BACKGROUND
boar taint is an off-odour and off-flavour in pig carcasses that is primarily caused by high levels of 16-androstene steroids  <cit>  and/or skatole  <cit>  in adipose tissue.
male pigs used for meat production are normally castrated very early in life to prevent boar taint in the meat.
however, castration also removes the source of natural anabolic androgens that stimulate lean growth and, as a result, uncastrated males have improved feed efficiency and greater lean yield of the carcass compared to barrows  <cit> .
detection of genetic factors influencing boar taint may facilitate implementation of selective breeding practices to produce pigs with little or no taint.
because of unfavourable correlations between androstenone and other sex steroids  <cit> , a direct selection against high levels of androstenone would result in decreased production of testosterone and estrogens, with associated negative effects on performance and sexual maturation.
therefore, a comprehensive understanding of the complex genetic system controlling boar taint is required before genetic improvement can be achieved.
androstenone  is produced in the testis and is transported by blood to the salivary gland where it functions as a pheromone to stimulate mating stance in gilts  <cit> .
it is metabolised in the liver, producing α-androstenol and β-androstenol  <cit> , and deficient degradation may lead to the accumulation of androstenone in adipose tissue.
skatole  is produced by the metabolism of tryptophan from the gut and is also catabolised in liver.
diaz et al.
<cit>  identified seven metabolites of skatole in pig liver microsomes.
an increase in levels of skatole in adipose tissue occurs in boars around puberty, but not in barrows or sows, indicating that skatole metabolism is regulated by testicular steroids  <cit> .
enzymes responsible for metabolism of androstenone and skatole in liver have been identified but relatively few genes have been investigated.
several studies have shown that 3β-hydroxysteroid dehydrogenase  is involved in androstenone metabolism, with low mrna, protein and enzyme expression levels correlated to high androstenone levels in adipose tissue  <cit> .
cytochrome p <dig> 2e <dig>  is involved in metabolism of skatole  <cit>  and deficient cyp2e <dig> induction is suggested to be a major cause of high levels of skatole in adipose tissue  <cit> .
a relationship between metabolism of androstenone and skatole in liver has been observed  <cit> , with evidence to suggest that androstenone blocks cyp2e <dig> induction by skatole  <cit> .
sulfotransferase enzymes including sult2a <dig>  sult2b <dig> and sult1a <dig> are associated with metabolic clearance of androstenone and skatole since they are known to conjugate steroid hormones and drugs into more water soluble compounds that facilitates excretion  <cit> .
although both androstenone and skatole are responsible for boar taint, androstenone seems to be the biggest problem in the duroc and norwegian landrace populations  <cit> .
levels of androstenone are also predominantly genetically determined whereas levels of skatole are more heavily influenced by feeding and environmental factors.
levels of androstenone will be a function of both production in testes and elimination in liver.
we have previously studied differentially expressed genes related to the production of androstenone in boar testis  <cit> , and the objective of this study was to investigate global gene expression profiles related to the degradation of androstenone in pig livers.
microarray technology was used to obtain gene expression profiles from liver samples of duroc  and norwegian landrace  boars with extreme high or low levels of androstenone in adipose tissue.
these samples were hybridised together with a common reference sample to cdna microarrays representing approximately  <dig>  porcine gene transcripts.
a total of  <dig> animals were selected from each group, resulting in a total of  <dig> arrays.
a subset of the differentially expressed genes was verified using a quantitative pcr-based method.
RESULTS
porcine cdna microarrays were used to obtain global gene expression profiles in liver tissues sampled from  <dig> d and  <dig> nl boars with extreme high and low levels of androstenone.
average levels of androstenone were  <dig>  ±  <dig>  ppm for d high  and  <dig>  ±  <dig>  ppm for d low  boars.
average levels of androstenone were  <dig>  ±  <dig>  ppm for nl high  and  <dig>  ±  <dig>  ppm for nl low  boars.
linear models were used to identify significantly differentially expressed genes and the top 1%  most significant genes were subsequently inspected as possible candidate genes affecting androstenone levels .
the top  <dig> genes of d and nl are presented in table  <dig> and  <dig>  respectively.
among the  <dig> affected genes,  <dig> were found to be in common for the two breeds and expression differences were generally more significant in d compared to nl.
gene expression profiles were identified in limma using empirical bayes moderated t-statistics.
fold change and statistical significance is shown .
the clone names refer to hits to pig, human, mouse or bovine sequences.
some genes are represented by several different clones on the array and may therefore show up more than once in the table, while others have no hits to the abovementioned species.
gene expression profiles were identified in limma using empirical bayes moderated t-statistics.
fold change and statistical significance is shown .
the clone names refer to hits to pig, human, mouse or bovine sequences.
some genes are represented by several different clones on the array and may therefore show up more than once in the table, while others have no hits to the abovementioned species.
testing for overrepresentation of gene ontology  terms among the  <dig> affected genes was performed relative to the global representation of go terms on the microarray.
a majority of the significant genes were related to oxidoreductase activities, glucuronosyltransferase activity and the binding of iron ion, haem, dna and oxygen .
additionally, cellular metabolic process pathways such as amino acid metabolism and catabolism, electron transport and inflammatory response were overrepresented .
furthermore, the genes were classified according to their cellular component ontology  results for the cellular component ontology in duroc, additional file 4: gene ontology  results for the cellular component ontology in norwegian landrace).
for d, cytoplasm, microsomes and endoplasmatic reticulum were among the important cellular components, none of the cellular components reached the level of significance, i.e.
had more than  <dig> significant genes related to the cc terms.
elevated levels of androstenone in adipose tissue can result from both increased biosynthesis in the testes and deficient metabolism in liver.
we have previously conducted a microarray experiment investigating gene expression profiles in the testes of boars with extreme levels of androstenone, and in this study we have examined gene expression profiles in the liver of some representatives from that study along with new individuals.
liver metabolism can be divided into phase i and phase ii reactions  <cit> .
phase i metabolism involves oxidation and hydroxylation reactions that make the substrate more water soluble.
phase ii conjugation reactions further increase hydrophilicity by adding polar groups.
as a result of these metabolic reactions, compounds including endogenous steroids, fatty acids and drugs are inactivated and eliminated.
we have identified a number of differentially expressed genes that function in pathways affecting both phase i and phase ii reactions involved in metabolism of androstenone in the liver.
phase i metabolism
the most significantly differentially expressed gene involved in phase i oxidation reactions was cytochrome p <dig> 2c <dig> .
this gene is a member of the cyp2c family  <cit>  and genes of this family encode monooxygenases that catalyse several reactions involved in the metabolism of drugs and endogenous compounds.
different cyp2c isoforms show some cross reactivity towards substrates which makes it difficult to differentiate cyp2c activities, and substrate specificity for cyp2c <dig> is not known  <cit> .
in our study, cyp2c <dig> transcripts were shown to have a significant up-regulation in boars with high levels of androstenone in both d and nl lines  but differences were clearly most prominent in the d breed.
another member of the cytochrome p <dig> superfamily, that was differentially expressed in our study, is cytochrome p <dig> 2e <dig> .
in contrast to expression of the cyp2c <dig> gene, cyp2e <dig> showed a down-regulation in dh and nlh boars.
cyp2e <dig> encodes an enzyme that metabolises many endogenous and exogenous substrates such as alcohols, ketones and drugs  <cit> .
androstenone has been shown to block skatole induced expression of cyp2e <dig> in pig hepatocytes and to inhibit activity of cyp2e <dig> in porcine liver microsomes  <cit> .
liver metabolism of skatole also involves this gene and enzyme  <cit> , and the observed pubertal increase in skatole levels has been attributed to the inhibition of cyp2e <dig> by the sex steroids androstenone and 17β-estradiol  <cit> .
this is the first study, however, to show gene expression differences for cyp2e <dig> in relation to androstenone.
cytochrome p <dig> member 2a <dig> , which is the pig ortholog of human cyp2a <dig>  catalyses 7-hydroxylation of coumarin in pigs  <cit> , in addition to being involved in skatole metabolism  <cit> .
in this study, cyp2a <dig> was significantly down-regulated in dh boars.
gene expression differences in animals with high and low androstenone levels have not previously been reported for porcine cyp2a <dig>  however its protein expression has been shown to be inhibited by androstenone  <cit> .
diaz and squires  <cit>  found that both cyp2a <dig> protein content and its enzymatic activity were negatively correlated with skatole levels in adipose tissue.
in contrast, results presented by terner et al.
