Pyrrolooxygenases from Argentine wheat varieties

Pyrrolooxygenase activities were examined in different varieties of Argentine wheat (Triticum aestivum) which included the traditional Klein varieties and the new mixed Mexican and traditional varieties (DeKalb and Cargill). The enzymatic activities were variety-dependent and were more inhibited in some varieties than in others, while some (Cargill) were devoid of the proteic inhibitor. The enzymes were isolated from the flours as two isoenzymes of different charge whose relative proportions were dependent on the variety of wheat used. The more cationic isoenzymes were eluted with 10 mM Tris-HCl buffer (pH 7.6 from DEAE-cellulose and the less cationic were eluted with 50 mM NACl in the same buffer. The protein inhibitor, when present, was associated with the more cationic isoenzymes. Porphobilinogen oxygenase and skatole pyrrolooxygenase activities were higher in the endosperm, while tryptophan pyrrolooxygenase activity was higher in the embryo. The proteic inhibitors were mainly concentrated in the embryo.     

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.     

Pyrrolooxygenase isoenzymes from wheat germ

Both porphobilinogen oxygenase and skatole pyrrolooxygenase of wheat germ have isoenzyme forms of different charge. The more cationic isoenzymes were eluted from DEAE-cellulose with 10 mM Tris-HCl buffer (pH 7.6) and the less cationic were eluted with 50 mM NACl in the same buffer. The former had almost twice as many free amino groups (per mg of protein) as the latter. The more cationic isoenzyme was more sensitive to chelating agents and to acid treatment. They were differently inhibited by sodium dodecyl treatment and by temperature inactivation. Porphobilinogen oxygenase isoenzymes showed different activities with different buffers and also differed in their kinetics.     

How does barley supplementation in lambs grazing alfalfa affect meat sensory quality and authentication?

Excessive flavour in lamb meat is undesirable for consumers and can prompt purchase resistance. Volatile indoles responsible for off-flavours accumulate more in the fat of lambs on pasture than on grain and are enhanced when lambs graze alfalfa. Here, we investigated whether barley supplementation of lambs grazing alfalfa influences meat sensory quality. Using three groups of 12 male Romane lambs, we compared three feeding regimes: alfalfa grazing (AG), alfalfa grazing + daily supplementation with barley (29 g/kg live weight^0.75, AGS) and stall feeding with concentrate and hay (SF). As some of the compounds involved in meat sensory traits may act as dietary biomarkers, we also investigated potential implications for meat authentication. Although barley represented 38% of the diet in AGS lambs, it did not offer any advantage for animal average daily gain or parasitism level. Animal performance, carcass weight and fatness did not differ between feeding regimes. Dorsal fat firmness tended to be greater in AG than AGS and greater in AGS than SF. Skatole and indole concentrations in perirenal and dorsal fat were lower in SF lambs than in AG and AGS lambs (P<0.01 to P<0.0001), but did not differ between AG and AGS lambs. Yellowness, chroma and hue angle of perirenal fat were lower in SF lambs than in AG and AGS lambs (P<0.001), but did not differ between AG and AGS lambs. Absolute value of the mean integral for both perirenal fat and subcutaneous caudal fat (AVMI_PF and AVMI_SC), quantifying the intensity of light absorption by carotenoids in perirenal and subcutaneous caudal fat, respectively, were lower in SF lambs than in AG and AGS lambs (P<0.0001 for both comparisons), but did not differ between AG and AGS lambs. Meat colour was unaffected by the treatment. We confirm that lambs grazing alfalfa accumulate high levels of volatile indoles in their fat, but we show that barley supplementation to lambs grazing alfalfa is not effective in reducing fat volatile indoles concentration and excessive odour/flavour in the meat. We also confirm that both perirenal fat skatole concentration and AVMI_PF are of interest for discriminating lambs that grazed alfalfa from lambs that were stall-fed, and we show that they are not effective for discriminating supplemented from non-supplemented grazing lambs.     

Olfactory memory in the mosquito Culex quinquefasciatus

The cosmotropical urban mosquito Culex quinquefasciatus Say (Diptera: Culicidae) uses chemical cues to locate suitable water pools for oviposition. Although gravid females are innately attracted to or repelled by certain compounds, this study found that an individual mosquito's preferences for these odours could be altered greatly by prior experience. Mosquitoes reared in water containing skatole, at a level normally repellent to ovipositing females, preferred to oviposit in water containing that compound rather than in water with an otherwise attractive odour compound (P-cresol). This behaviour occurred regardless of whether mosquitoes were tested individually or in groups of up to 50 per cage. The F1 progeny of conditioned mosquitoes did not exhibit the parental preference, but were as susceptible to conditioning as their parents. Moreover, rearing mosquitoes in infusions of hay or animal (guinea-pig) faeces produced a similar although less dramatic change, such that the innate propensity for hay infusion could be cancelled by rearing in guinea-pig faeces infusion. The results demonstrated a change in odour preference by Cx. quinquefasciatus following exposure to the odour during development or pupal eclosion, suggesting that some form of larval conditioning or early adult imprinting occurred. Precisely when that conditioning occurred remains to be determined.     

