Development of a quantitative method for the simultaneous analysis of the boar taint compounds androstenone, skatole and indole in porcine serum and plasma by means of ultra-high performance liquid chromatography coupled to high resolution mass spectrometry.

Boar taint is an off-odour occurring while heating meat or fat from boars. A method detecting the three compounds (androstenone, skatole and indole) simultaneously in blood would offer substantial advantages since it would allow monitoring the impact of rearing strategies. Therefore, a UHPLC-HR-Orbitrap-MS analysis method is optimized and validated for the quantification of these compounds in plasma or serum. Sample pre-treatment involved an extraction with diethylether followed by a centrifugal filtration (30 kDa). Limits of detection and quantification varied between 0.5 and 1 µg L(-1) and 2 and 3 µg L(-1) for the three compounds, respectively. Besides, an excellent repeatability (RSD < 7.6%), within-laboratory reproducibility (RSD<10.5%), recovery (87-97%) and linearity (R(2)>0.99) were recorded. Correlations between serum/plasma and fat levels of the boar taint compounds were positive for skatole (r(serum) = 0.39 and r(plasma) = 0.84) and androstenone (r(serum) = 0.73-0.78 and r(plasma) = 0.32-0.80).

The MC4R c.893G>A mutation: a marker for growth and leanness associated with boar taint odour in Belgian pig breeds.

Since surgical castration of male piglets without anaesthesia is under heavy societal pressure, finding a sustainable solution to reduce boar taint has become urgent. One way to circumvent this animal welfare violation is raising entire male pigs whilst selecting against the tainted phenotype through marker-assisted selection. Since slaughtering at a lower weight is often suggested to reduce boar taint, selection using a marker for that trait could be a promising strategy. Therefore, in this study a melanocortin 4 receptor (MC4R) mutation, frequently described in different pig breeds as marker for fat content, weight gain and feed intake, was examined in relation to boar taint in pig breeds used in Belgian pig farms. Although results suggest an association between this mutation and a boar taint odour score assigned by experts, no association was found between the mutation and the concentration of the individual chemical boar taint components androstenone, skatole and indole.

Quality assessment of the blue mussel (Mytilus edulis): comparison between commercial and wild types.

This study compared species identity, microplastics, chemical and microbial contamination between consumption mussels and wild type mussels, collected at Belgian department stores and Belgian groynes and quaysides, respectively. Species identification based on genetic analysis showed a high number of Mytilus (M.) edulis compared to M. galloprovincialis and M. edulis/galloprovincialis hybrid mussels. The number of total microplastics varied from 2.6 to 5.1 fibres/10 g of mussel. A higher prevalence of orange fibres at quaysides is related to fisheries activities. Chemical contamination of polycyclic aromatic hydrocarbons and polychlorobiphenyls could be related to industrial activities and water turbidity, with maximum concentrations at the quayside of port Zeebrugge. The inverse was noted for Escherichia coli contamination, which was relatively low at Zeebrugge quayside with a total count of 3.9 × 10(2)CFU/100 g tissue, due to limited agricultural effluents. Results of this complementary analysis stress the importance of integrated monitoring and quality assessment.

Effect of surgical castration, immunocastration and chicory-diet on the meat quality and palatability of boars.

This study evaluates 1) carcass quality, meat quality and palatability for barrows, immunocastrates and boars and 2) the effect of chicory supplemented feed during 10 days before slaughter on boar meat quality. At comparable carcass weights, estimated carcass lean meat percentage was higher in immunocastrates and boars than in barrows. Muscle thickness was higher for immunocastrates and barrows compared to boars, while fat thickness was lowest for immunocastrates and boars. Barrows, immunocastrates and boars differed in water holding capacity and boar taint. Home consumer panels were conducted to evaluate palatability. The consumers did detect differences in tenderness and juiciness, but not for boar taint. The chicory feed supplemented in boar feed decreased skatole concentration in backfat, without largely influencing meat quality or palatability. Not only boar taint, but also carcass and meat quality should be considered when evaluating alternatives for surgical castration.

Evaluation of different heating methods for the detection of boar taint by means of the human nose.

No automated detection system for boar taint detection is currently available, thus boar taint at the slaughterline can currently only be assessed using the singeing method (olfactory scoring). This study compares several heating methods (microwave, soldering iron and pyropen) and evaluates the effect of habituation, cleaning the soldering iron, singeing the fat twice in the same place, and variations in the technical procedures. All methods seem to be suitable for detecting boar taint but the choice of heating method for sensory scoring of boar taint depends on habituation of the trained assessor and specific conditions applied. The pyropen seems to be most suitable because it does not contact the fat and is easy to handle (wireless). Finally, the intensity score may also be influenced by: contamination from not cleaning the soldering iron, singeing the fat twice in the same place, and the effect of habituation.

