Dual UHPLC-HRMS Metabolomics and Lipidomics and Automated Data Processing Workflow for Comprehensive High-Throughput Gut Phenotyping.

In recent years, feces has surfaced as the matrix of choice for investigating the gut microbiome-health axis because of its non-invasive sampling and the unique reflection it offers of an individual's lifestyle. In cohort studies where the number of samples required is large, but availability is scarce, a clear need exists for high-throughput analyses. Such analyses should combine a wide physicochemical range of molecules with a minimal amount of sample and resources and downstream data processing workflows that are as automated and time efficient as possible. We present a dual fecal extraction and ultra high performance liquid chromatography-high resolution-quadrupole-orbitrap-mass spectrometry (UHPLC-HR-Q-Orbitrap-MS)-based workflow that enables widely targeted and untargeted metabolome and lipidome analysis. A total of 836 in-house standards were analyzed, of which 360 metabolites and 132 lipids were consequently detected in feces. Their targeted profiling was validated successfully with respect to repeatability (78% CV < 20%), reproducibility (82% CV < 20%), and linearity (81% R2 > 0.9), while also enabling holistic untargeted fingerprinting (15,319 features, CV < 30%). To automate targeted processing, we optimized an R-based targeted peak extraction (TaPEx) algorithm relying on a database comprising retention time and mass-to-charge ratio (360 metabolites and 132 lipids), with batch-specific quality control curation. The latter was benchmarked toward vendor-specific targeted and untargeted software and our isotopologue parameter optimization/XCMS-based untargeted pipeline in LifeLines Deep cohort samples (n = 97). TaPEx clearly outperformed the untargeted approaches (81.3 vs 56.7-66.0% compounds detected). Finally, our novel dual fecal metabolomics-lipidomics-TaPEx method was successfully applied to Flemish Gut Flora Project cohort (n = 292) samples, leading to a sample-to-result time reduction of 60%.

Agricultural contaminants in amphibian breeding ponds: Occurrence, risk and correlation with agricultural land use.

Anthropogenic pressure such as agricultural pollution globally affects amphibian populations. In this study, a total of 178 different compounds from five agrochemical groups (i.e. antimicrobial drugs residues (ADRs), coccidiostats and anthelmintics, heavy metals, mycotoxins and pesticides) were determined monthly, from March until June 2019 in 26 amphibian breeding ponds in Flanders, Belgium. Furthermore, a possible correlation between the number and concentration of selected contaminants that were found and the percentage of arable land within a 200 m radius was studied. Within each group, the highest detected concentrations were obtained for 4-epioxytetracycline (0.422 µg L-1), levamisole (0.550 µg L-1), zinc (333.1 µg L-1), 3-acetyldeoxynivalenol (0.013 µg L-1), and terbuthylazine (38.7 µg L-1), respectively, with detection frequencies ranging from 1 (i.e. 3-acetyldeoxynivalenol) to 26 (i.e. zinc) out of 26 ponds. Based on reported acute and chronic ecotoxicological endpoints, detected concentrations of bifenthrin, cadmium, copper, cypermethrin, hexachlorobenzene, mercury, terbuthylazine, and zinc pose a substantial ecological risk to aquatic invertebrates such as Daphnia magna and Ceriodaphnia dubia, which both play a role in the food web and potentially in amphibian disease dynamics. Additionally, the detected concentrations of copper were high enough to exert chronic toxicity in the gray treefrog (Hyla versicolor). The number of detected compounds per pond ranged between 0 and 5 (ADRs), 0 - 2 (coccidiostats and anthelmintics), 1 - 7 (heavy metals), 0 - 4 (mycotoxins), and 0 - 12 (pesticides) across the four months. Furthermore, no significant correlation was demonstrated between the number of detected compounds per pond, as well as the detected concentrations of 4-epioxytetracycline, levamisole, copper, zinc, enniatin B and terbuthylazine, and the percentage of arable land within a 200 m radius. For heavy metals and pesticides, the number of compounds per pond varied significantly between months. Conclusively, amphibian breeding ponds in Flanders were frequently contaminated with agrochemicals, yielding concentrations up to the high µg per liter level, regardless of the percentage surrounding arable land, however showing temporal variation for heavy metals and pesticides. This research also identifies potential hazardous substances which may be added to the European watch list (CD 2018/408/EC) in the future.

