Effects of dietary selenized glucose on intestinal microbiota and tryptophan metabolism in rats: Assessing skatole reduction potential.

3-Methylindole (Skatole), a degradation product of tryptophan produced by intestinal microbial activity, significantly contributes to odor nuisance. Its adverse effects on animal welfare, human health, and environmental pollution have been noted. However, it is still unclear whether the intestinal microbiota mediates the impact of selenium (Se) on skatole production and what the underlying mechanisms remain elusive. A selenized glucose (SeGlu) derivative is a novel organic selenium compound. In this study, a diverse range of dietary SeGlu-treated levels, including SeGlu-deficient (CK), SeGlu-adequate (0.15 mg Se per L), and SeGlu-supranutritional (0.4 mg Se per L) conditions, were used to investigate the complex interaction of SeGlu on intestinal microbiome and serum metabolome changes in male Sprague-Dawley (SD) rats. The study showed that SeGlu supplementation enhanced the antioxidant ability in rats, significantly manifested in the increases of the activity of catalase (CAT) and glutathione peroxidase (GSH-Px), while no change in the level of malonaldehyde (MDA). Metagenomic sequencing analysis verified that the SeGlu treatment group significantly increased the abundance of beneficial microorganisms such as Clostridium, Ruminococcus, Faecalibacterium, Lactobacillus, and Alloprevotella while reducing the abundance of opportunistic pathogens such as Bacteroides and Alistipes significantly. Further metabolomic analysis revealed phenylalanine, tyrosine, and tryptophan biosynthesis changes in the SeGlu treatment group. Notably, the biosynthesis of indole, a critical pathway, was affected by SeGlu treatment, with several crucial enzymes implicated. Correlation analysis demonstrated strong associations between specific bacterial species - Treponema, Bacteroides, and Ruminococcus, and changes in indole and derivative concentrations. Moreover, the efficacy of SeGlu-treated fecal microbiota was confirmed through fecal microbiota transplantation, leading to a decrease in the concentration of skatole in rats. Collectively, the analysis of microbiota and metabolome response to diverse SeGlu levels suggests that SeGlu is a promising dietary additive in modulating intestinal microbiota and reducing odor nuisance in the livestock and poultry industry.

A study on the association between gut microbiota, inflammation, and type 2 diabetes.

Type 2 diabetes mellitus (T2DM) was reported to be associated with impaired immune response and alterations in microbial composition and function. However, the underlying mechanism remains elusive. To investigate the association among retinoic acid-inducible gene-I-like receptors (RLRs) signaling pathway, intestinal bacterial microbiome, microbial tryptophan metabolites, inflammation, and a longer course of T2DM, 14 patients with T2DM and 7 healthy controls were enrolled. 16S rRNA amplicon sequencing and untargeted metabolomics were utilized to analyze the stool samples. RNA sequencing (RNA-seq) was carried out on the peripheral blood samples. Additionally, C57BL/6J specific pathogen-free (SPF) mice were used. It was found that the longer course of T2DM could lead to a decrease in the abundance of probiotics in the intestinal microbiome. In addition, the production of microbial tryptophan derivative skatole declined as a consequence of the reduced abundance of related intestinal microbes. Furthermore, low abundances of probiotics, such as Bacteroides and Faecalibacterium, could trigger the inflammatory response by activating the RLRs signaling pathway. The increased level of the member of TNF receptor-associated factors (TRAF) family, nuclear factor kappa-B (NF-κB) activator (TANK), in the animal colon activated nuclear factor kappa B subunit 2 (NFκB2), resulting in inflammatory damage. In summary, it was revealed that the low abundances of probiotics could activate the RLR signaling pathway, which could in turn activate its downstream signaling pathway, NF-κB, highlighting a relationship among gut microbes, inflammation, and a longer course of T2DM. KEY POINTS: Hyperglycemia may suppress tryptophanase activity. The low abundance of Bacteroides combined with the decrease of Dopa decarboxylase (DDC) activity may lead to the decrease of the production of tryptophan microbial derivative skatole, and the low abundance of Bacteroides or reduced skatole may further lead to the increase of blood glucose by downregulating the expression of glucagon-like peptide-1 (GLP1). A low abundance of anti-inflammatory bacteria may induce an inflammatory response by triggering the RLR signaling pathway and then activating its downstream NF-κB signaling pathway in prolonged T2DM.

