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Boar taint is an unpleasant odour found in the carcasses of entire male pigs, resulting from androstenone and skatole accumulation during pubertal development, and impacting pork quality. This study proposes the validation of an adapted chromatographic method for quantifying skatole and androstenone in the pigs' liquid fat using fluorescence detection. A good chromatographic separation was achieved, with skatole (SKA) and androstenone (AND) elution at 4.4 and 9.9 min., respectively. An external calibration method was applied, with calibration curves correlation coefficient of 0.9999 for both analytes. Detection limit values were 1.53 and 16.02 ng/g for SKA and AND, respectively. SKA recovery was 99.72±2.34 % (2.34 % RSD) and 102.84±1.62 % (1.57 % RSD) for AND. Results showed good precision values (repeatability <2.46 % RSD for SKA, <6.85 % RSD for AND; intermediate precision <2.87 % RSD for SKA, <6.98 % RSD for AND). The method's robustness was tested and the values were within the reference ranges. The validation results proved that the adaptation of an existing method resulted in good assessments of robustness, reliability and accuracy., (© 2024 The Authors. ChemistryOpen published by Wiley-VCH GmbH.)

Mainly skatole and androstenone have so far been considered causative for boar taint. Using a mixed methods approach it is shown herein that 2-aminoacetophenone (AAP) affects human perception of pork, too. We explored the importance of AAP in four trials: (1) chemical analyses of 221 fat samples from boar carcasses revealed that AAP occurs, on average, in similar quantities as skatole while the levels of androstenone being four-fold. (2) ranking tests with mixtures of androstenone and/or skatole with AAP presented on smell strips to trained sensory assessors showed that AAP amplifies boar odour. In order to study AAP's importance in meat products, four experimental variants of Lyon type sausage were then produced: a control, a product with added skatole (0.075 μg/g fat tissue), with added AAP (0.075 μg/g fat tissue), and with addition of both compounds. (3) results of a consumer discrimination test panel (n = 71) showed that, when added to a sausage system, APP causes a sensory difference of similar size as skatole while the methodology chosen affects the effect size: tetrad tests proved to be more sensitive than duo trio difference tests, in the tetrad test a sensory difference expressed as d' (d-prime) of 1.0 was reached. (4) a hedonic consumer test (n = 121) finally revealed that APP decreased consumer liking of the APP-spiked sausage - even to a stronger extent than skatole. APP caused significant drops in smell, taste, mouth-feel, after-taste and overall liking in Lyoner. Overall the findings suggest that, in the context of pork meat, AAP is of similar olfactory importance as skatole., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The testing strategy for the detection of testosterone (T) or T-prohormones is based on the longitudinal evaluation of urinary steroid concentrations accompanied by subsequent isotope ratio mass spectrometry (IRMS)-based confirmation of samples showing atypical concentrations or concentration ratios. In recent years, the IRMS methodology focussed more and more on T itself and on the metabolites of T, 5α- and 5β-androstanediol. These target analytes showed the best sensitivity and retrospectivity, but their use has occasionally been challenging due to their comparably low urinary concentrations. Conversely, the carbon isotope ratios (CIR) of the main urinary metabolites of T, androsterone (A) and etiocholanolone (EITO), can readily be measured even from low urine volumes; those however, commonly offer a lower sensitivity and shorter retrospectivity in uncovering T misuse. Within this study, the CIRs of A and ETIO were combined with their urinary concentrations, resulting in a single parameter referred to as 'difference from weighted mean' (DWM). Both glucuronidated and sulfated steroids were investigated, encompassing a reference population (n = 110), longitudinal studies on three individuals, influence of ethanol in two individuals, and re-analysis of several administration studies including T, dihydrotestosterone, androstenedione, epiandrosterone, dehydroepiandrosterone, and T-gel. Especially DWM calculated for the sulfoconjugated steroids significantly prolonged the detection time of steroid hormone administrations when individual reference ranges were applied. Administration studies employing T encompassing CIR common for Europe (-23.8‰ and -24.4‰) were investigated and, even though for a significantly shorter time period and less pronounced, DWM could demonstrate the exogenous source of T metabolites., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Intact phase II steroid metabolites have poor product ion mass spectra under collision-induced dissociation (CID) conditions. Therefore, we present herein the liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/(MS)) behavior of intact phase II metabolites of oxosteroids after derivatization. Based on the fact that Girard's reagent T (GRT), as derivatization reagent, was both convenient and efficient in terms of the enhancement in the ionization efficiency and the production of diagnostic product ions related to the steroid moiety, the latter was preferably selected between methoxamine and hydroxylamine upon the model compounds of androsterone glucuronide and androsterone sulfate. Sixteen different glucuronides and 29 sulfate conjugated metabolites of anabolic androgenic steroids (AASs), available either as pure reference materials or synthesized/extracted from administration studies, were derivatized with GRT, and their product ion spectra are presented. Product ion spectra include in all cases high number of product ions that in some cases are characteristic for certain structures of the steroid backbone. More specifically, preliminary results have shown major differences in fragmentation pattern for 17α/17β-isomers of the sulfate conjugates, but limited differentiation for 17α/17β-isomers of glucuronide conjugates and for 3α/3β- and 5α/5β-stereoisomers of both sulfate and glucuronide conjugates. Further to the suggestion of the current work, application on mesterolone administration studies confirmed-according to the World Anti-Doping Agency (WADA) TD2015IDCR-the presence of seven intact phase II metabolites, one glucuronide and six sulfates with use of LC-ESI-MS/(MS)., (© 2021 John Wiley & Sons, Ltd.)

The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography-tandem mass spectrometry (GC-MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/ml and not originated from pregnant female athletes or female treated with 19-norethisterone. 19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TCs), whereas androsterone (A), pregnanediol (PD), and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone-based formulations. Before the instrumental analysis, urine samples (<25 ml) were hydrolyzed with β-glucuronidase from Escherichia coli and extracted with n-pentane. Compounds of interest were purified through a single (for PT) or double (for 19-NE, 19-NA, A, and PD) liquid chromatographic step, to reduce the background noise and eliminate interferences that could have affect the accuracy of δ 13 C values. The limit of quantification (LOQ) of 2 ng/ml was ensured for both 19-NA and 19-NE. The 19-NE determination could be helpful in case of "unstable" urine samples, in late excretion phases or when coadministration with 5α-reductase inhibitors occur., (© 2020 John Wiley & Sons, Ltd.)

