Characterisation of androstenone metabolism in pig liver microsomes

Androstenone (5 alpha-androst-16-en-3-one) is a steroid pheromone produced in the testis. Excessive accumulation of androstenone together with skatole (3-methyl-indole) in the adipose tissue of some male pigs leads to "boar taint". In isolated pig hepatocytes androstenone represses the expression of cytochrome P450IIE1 (CYP2E1), the enzyme principally responsible for skatole metabolism. Androstenone can be metabolised in liver microsomes but the pathway has not been established. We have investigated androstenone metabolism in liver microsomes from two breeds of pigs exhibiting low and high levels of androstenone in adipose tissue-Large White (LW) and Meishan (M), respectively. Androstenone was reduced in isolated liver microsomes mainly to beta-androstenol using NADH as a co-factor. The rate of beta-androstenol formation in the presence of NADPH was very low. In microsomes from LW pigs the rate of beta-androstenol formation from androstenone was six times higher than in M pigs. 3beta-hydroxysteroid dehydrogenase (3beta-HSD) was investigated as a likely candidate for the enzyme catalysing androstenone reduction in pig liver. RT-PCR analysis showed that there was no sequence difference in the cDNA encoding 3beta-hydroxysteroid dehydrogenase from LW and M pigs. However, competitive RT-PCR analysis showed that the expression of 3beta-hydroxysteroid dehydrogenase mRNA was about 12 times higher in the case of LW compared to M pigs. It is concluded that the rate of androstenone metabolism in pig liver microsomes is determined by the level of expression of hepatic 3beta-hydroxysteroid dehydrogenase. The differential expression of this enzyme could be a factor affecting the rate of hepatic androstenone metabolism which in turn may influence the level of hepatic CYP2E1 expression and hence the rate of hepatic skatole metabolism.     

Skatole metabolites in urine as a biological marker of pigs with enhanced hepatic metabolism

Boar taint is a quality defect in meat, related to accumulation of skatole and androstenone in male pigs. The levels of skatole and its main metabolites in plasma and urine samples were measured with a validated liquid chromatography-MS method and related to activity of hepatic cytochrome P450 (CYP450) in order to identify ‘fast metabolizing’ pigs. Urine (n=46), blood (n=12), liver (n=25) and adipose tissue (n=46) were sampled from a total of 46 entire male pigs. Skatole levels in fat were negatively correlated to CYP2E1 activity and positively to 3-hydroxy-3-methyloxindole (HMOI), indole-3-carboxylic acid (ICA) and 2-aminoacetophenone in urine. HMOI and ICA levels in urine were the best predictors of high skatole levels in fat. In summary, the present study provided further evidence for the key role of CYP2E1 in skatole metabolism and suggested that measurement of HMOI and/or ICA in urine might provide information about skatole levels in live pigs.     

Hydrolysable tannin-based diet rich in gallotannins has a minimal impact on pig performance but significantly reduces salivary and bulbourethral gland size

Tannins have long been considered ‘anti-nutritional’ factors in monogastric nutrition, shown to reduce feed intake and palatability. However, recent studies revealed that compared with condensed tannins, hydrolysable tannins (HT) appear to have far less impact on growth performance, but may be inhibitory to the total activity of caecal bacteria. This in turn could reduce microbial synthesis of skatole and indole in the hindgut of entire male pigs (EM). Thus, the objective of this study was to determine the impact of a group of dietary HT on growth performance, carcass traits and boar taint compounds of group housed EM. For the study, 36 Swiss Large White boars were assigned within litter to three treatment groups. Boars were offered ad libitum one of three finisher diets supplemented with 0 (C), 15 (T15) or 30 g/kg (T30) of HT from day 105 to 165 of age. Growth performance, carcass characteristics, boar taint compounds in the adipose tissue and cytochrome P450 (CYP) isoenzymes CYP2E1, CYP1A2 and CYP2A19 gene expression in the liver was assessed. Compared with C, feed efficiency but not daily gain and daily feed intake was lower (P<0.05) in T15 and T30 boars. Except for the percentage carcass weight loss during cooling, which tended (P<0.10) to be greater in T30 than C and T15, carcass characteristics were not affected by the diets. In line with the numerically lower androstenone level, bulbourethral and salivary glands of T30 boars were lighter (P<0.05) than of T15 with intermediate values for C. Indole level was lower (P<0.05) in the adipose tissue of T30 than C pigs with intermediate levels in T15. Skatole levels tended (P<0.10) to be lower in T30 and C than T15 pigs. Hepatic gene expression of CYP isoenzymes did not differ between-treatment groups, but was negatively correlated (P<0.05) with androstenone (CYP2E1 and CYP1A2), skatole (CYP2E1, CYP2A) and indole (CYP2A) level. In line with the numerically highest androstenone and skatole concentrations, boar taint odour but not flavour was detected by the panellists in loins from T15 compared with loins from C and T30 boars. These results provide evidence that HT affected metabolism of indolic compounds and androstenone and that they affected the development of accessory sex glands. However, the effects were too small to be detected by sensory evaluation.     

