- Research article
- Open Access
Association between SNPs within candidate genes and compounds related to boar taint and reproduction
BMC Genetics volume 10, Article number: 32 (2009)
Boar taint is an unpleasant odour and flavour of the meat from some uncastrated male pigs primarily caused by elevated levels of androstenone and skatole in adipose tissue. Androstenone is produced in the same biochemical pathway as testosterone and estrogens, which represents a particular challenge when selecting against high levels of androstenone in the breeding programme, without simultaneously decreasing levels of other steroids. Detection of single nucleotide polymorphisms (SNPs) associated with compounds affecting boar taint is important both for gaining a better understanding of the complex regulation of the trait and for the purpose of identifying markers that can be used to improve the gain of breeding. The beneficial SNPs to be used in breeding would have the combinational effects of reducing levels of boar taint without affecting fertility of the animals. The aim of this study was to detect SNPs in boar taint candidate genes and to perform association studies for both single SNPs and haplotypes with levels of boar taint compounds and phenotypes related to reproduction.
An association study involving 275 SNPs in 121 genes and compounds related to boar taint and reproduction were carried out in Duroc and Norwegian Landrace boars. Phenotypes investigated were levels of androstenone, skatole and indole in adipose tissue, levels of androstenone, testosterone, estrone sulphate and 17β-estradiol in plasma, and length of bulbo urethralis gland. The SNPs were genotyped in more than 2800 individuals and several SNPs were found to be significantly (LRT > 5.4) associated with the different phenotypes. Genes with significant SNPs in either of the traits investigated include cytochrome P450 members CYP2E1, CYP21, CYP2D6 and CYP2C49, steroid 5α-reductase SRD5A2, nuclear receptor NGFIB, catenin CTNND1, BRCA1 associated protein BAP1 and hyaluronoglucosaminidase HYAL2. Haplotype analysis provided additional evidence for an effect of CYP2E1 on levels of skatole and indole, and for BAP1, HYAL2 and SRD5A2 on levels of androstenone.
The findings in this study indicate that polymorphisms in CYP2E1, CYP21, CYP2D6, CYP2C49, NGFIB and CTNND1 might be used to reduce levels of boar taint without affecting levels of testosterone, estrone sulphate, 17β-estradiol or length of bulbo urethralis gland.
Male pigs used for meat production are castrated at an early age to avoid boar taint, which is an unpleasant odour and flavour of the meat from some boars. Due to animal welfare concerns, castration will be prohibited in Norway, and possibly EU countries and others. Alternative methods are therefore needed to prevent tainted meat. Because of available testicular steroids, entire male pigs also have better feed conversion and carcass traits compared to barrows and this makes them more advantageous for the pig industry . Reduction of boar taint levels without castration is therefore of interest for pig breeders worldwide. Identification of genetic factors controlling boar taint may be implemented in breeding programmes to select animals that produce low levels of taint. However, selection for low boar taint generally coincides with selection for low androgen production . Before starting selection it is therefore important to understand the complex genetic system controlling boar taint and to take into account possible correlated effects on other traits in the breeding goal.
Boar taint is mainly caused by elevated levels of the compounds androstenone  and/or skatole  in adipose tissue. Androstenone (5α-androst-16-en-3-one) is a 16-androstene steroid metabolised from cholesterol through the C21 steroids pregnenolone and progesterone in boar testis . It is further reduced to its alcohols α-androstenol and β-androstenol , which also, in a lesser degree, contribute to tainted meat . Skatole (3-methylindole) is a metabolite of the amino acid tryptophan and is produced by intestinal bacteria in the gut [8, 9]. Skatole is only a problem in intact male pigs and not in gilts or barrows, and its levels increase at sexual maturity [10, 11]. Indole is another metabolite from tryptophan. It also contributes to boar taint levels, although to less extent than androstenone and skatole . Both androstenone and skatole are degraded in the liver and a relationship between their metabolism has been found .
Pregnenolone and progesterone are not only precursors of androstenone, but also of testosterone and estrogens . The influence of other sex steroids on levels of androstenone and skatole has been studied with ambiguous results. Most studies have found levels of testosterone in plasma not to be correlated to levels of androstenone in adipose tissue [13, 15, 16] or levels of skatole in adipose tissue [13, 15, 17, 18]. In some studies, however, levels of androstenone in adipose tissue [18, 19] have been found to be correlated (0.26 – 0.64) to levels of testosterone. Results for estrogens are more consistent, showing positive correlations to levels of both androstenone (0.42 – 0.93) [13, 15, 16, 18–20] and skatole (0.29 – 0.53) [13, 15, 17, 20] in adipose tissue. No correlation between levels of skatole and estrone sulphate was, however, found in one study . The correlations between levels of androstenone in plasma and adipose tissue diverge from high (0.46–0.94) [18, 19, 21–23] to not significant [24, 25]. Levels of skatole and indole in adipose tissue are shown to be highly correlated (0.46–0.75) [26, 10]. Studies on correlations between levels of androstenone and skatole in adipose tissue show inconsistent results, from medium correlations around 0.3 [18, 27, 28] to higher correlations between 0.45 and 0.68 [15, 23]. Levels of androstenone in plasma has been found correlated (0.44)  and not correlated [18, 23] to levels of skatole, while levels of skatole in plasma has been shown correlated (0.76)  and not correlated  to levels of androstenone in adipose tissue. Diverging results might be explained by breed effects, which significantly affect levels of boar taint, e.g. . Moreover, levels of androstenone, testosterone and estrogens in plasma can also be affected by diurnal variations during the day, e.g. [30, 25, 31].