Effect of nanocurcumin and fish oil as natural anti-inflammatory compounds vs. glucocorticoids in a lipopolysaccharide inflammation model on Holstein calves' health status.

Curcumin (CUR) and fish oil (FO) are among the most well-known types of natural anti-inflammatory compounds. The aim of this study was to assess the effect of nanocurcumin and fish oil vs. glucocorticoids on Holstein calves' health status. A lipopolysaccharide (LPS) challenge (0.5 µg kg-1 BW) was used to induce an acute phase response. A total of 42 male Holstein calves were randomized into 7 groups: negative control (CON), positive control (LPS, injected once), 250 mg/kg BW per day fish oil + LPS (FO250), 350 mg/kg BW per day fish oil + LPS (FO350), 2 mg/kg BW per day nanocurcumin + LPS (NCUR2), 4 mg/kg BW per day nanocurcumin + LPS (NCUR4), and 0.3 mg/kg BW dexamethasone (injected once) + LPS (DEX). The duration of this experiment was 11 days, with application of the LPS challenge on day 8. Calves were weighed on days 0, 7, 9, 10, and 11 to record the average daily weight gain; diets offered and refused were recorded daily throughout the experiment. Blood collection and clinical scoring were conducted at successive time points until 72 h post LPS challenge. The data obtained also comprised rectal temperature (RT), respiratory rate (RR), heart rate (HR), concentrations of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), serum amyloid A (SAA), and haptoglobin (Hp). This experiment could not uncover significant effects of LPS, FO, NCUR, and DEX on the area under the curve (AUC) of the RT, HR, and RR; in addition, there was no difference between FO and NCUR vs. LPS in sickness behavior, however, DEX group significantly recovered faster than others (P < 0.01). There was no significant difference between groups in dry matter intake (DMI) and average daily gain (ADG) during three days post LPS challenge. The concentrations of TNF-⍺, IL-6, and SAA were lower in the DEX group (P < 0.05). Finally, no effects of FO and NCUR on cytokines and acute phase proteins (APPs) could be observed in this study. In conclusion, supplementation of FO and NCUR was not able to impact the acute phase response (APR) in calves, as levels of inflammatory cytokines and APPs as well as sickness behavior remained unchanged. It seems that the anti-inflammatory effects of FO and CUR on APR, as has been observed for other animal species, do not manifest that clearly in calves.

On-farm prevalence of and potential risk factors for boar taint.

Boar taint is an unpleasant taste and odor that can occur in entire male pigs and is caused by androstenone, skatole, and to a lesser extent indole accumulating in fat tissue. In the present observational study, we evaluated an extensive list of such potential risk factors which influence boar taint: social hierarchy and puberty attainment, housing, health, preslaughter conditions, season, feed, carcass composition, slaughter weight or age, and breed. Details on these factors were collected by interviews with the participating farmers, observations on each farm by trained observers and farmers, as well as slaughterhouse data. Twenty-two farms (in West- and East-Flanders, ranging from 160 to 600 sows, selected on suitability) raising entire male pigs were included in the study to evaluate the link between boar taint and potential risk factors related to the farm and slaughter batch (114 slaughter batches and 16 791 entire male pigs in total). Average olfactory boar taint prevalence was 1.8 ±â€¯0.8%. Boar taint prevalence varied also within farms up to a maximum range between slaughter batches of 9.1% which suggests an effect of factors varying between slaughter batches such as season or other variables varying between slaughter batches. Less aggressive behavior at the end of fattening as well as lower skin lesion scores at fattening as well as at slaughter could be associated with less boar taint. The same might be said for sexual behavior, though less convincingly from this study. Measures that reduce aggression and stress have therefore have the potential to lower boar taint prevalence. The same might be said for sexual behavior, though less convincingly from this study. Furthermore, boar taint prevalence was generally higher in winter than in summer, which is relevant from a planning perspective for the slaughterhouses to seek alternative markets. Finally, increased CP gave significantly lower boar taint prevalences. This may to some extent be explained by the negative association between boar taint and lean meat percentage, as increased dietary CP levels promote the carcass lean meat percentages which can then be associated with lower boar taint levels.