Feeding dried chicory root to pigs decrease androstenone accumulation in fat by increasing hepatic 3β hydroxysteroid dehydrogenase expression

The present study investigated the in vivo effect of chicory root on testicular steroid concentrations and androstenone metabolizing enzymes in entire male pigs. Furthermore, the effect on skatole and indole concentrations in plasma and adipose tissue was investigated. The pigs were divided into two groups; one receiving experimental feed containing 10% dried chicory root for 16 days before slaughter, the control group was fed a standard diet. Plasma, adipose and liver tissue samples were collected at slaughter. Plasma was analyzed for the concentration of testosterone, estradiol, insulin-like growth factor 1 (IGF-1), skatole and indole. Adipose tissue was analyzed for the concentration of androstenone, skatole and indole, while the liver tissue was analyzed for mRNA and protein expressions of 3β-hydroxysteroid dehydrogenase (3β-HSD), sulfotransferase 2A1 and heat-shock protein 70 (HSP70). The results showed that the androstenone concentrations in the adipose tissue of chicory fed pigs were significantly (p<0.05) lower and indole concentrations were higher (p<0.05) compared to control fed pigs. Moreover the chicory root fed pigs had increased mRNA and protein expression of 3β-HSD and decreased HSP70 expression (p<0.05). Testosterone and IGF-1 concentrations in plasma as well as skatole concentrations in adipose tissue were not altered by dietary intake of chicory root. It is concluded that chicory root in the diet reduces the concentration of androstenone in adipose tissue via induction of 3β-HSD, and that these changes were not due to increased cellular stress.     

Relationship between carcass weight, adipose tissue androstenone level and expression of the hepatic 3β-hydroxysteroid dehydrogenase in entire commercial pigs

Boar taint is a major meat-quality defect in pigs and is due to excessive accumulation of skatole and androstenone in adipose tissue. The present work investigated the relationship between carcass weight, levels of skatole and androstenone in adipose tissue, and expression of the hepatic androstenone-metabolising enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD), in 22 entire male and 22 entire female crossbred pigs (Large White (40%) × Landrace (40%) × Duroc (20%)). Animals of each gender were divided into two subgroups (11 pigs in each subgroup): (i) conventional weight (carcass weight 59 to 77 kg) and (ii) heavy weight (carcass weight 84 to 95 kg). No relationship between carcass weight and adipose tissue skatole level was found for entire male pigs (r2 = 0.013, P > 0.05). There was a significant negative relationship between carcass weight and expression of the hepatic 3β-HSD protein (r2 = 0.502, P < 0.001) and a significant negative relationship between 3β-HSD protein expression and androstenone level in adipose tissue (r2 = 0.24, P < 0.05) in entire males. No relationship was found between carcass weight and 3β-HSD protein expression in female pigs (r2 = 0.001, P > 0.05). 3β-HSD expression was 59% higher in conventional-weight male pigs when compared with heavy-weight animals (P < 0.05) and 36% higher in heavy-weight females when compared with heavy-weight males (P < 0.05). It is concluded that an increase in slaughter weight of entire commercial crossbred Large White pigs is accompanied by inhibition of expression of the hepatic 3β-HSD protein, which might result in a reduced rate of hepatic androstenone clearance with its subsequent accumulation in adipose tissue. It is suggested that regulation of pig hepatic 3β-HSD expression is under the control of sex hormones.     