Microbiome-metabolomics analysis reveals abatement effects of itaconic acid on odorous compound production in Arbor Acre broilers.

BACKGROUND: Public complaints concerning odor emissions from intensive livestock and poultry farms continue to grow, as nauseous odorous compounds have adverse impacts on the environment and human health. Itaconic acid is a metabolite from the citric acid cycle of the host and shows volatile odor-reducing effects during animal production operations. However, the specific role of itaconic acid in decreasing intestinal odorous compound production remains unclear. A total of 360 one-day-old chicks were randomly divided into 6 treatment groups: control group (basal diet) and itaconic acid groups (basal diet + 2, 4, 6, 8 and 10 g/kg itaconic acid). The feeding experiment lasted for 42 d. RESULTS: Dietary itaconic acid supplementation linearly and quadratically decreased (P < 0.05) the cecal concentrations of indole and skatole but did not affect (P > 0.05) those of lactic, acetic, propionic and butyric acids. The cecal microbial shift was significant in response to 6 g/kg itaconic acid supplementation, in that the abundances of Firmicutes, Ruminococcus and Clostridium were increased (P < 0.05), while those of Bacteroidetes, Escherichia-Shigella and Bacteroides were decreased (P < 0.05), indicative of increased microbial richness and diversity. Furthermore, a total of 35 significantly (P < 0.05) modified metabolites were obtained by metabolomic analysis. Itaconic acid decreased (P < 0.05) the levels of nicotinic acid, nicotinamide, glucose-6-phosphate, fumatic acid and malic acid and increased (P < 0.05) 5-methoxytroptomine, dodecanoic acid and stearic acid, which are connected with the glycolytic pathway, citrate acid cycle and tryptophan metabolism. Correlation analysis indicated significant correlations between the altered cecal microbiota and metabolites; Firmicutes, Ruminococcus and Clostridium were shown to be negatively correlated with indole and skatole production, while Bacteroidetes, Escherichia-Shigella and Bacteroides were positively correlated with indole and skatole production. CONCLUSIONS: Itaconic acid decreased cecal indole and skatole levels and altered the microbiome and metabolome in favor of odorous compound reduction. These findings provide new insight into the role of itaconic acid and expand its application potential in broilers.

Activation of PGRN/MAPK axis stimulated by the hypoxia-conditioned mesenchymal stem cell-derived HIF-1α facilitates osteosarcoma progression.

BACKGROUND: Progranulin (PGRN) is an important survival factor in the progression of multiple cancers. PURPOSE: To explore the effects and mechanisms of PGRN on malignant biological behavior of osteosarcoma (OS) cells and the effects of mesenchymal stem cells (MSCs) and the hypoxic microenvironment on PGRN alteration. MATERIAL AND METHODS: The expression pattern of PGRN in OS were evaluated in OS tissues and cell lines. Next, a loss-of-function assay investigated the function of PGRN on the proliferation, migration and cell death of OS cells. The activation of MAPK signaling in the process was examined by western blot and functional experiments accompanied by skatole. Additionally, we internally silenced hypoxia-inducible factor-1α (HIF-1α) in MSCs along with exogenously added HIF-1α (exo-HIF-1α) to explore how MSCs affect PGRN alteration and the malignant behavior of OS cells. RESULTS: An aberrantly high expression of PGRN was observed in OS and associated with the poor prognosis of OS patients. PGRN knockdown repressed the proliferation, migration and induced cell death of OS cells, and activating MAPK pathway reversed these effects. Further evidence showed that MSCs regulated PGRN to mediate the malignant biological behavior of OS cells. Hypoxia enhanced HIF-1α expression in MSCs. HIF-1α silencing in MSCs under hypoxia suppressed the oncogenic effects of MSCs and reduced PGRN expression in OS cells, while the treatment of exo-HIF-1α reversed the depressive effects of HIF1α silencing on OS progression. CONCLUSION: Overall, we concluded that PGRN, which was activated by the increase of hypoxic-MSCs-derived HIF-1α, promoted OS progression through the activation of MAPK signaling.