Detection of single nucleotide polymorphisms (SNPs) associated with boar taint compounds may be applied in practical breeding to reduce boar taint in intact boars. However, before implementation, it is of importance to examine associations of these SNPs with phenotypes related to reproduction because of possible unfavourable correlations. Breed specific variations has been observed for both androstenone  and skatole  and it is therefore of interest to include different breeds in such an examination. The objective of this study was to genotype candidate gene SNPs in Duroc and Norwegian Landrace boars and to study associations with traits related to boar taint and reproduction. The phenotypic traits included were levels of androstenone, skatole and indole in adipose tissue, levels androstenone, testosterone, 17β-estradiol and estrone sulphate in plasma, and the length of bulbo urethralis gland. Estrogens and the length of bulbo urethralis gland are also indicators of sexual maturity, e.g. [32, 17, 33]. Moreover, haplotype analyses were carried out in genes with several SNPs.
A total of 1102 Duroc and 1726 Norwegian Landrace boars were included in this study. The boars were the sons of 81 Duroc and 90 Norwegian Landrace sires. The boars were raised at NORSVIN's boar test stations until 100 kg live weight, on average 156 and 143 days for Duroc and Norwegian Landrace, respectively, and slaughtered on average 15 days later. The days between 100 kg live weight and slaughter are due to the boar selection process, where some animals wait for their destiny as elite boars. Blood samples were taken three days before slaughter for plasma suspension and DNA extraction. Samples from subcutaneous fat were taken at the slaughter line. All the samples were stored at -20°C until chemical analyses or DNA extraction was performed. The length of bulbo urethralis gland was measured at the slaughter line.
Levels of androstenone, skatole and indole in adipose tissue and plasma were analysed at the hormone laboratory at the Norwegian School of Veterinary Sciences (NVH). Levels of androstenone were analysed by a modified time-resolved fluoroimmunoassay , using an antibody produced at NVH , whereas levels of skatole and indole in adipose tissue were analysed using high performance liquid chromatography . Levels of testosterone, 17β-estradiol and estrone sulphate in plasma were analysed at the hormone laboratory at Aker University Hospital. Plasma levels of testosterone were measured by a radioimmunoassay (Orion Diagnostica, Espoo, Finland). The intra- and total assay coefficients of variation (CVs) were 7% and 9%, respectively. Plasma levels of estradiol were measured by a fluoroimmunoassay (Perkin Elmer, Turku, Finland). The intra- and total assay coefficients of variation (CVs) were 3% and 7%, respectively. Plasma levels of estrone sulphate were measured by a radioimmunoassay (Diagnostic System Laboratories, Inc., Webster, TX, USA). The intra- and total assay coefficients of variation (CVs) were 5% and 7%, respectively.
DNA was isolated from leukocytes using the automated DNA extractor Bio Robot M48 from Qiagen (CA, USA) and the supplementary MagAttract DNA Blood Midi M48 protocol. Concentrations were measured on a 1420 Victor plate reader (Turku, Finland) using PicoGreen fluorescence (Molecular Probes, OR, USA) or on a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, DE, USA). Normalisation of DNA samples was done using the Biomek FX robot from Beckman (Beckman Coulter, CA, USA).
SNP discovery and validation
On the basis of their known or putative role in boar taint, candidate genes were chosen based on literature studies and on results from our previously published microarray results [36, 37]. SNP discovery was performed by PCR resequencing of genomic DNA and cDNA from Duroc and Norwegian Landrace boars. Primers were designed using Primer3  or Oligo primer analysis software v.6 (Molecular Biology Insights, Inc., CO, USA). The programmes Phred, Phrap and PolyPhred (v.4.06) were used to identify putative SNPs from the PCR resequencing chromatograms [39, 40] and the Consed programme was used to visually confirm the putative SNPs . Additionally, SNPs were provided from alignment of EST sequences produced in the Sino-Danish sequencing project . The genotyping was done in a two-step approach. Before high throughput genotyping, all the SNPs (a total of 275 SNPs in 121 genes) were validated on 380 animals from each breed using the genotyping procedure described below (see additional file 1: Genotyped SNPs). The SNPs that were significant in at least one of the phenotypes using a non-stringent significance level (p < 0.1) were used for genotyping of all the boars. Of the selected SNPs, 135 were in successful assays (see additional file 1: Genotyped SNPs).
SNPs were genotyped using matrix-assisted laser desorption/ionisation time-of-flight mass spectroscopy (MALDI-TOF MS) assays. Multiplex assays for use in the Sequenom MassARRAY system were designed using MassARRAY Assay Design software (Sequenom, San Diego, USA) at multiplexing levels between 7 and 35. Primers for the genotyping can be found in additional file 2: Primer sequences for genotyping. Genotyping was done by the IPLEX protocol using manufacturer's instructions (Sequenom, San Diego, USA) . The MassARRAY Typer software was used for automated genotype calling.
Genes with more than one SNP were used for haplotype analyses if one of the SNPs was significant for one of the phenotypes. Haplotype construction and frequency estimation were done using two programmes in combination. CRIMAP v2.4  used pedigree information while PHASE v2.1.1 [45, 46] used linkage disequilibrium information to determine haplotypes.