Functional polymorphism in porcine CYP2E1 gene: Its association with skatole levels

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 intact male pigs could be highly objectionable to consumer. The excessive accumulation of skatole in fat is a major cause of boar taint, and is associated with defective expression of cytochrome P4502E1 (CYP2E1). In pigs, it has been found that CYP2E1 is negatively correlated with accumulation of skatole. The searching for polymorphism of CYP2E1 and the relevant functional analysis would help develop a genetic marker for the selection of pigs with low skatole levels in fat. The aim of this study was to measure the expression pattern of CYP2E1 mRNA in various tissues of the pig, to identify genetic polymorphisms, and to evaluate the functional relevance of polymorphic sites with respect to the skatole level in fat. We show herein that a substitution of G → A at base 1423 of the CYP2E1 gene in the liver causes a significant decrease in the expressed CYP2E1 level. Our data suggest that the G → A substitute might be at least partially responsible for a high level of skatole in pigs. We believe that this is an important step toward the selection of genetic markers for boar taint by lowering fat levels of skatole in fat.     

Effect of faecal soiling on skatole and androstenone occurrence in organic entire male pigs

Production of entire male pigs could be a future strategy for organic pig production. However, production of entire males leads to increased risk of carcasses with elevated boar taint levels. It is hypothesized that skatole levels in pig meat are affected by faecal soiling and that organic housing facilities can increase the risk of pigs being heavily soiled. Therefore, the overall aim of this study was to investigate if increased pig and pen soiling increases skatole concentration in entire male pigs. In five herds, 1174 organic entire male pigs were reared in four batches across two seasons, summer and winter. Measurements of pig and pen soiling, as well as fat skatole and androstenone concentration and human nose sensory tests of fat odour, were performed. Skatole and androstenone concentrations varied greatly within and between herds with a 10% and 90% percentile for the overall population of 0.02 and 2.25 µg/g for skatole and 0.53 and 4.84 µg/g for androstenone. Human nose positive tests averaged 18.3% with great variation between herds and seasons. Pen soiling had significant effects on pig soiling. Moreover, outdoor pen soiling significantly affected skatole concentration in interactions with herd and season (P<0.001 and P=0.003) and affected human nose positive risk in interaction with herd (P=0.005). Soiling on indoor pen areas did not affect skatole levels and no effect on androstenone was found for any pen area. Soiling of pigs affected both skatole and androstenone levels, with the size of the head and abdomen body areas covered in manure showing significant positive effects on skatole concentration. No effect of density of the manure layer was found on either boar taint measure. Herd significantly affected both skatole and androstenone in fat as well as the human nose positive risk. The human nose test revealed no effect from pig soiling. A large variation in the different boar taint measures was found for both high and low scores of pen and pig soiling, and only a small difference in skatole and androstenone concentrations between the high and low soiling categories was found. Therefore, while increasing the hygiene management could be a strategy for reducing boar taint in production of organic entire male pigs, it should be emphasized that other factors would also need to be considered.     

