Accumulation Of Androstenone Research Articles

230 The relationship between hormone status and the metabolism of androstenone and skatole in isolated porcine hepatocytes

Abstract Surgical castration is a significant welfare concern within the swine industry; however, it is currently the most effective method to prevent boar taint, which is a meat quality issue that develops in entire male pigs due to the accumulation of androstenone and skatole in the fat. Metabolism of the boar taint compounds occurs within the liver and promotes their excretion, ultimately reducing the extent of their accumulation in the fat. Previous research has demonstrated that plasma concentrations of estrone sulfate (E1S), which are indicative of steroid hormone status in the boar, are highly variable between individual animals of similar live body weights at slaughter. Testicular steroids are known to influence the activity and expression of some enzymes regulating the metabolism of skatole, but the effect of hormone concentrations on the entire expression profile of enzymes responsible for boar taint metabolism has not been characterized. Therefore, the objective of this study was to relate plasma E1S concentrations at slaughter to expression levels of genes regulating the metabolism of androstenone and skatole and their respective metabolite profiles in isolated hepatocytes, to further characterize the relationship between hormone status and boar taint metabolism. Blood and liver samples were collected from 5-mo-old crossbred [(Yorkshire x Landrace) x Duroc] boars (n = 8)] at slaughter. Plasma E1S concentrations were quantified using a radioimmunoassay and gene expression in the liver was evaluated by RT-qPCR. Hepatocytes were also isolated and incubated with androstenone or skatole, with metabolite concentrations in the incubation media quantified by high-performance liquid chromatography. Statistical analysis was performed using Pearson correlation. Plasma E1S concentrations at slaughter ranged from 2.2-108.5 ng/mL and were positively correlated with overall skatole metabolism (r = 0.77, P = 0.0265), the production of the Phase I skatole metabolites 3-methyloxindole (3MOI; r = 0.80, P = 0.0183), and 3-hydroxy-3-methyloxindole (HMOI; r = 0.73, P = 0.0413), as well as expression levels of CYP2C33 (r = 0.88, P = 0.0042), CYP2C49 (r = 0.78, P = 0.0221), and CYB5R1 (r = 0.80, P = 0.0165), which regulate skatole metabolism. A strong positive relationship was also identified between overall skatole metabolism and the production of 3MOI (r = 0.96, P = 0.0001) and HMOI (r = 0.93, P = 0.001). Androstenone metabolism primarily resulted in the production of Phase II glucuronide metabolites, but the levels produced were not related to E1S concentrations or gene expression levels. Taken together, these results demonstrate that plasma E1S concentrations are related to the rate of skatole, but not androstenone, metabolism in mature boars. Additional research is required to assess the potential of E1S concentrations as a biomarker for the rate of skatole metabolism in vivo and establish thresholds that could be used to identify boars with high and low rates of metabolism. This may provide an opportunity to develop targeted treatments for boar taint based on the metabolic status of the animal.

505 Utilizing porcine hepatocyte cultures to test the effect of short chain fatty acids on androstenone metabolism

Abstract Boar taint is an off-putting odor found in the meat of entire male pigs due to the accumulation of androstenone and skatole in the fat. Development of boar taint is dependent on the balance between the rate of synthesis and metabolism of these compounds. Prior studies have shown that feed ingredients high in fermentable carbohydrates, including chicory root, sugar beet pulp, and Jerusalem artichoke decreased the synthesis of skatole in the hindgut and altered the metabolism in the liver, resulting in a skatole concentrations comparable with castrated males. However, the mechanism of how these dietary fermentable carbohydrates affect liver metabolism is not well understood. Metabolism of boar taint compounds takes place in a two-phase process, regulated by various enzymes and cytochrome families. Short-chain fatty acids (SCFAs) are produced from the microbial fermentation of fiber and have been shown to have regulatory effects on histone deacetylases and gene expression in humans, providing a potential mechanism in which feed high in dietary fiber reduces boar taint. The purpose of this study was to assess if SCFA directly affect the metabolism of androstenone in liver hepatocytes. Primary hepatocytes were isolated from the liver of 6 [(Yorkshire x Landrace) x Duroc] boars weighing around 90 kg. Cells were treated in triplicate with acetate, butyrate, propionate, and various combinations for 24 h to alter gene expression. Two sets of wells acted as a control, not being treated with SCFAs. The cells were then incubated with androstenone for 3 h, and media was extracted, and metabolite production was analyzed using high-performance liquid chromatography. The effects of treatments were using a one-way ANOVA with a Dunnet’s adjustment for multiple comparisons to a control. Results showed the combined acetate, butyrate, and propionate treatment had the greatest effect on androstenone glucuronide production when compared with the control, resulting in significant increases (P < 0.05) ranging from 82 ± 1.6% to 1 ± 2.4% in individual boars. Other treatments had a similar effect, significantly increasing the production of androstenone glucuronide but to a lesser degree. Butyrate increased production ranging from 40 ± 2.2% to 5±1.5% (P < 0.05), propionate from 38 ± 3.6% to 3 ± 2.6% (P < 0.05), acetate and butyrate from 61±3.9% to 1 ± 2.8% (P < 0.05), acetate and propionate from 70±3.5% to 5±1.7% (P < 0.05), and butyrate and propionate from 82 ± 2.5% to 4 ± 1.1% (P < 0.05) when compared with the control. In conclusion, this trial provided evidence that SCFAs produced by the microbial fermentation of fiber can significantly increase hepatic androstenone metabolism in some animals, with a wide range in these effects among individual boars. Future research will involve assessing the effects of SCFAs on hepatic skatole metabolism.