Multi-class analysis of 46 antimicrobial drug residues in pond water using UHPLC-Orbitrap-HRMS and application to freshwater ponds in Flanders, Belgium.

Increasing anthropogenic pressure and agricultural pollution raises concerns regarding antimicrobial resistance and biodiversity loss in aquatic environments. In order to protect and restore water resources and biodiversity, antimicrobial drug residues should be monitored in all aquatic environments including pond water. Consequently, the objective of this research was to develop and validate a novel multi-residue method for the simultaneous quantification of 46 targeted human and veterinary antimicrobial drugs in pond water. A suitable extraction method based on solid-phase extraction (SPE) was developed, assisted by a fractional factorial design. A broad polarity range of compounds was covered (log P from -4.05 to 4.38), including major representatives of the following classes: sulfonamides, tetracyclines, quinolones, macrolides, lincosamides, nitrofurans, penicillins, cephalosporins, diaminopyrimidines, pleuromutilins and phenicols. All analytes were separated using ultra-high performance liquid chromatography (UHPLC) and detected in full-scan by Orbitrap high resolution mass spectrometry (Orbitrap-HRMS). Good linearity was obtained for all compounds with R2 ≥ 0.993 and goodness-of-fit coefficient (g) ≤ 11.56%. Method detection limits ranged from 10 to 50 ng L-1 and method quantification limits were 50 ng L-1 for all compounds. Acceptable values were obtained for within-day and between-day apparent recoveries (i.e. between 50 and 120%), precision (< 30% and < 45%) and measurement uncertainty (< 50%). Targeted analysis of 18 freshwater ponds throughout Flanders was performed to demonstrate the applicability of the newly developed UHPLC-HRMS method. Overall, 20 antimicrobial drugs were detected with highest concentrations observed for tetracyclines and their transformation products ranging between 51 and 248 ng L-1. Finally, suspect screening was performed suggesting the presence of 14 additional pharmaceuticals including 3 antimicrobial degradation products (e.g. apo-oxytetracycline, amoxicillin penicilloic acid and penilloic acid) and 11 pesticides.

Monitoring of behavior, sex hormones and boar taint compounds during the vaccination program for immunocastration in three sire lines.

Immunocastration (vaccination against boar taint) is an alternative method to prevent boar taint without the need for surgical castration. This study investigates the evolution of boar taint compounds in serum and fat, serum steroid compounds as well as behavior in immunocastrated pigs from 3 sire lines: 15 stress positive Belgian Piétrain (BP), 20 stress negative French Piétrain (FP), and 20 stress negative Canadian Duroc (CD). Hormone and boar taint compounds in serum were determined at 4 time points; boar taint compounds in fat were determined at 3 time points. Behavior, skin lesions, animal and pen fouling were also recorded before the first vaccination (V2). Aggressiveness, eating and drinking and general activity behavior declined from  V2 for all sire lines. Pigs from BP were cleaner than FP and CD pigs. Even though immunocastration was effective in general (reduced testosterone, estradiol as well as androstenone in serum) for all sire lines, some individual pigs showed either androstenone or skatole levels in fat above cutoff values. While the immunocastration mechanism works as intended for androstenone, and also for skatole for the three sire lines, the risk of carcasses with boar taint compounds above cutoff levels (respectively 1.9 and 3.7%) still remains to some extent.