Biochemical, nutritional and genetic effects on boar taint in entire male pigs

Pork odour is to a great extent affected by the presence of malodorous compounds, mainly androstenone and skatole. The present review outlines the current state of knowledge about factors involved in the regulation of androstenone and skatole in entire male pigs. Androstenone is a pheromonal steroid synthesised in the testes and metabolised in the liver. Part of androstenone accumulates in adipose tissue causing a urine-like odour. Skatole is produced in the large intestine by bacterial degradation of tryptophan and metabolised by hepatic cytochrome P450 enzymes and sulphotransferase. The un-metabolised part accumulates in adipose tissue, causing faecal-like odour. Androstenone levels are mostly determined by genetic factors and stage of puberty, whereas skatole levels in addition to genetic background and hormonal status of the pigs are also controlled by nutritional and environmental factors. To reduce the risk of tainted carcasses entering the market, male pigs are surgically castrated in many countries. However, entire males compared to castrates have superior production characteristics: higher growth rate, better feed efficiency and leaner carcasses. Additionally, animal welfare aspects are currently of particular importance in light of increasing consumers' concerns. Nutrition, hormonal status, genetic influence on boar taint compounds and the methods to develop genetic markers are discussed. Boar taint due to high levels of skatole and androstenone is moderately heritable and not all market weight entire males have boar taint; it should thus be possible to select for pigs that do not have boar taint. In these studies, it is critical to assess the steroidogenic potential of the pigs in order to separate late-maturing pigs from those with a low genetic potential for boar taint. A number of candidate genes for boar taint have been identified and work is continuing to develop genetic markers for low boar taint. More research is needed to clarify the factors involved in the development of boar taint and to develop additional methods to prevent the accumulation of high concentrations of skatole and androstenone in fat. This review proposes those areas requiring further research.     

Estimation of genetic parameters of boar taint; skatole and androstenone and their correlations with sexual maturation

Boar taint is mainly caused by two components; skatole (3-methylindole) and androstenone. By castrating the male pigs, boar taint will be avoided. In Norway, castration of pigs will no longer be permitted after 2009. This represents a substantial cost for the Norwegian swine production. Other Norwegian studies have shown that a large proportion of pigs are above the consumer detection limits for these two chemical components. The obvious question for the geneticist arises: Is it possible to select against skatole and androstenone in a breeding programme? Skatole is produced in the gut by bacteria. It is then absorbed in the blood stream. Skatole is either metabolised in the liver or transported and stored in fatty tissue. Androstenone is produced in the testis, and its biochemical pathway is related to the pathway of testosterone. In this study, fatty tissue was collected from the carcasses of Norwegian Landrace and Duroc boars, and analysed for androstenone and skatole. The length of glandula bulbourethralis was measured on the same animals, as this is regarded as a good indicator of sexual maturation. Heritabilities of androstenone and skatole were substantial. The two components were genetically correlated. Sexual maturation was also highly heritable. However, correlations to both androstenone and skatole were significantly unfavourable.     

Effect of polymorphism in the porcine cytochrome b5 ( CYB5A) gene on androstenone and skatole concentrations and sexual development in Swedish pig populations

The present study investigated the presence of a single-nucleotide polymorphism (G > T) at base -8 upstream of ATG in 5' 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 × Landrace crossbred pigs (n = 245), 6.5% for Swedish Yorkshire (n = 99) and 12.8% for Landrace breed (n = 74). No deviations from Hardy-Weinberg equilibrium were observed in the investigated populations. In Swedish Yorkshire × Landrace crossbred entire male pigs (n = 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 = 0.006). In heavier pigs, plasma androstenone levels did not differ between genotypes (P = 0.382). Fat androstenone levels were not affected by CYB5A genotype (P = 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 = 0.048) and fat (P = 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.     

Analysis of the genetics of boar taint reveals both single SNPs and regional effects

Background

Boar taint is an offensive urine or faecal-like odour, affecting the smell and taste of cooked pork from some mature non-castrated male pigs. Androstenone and skatole in fat are the molecules responsible. In most pig production systems, males, which are not required for breeding, are castrated shortly after birth to reduce the risk of boar taint. There is evidence for genetic variation in the predisposition to boar taint.A genome-wide association study (GWAS) was performed to identify loci with effects on boar taint. Five hundred Danish Landrace boars with high levels of skatole in fat (>0.3 μg/g), were each matched with a litter mate with low levels of skatole and measured for androstenone. DNA from these 1,000 non-castrated boars was genotyped using the Illumina PorcineSNP60 Beadchip. After quality control, tests for SNPs associated with boar taint were performed on 938 phenotyped individuals and 44,648 SNPs. Empirical significance thresholds were set by permutation (100,000). For androstenone, a ‘regional heritability approach’ combining information from multiple SNPs was used to estimate the genetic variation attributable to individual autosomes.