382 The Effect of Natural Products on the Metabolism of Boar Taint Compounds in Porcine Hepatocytes

Abstract Boar taint is a meat quality issue that develops in entire male pigs resulting from the accumulation of androstenone and skatole in the fat, which is dependent on the balance between their rate of synthesis and hepatic metabolism. Prior research from our lab showed that transactivation of the nuclear receptors pregnane X receptor (PXR), constitutive androstane receptor (CAR), and farnesoid X receptor (FXR) can alter gene expression in porcine hepatocytes to increase the metabolism androstenone and skatole. Many plant species and herbal medicines contain active compounds that function as ligands for these nuclear receptors and could be developed into dietary treatments for boar taint unlike the classic agonists used in our previous work, which have known cytotoxic effects in vivo. Therefore, the purpose of this study was to evaluate the effect of different natural product (NP) treatments on the hepatic metabolism of androstenone and skatole. Hepatocytes were isolated from 5-month-old crossbred [(Yorkshire x Landrace) x Duroc] boars (n = 7) and pretreated for 24 hours with one of three NP treatments, which included the PXR agonist hyperforin (HYP) from Hypericum perforatum or St. John’s Wort, diallyl sulfide (DAS), an active ingredient found in garlic and an agonist of CAR, and oleanolic acid (OA), a selective modulator of FXR present in various plant species. Control incubations were pretreated with dimethyl sulfoxide (DMSO). Next, all pretreated hepatocytes were treated with androstenone or skatole for 3 hours. The metabolism of androstenone and skatole was evaluated using high-performance liquid chromatography and boars were classified as rapid (RM, n = 4) or slow (SM, n = 3) metabolizers according to natural threshold values established from the percentage of androstenone (< 5% RM; >5% SM) or skatole (< 50% RM; >50% SM) remaining in control incubations with DMSO. Statistical analysis was conducted using a two-way ANOVA in SAS with Dunnett’s post hoc test. NP treatments differentially regulated androstenone metabolism in SM and RM boars but had no effect (P > 0.05) on skatole metabolism. In SM boars, androstenone metabolism was increased 1.43, 1.75, and 1.67-fold by HYP, DAS, and OA, respectively, but was significantly decreased (P < 0.05) by all NPs in RM boars relative to control incubations with DMSO. These results indicate that SM boars respond more favorably to NP treatments than RM boars suggesting that these NPs may be a viable dietary treatment for boar taint if provided exclusively to SM boars. This targeted treatment approach will require additional research to identify predictive biomarkers associated with the SM profile.

The Uptake and Deconjugation of Androstenone Sulfate in the Adipose Tissue of the Boar.

