small to perform a gene expression experiment on, so it was not used. there was no difference in body weight between the groups. gene expression analysis in additional file the de genes for the comparison between p&r and r are presented. the logfc was in the range of to -. of the genes, had a gene annotation, and were mapped to the chicken genome. however, none of these genes were significant at the false discovery rate p <. in additional file the genes for the comparison between p&r and p are presented. the logfc was in the range of to - of the genes, had a gene annotation, were mapped to the chicken genome, and were significant at the fdr p <. in additional file the genes of interest from the comparison between r and p are presented. the logfc was in the range of to - of the genes, had a gene annotation, were mapped to the chicken genome, and were significant at the fdr p <. figure shows the logfc distribution of the significant genes for the p&r vs. p and r vs. p comparisons. figure shows a venn diagram of the overlapping genes in the comparisons. there were genes in common between the p&r vs. p and r vs. p comparisons. a heatmap of the de genes in the r vs. p comparison is shown in figure clustering of the individuals based on their de genes showed that three out of the five peckers clustered together. the other two animals were assigned to two different clusters that contained receiver animals. clustering of the individuals in the p&r vs. p comparison showed that the animals were mixed. we tested for overrepresentation of gene sets representing biological processes, cellular components, and the molecular function of gene ontology in the p&r vs. p and r vs. p comparisons. for the r vs. p comparison, go identifiers were significant. of these go identifiers, belonged to the bp set, belonged to the cc set, and six belonged to the mf gene set. the go identifiers that could potentially be involved in behavioural processes were related to synaptosomes, the regulation of excitatory postsynaptic membrane potential, the regulation of membrane potential, and glutamate receptor binding. the go: go: go: genes were the glutamate receptors. the go: genes were mainly acetylcholine receptors. go identifiers that were involved in muscle development and lipid biosynthesis were also identified. the go identifiers detected from the p&r vs. p comparison are shown in additional file 5: figure s discussion this study identified genes that are involved in aggressive pecking behaviour in chickens, which is a behaviour related to social dominance. this research will help gain a better understanding of the underlying genetics of this behaviour. our results showed that there was no association between number of pecks given or received and the gene expression level. however, comparison between the pecker and receiver animals showed that some genes were differentially expressed in the two groups. grouping of animals the aim of this study was to detect differences between pecker and receiver animals, and we assumed that the peckers were the aggressive animals and the receivers were the submissive animals or victims. in our study, we clearly divided groups of pecker and receiver animals. we are aware that there are animals in the pecker and receiver group that have phenotypic profiles that may match either the pecker group or receiver group. however, it is difficult to assign a clear cut-off point to define an animal as a pecker or receiver when it both performs and receives aggressive pecks. some researchers have used aggressive encounters to rank animals in a social dominance hierarchy. these approaches have varied, with some researchers counting only the number of pecks and receiving pecks, while others have taken the interactions between animals into account. nevertheless, there are no major differences between the animal rankings in groups when the different ranking methods are used. in most cases, the animals defined as'pure' peckers or receivers in our study were also among the top ranking or bottom ranking animals using the previously proposed formulas to rank animals in groups. these similarities indicate that the groupings used in this study represent aggressive and submissive animals. genes and behaviour the p&r group had great variability in terms of the number of pecks given and received and is large compared to both the r and the p groups. this may explain why there were no significant genes at the fdr p < level for the p&r vs. r comparison. the p&r vs. p comparison detected significant genes, and of these overlapped with significant genes from the r vs. p comparison. this suggests that in both of these cases, the gene expression differences were caused by the p group. interpreting the de genes in the r vs. p comparison is a first step towards understanding the underlying genetic mechanism behind aggressive behaviour in chickens. the r vs. p comparison identified significant genes. it was not fully clear how the annotated genes were involved in regulating aggressive behaviour; however, as the annotation of the chicken genome improves, this may provide a clearer picture of how the genes are related to aggressive behaviour. none of the most obvious candidate genes for aggressive behaviour were identified in our comparisons. for example, it was previously shown that serotonergic receptors play a role in aggression in chickens. it is possible that these genes were up- or down-regulated in a specific part of the brain, but could not be detected in our study of whole brain gene expression because the effect was diluted. another explanation is that these genes influence aggressive behaviour via different allelic forms. the gene set enrichment analysis, which involved all of the genes on the array, demonstrated that genes involved with muscle development were among the significant go identifiers. in drosophila tested for aggressive behaviour, genes involved in muscle contraction were among the significant go identifiers. it is not clear whether genes related to muscle development have the same function in the brain; however, in chickens, the largest bird usually ranks the highest. in spite of this, we observed no difference in body weight between the pecking and receiver animals in our study. other go identifiers detected in this study were involved in lipid metabolism, lipid synthesis and low density lipo-protein binding. fatty acid binding proteins have been shown to play a role in the differentiation of neurons and glial cells in rats. as a consequence of improved neural development, subsequent changes in the brain and behaviour could occur between low and high ranking animals, such as differences in the development of memory functions. in our study, some of the significant go identifiers coded for synaptosomes, glutamate receptor binding, and the regulation of excitatory postsynaptic membrane potentials. glutamate receptors, which were detected in the go identifiers, play an important role in the development of memory formation following passive avoidance training in young chickens. therefore, memory may play a role in remembering the social hierarchy of the group.  in conclusion, our study provides a first insight into which genes are involved in aggressive behaviour in chickens. the results of our study showed that the level of expression is not dependent on the number of pecks given or received. it was not fully clear how the de genes were involved in regulating aggressive behaviour. however, the gene set enrichment test showed that the de genes coded for synaptosomes, and genes involved with lipid metabolism and memory formation. when confirmed in future studies, the de genes may help scientists understand how the pecking order forms in laying hens at a molecular level. 
aggressive animals have advantages, such as better access to food or territories, and they produce more offspring than low ranking animals. the social hierarchy in chickens is measured using the'pecking order' concept, which counts the number of aggressive pecks given and received. following confirmation, the identification of differentially expressed genes may elucidate how the pecking order forms in laying hens at a molecular level. in comparing the r and p groups, we observed that there were differentially expressed genes. these genes code for synaptosomes, and proteins involved in the regulation of the excitatory postsynaptic membrane potential, the regulation of the membrane potential, and glutamate receptor binding. in conclusion, our study provides new insights into which genes are involved in aggressive behaviours in chickens. aggressive behaviour is an important aspect in the daily lives of animals living in groups. it was not fully clear how the genes regulated aggressive behaviour; however, gene set analysis detected a number of go identifiers, which were potentially involved in aggressive behavioural processes.
 aggressive behaviour is an important aspect in the daily lives of animals living in groups. aggressive animals have advantages, such as better access to food or territories, and they produce more offspring than low ranking animals. the social hierarchy in chickens is measured using the'pecking order' concept, which counts the number of aggressive pecks given and received. to date, little is known about the underlying genetics of the'pecking order'.  a total of hens from a high feather pecking selection line were divided into three groups: only receivers, only peckers and mixed peckers and receivers. in comparing the r and p groups, we observed that there were differentially expressed genes. it was not fully clear how the genes regulated aggressive behaviour; however, gene set analysis detected a number of go identifiers, which were potentially involved in aggressive behavioural processes. these genes code for synaptosomes, and proteins involved in the regulation of the excitatory postsynaptic membrane potential, the regulation of the membrane potential, and glutamate receptor binding.  in conclusion, our study provides new insights into which genes are involved in aggressive behaviours in chickens. pecking and receiving hens exhibited different gene expression profiles in their brains. following confirmation, the identification of differentially expressed genes may elucidate how the pecking order forms in laying hens at a molecular level. 