Predicting the likelihood of developing boar taint: early physical indicators in entire male pigs.

Three potential early-age predictors of which boars are likely to develop boar taint (testes volume, skin lesions and dirtiness) were measured on 102 boars every fortnight from 10 weeks of age until slaughter. These predictors were correlated with the level of boar taint according to the hot iron method and the concentrations of skatole and androstenone as determined by chemical analysis. The chance of no/low boar taint according to the hot iron method decreased with higher testes volume (weeks 22 and 24) and increased with skin lesion score (weeks 12, 16 and 18). For the concentrations of androstenone and skatole, the strongest correlation was found with testes volume in week 12. Skin lesions in week 16 were negatively correlated with skatole levels. Dirtiness was negatively correlated with skatole concentrations (week 18) but positively correlated with androstenone concentrations (weeks 20 and 22). Testes volume has the greatest potential for predicting the likelihood of developing boar taint.

Evaluation of various boar taint detection methods.

The aim of this study was to evaluate the performance of various boar taint detection methods, measure the relationship between them and identify possible points of improvement for boar taint detection. The methods used to evaluate boar taint in the carcasses of 448 entire male pigs and 17 barrows were the hot iron method (n = 442), a standardised (n = 323) and home (n = 58) consumer meat-evaluation panel, an expert panel assessment of meat and fat (n = 464) and laboratory analysis of skatole, androstenone and indole in fat (n = 464). The axillary odour of a number of slaughtered entire male pigs was also investigated (n = 231). As correlation coefficients were generally weak, a positive result for one of these detection methods did not per se result in a positive result for all other methods. Results of one detection method could not be generalised. The choice to use one or more detection methods deserves consideration depending on the aim of the study. In this paper, we suggest some possible improvements for evaluating boar taint with a consumer panel based on our results and experience. The home consumer evaluation was correlated with the concentration of indole (r = 0.27) but not with skatole or androstenone. We therefore recommend that lab analyses include indole testing. The hot iron method seems to be an easy and fast detection method, which yields comparable or better correlation coefficients with the other detection methods than an expert panel evaluating fat samples. However, the reliability of the hot iron method depends on the training and reliability of one or two assessors. Efforts should be made to further optimise this method by evaluating the effect of testing conditions. The axillary odour score was moderately correlated with the other detection methods (up to 0.32). More research is needed to evaluate the possibilities of axillary odour as a boar taint detection method.

A validated ultra-high performance liquid chromatography coupled to high resolution mass spectrometry analysis for the simultaneous quantification of the three known boar taint compounds.

Boar taint is an off-odour that can occur when meat or fat from entire male pigs is heated. Most of the currently available analytical methods are not capable of detecting the three known boar taint compounds (indole, skatole and androstenone) simultaneously, which renders their analysis often labour-intensive and time-consuming as separate analyses are required. In this study a validated U-HPLC-HR-Orbitrap-MS analysis method is described for the quantitative determination of the three boar taint compounds in fat. The sample pre-treatment involves a melting step followed by extraction with methanol and clean-up consisting of a freezing step and solid phase extraction (HLB cartridges). The analytes are then chromatographically separated and detected with an Exactive high-resolution mass spectrometer. Due to the absence of guidelines for the analysis of boar taint in fat, the Commission Decision 2002/657/EC [18] and ISO 17025 [19] guidelines were used as guideline for validation of the developed detection method. This resulted in limits of detection and limits of quantification between 2.5 and 7 µg kg(-1) and between 5 and 10 µg kg(-1) for the three compounds, respectively, which is far below the threshold values set at 100 µg L(-1) for indole, 200 µg L(-1) for skatole and 1000 µg L(-1) for androstenone in pig fat samples. The method obtained for the three compounds a repeatability (RSD) lower then 12.7% and a within-laboratory reproducibility (RSD) lower than 16.9%. The recovery of the three compounds ranged between 99 and 112 and an excellent linearity (R(2) ≥ 0.99) was found. In the future, this method may be extended with other compounds that turn out to be correlated with boar taint.

The sensitivity of Flemish citizens to androstenone: influence of gender, age, location and smoking habits.