2-Oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole, a contributor to the odour of boar-tainted pork meat, mediated by porcine liver microsomal cytochromes P450 1A2, 2A19, 2E1, and 3A22.

The 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole (a contributor to boar taint) mediated by minipig liver microsomes and recombinant P450 enzymes expressed in bacterial membranes were investigated.At low substrate concentrations of 10 µM, the formation rates of indole-3-carbinol, 6-hydroxyskatole, and the sum of 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole in male minipig liver microsomes were significantly lower than those in female minipig liver microsomes.Compensatory 3-methyloxindole and indole-3-carbinol formation in minipig liver microsomes, which lack 6-hydroxyskatole formation in males, was mediated partly by liver microsomal P450 1A2 and P450 1A2/2E1, respectively. These enzymes were suppressed by typical P450 inhibitors in female minipig liver microsomes.Among the 14 pig P450 forms evaluated, P450 2A19 was the dominant form mediating 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole formation from skatole at substrate concentrations of 100 µM. Positive cooperativity was observed in 3-methyloxindole formation from skatole mediated by male minipig liver microsomes and by pig P450 3A22 with Hill coefficients of 1.2-1.5.These results suggest high skatole 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation activities of pig P450 2A19 and compensatory skatole oxidations mediated by pig P450 1A2, 2E1, or 3A22 in male minipig liver microsomes.

Skatole-induced p38 and JNK activation coordinately upregulates, whereas AhR activation partially attenuates TNFα expression in intestinal epithelial cells.

Increased tumor necrosis factor α (TNFα) expression in intestinal epithelial cells (IECs) plays a major role in the development and progression of inflammatory bowel disease (IBD) and colorectal cancer (CRC). The present study aimed to clarify the relationship between TNFα and skatole, a tryptophan-derived gut microbiota metabolite. The aryl hydrocarbon receptor (AhR) antagonist CH223191 promoted, whereas the p38 inhibitor SB203580 suppressed the increase in TNFα mRNA and protein expression induced by skatole in intestinal epithelial Caco-2 cells. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 repressed only the increased TNFα protein expression, whereas the extracellular signal-regulated kinase (ERK) pathway inhibitor U0126 did not affect increased TNFα expression at any level. A neutralizing antibody against TNFα partially inhibited skatole-induced cell death. Overall, these results suggested that TNFα expression is increased by the concerted actions of skatole-activated p38 and JNK, and that TNFα exerts autocrine/paracrine actions on IECs despite partial suppression by activated AhR. Therefore, skatole might play an important role in the development and progression of IBD and CRC via increased TNFα expression.

Transcriptomic profiling reveals the molecular responses of Rhodococcus aetherivorans DMU1 to skatole stress.

Skatole is a typical malodor compound in animal wastes. Several skatole-degrading bacterial strains have been obtained, whereas the molecular response of strains to skatole stress has not been well elucidated. Herein, the skatole degradation by a Gram-positive strain Rhodococcus aetherivorans DMU1 was investigated. Strain DMU1 showed high efficiency in skatole degradation under the conditions of 25-40 °C and pH 7.0-10.0. It could utilize various aromatics, including cresols, phenol, and methylindoles, as the sole carbon source for growth, implying its potential in the bioremediation application of animal wastes. Transcriptomic sequencing revealed that 328 genes were up-regulated and 640 genes were down-regulated in strain DMU1 when grown in the skatole-containing medium. Skatole increased the gene expression levels of antioxidant defense systems and heat shock proteins. The expression of ribosome-related genes was significantly inhibited which implied the growth inhibition of skatole. A rich set of oxidoreductases were changed, and a novel gene cluster containing the flavoprotein monooxygenase and ring-hydroxylating oxygenase genes was highly up-regulated, which was probably involved in skatole upstream degradation. The upregulation pattern of this gene cluster was further verified by qRT-PCR assay. Furthermore, skatole should be mainly degraded via the catechol ortho-cleavage pathway with cat25170 as the functional gene. The gene cat25170 was cloned and expressed in E. coli BL21(DE3). Pure enzyme assays showed that Cat25170 could catalyze catechol with Km 9.96 µmol/L and kcat 12.36 s-1.