Molecular cloning, expression and functional characterization of the cytochrome P450 2A6 gene in pig liver

Raising intact male pigs would have a significant economic impact on the pork industry because intact males have improved feed efficiency and a greater lean yield of the carcass compared with barrows. However, the presence of skatole, a major cause of boar taint, in meat from intact male pigs could be highly objectionable to consumers. It has been shown that CYP2A6 is a key enzyme in the hepatic metabolism of skatole and that the activity of CYP2A6 is negatively correlated with skatole accumulation in fat. The aim of this study was to isolate and characterize CYP2A6 from pig liver, as well as identify genetic polymorphisms in the CYP2A6 gene, and examine the association between these polymorphisms and skatole level. We identified a single base deletion in CYP2A6, resulting in a frame shift in the coding region that produces a non-functional enzyme, which was associated with high levels of skatole in fat tissue.     

Production of indolic compounds by rumen bacteria isolated from grazing ruminants

AIM: To screen rumen bacterial cultures and fresh ruminal isolates for indole and skatole production. METHODS AND RESULTS: Culture collection strains and fresh bacterial isolates from rumen contents of sheep and dairy cows were screened for the production of indolic compounds. Clostridium aminophilum FT, Peptostreptococcus ssp. S1, Fusobacterium necrophorum D4 produced indole and Clostridium sticklandii SR produced indoleacetic acid. Fresh isolates from sheep (TrE9262 and TrE7262) and dairy cows (152R-1a, 152R-1b, 152R-3 and 152R-4) produced indole, indolepropionic acid, tryptophol and skatole from the fermentation of tryptophan and indoleacetic acid. Glucose altered the indolic compounds produced in some, but not all, isolates. TrE7262 and 152R-4 were identified as Clostridium sporogenes and 152R-1b as a new Cl. aminophilum strain. Isolates TrE9262, 152R-1a and 152R-3 were not closely related to any described species but belong to Megasphaera, Prevotella and Actinomyces genera, respectively. CONCLUSIONS: Rumen bacteria that produced a range of indolic compounds were identified. Some isolates are distinct from the previously described bacteria and may represent novel species. SIGNIFICANCE AND IMPACT OF THE STUDY: These observations will contribute to understanding skatole and indole formation in the rumen and will lead to methods that control the formation of indolic compounds in pasture-grazed ruminants.     

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.     

Efficacy of non-nutritive sorbent materials as intestinal-binding agents for the control of boar taint

In many countries, male pigs are castrated to prevent boar taint, but this practice raises concerns about animal welfare and reduces the production efficiency of pork. The objective of this study was to develop dietary manipulations to prevent boar taint. We evaluated the effectiveness of adding activated carbon (AC) or Tween-60 (Tween; polyoxyethylene sorbitan monostearate) to pig finishing diets to reduce levels of androstenone (AND) and skatole in plasma and fat of entire male pigs. Boars (159 ± 2 days of age at the start of the experiment) were fed diets supplemented with either 5% AC or 5% Tween for 28 days followed by either 14 or 28 days of recovery. Plasma samples were collected at experimental days 0, 7, 14, 21, 28, 42 and 56, and backfat biopsies were taken at experimental days 0, 28, 42 and 56. Feeding AC significantly (P < 0.05) reduced the levels of AND in plasma by day 28 compared to day 0 and by day 42 in fat compared to day 0. AC treatment also decreased levels of oestrone sulphate (E(1)S) in plasma by day 7 compared to day 0. Treatment with Tween significantly decreased (P < 0.05) the levels of plasma AND by day 28 from levels at day 0. Tween treatment did not significantly affect levels of fat AND or plasma E(1)S compared to day 0; however, fat AND levels decreased between days 28 and 42 following treatment with Tween (P < 0.05). Levels of plasma E(1)S, plasma AND and fat AND for control boars remained constant throughout the experiment. Skatole plasma concentrations were very low and did not vary significantly (P > 0.05) from day 0 for any treatment, but fat skatole levels decreased by day 42 in the Tween treatment group. Importantly, there was no difference in growth rate between the control and experimental groups. We conclude that adding AC or Tween to finishing diets for boars can reduce the levels of plasma and fat AND, but further work is needed to confirm the effects of these treatments on reducing fat skatole levels.