Results

A highly significant association was found between variation in skatole levels and SNPs within the CYP2E1 gene on chromosome 14 (SSC14), which encodes an enzyme involved in degradation of skatole. Nominal significance was found for effects on skatole associated with 4 other SNPs including a region of SSC6 reported previously. Genome-wide significance was found for an association between SNPs on SSC5 and androstenone levels and nominal significance for associations with SNPs on SSC13 and SSC17. The regional analyses confirmed large effects on SSC5 for androstenone and suggest that SSC5 explains 23% of the genetic variation in androstenone. The autosomal heritability analyses also suggest that there is a large effect associated with androstenone on SSC2, not detected using GWAS.

Conclusions

Significant SNP associations were found for skatole on SSC14 and for androstenone on SSC5 in Landrace pigs. The study agrees with evidence that the CYP2E1 gene has effects on skatole breakdown in the liver. Autosomal heritability estimates can uncover clusters of smaller genetic effects that individually do not exceed the threshold for GWAS significance.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-424) contains supplementary material, which is available to authorized users.     

Cloning and mapping of the porcine cytochrome-p450 2E1 gene and its association with skatole levels in the domestic pig

The porcine cytochrome-p450 2E1 (CYP2E1) gene was isolated by screening a pig BAC library and partially sequenced. This sequence information was used to identify six single nucleotide polymorphisms (SNPs) within the CYP2E1 gene and its promoter. In addition, a microsatellite marker tightly linked to the CYP2E1 gene was subcloned from the BAC. One of these markers was used to map the CYP2E1 gene distal of SWC27 on SSC14, well outside reported quantitative trait loci on SSC14 for skatole, indole and taste test measures of boar taint. However, in a population of commercial pigs scored for backfat skatole levels, there was evidence of association between a SNP in the CYP2E1 promoter and skatole deposition, although there was no significant association between this SNP and skatole levels in the experimental cross.     

Gender-related differences in the formation of skatole metabolites by specific CYP450 in porcine hepatic S9 fractions

Higher accumulation of skatole in the fat of male pigs compared with female pigs might be due to gender-related differences in the rate of skatole degradation. In the present study, skatole metabolites and cytochrome P450 (CYP450) isoforms involved in skatole metabolism were for the first time investigated in hepatic S9 fractions from six male and four female pigs (crossbred Landrace×Yorkshire dams and Duroc boar). Surprisingly, the rates of production of major skatole metabolites were similar in male and female pigs. The most abundant metabolite of skatole was 3-hydroxy-3-methyloxindole (HMOI) followed by 3-methyloxindole and indole-3-carbinol in both male and female S9 fractions. Concentrations of formed HMOI and 3-methyloxindole did not differ between the genders (P=0.124 for HMOI, and P=0.575 for 3-methyloxindole). Indole-3-carbinol formation was higher in S9 fractions from the females compared with male pigs (P=0.0001). Enzyme kinetic parameters were similar for both genders (P>0.05). In both male and female pigs, ellipticine, diallyl sulphide (DAS) and quercetin inhibited HMOI formation, confirming the involvement of CYP1A1 and CYP2E1. The formation of 3-methyloxindole was reduced in the presence of the CYP2E1 inhibitor DAS, and formation of indole-3-carbinol was reduced in the presence of CYP1A1 and CYP2A19 inhibitors. We found only minor differences in skatole metabolism between male and female pigs, particularly the involvement of CYP2C and CYP3A in indole-3-carbinol formation in female but not in male pigs. This is a very essential finding, suggesting the involvement of larger number of CYP450 isoforms in female pigs. On the other hand, indole-3-carbinol is a minor skatole metabolite, and the physiological significance of CYP2C and CYP3A involvement in its formation in female pigs, but not in male pigs, needs to be elucidated. Our results, however, should be interpreted with caution because of the low number of animals and possibility of breed and age effects on skatole metabolism.     

Use of data mining techniques to investigate disease risk classification as a proxy for compromised biosecurity of cattle herds in Wales

Background  

Boar taint is the unpleasant odour and flavour of the meat of uncastrated male pigs that is primarily caused by high levels of androstenone and skatole in adipose tissue. Androstenone is a steroid and its levels are mainly genetically determined. Studies on androstenone metabolism have, however, focused on a limited number of genes. Identification of additional genes influencing levels of androstenone may facilitate implementation of marker assisted breeding practices. In this study, microarrays were used to identify differentially expressed genes and pathways related to androstenone metabolism in the liver from boars with extreme levels of androstenone in adipose tissue.     

