Quantifying maternal investment in mammals using allometry.

Maternal investment influences the survival and reproduction of both mothers and their progeny and plays a crucial role in understanding individuals' life-history and population ecology. To reveal the complex mechanisms associated with reproduction and investment, it is necessary to examine variations in maternal investment across species. Comparisons across species call for a standardised method to quantify maternal investment, which remained to be developed. This paper addresses this limitation by introducing the maternal investment metric - MI - for mammalian species, established through the allometric scaling of the litter mass at weaning age by the adult mass and investment duration (i.e. gestation + lactation duration) of a species. Using a database encompassing hundreds of mammalian species, we show that the metric is not highly sensitive to the regression method used to fit the allometric relationship or to the proxy used for adult body mass. The comparison of the maternal investment metric between mammalian subclasses and orders reveals strong differences across taxa. For example, our metric confirms that Eutheria have a higher maternal investment than Metatheria. We discuss how further research could use the maternal investment metric as a valuable tool to understand variation in reproductive strategies.

Development and validation of a versatile non-invasive urinary steroidomics method for wildlife biomonitoring.

Wildlife conservation is often challenged by a lack of knowledge about the reproduction biology and adaptability of endangered species. Although monitoring steroids and related molecules can increase this knowledge, the applicability of current techniques (e.g. immunoassays) is hampered by species-specific steroid metabolism and the requisite to avoid invasive sampling. This study presents a validated steroidomics method for the (un)targeted screening of a wide range of sex and stress steroids and related molecules in urine using ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). In total, 50 steroids (conjugated and non-conjugated androgens, estrogens, progestogens and glucocorticoids) and 6 prostaglandins could be uniquely detected. A total of 45 out of 56 compounds demonstrated a detection limit below 0.01 ng µL-1. Excellent linearity (R2 > 0.99), precision (CV < 20 %), and recovery (80-120 %) were observed for 46, 41, and 39 compounds, respectively. Untargeted screening of pooled giant panda and human samples yielded 9691 and 8366 features with CV < 30 %, from which 84.1 % and 83.0 %, respectively, also demonstrated excellent linearity (R2 > 0.90). The biological validity of the method was investigated on male and female giant panda urine (n = 20), as well as pooled human samples (n = 10). A total of 24 different steroids were detected with clear qualitative and quantitative differences between human and giant panda samples. Furthermore, expected differences were revealed between female giant panda samples from different reproductive phases. In contrast to traditional biomonitoring techniques, the developed steroidomics method was able to screen a wide range of compounds and provide information on the putative identities of metabolites potentially important for reproductive monitoring in giant pandas. These results illustrate the advancements steroidomics brings to the field of wildlife biomonitoring in the pursuit to better understand the biology of endangered species.

The novel use of urinary androgens to optimise detection of the fertile window in giant pandas.

Abstract: Giant pandas are mono-estrus seasonal breeders, with the breeding season typically occurring in the spring. Successful fertilization is followed by an embryonic diapause, of variable length, with birth in the late summer/autumn. There is a need for additional understanding of giant panda reproductive physiology, and the development of enhanced biomarkers for impending proestrus and peak fertility. We aimed to determine the utility of non-invasive androgen measurements in the detection of both proestrus and estrus. Urine from 20 cycles (-40 days to +10 days from peak estrus) from 5 female giant pandas was analyzed for estrogen, progestogens and androgens (via testosterone and DHEA assays), and hormone concentrations were corrected against urinary specific gravity. Across proestrus, estrogens increased while progestogens and androgens decreased - at the point of entry into proestrus, androgens (as detected by the testosterone assay) decreased prior to progestogens and gave 4 days advanced warning of proestrus. At the time of peak estrus, androgens (as detected by the DHEA assay) were significantly increased at the time of the decrease in estrogen metabolites from the peak, acting as an alternative confirmatory indicator of the fertile window. This novel finding allows for enlargement of the preparative window for captive breeding and facilitates panda management within breeding programmes. Androgens allow an enhanced monitoring of giant panda estrus, not only advancing the warning of impending proestrus, but also prospectively identifying peak fertility. Lay summary: Giant pandas have one chance at pregnancy per year. The 2-day fertile window timing varies by year and panda. This is monitored by measuring the level of estrogens in the urine, which increase, indicating an upcoming fertile period. After 1-2 weeks of increase, estrogens peak and fall, marking the optimal fertile time. We tested other hormones to see if we can predict the fertile window in advance, and the specific fertile time with more accuracy. In 20 breeding seasons from 5 females, we found androgens, usually thought of as male hormones, had an important role. Testosterone gives 4 days advanced warning of estrogens increasing. DHEA identified peak estrogen and the fertile time before needing to see a confirmed decrease in estrogen itself. Therefore, androgens help improve monitoring of the giant panda breeding season, giving early warning of fertility, key in facilitating captive breeding and giant panda conservation.