Statistical analyses were performed for the two breeds Duroc and Norwegian Landrace separately. Association studies were done using the likelihood ratio test  by average information restricted maximum likelihood (AI-REML)  combined with expectation maximisation (EM-REML) if an update goes outside parameter space . The procedure is part of the package DMU, v.6 release 4.7 . The fixed effects fitted were sire, herd-year-season, waiting in boar test station before slaughter or not, and pen. By having sire as a fixed effect we make sure that the sire family effect does not affect the SNP estimate. Covariates used were age at 25 kg (start of boar test), days from 25 kg to 100 kg (days in boar test), days from 100 kg to slaughter (days in waiting station) and number of live born in same litter. Number of live born was also included as a squared term as this was shown to have a significant effect in the model. Animal ID, sample date for adipose tissue or plasma, and SNP-genotype or haplotype-genotype were fitted as random effects. The model used was as follows
where trait is bulbo urethralis gland length (cm), ln(ppm levels of androstenone in adipose tissue), ln(ppm levels of skatole in adipose tissue), ln(ppm levels of indole in adipose tissue), ln(ppm levels of androstenone in plasma), ln(ppm levels of testosterone in plasma), ln(ppm levels of estrone sulphate in plasma) or ln(ppm levels of 17β-estradiol in plasma). The linear model used assumes normality. The distributions were, however, skewed for all the chemical compounds in the study and the data was log-transformed to reduce this problem.
In the haplotype analyses, SNP was replaced by haplotype. SNPs with a genotyping success rate of less than 90% were excluded from further analyses and missing data was considered as a separate class in the random effect of SNP or haplotype. For each trait the model was run without SNPs or haplotypes for log likelihood comparison. A log likelihood ratio (LRT) exceeding 5.4 units, corresponding p < 0.001, was considered significant (assuming that 2*LRT is approximately chi-squared distributed with one degree of freedom).
A total of 135 SNPs from 57 candidate genes for boar taint were included in this study. The SNPs were distributed in exons, introns and untranslated regions of the genes and were genotyped in the two breeds Duroc (D) and Norwegian Landrace (NL). Out of these, 9 and 4 SNPs were monomorphic in D and NL, respectively (Table 1). SNPs with an estimated minor allele frequency (MAF) of less than 1% were excluded from further analyses. For the D breed, 19 SNPs were excluded due to low MAF while for NL this number was 14 (Table 1). The resulting SNPs with a genotyping success rate of more than 90% were used for association studies. A summary of these SNPs and their genes, alleles and frequencies are presented for D in Table 2 and for NL in Table 3. Descriptive statistics for the different phenotypes used in association analyses are presented for D in Table 4 and for NL in Table 5. Significant effects of SNPs on these phenotypes are shown in Table 6.
In Duroc, 4 SNPs were associated with levels of androstenone in adipose tissue and they were found to explain 5.1% (NGFIB_in4), 5.6% (CYP2D6_1276(ex7)), 12.3% (CYP2C49_1083(ex7)) and 16.3% (CTNND1_3'UTRa) of the total variation. In NL, none of the SNPs examined in this study was significantly associated with levels of androstenone in adipose tissue, but 4 SNPs were significantly associated with levels of androstenone in plasma. These SNPs explain 1.3% (BAP1_3'UTRb), 1.6% (HYAL2_583(ex1)), 2% (BAP1_3'UTRa) and 2.1% (SRD5A2_3'UTRd) of the total variation. Levels of skatole in adipose tissue were significantly associated with 5 SNPs within two different genes in D and 3 SNPs from one gene in NL. In D, SNPs within CYP21 explained 12.4% and 13.4% of the total variation while SNPs within CYP2E1 explained between 2.5% and 3.1% of the total variation. CYP2E1 SNPs in NL explained between 2.1% and 3% of the total skatole variation. Association results for levels of indole in adipose tissue show that 7 SNPs from two genes (CYP21 and CYP2E1) explain between 6.3% and 7.5% of the total variation in D and that 5 SNPs from CYP2E1 explain between 3.1% and 4.9% of the total variation in NL. Some SNPs were also associated with levels of other steroids but none of them were associated with the length of bulbo urethral gland (Table 6).
Haplotypes were constructed for genes with more than one SNP and an additional association analysis was performed on the phenotypes found to be significant in the single SNP analysis. This resulted in 5 significant genes for D (Table 7) and 5 for NL (Table 8). Only haplotypes with frequencies of more than 1% were included in the statistical analyses. Significant haplotype effects on the phenotypes investigated are shown in Table 9. The haplotype analyses of CYP2E1 were significant in both breeds for levels of skatole and indole and analyses suggest that as much as 12% of the total variation in indole and 6% of the total variation in skatole can be explained by this gene in D. In NL, 9.5% and 4.6% of the total variation in indole and skatole, respectively, was explained by CYP2E1 haplotypes. The effect of different CYP2E1 haplotypes is presented in Figure 1a for D and in Figure 1b for NL. Significant effects were also found for BAP1, HYAL2 and SRD5A2 and levels of androstenone in Norwegian Landrace and these haplotypes are presented in Figure 1c, d and 1e, respectively.
Unfavourable correlations between boar taint compounds and phenotypes related to reproduction make selection against high levels of boar taint challenging. Analysing the effect of candidate genes on androstenone, skatole and sex steroids will give new information about which genes that affect different compounds. It may also reveal polymorphisms that can be used for breeding against high levels of boar taint without simultaneously affecting fertility and reproduction in the pigs. In this study we report a larger set of SNPs in selected genes involved in biosynthesis and metabolism of androstenone and skatole, in addition to regulatory factors likely involved in boar taint. The SNPs were genotyped in boars from Duroc (D) and Norwegian Landrace (NL) and tested for associations to 8 different phenotypes related to boar taint and reproduction.