<cit>  indicate that the cyp2a <dig> enzyme is not important for metabolism of skatole in primary cultured porcine hepatocytes.
two other members of the cytochrome p <dig> family, cyp27a <dig> and cyp2c <dig>  were also up-regulated in dh.
cyp27a <dig> is a multifunctional enzyme that oxygenates cholesterol, bile acids and vitamin d. it has not previously been identified as a candidate gene associated with androstenone, but it is regulated by other sex steroids in human cells  <cit> .
cyp2c <dig> belongs to the same sub-family as cyp2c <dig>  but its function has yet to be described in the literature.
like the cyp2c <dig> gene, cyp2c <dig> was found to be up-regulated in dh and nlh boars.
the differential expression of cyp2a <dig>  cyp27a <dig> and cyp2c <dig> in d and not nl boars might suggest that more genes are involved in phase i reactions in d. this is supported by a high number of oxidoreductase pathways significant in d .
monooxygenase reactions are, however, clearly important in both breeds according to our gene ontology results .
down-regulation of cyp2e <dig> and cyp2a <dig> might suggest a different role for these genes compared to the up-regulated cyp2c <dig>  cyp2c <dig> and cyp27a <dig>
our study implicates another class of genes involved in phase i oxidation reactions, namely the flavin-containing monooxygenases .
the fmo family of enzymes converts lipophilic compounds into more polar metabolites and decreases activity of the compounds, a similar activity to that of the cytochrome p450s  <cit> .
microarray expression results show that flavin-containing monooxygenase  <dig>  was significantly up-regulated in dh in the microarray results.
the expression pattern in dh boars was confirmed by rcpcr and the rcpcr analysis also revealed that fmo <dig> was up-regulated in nlh boars.
as shown in figure  <dig>  the fold change in nlh was only half of dh, which might explain why this gene was not found to be significantly differentially expressed in the microarray results.
regulation of fmo involves sex steroids.
in male mice, castration was reported to increase fmo <dig> expression  <cit> , while in rats the opposite effect has been shown, with positive regulation of fmo by testosterone and negative by estradiol  <cit> .
the monooxygenase activity of fmo <dig> in addition to its regulation by steroid hormones makes it an interesting candidate gene for boar taint.
phase ii metabolism
phase i oxidation reactions of androstenone by enzymes including cytochrome p450s and fmos are often followed by phase ii conjugation reactions that are catalysed by glucuronosyltransferases, sulfotransferases, acetyltransferases and glutathione s-transferases  <cit> .
through the addition of polar moieties, these enzymes increase substrate solubility.
conjugation reactions are an important means of excreting steroid hormones and other compounds, these reactions have also been proposed to keep inactive steroids easily available in cells.
the hydroxysteroid sulfotransferases sult2a <dig> and sult2b <dig> have been associated with androstenone levels in previous studies  <cit> .
these genes were not found to be significantly differentially expressed in our study, however the estrogen sulfotransferase ste  was found to be up-regulated in dh boars and down-regulated in nlh boars.
a previous microarray study performed by our group showed an up-regulation of cyp <dig> gene expression in the testis of d and nl boars with high androstenone levels  <cit> .
these genes encode enzymes biosynthesising estrogens and might explain why an estrogen conjugation enzyme is differentially expressed in livers of the same pigs.
furthermore, boars secrete large quantities of estrogens from the testis  <cit> , and both fat and plasma estrogen levels have previously been shown to be highly correlated with levels of androstenone in adipose tissue  <cit> .
phase ii conjugation by glucuronidation is another major pathway of liver elimination of endogenous and exogenous compounds, and in d this molecular function was significantly overrepresented.
it is catalysed by uridine diphospho-glucuronosyltransferases  which transfer glucuronic acid to substrates to increase solubility  <cit> .
the ugts have been divided into two subfamilies, the ugt1s and the ugt2s, and in this study the family member ugt1a <dig> was found to be up-regulated in dh boars.
furthermore, a transcript similar to ugt2b <dig> was up-regulated in d and a transcript similar to ugt2a <dig> was down-regulated in nlh.
ugt2b <dig> has been shown to conjugate several androgens in humans  <cit> , whereas ugt1a <dig> has been found to be catalytically active against some exogenous compounds  <cit> .
ugt2a <dig> contributes to glucuronidation of steroids and phenolic compounds in olfactory tissue and has been found to eliminate odourants  <cit> .
the ugt family of conjugation enzymes have previously been associated with the androstenone metabolites α-androstenol and β-androstenol  <cit> .
n-acetyltransferases  are another family of conjugation enzymes and act by transferring the acetyl group of acetyl coenzyme a to aromatic amines to increase their water solubility  <cit> .
nat type  <dig>  was found to be up-regulated in nlh based on the microarray results and this was verified by rcpcr.
the rcpcr results also revealed an up-regulation in dh boars.
androgens have been shown to increase expression of nat type  <dig> in human cancer cells  <cit> , but the enzyme family has not previously been associated with levels of androstenone.
tryptophan, the precursor of skatole, contains an aromatic, suggesting a role for this family of conjugation enzymes in the control of skatole levels, possibly through regulation by steroid hormones.
glutathione s-transferases  are functionally diverse enzymes mostly known to catalyse conjugation reactions of endogenous substances, haem, fatty acids, xenobiotics and products of oxidative processes  <cit> .
they have also been implicated in the intracellular transport of steroids to their site of action  <cit> .
in this study, a glutathione s-transferase gene was found to be down-regulated in nlh.
the gene is described in the database as loc <dig>  we have previously described up-regulation of two gst genes, gsto <dig> and mgst <dig>  in association with testicular androstenone levels  <cit> , and here we additionally propose a role for glucuronidation in liver metabolism.
expression of phase ii metabolic genes in d and nl pigs suggest breed specific mechanisms.
we found differential expression of a gst variant  in nl but not d boars, up-regulation of ste in dh but down-regulation in nlh, and up-regulation of two ugt genes in dh but down-regulation of another family member in nlh.
breed specific phase ii mechanisms for these breeds are in agreement with our previous finding for the protein sult2b <dig>  <cit> .
regulation of steroid availability
17β-hydroxysteroid dehydrogenases  regulate the availability of androgens and estrogens in tissues by catalysing interconvertion of active and inactive forms of steroids  <cit> .
they do so by regulating the occupancy of steroid nuclear hormone receptors like androgen receptor, estrogen receptor and progesterone receptor  <cit> .
gene expression of 17β-hsd in porcine liver has been shown to be negatively associated with levels of androstenone in adipose tissue  <cit> .
in a previous study, we found that the hsd17b <dig> gene was significantly up-regulated in testicle samples from dh and nlh  <cit> , and in this study hsd17b <dig> was up-regulated in the liver of dh boars.
additionally, we found that hsd17b <dig> was down-regulated in dh and that the isoform hsd17b <dig> was down-regulated in both dh and nlh.
differential expression of both hsd17b <dig> and hsd17b <dig> was verified by rcpcr, which also showed significant down-regulation of hsd17b <dig> in nlh boars.
the hsd17b <dig> enzyme catalyses the interconversion of testosterone and androstenedione, as well as estradiol and estrone  <cit> .
the function of isoform hsd17b <dig>  also known as short-chain dehydrogenase/reductase  <dig>  has yet to be described but its expression has been characterised in human liver  <cit> .
the short-chain dehydrogenase/reductase  <dig> gene , also known as hsd17b <dig>  was found to be down-regulated in nlh boars.
hsd17b <dig> is involved in androgen metabolism  <cit>  and we previously found this gene to be down-regulated in the testis of dh boars  <cit> .
the hsds belong to two superfamilies: the short-chain dehydrogenase/reductases  and the aldo-keto reductases   <cit> .
the four hsds described above all belong to the sdr superfamily but a gene from the akrs was also found to be differentially expressed.
the aldo-keto reductase family member 1d <dig>  was down-regulated in nlh animals.
akr1d <dig> is a liver specific enzyme that regulates the hormone levels of several steroids  <cit> .
the enzyme has a 5β-reductase activity and is necessary for the catabolism of aldosterone, cortisol and androgens  <cit> .
aldosterone, cortisol and androgens are three of four metabolic compounds formed from pregnenolone, with the fourth compound being androstenone  <cit> .
a role for akr1d <dig> in androstenone catabolism might therefore be possible.
we previously reported up-regulation of the family member akr1c <dig> in the testis of dh and nlh boars  <cit> , but akr1d <dig> has not formerly been associated with porcine androstenone levels.
another class of enzymes that regulate the availability of steroids is the plasma proteins.
binding of plasma protein is a reversible reaction that has no physiological effect, but it controls the amount of free drugs and hormones available to tissues by protecting them from metabolism  <cit> .
alpha-1-acid glycoprotein  is an acute phase serum protein synthesised in the liver and secreted to plasma where it binds and carries drugs and steroid hormones  <cit> .