Simple SummaryBoar taint is a meat quality issue that results from the accumulation of androstenone in the adipose tissue. During steroid synthesis, steroids such as androstenone undergo sulfoconjugation, a process that involves the attachment of a sulfonate group to enhance polarity. Androstenone sulfate is more abundant in the plasma than free androstenone and is suspected to enzymatically regenerate free androstenone in peripheral tissues such as the fat to indirectly contribute to boar taint development. In this article, we identified a specific membrane transporter that is responsible for the uptake of androstenone sulfate into the fat and confirmed that androstenone sulfate can enzymatically regenerate free androstenone within the adipose tissue. We also identified a positive relationship between the quantity of free androstenone enzymatically produced from androstenone sulfate and fat androstenone concentrations in early maturing boars. These results suggest that the production of free androstenone from androstenone sulfate may contribute to the development of boar taint in early maturing animals.Boars express high testicular levels of sulfotransferase enzymes, and consequently, the boar taint causing compound androstenone predominantly circulates as a steroid sulfate. Androstenone sulfate is suspected to function as a steroid reservoir that can be deconjugated to provide a source of free androstenone for accumulation. Therefore, the purpose of this study was to characterize the uptake and deconjugation of androstenone sulfate in the adipose tissue of the boar. Real-time PCR was used to quantify the expression of steroid sulfatase (STS) and several organic anion transporting polypeptides (OATPs) in the adipose tissue. Additionally, [3H]-androstenone sulfate was incubated with adipocytes or supernatant from homogenized fat to assess steroid uptake and conversion, respectively. A positive correlation existed between OATP-B expression and androstenone sulfate uptake (r = 0.86, p = 0.03), as well as between STS expression and androstenone sulfate conversion (r = 0.76, p < 0.001). Moreover, fat androstenone concentrations were positively correlated (r = 0.85, p < 0.001) with androstenone sulfate conversion and tended to increase with STS expression in early maturing boars. This suggests that androstenone sulfate uptake and deconjugation are mediated by OATP-B and STS, respectively, which may influence the development of boar taint in early maturing animals.

PSX-A-27 Late-Breaking: The sulfatase mediated regeneration of androstenone from androstenone sulfate in boar fat

Abstract Boar taint is an off-odour or off-flavour that develops in heated pork products from entire male pigs, which is caused by the accumulation of androstenone, a sex pheromone, in the fat. However, we have previously demonstrated that a significant amount of androstenone undergoes sulfoconjugation upon synthesis in the Leydig cells and circulates in the plasma primarily as a polar steroid sulfate. Therefore, the purpose of this study was to determine if androstenone sulfate can be deconjugated within the adipose tissue by the sulfatase enzyme to return free androstenone and indirectly contribute to the development of boar taint. Backfat was obtained from 6-month-old terminal cross [Duroc x (Landrace x Yorkshire)] boars that had high (n=4) or low (n=4) sulfatase expression as determined by RT-PCR. Sulfatase activity in the fat was measured by quantifying the conversion of androstenone sulfate to free androstenone. Backfat was homogenized and the supernatant was incubated with [3H]-androstenone sulfate for 24-hours. Androstenone was extracted from the incubation using ether and steroid conversion was quantified using high-performance liquid chromatography (HPLC). Additionally, fat androstenone concentrations were quantified using an established HPLC procedure. Statistical analysis was conducted using a Student’s t-test. There was a significant difference (p=0.04) in the expression of sulfatase between the high (2.99 ± 0.67) and low (1.21 ± 0.19) sulfatase boars and the percentage of androstenone sulfate that was converted to free androstenone was proportional to the expression of sulfatase. Interestingly, the expression of sulfatase was positively related to the concentration of androstenone in the fat in boars with high sulfatase expression; however, this relation was not as strong in animals with low sulfatase expression. These preliminary results suggest that the development of boar taint may occur indirectly through the deconjugation of androstenone sulfate in boars with high expression of sulfatase in the fat.

The Binding of Free and Sulfated Androstenone in the Plasma of the Boar.

Simple SummaryBoar taint is characterized by an off-odor or off-flavor in heated pork products that is caused by the accumulation of androstenone in the fat. We have previously demonstrated that androstenone is transported to the fat bound by the plasma protein albumin; however, it is unclear if androstenone sulfate, which is more abundant in the circulation, is transported in the same manner and if the transport of androstenone in the plasma influences the degree of accumulation in the fat. In this article, we determined that androstenone sulfate bound minimally in the plasma of the boar and suggested that this may leave it readily available to enter peripheral tissues, such as the fat where it may enzymatically return free androstenone. Additionally, we demonstrated that the binding of androstenone in the plasma varies significantly between boars with high and low concentrations of androstenone in the fat. This suggests that the binding of androstenone to albumin in the plasma affects the transport and distribution of androstenone within the boar.Androstenone circulates in the plasma bound to albumin before accumulating in the fat, resulting in the development of boar taint. Androstenone sulfate is more abundant in the circulation than free androstenone; however, it is unclear how androstenone sulfate is transported in the plasma and if steroid transport affects the development of boar taint. Therefore, the purpose of this study was to characterize the binding of androstenone sulfate in boar plasma and determine if variability in steroid binding affects the accumulation of androstenone in the fat. [3H]-androstenone sulfate was incubated with plasma and the steroid binding was quantified using gel filtration chromatography. Inter-animal variability was assessed by quantifying androstenone binding specificity in plasma obtained from boars that had high or low fat androstenone concentrations at slaughter. Androstenone sulfate bound minimally in the plasma and to isolated albumin, which suggests that it is transported primarily in solution. The specific binding of androstenone quantified in plasma and isolated albumin from low fat androstenone animals was significantly higher (p = 0.01) than in high fat androstenone boars. These results indicate that the binding of androstenone to albumin varies amongst individual animals and affects the transport of androstenone in the plasma and accumulation in the fat of the boar.

