A total of 228 intact male pigs form Duroc, Hampshire, Landrace, and Yorkshire breeds were used in the experiment. Samples of salivary gland and backfat were collected at slaughter for colorimetric assay of salivary and fat 16-androstene levels and fat skatole levels. Fat levels also were tested by a sensory panel using an R-index technique for detecting the presence of boar taint. The proportion of tainted carcasses determined by the sensory panel was 5.0% for androstenone and 11.4% for skatole, with a combined total of 15.0% tainted from either source. Sensory analysis of taint showed a lower proportion (P < .05) of tainted carcasses in Hampshire, with no difference in taint across the other three breeds. Analysis of taint compounds indicated that overall 14.5% of pigs had salivary gland 16-androstene levels and 20.9% had fat 16-androstene levels above acceptable limits. There was a higher (P < .05) proportion of Duroc pigs above the threshold levels for 16-androstenes in both salivary gland and fat. Landrace pigs had the lowest (P < .05) average tissue concentrations of steroids and skatole. Across breeds, only 1.8% of pigs had fat skatole concentrations above .25 ppm, which has been suggested as threshold levels of skatole for taint. The canonical correlation coefficient between fat compound levels and the R-indices of fat 16-androstenes and skatole was .40 (P < .001). Our results indicate breed differences in tissue levels of taint compounds and in taint assessed by a sensory panel. Levels of 16-androstene steroids were highly associated with taint, but more pigs had measured levels above the threshold than those identified as tainted by sensory analysis. Levels of fat skatole were low overall and did not account for all the pigs judged as tainted from skatole by sensory analysis.

Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.Keywords: boar taint, skatole, 3-methylindole, cytochrome b5A, cytochrome P450 ImplicationsStudies on the metabolism and clearance of skatole, a major component of boar taint, have been hampered by the lack of specific tools to measure the activity of the cytochrome P450 (CYP450) enzymes involved. Here we have used a defined cellbased system to individually express six different isoforms of porcine CYP450 and cytochrome b5A, and measure their role in the metabolism of skatole. Our results show that CYP2A19 is the primary isoform responsible for the formation of 6-hydroxyskatole, although its importance to in vivo clearance will also be affected by its level of expression in vivo.

The characterization of the SULT1A1 gene and its variants should have an important impact on the efforts to develop genetic markers to select for low skatole in pigs. Raising intact male pigs would have a significant economic impact on the pork industry; however, the presence of skatole (a major cause of boar taint) in meat from male pigs would be highly objectionable to consumers. It has been shown that the phase II metabolism of skatole metabolites by phenol sulfotransferase is related to the clearance of skatole in the liver. The aim of this study was to isolate and characterize the SULT1A1 gene from pig liver, examine its expression, identify genetic polymorphisms, and study how a genetic variation in this enzyme translates into interindividual variation in skatole levels. We show here that a substitution of A-->G at base 546 of SULT1A1 causes a significant decrease in its sulfation activity and thus may be at least partially responsible for a higher level of skatole in pigs. Our findings provide an important basis toward the goal of making it possible to predict the sulfation status in pigs and the development of genetic markers for low skatole.

Raising intact male pigs would have a significant economic impact on the pork industry; however, the presence of 16-androstene (a major cause of boar taint) in meat from male pigs would be highly objectionable to consumers. In pigs, a positive correlation has been found between cytochrome b5 (CYB5) and production of 16-androstene. The search for polymorphism of CYB5 and functional analysis of polymorphism found should have an important impact on the efforts to develop genetic markers to select for low androstenone levels in fat from pigs. The aim of this study was to search the porcine CYB5 gene for mutations, examine its expression, identify genetic polymorphisms, and study how a genetic variation in this enzyme translates into interindividual variation in androstenone levels in fat from pig testis. We have identified a single nucleotide polymorphism (SNP) (G --> T) at base 8 up-stream of ATG in the CYB5 5' untranslated region which is associated with a lower fat androstenone level. Of the 229 testis samples tested, 84.8% were homozygous for the variant G, 12.4% were heterozygous, and 2.8% were homozygous for the variant T. Functional analysis of this mutation revealed that an individual homozygous for the T allele showed significantly lower CYB5 activity than an individual homozygous for the G allele. Thus, this may be at least partially responsible for a lower level of androstenone in pigs. Our findings provide an important genetic basis toward the goal of predicting the androstenone status in pigs and developing genetic markers for low androstenone.

Hydroxysteroid sulfotransferase (SULT2A1) is a key enzyme in the testicular and hepatic metabolism of 5 -androstenone, which is a major component of the off-odor and off-flavor in pork known as boar taint. The goals of this study were to determine the role of testicular and hepatic SULT2A1 activity on plasma 5 -androstenone sulfate levels, the accumulation of 5 -androstenone in adipose tissue, and to gain insight into the regulatory control of SULT2A1. Testicular SULT2A1 activity was negatively correlated (r= −0·57; P,0·01) with 5 -androstenone concentrations in fat. The differences observed in SULT2A1 activity warranted investigation into potential genetic variation within porcine SULT2A1. The cDNA sequence of porcine Sult2A1 was determined to be .82% homologous to the human, mouse, and rat Sult2A1 genes. A single nucleotide polymorphism was detected within the coding region of the Sult2A1 from individual testes and liver samples; however, this did not affect the amino acid sequence of the enzyme. Western blot analysis determined that animals with high concentrations of 5 -androstenone in fat and low SULT2A1 activity had corresponding low levels of SULT2A1 protein compared with animals with low levels of 5 -androstenone in fat. Real-time PCR analysis indicated that Sult2A1 mRNA was increased 2·8-fold in animals with high levels of the protein relative to animals with low levels of the protein. Furthermore, we demonstrated the positive role of the nuclear receptors constitutive androstane receptor and pregnane X receptor, as well as the possible role of farnesoid X receptor in the regulation of testicular SULT2A1 activity. Together, the results of this study suggest that differences in SULT2A1 expression can influence 5 -androstenone accumulation in fat.