Boar taint detection using parasitoid biosensors

The off-flavor boar taint associated with the substances skatole, androstenone, and possibly indole represents a significant problem in the pig husbandry industry. Boar taint may occur in meat from uncastrated sexually mature male pigs; consumers commonly show a strong aversion to tainted meat. Consequently, there is a need for rapid methods to sort out and remove tainted carcasses at the slaughterline. We tested the ability of wasps, Microplitis croceipes to perceive and learn the 3 boar taint compounds both individually and in combination using classical conditioning paradigms. We also established the effectiveness and reliability of boar taint odor detection when wasps were used as biosensors in a contained system called the "wasp hound" using a cohort of trained wasps. We found that the wasps are able to successfully learn indole, skatole and to also detect them when presented a 1:1:1 mixture of all 3 compounds. This was shown for both a single hand-manipulated wasp bioassay and when using the "wasp hound" detector device. In contrast, the wasps showed a weak conditioned response to androstenone at the concentration tested. The estimated gas phase concentrations that the wasps perceived during training were in the range of 10 ± 0.4 pg/s for skatole and indole, and 2 ± 0.5 pg/s for androstenone. We conclude that use of these wasps as biosensors presents a promising method for boar taint detection and discuss future training paradigms that may improve their responses to compounds such as androstenone. Practical Application: The development of a perceptive, inexpensive, and reliable means of detecting boar taint before the product is presented to sensitive consumers.     

Threshold Detection of Boar Taint Chemicals Using Parasitic Wasps

Abstract: 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.     

Supercritical Carbon Dioxide Extraction of Androstenone and Skatole from Pork Fat

Boar taint, an unpleasant odor observed in pork from some mature intact male pigs, is attributed primarily to the presence of two compounds in boar fat, androstenone (Sα-androst-16-ene-3-one) and skatole (3-methyl indole). A rapid extraction method for the preparation of fat samples for androstenone/skatole screening assays would lead to more efficient use of boar carcasses for pork production. Supercritical carbon dioxide removed 97 ± 2% of the androstenone (extraction at 40°C; 5 min) and 65 ± 3% of the skatole (extraction at 40°C; 20 min) from 0.5 g of boar backfat. Supercritical CO₂ provides the basis for a fairly rapid extraction method to remove compounds associated with boar taint.     

Marinating as a technology to shift sensory thresholds in ready-to-eat entire male pork meat

This study investigated the effect of marinades in improving the eating quality in ready-to-eat boar meat. Neck chops with fat content below 18.9%, skatole 1.1 ppm (range 0.03–1.1) and androstenone 5.6 ppm (range 0.01–5.6) were used. In a screening experiment different marinades were tested for their ability to mask boar taint (defined as manure and urine odour and flavour). Liquid smoke and oregano extracts appeared to have the best potential for masking, and were studied in detail. Results from the study indicated that marinated chops with skatole content of approximately 0.4 ppm appeared similar to castrates in boar taint. Chops with skatole contents above 0.7 ppm remained unmasked despite the use of strongly flavoured marinades. Unmarinated chops served at 60 °C were more tainted than those served at 15 °C, but scored lower for boar taint when reheated, although the concentrations of androstenone and skatole remained the same. The fat content of the chops was not well correlated to the perception of boar taint. The attributes manure and urine were correlated with the level of skatole, but urine attribute was not a good indicator of the androstenone level.     

Comparison of heating methods and the use of different tissues for sensory assessment of abnormal odours (boar taint) in pig meat

Five heating methods (microwave, hotwire, boiling at 25 °C and 75 °C and melting) were used to generate cooking odours from backfat of entire male pigs and a 'composite' sample consisting of fat and muscle from the head along with salivary glands. The methods elicited significantly different scores for odours from 4 groups of 10 samples differing in their concentrations and ratios of skatole and androstenone. The odours (pork odour, abnormal odour, skatole odour and androstenone odour) were assessed by 3 experienced assessors. Correlations between skatole and androstenone concentrations and abnormal odour score in backfat were higher for skatole, suggesting it is the more important boar taint compound. In the composite sample, androstenone concentration was much higher than in backfat and androstenone was a more important contributor to boar taint. The microwave, hotwire and boiling (75 °C) methods produced the clearest separation between samples and the microwave method was considered the most suitable for on-line use.     