Relations between the time of ovulation and fecal estrogen concentration in sows.

Artificial insemination (AI) is the most important biotechnology in pig reproduction. To achieve the best possible fertility results, appropriate timing of the insemination is essential. The optimal time for AI is 12 h before to 4 h after ovulation. This time-frame, unlike in estrus, is not recognizable through external indicators. It would, therefore, be beneficial to find simple and economical methods that support manual estrus checks and are able to determine the time of ovulation more accurately. On this basis, starting 80 h after weaning, 14 DanBred sows (parity: 5.2 ± 2.4) were checked for ovulation via ultrasound scans every 8 h over a period of 72 h. Additionally, rectal fecal samples were taken and analyzed for their estrogen concentration to assess possible relations to ovulation time. On average, sows ovulated 121 ± 10 h after weaning and 16 ± 9 h after onset of heat. There was a prominent drop in fecal estrogen levels 4 h before ovulation when compared to almost all other points in time (before ovulation: 20 h (P = 0.056), 12 h (P = 0.006); after ovulation: 4 h and 12 h (P < 0.001)). There are, however, significant differences in the sow-individual fecal estrogen concentrations for which several influencing factors must be considered.

Decreasing glucocorticoid levels towards the expansion front suggest ongoing expansion in a terrestrial mammal.

Understanding the causes of range expansions in abundant species can help predict future species distributions. During range expansions, animals are exposed to novel environments and are required to cope with new and unpredictable stressors. Glucocorticoids (GCs) are mediators of the hormonal and behavioural mechanisms allowing animals to cope with unpredictable changes in the environment and are therefore expected to differ between populations at expansion edge and the historic range. However, to date, very few studies have evaluated the relationship between GCs and range expansion. The Egyptian mongoose has been rapidly expanding its range in Portugal over the past 30 years. In this study, we applied an information theoretic approach to determine the most important spatial and environmental predictors of hair GCs (hGCs) in the population, after controlling for normal patterns of hGC variation in the species. We observed a decrease in hGC as distance from the historic range increased (i.e. closer to the expansion front). This distance term was present in all of the top models and had a 95% confidence interval (95% CI) that did not overlap with zero, strongly supporting its influence on hGC. We estimated a 0.031 pg/mg (95% CI: -0.057, -0.004) decrease in hGCs for each kilometre distance to the Tagus River, which was once the limit of the species' distribution. Our results indicate that the species' expansion is unlikely to be limited by mechanisms related to or mediated by the physiological stress response. The decrease in hGC levels towards the expansion edge coupled with limited evidence of a negative effect of human population density suggests that the species' northward expansion in Portugal could continue.

The female effect-how female receptivity influences faecal testosterone metabolite levels, socio-positive behaviour and vocalization in male Southern white rhinoceroses.