the protein has been shown to interact with cyp3a and cyp2c <dig> in humans and to inhibit cytochrome p <dig> activity  <cit> .
it is suggested that serum protein interaction is an important factor for cytochrome p <dig> mediated metabolism, and that the interaction is isoform specific  <cit> .
agp has also been studied in pigs as a binding factor of pheromones, but has not been found to bind progesterone in pig nasal mucosa  <cit> .
agp has two variants in humans: orosomucoid  <dig> and  <dig>   <cit> .
in this study we found that porcine transcript homologous to bovine agp and human orm <dig> were significantly down-regulated in dh and nlh boars.
significant expression of these genes might explain the inflammatory response ontology term in figure  <dig> as these genes have mostly been described in relation to inflammatory response.
agp has not been investigated as a binding protein and transporter for androstenone, but different levels of androstenone could be related to differences in its availability through plasma binding by agp.
regulatory factors
one of the most significant molecular function terms in nl was nucleic acid binding.
this term includes transcription factor binding and several transcription factors were found to be differentially expressed in this study.
the gata factor  <dig>  was found to be down-regulated based on the microarray data, but we were not able to confirm this result with rcpcr.
other transcription factors identified in this study should be further investigated, including rar-related orphan receptor a , transcription elongation factor b <dig> , nuclear factor i/x , transcription factor  <dig>  and heat shock transcription factor  <dig> .
we have previously identified iron ion binding, ferric ion binding and electron transport as being associated with levels of androstenone  <cit> , possibly through interaction with the haem-containing cytochrome p450s.
in this study we also found iron ion binding and electron transport, in addition to haem binding, to be important pathways, supporting our previous findings.
cytochrome b <dig>  is involved in electron transfer to cytochrome p450s and has been proposed as a candidate gene for androstenone through its interaction with cytochrome p <dig> c <dig>   <cit> .
in this study we found cyb <dig> to be down-regulated in nlh boars, however we previously identified cyb <dig> as being up-regulated in the testis of both dh and nlh  <cit> .
both cyp2e <dig> and cyp2a <dig> have been found to be activated by cyb <dig> in humans  <cit> , supporting the down-regulation of cyb <dig> together with cyp2e <dig> and cyp2a <dig> in this study.
up-regulation in testis and down-regulation in liver might also support a key regulatory role of cyb <dig> in both tissues.
breed differences
breed differences in boar taint candidate genes have previously been shown between d and nl  <cit> .
consistent with these findings, we found breed differences in the expression profiles of genes involved in both phase i metabolism and phase ii metabolism.
however, rcpcr results might suggest that genes appearing in one breed can be differentially expressed also in the other breed.
additional and more comprehensive rcpcr studies are planned to clarify this.
in general, the d breed showed higher levels of significance compared to the nl breed, potentially due to the larger contrasts between high and low androstenone groups in this breed.
a number of new candidate genes have been identified in this study, however, we do not know their effect on performance and sexual maturation in pig.
additional studies are needed to investigate potential roles of the genes identified for phenotypes related to fertility.
relationship with skatole
pigs in this study were selected based on extreme androstenone values.
however, levels of androstenone and skatole have previously been found to be highly correlated, showing genetic correlations of  <dig>  for d and  <dig>  for nl  <cit> .
some of the genes found differentially expressed in this study have previously been found to be associated with tryptophan or skatole and this could be explained by high correlations with androstenone.
this might also be the reason for the significance of go terms such as aromatic amino acid metabolism and catabolism, since the skatole precursor tryptophan belongs to this family of amino acids.
the tryptophan  <dig>  3-dioxygenase gene , which oxidises tryptophan, was differentially expressed in dh.
a gene of the fmo family, kynurenine 3-monooxygenase , was found to be down-regulated in nlh.
kmo is involved in tryptophan degradation and might therefore be interesting in regards to skatole  <cit> .
the aldehyde oxidase  gene was significantly down-regulated in high androstenone animals of both breeds and this gene has not previously been associated with androstenone levels.
it has, however, been shown to play an important role in skatole metabolism in several species including pigs  <cit> .
CONCLUSIONS
in this study we compared global gene expression profiles from the livers of boars with extreme high and low levels of androstenone from two breeds, duroc and norwegian landrace.
breed differences are evident for molecular functions and biological processes involved in metabolism of androstenone, however many of the same genes are differentially expressed in the two breeds as well.
genes encoding different oxidising enzymes including the cytochrome p <dig> family  and the flavin-containing monooxygenase family  were significantly differentially expressed.
furthermore, genes involved in conjugation reactions, including the udp-glucuronosyltransferases , sulfotransferases , n-acetyltransferases  and glutathione s-transferase were significant, in addition to genes of the 17β-hydroxysteroid dehydrogenase family , which are known to regulate availability of active steroids.
we suggest a novel role for plasma proteins including agp and orm <dig> in regulating availability of androstenone in pigs.
this is the first published microarray experiment describing liver metabolism of androstenone.
a number of new candidate genes have been identified, both from phase i and phase ii metabolism as well as pathways regulating steroid availability.
materials and methods
animals
animals used in this study were duroc  and norwegian landrace  boars from norsvin's three boar testing stations.
the d boars were on average  <dig> days old at  <dig> kg live weight compared to the nl boars that were on average  <dig> days old at  <dig> kg live weight.
the boars were slaughtered on average  <dig> days later.
tissue samples from liver were frozen in liquid n <dig> immediately after slaughter and stored at -80°c until used for rna extraction as described below.
samples from adipose tissue were collected from the neck at slaughter and stored at -20°c until used for androstenone measurements.
androstenone levels were measured by a modified time-resolved fluoroimmunoassay at the hormone laboratory, norwegian school of veterinary science   <cit>  using an antiserum produced at nvh  <cit> .
the androstenone measurements were performed on more than  <dig> boars, and statistical power calculations showed that selecting animals from each tail of the androstenone distribution would yield sufficient power to detect differentially expressed genes with a limited number of arrays.
the  <dig> most extreme boars from each tail of the androstenone level distribution were therefore selected.
due to poor rna quality for two of the samples,  <dig> samples were subsequently used from each group.
<dig> of the animals used were the same individuals as those used in our previous study examining gene expression in boar testis  <cit> , with  <dig> being new animals as liver samples were not available for all of the previously studied animals.
average androstenone levels for the selected boars were  <dig>  ppm and  <dig>  ppm for nl and d, respectively.
.
average values for the groups were  <dig>  ±  <dig>  ppm for nl high ,  <dig>  ±  <dig>  ppm for nl low ,  <dig>  ±  <dig>  ppm for d high  and  <dig>  ±  <dig>  ppm for d low .
in order to reduce family effects, a maximum of two and three half sibs were chosen from nl and d, respectively.
the selected animals were used for expression profiling by microarrays and for the following verification of selected genes by rcpcr.
expression profiling using microarrays
the present work utilises and extends methods described in our previous microarray experiment  <cit> .
the porcine cdna microarrays were produced at the faculty of agricultural sciences, university of aarhus and contained  <dig>  features printed in duplicates.
<dig>  features were pcr products amplified from cdna clones produced by the sino-danish porcine genome sequencing project  <cit> , and  <dig> were control features.
the  <dig>  features represent approximately 20k gene transcripts.
additional information about the porcine cdna microarray can be found at ncbis gene expression omnibus  using the platform accession number gpl <dig>  this is a different batch of microarrays compared to the one we used in the testis experiment.
total rna was extracted from liver tissue using qiagen's rneasy midi kit according to manufacturer's instruction .
rna quantity was measured on a nanodrop nd- <dig> spectrophotometer  and rna quality was evaluated by the 28s:18s rrna ratio using a rna  <dig> nano labchip® kit on  <dig> bioanalyzer .
the microarray experiment was conducted as a common reference design using rna purified from liver tissue sampled from an unrelated danish landrace × hampshire pig as a reference.
aminoallyl-cdna was synthesised from  <dig> μg of total rna using the superscript indirect cdna labelling system  and labelled using ares alexa fluor labelling kits .
amino-modified and fluorescently labelled cdna was purified using nucleospin  <dig> extract ii pcr clean-up kits .
the individual samples were labelled with alexa fluor  <dig> and the reference was labelled with alexa fluor  <dig>  "green" spike-in rna from the lucidea universal scorecard  was added to the individuals and "red" spike-in rna was added to the reference.
hybridisation was performed in a discovery xt hybridisation station , followed by manual washing and drying by centrifugation.
the microarrays were scanned using a scanarray express scanner  and image analyses were conducted using genepix pro  <dig> software .
statistical analyses were carried out in r version  <dig> .