PSVII-33 Inter-Animal Variability in Androstenone Transport and the Potential Effect on the Development of Boar Taint

Abstract Boar taint is a meat quality issue characterized by an off-odour or off-flavour in pork caused by the accumulation of androstenone in the fat. Our previous work demonstrated that androstenone binds non-specifically to albumin in the plasma and suggested that the binding affinity of androstenone might vary between individual animals, which may affect the subsequent development of boar taint. Therefore, the purpose of this study was to characterize the binding of androstenone in the plasma of animals with high and low fat androstenone concentrations to determine the effect of androstenone binding affinity on the development of boar taint. Plasma and backfat samples were obtained from 5-month-old terminal cross [Duroc x (Landrace x Yorkshire)] (n = 8) boars. An androstenone specific enzyme-linked immunosorbent assay (ELISA) was used to determine animals with high (n = 4) or low (n = 4) fat androstenone concentrations. The plasma from each boar was incubated with radiolabeled [3H]-androstenone in the presence or absence of excess unlabeled androstenone. Excess unlabeled androstenone created competition for binding sites allowing the displacement of [3H]-androstenone from albumin to be quantified. Incubations were analyzed by a novel high-performance liquid chromatography (HPLC) method we previously developed to assess androstenone binding. Statistical analysis was conducted using a two-way ANOVA. The plasma albumin concentrations were similar across individual boars; however, the percentage of androstenone that could be displaced from albumin in boars with high fat androstenone concentrations ranged from 7.1 ± 2.4% to 21.9 ± 5.2%, and was significantly lower (P = 0.01) than the percentage of androstenone displaced in the animals with low fat androstenone concentrations. These results suggest that the binding affinity of androstenone is inversely related with fat androstenone concentrations, demonstrating for the first time that the transport of androstenone in the plasma may contribute to the development of boar taint.

Pork Production with Entire Males: Directions for Control of Boar Taint.

Boar taint is caused by the accumulation of androstenone and skatole and other indoles in the fat; this is regulated by the balance between synthesis and degradation of these compounds and can be affected by a number of factors, including environment and management practices, sexual maturity, nutrition, and genetics. Boar taint can be controlled by immunocastration, but this practice has not been accepted in some countries. Genetics offers a long-term solution to the boar taint problem via selective breeding or genome editing. A number of short-term strategies to control boar taint have been proposed, but these can have inconsistent effects and there is too much variability between breeds and individuals to implement a blanket solution for boar taint. Therefore, we propose a precision livestock management approach to developing solutions for controlling taint. This involves determining the differences in metabolic processes and the genetic variations that cause boar taint in specific groups of pigs and using this information to design custom treatments based on the cause of boar taint. Genetic, proteomic or metabolomic profiling can then be used to identify and implement effective solutions for boar taint for specific populations of animals.

91 The production of sulfated 16-androstene steroids by porcine Leydig cells and their potential role in the development of boar taint

