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.

Porcine constitutive androstane receptor (CAR; NR1I3) was cloned and compared for homology and activity with mouse and human CAR (mCAR, hCAR). Porcine CAR (pgCAR) was 86% and 75% homologous to hCAR at the nucleotide and protein levels. Five alternatively spliced variants of pgCAR were identified, each of which generated a truncated protein product. Real-time polymerase chain reaction (PCR) analyses showed that these variants were present in pig liver cDNA samples from 4.61% to 9.20% of total pgCAR. pgCAR and hCAR responded similarly to more ligands than did hCAR and mCAR. The known hCAR agonist (6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) activated pgCAR, while the murine agonist 1,4 bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) had no effect. 5beta-dihydrotestosterone was identified as a novel inverse agonist of both pgCAR and hCAR. pgCAR splice variant 2 (SV2) had a dose-dependent dominant negative effect on the activity of wild-type pgCAR in dual luciferase assays. SV2 had no effect against pgPXR (pregnane X receptor) or pgFXR (farnesoid X receptor) activity when using PXR- or FXR-specific reporters.

The level of cytochrome b5A (CYB5A) in pig testis is correlated with boar taint from androstenone and an AF016388:c.-8G>T polymorphism in CYB5A has been linked with low androstenone levels in the fat of pigs. In this study, we developed a polymerase chain reaction-based assay to genotype 1242 boars from eight lines for the c.-8G>T SNP. The c.-8T allele was found in all eight lines at a frequency ranging from 1.8% to 20.3% with an overall frequency of 8.6%. Significant deviations from Hardy-Weinberg equilibrium were found in the Hampshire, Landrace and Yorkshire breeds. The homozygous mutant c.-8TT occurred infrequently and was not found in some lines, but was consistently associated with low androstenone levels in fat. Both CYB5A mRNA and CYB5A protein levels were decreased in the c.-8TT genotype in a subset of Yorkshire boars, suggesting that low levels of CYB5A protein in the c.-8TT mutant were not due to inefficient translation of CYB5A mRNA. There were significant but modest marker effects on fat androstenone levels in Landrace, Yorkshire and a Large White/Duroc cross and fat skatole in Duroc and Sire Line breeds. There was no effect of CYB5A genotype on bulbourethral gland length, suggesting that this SNP will not affect reproductive traits. We conclude that the c.-8G>T SNP in the CYB5A gene has a significant but modest effect on boar taint in male pigs and could be useful in some breeds as part of a panel of SNP markers in a marker-assisted selection programme to produce low boar taint pigs.

The present study investigated the presence of a single-nucleotide polymorphism (G . T) at base 28 upstream of ATG in 5 0 untranslated region of cytochrome b5 (CYB5A) gene in Swedish pig populations and evaluated the significance of this polymorphism for androstenone and skatole levels, sexual development and performance parameters in pigs. Frequencies of the T allele were 6.7% for Swedish Yorkshire 3 Landrace crossbred pigs (n 5 245), 6.5% for Swedish Yorkshire (n 5 99) and 12.8% for Landrace breed (n 5 74). No deviations from Hardy-Weinberg equilibrium were observed in the investigated populations. In Swedish Yorkshire 3 Landrace crossbred entire male pigs (n 5 193), plasma samples were analysed for skatole, androstenone, testosterone and oestrone sulphate, and fat samples were analysed for androstenone, skatole and free oestrone. Additionally, testis weight and bulbourethral gland length for crossbred pigs were recorded. Plasma androstenone levels were significantly lower in the G/T genotype at 90 kg live weight compared with the wild G/G genotype at the same live weight (P 5 0.006). In heavier pigs, plasma androstenone levels did not differ between genotypes (P 5 0.382). Fat androstenone levels were not affected by CYB5A genotype (P 5 0.252). Skatole levels in the G/T genotype at 115 kg live weight were lower compared with those in the G/G genotype in plasma (P 5 0.048) and fat (P 5 0.028), although no differences were observed in lighter pigs. Testis weight, bulbourethral gland length, testosterone and oestrone sulphate levels in plasma, and oestrone levels in fat were not affected by genotype. We concluded that the presence of the T allele in the CYB5A gene resulted in lower androstenone levels in plasma, and lower skatole levels in fat and plasma; this reduction, however, was dependent on the live weight of the animals. Reproductive hormones and growth rate did not differ between the pigs of different genotypes, whereas a higher lean meat content was found in the G/T genotype in comparison with the G/G genotype. The practical application of those results in Sweden is doubtful because of lack of the effect on androstenone in fat and the low frequency of the T allele in the studied Swedish pig populations.