<dig>  <cit>  using the software package linear models for microarray analysis   <cit>  which is part of the bioconductor package  <cit> .
log-transformed ratios of mean foreground intensities  were print tip loess normalised.
the duplicate correlation function in limma was used to consider duplicate printing of each feature.
to evaluate the analyses, ma-plots /2), image plots and box plots were constructed using the limma tools for visualisation both before and after normalisation .
to assess differential expression, limma uses linear models in combination with an empirical bayes method to moderate the standard errors of the estimated log-fold changes  <cit> .
the nominal p-values were corrected for multiple testing by false discovery rates  using benjamini and hochberg approach  <cit> .
each of the groups dh, dl, nlh and nll animals were hybridised in individual batches, causing some confounding between androstenone levels and hybridisation batch.
however, as neither boxplots nor ma-plots were found to differ between hybridisation batches, it was assumed that the impact of hybridisation batch on the obtained data can be ignored.
the top 1% of the genes was considered for further analyses.
the array features were mapped to a locuslink identifier and an annotation package was built using the bioconductor package annbuilder .
go terms  were analysed for overrepresentation using the gohyperg function of the bioconductor package gostats   <cit> .
more detailed descriptions of the microarray experiments are available at the geo database through the series accession number gse  <dig>
quantitative real competitive pcr analysis
a real competitive pcr  gene expression analysis was used to verify a subset of the results from the microarray study.
quantitative gene expression  was performed using massarray methodology and the iplex protocol .
total rna was isolated from liver using an automatic dna/rna extractor .
total rna was treated with turbo dna-free™  for removal of contaminating dna and first strand cdna synthesis was conducted on  <dig>  μg total rna using superscript™-ii rnase h- reverse transcriptase .
assays for the genes included in this study  were designed and multiplexed into a single reaction using massarray qge assay design software .
the competitor, a synthetic dna molecule matching the targeted cdna sequence at all positions except for one single base, served as an internal standard for each transcript.
a 10-fold competitor dilution was initially used over a wide range of concentrations to determine an approximate equivalence point.
following this, a 7-fold dilution of competitor from  <dig>  × 10- <dig> to  <dig>  × 10- <dig> was used to achieve accurate quantification.
the cdna and competitor were co-amplified in the same pcr reaction with the following conditions: 95°c for  <dig> minutes,  <dig> cycles each of 95°c for  <dig> second, 56°c for  <dig> seconds and 72°c for  <dig> minute, and 72°c for  <dig> minutes.
a clean-up step was performed to remove unincorporated nucleotides.
the iplex reaction cocktail mix and pcr conditions were according to manufacturer's instructions  <cit> .
parallel pcr reactions were performed for all samples and the products were printed with two replicates on a spectrochip.
the primer extension reaction uses pcr products as templates and generates distinct mass signals for competitor and cdna-derived products.
mass spectrometric analysis generated signals from which peak areas were calculated.
gene expression levels were analysed using titan software version  <dig> – <dig>  <cit>  that runs in the r statistical environment.
raw data from the genotype analyzer software  were imported into titan and analysed using the default values of linear least squares polynomial regression and  <dig> bootstrap replicates.
cdna concentrations were corrected with respect to the housekeeping gene , and p-values and confidence intervals for fold changes were calculated.
list of abbreviations
d: duroc; nl: norwegian landrace; dh: duroc high androstenone; nlh: norwegian landrace high androstenone; dl: duroc low androstenone; nll: norwegian landrace high androstenone; 17β-hsd: 17β-hydroxysteroid dehydrogenase; 3β-hsd: 3β-hydroxysteroid dehydrogenase; agp: alpha-1-acid glycoprotein; akr: aldo-keto reductase; akr1c4: aldo-keto reductase family member 1c4; akr1d1: aldo-keto reductase family member 1d1; aox1: aldehyde oxidase; cyb5: cytochrome b5; cyp17: cytochrome p <dig> family 17; cyp19: cytochrome p <dig> family 19; cyp27a1: cytochrome p <dig> family member 27a1; cyp2a: cytochrome p <dig> family 2a; cyp2a19: cytochrome p <dig> family member 2a19; cyp2a6: cytochrome p <dig> family member 2a6; cyp2c19: cytochrome p <dig> family member 2c19; cyp2c33: cytochrome p <dig> family member 2c33; cyp2c49: cytochrome p <dig> family member 2c49; cyp2e1: cytochrome p <dig> family member 2e1; cyp3a: cytochrome p <dig> family member 3a; dhrs8: short-chain dehydrogenase/reductase 8; fmo: flavin-containing monooxygenase; fmo1: flavin-containing monooxygenase; gata4: transcription factor gata-4; go: gene ontology; gst: glutathione s-transferase; gsto1: glutathione s-transferase omega; hsds: hydroxysteroid dehydrogenases; hsd17b2: hydroxysteroid 17β dehydrogenase 2; hsd17b4: hydroxysteroid 17β dehydrogenase 4; hsd17b11: hydroxysteroid 17β dehydrogenase 11; hsd17b13: hydroxysteroid 17β dehydrogenase 13; hsf1: heat shock transcription factor 1; kmo: kynurenine 3-monooxygenase; mgst1: glutathione s-transferase; nat: n-acetyltransferase; nat12: n-acetyltransferase family member 12; nfix: nuclear factor i/x; orm1: orosomucoid 1; orm2: orosomucoid 2; qge: quantitative gene expression; rora: rar-related orphan receptor a; rcpcr: real competitive pcr; sdr: short-chain dehydrogenase/reductase; ste: estrogen sulfotransferase; sult1a1: sulfotransferase family member 1a1; sult2e1: sulfotransferase family member 1e1; sult2a1: sulfotransferase family member 2a1; sult2b1: sulfotransferase family member 2b1; tceb3: transcription elongation factor b3; tcf8: transcription factor 8; tdo2: tryptophan  <dig> -dioxygenase; tfrc: transferrin receptor; ugt: uridine diphospho-glucuronosyltransferase; ugt1a5: udp-glucuronosyltransferase family member 1a5; ugt2a1: udp-glucuronosyltransferase family member 2a1; ugt2b15: udp-glucuronosyltransferase family member 2b15
authors' contributions
mm carried out the microarray experimental work, performed statistical analysis and drafted the paper.
sl was involved in planning the project, provided laboratory facilities for rcpcr work and took part in writing the paper.
cb was involved in planning the project and was in charge of lab facilities for microarray studies.
jh was involved in microarray experimental work and took part in writing the paper.
hh carried out bioinformatics work.
ib was involved in statistical analysis.
tm was involved in power calculations and statistical supervision.
eg coordinated the study, was involved in planning the project, carried out rcpcr molecular work and took part in writing the paper.
supplementary material
additional file 1
microarray results for duroc.
gene expression profiling was performed using  <dig> arrays and the 1% most differentially expressed genes were considered significant.
the clone names are sequences with a hit to pig, human, mouse or bovine genes.
some genes are represented by several different clones on the array and may therefore show up more than once in the table, while some have no hits to the abovementioned species.
the id column gives the feature id on the microarray, m value indicates fold change, t gives the t-statistics, p.value is the nominal p-value and adj.p.value is the fdr corrected p-value.
click here for file
additional file 2
microarray results for norwegian landrace.
gene expression profiling was performed using  <dig> arrays and the 1% most differentially expressed genes were considered significant.
the clone names are sequences with a hit to pig, human, mouse or bovine genes.
some genes are represented by several different clones on the array and may therefore show up more than once in the table, while some have no hits to the abovementioned species.
the id column gives the feature id on the microarray, m value indicates fold change, t gives the t-statistics, p.value is the nominal p-value and adj.p.value is the fdr corrected p-value.
click here for file
additional file 3
gene ontology  results for the cellular component ontology in duroc.
the top 1% differentially expressed genes in duroc were analysed for over-represented  go terms in the cellular component ontology.
click here for file
additional file 4
gene ontology  results for the cellular component ontology in norwegian landrace.
the top 1% differentially expressed genes in norwegian landrace were analysed for over-represented  go terms in the cellular component ontology.
click here for file
additional file 5
androstenone values.
the androstenone values  in duroc high , duroc low , landrace high  and landrace low  animals used in this study.
click here for file
additional file 6
boxplots.
boxplots of normalised arrays for duroc  and norwegian landrace .
click here for file
additional file 7
gene transcripts included in the rcpcr analyses.
click here for file
acknowledgements
this study was financed by the norwegian pig breeders association  and the research council of norway.
the work conducted at university of aarhus was supported by the eu-project sabre.
we want to thank ellen dahl and Øystein andresen for being in charge of the androstenone analyses performed at the hormone laboratory at the norwegian school of veterinary science .
we also thank elin bergseth at norsvin for collecting samples, dr. peter sørensen and helle jensen at the microarray platform at university of aarhus for kind assistance, dr. paul oeth at sequenom for carrying out the rcpcr primer design and dr. matthew baranski for valuable help with english grammar.