Unraveling the skatole biodegradation process in an enrichment consortium using integrated omics and culture-dependent strategies.

3-Methylindole (skatole) is regarded as one of the most offensive compounds in odor emission. Biodegradation is feasible for skatole removal but the functional species and genes responsible for skatole degradation remain enigmatic. In this study, an efficient aerobic skatole-degrading consortium was obtained. Rhodococcus and Pseudomonas were identified as the two major and active populations by integrated metagenomic and metatranscriptomic analyses. Bioinformatic analyses indicated that the skatole downstream degradation was mainly via the catechol pathway, and upstream degradation was likely catalyzed by the aromatic ring-hydroxylating oxygenase and flavin monooxygenase. Genome binning and gene analyses indicated that Pseudomonas, Pseudoclavibacter, and Raineyella should cooperate with Rhodococcus for the skatole degradation process. Moreover, a pure strain Rhodococcus sp. DMU1 was successfully obtained which could utilize skatole as the sole carbon source. Complete genome sequencing showed that strain DMU1 was the predominant population in the consortium. Further crude enzyme and RT-qPCR assays indicated that strain DMU1 degraded skatole through the catechol ortho-cleavage pathway. Collectively, our results suggested that synergistic degradation of skatole in the consortium should be performed by diverse bacteria with Rhodococcus as the primary degrader, and the degradation mainly proceeded via the catechol pathway.

Mechanistic Studies of a Skatole-Forming Glycyl Radical Enzyme Suggest Reaction Initiation via Hydrogen Atom Transfer.

Gut microbial decarboxylation of amino acid-derived arylacetates is a chemically challenging enzymatic transformation which generates small molecules that impact host physiology. The glycyl radical enzyme (GRE) indoleacetate decarboxylase from Olsenella uli (Ou IAD) performs the non-oxidative radical decarboxylation of indole-3-acetate (I3A) to yield skatole, a disease-associated metabolite produced in the guts of swine and ruminants. Despite the importance of IAD, our understanding of its mechanism is limited. Here, we characterize the mechanism of Ou IAD, evaluating previously proposed hypotheses of: (1) a Kolbe-type decarboxylation reaction involving an initial 1-e- oxidation of the carboxylate of I3A or (2) a hydrogen atom abstraction from the α-carbon of I3A to generate an initial carbon-centered radical. Site-directed mutagenesis, kinetic isotope effect experiments, analysis of reactions performed in D2O, and computational modeling are consistent with a mechanism involving initial hydrogen atom transfer. This finding expands the types of radical mechanisms employed by GRE decarboxylases and non-oxidative decarboxylases, more broadly. Elucidating the mechanism of IAD decarboxylation enhances our understanding of radical enzymes and may inform downstream efforts to modulate this disease-associated metabolism.

Novel ECL Method for the Determination of Skatole in Porcine Adipose Tissue.

A new method for the determination of skatole present in porcine adipose tissue samples utilizing the electrochemiluminescence of skatole is presented. It has been observed that oxygen radicals produced at a high cathodic voltage can react with oxidized skatole to create an excited intermediate molecule that then relaxes, generating peak photon emission at around 480 nm. A strong electrochemiluminescence or electrogenerated chemiluminescence (ECL) signal using boron-doped diamond (BDD) electrodes was observed optimally when a reduction potential of -1.8 V was applied, held for 40 s, before holding an oxidation potential of 0.8 V for 10 s. Using this principle, a calibration curve using known concentrations of skatole showed good linearity (range 0.025-2 µM) and a very low detection limit (LOD, 0.7 nM). A method that demonstrates for the first time an approach that utilizes this ECL reaction, and has the potential to be developed into an analytical device for use in the slaughterhouse, has been developed. This was achieved by extracting skatole out of the porcine adipose tissues into acetonitrile - giving an extraction efficiency of 67.6%. This method was then validated by analyzing the skatole content of 33 pig fat samples that had been previously tested using a standard technique, high-performance liquid chromatography (HPLC), containing a range of concentrations (0.02-2.58 µg/g). This ECL method exhibited excellent reliability and correlation with HPLC, giving a R2 coefficient of 0.911, thus demonstrating the potential for this method to be developed for an on-line skatole detector.