One of the key enzymes in the metabolism of skatole is cytochrome P450 family member CYP2E1 [15, 50]. A total of 7 metabolites have been identified for skatole  and 5 of them were produced at decreased levels when a CYP2E1 inhibitor was present . Moreover, levels of CYP2E1 mRNA , protein [15, 53], and enzyme activity  have been found negatively associated with levels of skatole in adipose tissue of boars. It has also been shown that CYP2E1 is induced by skatole  and inhibited by androstenone and 17β-estradiol . In our study we found no association of CYP2E1 and androstenone. This is in accordance with the results of Zamaratskaia et al. . We did, however, find significant associations between SNPs and haplotypes within CYP2E1 and levels of skatole and indole in both breeds. These results are in agreement with associations previously found between CYP2E1_1423(ex9) and levels of skatole , although in another study there was no significant associations detected in Large White × Meishan crossbreds . Both SNP and haplotype analyses showed that the extent of variation in skatole and indole explained by CYP2E1 is higher in D than NL. Breed specific differences for CYP2E1 is in agreement with Doran et al. . In both breeds we identified a haplotype that was associated with reduced levels of both skatole and indole, and this haplotype contains the favourable alleles from the SNP analysis.
Cytochrome P450 family member CYP21 is involved in the steroid biosynthesis pathway . It has previously been suggested as a positional candidate gene for androstenone . We identified 7 SNPs in the introns of this gene. None of these SNPs were found to be associated with levels of any of the steroids included in this study. On the contrary, 3 SNPs were significantly associated with levels of skatole and indole in D, but not in NL. Two haplotypes containing all the favourable alleles have an advantageous effect on levels of indole and skatole in D. This effect was, however, not significant (LRT of 3.4 (p < 0.01) for indole and 1.6 (p < 0.1) for skatole). The role of CYP21 regarding to levels of skatole and indole needs to be clarified.
Cytochrome P450 porcine isoform CYP2C49 is a member of the subfamily CYP2C, which metabolise drugs and steroids . We previously identified this isoform as the most significant gene in a gene expression study including pigs with extreme high and low levels of androstenone  and in the current study we identified 4 SNPs in the CYP2C49 gene. A significant association was shown between CYP2C49_1083(ex7) and levels of androstenone in adipose tissue in D, and this SNP explain 12.3% of the total variation. This result suggests that the level of gene expression is regulated within the gene. The SNP was not significantly associated with any of the other phenotypes in the study. A microsatellite linked to CYP2C18, which is the human ortholog of CYP2C49, has previously been tested for association with levels of skatole in adipose tissue, in accordance with our results, no significant results were obtained .
Cytochrome P450 member CYP2D6 is involved in drug metabolism and has a broad substrate specificity . Some studies have also suggested that CYP2D6 is regulated by steroid hormones [65, 66]. In this study we found significant association between the SNP CYP2D6_1276(ex7) and level of androstenone in adipose tissue of D boars. SNPs in CYP2D6 showed no significant associations to levels of skatole or indole. This result is supported by the study of Diaz and Squires , showing that inhibition of CYP2D6 do not to affect production of skatole metabolites. Moreover, we found no associations with levels of testosterone or estrogens.
Catenin delta, CTNND1, is part of the catenin family that functions in intracellular signalling and transcriptional regulation  and phosporylation of CTNND1 leads to regulation of several transcription factors . A SNP in CTNND1 was significantly associated with levels of androstenone in adipose tissue of D boars. No associations were found for any of the other phenotypes analysed, which makes CTNND1 interesting for selection against high levels of androstenone in D. Such regulators, however, will likely affect several other biological processes and this need to be considered before implementation in the selection scheme.
Orphan nuclear receptor family member NGFI-B, also known as Nurr77, is involved in transcriptional regulation of several steroidogenic genes, including steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 c17 (CYP17), and CYP21 [68, 69]. A SNP in NGFI-B was significantly associated with levels of androstenone in adipose tissue of D boars. It was not associated with any of the other phenotypes examined and is therefore also interesting as a possible genetic marker for selection against androstenone.
Short-chain dehydrogenase/reductase family members SRD5A1 and SRD5A2 are enzymes that catabolise a number of steroids into their 5α-reduced metabolites . SRD5A2 also catalyses the final step of androstenone formation . No relationship was found between genetic variation of SRD5A2 and androstenone in a Large White × Meishan crossbreed . In our study, however, 6 SNPs were detected in the 3' UTR region of porcine SRD5A2 and the SNP SRD5A2_3'UTRd was significantly associated with levels of androstenone and estrone sulphate in plasma of NL boars. The haplotype analyses confirmed these associations. The haplotype associated with reduced levels of androstenone, however, was also associated with reduced levels of estrone sulphate, which makes it less desirable for selection purposes. Aldo-keto reductase AKR1C isoforms work together with SRD5As in liver catabolism of steroids  and we previously found the isoform AKR1C4 differentially expressed regarding to levels of androstenone in pig testes . SNPs in isoform AKR1C3 were associated with levels of both estrone sulphate and estradiol in D boars, but no association with levels of androstenone suggest different functions for the two isoforms AKR1C3 and AKR1C4.
The breast/ovarian cancer susceptibility associated protein-1 (BAP1) has been implied to alter substrate function through post-translational modifications . It has the same structure and functions as the ubiquitin carboxy-terminal hydrolase (UCH) family, which are involved in ubiquitin-mediated regulatory pathways . Two SNPs in BAP1 were significantly associated with levels of androstenone in plasma of NL boars. Moreover, a BAP1 haplotye was associated with decreased levels of androstenone. One of the SNPs in BAP1 was, however, also associated with levels of testosterone in plasma in the same breed. The favourable allele with respect to androstenone was also associated with reduced levels of testosterone, which suggest that this SNP is not appropriate for selection purposes.