Skatole and androstenone are the main boar taint compounds. Whereas nearly everybody is sensitive to skatole, the sensitivity to androstenone is genetically determined and differs between countries. In this study the methodology for testing androstenone sensitivity was refined and applied to 1569 consumers that were approached at six shopping malls in Flanders. Participants were asked to smell the contents of four bottles (three were filled with water and one with androstenone solved in water) and to identify and describe the odour of the strongest smelling bottle. This test was performed twice. 45.3% of the respondents were classified as sensitive to androstenone (i.e. the percentage of participants that identified the correct bottle in both tests minus a guess correction). Sensitivity differed between sexes (men: 38.3%-women: 51.1%, P<0.001), according to age (older people were less sensitive, P<0.001), and between the test locations (P<0.001), but not between smokers versus non-smokers.

Ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry detection of naturally occurring thiouracil in urine of untreated livestock, domesticated animals and humans.

Thiouracil belongs to the xenobiotic thyreostats, which are growth-promoting agents illegally used in animal production. Recently it has been reported that thiouracil is suspected to have a natural origin. The European Union of Reference Laboratory guidance paper of 2007 acknowledged this by stating that thiouracil concentrations below 10 µg l⁻¹ might have a natural origin derived from the consumption of Brassicaceae. The present research aimed at endorsing this possible natural occurrence. Urine samples of animals (livestock and domesticated) with known and unknown clinical backgrounds were analysed for thiouracil with a newly developed ultra-high performance liquid chromatography coupled to a triple quadrupole mass spectrometric analysis method without derivatisation. In addition, a small-scale 9-day human experiment with Brassicaceae vegetables was performed to investigate if this natural prevalence could be extrapolated to the human population. The untreated animals had thiouracil concentrations below 10 µg l⁻¹ acknowledging the alleged natural occurrence of thiouracil. As for the humans, in 66.7% of the urine samples thiouracil was found above the CC(α) of 2.2 µg l⁻¹. However, the correlation with the Brassicaceae diet proved to be non-significant (p = 0.095). Nevertheless, these results clearly demonstrate the natural occurrence of thiouracil in urine of animals and humans. The exact origin of this natural thiouracil trace still needs to be identified.

Influence of breed and slaughter weight on boar taint prevalence in entire male pigs.

Piétrain (P), Large White (LW) and Belgian Landrace stress negative (BN) boars were slaughtered at 50, 70, 90 or 110 kg live weight to investigate breed differences and the effect of slaughter weight on boar taint prevalence. Boar taint was quantified by four different methodologies: sensory evaluation of neckfat heated with a hot iron in the slaughterhouse, sensory evaluation of meat by consumer panels, sensory evaluation of fat and meat by expert panels and laboratory analysis of indole, skatole and androstenone in backfat. Skatole levels in backfat were significantly higher for LW and BN than for P boars. The androstenone levels and the hot iron method revealed a significant interaction between breed and slaughter weight. On the other hand, experts detected an effect of weight on the androstenone odour perception, which was significantly higher in fat from boars slaughtered at 90 kg compared with 50 kg, and significantly higher in meat from boars slaughtered at 110 kg compared with 50 kg. Consumers did not detect differences in the sensory characteristics among breeds or slaughter weight. These results indicate opportunities to minimise the risk of boar taint in entire male pigs by carefully selecting a combination of breed and slaughter weight. Along with the optimal slaughter weight, the effectiveness of reducing boar taint by lowering slaughter weight appeared to be breed dependent.

Influence of soiling on boar taint in boars.

It has been suggested that skatole, one of the main compounds responsible for boar taint, can be lowered by keeping pigs clean, as skatole can be absorbed through skin and/or lungs (Hansen, Larsen, Jensen, HansenMoller & Bartongade, 1994). With this experiment, we further investigated this hypothesis by comparing extremely clean with extremely dirty animals with regard to the occurrence of boar taint. One group of boars was washed daily and pens were mucked on and littered down daily (CLEAN), a second group of boars was rubbed with faeces daily (DIRTY) and a third group of boars was kept in control conditions (CONTROL). The treatment was performed during the last four weeks before slaughter. According to the standardised consumer panel evaluations, boars subjected to extra soiling had a higher concentration of boar taint than boars that were kept extra clean. In contrast, expert panels judged general meat flavour to be inferior in CLEAN than CONTROL pigs. The home consumer panel, the hot iron method, and laboratory analyses, i.e., the presence of indole, skatole and androstenone in fat and serum, all showed no significant differences. So no clear indications towards skatole reduction by improving cleanliness of pigs were found.

The state of the art of residue analysis: the 6th VDRA symposium 2010.