Development of an analytical method for the determination of sterol compounds in boars' saliva.

"Boar taint" compounds influence the sexual behavioral responses of sows and stimulate their reproduction. This paper reports a fast, easier, and a non-invasive analytical method for the analysis of three "boar taint" compounds in boar' saliva samples: androstenone, androsten-3α-ol, and androsten-3ß-ol. This method was developed and validated based on solid-phase extraction (SPE) and multidimensional gas chromatography-mass spectrometry (MDGC-MS). All the compounds were detected without derivatization. This method affords good reproducibility (4%-8%), accuracy (80%-105%), precision (5.5%-9.1%), linearity (R2 = 0.98-0.99), and lower limits of quantitation (LLOQ) (0.1-0.2 µg/L). Although the presence of these compounds in saliva has been known for a long time, no simple and easy analytical method has been developed.

Dysregulation of murine immune functions on inhalational exposure to ammonia, dimethyl disulfide, 3-methylindole, or propionic acid.

Animal husbandry workers are exposed to various malodorous compounds in the workplace. Although these compounds cause severe nuisance, no systemic investigation of their effects on the immune system has been conducted. To address this issue, we evaluated the effects of inhalational exposure to ammonia, dimethyl disulfide, 3-methylindole (3-MI), and propionic acid (PA), representing four major groups of malodorous compounds, on humoral and cellular immunity in mice. Mice were exposed to the substances (low dose: 10 µL and high dose: 200 µL) for 10 min/day for 4 weeks in a modified standard mouse cage. Neutrophil% and splenic cytotoxic T cell% were significantly lower in the high-dose ammonia group than in the vehicle control. Exposure to ammonia and 3-MI increased immature thymic T lymphocyte% relative to control and concomitantly decreased both mature helper and cytotoxic T-cell populations in the thymus. In the ammonia exposure group, levels of serum immunoglobulin E and immunoglobulin A were elevated, and the IgG2a:IgG1 ratio in the serum was reduced in a dose-dependent manner. Splenic natural killer cell activity was significantly less in the PA exposure group than in the control. Overall, our findings suggest that inhalational exposure to these malodorous substances disturbs immune homeostasis in vivo.

Applying an association weight matrix in weighted genomic prediction of boar taint compounds.

Biological information regarding markers and gene association may be used to attribute different weights for single nucleotide polymorphism (SNP) in genome-wide selection. Therefore, we aimed to evaluate the predictive ability and the bias of genomic prediction using models that allow SNP weighting in the genomic relationship matrix (G) building, with and without incorporating biological information to obtain the weights. Firstly, we performed a genome-wide association studies (GWAS) in data set containing single- (SL) or a multi-line (ML) pig population for androstenone, skatole and indole levels. Secondly, 1%, 2%, 5%, 10%, 30% and 50% of the markers explaining the highest proportions of the genetic variance for each trait were selected to build gene networks through the association weight matrix (AWM) approach. The number of edges in the network was computed and used to derive weights for G (AWM-WssGBLUP). The single-step GBLUP (ssGBLUP) and weighted ssGBLUP (WssGBLUP) were used as standard scenarios. All scenarios presented predictive abilities different from zero; however, the great overlap in their confidences interval suggests no differences among scenarios. Most of scenarios of based on AWM provide overestimations for skatole in both SL and ML populations. On the other hand, the skatole and indole prediction were no biased in the ssGBLUP (S1) in both SL and ML populations. Most of scenarios based on AWM provide no biased predictions for indole in both SL and ML populations. In summary, using biological information through AWM matrix and gene networks to derive weights for genomic prediction resulted in no increase in predictive ability for boar taint compounds. In addition, this approach increased the number of analyses steps. Thus, we can conclude that ssGBLUP is most appropriate for the analysis of boar taint compounds in comparison with the weighted strategies used in the present work.

Genomic background and genetic relationships between boar taint and fertility traits in German Landrace and Large White.