On farm intervention studies on reduction of boar taint prevalence: Feeding strategies, presence of gilts and time in lairage.

One of the challenges in the production of entire male pigs is the occurrence of boar taint. We separately tested the effect of 3 management strategies to reduce boar taint on respectively 2, 3, and 6 Flemish pig farms: 1) adapted feeding strategies, 2) presence of gilts in the compartment, and 3) varying lairage duration at the slaughterhouse. A commercialized feed concept resulted in a significant reduction of olfactory boar taint prevalence when fed for 2 weeks (T2W) compared to control (T-CON) (P = 0.030). For T2W and when fed for 3 weeks (T3W), androstenone (AND) (P = 0.002 for T2W, P = 0.029 for T3W) and skatole (SKA) (P < 0.001 for T2W and T3W) were significantly reduced compared to T-CON. Olfactory boar taint prevalence was significantly reduced when feeding 5% dried chicory roots (FI5%) (P = 0.032), but not for 3% dried chicory roots (FI3%) (P = 0.958). SKA concentration was significantly lower when feeding FI5% (P < 0.001) and when feeding FI3% (P = 0.034). Rearing entire male pigs separately from gilts and increasing lairage duration from <1 h to >3 h did not significantly affect boar taint.

Detection of prohibited substances in equine hair by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry - application to doping control samples.

The detection of drugs in human hair samples has been performed by laboratories around the world for many years and the matrix is popular in disciplines, such as workplace drug testing. To date, however, hair has not become a routinely utilised matrix in sports drug detection. The analysis of hair samples offers several potential advantages to doping control laboratories, not least of which are the greatly extended detection window and the ease of sample collection and storage. This article describes the development, validation, and utilisation of a sensitive ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS/MS) method for the detection of 50 compounds. This provides significantly improved coverage for those analytes which would be of particular interest if detected in hair, such as anabolic steroid esters and selective androgen receptor modulators (SARMs). Qualitative validation of the method resulted in estimated limits of detection as low as 0.1 pg/mg for the majority of compounds, with all being detected at 2 pg/mg or below. The suitability of the method for the detection of prohibited substances in incurred material was demonstrated by the successful detection of several compounds, such as stanozolol, boldenone undecylenate, clenbuterol, and GW-501516, in genuine equine hair samples. Estimated concentrations of the detected substances ranged from 0.27 to 8.6 pg/mg. The method has been shown to be fit-for-purpose for routine screening of equine hair samples by the analysis of over 400 genuine hair samples.

Cofactor F430 as a biomarker for methanogenic activity: application to an anaerobic bioreactor system.

Over the last decades, anaerobic bioreactor technology proved to be a competitive technology for purifying wastewater while producing biogas. Methanogens perform the crucial final step in methane production, and monitoring their activity is of paramount importance for system understanding and management. Cofactor F430 is an essential prosthetic group of the methyl-coenzyme M reductase (MCR) enzyme catalysing this final step. This research investigates whether the quantification of cofactor F430 in bioreactor systems is a viable intermediate-complexity monitoring tool in comparison to the conventional biogas and volatile fatty acid (VFA) concentration follow-up and molecular genetic techniques targeting the mcrA gene encoding the MCR protein or its transcripts. Cofactor F430 was quantified in a lab-scale anaerobic membrane bioreactor (AnMBR) using liquid chromatography. The system was subjected to two organic loading rate shocks, and the F430 content of the sludge was followed up alongside mcrA gene copy and transcript numbers and classical performance monitoring tools. The research showed for the first time the combined mcrA gene transcript and F430 content dynamics in an anaerobic bioreactor system and reveals their significant positive correlation with in situ methane production rate. The main difference between the two monitoring methods relates to the cofactor's slower degradation kinetics. The work introduces the use of cofactor F430 as a biomarker for methanogenic activity and, hence, as a monitoring tool that can be quantified within half a working day, yielding information directly related to in situ methanogenic activity in methanogenic reactors.