Abstract Boar taint is a meat quality issue characterized by an off-odour or off-flavour in pork caused primarily by the accumulation of androstenone in the fat, but sensory estimates of boar taint do not always correlate with fat androstenone concentrations. However, these evaluations examine the sensory qualities of both the fat and lean tissue of heated pork products. Sulfated metabolites of androstenone are polar compounds that are abundantly produced by the Leydig cells of the testes, which may accumulate in more hydrophilic lean tissue. Therefore, the purpose of this study was to investigate the testicular metabolism of androstenone, which is responsible for the high production of sulfated androstenone metabolites. Leydig cells were isolated from 7-month-old Yorkshire, Duroc and terminal cross [Duroc x (Landrace x Yorkshire)] (n = 3) boars and incubated with radiolabeled androstenone for 8 hours. The proportion of sulfated metabolites produced was quantified using reverse phase high performance liquid chromatography (HPLC) and radioisotope detection. The sulfated metabolites were subsequently identified using liquid chromatography-mass spectrometry (LC-MS/MS). Statistical analysis was conducted using a one-way ANOVA in SAS. Following isolation and analysis with LC-MS/MS, the sulfated metabolites were identified as androstenol-3-sulfate and two major sulfated forms of androstenone. Additionally, removal of the sulfate group from these two sulfated forms of androstenone returned the parent compound androstenone, and not a hydroxylated metabolite. The average production of sulfated androstenol produced across all boars was 52.1±8.6%, which was not significantly different (P = 0.7) from the average production of sulfated androstenone (47.9±8.6%). The results of this study indicate that androstenone is directly sulfated, which allows these sulfated metabolites to function as steroid reservoirs that can enzymatically regenerate free androstenone within hydrophilic lean tissue. Alternatively, these metabolites may accumulate in the lean tissue, which we are proposing as a novel mechanism that contributes to the development of boar taint.

Consumers' perception and acceptance of boiled and fermented sausages from strongly boar tainted meat

Characteristic off-flavours may occur in uncastrated male pigs depending on the accumulation of androstenone and skatole. Feasible processing of strongly tainted carcasses is challenging but gains in importance due to the European ban on piglet castration in 2018. This paper investigates consumers' acceptability of two sausage types: (a) emulsion-type (BOILED) and (b) smoked raw-fermented (FERM). Liking (9 point scales) and flavour perception (check-all-that-apply with both, typical and negatively connoted sensory terms) were evaluated by 120 consumers (within-subject design). Proportion of tainted boar meat (0, 50, 100%) affected overall liking of BOILED, F (2, 238)=23.22, P<.001, but not of FERM sausages, F (2, 238)=0.89, P=.414. Consumers described the flavour of BOILED-100 as strong and sweaty. In conclusion, FERM products seem promising for processing of tainted carcasses whereas formulations must be optimized for BOILED in order to eliminate perceptible off-flavours. Boar taint rejection thresholds may be higher for processed than those suggested for unprocessed meat cuts.

Identification of gene co-expression clusters in liver tissues from multiple porcine populations with high and low backfat androstenone phenotype.

BackgroundBoar taint is principally caused by accumulation of androstenone and skatole in adipose tissues. Studies have shown high heritability estimates for androstenone whereas skatole production is mainly dependent on nutritional factors. Androstenone is a lipophilic steroid mainly metabolized in liver. Majority of the studies on hepatic androstenone metabolism focus only on a single breed and very few studies account for population similarities/differences in gene expression patterns. In this work, we concentrated on population similarities in gene expression to identify the common genes involved in hepatic androstenone metabolism of multiple pig populations. Based on androstenone measurements, publicly available gene expression datasets from three porcine populations were compiled into either low or high androstenone dataset. Gene expression correlation coefficients from these datasets were converted to rank ratios and joint probabilities of these rank ratios were used to generate dataset specific co-expression clusters. Finally, these networks were clustered using a graph clustering technique.ResultsCluster analysis identified a number of statistically significant co-expression clusters in the dataset. Further enrichment analysis of these clusters showed that one of the clusters from low androstenone dataset was highly enriched for xenobiotic, drug, cholesterol and lipid metabolism and cytochrome P450 associated metabolism of drugs and xenobiotics. Literature references revealed that a number of genes in this cluster were involved in phase I and phase II metabolism. Physical and functional similarity assessment showed that the members of this cluster were dispersed across multiple clusters in high androstenone dataset, possibly indicating a weak co-expression of these genes in high androstenone dataset.ConclusionsBased on these results we hypothesize that majority of the genes in this cluster forms a signature co-expression cluster in low androstenone dataset in our experiment and that majority of the members of this cluster might be responsible for hepatic androstenone metabolism across all the three populations used in our study. We propose these results as a background work towards understanding breed similarities in hepatic androstenone metabolism. Additional large scale experiments using data from multiple porcine breeds are necessary to validate these findings.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-014-0158-8) contains supplementary material, which is available to authorized users.

Identification of the novel candidate genes and variants in boar liver tissues with divergent skatole levels using RNA deep sequencing.