Testosterone is known to be essential for sexual maturation as well as for the display of behavioural traits linked to reproduction. At the same time, external factors such as the presence of receptive females may affect testosterone levels, stressing the hormone's substantial role in reproductive success. It is therefore of major interest to investigate the links between androgens, behaviour and the social environment especially in species that rely on a resilient reproduction rate, such as the white rhinoceros (WR). We collected faecal samples of 16 male Southern WR (Ceratotherium simum simum) aged between 1 and 44 years from 11 European zoos. Audio and video recordings were simultaneously taken from five of the study males that were sexually mature and had direct contact with receptive females. Our results showed a positive correlation of faecal testosterone metabolite (fTM) concentrations and progressing age up until adulthood followed by a decline in older males. While previous reproductive success did not show any effect, the access to receptive females resulted in higher fTM levels. Thereby, fTM concentrations remained at the same level regardless of the receptivity phase, while social cohesion with respective females, affiliative behaviour as well as call rates of Pant and Hiss distinctly peaked during the receptive compared to the non-receptive periods. Conclusively, the immediate presence of receptive females poses a female effect that enhances the overall androgen levels in males and, thus, might facilitate their reproductive success. However, androgens do not seem to be the main driver of behavioural changes during courtship or mating. By linking endocrinological and socio-behavioural factors, we were able to provide an applicable basis for non-invasive monitoring of reproductive behaviour in male WR in captivity, thereby contributing to deeper understanding of potential reproduction impairments in a species whose population in captivity remains not fully self-sustaining.

Linking socio-sexual and vocal behaviour with faecal progesterone and oestrogen metabolite levels in Southern white rhinoceros females.

Progesterone and oestrogen are the main gonadal steroid hormones that regulate the ovarian activity and induce the fertile oestrus period in females. The monitoring of this receptive phase is particularly decisive for captive breeding and is commonly based on the observation of female behavioural patterns that coincide with their hormonal oestrus. However, in the white rhinoceros (WR), a species that is well known for its impaired reproductive rate in captivity, the female behavioural and vocal indicators of receptivity have not been systematically investigated or linked to their hormonal states so far. In order to close this gap, we combined behavioural and acoustic recordings, conducted over an average period of 32 days, with the analysis of faecal progesterone and oestrogen metabolite concentrations (fPM and fEM) in 27 adult Southern white rhinoceros (Ceratotherium simum simum; SWR) females from 10 European zoos. For eight of the study females, we were able to detect a receptive period indicated by their acceptance of sexual behaviour from the bulls. The comparison of behaviour and vocalization between receptive and non-receptive periods in these females demonstrated that particularly presenting and marking behaviour distinctly peaked during the receptive period, indicating the significance of olfactory signalling for female reproductive behaviour. Based on the analysis of fPM profiles, we were able to identify different reproductive states (cycling, non-cycling, pregnant) in 21 of 27 study females. In contrast, fEM profiles proved to be unsuitable for the detection of ovarian activity. For the majority (five of six females for which behavioural receptivity and endocrine cyclicity were determined), a coincidence of their receptive period and the hormonal oestrus, indicated by a nadir in fPM levels, could be detected. Conclusively, this study revealed a comprehensive behavioural repertoire that reflects the hormonal oestrus in SWR females and can therefore be reliably used for non-invasive ex situ reproduction monitoring.

Instant killing of pathogenic chytrid fungi by disposable nitrile gloves prevents disease transmission between amphibians.

To prevent transmission of the pathogenic chytrid fungi Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal), hygiene protocols prescribe the single use of disposable gloves for handling amphibians. We discovered that rinse water from nitrile gloves instantly kills 99% of Bd and Bsal zoospores. Transmission experiments using midwife toads (Alytes obstetricans) and Bd, and Alpine newts (Ichthyosaura alpestris) and Bsal, show that the use of the same pair of gloves for 2 subsequent individuals does not result in significant transmission of any chytrid fungus. In contrast, handling infected amphibians bare-handed caused transmission of Bsal in 4 out of 10 replicates, but did not result in transmission of Bd. Based on the manufacturer's information, high resolution mass spectrometry (HRMS) and colorimetric tests, calcium lactate and calcium nitrate were identified as compounds with antifungal activity against both Bd and Bsal. These findings corroborate the importance of wearing gloves as an important sanitary measure in amphibian disease prevention. If the highly recommended single use of gloves is not possible, handling multiple post-metamorphic amphibians with the same pair of nitrile gloves should still be preferred above bare-handed manipulation.

Developing and Understanding Olfactory Evaluation of Boar Taint.