BACKGROUND: Due to ethical reasons, surgical castration of young male piglets in their first week of life without anesthesia will be banned in Germany from 2021. Breeding against boar taint is already implemented in sire breeds of breeding organizations but in recent years a low demand made this trait economically less important. The objective of this study was to estimate heritabilities and genetic relationships between boar taint compounds androstenone and skatole and maternal/paternal reproduction traits in 4'924 Landrace (LR) and 4'299 Large White (LW) animals from nucleus populations. Additionally, genome wide association analysis (GWAS) was performed per trait and breed to detect SNP marker with possible pleiotropic effects that are associated with boar taint and fertility. RESULTS: Estimated heritabilities (h2) were 0.48 (±0.08) for LR (0.39 ± 0.07 for LW) for androstenone and 0.52 (±0.08) for LR (0.32 ± 0.07 for LW) for skatole. Heritabilities for reproduction did not differ between breeds except age at first insemination (LR: h2 = 0.27 (±0.05), LW: h2 = 0.34 (±0.05)). Estimates of genetic correlation (rg) between boar taint and fertility were different in LR and LW breeds. In LR an unfavorable rg of 0.31 (±0.15) was observed between androstenone and number of piglets born alive, whereas this rg in LW (- 0.15 (±0.16)) had an opposite sign. A similar breed-specific difference is observed between skatole and sperm count. Within LR, the rg of 0.08 (±0.13) indicates no relationship between the traits, whereas the rg of - 0.37 (±0.14) in LW points to an unfavorable relationship. In LR GWAS identified QTL regions on SSC5 (21.1-22.3 Mb) for androstenone and on SSC6 (5.5-7.5 Mb) and SSC14 (141.1-141.6 Mb) for skatole. For LW, one marker was found on SSC17 at 48.1 Mb for androstenone and one QTL on SSC14 between 140.5 Mb and 141.6 Mb for skatole. CONCLUSION: Knowledge about such genetic correlations could help to balance conventional breeding programs with boar taint in maternal breeds. QTL regions with unfavorable pleiotropic effects on boar taint and fertility could have deleterious consequences in genomic selection programs. Constraining the weighting of these QTL in the genomic selection formulae may be a useful strategy to avoid physiological imbalances.

Effects of dietary stachyose levels on caecal skatole concentration, hepatic cytochrome P450 mRNA expressions and enzymatic activities in broilers.

Effects of dietary supplemental stachyose on caecal skatole concentration, hepatic cytochrome P450 (CYP450, CYP) mRNA expressions and enzymatic activities in broilers were evaluated. Arbor Acre commercial mixed male and female chicks were assigned randomly into six treatments. The positive control (PC) diet was based on maize-soyabean meal, and the negative control (NC) diet was based on maize-non-soyabean meal. The NC diet was then supplemented with 4, 5, 6 and 7 g/kg stachyose to create experimental diets, named S-4, S-5, S-6 and S-7, respectively. Each diet was fed to six replicates of ten birds from days 1 to 49. On day 49, the caecal skatole concentrations in the PC, S-4, S-5, S-6 and S-7 groups were lower than those in the NC group by 42·28, 23·68, 46·09, 15·31 and 45·14 % (P < 0·01), respectively. The lowest pH value was observed in the S-5 group (P < 0·05). The stachyose-fed groups of broilers had higher caecal acetate and propionate levels compared with control groups, and propionate levels in the S-6 and S-7 groups were higher than those in the S-4 and S-5 groups (P < 0·001). The highest CYP3A4 expression was found in the S-7 group (P < 0·05), but this was not different from PC, S-4, S-5 and S-6 treatments. There was no significant difference in CYP450 (1A2, 2D6 and 3A4) enzymatic activities among the groups (P > 0·05). In conclusion, caecal skatole levels can be influenced by dietary stachyose levels, and 5 g/kg of stachyose in the diet was suggested.

Biodegradation of skatole by Burkholderia sp. IDO3 and its successful bioaugmentation in activated sludge systems.