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism

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.     

Weight and season affects androstenone and skatole occurrence in entire male pigs in organic pig production

To investigate the extent to which the level of androstenone and skatole decreases with a decrease in live weight and/or age at slaughter of entire male pigs produced under organic standards, 1174 entire male pigs were raised in parallel in five organic herds, distributed across four batches in summer and winter. The median androstenone level was high for organic entire male pigs (1.9 µg/g), but varied greatly both within and between herds. Median skatole level was 0.05 µg/g, also with a wide range both within and between herds. Decreasing live weight over the range of 110±15.6 kg s.d. was found to decrease androstenone as well as skatole concentration, however, with different patterns of association. Age did not have significant direct effect on either androstenone or skatole levels. Androstenone levels were higher during winter than summer (P<0.0001), but no difference in skatole was found between seasons. The study concludes that decreasing live weight at slaughter could be an applicable management tool to reduce risk of boar taint and the level of tainted carcasses for a future production of entire male pigs within the organic pig production system, although further studies are needed as great variation in boar taint was found also for low weight animals.     

Rapid high-performance liquid chromatographic method for simultaneous determination of androstenone, skatole and indole in back fat from pigs

A rapid high-performance reversed-phase liquid chromatographic method for the simultaneous quantitative determination of the main boar taint compounds androstenone, skatole (3-methylindole) and indole, in back fat from pigs has been developed. The compounds are extracted by a simple homogenisation of adipose tissue in methanol; interfering lipids are removed by precipitation after cooling and centrifugation. Androstenone is derivatized pre-column with dansylhydrazine (5 min at ambient temperature) using BF₃ as catalyst. The compounds are separated on a 60 × 4.6 mm I.D., 3 μm Hypersil ODS column (Hewlett-Packard) using a step-gradient; total time for the separation is 15 min. Fluorescence is used for selective detection. The limit of quantitation for indole and skatole is 30 ng/g and for androstenone 200 ng/g back fat. The results for skatole obtained by the present method were compared with those of a colorimetric method, while androstenone determinations were compared with two GC—MS methods and a RIA method. The correlations observed were in the range of 0.946–0.993. The average contents of androstenone and skatole were 640 ± 700 and 78 ± 113 ng/g (n = 1162 male pigs), respectively.     

猪Gli1基因的克隆、表达谱分析及脂肪组织特异性表达载体的构建

Hedgehog(Hh)信号通路对动物脂肪沉积具有抑制作用,并且从果蝇到脊椎动物具有高度保守性,但在家猪研究中鲜见报道。文章选择家猪Hh通路的转录激活因子Gli1进行研究,通过RT-PCR结合RACE技术,首次获得家猪Gli1基因cDNA全长,利用Real-time PCR对家猪Gli1基因在不同组织中的表达丰度进行了分析,并构建了真核表达载体和脂肪组织特异性表达载体。结果表明:猪Gli1基因cDNA全长3 576 bp,基因组序列全长10 715 bp,共12个外显子,编码1 106个氨基酸。生物信息学分析表明,猪Gli1为不稳定亲水性蛋白,不具有跨膜结构域和信号肽序列,但具有锌指结构与核定位序列。对7个物种的Gli1蛋白序列和基因组序列相似性进行分析,发现各物种间序列相似性均在80%以上,说明Gli1在物种间高度保守。组织表达谱分析表明,Gli1仅在成体猪舌组织中表达;在家猪脂肪组织发育进程中,Gli1仅在出生1周的猪脂肪组织中检测到微弱表达,但1月龄及3月龄猪脂肪组织中均检测不到表达,由此推断猪Gli1表达与脂肪组织发育呈负相关。最后,将猪Gli1编码区克隆到真核表达载体pIRES2-EGFP,体外转染实验证明该载体能够正确表达猪Gli1,另外还构建了脂肪组织特异性表达载体,为构建脂肪组织特异性转基因动物奠定基础。