Boar taint is the unpleasant odour of meat derived from non-castrated male pigs, caused by the accumulation of androstenone and skatole in fat. Skatole is a tryptophan metabolite produced by intestinal bacteria in gut and catabolised in liver. Since boar taint affects consumer’s preference, the aim of this study was to perform transcriptome profiling in liver of boars with divergent skatole levels in backfat by using RNA-Seq. The total number of reads produced for each liver sample ranged from 11.8 to 39.0 million. Approximately 448 genes were differentially regulated (p-adjusted <0.05). Among them, 383 genes were up-regulated in higher skatole group and 65 were down-regulated (p<0.01, FC>1.5). Differentially regulated genes in the high skatole liver samples were enriched in metabolic processes such as small molecule biochemistry, protein synthesis, lipid and amino acid metabolism. Pathway analysis identified the remodeling of epithelial adherens junction and TCA cycle as the most dominant pathways which may play important roles in skatole metabolism. Differential gene expression analysis identified candidate genes in ATP synthesis, cytochrome P450, keratin, phosphoglucomutase, isocitrate dehydrogenase and solute carrier family. Additionally, polymorphism and association analysis revealed that mutations in ATP5B, KRT8, PGM1, SLC22A7 and IDH1 genes could be potential markers for skatole levels in boars. Furthermore, expression analysis of exon usage of three genes (ATP5B, KRT8 and PGM1) revealed significant differential expression of exons of these genes in different skatole levels. These polymorphisms and exon expression differences may have impacts on the gene activity ultimately leading to skatole variation and could be used as genetic marker for boar taint related traits. However, further validation is required to confirm the effect of these genetic markers in other pig populations in order to be used in genomic selection against boar taint in pig breeding programs.

Molecular characterization of the porcine TEAD3 (TEF‐5) gene: examination of a promoter mutation as the causal mutation of a quantitative trait loci affecting the androstenone level in boar fat

A quantitative trait loci (QTL) for accumulation of androstenone in fat has been identified in an Large White × Meishan cross in a region of SSC7-containing TEAD3. In humans, TEAD3 is a transcription activator, known to be able to regulate the transcription of HSD3B. This enzyme is involved in the degradation of androstenone in the liver. In this study, porcine transcripts of TEAD3 were characterized and compared with mammalian transcripts. The complete structure of porcine TEAD3 gene was characterized including two 5' non-coding exons and one exon 5 not used in porcine transcripts. Variations were screened in sequences related to TEAD3: in exons, in flanking sequences of exons and in the promoter region. A SNP characterized at 726 bp at 5' of the first exon was tested on several pig populations without coherent and convincing results concerning its association with androstenone levels. We showed that in the liver of adult boars, the transcripts levels of TEAD3 and HSD3B were correlated. As in humans, it is possible that HSD3B is a target gene of TEAD3 in porcine liver. Nevertheless, no expression variation was observed for TEAD3 or HSD3B in liver between animals with different genotypes at the SNP. We concluded that this SNP was not the causal mutation of this QTL.

The distal end of porcine chromosome 6p is involved in the regulation of skatole levels in boars

BackgroundBoar taint is an unpleasant condition of pork, mainly due to the accumulation of androstenone and skatole in male pigs at onset of puberty. This condition is the cause of considerable economic losses in the pig industry and the most common practice to control it is to castrate male piglets. Because of the economic and animal welfare concerns there is interest in developing genetic markers that could be used in selection schemes to decrease the incidence of boar taint. The Porcine 60 K SNP Beadchip was used to genotype 891 pigs from a composite Duroc sire line, for which skatole levels in fat had been collected.ResultsThe genome-wide association study revealed that 16 SNPs (single nucleotide polymorphisms) located on the proximal region of chromosome 6 were significantly associated with skatole levels. These SNPs are grouped in three separate clusters located in the initial 6 Mb region of chromosome 6. The differences observed between the homozygote genotypes for SNPs in the three clusters were substantial, including a difference of 102.8 ng/g skatole in melted fat between the homozygotes for the ALGA0107039 marker. Single SNPs explain up to 22% of the phenotypic variance. No obvious candidate genes could be pinpointed in the region, which may be due to the need of further annotation of the pig genome.ConclusionsThis study demonstrated new SNP markers significantly associated with skatole levels in the distal region of chromosome 6p. These markers defined three independent clusters in the region, which contain a low number of protein-coding genes. The considerable differences observed between the homozygous genotypes for several SNPs may be used in future selection schemes to reduce skatole levels in pigs

Erratum to Genetic and metabolic aspects of androstenone and skatole deposition in pig adipose tissue: A review