Hyaluronoglucosaminidases are enzymes responsible for hyaluronan metabolism. It has been shown that hyaluronan metabolic products can induce expression of heat-shock proteins , which are known to activate for example steroid hormone receptors . Metabolism of hyaluronan has also been shown to change during testicular development and is associated with testicular descent . We have identified SNPs in HYAL1, HYAL2 and HYAL3. Among these a SNP in HAYL2 was associated with levels of androstenone in plasma of NL boars, and a haplotype with negative effect on the levels of androstenone was also identified. This HYAL2 SNP and the haplotype were also associated with levels of testosterone in NL. The favourable haplotype for levels of androstenone is also associated with reduced levels of testosterone, implying that it is not so suitable for breeding purposes.
Except from the associations found for SNPs in CYP2E1 with levels of skatole and indole in adipose tissue, our results show breed differences with respect to significantly associated SNPs. The breed differences are in agreement with results from our previous expression profiling for D and NL breeds [36, 37, 77]. Different associations suggest that D and NL have different linkage disequilibrium (LD) with the causative mutation. It is therefore likely that there is some distance between the SNP and the causative mutation. Moreover, fixation or nearly fixation was observed for many candidate gene SNPs and different SNPs were monomorphic in D and NL. It emphasises the importance of testing polymorphisms in the population in question before using them for breeding purposes. When including all the significant SNPs for a trait in the same model there were some problems of convergence, which might indicate that the SNP effects are correlated and partly explain the same variation. Although a rather strict significance threshold corresponding to p < 0.001 was used in this study, the large number of tests performed suggests that there might still be false positive results and the findings should be confirmed in another study. Moreover, SNPs that are significant for boar taint compounds and not for phenotypes related to reproduction might still have a small effect on reproduction even though this study was not able to detect it.
This study reports significant associations between SNPs within CYP21 and CYP2E1 and reduced levels of both skatole and indole. Moreover, a number of SNPs within CYP2C49, CYP2D6, NGFIB and CTNND1 were found significantly associated with levels of androstenone in adipose tissue. These SNPs did not reveal obvious associations with levels of testosterone or estrogens, which might indicate that they can be implemented in practical breeding to reduce levels of androstenone and skatole without causing simultaneous negative consequences on phenotypes related to reproduction.
aldo-keto reductase family member 1C3
aldo-keto reductase family member 1C4
BRCA1 associated protein
cytochrome P450 family member 17
cytochrome P450 family member 21
cytochrome P450 family member 2C49
cytochrome P450 family member 2D6
cytochrome P450 family member 2E1
short-chain dehydrogenase/reductase member 8
ferritin heavy polypeptide
minor allele frequency
orphan nuclear receptor member NGFI-B
single nucleotide polymorphism
steroidogenic acute regulatory protein
ubiquitin carboxy-terminal hydrolase.
Babol J, Squires EJ: Quality of meat from entire male pigs. Food research international. 1995, 28: 201-212. 10.1016/0963-9969(95)93528-3.
Willeke H, Claus R, Müller E, Pirchner F, Karg H: Selection for high and low level of 5α-androst-16-en-3-one in boars I. Direct and correlated response of endocrinological traits. Journal of animal breeding and genetics. 1986, 104: 64-73.
Patterson RLS: 5alpha-androst-16-ene-3-one: – Compound responsible for taint in boar fat. Journal of the science of food and agriculture. 1968, 19: 3-10.1002/jsfa.2740191301.
Vold E: Fleischproduktionseigenschaften bei ebern und kastraten. Meldinger fra Norges Landbrukshøgskole. 1970, 49: 1-25.
Ahmad N, Gower DB: The biosynthesis of som e androst-16-enes from C21 and C19 steroids in boar testicular and adrenal tissue. Biochemical journal. 1968, 108: 233-241.
Brophy PJ, Gower DB: 16-unsaturated C19 3-oxo steroids as metabolic intermediates in boar testis. Biochemical journal. 1972, 128: 945-952.
Thompson RH, Pearson AM, Banks KA: Identification of some C19 – Δ16 steroids contributing to sex odor in pork. Journal of agricultural food chemistry. 1972, 20: 185-189. 10.1021/jf60180a020.
Yokoyama MT, Carlson JR: Dissimilation of tryptophan and related indolic compounds by ruminal microorganisms in vitro. Applied microbiology. 1974, 27: 540-548.
Jensen MT, Cox RP, Jensen BB: 3-methylindole (skatole) and indole production by mixed populations of pig faecal bacteria. Appl Environ Microbiol. 1995, 61 (8): 3180-4.
Babol J, Zamaratskaia G, Juneja RK, Lundström K: The effect of age on distribution of skatole and indole levels in entire male pigs in four breeds: Yorkshire, Landrace, Hampshire and Duroc. Meat science. 2004, 67: 351-358. 10.1016/j.meatsci.2003.11.008.
Zamaratskaia G, Babol J, Madej A, Squires EJ, Lundström K: Age-related variation of plasma concentrations of skatole, androstenone, testosterone, oestradiol-17beta, oestrone sulphate, dehydroepiandrostenone sulphate, triiodothyronine and IGF-1 in six entire male pigs. Reproduction domestic animals. 2004, 39: 168-172. 10.1111/j.1439-0531.2004.00496.x.
Hansson K-E, Lundstrom K, Fjelkner-Modig S, Persson J: The importance of androstenone and skatole for boar taint. Swedish Journal of Agricultural Research. 1980, 10: 167-173.
Babol J, Squires EJ, Lundstrom K: Relationship between metabolism of androstenone and skatole in intact male pigs. Journal of animal science. 1999, 77: 84-92.
Brooks RI, Pearson AM: Steroid hormone pathways in the pig, with special emphasis on boar odor: a review. Journal of animal science. 1986, 62: 632-645.