The peripheral zone (PZ) and transition zone (TZ) represent about 70% of the human prostate gland with each zone having differential ability to develop prostate cancer. Androgens and their receptor are the primary driving cause of prostate cancer growth and eventually castration-resistant prostate cancer (CRPC). De novo steroidogenesis has been identified as a key mechanism that develops during CRPC. Currently, there is very limited information available on human prostate tissue steroidogenesis. The purpose of the present study was to investigate steroid metabolism in human prostate cancer tissues with comparison between PZ and TZ. Human prostate cancer tumors were procured from the patients who underwent radical prostatectomy without any neoadjuvant therapy. Human prostate homogenates were used to quantify steroid levels intrinsically present in the tissues as well as formed after incubation with 2 µg/mL of 17-hydroxypregnenolone (17-OH-pregnenolone) or progesterone. A Waters Acquity ultraperformance liquid chromatography coupled to a Quattro Premier XE tandem quadrupole mass spectrometer using a C 18 column was used to measure thirteen steroids from the classical and backdoor steroidogenesis pathways. The intrinsic prostate tissue steroid levels were similar between PZ and TZ with dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), pregnenolone and 17-OH-pregnenolone levels higher than the other steroids measured. Interestingly, 5-pregnan-3,20-dione, 5-pregnan-3-ol-20-one, and 5-pregnan-17-ol-3,20-dione formation was significantly higher in both the zones of prostate tissues, whereas, androstenedione, testosterone, DHT, and progesterone levels were significantly lower after 60 min incubation compared to the 0 min control incubations. The incubations with progesterone had a similar outcome with 5-pregnan-3,20-dione and 5-pregnan-3-ol-20-one levels were elevated and the levels of DHT were lower in both PZ and TZ tissues. The net changes in steroid formation after the incubation were more observable with 17-OH-pregnenolone than with progesterone. In our knowledge, this is the first report of comprehensive analyses of intrinsic prostate tissue steroids and precursor-driven steroid metabolism using a sensitive liquid chromatography-mass spectrometry assay. In summary, the PZ and TZ of human prostate exhibited similar steroidogenic ability with distinction in the manner each zone utilizes the steroid precursors to divert the activity towards backdoor pathway through a complex matrix of steroidogenic mechanisms.

Identification and evaluation of long-term markers is crucial in prolonging the detection window for anabolic steroid abuse in sport. Recently, sulfoconjugated epiandrosterone was identified as a potential long-term marker for the abuse of certain endogenous anabolic agents, including testosterone, which continues to be widely used as a performance enhancing agent in sport. To evaluate the applicability of epiandrosterone sulfate as a marker for testosterone use, administration studies were conducted with multiple modes of testosterone administration - transdermal, intramuscular, and subcutaneous. A modified sample preparation method was used to collect both glucuronidated and sulfoconjugated analytes of interest. Carbon isotope ratio measurements from the administration studies are presented here. Epiandrosterone was less effective than the conventionally used target compounds for detection of the low dose application (transdermal gel). With intramuscular administration, epiandrosterone was more diagnostic than with transdermal administration, but it did not prolong the detection window more than the conventional target compounds. With subcutaneous administration, the doses administered to the subjects were varied and the effect on the epiandrosterone values was dependent on the magnitude of the dose administered. Epiandrosterone does not appear to be a useful marker in the detection of low dose testosterone administration. It is responsive to higher dose administration, but it does not provide an extension of the detection window relative to conventional target compounds., (© 2020 John Wiley & Sons, Ltd.)

Background: Urinary sulfate fraction of the anabolic androgenic steroids is not analyzed routinely in anti-doping analyses but has demonstrated in the last years an increasing interest among the anti-doping community. Sulfate conjugates are linked to plasma proteins increasing the residence time in the body compared to glucuro-conjugated metabolites, and then their analyses may allow improving the detection time window of specific metabolites. Hydrolysis of sulfates can be made enzymatically or chemically and can be challenging, depending on the strategy selected., Methods: Hydrolysis by solvolysis was validated for metabolic studies, focusing on setting a quality control able to assess the hydrolytic step. To the internal standards mixture, androsterone-D4 and etiocholanolone-D5 sulfate were added. The proposed protocol was applied over samples collected after dehydroepiandrosterone (DHEA) administrations., Results: The stability of the structures showed good results, and no evident formation of degradation products was observed. Internal standard to monitor the efficiency of hydrolysis, recovery, and retention time was successfully introduced. Additional analytes (4β-hydroxy-DHEA, 5-androstene-3β,17β-diol and 5α-androstane-3β,17β-diol) were found to be affected besides of DHEA and epiandrosterone (epiA) as previously described., Conclusions: Results in terms of linearity, precision, and accuracy, showed that the method is suitable to quantify seven analytes in urine in the sulfated fraction. The validated method was successfully applied to urine samples after administration of DHEA to detect this compound in the sulfate fraction and preliminarily to negative samples from athletes of both sexes, to determine Q1 and Q3 inter-quartiles. A quality control assessment for the hydrolysis efficiency was established for every individual sample., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Public concerns about potential ecological risks of androgens discharged to the environment through wastewater treatment plants (WWTPs) has resulted in an increased interest regarding the occurrence and fate of androgens in WWTPs. In this study, the occurrence and removal of eight androgens from 12 municipal WWTPs distributed in eleven cities in China were investigated. The composition profiles of eight androgens in influent, effluent, and excess sludge were studied. Multiple factor analyses were performed to reveal the factors affecting the distribution of androgens in WWTP influent. Results showed similar composition profiles of androgens in the studied WWTPs, with androsterone and dehydroepiandrosterone confirmed as the dominant androgens. The distributions of androgens in WWTP influent were related to the chemical oxygen demand in influent and the gross domestic product (GDP) of WWTP-associated cities. The target androgens have high aqueous removal rates, with a mean removal rate of >90%. Additionally, the behaviors of androgens were evaluated by mass balance along anaerobic-anoxic-oxic (AAO) processes in a WWTP, in which many of the androgens were eliminated mainly in the anaerobic tank. Further, 15 biotransformation products of testosterone were identified under anaerobic, anoxic, and aerobic sludge, respectively. Based on these metabolites, a general biotransformation pathway of testosterone under anaerobic, anoxic, and aerobic sludge is presented., (Copyright © 2019. Published by Elsevier Ltd.)

While forming mixtures is a widely used approach for other raw materials in food industry, it has not yet been systematically analyzed for boar tainted meat. That is why we simultaneously studied four factors relevant for the production of emulsion-type sausages: percentage boar meat (skatole concentrations up to 0.3 μg/g, androstenone up to 3.8 μg/g in melted backfat), duration of traditional smoke and concentration levels of two spices. 16 variants of Frankfurters were produced in two independent studies and evaluated by in total 211 consumers. A linear mixed effects model revealed that increased levels of boar tainted meat significantly reduced consumer acceptance which could not be compensated by increased smoke or spice levels. We propose a non-inferiority test to identify the mixture which is similarly accepted as the reference made without boar tainted meat. Up to 33% tainted boar meat is proposed, assuming a liking drop of 0.5 on a 9 point liking scale as benchmark for an inferior product., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Growth and development of an embryo or fetus during human pregnancy mainly depend on intact hormone biosynthesis and metabolism in maternal amniotic fluid (AF). We investigated the hormonal milieu in AF and developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of 14 sulfated and 6 unconjugated steroids in AF. 65 A F samples (male: female = 35: 30) of mid-gestation ranging from 16 th week of gestation to 25 th week of gestation were analyzed. Reference data of 20 steroid levels in AF of healthy women were provided. 13 sulfated and 3 unconjugated steroids were for the first time quantified in AF by LC-MS/MS. Highest concentrations were found for pregnenolone sulfate (PregS: mean ± SD, 8.6 ± 3.7 ng/mL), 17α-hydroxypregnenolone sulfate (17OHPregS: 4.9 ± 2.0 ng/mL), epitestosterone sulfate (eTS: 7.3 ± 3.6 ng/mL), 16α-hydroxydehydroepiandrosterone sulfate (16OH-DHEAS: 21.5 ± 10.7 ng/mL), androsterone sulfate (AnS: 9.2 ± 7.4 ng/mL), estrone sulfate (E1S: 3.0 ± 3.0 ng/mL), estriol 3-sulfate (E3S: 8.1 ± 4.0 ng/mL) and estriol (E3: 1.2 ± 0.4 ng/mL). Only testosterone (T) showed a significant sex difference (p < 0.0001). Correlations between AF steroids mirrored the steroid metabolism of the feto-placental unit, and not only confirmed the classical steroid pathway, but also pointed to a sulfated steroid pathway., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Smoke has often been recommended as a masking agent for boar taint. However, guidelines on how much smoke is necessary have not been established. We compared different smoking parameters in bacon (smoking times) and sausages (smoking times and use of liquid smoke). In streaky bacon from entire male pigs with skatole concentrations of up to 0.6 μg/g and androstenone concentrations of up to 5.8 μg/g in the neck fat, three smoking times were compared: 10, 30 and 60 min. Boar taint was partially, but not fully, masked. The longer the smoking time, the better the masking effect. In sausages from entire male pigs with an average skatole concentration of up to 0.6 μg/g and androstenone concentration of up to 3.6 μg/g (the meat part) or 2.4 μg/g (the fat part) in the neck fat, smoking for 40 and 80 min fully masked the boar taint, whereas only a minor effect was seen after 10 and 20 min smoking. Liquid smoke (0.1%) did not mask boar taint when added to sausages from entire male pigs with an average skatole concentration of 0.36 μg/g (meat) or 0.38 μg/g (fat) and androstenone concentration of 2.3 μg/g (meat) and 2.9 μg/g (fat). To effectively mask boar taint, an intense smoked flavour is therefore necessary, and the longer the smoking time, the better. In contrast, the use of liquid smoke mixed into the product was not effective in the concentrations used in the current study., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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