Discrimination between Synthetically Administered and Endogenous Thiouracil Based on Monitoring of Urine, Muscle, and Thyroid Tissue: An in Vivo Study in Young and Adult Bovines.

Thiouracil (TU), synthesized for its thyroid-regulating capacities and alternatively misused in livestock for its weight-gaining effects, is acknowledged to have an endogenous origin. Discrimination between low-level abuse and endogenous occurrence is challenging and unexplored in an experimental setting. Therefore, cows (n = 16) and calves (n = 18) were subjected to a rapeseed-supplemented diet or treated with synthetic TU. Significant higher urinary TU levels were recorded after TU administration (

Olfactory evaluation of boar taint: effect of factors measured at slaughter and link with boar taint compounds.

There is a commitment by the European pig sector to ban surgical castration of male piglets in the European Union in 2018. One alternative to castration is to raise entire male pigs, with an increased risk of boar taint. A field study was performed to: (1) evaluate inter- and intra-farm variation in boar taint prevalence, (2) investigate factors measured at slaughter influencing boar taint and (3) evaluate the relationship between sensorial scoring by a trained panel and the concentration of boar taint components. From 34 farms, neck fat samples were collected from all entire male pigs in at least two slaughter batches per farm (78 batches; 9167 animals). In addition to olfactory boar taint analysis, data were also collected on fresh skin lesions (score 0 to 3) at the slaughter line, slaughter weight, lean meat percentage, duration of transport, time spent in lairage, total delivery duration, day length, shortening of days and outdoor mean temperature. Using the hot iron method, neck fat samples were scored (eight-point scale) for boar taint. Average boar taint prevalence (score ≥3) was 5.6±2.5% and the mean difference between the maximum and minimum prevalence per farm was 4.3±3.2%. Androstenone (AND), skatole (SKA) and indole concentrations were measured for a subset (n=254) of the samples. According to binomial univariate mixed models, entire male pigs with a higher skin lesion score had higher odds of having boar taint (P=0.031), as did fatter entire male pigs (P<0.001). In the binomial multivariate mixed model lean meat percentage (P<0.001) and outdoor mean temperature (P=0.005) remained as only significant factors. Based on our results, we can conclude that these statistically significant at least partially influence the prevalence of boar taint. According to the binomial univariate mixed models SKA concentration in liquid fat seems a better predictor for boar taint than AND. There were no significant synergetic effects between boar taint compounds.

Mouldy feed: A possible explanation for the excretion of anabolic-androgenic steroids in horses.

To ensure fair competition and to protect the horse's welfare, horses have to compete on their own merits, without any unfair advantage that might follow the use of drugs. Therefore, regulatory authorities list all substances that are not allowed in competition, including most anabolic-androgenic steroids. As zero-tolerance is retained, the question arose whether the consumption of mouldy feed could lead to the excretion of steroids, due to the biotransformation of plant phytosterols to steroids. A rapid ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analytical method, previously validated according to AORC (Association of Official Racing Chemists) and EC (European Commission) guidelines, was used to measure steroids in different sample types. Multiple mouldy feed samples were tested for the presence of steroids. The effect of digestion was tested by in vitro simulation of the horse's hindgut in batch incubations. In most feed samples no steroids were detected, even when the products were mouldy. Mouldy corn however showed to contain up to 3.0 ± 0.4 µg/kg AED (4-androstenedione), the main testosterone precursor. This concentration increased when mouldy corn (with added phytosterols) was digested in vitro. An herbal phytosupplement also showed to contain α-testosterone. These results demonstrate that it is important to caution against the consumption of any feed or (herbal) supplement of which the detailed ingredients and quantitative analysis are unknown. The consumption of mouldy corn should especially be avoided, not only from a horse health and welfare point of view, but also to avoid possible inadvertent positive doping results. Copyright © 2016 John Wiley & Sons, Ltd.