Squires EJ, Lundstrom K: Relationship between cytochrome P450IIE1 in liver and levels of skatole and its metabolites in intact male pigs. J Anim Sci. 1997, 75: 2506-2511.
Squires EJ, Gullett EA, Fisher KRS, Partlow GD: Comparison of androst-16-ene steroid levels determined by a colorimetric assay with boar taint estimated by a trained sensory panel. Journal of animal science. 1991, 69: 1092-1100.
Zamaratskaia G, Rydhmer L, Chen G, Madej A, Andersson HK, Lundstrom K: Boar taint is related to endocrine and anatomical changes at puberty but not to aggressive behaviour in entire male pigs. Reproduction in domestic animals. 2005, 40: 500-506. 10.1111/j.1439-0531.2005.00613.x.
Chen G, Zamaratskaia G, Madej A, Lundström K: Effect of hCG administration on the relationship between testicular steroids and indolic compounds in fat and plasma in entire male pigs. Meat science. 2006, 72: 339-347. 10.1016/j.meatsci.2005.08.004.
Sinclair PA, Squires EJ, Raeside JI: Early postnatal plasma concentrations of testicular steroid hormones, pubertal development, and carcass leanness as potential indicators of taint in market weight intact male pigs. Journal of animal science. 2001, 79: 1868-1876.
Zamaratskaia G, Madej A, Babol J, Squires E, Lundstrom K: Free oestrone in adipose tissue and its relation to androstenone and skatole in entire male pigs. Reprod Domest Anim. 2005, 40: 156-160. 10.1111/j.1439-0531.2005.00573.x.
Andresen Ø: Concentrations of fat and plasma 5-alpha-androstenone and plasma testosterone in boars selected for rate of body weight gain and thickness of back fat during growth, sexual maturation and after mating. Journal of Reproduction and Fertility. 1976, 48: 51-59.
Booth WD, Williamson ED, Patterson RLS: 16-androstene steroids in the submaxillary salivary gland of the boar in relation to measures of boar taint in carcasses. Animal production. 1986, 42: 145-152.
Zamaratskaia G, Babol J, Andersson H, Lundström K: Plasma skatole and androstenone levels in entire male pigs and relationship between boar taint compounds, sex steroids and thyroxine at various ages. Livestock production science. 2004, 87: 91-98. 10.1016/j.livprodsci.2003.09.022.
Malmfors B, Andresen Ø: Relationship between boar taint intensity and concentratino of 5α-androst-16-en-3-one in boar peripheral plasma and back fat. Acta Agriculturæ Scandinavica. 1975, 25: 92-96.
Lundstrom K, Malmfors B, Hansson I, Edqvist L-E, Gahne B: 5α-androstenone and testosterone in boars. Early testing with HCG, sexual stimulation and diurnal variation. Swedish Journal of Agricultural Research. 1978, 8: 171-180.
Tuomola M, Vahva M, Kallio H: High-Performance liquid chromatography determination of skatole and indole levels in pig serum, subcutaneous fat and submaxillary salivary glands. Journal of agricultur food chemistry. 1996, 44: 1265-1270. 10.1021/jf950796z.
Lundström K, Malmfors B, Malmfors G, Stern S, Petersson H, Mortensen AB, et al: Skatole, androstenone and taint in boars fed two different diets. Livestock production science. 1988, 18: 55-67. 10.1016/0301-6226(88)90067-X.
Walstra P, Claudi-Magnussen C, Chevillon P, von Seth G, Diestre A, Matthews KR, et al: An international study on the importance of androstenone and skatole for boar taint: levels of androstenone and skatole by country and season. Livestock production science. 1999, 62: 15-28. 10.1016/S0301-6226(99)00054-8.
Xue J, Dial GD, Holton EE, Vickers Z, Squires EJ, Lou Y, et al: Breed differences in boar taint: relationship between tissue levels of boar taint compounds and sensory analysis of taint. Journal of animal science. 1996, 74: 2170-2177.
Claus R, Gimènez T: Diurnal rhythm of 5α-androst-16-en-3-one and testosterone in peripheral plasma of boars. Acta Endocrinologica. 1977, 84: 200-206.
Claus R, Hoffman B: Oestrogens, compared to other steroids of testicular origin, in bloodplasma of boars. Acta Endocrinologica. 1980, 94: 404-411.
Andersson HK, Hullberg A, Malmgren L, Lundström K, Rydhmer L, Squires EJ: Sexual maturity in entire male pigs – Environmental effects, relations to skatole level and female puberty. Acta Agriculturæ Scandinavica Section A Animal Science. 1999, 49: 103-112. 10.1080/090647099424169.
Fouilloux MN, LeRoy P, Gruand J, Renard C, Sellier P, Bonneau M: Support for single major genes influencing fat androstenone level and development of bulbo-urethral glands in young boars. genetics selection evolution. 1997, 29: 357-366. 10.1051/gse:19970304.
Tuomola M, Harpio R, Knuuttila P, Mikola H, Løvgren T: Time-resolved fluoroimmunoassay for the measurement of androstenone in porcine serum and fat samples. Journal of agricultural food chemistry. 1997, 45: 3529-3534. 10.1021/jf9702398.
Andresen Ø: Development of radioimunoassay for 5alpha-adrost-16-en-3-one in pig peripheral plasma. Acta endochrinologia. 1974, 76: 377-387.
Moe M, Meuwissen T, Lien S, Bendixen C, Wang X, Conley LN, et al: Gene expression profiles in testis of pigs with extreme high and low levels of androstenone. BMC Genomics. 2007, 8: 405-10.1186/1471-2164-8-405.