This study investigated the effects of ageing period (2 or 7days), endpoint temperature (70 or 75°C), cut type (loin (M. longissimus thoracis et lumborum), silverside (M. biceps femoris), blade (M. triceps brachii) and chuck tender (M. supraspinatus)), cooking method (roast and stir fry (all cuts) and steak (loin only)) on eating quality attributes of pork from entire and immunocastrated male pigs. Higher intramuscular fat levels were found in all cuts from immunocastrated males compared with entire males. Of the seven cut type×cooking method combinations evaluated, shoulder cuts had the lowest fail rate (P<0.001) and loin steak and silverside cuts scored lowest (P<0.001) for tenderness, juiciness, overall liking, quality grade and re-purchase intention. Although no differences in sensory scores due to gender were observed, boar taint was identified in 10% of entire males. Minimising adverse risks in eating quality due to boar taint in males remains a key industry issue to ensure consistent delivery of high quality pork., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidation of androsterone with NAD + to form androstanedione and NADH with the rate limiting step being the release of NADH. In this study, we elucidate the role of remote substrate binding interactions contributing to the rate enhancement by 3α-HSD/CR through steady-state kinetic studies with the truncated substrate analogs. No enzyme activity was detected for methanol, ethanol, and 2-propanol, which lack the steroid scaffold of androsterone, implying that the steroid scaffold plays an important role in enzyme catalytic specificity. As compared to cyclohexanol, the activity for 2-decalol, androstenol, and androsterone increases by 0.9-, 90-, and 200-fold in k cat , and 37-, 1.9 × 10 6 -, and 1.8 × 10 6 -fold in k cat /K B , respectively. The rate limiting step is hydride transfer for 3α-HSD/CR catalyzing the reaction of cyclohexanol with NAD + based on the observed rapid equilibrium ordered mechanism and equal deuterium isotope effects of 3.9 on V and V/K for cyclohexanol. The k cat /K B value results in ΔG ‡ of 14.7, 12.6, 6.2, and 6.2 kcal/mol for the 3α-HSD/CR catalyzed reaction of cyclohexanol, 2-decalol, androstenol, and androsterone, respectively. Thus, the uniform binding energy from the B-ring of steroids with the active site of 3α-HSD/CR equally contributes 2.1 kcal/mol to stabilize both the transition state and ground state of the ternary complex, leading to the similarity in k cat for 2-decalol and cyclohexanol. Differential binding interactions of the remote BCD-ring and CD-ring of androsterone with the active site of 3α-HSD/CR contribute 8.5 and 6.4 kcal/mol to the stabilization of the transition state, respectively. The removal of the carbonyl group at C17 of androsterone has small effects on catalysis. Both uniform and differential binding energies from the remote sites of androsterone compared to cyclohexanol contribute to the 3α-HSD/CR catalysis, resulting in the increases in k cat and k cat /K B ., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Boar taint (BT) is an offensive odour or taste observed in pork from a proportion of non-castrated male pigs. Surgical castration is effective in avoiding BT, but animal welfare issues have created an incentive for alternatives such as genomic selection. In order to find candidate biomarkers, gene expression profiles were analysed from tissues of non-castrated pigs grouped by their genetic merit of BT. Differential expression analysis revealed substantial changes with log-transformed fold changes of liver and testis from -3.39 to 2.96 and -7.51 to 3.53, respectively. Co-expression network analysis revealed one module with a correlation of -0.27 in liver and three modules with correlations of 0.31, -0.44 and -0.49 in testis. Differential expression and co-expression analysis revealed candidate biomarkers with varying biological functions: phase I (COQ3, COX6C, CYP2J2, CYP2B6, ACOX2) and phase II metabolism (GSTO1, GSR, FMO3) of skatole and androstenone in liver to steroidgenesis (HSD17B7, HSD17B8, CYP27A1), regulation of steroidgenesis (STARD10, CYB5R3) and GnRH signalling (MAPK3, MAP2K2, MAP3K2) in testis. Overrepresented pathways included "Ribosome", "Protein export" and "Oxidative phosphorylation" in liver and "Steroid hormone biosynthesis" and "Gap junction" in testis. Future work should evaluate the biomarkers in large populations to ensure their usefulness in genomic selection programs.

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 (R 2 (Y)=0.872-0.969; Q 2 (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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

While recent studies suggest an influence of noise on olfactory performance, it is unclear as to what extent the influence varies between subjects who are accustomed to noise and those who are not. Two groups of panelists were selected: a University panel usually working under silent conditions and an abattoir panel usually working on the slaughter line with abattoir noise. Odor discrimination, odor identification, and odor detection thresholds were studied. Furthermore, a sensory quality control task using 40 boar samples was performed. All tests were accomplished both with and without extraneous noise recorded at an abattoir (70 dB) using headphones. Contrary to the researchers' expectations, abattoir noise hardly affected the olfactory tests nor was the quality control task impaired. Abattoir noise did not influence the perceived intensity of boar taint and the classification results of the testers, regardless of whether they were accustomed to such noise or not. The results indicate that sensory quality control can be conducted in a manufacturing environment with constant noise without diminishing the assessors' performance.