The effect of feed water dissolved organic carbon concentration and composition on organic micropollutant removal and microbial diversity in soil columns simulating river bank filtration.

This study investigated organic micropollutant (OMP) biodegradation rates in laboratory-scale soil columns simulating river bank filtration (RBF) processes. The dosed OMP mixture consisted of 11 pharmaceuticals, 6 herbicides, 2 insecticides and 1 solvent. Columns were filled with soil from a RBF site and were fed with four different organic carbon fractions (hydrophilic, hydrophobic, transphilic and river water organic matter (RWOM)). Additionally, the effect of a short-term OMP/dissolved organic carbon (DOC) shock-load (e.g. quadrupling the OMP concentrations and doubling the DOC concentration) on OMP biodegradation rates was investigated to assess the resilience of RBF systems. The results obtained in this study imply that - in contrast to what is observed for managed aquifer recharge systems operating on wastewater effluent - OMP biodegradation rates are not affected by the type of organic carbon fraction fed to the soil column, in case of stable operation. No effect of a short-term DOC shock-load on OMP biodegradation rates between the different organic carbon fractions was observed. This means that the RBF site simulated in this study is resilient towards transient higher DOC concentrations in the river water. However, a temporary OMP shock-load affected OMP biodegradation rates observed for the columns fed with the river water organic matter (RWOM) and the hydrophilic fraction of the river water organic matter. These different biodegradation rates did not correlate with any of the parameters investigated in this study (cellular adenosine triphosphate (cATP), DOC removal, specific ultraviolet absorbance (SUVA), richness/evenness of the soil microbial population or OMP category (hydrophobicity/charge).

The effect of redox conditions and adaptation time on organic micropollutant removal during river bank filtration: A laboratory-scale column study.

This study investigated the redox dependent removal and adaptive behaviour of a mixture of 15 organic micropollutants (OMPs) in laboratory-scale soil columns fed with river water. Three separate pilot systems were used consisting of: (1) two columns, (2) ten columns and (3) twenty two columns to create oxic, suboxic (partial nitrate removal) and anoxic (complete nitrate removal). The pilot set-up has some unique features--it can simulate fairly long residence times (e.g., 45 days using the 22 column system) and reduced conditions developed naturally within the system. Dimethoate, diuron, and metoprolol showed redox dependent removal behaviour with higher biodegradation rates in the oxic zone compared to the suboxic/anoxic zone. The redox dependent behaviour of these three OMPs could not be explained based on their physico-chemical properties (hydrophobicity, charge and molecular weight) or functional groups present in the molecular structure. OMPs that showed persistent behaviour in the oxic zone (atrazine, carbamazepine, hydrochlorothiazide and simazine) were also not removed under more reduced conditions. Adaptive behaviour was observed for five OMPs: dimethoate, chloridazon, lincomycin, sulfamethoxazole and phenazone. However, the adaptive behaviour could not be explained by the physico-chemical properties (hydrophobicity, charge and molecular weight) investigated in this study and only rough trends were observed with specific functional groups (e.g. ethers, sulphur, primary and secondary amines). Finally, the adaptive behaviour of OMPs was found to be an important factor that should be incorporated in predictive models for OMP removal during river bank filtration.

Common European harmful algal blooms affect the viability and innate immune responses of Mytilus edulis larvae.