Skatole is the key malodorous compound in livestock and poultry waste and wastewater with a low odor threshold. It not only causes serious nuisance to residents and workers, but also poses threat to the environment and human health due to its biotoxicity and recalcitrant nature. Biological treatment is an eco-friendly and cost-effective approach for skatole removal, while the bacterial resources are scarce. Herein, the Burkholderia strain was reported to efficiently degrade skatole for the first time. Results showed that strain IDO3 maintained high skatole-degrading performance under the conditions of pH 4.0-9.0, rotate speed 0-250 rpm, and temperature 30-35 °C. RNA-seq analysis indicated that skatole activated the oxidative phosphorylation and ATP production levels in strain IDO3. The oxidoreductase activity item which contained 373 differently expressed genes was significantly impacted by Gene Ontology analysis. Furthermore, the bioaugmentation experiment demonstrated that strain IDO3 could notably increase the removal of skatole in activated sludge systems. High-throughput 16S rRNA gene sequencing data indicated that the alpha-diversity and bacterial community tended to be stable in the bioaugmented group after 8 days operation. PICRUSt analysis indicated that xenobiotics biodegradation and metabolism, and membrane transport categories significantly increased, consistent with the improved skatole removal performance in the bioaugmented group. Burkholderia was survived and colonized to be the predominant population during the whole operation process (34.19-64.00%), confirming the feasibility of Burkholderia sp. IDO3 as the bioaugmentation agent in complex systems.

Skatole regulates intestinal epithelial cellular functions through activating aryl hydrocarbon receptors and p38.

Intestinal bacteria produce skatole (3-methylindole) from tryptophan in dietary proteins and ingesting large quantities of animal protein is associated with increased fecal skatole concentrations. Although possibly associated with disrupted intestinal homeostasis, the influence of skatole on intestinal epithelial cellular function has not been characterized in detail. The present study aimed to determine whether skatole induces intestinal epithelial cell (IEC) dysfunction. We found that skatole dose-dependently caused IEC death and time-dependently induced IEC apoptosis. Since skatole directly interacts with aryl hydrocarbon receptors (AhR), we investigated whether these receptors influence the skatole-induced death of IEC. In addition to increased AhR transcriptional activity induced by skatole, the AhR antagonist CH223191 partially suppressed of skatole-induced IEC death. Extracellular signal-related kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) are mitogen-activated protein kinases (MAPK) induced by skatole. None of them were repressed by CH223191, whereas the p38 inhibitor SB203580 promoted skatole-induced IEC death. These findings together indicated that skatole induces both AhR-dependent activation pathways and the AhR-independent activation of p38, consequently regulating the amount of IEC death. Accumulating evidence indicates that consuming large amounts of animal protein is associated with the pathogenesis and progression of inflammatory bowel diseases (IBD). Thus, intestinal skatole production induced by large amounts of dietary animal protein might be associated via IEC death with intestinal pathologies such as IBD.

The Mode of Action of Chicory Roots on Skatole Production in Entire Male Pigs Is neither via Reducing the Population of Skatole-Producing Bacteria nor via Increased Butyrate Production in the Hindgut.

The effect of high levels of dietary chicory roots (25%) and intracecal exogenous butyrate infusion on skatole formation and gut microbiota was investigated in order to clarify the mechanisms underlying the known reducing effect of chicory roots on skatole production in entire male pigs. A Latin square design with 3 treatments (control, chicory, and butyrate), 3 periods, and 6 animals was carried out. Chicory roots showed the lowest numerical levels of skatole in both feces and plasma and butyrate infusion the highest. In the chicory group, an increased abundance of the skatole-producing bacterium Olsenella scatoligenes compared to the control group (P = 0.06), and a numerically higher relative abundance of Olsenella than for the control and butyrate groups, was observed. Regarding butyrate-producing bacteria, the chicory group had lower abundance of Roseburia but a numerically higher abundance of Megasphaera than the control group. Lower species richness was found in the chicory group than in the butyrate group. Moreover, beta diversity revealed that the chicory group formed a distinct cluster, whereas the control and butyrate groups clustered more closely to each other. The current data indicated that the skatole-reducing effect of chicory roots is neither via inhibition of cell apoptosis by butyrate nor via suppression of skatole-producing bacteria in the pig hindgut. Thus, the mode of action is most likely through increased microbial activity with a corresponding high incorporation of amino acids into bacterial biomass, and thereby suppressed conversion of tryptophan into skatole, as indicated in the literature.IMPORTANCE Castration is practiced to avoid the development of boar taint, which negatively affects the taste and odor of pork, and undesirable aggressive behavior. Due to animal welfare issues, alternatives to surgical castration are sought, though. Boar taint is a result of high concentrations of skatole and androstenone in back fat. Skatole is produced by microbial fermentation in the large intestine, and therefore, its production can be influenced by manipulation of the microbiota. Highly fermentable dietary fiber reduces skatole production. However, various theories have been proposed to explain the mode of action. In order to search for other alternatives, more efficient or less expensive, to reduce skatole via feeding, it is important to elucidate the mechanism behind the observed effect of highly fermentable dietary fiber on skatole. Our results indicate that highly fermentable dietary fiber does not affect skatole production by reducing the number of skatole-producing bacteria or stimulating butyrate production in the large intestine.