 boar taint is an off-odour and off-flavour in pig carcasses that is primarily caused by high levels of 16-androstene steroids and/or skatole in adipose tissue. male pigs used for meat production are normally castrated very early in life to prevent boar taint in the meat. however, castration also removes the source of natural anabolic androgens that stimulate lean growth and, as a result, uncastrated males have improved feed efficiency and greater lean yield of the carcass compared to barrows. detection of genetic factors influencing boar taint may facilitate implementation of selective breeding practices to produce pigs with little or no taint. because of unfavourable correlations between androstenone and other sex steroids, a direct selection against high levels of androstenone would result in decreased production of testosterone and estrogens, with associated negative effects on performance and sexual maturation. therefore, a comprehensive understanding of the complex genetic system controlling boar taint is required before genetic improvement can be achieved. androstenone is produced in the testis and is transported by blood to the salivary gland where it functions as a pheromone to stimulate mating stance in gilts. it is metabolised in the liver, producing α-androstenol and β-androstenol, and deficient degradation may lead to the accumulation of androstenone in adipose tissue. skatole is produced by the metabolism of tryptophan from the gut and is also catabolised in liver. diaz et al. identified seven metabolites of skatole in pig liver microsomes. an increase in levels of skatole in adipose tissue occurs in boars around puberty, but not in barrows or sows, indicating that skatole metabolism is regulated by testicular steroids. enzymes responsible for metabolism of androstenone and skatole in liver have been identified but relatively few genes have been investigated. several studies have shown that 3β-hydroxysteroid dehydrogenase is involved in androstenone metabolism, with low mrna, protein and enzyme expression levels correlated to high androstenone levels in adipose tissue. cytochrome p 2e is involved in metabolism of skatole and deficient cyp2e induction is suggested to be a major cause of high levels of skatole in adipose tissue. a relationship between metabolism of androstenone and skatole in liver has been observed, with evidence to suggest that androstenone blocks cyp2e induction by skatole. sulfotransferase enzymes including sult2a sult2b and sult1a are associated with metabolic clearance of androstenone and skatole since they are known to conjugate steroid hormones and drugs into more water soluble compounds that facilitates excretion. although both androstenone and skatole are responsible for boar taint, androstenone seems to be the biggest problem in the duroc and norwegian landrace populations. levels of androstenone are also predominantly genetically determined whereas levels of skatole are more heavily influenced by feeding and environmental factors. levels of androstenone will be a function of both production in testes and elimination in liver. we have previously studied differentially expressed genes related to the production of androstenone in boar testis, and the objective of this study was to investigate global gene expression profiles related to the degradation of androstenone in pig livers. microarray technology was used to obtain gene expression profiles from liver samples of duroc and norwegian landrace boars with extreme high or low levels of androstenone in adipose tissue. these samples were hybridised together with a common reference sample to cdna microarrays representing approximately porcine gene transcripts. a total of animals were selected from each group, resulting in a total of arrays. a subset of the differentially expressed genes was verified using a quantitative pcr-based method.  porcine cdna microarrays were used to obtain global gene expression profiles in liver tissues sampled from d and nl boars with extreme high and low levels of androstenone. average levels of androstenone were ± ppm for d high and ± ppm for d low boars. average levels of androstenone were ± ppm for nl high and ± ppm for nl low boars. linear models were used to identify significantly differentially expressed genes and the top 1% most significant genes were subsequently inspected as possible candidate genes affecting androstenone levels. the top genes of d and nl are presented in table and respectively. among the affected genes, were found to be in common for the two breeds and expression differences were generally more significant in d compared to nl. gene expression profiles were identified in limma using empirical bayes moderated t-statistics. fold change and statistical significance is shown. the clone names refer to hits to pig, human, mouse or bovine sequences. some genes are represented by several different clones on the array and may therefore show up more than once in the table, while others have no hits to the abovementioned species. gene expression profiles were identified in limma using empirical bayes moderated t-statistics. fold change and statistical significance is shown. the clone names refer to hits to pig, human, mouse or bovine sequences. some genes are represented by several different clones on the array and may therefore show up more than once in the table, while others have no hits to the abovementioned species. testing for overrepresentation of gene ontology terms among the affected genes was performed relative to the global representation of go terms on the microarray. a majority of the significant genes were related to oxidoreductase activities, glucuronosyltransferase activity and the binding of iron ion, haem, dna and oxygen. additionally, cellular metabolic process pathways such as amino acid metabolism and catabolism, electron transport and inflammatory response were overrepresented. furthermore, the genes were classified according to their cellular component ontology results for the cellular component ontology in duroc, additional file 4: gene ontology results for the cellular component ontology in norwegian landrace). for d, cytoplasm, microsomes and endoplasmatic reticulum were among the important cellular components, none of the cellular components reached the level of significance, i.e. had more than significant genes related to the cc terms. elevated levels of androstenone in adipose tissue can result from both increased biosynthesis in the testes and deficient metabolism in liver. we have previously conducted a microarray experiment investigating gene expression profiles in the testes of boars with extreme levels of androstenone, and in this study we have examined gene expression profiles in the liver of some representatives from that study along with new individuals. liver metabolism can be divided into phase i and phase ii reactions. phase i metabolism involves oxidation and hydroxylation reactions that make the substrate more water soluble. phase ii conjugation reactions further increase hydrophilicity by adding polar groups. as a result of these metabolic reactions, compounds including endogenous steroids, fatty acids and drugs are inactivated and eliminated. we have identified a number of differentially expressed genes that function in pathways affecting both phase i and phase ii reactions involved in metabolism of androstenone in the liver. phase i metabolism the most significantly differentially expressed gene involved in phase i oxidation reactions was cytochrome p 2c. this gene is a member of the cyp2c family and genes of this family encode monooxygenases that catalyse several reactions involved in the metabolism of drugs and endogenous compounds. different cyp2c isoforms show some cross reactivity towards substrates which makes it difficult to differentiate cyp2c activities, and substrate specificity for cyp2c is not known. in our study, cyp2c transcripts were shown to have a significant up-regulation in boars with high levels of androstenone in both d and nl lines but differences were clearly most prominent in the d breed. another member of the cytochrome p superfamily, that was differentially expressed in our study, is cytochrome p 2e. in contrast to expression of the cyp2c gene, cyp2e showed a down-regulation in dh and nlh boars. cyp2e encodes an enzyme that metabolises many endogenous and exogenous substrates such as alcohols, ketones and drugs. androstenone has been shown to block skatole induced expression of cyp2e in pig hepatocytes and to inhibit activity of cyp2e in porcine liver microsomes. liver metabolism of skatole also involves this gene and enzyme, and the observed pubertal increase in skatole levels has been attributed to the inhibition of cyp2e by the sex steroids androstenone and 17β-estradiol. this is the first study, however, to show gene expression differences for cyp2e in relation to androstenone. cytochrome p member 2a, which is the pig ortholog of human cyp2a catalyses 7-hydroxylation of coumarin in pigs, in addition to being involved in skatole metabolism. in this study, cyp2a was significantly down-regulated in dh boars. gene expression differences in animals with high and low androstenone levels have not previously been reported for porcine cyp2a however its protein expression has been shown to be inhibited by androstenone. diaz and squires found that both cyp2a protein content and its enzymatic activity were negatively correlated with skatole levels in adipose tissue. in contrast, results presented by terner et al. indicate that the cyp2a enzyme is not important for metabolism of skatole in primary cultured porcine hepatocytes. two other members of the cytochrome p family, cyp27a and cyp2c were also up-regulated in dh. cyp27a is a multifunctional enzyme that oxygenates cholesterol, bile acids and vitamin d. it has not previously been identified as a candidate gene associated with androstenone, but it is regulated by other sex steroids in human cells. cyp2c belongs to the same sub-family as cyp2c but its function has yet to be described in the literature. like the cyp2c gene, cyp2c was found to be up-regulated in dh and nlh boars. the differential expression of cyp2a cyp27a and cyp2c in d and not nl boars might suggest that more genes are involved in phase i reactions in d. this is supported by a high number of oxidoreductase pathways significant in d. monooxygenase reactions are, however, clearly important in both breeds according to our gene ontology results. down-regulation of cyp2e and cyp2a might suggest a different role for these genes compared to the up-regulated cyp2c cyp2c and cyp27a our study implicates another class of genes involved in phase i oxidation reactions, namely the