Like marine diseases, harmful algal blooms (HABs) are globally increasing in frequency, severity and geographical scale. As a result, bivalves will have to face the combined threat of toxic algae and marine pathogens more frequently in the (near) future. These stressors combined may further affect the recruitment of ecologically and economically important bivalve species as HABs can affect the growth, viability and development of their larvae. To date, little is known on the specific effects of HABs on the innate immune system of bivalve larvae. This study therefore investigates whether two common harmful algae can influence the larval viability, development and immunological resilience of the blue mussel Mytilus edulis. Embryos of this model organism were exposed (48 h) to five densities of Pseudo-nitzschia multiseries or Prorocentrum lima cells. In addition, the effect of six concentrations of their respective toxins: domoic acid (DA) and okadaic acid (OA) were assessed. OA was found to significantly reduce larval protein phosphatase activity (p < 0.001) and larval viability (p < 0.01) at concentrations as low as 37.8 µg l(-1). P. multiseries (1400 cells ml(-1)), P. lima (150 cells ml(-1)) and DA (dosed five times higher than typical environmental conditions i.e. 623.2 µg l(-1)) increased the phenoloxidase (PO) innate immune activity of the mussel larvae. These results suggest that the innate immune response of even the earliest life stages of bivalves is susceptible to the presence of HABs.

A laboratory-scale column study comparing organic micropollutant removal and microbial diversity for two soil types.

This study investigated sorption and biodegradation behaviour of 20 organic micropollutants (OMPs) in lab-scale columns filled with two types of soil (fed with the same water quality) simulating river bank filtration (RBF) under oxic conditions. Retardation factors and OMP biodegradation rates were similar for the two soils that were characterised by a different cationic exchange capacity, organic matter and sand/silt/clay content. This result was supported by the microbial community composition (richness, evenness) of the two soils that became more similar as a result of feeding both columns with the same water quality. This indicates that microbial community composition and thereby OMP removal in soils is primarily determined by the composition of the aqueous phase (organic matter quantity and quality, nutrients) rather than the soil phase. These results indicate that different RBF sites located along the same river may show similar OMP removal (in case of similar water quality and residence time). CAPSULE: This study shows that the microbial community composition and thus OMP removal is primarily determined by the aqueous phase (water quality) rather than the soil phase.

Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster.

The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.

The effect of the MC4R gene on boar taint compounds, sexual maturity and behaviour in growing-finishing boars and gilts.

Societal pressure to ban surgical castration of male piglets is rising due to animal welfare concerns, thus other methods to prevent boar taint need to be explored. Genetic selection against boar taint appears to be a long-term sustainable alternative. However, as boar taint is linked to reproductive hormones, it is important to consider possible negative side effects such as delayed sexual maturity or changes in behaviour. We reported earlier that the melanocortin-4 receptor (MC4R) marker can be used to reduce boar taint levels in fat of boars. The objective of this study was to evaluate whether MC4R marker-assisted selection for lower boar taint prevalence affects plasma levels of boar taint compounds and testosterone; sexual maturity; behaviour; skin lesions; and lameness in boars and gilts. Using an intervention study with a 2×2 design, 264 boars and gilts differing on position 893 of the MC4R gene (AA v. GG) were compared. The MC4R polymorphism did not affect the plasma concentration of either androstenone or testosterone at different time points, whereas the concentration of skatole was significantly lower (P=0.003) and the concentration of indole tended to be lower (P=0.074) in GG compared with AA boars. A higher percentage of gilts of the GG genotype were in puberty at slaughter age compared with AA gilts (P<0.001). The age of the boars at sexual maturity (as indicated by the first positive preputial smear test) did not differ between AA and GG boars. In contrast, weight of GG boars at sexual maturity tended to be lower (P=0.065). During the period from 6 weeks of age to slaughter, boars and gilts of the GG genotype showed more playing behaviour (P=0.015) and less passive and feeding behaviour (P=0.003). They showed more skin lesions on their back and caudal area (P=0.022), and tended to show more skin lesions on their head and anterior area (P=0.093) compared with AA animals. In conclusion, the polymorphism in the MC4R gene can be used as a marker without negative effects on reproduction characteristics in boars and gilts. Genetic selection towards a lower prevalence of boar taint will lead to more active pigs with more skin lesions. Management strategies may therefore be necessary to reduce skin lesions in the selected animals.