The aim of this study was to determine whether concentrations of reproductive steroid hormone metabolites significantly differed between harem stallions and bachelor stallions in the free ranging group of Przewalski's horses (Equus ferus przewalskii) at the Hortobágy National Park in Hungary. Throughout the study, fecal samples were collected from 21 harem stallions and 15 bachelor stallions and analyzed for immunoreactive estrogen and androgen metabolites. Harem stallions demonstrated significantly higher concentrations of estrogen (P < 0.001) and epi-androsterone (P < 0.001), but not testosterone (P = 0.426). These findings confirm that sociosexual status has a significant effect on androgen concentrations in individual Przewalski stallions. Zoo Biol. 36:127-131, 2017. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Yeast inoculation of dry fermented sausages manufactured with entire male fat was evaluated as a strategy to improve sausage quality. Four different formulations with entire male/gilt back fat and inoculated/non-inoculated with Debaryomyces hansenii were manufactured. The use of entire male back fat produced the highest weight losses, hardness and chewiness in dry sausages. Consumers clearly distinguished samples according to drying time and D. hansenii inoculation while the use of entire/gilt back fat was not highly perceived. The presence of androstenone and skatole was close to their sensory thresholds. Androstenone was not degraded during the process but skatole was affected by yeast inoculation. D. hansenii growth on the surface regulated water release during ripening, reduced hardness and chewiness in entire male sausages and resulted with similar texture to gilt sausages. Yeast inoculation inhibited lipid oxidation providing fruity odours and less oxidized fatty sausages in the sensory analysis. The effectiveness of yeast to mask boar taint was demonstrated by sensory analysis., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Testosterone (T) has traditionally been the most commonly reported doping agent by doping control laboratories. The screening of T misuse is performed by the quantification of six endogenous androgenic steroids and the ratio T/E included in the Athlete Biological Passport (ABP). The inclusion of additional metabolites can improve the screening capabilities of ABP. In this study, the potential of 3α-glucuronide-6β-hydroxyandrosterone (6OH-Andros3G) and 3α-glucuronide-6β-hydroxyetiocholanolone (6OH-Etio3G) as markers of T oral administration was evaluated. These glucuronides have been shown to be resistant to enzymatic hydrolysis and their quantification by means of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) was reported as the only way to obtain feasible results. Urine samples were collected from five volunteers before and after the oral administration of 40mg of T undecanoate and were analyzed by a LC-MS/MS method recently developed. Concentration of 6OH-Andros3G and 6OH-Etio3G compounds and those of the glucuronides of T (TG), epitestosterone (EG), androsterone and etiocholanolone were established and different concentration ratios were calculated. The detection windows (DWs) for the T administration obtained by each selected ratio were compared to the one of TG/EG. The results showed that four out of the nine tested markers presented DWs much larger for all volunteers than those obtained by the World Anti-Doping Agency established T/E marker or other alternative markers. The 6OH-Andros3G/EG, 6OH-Etio3G/EG, 6OH-Andros3G/TG and 6OH-Etio3G/TG markers were able to identify the T abuse up to 96h after the administration, extending our detection capability for the misuse up to 84h more than the classic marker. The importance of these markers was also highlighted by their prolonged capacity to detect the T misuse in the case of one volunteer whose TG/EG barely exceeded his individual threshold. As a consequence, the four markers presented in this study seem to have an exceptional potential as biomarkers of T oral administration., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Direct electrochemistry and bioelectrocatalysis of a newly discovered C-19 steroid 1α-hydroxylase (CYP260A1) from the myxobacterium Sorangium cellulosum So ce56 were investigated. CYP260A1 was immobilized on screen-printed graphite electrodes (SPE) modified with gold nanoparticles, stabilized by didodecyldimethylammonium bromide (SPE/DDAB/Au). Cyclic voltammograms in argon-saturated substrate free 0.1 M potassium phosphate buffer, pH 7.4, and in enzyme-substrate complex with androstenedione demonstrated a redox processes with a single redox couple of E(0') of -299 ± 16 mV and -297.5 ± 21 mV (vs. Ag/AgCl), respectively. CYP260A1 exhibited an electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen and upon addition of the substrate (androstenedione) in the air-saturated buffer. The catalytic current of the enzyme correlated with substrate concentration in the electrochemical system and this dependence can be described by electrochemical Michaelis-Menten model. The products of CYP260A1-depended electrolysis at controlled working electrode potential of androstenedione were analyzed by mass-spectrometry. MS analysis revealed a mono-hydroxylated product of CYP260A1-dependent electrocatalytic reaction towards androstenedione., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Measuring carbon isotope ratios (CIRs) of urinary analytes represents a cornerstone of doping control analysis and has been particularly optimized for the detection of the misuse of endogenous steroids. Isotope ratio mass spectrometry (IRMS) of appropriate quality, however, necessitates adequate purities of the investigated steroids, which requires extensive pre-analytical sample clean-up steps due to both the natural presence of the target analytes and the high complexity of the matrix. In order to accelerate the sample preparation and increase the automation of the process, the use of multidimensional gas chromatography (MDGC) prior to IRMS experiments, was investigated. A well-established instrumental configuration based on two independent GC ovens and one heart-cutting device was optimized. The first dimension (1D) separation was obtained by a non-polar column which assured high efficiency and good loading capacity, while the second dimension (2D), based on a mid-polar stationary phase, provided good selectivity. A flame ionization detector monitored the 1D, and the 2D was simultaneously recorded by isotope ratio and quadrupole mass spectrometry. The assembled MDGC set-up was applied for measuring testosterone, 5α- and 5β-androstanediol, androsterone, and etiocholanolone as target compounds and pregnanediol as endogenous reference compound. The urine sample were pretreated by conventional sample preparation steps comprising solid-phase extraction, hydrolysis, and liquid-liquid extraction. The extract obtained was acetylated and different aliquots were injected into the MDGC system. Two high performance liquid chromatography steps, conventionally adopted prior to CIR measurements, were replaced by the MDGC approach. The obtained values were consistent with the conventional ones. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The laboratory profile of intranasal testosterone gel has not been previously reported from an anti-doping perspective. Because intranasal testosterone gel is newly available as a commercial product, we sought to examine the laboratory parameters following administration of this formulation, with particular attention to anti-doping guidelines. Five healthy and active male subjects were administered testosterone intranasal gel three times daily for four weeks, using a pattern of five consecutive days on, two days off. Urine was collected after each five-day round of drug administration and analyzed using a full steroid screen and isotope ratio mass spectrometry (IRMS). Windows of detection for elevated testosterone/epitestosterone (T/E) and other steroid ratios, World Anti-Doping Agency (WADA) athlete biological passport (ABP) findings, and IRMS results were analyzed in this study. In the 0-24 h window post-administration, 70% of samples were flagged with a suspicious steroid profile and 85% were flagged as atypical passport findings according to the WADA ABP steroid module. In the 24-48 h window, 0% of samples displayed suspicious steroid profiles while 40% resulted in atypical passport findings. IRMS testing confirmed the presence of exogenous testosterone in 90% and 40% of samples in the 0-24 h and 24-48 h windows post-administration, respectively. Additionally, IRMS data were analyzed to determine commonalities in the population changes in δ 13 C values of testosterone, androsterone, etiocholanolone, 5αAdiol, and 5βAdiol. Though no discernible metabolic trend of the route of administration was identified, we discovered that intranasal gel testosterone is detectable using conventional anti-doping tests. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Characteristic off-flavours may occur in uncastrated male pigs depending on the accumulation of androstenone and skatole. Feasible processing of strongly tainted carcasses is challenging but gains in importance due to the European ban on piglet castration in 2018. This paper investigates consumers' acceptability of two sausage types: (a) emulsion-type (BOILED) and (b) smoked raw-fermented (FERM). Liking (9 point scales) and flavour perception (check-all-that-apply with both, typical and negatively connoted sensory terms) were evaluated by 120 consumers (within-subject design). Proportion of tainted boar meat (0, 50, 100%) affected overall liking of BOILED, F (2, 238)=23.22, P<.001, but not of FERM sausages, F (2, 238)=0.89, P=.414. Consumers described the flavour of BOILED-100 as strong and sweaty. In conclusion, FERM products seem promising for processing of tainted carcasses whereas formulations must be optimized for BOILED in order to eliminate perceptible off-flavours. Boar taint rejection thresholds may be higher for processed than those suggested for unprocessed meat cuts., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