High levels of androstenone and skatole in fat tissues are considered the primary causes of boar taint, an unpleasant odour and flavour of the meat from non-castrated male pigs. The aim of this article is to review our current knowledge of the biology and genetic control of the accumulation of androstenone and skatole in fat tissue. Two QTL mapping studies have shown the complexity of the genetic control of these traits. During the last ten years, several authors have taken a more physiological approach to investigate the involvement of genes controlling the metabolism of androstenone and skatole. Although some authors have claimed the identification of candidate genes, it is more appropriate to talk about target genes. This suggests that genes affecting androstenone and skatole levels will have to be sought for among specific or non-specific transcription factors interacting with these target genes.

Effect of sex steroids on expression of sulfotransferase 2B1 immunoreactive protein in primary cultured porcine hepatocytes

An excessive accumulation of androstenone (5α-androst-16-en-3-one) in pig adipose tissue is one of the two major contributors to the phenomenon of boar taint. High levels of adipose tissue androstenone have been related to a low rate of hepatic androstenone metabolism, which includes two stages: oxidative and conjugative. Sulfotransferases (SULTs), alongside with other specific enzymes, play the key role in the conjugative stage of androstenone metabolism. The present study investigated the mechanism regulating expression of sulfotransferase 2B1 (SULT2B1) immunoreactive protein using primary cultured pig hepatocytes as a model system. A specific objective was to determine whether the expression of pig hepatic SULT2B1 is regulated by the sex steroids; androstenone, testosterone and estrone sulphate. The study was performed on entire male pigs of a Large White (40%) × Landrace (40%) × Duroc (20%) cross-breed, average carcass weight 72.2 kg. The study shows that SULT2B1 immunoreactive protein expression can be induced by testosterone (final concentrations, 10 and 500 nM) and repressed by estrone sulphate (final concentration, 100 nM). Androstenone had no significant effect on SULT2B1 immunoreactive protein expression in the range of concentration, 10 nM to 1 μM. Time-courses (0 to 48 h) of steroid effects were investigated. The maximum effects of testosterone and estrone sulphate were observed in 24 h after the steroid treatments. This study provides direct evidence for involvement of sex steroids in the regulation of porcine hepatic SULTs.

Genetic and metabolic aspects of androstenone and skatole deposition in pig adipose tissue: A review (Open Access publication)

High levels of androstenone and skatole in fat tissues are considered the primary causes of boar taint, an unpleasant odour and flavour of the meat from non-castrated male pigs. The aim of this article is to review our current knowledge of the biology and genetic control of the accumulation of androstenone and skatole in fat tissue. Two QTL mapping studies have shown the complexity of the genetic control of these traits. During the last ten years, several authors have taken a more physiological approach to investigate the involvement of genes controlling the metabolism of androstenone and skatole. Although some authors have claimed the identification of candidate genes, it is more appropriate to talk about target genes. This suggests that genes affecting androstenone and skatole levels will have to be sought for among specific or non-specific transcription factors interacting with these target genes.

Expression of 3β-hydroxysteroid dehydrogenase, cytochrome P450-c17, and sulfotransferase 2B1 proteins in liver and testis of pigs of two breeds: Relationship with adipose tissue androstenone concentration1

An excessive accumulation of androstenone in pig adipose tissue is a major contributor to the phenomenon of boar taint. Androstenone deposition is dependent on the rate of androstenone biosynthesis in testis and androstenone degradation in liver. The aim of the current study was to examine the possibility of the existence of breed-specific mechanisms controlling androstenone accumulation in pig adipose tissue. The specific objective was to investigate the expression of some of the key enzymes involved in testicular and hepatic androstenone metabolism in pigs of 2 breeds by using animals with high and low androstenone concentrations within each breed. The study was conducted with Norwegian Landrace (N. Landrace) and Duroc boars. The mean androstenone values for the low- and high-androstenone groups were 0.1 +/- 0.01 microg/g and 7.58 +/- 0.68 microg/g for N. Landrace boars, and 0.22 +/- 0.04 microg/g and 13.55 +/- 1.14 microg/g for Duroc boars. The enzymes investigated were 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450-c17, and sulfotransferase 2B1 (SULT2B1). Expression of cytochrome P450-c17 in liver and testis did not differ between animals with high and low androstenone concentrations in either the N. Landrace or Duroc breed. Expression of hepatic 3beta-HSD, which catalyzes the first stage of androstenone degradation, was decreased in high-androstenone N. Landrace boars (P < 0.01), but not in high-androstenone Duroc boars. In contrast, the expression of hepatic SULT2B1, which catalyzes the second stage of steroid catabolism, was decreased in high-androstenone Duroc animals (P < 0.05), but not in high-androstenone N. Landrace animals. Sulfotransferase 2B1 was also inhibited in testis of high-androstenone pigs of both breeds compared with low-androstenone animals. We report breed differences in expression of the androstenone-metabolizing enzymes 3beta-HSD and SULT2B1 in the liver of high- and low-androstenone pigs. It is suggested that accumulation of androstenone in adipose tissue of N. Landrace boars might be related to a low rate of hepatic androstenone degradation in metabolic stage I, whereas the high androstenone concentration in Duroc boars might be related to a low rate of androstenone metabolism in metabolic stage II.