In vitro simulation of the equine hindgut as a tool to study the influence of phytosterol consumption on the excretion of anabolic-androgenic steroids in horses.

Traditionally, steroids other than testosterone are considered to be synthetic, anabolic steroids. Nevertheless, in stallions, it has been shown that ß-Bol can originate from naturally present testosterone. Other precursors, including phytosterols from feed, have been put forward to explain the prevalence of low levels of steroids (including ß-Bol and ADD) in urine of mares and geldings. However, the possible biotransformation and identification of the precursors has thus far not been investigated in horses. To study the possible endogenous digestive transformation, in vitro simulations of the horse hindgut were set up, using fecal inocula obtained from eight different horses. The functionality of the in vitro model was confirmed by monitoring the formation of short-chain fatty acids and the consumption of amino acids and carbohydrates throughout the digestion process. In vitro digestion samples were analyzed with a validated UHPLC-MS/MS method. The addition of ß-Bol gave rise to the formation of ADD (androsta-1,4-diene-3,17-dione) or αT. Upon addition of ADD to the in vitro digestions, the transformation of ADD to ß-Bol was observed and this for all eight horses' inocula, in line with previously obtained in vivo results, again confirming the functionality of the in vitro model. The transformation ratio proved to be inoculum and thus horse dependent. The addition of pure phytosterols (50% ß-sitosterol) or phytosterol-rich herbal supplements on the other hand, did not induce the detection of ß-Bol, only low concentrations of AED, a testosterone precursor, could be found (0.1 ng/mL). As such, the digestive transformation of ADD could be linked to the detection of ß-Bol, and the consumption of phytosterols to low concentrations of AED, but there is no direct link between phytosterols and ß-Bol.

An intervention study demonstrates effects of MC4R genotype on boar taint and performances of growing-finishing pigs.

The Asp298Asn polymorphism of the melanocortin-4 receptor (MC4R) in pigs is known to affect economically important traits such as growth rate and backfat thickness. We have assessed the possible use of this polymorphism as a molecular marker to perform genetic selection toward lower boar taint levels without compromising growth performance and carcass and meat quality in commercial boars and gilts. Homozygous boars and gilts of the AA genotype and GG genotype were compared in an intervention study with a 2 × 2 design to assess main effects and possible interactions between sex and genotype. The concentrations of the 3 boar taint compounds androstenone ( = 0.044), skatole ( = 0.049), and indole ( = 0.006) were significantly higher in fat of AA boars compared to GG boars. However, no effect on the sensory analysis of the fat samples could be observed. Between 20 and 115 kg BW, AA pigs showed higher ADFI than GG pigs ( < 0.001). An interaction between genotype and sex was observed for ADG ( = 0.044): AA boars had a significantly higher ADG than GG boars but there was no significant difference between the gilts. Daily lean meat gain tended to be higher in boars compared to gilts ( = 0.051), independent of genotype. Similarly, boars showed higher G:F compared to gilts ( < 0.001), without effect of genotype. Genotype and sex affected several carcass quality parameters but there was no interaction. Pigs of the AA genotype displayed a lower dressing percentage ( = 0.005), lower ham width ( = 0.024), lower muscle thickness ( = 0.011), and higher fat thickness ( < 0.001), resulting in a lower lean meat percentage ( < 0.001) in comparison with GG pigs. Gilts had a significantly higher dressing percentage ( < 0.001), higher muscle thickness ( < 0.001), higher ham width ( < 0.001), and lower ham angle ( < 0.001) compared to boars. Other than the boar taint compounds, meat quality was not affected by genotype. Pork of gilts was darker ( = 0.014) and less exudative during cooking ( < 0.001) and contained more intramuscular fat ( = 0.013). These results indicate that genetic selection against boar taint is possible using this marker. This will also result in lower feed intake and ADG and, consequently, better carcass quality.