Trained expert panels are used routinely in boar taint research, with varying protocols for training of panelists and scoring methods. We describe a standardized process for training and scoring, to contribute to standardize the olfactory detection of boar taint. Three experiments are described in which we (1) evaluate the importance of training and the effect of the previous sample, (2) determine detection thresholds on strips and in fat for our panel, and (3) test priming panelists before boar taint evaluation. For the final evaluation of boar taint, we propose a consistent three-person evaluation scoring on a 0-4 scale using a final mean score of 0.5 as the cut-off for boar taint. This gave an optimal sensitivity of 0.81 and a specificity of 0.56 compared to chemical cut-offs. Even limited training proved useful, but priming assessors with strips did not improve the evaluation of fat samples. Detection thresholds were higher in fat compared to strips, except for indole. We recommend panelists to always smell a non-tainted control sample after a tainted one as a 'reset' mechanism, before continuing. For longitudinal studies, we additionally advise to set up an expert panel with a fixed number of assessors performing each evaluation in duplicate.

Sex steroids and glucocorticoid ratios in Iberian lynx hair.

Knowledge on species' reproductive biology is a fundamental pre-requisite of every conservation effort, but is often lacking. Sex steroids can provide valuable information for the assessment of reproductive success, whereas glucocorticoids are used to assess adrenocortical activity and stress-related bodily adaption. However, due to their perilous condition, access to animals is often difficult, which makes hormone measurement in non-invasively collected hair samples an attractive option. We determined cortisol, cortisone, corticosterone, testosterone and progesterone in Iberian lynx hair using enzyme immunoassay (EIA). Cross-validation was performed with high-performance liquid chromatography (HPLC) and high-performance liquid chromatography coupled to tandem mass spectrometry (HPLC-MS/MS). Finally, we statistically evaluated the variations of sex steroids and glucocorticoids according to age, sex, origin, behavior and management. All steroids except corticosterone were detectable in Iberian lynx hair. Hair progesterone measured by EIA was overestimated by cross-reaction with 5α-dihydroprogesterone, a biologically active gestagene, and was highly correlated with HPLC-MS/MS results. Progesterone was higher in adult females compared to all other age-sex groups. Cortisol measured by EIA was overestimated due to antibody cross-reactivity with cortisone and was correlated to the sum of HPLC-MS/MS measurements for cortisol and cortisone. Cortisol was higher in females than in males measured by HPLC-MS/MS, but the EIA results were confounded by the lack of specificity. When using cortisol-cortisone and cortisol-dihydroepiandrosterone ratios, differences were noted between wild-caught and captive-bred lynxes. Additionally, longitudinal EIA measurements of an Iberian lynx after a wildfire showed an inversion of the cortisol-cortisone ratio that later subsided. These results validate the use of hair progesterone measurement for Iberian lynx reproductive monitoring and add to the growing evidence supporting the need for a more comprehensive approach to hair steroid measurement that accounts for local interconversion and co-regulation mechanisms.

Valorisation of tainted boar meat in patties, frankfurter sausages and cooked ham by means of targeted dilution, cooking and smoking.

Because of the need to abolish the castration of piglets without anaesthesia/analgesia, the pig industry is searching for a mode of action for the valorisation of meat with boar taint, an off-odour in entire male pigs. Carcasses with boar taint were selected by means of sensory and chemical analysis, after which patties with different levels of tainted boar meat were produced, as well as cooked ham and Frankfurter sausages using different smoke condensates and cooking temperatures. For these products orthonasal and retronasal boar taint odour were assessed by a trained expert panel. The results offer guidance regarding dilution of tainted meat (with <400 µg/kg androstenone if skatole is low or <200 µg/kg androstenone in concurrence with ≥37 µg/kg skatole) and the potential application of smoke condensates (e.g., Rudinsmoke C for sausages and Smokez LFBN for ham) as promising boar taint masking strategies.

Urinary estrogens as a non-invasive biomarker of viable pregnancy in the giant panda (Ailuropoda melanoleuca).