An international study on the importance of androstenone and skatole for boar taint: I. Presentation of the programme and measurement of boar taint compounds with different analytical procedures

An international study, involving 11 participants in 7 European countries, was conducted to provide scientific evidence for an objective measurement of boar taint in entire male pigs and its possible variation between countries. The specific objectives were to determine the respective contributions of androstenone and skatole to boar taint and their possible variations according to production systems and consumer populations. Over 4000 entire male pigs and 200 gilts were raised and slaughtered in 6 countries. Meat samples were taken from the loin and backfat samples were used for the rapid measurement of androstenone and skatole. A sub-population of 377 entire males and 42 gilts was then selected in such a way as to represent all combinations of skatole and androstenone levels. Androstenone and skatole levels in the selected samples were checked, using established reference methods. Meat samples from the selected animals were used for sensory evaluation by trained panels and for consumer surveys in 7 European countries. The present paper gives a general presentation of the programme and reports the main characteristics of the samples. Three companion papers present the results of the evaluation by trained sensory panels [Dijksterhuis, G., Engel, B., Walstra, P., Font i Furnols, M., Agerhem, H., Fisher, K., Oliver, M. A., Claudi-Magnussen, C., Siret, F., Béague, M. P., Homer, D. B., & Bonneau, M. (2000). An international study on the importance of androstenone and skatole for boar taint: II. Sensory evaluation by trained panels in seven European countries. Meat Science54, 261-269], the results of the consumer surveys [Matthews, K. R., Homer, D. B., Punter, P., Béague, M. P., Gispert, M., Kemspter, A. J., Agerhem, H., Claudi-Magnussen, C., Fischer, K., Siret, F., Leask, H., Font i Furnols, M., & Bonneau, M. (2000). An international study on the importance of androstenone and skatole for boar taint: III. Consumer survey in seven European countries. Meat Science, 54, 271-283] and the main conclusions and recommendations [Bonneau, M., Walstra, P., Claudi-Magnussen, C., Kempster, A. J., Tornberg, E., Fischer, K., Diestre, A., Siret, F., Chevillon, P., Claus, R., Dijksterhuis, G., Punter, P., Matthews, K. R., Agerhem, H., Béague, M. P., Oliver, M. A., Gispert, M., Weiler, U., von Seth, G., Leask, H., Font i Furnols, M., Homer, D. B., & Cook, G. L. (2000). An international study on the importance of androstenone and skatole for boar taint: IV. Simulation studies on consumer dissatisfaction with entire male pork and the effect of sorting out carcasses on the slaughter line, main conclusions and recommendations. Meat Science, 54, 285-295]. Seasonal effects and differences between countries in skatole and androstenone levels are presented elsewhere [Walstra, P., Claudi-Magnussen, C., Chevillon, P., von-Seth, G., Diestre, A., Matthews, K. R., Homer, D. B., & Bonneau, M. (in press). An international study on the importance of androstenone and skatole for boar taint: Levels of androstenone and skatole according to country and season. Livestock Production Science]. A supplementary paper considers the effects of human sensitivity to androstenone and skatole on the consumer acceptance of entire male pork [Weiler, U., Font i Furnols, M., Fischer, K., Kemmer, H., Oliver, M. A., Gispert, M., Dobrowolski, A., & Claus, R. (in press). Influence of differences in sensitivity of Spanish and German consumers to perceive androstenone on the acceptance of boar meat differing in skatole and androstenone concentrations. Meat Science]. A study of possible other compounds contributing to boar taint was also carried out within this programme.     

Prevalence and relationships of sensory taint, 5α-androstenone and skatole in fat and lean tissue from the loin (Longissimus dorsi) of barrows, gilts, sows, and boars from selected abattoirs in the United States

This study assessed prevalence of boar taint in backfat and lean of barrows, gilts, sows, and boars, collected from abattoirs, without knowledge of the farms of origin, in different regions in the United States. Concentrations of 5α-androstenone (liquid chromatography/mass spectroscopy) and skatole (liquid chromatography with fluorescent detection) in backfat were measured. A trained panel evaluated boar taint aroma in heated samples. Mean 5α-androstenone and skatole levels were low among barrows, gilts, and sows, whereas 55.8% of boars scored above a 1.0 μg/g threshold for 5α-androstenone concentrations and 34.2% were above a 0.2 μg/g threshold for skatole concentrations. Mean aroma scores for backfat and lean from barrows, gilts, and sows were low. In comparison, 59.2% of boars had elevated mean aroma scores from fat samples and 31.7% from lean. Importantly, boar taint aroma was detectable by the trained panel in at least some animals in each of the sex classes.     