Moe M, Lien S, Bendixen C, Hedegaard J, Hornshøj H, Berget I, et al: Gene expression profiles in liver of pigs with extreme high and low levels of androstenone. BMC Veterinary Research. 2008, 4: 29-10.1186/1746-6148-4-29.
Rozen S, Skaletsky HJ: Primer3 on the WWW for general users and biologist programmers. Bioinformatic methods and protocols: Methods in molecular biology. Edited by: Krawetz S, Misener S. 2000, Totowa, NJ: Humana Press, 365-386.
Phred, Phrap, Consed. 2008, [http://www.phrap.org/phredphrapconsed.html]
PolyPhred. 2008, [http://droog.mbt.washington.edu/PolyPhred.html]
Gordon D, Abajian C, Green P: Consed: A graphical tool for sequence finishing. Genome Research. 1998, 8: 195-202.
Gorodkin J, Cirera S, Hedegaard J, Gilchrist MJ, Panitz F, Jørgensen C, et al: Porcine transcriptome analysis based on 97 non-normalized cDNA libraries and assembly of 1,021,891 expressed sequence tags. Genome biology. 2007, 8: R45-10.1186/gb-2007-8-4-r45.
Sequenom. 2008, [http://www.sequenom.com/]
Green P, Falls K, Crooks S: Documentation for CRIMAP, version 2.4. 1990, Washington University School of Medicine, St. Louis, MO, USA
Stephens M, Smith NJ, Donnelly P: A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics. 2001, 68: 978-989. 10.1086/319501.
Stephens M, Scheet P: Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. American Journal of Human Genetics. 2005, 76: 449-462. 10.1086/428594.
Madsen P, Jensen J: A user's guide to DMU. A package for analysing multivariate mixed models. Version 6, release 4.7. 2008, University of Aarhus, Faculty of Agricultural Sciences (DJF), Dept of Genetics and Biotechnology, Research Centre Foulum, Tjele, Denmark, 1-33.
Jensen J, Mantysaari EA, Madsen P, Thompson R: Residual Maximum Likelihood Estimation of (Co) Variance Components in Multivariate Mixed Linear Models using Average Information. Journal of Indian Society of Agricultural Statistics. 1997, 49: 215-236.
Dempster AP, Laird NM, Rubin DB: Maximum likelihood from incomplete data via the EM algorithm. Journal Of The Royal Statistical Society Series B. 1977, 39: 1-38.
Babol J, Squires E, Lundstrom K: Hepatic metabolism of skatole in pigs by cytochrome P4502E1. Journal of animal science. 1998, 76: 822-828.
Diaz GJ, Skordos KW, Yost GS, Squires EJ: Identification of phase I metabolites of 3-methylindole produced by pig liver microsomes. Drug Metab Dispos. 1999 Oct;27(10):1150-6. 1999, 27 (10): 1150-6.
Diaz GJ, Squires EJ: Metabolism of 3-methylindole by porcine liver microsomes: responsible cytochrome P450 enzymes. Toxicology science. 2000, 55: 284-292. 10.1093/toxsci/55.2.284.
Doran E, Whittington FM, Wood JD, McGivan JD: The relationship between adipose tissue skatole levels, rates of hepatic microsomal skatole metabolism and hepatic cytochrome P450IIE1 expression in two breeds of pig. Animal science. 2002, 74: 461-468.
Zamaratskaia G, Squires EJ, Babol J, Andersson HK, Andersson K, Lundström K: Relationship between the activities of cytochrome P4502E1 and P4502A6 and skatole content in fat in entire male pigs fed with and without raw potato starch. Livestock production science. 2005, 95: 83-88. 10.1016/j.livprodsci.2004.12.012.
Doran E, Whittington FM, Wood JD, McGivan JD: Cytochrome P450IIE1 (CYP2E1) is induced by skatole and this induction is blocked by androstenone in isolated pig hepatocytes. Chemico-biological interactions. 2002, 140: 81-92. 10.1016/S0009-2797(02)00015-7.
Zamaratskaia G, Gilmore WJ, Lundstrom K, Squires EJ: Effect of testicular steroids on catalytic activities of cytochrome P450 enzymes in porcine liver microsomes. Food and Chemical Toxicology. 2007, 45: 676-681. 10.1016/j.fct.2006.10.023.
Zamaratskaia G, Chen G, Lundström K: Effects of sex, weight, diet and hCG administration on levels of skatole and indole in the liver and hepatic activities of cytochromes P4502E1 and P4502A6 in pigs. Meat science. 2006, 72: 331-338. 10.1016/j.meatsci.2005.07.020.
Lin Z, Lou Y, Squires EJ: Functional ploymorphism in porcine CYP2E1 gene: Its association with skatole levels. Journal of steroid biochemistry and molecular biology. 2006, 99: 231-237. 10.1016/j.jsbmb.2005.07.001.
Skinner TM, Doran E, McGivan JD, Haley CS, Archibald AL: Cloning and mapping of the porcine cytochrome-p450 2E1 gene and its association with skatole levels in the domestic pig. Animal genetics. 2005, 36: 417-422. 10.1111/j.1365-2052.2005.01342.x.
Payne AH, Hales DB: Overview of steroidogenic enzymes in the pathway from cholesterol to active steroid hormones. Endocrine Reviews. 2004, 25: 947-970. 10.1210/er.2003-0030.
Quintanilla R, Demeure O, Bidanel JP, Milan D, Iannuccelli N, Amigues Y, et al: Detection of quantitative trait loci for fat androstenone levels in pigs. J Anim Sci. 2003, 81: 385-394.