Female giant pandas show complex reproductive traits, being seasonally monoestrus, displaying a variable length embryonic diapause and exhibiting pseudopregnancy. Currently, there is no confirmatory non-invasive biomarker of blastocyst implantation or pregnancy. This study aimed to monitor urinary estrogens across gestation in pregnancy (n = 4), pseudopregnancy (n = 4) and non-birth cycles (n = 5) in the giant panda. A pregnancy-specific profile of estrogens corrected for urinary specific gravity was identified during the gestation period. Pregnant females showed increasing concentrations of estrogens for 29 days until birth, no increase was observed during pseudopregnancy and the two profiles were distinguishable from each other for the final 2 weeks of the cycle suggesting the estrogens are of placental origin. This allowed a nomogram, starting at a known fixed point during the cycle, to be created and tested with cycles of known outcome, and cycles which were inseminated but did not result in a birth. Non-birth profiles showed deviations from that of pregnancy. We believe these deviations indicate the point of failure of the placenta to support a developing cub. Non-invasive longitudinal monitoring of estrogen concentrations therefore has the potential to be developed as a panda pregnancy test to predict viable cub development.

Ultra-high-performance liquid chromatography coupled to quadrupole orbitrap high-resolution mass spectrometry for multi-residue screening of pesticides, (veterinary) drugs and mycotoxins in edible insects.

A generic extraction and UHPLC-Q-Orbitrap™-HRMS method was developed for four insect species (mealworm, grasshopper, house cricket and black soldier fly) analyzing a large spectrum of organic chemical contaminants, including pesticides (n = 25), (veterinary) drugs (n = 29), and mycotoxins (n = 23). To prove the method as 'fit-for-purpose', a successful validation was performed, both qualitatively, by determining the screening detection limit (SDL), selectivity and specificity, as well as semi-quantitatively, by assessing the within-day precision (relative standard deviation (RSD)) and recovery. For both the mealworm, grasshopper, house cricket and black soldier fly, 64, 61, 59 and 62 compounds were detected at the respective SDL levels (1-100 µg kg-1), predominantly below existing maximum residue limits for other edible matrices. Mean recoveries ranged between 70% and 120% and RSD-values were in line with European regulations (CD 2002/657/EC; SANCO). Finally, the potential of the screening methodology was demonstrated on real insect samples, revealing minor to no contamination.

A validated multi-matrix platform for metabolomic fingerprinting of human urine, feces and plasma using ultra-high performance liquid-chromatography coupled to hybrid orbitrap high-resolution mass spectrometry.

In recent years, metabolomics has surfaced as an innovative research strategy in human metabolism, whereby selection of the biological matrix and its inherent metabolome is of crucial importance. However, focusing on a single matrix may imply that relevant molecules of complementary physiological pathways, covered by other matrices, are missed. To address this problem, this study presents a unique multi-matrix platform for polar metabolic fingerprinting of feces, plasma and urine, applying ultra-high performance liquid-chromatography coupled to hybrid quadrupole-Orbitrap high-resolution mass spectrometry, that is able to achieve a significantly higher coverage of the system's metabolome and reveal more significant results and interesting correlations in comparison with single-matrix analyses. All three fingerprinting approaches were proven 'fit-for-purpose' through extensive validation in which a number of endogenous metabolites were measured in representative quality control samples. For targeted and untargeted validation of all three matrices, excellent linearity (coefficients of determination R2 ≥ 0.99 or 0.90 respectively), recovery and precision (coefficients of variance ≤ 15% or 30% respectively) were observed. The potential of the platform was demonstrated by subjecting fecal, urine and plasma samples (collected within one day) from ten healthy volunteers to metabolic fingerprinting, yielding respectively 9 672, 9 647, and 6122 components. Orthogonal partial least-squares discriminant analysis provided similar results for feces and plasma to discriminate according to gender (p-value, R2(X), R2(Y) and Q2(Y)), suggesting feces as an excellent alternative biofluid to plasma. Moreover, combining the different matrices improved the model's predictivity, indicating the superiority of multi-matrix platforms for research purposes in biomarker detection or pathway elucidation and in the selection of the most optimal matrix for future clinical purposes.