This study analyzed odor-odor interactions of two malodorous volatile substances, androstenone and skatole, that may accumulate in fat and meat of uncastrated male (boar) pigs. Therefore, fat samples were collected from 1000+ entire male pig carcasses for sensory evaluation and quantification of boar taint compounds using gas chromatography-mass spectrometry (GC-MS). Each sample was sniffed by 10 trained assessors, resulting in 11 000+ individual ratings, which were subjected to statistical analysis. Pearson correlations of chemical traits and sensory traits (panel average) were higher for skatole [r(1029) = 0.59; p < 0.001] than for androstenone [r(1029) = 0.44; p < 0.001]. Linear terms of androstenone and skatole as well as their interaction significantly (p < 0.05) contributed to perception of deviant smell (R(2) = 0.43). Standardized regression coefficients illustrate the higher importance of skatole (β = 0.68) than androstenone (β = 0.39). Interindividual differences in the responses of assessors to androstenone and skatole are confirmed. A new curved approach is suggested because it better accounts for the interaction of androstenone and skatole than the "safe box" approach. On the basis of these data, sorting strategies using instrumental measurements are discussed. An automated detection based on only skatole measurements is recommended because its performance is only slightly inferior to a sorting based on both androstenone and skatole. Sorting thresholds need to be calibrated against consumer acceptance though.

In mammals, glucocorticoid (i.e. GC) levels have been associated with specific life-history stages and transitions, reproductive strategies, and a plethora of behaviors. Assessment of adrenocortical activity via measurement of glucocorticoid metabolites in feces (FGCM) has greatly facilitated data collection from wild animals, due to its non-invasive nature, and thus has become an established tool in behavioral ecology and conservation biology. The aim of our study was to validate a fecal glucocorticoid assay for assessing adrenocortical activity in meerkats (Suricata suricatta), by comparing the suitability of three GC enzyme immunoassays (corticosterone, 11β-hydroxyetiocholanolone and 11oxo-etiocholanolone) in detecting FGCM increases in adult males and females following a pharmacological challenge with adrenocorticotropic hormone (ACTH) and biological stimuli. In addition, we investigated the time course characterizing FGCM excretion, the effect of age, sex and time of day on FGCM levels and assessed the potential effects of soil contamination (sand) on FGCM patterns. Our results show that the group specific 11β-hydroxyetiocholanolone assay was most sensitive to FGCM alterations, detecting significant and most distinctive elevations in FGCM levels around 25 h after ACTH administration. We found no age and sex differences in basal FGCM or on peak response levels to ACTH, but a marked diurnal pattern, with FGCM levels being substantially higher in the morning than later during the day. Soil contamination did not significantly affect FGCM patterns. Our results emphasize the importance of conducting assay validations to characterize species-specific endocrine excretion patterns, a crucial step to all animal endocrinology studies using a non-invasive approach.

In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Consumer liking was assessed for streaky bacon and pork belly roll from entire male pigs with an androstenone (AND) content of up to 9.4 ppm and a skatole (SKA) content of up to 0.92 ppm in the back fat and castrates. No clear effect of either AND or SKA was seen in consumer liking, although an insignificant tendency was seen for SKA. A sensory profile analysis showed that AND increased the boar taint of bacon, while both AND and SKA increased the boar taint of the pork belly roll. Consumer sensitivity towards AND and SKA did not affect liking of the meat products. The lack of effect of AND and SKA on consumer liking could be due to a masking effect of the spices and smoke. Three consecutive weeks' exposure to bacon did not change the liking score, irrespective of the AND and SKA content. This indicates that the consumers did not become more sensitive towards boar taint., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Thirteen unilaterally cryptorchid Large White pigs, which had been immunized at 4 and 8 weeks of age and a third time at 64 ± 4 kg body weight against the gonadotropin releasing hormone with the vaccine Improvac®, were slaughtered at the age of 170 ± 9 days at a body weight of 102 ± 12 kg. Twelve pigs tested negative in the olfactory test of the salivary gland; their descended testicles were small and their fat androstenone concentration was low compared to normally developed boars of a previous experiment which had been vaccinated twice with Improvac® according the manufacturer's recommendation. One cryptorchid boar, which tested positive in the olfactory test and whose testicular weight and fat androstenone concentration corresponded to values of unvaccinated boars of the same age, obviously had not responded to the vaccination. It is an open question if the vaccination protocol for normal boars is sufficient to prevent boar taint in the majority of cryptorchid pigs, too.

Black tattoo inks are composed of carbon nanoparticles, additives and water and may contain polycyclic aromatic hydrocarbons (PAHs). We aimed to clarify whether reactive oxygen species (ROS) induced by black inks in vitro is related to pigment chemistry, physico-chemical properties of the ink particles and the content of chemical additives and contaminants including PAHs. The study included nine brands of tattoo inks of six colours each (black, red, yellow, blue, green and white) and two additional black inks of different brands (n = 56). The ROS formation potential was determined by the dichlorofluorescein (DCFH) assay. A semiquantitative method was developed for screening extractable organic compounds in tattoo ink based on gas chromatography-mass spectrometry (GC-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Two black inks produced high amounts of ROS. Peroxyl radicals accounted for up to 72% of the free radicals generated, whereas hydroxyl radicals and H₂O₂ accounted for <14% and 16%, respectively. The same two inks aggregated strongly in water in contrast to the other black inks. They did not exhibit any shared pattern in PAHs and other organic substances. Aggregation was exclusively shared by all ink colours belonging to the same two brands. Ten of 11 black inks had PAH concentrations exceeding the European Council's recommended level, and all 11 exceeded the recommended level for benzo(a)pyrene. It is a new finding that aggregation of tattoo pigment particles correlates with ROS production and brand, independently of chemical composition including PAHs. ROS is hypothesized to be implicated in minor clinical symptoms., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

An extensive body of research exists on androgen secretion in males. Although androgens are also known to drive patterns of female reproductive physiology and behavior, little attention has been paid to these "male" hormones in females. Here, we examined female fecal androgen excretion in strictly seasonally breeding wild Assamese macaques (Macaca assamensis) and tested the hypothesis that changes in premating season fecal androgens vary with female readiness to resume ovarian cycling and thus to conceive. Across 2 years, 155 fecal samples were collected from seven reproductively quiescent (i.e., acyclic) females during the premating season months, August and September. Samples were analyzed for immunoreactive epiandrosterone (iEA), a major fecal metabolite of testosterone in macaques. Changes in iEA concentrations during the premating season were significantly negatively associated with (1) the presence of dependent offspring, i.e., lactating females showed a smaller change, and (2) with the timing of conceptions in the mating seasons, i.e., females that conceived early exhibited the greatest changes. These results provide strong evidence for a relationship between changes in androgens during the premating season and female readiness to conceive., (Copyright © 2013 Wiley Periodicals, Inc.)

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.