Reduction of androstenone perception in pan-fried boar meat by different masking strategies.

BACKGROUND: Consumers highly sensitive to androstenone María (AND) will probably reject meat from entire male pigs, which tends to have high levels of this hormone. To avoid this, the effect of different masking strategies (sprinkling with mixed spices or fennel, marinating and breading with garlic-parsley or curry) on the sensory parameters of pork loin chops obtained from entire animals with high levels of AND (1.0-2.9 mg kg-1 AND in fat) and castrated animals (<0.4 mg kg-1 AND in fat), both with low levels of skatole (<0.1 mg kg-1 skatole in fat) was investigated. RESULTS: The garlic-parsley breadcrumbs led to the highest reduction in the perception of AND compared with the other masking strategies used, and preserved the juiciness of the product. There was a negative correlation between AND and fat content. CONCLUSION: AND odor and flavor can be reduced in meat from entire male pigs by using suitable strategies, the best strategy being the garlic-parsley breadcrumbs. © 2017 Society of Chemical Industry.

Dietary approaches reducing boar taint-Importance of Lawsonia intracellularis colonisation for interpreting results.

In the fattening of male pigs, boar odour is a major problem with regard to the acceptance of the meat by consumers. Skatole can be one cause. Tryptophan from non-digested feed ingredients and intestinal cell debris can be the precursor in skatole formation. Lawsonia intracellularis, one of the most widespread pathogens in swine, promotes the epithelial cell turnover and might favour the tryptophan influx into the hindgut. Therefore, the question arises how far the severity of a Lawsonia intracellularis infection has an effect on results of dietary experiments with specific issues. Fifty finishing boars from a specific pathogen-free farm were randomly allotted to ten boxes in five feeding groups. Natural developing Lawsonia intracellularis colonisation was monitored serologically (twice individually) and molecular biologically (weekly individually). Over 4 weeks, animals were fed either a finely ground pelleted diet (FP), a coarsely ground meal diet (CM), a meal diet either with 22% cracked corn (CORN), 16.9% dried whey (WHEY) or 30% raw potato starch (RPS). Fifty % of animals showing lower differences in serological Lawsonia intracellularis values between the start and the end of the trial were characterised by a higher dry matter content in faeces (256 ± 29.4 vs. 239 ± 23.6 g/kg). Lawsonia intracellularis-negative caecal samples showed the highest butyrate concentrations (27.2 ± 7.53 mmol/kg). Lawsonia intracellularis-negative faecal samples of group FP showed the highest DM levels in faeces (neg: 290 ± 46.1/pos: 250 ± 52.2 g/kg); negative samples from group RPS had the lowest values (217 ± 24.4 g/kg). Lawsonia intracellularis-negative faecal samples from the group CM were lower in skatole than positive samples (82.8 ± 32.8 vs. 119 ± 29.3 µg/g DM). RPS group samples without pathogen detection had the lowest skatole concentrations (30.5 ± 36.3 µg/g DM). This study provides first evidence that clinically unremarkable colonisation with intestinal pathogens might influence the results of dietary approaches.