Urinary specific gravity as an alternative for the normalisation of endocrine metabolite concentrations in giant panda (Ailuropoda melanoleuca) reproductive monitoring.

Reproductive monitoring for captive breeding in giant pandas is based on behavioural observation and non-invasive hormone analysis. In urine, interpretation of results requires normalisation due to an animal's changing hydration. Correction of urinary concentrations based on creatinine is the gold standard. In this study, a largely unexplored, easy-to-perform normalisation technique, based on urinary specific gravity (USpG), was examined and compared to creatinine. To this extent, six cycles from two female pandas (SB741(1) and SB569(5)) were monitored through urine analysis for oestrogen, progesterone, ceruloplasmin and 13,14-dihydro-15-keto-PGF2a (PGFM). The Pearson's correlation between creatinine and USpG was high (r = 0.805-0.894; p < 0.01), indicative for a similar performance of both normalisation methods. However, generally lower values were observed during pro-oestrus and primary (progesterone) rise. This could be associated with huge shifts in appetite, monitored by faecal output (kg) with an averaged > 50% decrease during oestrus and >50% increase during primary progesterone rise. In parallel, respectively highest and lowest creatinine and USpG levels, were measured, with creatinine obviously more affected as a result of linkage with muscle tissue metabolism affected by reproductive hormones. As a consequence, metabolite levels were significantly different between both corrected datasets with significantly higher oestrogen peak levels during oestrus ranging from 2.13-86.93 and 31.61-306.45 ng/mL (USpG correction) versus 2.33-31.20 and 36.36-249.05 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively, and significant lower progesterone levels during primary progesterone rise ranging from 0.35-3.21 and 0.85-6.80 ng/mL (USpG correction) versus 0.52-10.31 and 2.10-272.74 ng/mL Cr (creatinine correction) for SB569 and SB741 respectively. Consequently, USpG correction rendered unbiased profiles, less subject to variation and metabolic artefacts and therefore allowed a more straightforward identification of peak oestrogen and onset of secondary progesterone rise, being potentially advantageous for future studies unravelling key giant panda reproductive events, including (delayed) implantation. The alternative application of USpG as a normalisation factor was further supported by its easy application and environmental and technical robustness.

Discovery of urinary biomarkers to discriminate between exogenous and semi-endogenous thiouracil in cattle: A parallel-like randomized design.

In the European Union, the use of thyreostats for animal fattening purposes has been banned and monitoring plans have been established to detect potential abuse. However, this is not always straightforward as thyreostats such as thiouracil may also have a semi-endogenous origin. Therefore, this study aimed at defining urinary metabolites, which may aid in defining the origin of detected thiouracil. Hereto, a parallel-like randomized in vivo study was conducted in which calves (n = 8) and cows (n = 8) were subjected to either a control treatment, rapeseed-enriched diet to induce semi-endogenous formation, or thiouracil treatment. Urine samples (n = 330) were assessed through metabolic fingerprinting, employing liquid-chromatography and Q-ExactiveTM Orbitrap mass spectrometry. Urinary fingerprints comprised up to 40,000 features whereby multivariate discriminant analysis was able to point out significant metabolome differences between treatments (Q2(Y) ≥ 0.873). Using the validated models, a total of twelve metabolites (including thiouracil) were assigned marker potential. Combining these markers into age-dependent biomarker panels rendered a tool by which sample classification could be improved in comparison with thiouracil-based thresholds, and this during on-going thiouracil treatment (specificities ≥ 95.2% and sensitivities ≥ 85.7%), post-treatment (sensitivities ≥ 80% for ≥ 24 h after last administration), and simulated low-dose thiouracil treatment (exogenous thiouracil below 30 ng µL-1). Moreover, the metabolic relevance of revealed markers was supported by the suggested identities, for which a structural link with thiouracil could be determined in most cases. The proposed biomarker panels may contribute to a more justified decision-making in monitoring thiouracil abuse.

Sensory evaluation of boar meat products by trained experts.