Unlabelled: Surgical castration has been long used to prevent consumers from experiencing taint in meat from male pigs, which is a large problem in the pig husbandry industry. Due to obvious animal welfare issues, the EU now wants an alternative for castration, suggesting an urgent need for novel methods of boar taint detection. As boar taint is only a problem when taint chemicals exceed a well-defined threshold, detection methods should be concentration-specific. The wasp, Microplitis croceipes' ability to learn and respond to particular concentrations of the boar taint compounds, skatole, androstenone, and indole was tested. Also tested was the wasps' ability to discriminate between known concentrations of indole, skatole, and androstenone in real boar fat samples at room temperature. Wasps were trained using associative learning by providing food-deprived wasps with sucrose-water in the presence of specific odor concentrations. Trained wasps' responses were tested to a range of concentrations of 3 compounds. Wasps showed unidirectional generalization of learned concentration responses, whereby the direction of concentration generalization was shown to be chemical-dependent. Through both positive (sucrose) and negative feeding experiences (water only) with varying compound concentrations, the wasps can also be conditioned to respond to concentrations exceeding a defined threshold, and they were successful in reporting low, medium, and high concentrations of indole, skatole, and androstenone in boar fat at room temperature. The need for threshold detection rather than simple detection of absence/presence applies to many food quality issues, including the detection of spoilage or pest damage in crops or stored foods., Practical Application: An inexpensive and reliable means of detecting boar tainted pork at slaughter to avoid tainted meat on the market and dissatisfied consumers., (Journal of Food Science © 2012 Institute of Food Technologists® No claim to original US government works.)

A 3α-hydrosteroid biosensor for androsterone determination has been prepared by immobilizing the enzyme 3α-hydroxysteroid dehydrogenase (3α-HSD) in a composite electrode platform constituted of a mixture of multi-walled carbon nanotubes (MWCNTs), octylpyridinium hexafluorophosphate (OPPF(6)) ionic liquid and NAD(+) cofactor. This configuration allowed the fast, sensitive and stable electrochemical detection of the NADH generated in the enzyme reaction. All the experimental variables involved in the preparation and performance of the enzyme biosensor were optimized. Amperometry in stirred solutions at +400 mV provided a linear calibration plot for androsterone in the 0.5-10 μM concentration range with a slope value more than 200-times higher than that previously reported. The detection limit achieved was 0.15 μM and a low value of the apparent Michaelis-Menten constant (K(app)(M)), 36.0 μM, similar to that reported for the enzyme in solution, was calculated. The 3α-HSD/MWCNTs/OPPF(6)/NAD(+) biosensor provided good results in the determination of androsterone in spiked human serum samples., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In placental mammals, pregnancy usually is associated with an increase in maternal androgens, which may significantly impact fetal growth and differentiation, and affect postnatal development and behavior. Owing to their slow life histories and challenging social conditions, determination of maternal androgens and potential interference effects of fetal androgen production are of particular interest in wild primates. However, androgen production has been rarely investigated in wild female primates, and studies on maternal androgens during gestation in particular often do not span the entire pregnancy. Here, we characterize fecal androgen production throughout gestation in wild Assamese macaques (Macaca assamensis) using noninvasive hormone analysis and, furthermore, examine fetal sex effects on maternal androgen excretion. A total of 207 fecal samples were analyzed from seven females for concentrations of immunoreactive epiandrosterone (iEA). Fecal iEA concentrations, as predicted based on cercopithecine blood-serum patterns, increased during early gestation and were significantly higher during the first trimester compared with preconception concentrations and those recorded during later stages of gestation. Further, during the third trimester, male-carrying mothers showed significantly higher iEA concentrations compared with female-carrying mothers. This first characterization of fecal androgen excretion during gestation in Assamese macaques indicates both a maternal and fetal effect on androgen production. Although our sample size is small, our results, nevertheless, provide the basis for assessing potential influences of maternal androgens on postnatal offspring development and behavior., (Copyright © 2011 Wiley Periodicals, Inc.)

Consumer acceptability of entire male pork at eating was assessed in three experiments. The 140 consumers involved in each experiment were classified as insensitive (INSENS) to the odor of pure androstenone or sensitive perceiving it as pleasant (SENS-PLEA) or unpleasant (SENS-UNPL). Entire male pork with very low skatole and androstenone levels (LS-LA) was as well accepted as gilt pork, whatever the consumer category. Entire male pork with elevated levels in both skatole and androstenone (HS-HA) was clearly differentiated from LS-LA pork by SENS-UNPL, but not by SENS-PLEA or INSENS consumers. Whatever the consumer category, entire male pork with elevated levels of androstenone and very low levels of skatole (LS-HA and LS-HHA) were not significantly differentiated from LS-LA pork. The results suggest that, in the conditions of the present experiment, androstenone and skatole totally explain boar taint at eating and that the acceptability threshold for androstenone, in the absence of skatole, is in the range of 2-3 μg/g liquid fat., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This comprehensive review describes the analytical methods developed for quantification of the boar taint compounds skatole and androstenone in porcine adipose tissue. The following parts are considered; sampling, sample preparation, calibration and instrumentation. Additionally, method performance characteristics and level of validation of the existing methodology are discussed. It is concluded that there is a need for further validation of existing methods and need for standardisation of methodology to quantify boar taint compounds. Facing a possible near future ban of castration of male piglets would enforce further method harmonisation in this field., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Due to the impact in the media and the requirements of sensitivity and robustness, the detection of the misuse of forbidden substances in sports is a really challenging area for analytical chemistry, where any study focused on enhancing the performance of the analytical methods will be of great interest. The aim of the present study was to evaluate the usefulness of using hydrogen instead of helium as a carrier gas for the analysis of anabolic steroids by gas chromatography-mass spectrometry with electron ionization. There are several drawbacks related with the use of helium as a carrier gas: it is expensive, is a non-renewable resource, and has limited availability in many parts of the world. In contrast, hydrogen is readily available using a hydrogen generator or high-pressure bottled gas, and allows a faster analysis without loss of efficiency; nevertheless it should not be forgotten that due to its explosiveness hydrogen must be handled with caution. Throughout the study the impact of the change of the carrier gas will be evaluated in terms of: performance of the chromatographic system, saving of time and money, impact on the high vacuum in the analyzer, changes in the fragmentation behaviour of the analytes, and finally consequences for the limits of detection achieved with the method., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The objective of this study was to evaluate whether altering the timing of the secondary anti-gonadotropin-releasing factor (GnRF) immunization closer to slaughter in male finishing pigs would reduce the increase in P2 fat depth (6.5 cm from the midline over the last rib), while still limiting the incidence of boar taint. Entire male pigs are immunized against GnRF to reduce the concentration of testicular steroids that in turn limits the incidence of boar taint. Additionally, testicle measurements and color measurements were taken to examine whether they could be used to differentiate nonimmunized entire males from immunized male pigs. A total of 175 Large White × Landrace entire male pigs aged 16 wk (59 kg of BW) were used in a completely randomized design with 5 treatment groups based on the time that pigs received the secondary immunization before slaughter. Pigs were housed in groups of 7 and randomly allocated to 1 of 5 treatments with 5 replicates per treatment. The treatment groups were as follows: no secondary immunization before slaughter, and the secondary immunization given at 2, 3, 4, or 6 wk before slaughter. The P2 fat depth levels were reduced (P = 0.054) with the secondary immunization closer to slaughter (11.7, 11.3, 12.8, 12.6, and 13.7 mm for no secondary immunization, secondary immunization at 2, 3, 4, and 6 wk before slaughter, respectively). Androstenone concentration did not exceed the generally accepted industry sensory threshold of 1.0 µg/g of fat, and both androstenone concentration in the adipose tissue and testosterone concentrations in the blood were suppressed (P < 0.001) in all immunized pigs regardless of timing of the secondary immunization compared with pigs that did not receive the secondary immunization. Skatole concentration of all pigs in the experiment did not exceed the generally accepted industry sensory threshold of 0.2 µg/g. Testes weight was reduced (P < 0.001) with increased time between slaughter and the secondary immunization. Immunized pigs, regardless of time before slaughter, had greater L* (lightness) and b* (yellowness) color of the testicle surface (P < 0.001 and P = 0.020, respectively), and less a* (redness) color compared with entire males (P < 0.001). The study provides further evidence of the efficacy of the anti-GnRF immunization and indicates that the secondary immunization can be moved closer to slaughter, while still limiting the incidence of boar taint. Testicle measurements and color measurements together could provide a method of discrimination between carcasses from immunized entire males clear of boar taint and tainted carcasses.