Skaanild MT, Friis Ch: Porcine CYP2A polymorphisms and activity. Basic and Clinical Pharmacology and Toxicology. 2005, 97: 115-121. 10.1111/j.1742-7843.2005.pto_148.x.
Skinner TM, Anderson JA, Haley CS, Archibald AL: Assessment of SULT1A1, CYP2A6 and CYP2C18 as candidate genes for elevated backfat skatole levels in commercial and experimental pig populations. Animal genetics. 2006, 37: 521-522. 10.1111/j.1365-2052.2006.01502.x.
Skaanild MT: Porcine cytochrome P450 and metabolism. Current Pharmaceutical Design. 2006, 12: 1421-1427. 10.2174/138161206776361183.
Baum LO, Strobel HW: Regulation of expression of cytochrome P-450 2D mRNA in rat brain with steroid hormones. Brain Research. 1997, 765: 67-73. 10.1016/S0006-8993(97)00428-9.
Wadelius M, Darj E, Frenne G, Rane A: Induction of CYP2D6 in pregnancy. Clin Pharmacol Ther. 1997, 62 (4): 400-7. 10.1016/S0009-9236(97)90118-1.
McCrea PD, Park J: Developmental functions of the P120-catenin sub-family. Biochimica et Biophysica Acta. 2007, 1773: 17-33.
Hong CY, Park JH, Ahn RS, Im SY, Choi H-S, Soh J, et al: Molecular mechanism of suppression of testicular steroidogenesis by proinflammatory cytokine tumor necrosis factor alpha. Mol Cell Biol. 2004, 24 (7): 2593-604. 10.1128/MCB.24.7.2593-2604.2004.
Wilson TE, Mouw AR, Weaver CA, Milbrandt J, Parker KL: The orphan nuclear receptor NGFI-B regulates expression of the gene encoding steroid 21-hydroxylase. Mol Cell Biol. 1993, 13 (2): 861-8.
Wilson JD, Leihy MW, Shaw G, Renfree MB: Androgen physiology: unsolved problems at the millennium. Mol Cell Endocrinol. 2002, 198 (1-2): 1-5. 10.1016/S0303-7207(02)00362-3.
Robic A, Larzul C, Bonneau M: Genetic and metabolic aspects of androstenone and skatole deposition in pig adipose tissue: a review. Genet Sel Evol. 2008, 40 (1): 129-43.
Bauman DR, Steckelbroeck S, Penning TM: The roles of aldo-keto reductases in steroid hormone action. Drug News Perspect. 2004, 17: 563-578. 10.1358/dnp.2004.17.9.872570.
Jensen DE, Rauscher III FJ: Defining biochemical functions for the BRCA1 tumor suppressor protein: analysis of the BRCA1 binding protein BAP1. Cancer Letters. 1999, 143: S13-S17. 10.1016/S0304-3835(99)90004-6.
Stern R: Hyaluronan catabolism: a new metabolic pathway. European Journal of Cell Biology. 2004, 83: 317-325. 10.1078/0171-9335-00392.
Picard D: Chaperoning steroid hormone action. Trends Endocrinol Metab. 2006, 17 (6): 229-35. 10.1016/j.tem.2006.06.003.
Fentener van Vlissingen JM, Koch CAM, Delpech B, Wensing CJG: Growth and differentiation of the gubernaculum testis during testicular descent in the pig: Changes in the extracellular matrix, DNA content, and hyaluronidase, β-glucuronidase, and β-N-acetylglucosaminidase activities. The Journal of Urology. 1989, 142: 837-845.
Moe M, Grindflek E, Doran O: Expression of 3β-hydroxysteroid dehydrogenase, cytochrome P450c17 and sulfotransferase 2B1 proteins in liver and testis of pigs of two breeds: relationship with adipose tissue androstenone level. Journal of animal science. 2007, 85: 2924-2931. 10.2527/jas.2007-0283.
This study was financed by the Norwegian pig breeders association (NORSVIN) and The Research Council of Norway. We want to thank Ellen Dahl and Øystein Andresen for being in charge of analyses done at the hormone laboratory at the Norwegian School of Veterinary Science (NVH) and Peter A. Torjesen for being in charge of the analyses done at the hormone laboratory at Aker University Hospital. We would also like to thank Elin Bergseth at NORSVIN/BioBank AS for collection and handling of samples.
MM conducted SNP molecular work, was involved in statistical analyses and drafted the paper. SL was involved in planning the project, provided laboratory facilities and took part in writing the paper. TA carried out statistical analyses. THEM was involved in statistical supervision. MHSH conducted SNP molecular work. CB provided SNPs from the Sino-Danish sequencing project. EG coordinated the study, was involved in planning the project, performed statistical analyses and took part in writing the paper. All authors have read and approved the final manuscript.
Electronic supplementary material
Additional file 1: Genotyped SNPs. Genotyped SNPs are presented with gene name and accession number. Position indicates base pairs from A TG start codon according to human reference sequence. Exon number is presented between brackets and if the SNP is in an intron, the intron number is indicated. An asterix is designated to SNP alleles causing amino acid change. Step 1+2 indicates the SNPs that were genotyped in all animals whereas step 1 indicates the SNPs that were initially genotyped in 380 animals per breed and discarded due to low significance levels (NS) or failing assays (-). (XLS 70 KB)
Additional file 2: Primer sequences for genotyping. Forward, reverse and extension primers for genotyping on the Sequenom MassARRAY system (Sequenom, San Diego, USA) are presented. The multiplexes are indicated in the first column. Multiplexes W1-W6 represents primers for the SNPs that were genotyped in all animals whereas 1 indicates primers for the SNPs that were initially genotyped in the first 760 animals and discarded. (DOC 260 KB)
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.