Since the onset of residue analysis (ca 40 years ago) a lot of attention has been paid to the amelioration of analytical methods, for example, lowering the limits of detection (LOD) and limits of quantification (LOQ) or decision limits (CCα) and detection capabilities (CCß), including an increase in the number of analytes, shortening runtimes, increasing sample throughput, amongst others. The state of the art in residue analysis, which was presented at the VDRA 2010 symposium (Hormone and Veterinary Drug Residue Analysis) in Ghent is reviewed in this article. From an analytical point of view, the use of ultra high performance liquid chromatography (UHPLC) hyphenated with accurate mass spectrometry is often used in combination with other (biological) detection systems and 'omic' approaches. Through these techniques more xenobiotic substances turn out to be naturally occurring in some matrices and/or circumstances (e.g. thiouracil, chloramphenicol and semicarbazide).

Development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method for quantifying thyreostats in urine without derivatisation.

Thyreostatic drugs, illegally administrated to livestock for fattening purposes, are banned in the European Union since 1981 (Council Directive 81/602/EC). For monitoring their illegal use, sensitive and specific analytical methods are required. In this study an UHPLC-MS/MS method was described for quantitative analysis of eight thyreostatic drugs in urine, this without a derivatisation step. The sample pretreatment involved a reduction step with dithiothreitol under denaturating conditions at 65 degrees C, followed by liquid-liquid extraction with ethyl acetate. This analytical procedure was subsequently validated according to the EU criteria (2002/657/EC Decision), resulting in decision limits and detection capabilities ranging between 1.1 and 5.5 microg L(-1) and 1.7 and 7.5 microg L(-1), respectively. The method obtained for all, xenobiotic thyreostats, a precision (relative standard deviation) lower than 15.5%, and the linearity ranged between 0.982 and 0.999. The performance characteristics fulfill not only the requirements of the EU regarding the provisional minimum required performance limit (100 microg L(-1)), but also the recommended concentration fixed at 10 microg L(-1) in urine set by the Community of Reference Laboratories. Future experiments applying this method should provide the answer to the alleged endogenous status of thiouracil.

Endogenous boldenone-formation in cattle: alternative invertebrate organisms to elucidate the enzymatic pathway and the potential role of edible fungi on cattle's feed.

Although beta-boldenone (bBol) used to be a marker of illegal steroid administration in calves, its endogenous formation has recently been demonstrated in these vertebrates. However, research on the pathway leading to bBol remains scarce. This study shows the usefulness of in vivo invertebrate models as alternatives to vertebrate animal experiments, using Neomysis integer and Lucilia sericata. In accordance with vertebrates, androstenedione (AED) was the main metabolite of beta-testosterone (bT) produced by these invertebrates, and bBol was also frequently detected. Moreover, in vitro experiments using feed-borne fungi and microsomes were useful to perform the pathway from bT to bBol. Even the conversion of phytosterols into steroids was shown in vitro. Both in vivo and in vitro, the conversion of bT into bBol could be demonstrated in this study. Metabolism of phytosterols by feed-borne fungi may be of particular importance to explain the endogenous bBol-formation by cattle. To the best of our knowledge, it is the first time the latter pathway is described in literature.

Characterisation of steroids in wooden crates of veal calves by accelerated solvent extraction (ASE) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-QqQ-MS-MS).

Illegal steroid administration to enhance growth performance in veal calves has long been, and still is, a serious issue facing regulatory agencies. Over the last years, stating undisputable markers of illegal treatment has become complex because of the endogenous origin of several anabolic steroids. Knowledge on the origin of an analyte is therefore of paramount importance. The present study shows the presence of steroid analytes in wooden crates used for housing veal calves. For this purpose, an analytical procedure using accelerated solvent extraction (ASE(R)), solid-phase extraction (SPE) and ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (U-HPLC-MS-MS) is developed for the characterisation of androstadienedione (ADD), boldenone (bBol), androstenedione (AED), beta-testosterone (bT), alpha-testosterone (aT), progesterone (P) and 17alpha-hydroxy-progesterone (OH-P) in wood samples. In samples of wooden crates used for housing veal calves, ADD, AED, aT and P could be identified. Using the standard addition approach concentrations of these analytes were determined ranging from 20 +/- 4 ppb to 32 +/- 4 ppb for ADD, from 19 +/- 5 ppb to 44 +/- 17 ppb for AED, from 11 +/- 6 ppb to 30 +/- 2 ppb for aT and from 14 +/- 1 ppb to 42 +/- 27 ppb for P, depending on the sample type. As exposure of veal calves to steroid hormones in their housing facilities might complicate decision-making on illegal hormone administration, inequitable slaughter of animals remains possible. Therefore, complete prohibition of wooden calf accommodation should be considered.