flavin-containing monooxygenases. the fmo family of enzymes converts lipophilic compounds into more polar metabolites and decreases activity of the compounds, a similar activity to that of the cytochrome p450s. microarray expression results show that flavin-containing monooxygenase was significantly up-regulated in dh in the microarray results. the expression pattern in dh boars was confirmed by rcpcr and the rcpcr analysis also revealed that fmo was up-regulated in nlh boars. as shown in figure the fold change in nlh was only half of dh, which might explain why this gene was not found to be significantly differentially expressed in the microarray results. regulation of fmo involves sex steroids. in male mice, castration was reported to increase fmo expression, while in rats the opposite effect has been shown, with positive regulation of fmo by testosterone and negative by estradiol. the monooxygenase activity of fmo in addition to its regulation by steroid hormones makes it an interesting candidate gene for boar taint. phase ii metabolism phase i oxidation reactions of androstenone by enzymes including cytochrome p450s and fmos are often followed by phase ii conjugation reactions that are catalysed by glucuronosyltransferases, sulfotransferases, acetyltransferases and glutathione s-transferases. through the addition of polar moieties, these enzymes increase substrate solubility. conjugation reactions are an important means of excreting steroid hormones and other compounds, these reactions have also been proposed to keep inactive steroids easily available in cells. the hydroxysteroid sulfotransferases sult2a and sult2b have been associated with androstenone levels in previous studies. these genes were not found to be significantly differentially expressed in our study, however the estrogen sulfotransferase ste was found to be up-regulated in dh boars and down-regulated in nlh boars. a previous microarray study performed by our group showed an up-regulation of cyp gene expression in the testis of d and nl boars with high androstenone levels. these genes encode enzymes biosynthesising estrogens and might explain why an estrogen conjugation enzyme is differentially expressed in livers of the same pigs. furthermore, boars secrete large quantities of estrogens from the testis, and both fat and plasma estrogen levels have previously been shown to be highly correlated with levels of androstenone in adipose tissue. phase ii conjugation by glucuronidation is another major pathway of liver elimination of endogenous and exogenous compounds, and in d this molecular function was significantly overrepresented. it is catalysed by uridine diphospho-glucuronosyltransferases which transfer glucuronic acid to substrates to increase solubility. the ugts have been divided into two subfamilies, the ugt1s and the ugt2s, and in this study the family member ugt1a was found to be up-regulated in dh boars. furthermore, a transcript similar to ugt2b was up-regulated in d and a transcript similar to ugt2a was down-regulated in nlh. ugt2b has been shown to conjugate several androgens in humans, whereas ugt1a has been found to be catalytically active against some exogenous compounds. ugt2a contributes to glucuronidation of steroids and phenolic compounds in olfactory tissue and has been found to eliminate odourants. the ugt family of conjugation enzymes have previously been associated with the androstenone metabolites α-androstenol and β-androstenol. n-acetyltransferases are another family of conjugation enzymes and act by transferring the acetyl group of acetyl coenzyme a to aromatic amines to increase their water solubility. nat type was found to be up-regulated in nlh based on the microarray results and this was verified by rcpcr. the rcpcr results also revealed an up-regulation in dh boars. androgens have been shown to increase expression of nat type in human cancer cells, but the enzyme family has not previously been associated with levels of androstenone. tryptophan, the precursor of skatole, contains an aromatic, suggesting a role for this family of conjugation enzymes in the control of skatole levels, possibly through regulation by steroid hormones. glutathione s-transferases are functionally diverse enzymes mostly known to catalyse conjugation reactions of endogenous substances, haem, fatty acids, xenobiotics and products of oxidative processes. they have also been implicated in the intracellular transport of steroids to their site of action. in this study, a glutathione s-transferase gene was found to be down-regulated in nlh. the gene is described in the database as loc we have previously described up-regulation of two gst genes, gsto and mgst in association with testicular androstenone levels, and here we additionally propose a role for glucuronidation in liver metabolism. expression of phase ii metabolic genes in d and nl pigs suggest breed specific mechanisms. we found differential expression of a gst variant in nl but not d boars, up-regulation of ste in dh but down-regulation in nlh, and up-regulation of two ugt genes in dh but down-regulation of another family member in nlh. breed specific phase ii mechanisms for these breeds are in agreement with our previous finding for the protein sult2b. regulation of steroid availability 17β-hydroxysteroid dehydrogenases regulate the availability of androgens and estrogens in tissues by catalysing interconvertion of active and inactive forms of steroids. they do so by regulating the occupancy of steroid nuclear hormone receptors like androgen receptor, estrogen receptor and progesterone receptor. gene expression of 17β-hsd in porcine liver has been shown to be negatively associated with levels of androstenone in adipose tissue. in a previous study, we found that the hsd17b gene was significantly up-regulated in testicle samples from dh and nlh, and in this study hsd17b was up-regulated in the liver of dh boars. additionally, we found that hsd17b was down-regulated in dh and that the isoform hsd17b was down-regulated in both dh and nlh. differential expression of both hsd17b and hsd17b was verified by rcpcr, which also showed significant down-regulation of hsd17b in nlh boars. the hsd17b enzyme catalyses the interconversion of testosterone and androstenedione, as well as estradiol and estrone. the function of isoform hsd17b also known as short-chain dehydrogenase/reductase has yet to be described but its expression has been characterised in human liver. the short-chain dehydrogenase/reductase gene, also known as hsd17b was found to be down-regulated in nlh boars. hsd17b is involved in androgen metabolism and we previously found this gene to be down-regulated in the testis of dh boars. the hsds belong to two superfamilies: the short-chain dehydrogenase/reductases and the aldo-keto reductases. the four hsds described above all belong to the sdr superfamily but a gene from the akrs was also found to be differentially expressed. the aldo-keto reductase family member 1d was down-regulated in nlh animals. akr1d is a liver specific enzyme that regulates the hormone levels of several steroids. the enzyme has a 5β-reductase activity and is necessary for the catabolism of aldosterone, cortisol and androgens. aldosterone, cortisol and androgens are three of four metabolic compounds formed from pregnenolone, with the fourth compound being androstenone. a role for akr1d in androstenone catabolism might therefore be possible. we previously reported up-regulation of the family member akr1c in the testis of dh and nlh boars, but akr1d has not formerly been associated with porcine androstenone levels. another class of enzymes that regulate the availability of steroids is the plasma proteins. binding of plasma protein is a reversible reaction that has no physiological effect, but it controls the amount of free drugs and hormones available to tissues by protecting them from metabolism. alpha-1-acid glycoprotein is an acute phase serum protein synthesised in the liver and secreted to plasma where it binds and carries drugs and steroid hormones. the protein has been shown to interact with cyp3a and cyp2c in humans and to inhibit cytochrome p activity. it is suggested that serum protein interaction is an important factor for cytochrome p mediated metabolism, and that the interaction is isoform specific. agp has also been studied in pigs as a binding factor of pheromones, but has not been found to bind progesterone in pig nasal mucosa. agp has two variants in humans: orosomucoid and. in this study we found that porcine transcript homologous to bovine agp and human orm were significantly down-regulated in dh and nlh boars. significant expression of these genes might explain the inflammatory response ontology term in figure as these genes have mostly been described in relation to inflammatory response. agp has not been investigated as a binding protein and transporter for androstenone, but different levels of androstenone could be related to differences in its availability through plasma binding by agp. regulatory factors one of the most significant molecular function terms in nl was nucleic acid binding. this term includes transcription factor binding and several transcription factors were found to be differentially expressed in this study. the gata factor was found to be down-regulated based on the microarray data, but we were not able to confirm this result with rcpcr. other transcription factors identified in this study should be further investigated, including rar-related orphan receptor a, transcription elongation factor b, nuclear factor i/x, transcription factor and heat shock transcription factor. we have previously identified iron ion binding, ferric ion binding and electron transport as being associated with levels of androstenone, possibly through interaction with the haem-containing cytochrome p450s. in this study we also found iron ion binding and electron transport, in addition to haem binding, to be important pathways, supporting our previous findings. cytochrome b is involved in electron transfer to cytochrome p450s and has been proposed as a candidate gene for androstenone through its interaction with cytochrome p c. in this study we found cyb to be down-regulated in nlh boars, however we previously identified cyb as being up-regulated in the testis of both dh and nlh. both cyp2e and cyp2a have been found to be activated by cyb in humans, supporting the down-regulation of cyb together with cyp2e and cyp2a in this study. up-regulation in testis and down-regulation in liver might also support a key regulatory role of cyb in both tissues. breed differences breed differences in boar taint candidate genes have previously been shown between d and nl. consistent with these