The objective of this study was to determine the effect of using a gonadotropin-releasing factor (GnRF) vaccine on follicle-stimulating hormone (FSH) and luteinizing hormone (LH) concentrations in plasma, the size of testicles, and the expression of boar taint in male pigs. Vaccinated pigs were compared with surgically castrated pigs and entire males. Pigs were randomly assigned to three treatment groups: surgically castrated during the first week of life (T01, n=274), immunized twice during the fattening period with a GnRF vaccine, the first when 13 to 14 wk of age and the second when 20 to 21 wk of age (T02, n=280), and entire males (T03, n=56). From a subgroup of both T01 and T02 and from all pigs of group T03, blood samples were collected immediately before second vaccination (T02) and again before slaughter at either 24 to 25 or 26 to 27 wk of life to determine the plasma concentrations of LH and FSH. Testicles were removed after slaughter and their size was determined. Meat and fat samples from all pigs of T02 and T03 as well as 25% of the pigs of T01 were examined with the cold cooking and fat melting test. Immediately before the second vaccination (T02 only), LH and FSH concentrations were not significantly different between T02 and T03. However, LH and FSH concentrations were significantly higher in T01 compared with T02 and T03. Before the first slaughter date, LH and FSH concentrations were significantly lower in T02 than in T03. Testicle size was significantly lower in T02 compared with that in T03. In T02, 98% (235 of 239) of the samples were rated negative for boar taint by the cooking test, whereas in T03, 94% (48 of 51) were rated positive. In the fat melting test, 97% of T02 were rated negative and 3% (7 pigs) were rated positive, including the pigs tested positive in the cold cooking test. In T03, 94% were rated positive. All pigs (7 of 239) in T02 that were positive for boar taint in the cooking or melting test and that were tested had androstenone and skatole concentrations in backfat below threshold levels of 1 microg/g and 0.2 microg/g, respectively.

The development and validation of liquid chromatography-electrospray ionization-tandem mass spectrometric (LC-ESI-MS/MS) methods that enable the quantification of neuroactive androgens, androsterone (5alpha-androstan-3alpha-ol-17-one, 3alpha,5alpha-A) and 5alpha-androstane-3alpha,17beta-diol (3alpha,5alpha-Adiol), in the rat brain and serum are presented. The androgens were extracted with methanol-acetic acid, purified using solid-phase extraction cartridges, derivatized with an ESI-active reagent, isonicotinoyl azide (INA), and then subjected to LC-ESI-MS/MS. The quantifications were based on selected reaction monitoring mode using the characteristic transitions of the INA derivatives. The methods allowed the reproducible and accurate quantification of the brain and serum neuroactive androgens using a 100 mg or 100 microL sample; the intra- and inter-assay relative standard deviations were below 3.6%, and the percentage accuracy values were 97.1-103.7% for both androgens. The animal study using the methods suggests that most of 3alpha,5alpha-Adiol found in the brain is derived from the periphery, while 3alpha,5alpha-A is not only transported from the periphery into the brain, but also synthesized in the brain by the oxidation of 3alpha,5alpha-Adiol. The androgens in the rats intraperitoneally administered finasteride, a 5alpha-reductatse inhibitor, were also measured; this treatment significantly reduced the brain 3alpha,5alpha-A and 3alpha,5alpha-Adiol levels and increased only the brain level of androstenedione, the precursor of 3alpha,5alpha-A.

The objective of this study was to evaluate the long-term effect of a gonadotropin-releasing hormone (GnRH) vaccine, Improvac (Pfizer Ltd.), on the levels of GnRH antibodies, testosterone, estrone sulphate (E1S) and androstenone, as well as skatole and indole in male pigs. Additionally, the long-term effect of immunocastration on social and sexual behaviour was studied. Male pigs were assigned to two treatment groups: a treatment group given two doses of Improvac (n=12) and a control group of entire male pigs (n=12). The pigs were kept either 16 or 22 weeks after vaccination. Blood samples were collected five or six times; prior to both first and second vaccination, then three or four times during the 16 or 22 week period after second vaccination. Immunocastration significantly reduced levels of testosterone and E1S in plasma, and levels of androstenone in fat (P<0.001 for all). Skatole and indole levels in plasma and fat were also lower in immunocastrated pigs than in entire male pigs. These effects lasted up to 22 weeks after the second vaccination. Testis weight and bulbourethral gland length were lower in immunocastrated pigs at slaughter and these pigs showed less social, manipulating and aggressive behaviour than entire male pigs. The immunocastrated pigs remained sexually inactive throughout the study. Our study represents a further step in the evaluation of the effectiveness of Improvac as an alternative to surgical castration of entire male pigs. It shows that Improvac may have an extended effect compared with that currently implied by the directions for use.

Together with steroid glucuronides, sulfoconjugates may be used as markers of steroid administration as well as endogenous steroid production. A fast and sensitive analytical procedure has been developed for the simultaneous separation, determination and quantification of sulfate and glucuronide derivatives of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio) and dehydroepiandrosterone (DHEA) in human urine. First, a weak anion-exchange solid-phase extraction support (SPE Oasis WAX) was used for complete and rapid separation of sulfates and glucuronides in two extracts after loading of urine sample (2 mL). Then sulfates were analyzed directly by high-performance liquid chromatography-ion-trap mass spectrometry (LC-MS/MS) with electrospray ionization in negative mode. Chromatographic separation of the targeted sulfoconjugates was achieved using a Waters XBridge C18 column (150 mm x 4.6 mm I.D., 5 microm) with gradient elution. Assay validation demonstrated good performance for instance for T sulfate (TS) and E sulfate (ES) in terms of trueness (89-107%), repeatability (3.4-22%) and intermediate precision (5.8-22%) over the range of 2-200 ng/mL (corresponding to 1.5-147 ng/mL as free steroids). Results obtained on biological samples demonstrated the suitability of this analytical strategy for direct measurement of androgen sulfoconjugates and glucuroconjugates in human urine.