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

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

Factors affecting the level of boar taint in entire male pigs as assessed by consumer sensory panel

A sensory study was conducted to determine consumer responses to meat from entire male pigs in order to evaluate different factors involved in causing boar taint, including 16-androstene steroids, skatole, size of accessory sexual glands and live weight. Pork chops from 33 entire Yorkshire male pigs were divided into three groups according to the levels of 16-androstene steroids in salivary glands, high (H; 56-114 μg/g), medium (M; 35-55 μg/g) and low (L; 26-6 μg/g), and were distributed among 59 families. Pork chops from gilts were used as control (C). The families were asked to rate liking of cooking aroma, flavour and texture, and overall liking. Consumer ratings were best related to the size of bulbourethral gland and salivary gland, followed by the levels of 16-androstene steroids in salivary gland. Meat from the L group received similar ratings to the C group, except the ratings for texture and overall liking were better than the C group. M samples were liked similarly to C, with the exception of some samples that had high fat skatole levels and which were liked less. H samples were liked less than C. The samples evaluated at the beginning of the experiment were liked more than those evaluated afterwards indicating sensitization of consumers to boar taint. The results indicate that levels of 16-androstenes in salivary glands can be used to estimate boar taint due to 16-androstenes; however, skatole measurements also need to be included. In addition, consumers are sensitive to some other maturity-related factors, which need to be considered when estimating the levels of boar taint. The effect of sensitization of consumers to boar taint requires more study.     

Influence of volatile compounds on the development of off-flavours in pig back fat samples classified with boar taint by a test panel

Dynamic Head Space methodology was applied to evaluate the possible contribution of some volatile compounds to the development of boar taint in pig backfat samples with low concentrations of skatole and androstenone, but which had previously been classified as tainted by a trained test panel. Volatile compounds were collected in a trap of graphited charcoal and analysed by GC–MS in Scan mode. Aldehydes and short chain fatty acids, compounds that play a significant role in the development of undesirable aromas in food products, were the main classes of compounds identified in this study, although the possible contribution of other compounds that were detected in a minor proportion – such as alcohols and ketones – was evaluated. Styrene and 1,4-dichlorobenzene, compounds that may have come from an external contamination, showed a high concentration in the samples classified with boar taint, so these compounds could have been responsible for the development of some off-flavours in the fat samples studied in this work. In the same study, skatole and androstenone were also determined by normal phase HPLC and GCMS, respectively.     

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

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

The measurement of the responses to different odour intensities of `boar taint' using a sensory panel and an electronic nose

This study explored the possibility of using an electronic nose (e-nose) with a 12-conducting-polymer sensor array combined with pattern recognition routines to discriminate between varying intensities of boar taint. A set of samples in a model system comprising a neutral lipid base containing varying combinations of androstenone and skatole were tested, as were pork fat samples. The e-nose responses for pork fat were calibrated against those given by a trained 10-member sensory panel for abnormal odour of the same samples from a total of 60 Large White cross-bred pigs. The e-nose responses related strongly to those of the sensory panel with a significant (p<0·01) canonical correlation of 0·78. The data set was used to develop a discriminant function for grouping pork samples into three `response classes': normal, doubtful and abnormal. Based on this, the e-nose identified all the abnormal samples correctly. However, 16% of the normal samples were also classified as abnormal. It was concluded that, in general, the electronic nose can discriminate between different levels of boar taint and that although a high specificity of sensors to androstenone and skatole may be desirable it may not be entirely important to the development and configuration of a boar taint sensor array.     