findings, we found breed differences in the expression profiles of genes involved in both phase i metabolism and phase ii metabolism. however, rcpcr results might suggest that genes appearing in one breed can be differentially expressed also in the other breed. additional and more comprehensive rcpcr studies are planned to clarify this. in general, the d breed showed higher levels of significance compared to the nl breed, potentially due to the larger contrasts between high and low androstenone groups in this breed. a number of new candidate genes have been identified in this study, however, we do not know their effect on performance and sexual maturation in pig. additional studies are needed to investigate potential roles of the genes identified for phenotypes related to fertility. relationship with skatole pigs in this study were selected based on extreme androstenone values. however, levels of androstenone and skatole have previously been found to be highly correlated, showing genetic correlations of for d and for nl. some of the genes found differentially expressed in this study have previously been found to be associated with tryptophan or skatole and this could be explained by high correlations with androstenone. this might also be the reason for the significance of go terms such as aromatic amino acid metabolism and catabolism, since the skatole precursor tryptophan belongs to this family of amino acids. the tryptophan 3-dioxygenase gene, which oxidises tryptophan, was differentially expressed in dh. a gene of the fmo family, kynurenine 3-monooxygenase, was found to be down-regulated in nlh. kmo is involved in tryptophan degradation and might therefore be interesting in regards to skatole. the aldehyde oxidase gene was significantly down-regulated in high androstenone animals of both breeds and this gene has not previously been associated with androstenone levels. it has, however, been shown to play an important role in skatole metabolism in several species including pigs.  in this study we compared global gene expression profiles from the livers of boars with extreme high and low levels of androstenone from two breeds, duroc and norwegian landrace. breed differences are evident for molecular functions and biological processes involved in metabolism of androstenone, however many of the same genes are differentially expressed in the two breeds as well. genes encoding different oxidising enzymes including the cytochrome p family and the flavin-containing monooxygenase family were significantly differentially expressed. furthermore, genes involved in conjugation reactions, including the udp-glucuronosyltransferases, sulfotransferases, n-acetyltransferases and glutathione s-transferase were significant, in addition to genes of the 17β-hydroxysteroid dehydrogenase family, which are known to regulate availability of active steroids. we suggest a novel role for plasma proteins including agp and orm in regulating availability of androstenone in pigs. this is the first published microarray experiment describing liver metabolism of androstenone. a number of new candidate genes have been identified, both from phase i and phase ii metabolism as well as pathways regulating steroid availability. materials and methods animals animals used in this study were duroc and norwegian landrace boars from norsvin's three boar testing stations. the d boars were on average days old at kg live weight compared to the nl boars that were on average days old at kg live weight. the boars were slaughtered on average days later. tissue samples from liver were frozen in liquid n immediately after slaughter and stored at -80°c until used for rna extraction as described below. samples from adipose tissue were collected from the neck at slaughter and stored at -20°c until used for androstenone measurements. androstenone levels were measured by a modified time-resolved fluoroimmunoassay at the hormone laboratory, norwegian school of veterinary science using an antiserum produced at nvh. the androstenone measurements were performed on more than boars, and statistical power calculations showed that selecting animals from each tail of the androstenone distribution would yield sufficient power to detect differentially expressed genes with a limited number of arrays. the most extreme boars from each tail of the androstenone level distribution were therefore selected. due to poor rna quality for two of the samples, samples were subsequently used from each group. of the animals used were the same individuals as those used in our previous study examining gene expression in boar testis, with being new animals as liver samples were not available for all of the previously studied animals. average androstenone levels for the selected boars were ppm and ppm for nl and d, respectively.. average values for the groups were ± ppm for nl high, ± ppm for nl low, ± ppm for d high and ± ppm for d low. in order to reduce family effects, a maximum of two and three half sibs were chosen from nl and d, respectively. the selected animals were used for expression profiling by microarrays and for the following verification of selected genes by rcpcr. expression profiling using microarrays the present work utilises and extends methods described in our previous microarray experiment. the porcine cdna microarrays were produced at the faculty of agricultural sciences, university of aarhus and contained features printed in duplicates. features were pcr products amplified from cdna clones produced by the sino-danish porcine genome sequencing project, and were control features. the features represent approximately 20k gene transcripts. additional information about the porcine cdna microarray can be found at ncbis gene expression omnibus using the platform accession number gpl this is a different batch of microarrays compared to the one we used in the testis experiment. total rna was extracted from liver tissue using qiagen's rneasy midi kit according to manufacturer's instruction. rna quantity was measured on a nanodrop nd- spectrophotometer and rna quality was evaluated by the 28s:18s rrna ratio using a rna nano labchip® kit on bioanalyzer. the microarray experiment was conducted as a common reference design using rna purified from liver tissue sampled from an unrelated danish landrace × hampshire pig as a reference. aminoallyl-cdna was synthesised from μg of total rna using the superscript indirect cdna labelling system and labelled using ares alexa fluor labelling kits. amino-modified and fluorescently labelled cdna was purified using nucleospin extract ii pcr clean-up kits. the individual samples were labelled with alexa fluor and the reference was labelled with alexa fluor "green" spike-in rna from the lucidea universal scorecard was added to the individuals and "red" spike-in rna was added to the reference. hybridisation was performed in a discovery xt hybridisation station, followed by manual washing and drying by centrifugation. the microarrays were scanned using a scanarray express scanner and image analyses were conducted using genepix pro software. statistical analyses were carried out in r version. using the software package linear models for microarray analysis which is part of the bioconductor package. log-transformed ratios of mean foreground intensities were print tip loess normalised. the duplicate correlation function in limma was used to consider duplicate printing of each feature. to evaluate the analyses, ma-plots /2), image plots and box plots were constructed using the limma tools for visualisation both before and after normalisation. to assess differential expression, limma uses linear models in combination with an empirical bayes method to moderate the standard errors of the estimated log-fold changes. the nominal p-values were corrected for multiple testing by false discovery rates using benjamini and hochberg approach. each of the groups dh, dl, nlh and nll animals were hybridised in individual batches, causing some confounding between androstenone levels and hybridisation batch. however, as neither boxplots nor ma-plots were found to differ between hybridisation batches, it was assumed that the impact of hybridisation batch on the obtained data can be ignored. the top 1% of the genes was considered for further analyses. the array features were mapped to a locuslink identifier and an annotation package was built using the bioconductor package annbuilder. go terms were analysed for overrepresentation using the gohyperg function of the bioconductor package gostats. more detailed descriptions of the microarray experiments are available at the geo database through the series accession number gse quantitative real competitive pcr analysis a real competitive pcr gene expression analysis was used to verify a subset of the results from the microarray study. quantitative gene expression was performed using massarray methodology and the iplex protocol. total rna was isolated from liver using an automatic dna/rna extractor. total rna was treated with turbo dna-free™ for removal of contaminating dna and first strand cdna synthesis was conducted on μg total rna using superscript™-ii rnase h- reverse transcriptase. assays for the genes included in this study were designed and multiplexed into a single reaction using massarray qge assay design software. the competitor, a synthetic dna molecule matching the targeted cdna sequence at all positions except for one single base, served as an internal standard for each transcript. a 10-fold competitor dilution was initially used over a wide range of concentrations to determine an approximate equivalence point. following this, a 7-fold dilution of competitor from × 10- to × 10- was used to achieve accurate quantification. the cdna and competitor were co-amplified in the same pcr reaction with the following conditions: 95°c for minutes, cycles each of 95°c for second, 56°c for seconds and 72°c for minute, and 72°c for minutes. a clean-up step was performed to remove unincorporated nucleotides. the iplex reaction cocktail mix and pcr conditions were according to manufacturer's instructions. parallel pcr reactions were performed for all samples and the products were printed with two replicates on a spectrochip. the primer extension reaction uses pcr products as templates and generates distinct mass signals for competitor and cdna-derived products. mass spectrometric analysis generated signals from which peak areas were calculated. gene expression levels were analysed using titan software version – that runs in the r statistical environment. raw data from the genotype analyzer software were imported into titan and analysed using the default values of linear least squares polynomial regression and