Dehydroepiandrosterone (DHEA) and androsterone (ADT) were detected in a traditional Chinese herbal product by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). DHEA and ADT were tentatively identified by comparing their electron ionization (EI) mass spectra with those in the GC-MS Wiley database. A multiple reaction monitoring (MRM) scan was performed in LC-MS/MS to confirm the presence of the DHEA and ADT in the herbal product extract. Both the [M + H]+ and the [M + NH4]+ of DHEA and ADT were selected as the precursor ions. DHEA was detected with ion transitions m/z 306.4 --> 271.2, 306.4 --> 253.3, 289.2 --> 270.9, 289.3 --> 253.1 while ADT was detected with ion transitions m/z 308.5 --> 273.6, 308.5 --> 255.3, 291.5 --> 273.5, 291.5 --> 255.2, which confirmed the presence of the two steroid hormones in the herbal product. Limits of detection (LODs) of 0.2 microg ml(-1) for DHEA and 0.3 microg ml(-1) for ADT were found in methanolic standard solutions when [M +NH4]+ of DHEA and ADT were selected as the precursor ions, which allowed the detection of DHEA and ADT at trace level without time-consuming derivatization.

An excessive accumulation of androstenone in pig adipose tissue is a major contributor to the phenomenon of boar taint. Androstenone deposition is dependent on the rate of androstenone biosynthesis in testis and androstenone degradation in liver. The aim of the current study was to examine the possibility of the existence of breed-specific mechanisms controlling androstenone accumulation in pig adipose tissue. The specific objective was to investigate the expression of some of the key enzymes involved in testicular and hepatic androstenone metabolism in pigs of 2 breeds by using animals with high and low androstenone concentrations within each breed. The study was conducted with Norwegian Landrace (N. Landrace) and Duroc boars. The mean androstenone values for the low- and high-androstenone groups were 0.1 +/- 0.01 microg/g and 7.58 +/- 0.68 microg/g for N. Landrace boars, and 0.22 +/- 0.04 microg/g and 13.55 +/- 1.14 microg/g for Duroc boars. The enzymes investigated were 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450-c17, and sulfotransferase 2B1 (SULT2B1). Expression of cytochrome P450-c17 in liver and testis did not differ between animals with high and low androstenone concentrations in either the N. Landrace or Duroc breed. Expression of hepatic 3beta-HSD, which catalyzes the first stage of androstenone degradation, was decreased in high-androstenone N. Landrace boars (P < 0.01), but not in high-androstenone Duroc boars. In contrast, the expression of hepatic SULT2B1, which catalyzes the second stage of steroid catabolism, was decreased in high-androstenone Duroc animals (P < 0.05), but not in high-androstenone N. Landrace animals. Sulfotransferase 2B1 was also inhibited in testis of high-androstenone pigs of both breeds compared with low-androstenone animals. We report breed differences in expression of the androstenone-metabolizing enzymes 3beta-HSD and SULT2B1 in the liver of high- and low-androstenone pigs. It is suggested that accumulation of androstenone in adipose tissue of N. Landrace boars might be related to a low rate of hepatic androstenone degradation in metabolic stage I, whereas the high androstenone concentration in Duroc boars might be related to a low rate of androstenone metabolism in metabolic stage II.

A short and efficient synthesis of pentadeuterated 2,2,3,4,4-d5-19-nor-5alpha-androsterone 7 starting from 19-norandrost-4-ene-3,17-dione 1 by a d1-L-Selectride mediated stereo- and regioselective reduction of the 3-keto group is presented. The use of compound 7 as internal standard for the detection of anabolic steroids via mass spectrometric techniques such as gas chromatography-mass spectrometry (GC-MS) is discussed.

A simple and sensitive liquid chromatography-electrospray ionization-mass spectrometric (LC-ESI-MS) method for the simultaneous quantification of representative 17-ketosteroid sulfates, dehydroepiandrosterone sulfate (DHEAS), androsterone sulfate and epiandrosterone sulfate, in human saliva has been developed and validated. The saliva was deproteinized with acetonitrile, purified using a solid-phase extraction cartridge and subjected to LC-MS. Deuterium-labeled DHEAS was used as the internal standard and quantification was based on the selected ion monitoring (SIM) mode of each deprotonated molecule. This method allowed the reproducible and accurate quantification of the salivary sulfates using a 100-microl sample; the intra- and inter-assay coefficients of variation were below 6.8 and 6.3%, respectively, and the % accuracy values were quantitative for all the sulfates. The limits of quantitation for all the sulfates were 100 pg/ml. No significant matrix effect or change in the measured value by freeze/thaw repetitions was observed. The developed method was applied to clinical studies, and produced satisfactory results.

This study investigated the relationship between expression of hepatic and testicular 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and accumulation of androstenone in adipose tissue because of its relation to boar taint. The experiments were performed on 13 Large White (50%) x Landrace (50%) and Meishan (25%) x Large White (25%) x Landrace (50%), pigs, which differed in the level of backfat androstenone. Our previous work showed that the major product of the hepatic androstenone metabolism is 3beta-androstenol. In this study, the formation of 3beta-androstenol was inhibited by the specific 3beta-HSD inhibitor trilostane. These results are the first direct confirmation that 3beta-HSD is the enzyme responsible for androstenone metabolism in the pig. The expression of the hepatic but not testicular 3beta-HSD protein showed a negative relationship with the level of backfat androstenone (r2 = 0.64; P < 0.001) and was accompanied by a reduced rate of the hepatic androstenone clearance. Low expression of 3beta-HSD protein in the liver of high androstenone pigs was also accompanied by a reduced level of 3beta-HSD mRNA (P < 0.001), which suggests a defective regulation of the hepatic 3beta-HSD expression at the level of transcription. In contrast, expression of the testicular 3beta-HSD protein did not differ between animals with high and low androstenone levels (P > 0.05) and was lower compared with the hepatic 3beta-HSD expression. Cloning and sequencing of the 3beta-HSD coding regions established that the hepatic and testicular 3beta-HSD cDNA have identical sequences, which were 98% similar to the human 3beta-HSD isoform I. It is suggested that expression of a single 3beta-HSD gene is regulated by different mechanisms in pig liver and testis. The liver-specific regulation of 3beta-HSD expression contributes to the low rate of hepatic androstenone metabolism and therefore can be considered as one of the factors regulating deposition of androstenone in pig adipose tissue and subsequent development of boar taint.

This study aimed to describe the association between incidence of boar taint and pubertal changes in gonadal hormones, size of reproductive organs and aggressive behaviour in entire male pigs. In total, 111 entire male pigs were included in the study. Sampling was performed first at 90 kg live weight (LW) and, then, at 115 kg LW. Variables measured were skatole and androstenone levels in plasma and fat, testosterone and oestrone sulphate in plasma, free oestrone in fat, weight of testes and length of bulbourethral glands. Aggressive interactions between pigs were registered when a limited amount of feed was provided to the pigs prior to routine feeding. The number of initiated interactions (attacks) and the difference between number of initiated and received interactions (relative attacks) were calculated for each pig. Multivariate analysis revealed that gonadal hormones and reproductive organ size influenced prevalence of boar taint, accounting for 30% of the variation in skatole levels in fat and for 37% of the variation in androstenone levels in fat. These relations were independent of aggression levels in entire male pigs. Skatole levels were influenced by the levels of oestrone sulphate in plasma and free oestrone in fat, but not levels of plasma testosterone. Pigs with testes weight below 565 g and a bulbourethral gland length <90 mm did not produce high amounts of skatole; therefore, these values can be used as a threshold level to detect pig carcasses with low skatole levels. High androstenone levels could not be predicted by measuring reproductive organ sizes. More research is required to develop a rapid and accurate method for the analysis of carcasses of entire male pigs.