Marinating as a technology to shift sensory thresholds in ready-to-eat entire male pork meat

This study investigated the effect of marinades in improving the eating quality in ready-to-eat boar meat. Neck chops with fat content below 18.9%, skatole ⩽1.1 ppm (range 0.03–1.1) and androstenone ⩽5.6 ppm (range 0.01–5.6) were used. In a screening experiment different marinades were tested for their ability to mask boar taint (defined as manure and urine odour and flavour). Liquid smoke and oregano extracts appeared to have the best potential for masking, and were studied in detail. Results from the study indicated that marinated chops with skatole content of approximately 0.4 ppm appeared similar to castrates in boar taint. Chops with skatole contents above 0.7 ppm remained unmasked despite the use of strongly flavoured marinades. Unmarinated chops served at 60 °C were more tainted than those served at 15 °C, but scored lower for boar taint when reheated, although the concentrations of androstenone and skatole remained the same. The fat content of the chops was not well correlated to the perception of boar taint. The attributes manure and urine were correlated with the level of skatole, but urine attribute was not a good indicator of the androstenone level.     

Effect of feeding fermentable fibre-rich feedstuffs on meat quality with emphasis on chemical and sensory boar taint in entire male and female pigs

Skatole, androstenone and other compounds such as indole cause boar taint in entire male pork. However, female pigs also produce skatole and indole. The purpose of this experiment was to minimise boar taint and increase overall impression of sensory quality by feeding entire male and female pigs with fibre-rich feedstuffs. The pigs have been fed three organic diets for either 1 or 2 weeks prior to slaughter of which two diets contained different fermentable fibre-rich feedstuffs – 10–13.3% dried chicory roots or 25% blue lupines. These two treatments were compared with pigs fed with an organic control diet for either 1 or 2 weeks prior to slaughter. Lupines significantly reduced skatole in blood and backfat for both genders after 1 week. Moreover, lupines showed negative impact on growth rate and feed conversion whilst chicory showed no significant differences in this respect. However, the indole concentration was significantly lower in chicory than lupine fed pigs. From a sensory perspective, chicory and lupine feeding reduced boar taint since odour and flavour of manure related to skatole and urine associated to androstenone were minimised. The level of boar taint in the entire male pigs was most effectively reduced after 14 days by both fibre-rich feeds while lupine had the largest influence on “boar” taint reduction in female pigs.     

Quantitative determination of the boar taint compounds androstenone,skatole, indole, 3α-androstenol and 3β-androstenol in wild boars (Sus scrofa) reveals extremely low levels of the tryptophan-related degradation products

The major boar taint compounds androstenone and skatole as well as the minor compounds indole, 3α-androstenol and 3β-androstenol were determined in back fat samples of 23 male wild boars by applying a recently published SIDA–HS-SPME–GC/MS method. The boar pheromones androstenone, 3α-androstenol and 3β-androstenol were found in extraordinary high concentrations, resulting in mean values of 3329 ng/g androstenone, 1273 ng/g 3α-androstenol and 545 ng/g 3β-androstenol. Interestingly, skatole was not detectable in about 50% of the boars and negligibly low in all other samples as expressed by a mean skatole value of only 14 ng/g. Indole was also found in every sample, but again in low concentrations with a mean value of 40 ng/g. Possible factors explaining this remarkably low skatole deposition in wild boars such as intestinal flora and anatomy, dietary composition, housing or genetic predisposition are discussed in this paper.     

Effect of feeding fermentable fibre-rich feedstuffs on meat quality with emphasis on chemical and sensory boar taint in entire male and female pigs

Skatole, androstenone and other compounds such as indole cause boar taint in entire male pork. However, female pigs also produce skatole and indole. The purpose of this experiment was to minimise boar taint and increase overall impression of sensory quality by feeding entire male and female pigs with fibre-rich feedstuffs. The pigs have been fed three organic diets for either 1 or 2 weeks prior to slaughter of which two diets contained different fermentable fibre-rich feedstuffs – 10–13.3% dried chicory roots or 25% blue lupines. These two treatments were compared with pigs fed with an organic control diet for either 1 or 2 weeks prior to slaughter. Lupines significantly reduced skatole in blood and backfat for both genders after 1 week. Moreover, lupines showed negative impact on growth rate and feed conversion whilst chicory showed no significant differences in this respect. However, the indole concentration was significantly lower in chicory than lupine fed pigs. From a sensory perspective, chicory and lupine feeding reduced boar taint since odour and flavour of manure related to skatole and urine associated to androstenone were minimised. The level of boar taint in the entire male pigs was most effectively reduced after 14 days by both fibre-rich feeds while lupine had the largest influence on “boar” taint reduction in female pigs.     