bootstrap replicates. cdna concentrations were corrected with respect to the housekeeping gene, and p-values and confidence intervals for fold changes were calculated. list of abbreviations d: duroc; nl: norwegian landrace; dh: duroc high androstenone; nlh: norwegian landrace high androstenone; dl: duroc low androstenone; nll: norwegian landrace high androstenone; 17β-hsd: 17β-hydroxysteroid dehydrogenase; 3β-hsd: 3β-hydroxysteroid dehydrogenase; agp: alpha-1-acid glycoprotein; akr: aldo-keto reductase; akr1c4: aldo-keto reductase family member 1c4; akr1d1: aldo-keto reductase family member 1d1; aox1: aldehyde oxidase; cyb5: cytochrome b5; cyp17: cytochrome p family 17; cyp19: cytochrome p family 19; cyp27a1: cytochrome p family member 27a1; cyp2a: cytochrome p family 2a; cyp2a19: cytochrome p family member 2a19; cyp2a6: cytochrome p family member 2a6; cyp2c19: cytochrome p family member 2c19; cyp2c33: cytochrome p family member 2c33; cyp2c49: cytochrome p family member 2c49; cyp2e1: cytochrome p family member 2e1; cyp3a: cytochrome p family member 3a; dhrs8: short-chain dehydrogenase/reductase 8; fmo: flavin-containing monooxygenase; fmo1: flavin-containing monooxygenase; gata4: transcription factor gata-4; go: gene ontology; gst: glutathione s-transferase; gsto1: glutathione s-transferase omega; hsds: hydroxysteroid dehydrogenases; hsd17b2: hydroxysteroid 17β dehydrogenase 2; hsd17b4: hydroxysteroid 17β dehydrogenase 4; hsd17b11: hydroxysteroid 17β dehydrogenase 11; hsd17b13: hydroxysteroid 17β dehydrogenase 13; hsf1: heat shock transcription factor 1; kmo: kynurenine 3-monooxygenase; mgst1: glutathione s-transferase; nat: n-acetyltransferase; nat12: n-acetyltransferase family member 12; nfix: nuclear factor i/x; orm1: orosomucoid 1; orm2: orosomucoid 2; qge: quantitative gene expression; rora: rar-related orphan receptor a; rcpcr: real competitive pcr; sdr: short-chain dehydrogenase/reductase; ste: estrogen sulfotransferase; sult1a1: sulfotransferase family member 1a1; sult2e1: sulfotransferase family member 1e1; sult2a1: sulfotransferase family member 2a1; sult2b1: sulfotransferase family member 2b1; tceb3: transcription elongation factor b3; tcf8: transcription factor 8; tdo2: tryptophan -dioxygenase; tfrc: transferrin receptor; ugt: uridine diphospho-glucuronosyltransferase; ugt1a5: udp-glucuronosyltransferase family member 1a5; ugt2a1: udp-glucuronosyltransferase family member 2a1; ugt2b15: udp-glucuronosyltransferase family member 2b15 authors' contributions mm carried out the microarray experimental work, performed statistical analysis and drafted the paper. sl was involved in planning the project, provided laboratory facilities for rcpcr work and took part in writing the paper. cb was involved in planning the project and was in charge of lab facilities for microarray studies. jh was involved in microarray experimental work and took part in writing the paper. hh carried out bioinformatics work. ib was involved in statistical analysis. tm was involved in power calculations and statistical supervision. eg coordinated the study, was involved in planning the project, carried out rcpcr molecular work and took part in writing the paper. supplementary material additional file 1 microarray results for duroc. gene expression profiling was performed using arrays and the 1% most differentially expressed genes were considered significant. the clone names are sequences with a hit to pig, human, mouse or bovine genes. some genes are represented by several different clones on the array and may therefore show up more than once in the table, while some have no hits to the abovementioned species. the id column gives the feature id on the microarray, m value indicates fold change, t gives the t-statistics, p.value is the nominal p-value and adj.p.value is the fdr corrected p-value. click here for file additional file 2 microarray results for norwegian landrace. gene expression profiling was performed using arrays and the 1% most differentially expressed genes were considered significant. the clone names are sequences with a hit to pig, human, mouse or bovine genes. some genes are represented by several different clones on the array and may therefore show up more than once in the table, while some have no hits to the abovementioned species. the id column gives the feature id on the microarray, m value indicates fold change, t gives the t-statistics, p.value is the nominal p-value and adj.p.value is the fdr corrected p-value. click here for file additional file 3 gene ontology results for the cellular component ontology in duroc. the top 1% differentially expressed genes in duroc were analysed for over-represented go terms in the cellular component ontology. click here for file additional file 4 gene ontology results for the cellular component ontology in norwegian landrace. the top 1% differentially expressed genes in norwegian landrace were analysed for over-represented go terms in the cellular component ontology. click here for file additional file 5 androstenone values. the androstenone values in duroc high, duroc low, landrace high and landrace low animals used in this study. click here for file additional file 6 boxplots. boxplots of normalised arrays for duroc and norwegian landrace. click here for file additional file 7 gene transcripts included in the rcpcr analyses. click here for file acknowledgements this study was financed by the norwegian pig breeders association and the research council of norway. the work conducted at university of aarhus was supported by the eu-project sabre. we want to thank ellen dahl and Øystein andresen for being in charge of the androstenone analyses performed at the hormone laboratory at the norwegian school of veterinary science. we also thank elin bergseth at norsvin for collecting samples, dr. peter sørensen and helle jensen at the microarray platform at university of aarhus for kind assistance, dr. paul oeth at sequenom for carrying out the rcpcr primer design and dr. matthew baranski for valuable help with english grammar.
boar taint is the unpleasant odour and flavour of the meat of uncastrated male pigs that is primarily caused by high levels of androstenone and skatole in adipose tissue. the study is a first step towards a deeper understanding of enzymes and regulators involved in pathways of androstenone metabolism and may ultimately lead to the discovery of markers to reduce boar taint. studies on androstenone metabolism have, however, focused on a limited number of genes. in this study, microarrays were used to identify differentially expressed genes and pathways related to androstenone metabolism in the liver from boars with extreme levels of androstenone in adipose tissue. identification of additional genes influencing levels of androstenone may facilitate implementation of marker assisted breeding practices. differences in the expression of fmo nat hsd17b and hsd17b were verified by quantitative real competitive pcr. a number of genes and pathways related to metabolism of androstenone in liver were identified, including new candidate genes involved in phase i oxidation metabolism, phase ii conjugation metabolism, and regulation of steroid availability. among the differentially expressed genes were metabolic phase i related genes belonging to the cytochrome p family and the flavin-containing monooxygenase fmo additionally, phase ii conjugation genes including udp-glucuronosyltransferases ugt1a ugt2a and ugt2b sulfotransferase ste, n-acetyltransferase nat and glutathione s-transferase were identified. phase i and phase ii metabolic reactions increase the water solubility of steroids and play a key role in their elimination. the samples were hybridised to porcine cdna microarrays and the 1% most significant differentially expressed genes were considered significant. differential expression was also found for genes encoding 17beta-hydroxysteroid dehydrogenases and plasma proteins alpha-1-acid glycoprotein and orosomucoid. 17beta-hydroxysteroid dehydrogenases and plasma proteins regulate the availability of steroids by controlling the amount of active steroids accessible to receptors and available for metabolism. liver tissue samples from boars of the two breeds duroc and norwegian landrace, with extreme high and with extreme low levels of androstenone, were selected from more than individuals.
 boar taint is the unpleasant odour and flavour of the meat of uncastrated male pigs that is primarily caused by high levels of androstenone and skatole in adipose tissue. androstenone is a steroid and its levels are mainly genetically determined. studies on androstenone metabolism have, however, focused on a limited number of genes. identification of additional genes influencing levels of androstenone may facilitate implementation of marker assisted breeding practices. in this study, microarrays were used to identify differentially expressed genes and pathways related to androstenone metabolism in the liver from boars with extreme levels of androstenone in adipose tissue.  liver tissue samples from boars of the two breeds duroc and norwegian landrace, with extreme high and with extreme low levels of androstenone, were selected from more than individuals. the samples were hybridised to porcine cdna microarrays and the 1% most significant differentially expressed genes were considered significant. among the differentially expressed genes were metabolic phase i related genes belonging to the cytochrome p family and the flavin-containing monooxygenase fmo additionally, phase ii conjugation genes including udp-glucuronosyltransferases ugt1a ugt2a and ugt2b sulfotransferase ste, n-acetyltransferase nat and glutathione s-transferase were identified. phase i and phase ii metabolic reactions increase the water solubility of steroids and play a key role in their elimination. differential expression was also found for genes encoding 17beta-hydroxysteroid dehydrogenases and plasma proteins alpha-1-acid glycoprotein and orosomucoid. 17beta-hydroxysteroid dehydrogenases and plasma proteins regulate the availability of steroids by controlling the amount of active steroids accessible to receptors and available for metabolism. differences in the expression of fmo nat hsd17b and hsd17b were verified by quantitative real competitive pcr.  a number of genes and pathways related to metabolism of androstenone in liver were identified, including new candidate genes involved in phase i oxidation metabolism, phase ii conjugation metabolism, and regulation of steroid availability. the study is a first step towards a deeper understanding of enzymes and regulators involved in pathways of androstenone metabolism and may ultimately lead to the discovery of markers to reduce boar taint. 