Effect of testosterone, estrone sulphate and androstenone on 3β-hydroxysteroid dehydrogenase protein expression in primary cultured hepatocytes

Androstenone (5α-androst-16-en-3-one) is a steroid pheromone, excessive accumulation of which in pig adipose tissue contributes to the phenomenon of “boar taint”. The incidence of boar taint increases with age and weight of entire male pigs. One of the reasons for the excessive accumulation of androstenone in adipose tissue of some pigs is a low rate of androstenone degradation in pig liver, which is related to defective expression of the androstenone-metabolising enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD). The mechanism regulating 3β-HSD expression in pig liver remains unclear. The present study investigated the effects of the sex steroids testosterone, estrone sulphate and androstenone on the expression of 3β-HSD protein using primary cultured hepatocytes as a model system. The study was performed on hepatocytes from pigs of two weight/age groups: “lighter weight” (average carcass weight 70 kg, age 21 weeks) and “heavier weight” (average carcass weight 92 kg, age 26 weeks). Testosterone at the range of concentrations from 5 to 100 nM induced 3β-HSD expression in “lighter weight” pigs but not in “heavier weight” animals. In contrast, estrone sulphate (5 to 100 nM) and androstenone (5 to 500 nM) induced 3β-HSD expression only in “heavier weight” but not “lighter weight” pigs. Induction of 3β-HSD expression by the sex steroids was protein-synthesis-dependent. Overall this study demonstrates for the first time that sex steroids are involved in regulation of 3β-HSD protein expression in primary cultured pig hepatocytes and that the effect of sex steroids depends on age and weight of animals. The results of this work might contribute to understanding the mechanism controlling androstenone deposition in pigs.

Relationship between the expression of hepatic but not testicular 3β-hydroxysteroid dehydrogenase with androstenone deposition in pig adipose tissue1

This study investigated the relationship between expression of hepatic and testicular 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and accumulation of androstenone in adipose tissue because of its relation to boar taint. The experiments were performed on 13 Large White (50%) x Landrace (50%) and Meishan (25%) x Large White (25%) x Landrace (50%), pigs, which differed in the level of backfat androstenone. Our previous work showed that the major product of the hepatic androstenone metabolism is 3beta-androstenol. In this study, the formation of 3beta-androstenol was inhibited by the specific 3beta-HSD inhibitor trilostane. These results are the first direct confirmation that 3beta-HSD is the enzyme responsible for androstenone metabolism in the pig. The expression of the hepatic but not testicular 3beta-HSD protein showed a negative relationship with the level of backfat androstenone (r2 = 0.64; P < 0.001) and was accompanied by a reduced rate of the hepatic androstenone clearance. Low expression of 3beta-HSD protein in the liver of high androstenone pigs was also accompanied by a reduced level of 3beta-HSD mRNA (P < 0.001), which suggests a defective regulation of the hepatic 3beta-HSD expression at the level of transcription. In contrast, expression of the testicular 3beta-HSD protein did not differ between animals with high and low androstenone levels (P > 0.05) and was lower compared with the hepatic 3beta-HSD expression. Cloning and sequencing of the 3beta-HSD coding regions established that the hepatic and testicular 3beta-HSD cDNA have identical sequences, which were 98% similar to the human 3beta-HSD isoform I. It is suggested that expression of a single 3beta-HSD gene is regulated by different mechanisms in pig liver and testis. The liver-specific regulation of 3beta-HSD expression contributes to the low rate of hepatic androstenone metabolism and therefore can be considered as one of the factors regulating deposition of androstenone in pig adipose tissue and subsequent development of boar taint.