Rearing entire male pigs, one of the alternatives for surgical castration, entails the possible occurrence of boar taint. This study aimed at the investigation of the acceptability of meat from entire male pigs in 8 different meat products (cutlets, bacon, blade loin, tenderloin, dry fermented sausage, cooked ham, dry-cured ham and minced meat) by trained assessors. Generally, the sensory evaluation of meat samples was affected the most in the androstenone (AEON) group, indicating that AEON is the most offensive boar taint compound for sensitive assessors. Differences between the meat products showed the highest potential for processing tainted meat in cold meat products, which was most likely due to the serving temperature on the one hand and production-related influences on the other. However, more insights regarding reducing and masking effects of production-related factors on boar taint are necessary.

Sensory evaluation of boar-taint-containing minced meat, dry-cured ham and dry fermented sausage by a trained expert panel and consumers.

One of the main issues related to entire male pigs is the occurrence of boar taint, an off-odour, which compromises meat consumability. In this study, odour thresholds (indole: 24-65µgkg-1, skatole: 44-89µgkg-1, androstenone: 121-342µgkg-1) for the boar taint compounds were estimated in minced meat, dry fermented sausage and dry-cured ham. Afterwards, sensory evaluation of these products containing 10% tainted meat (minced meat and dry fermented sausage) or moderate boar taint compound levels (dry-cured ham) occurred. The beneficial effect of diluting tainted meat was demonstrated, as no significant difference in consumability was observed between gilts and 10% tainted meat by experts as well as consumers. Also dry-curing proved a promising technique for masking boar taint and preventing consumer dissatisfaction. The obtained results demonstrate the applicability of the estimated thresholds in meat as a tool for identifying masking and reducing strategies on the perception of boar taint.

Rapid evaporative ionization mass spectrometry for high-throughput screening in food analysis: The case of boar taint.

Boar taint is a contemporary off-odor present in meat of uncastrated male pigs. As European Member States intend to abandon surgical castration of pigs by 2018, this off-odor has gained a lot of research interest. In this study, rapid evaporative ionization mass spectrometry (REIMS) was explored for the rapid detection of boar taint in neck fat. Untargeted screening of samples (n=150) enabled discrimination between sow, tainted and untainted boars. The obtained OPLS-DA models showed excellent classification accuracy, i.e. 99% and 100% for sow and boar samples or solely boar samples, respectively. Furthermore, the obtained models demonstrated excellent validation characteristics (R2(Y)=0.872-0.969; Q2(Y)=0.756-0.917), which were confirmed by CV-ANOVA (p<0.001) and permutation testing. In conclusion, in this work for the first time highly accurate and high-throughput (<10s) classification of tainted and untainted boar samples was achieved, rendering REIMS a promising technique for predictive modelling in food safety and quality applications.

Rapid method for the simultaneous detection of boar taint compounds by means of solid phase microextraction coupled to gas chromatography/mass spectrometry.

Because of animal welfare issues, the voluntary ban on surgical castration of male piglets, starting January 2018 was announced in a European Treaty. One viable alternative is the fattening of entire male pigs. However, this can cause negative consumer reactions due to the occurrence of boar taint and possibly lead to severe economic losses in pig husbandry. In this study, headspace solid phase microextraction (HS-SPME) coupled to GC-MS was used in the development and optimization of a candidate method for fast and accurate detection of the boar taint compounds. Remarkably fast extraction (45s) of the boar taint compounds from adipose tissue was achieved by singeing the fat with a soldering iron while released volatiles were extracted in-situ using HS-SPME. The obtained method showed good performance characteristics after validation according to CD 2002/657/EC and ISO/IEC 17025 guidelines. Moreover, cross-validation with an in-house UHPLC-HR-Orbitrap-MS method showed good agreement between an in-laboratory method and the new candidate method for the fast extraction and detection of skatole and androstenone, which emphasizes the accuracy of this new SPME-GC-MS method. Threshold detection of the boar taint compounds on a portable GC-MS could not be achieved. However, despite the lack of sensitivity obtained on the latter instrument, a very fast method with run-to-run time of 3.5min for the detection of the boar taint compounds was developed.