Investigation of factors responsible for the development of boar taint

The objective of this study was to evaluate the effect of 16-androstene steroids, skatole, size of accessory sexual glands and live weight on the development of boar taint in entire male pigs. In Trial I, 35 Yorkshire entire males were slaughtered at 116 ± 3.0 kg live weight. Levels of 16-androstene steroids in the salivary gland (STs), 16-androstene steroids in the fat (STf), skatole in the fat (SK), length of the bulbourethral gland (BG) and weight of the salivary gland (SG) were 44.2 ± 4.7 μg/g, 0.47 ± 0.06 μg/g, 0.20 ± 0.03 μg/g, 11.7 ± 2.2 cm and 63.50 ± 6.73 g, respectively. Samples of loin chops from the animals were evaluated by a trained sensory panel for the presence of boar odour and boar flavour. Sensory scores were correlated with all the investigated factors (P 0.05). Boar odour was particularly well correlated with SG (r = 0.61) and BG (r = 0.60); the correlation with STs was r = 0.53, with SK was r = 0.46 and with STf was r = 0.44. Similar results were obtained for boar flavour. Therefore, both skatole and 16-androstene steroids had significant effects on boar taint. The best explanation of boar taint was obtained by combining measurements of skatole levels in fat and bulbourethral gland length. Trial II involved 36 Yorkshire pigs, 12 each of entire males, gilts and castrates, slaughtered at 104.6 ± 1.1, 102.8 ± 1.8 and 101.0 ± 1.6 kg, respectively. Entire males were chosen which had low levels of 16-androstene steroids (in salivary gland: 13.4 ± 1.5 μg/g; in fat: 0.49 ± 0.09 μg/g) and low levels of skatole in the fat (0.10 ± 0.01 μg/g) but long bulbourethral glands (11.3 ± 0.2 cm). Sensory evaluation performed by a trained panel revealed stronger boar odour and boar flavour from meat from entire males than from other genders (P 0.05). The present results indicate that, although both 16-androstene steroids and skatole are important, they cannot completely account for the occurrence of boar taint detected by a trained sensory panel. Other factors, due to sexual maturity as indicated by the length of bulbourethral gland, are involved and should be considered when estimating boar taint in entire male pigs.     

The effect of age on distribution of skatole and indole levels in entire male pigs in four breeds: Yorkshire, Landrace, Hampshire and Duroc

High skatole and to a lesser degree indole levels in some entire male pigs result in occurrence of off-flavor in meat, called boar taint. In this study, plasma samples from 117 Yorkshire, 134 Landrace, 184 Hampshire and 75 Duroc entire male pigs collected at different ages were analyzed for skatole and slightly fewer samples for indole concentrations. For each breed, a distinct age-related distribution of plasma skatole levels was observed. Skatole levels were increased at approximately 180-200 days of age, reaching very high levels is some individuals. Afterwards, the levels decreased; in Yorkshire and Landrace at approximately 240-260 and in Hampshire and Duroc at 310-360 days of age. The decrease of skatole levels was also observed in samples collected from 15 Landrace boars at two different ages: 16.8 μg/L (SD=17.36) vs. 5.9 μg/L (SD=3.48, p=0.016) in samples taken at the mean age of 210 and 314 days, respectively. Indole levels showed similar age- and breed-related variations to skatole. At the time of increased skatole concentrations (age from 180 to 360 days, depending on breed) 25.5% of Yorkshire, 31.6% of Landrace, 20.3% of Hampshire and 61.1% of Duroc entire male pigs had skatole levels above 12.6 μg/L of plasma, which corresponds to the threshold level of 0.20 μg/g of fat that is used for selecting tainted carcasses. The results indicate that increased levels of skatole in entire male pigs are related to puberty and taking skatole measurements at that age could be advantageous when considering genetic selection to reduce boar taint level in entire male pigs. Breed differences should be also taken into account.     

Influence of soiling on boar taint in boars

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