Fatty acid composition of subcutaneous adipose tissue from entire male pigs with extremely divergent levels of boar taint compounds  — An exploratory study

Meat Science 99 (2015) 1–7

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Fatty acid composition of subcutaneous adipose tissue from entire male pigs with extremely divergent levels of boar taint compounds — An exploratory study Daniel Mörlein a,⁎, Ernst Tholen b a b

Department of Animal Sciences, University of Göttingen, D-37075 Göttingen, Germany Institute of Animal Science, University of Bonn, D-53115 Bonn, Germany

a r t i c l e

i n f o

Article history: Received 22 May 2014 Received in revised form 25 July 2014 Accepted 9 August 2014 Available online 19 August 2014 Keywords: Androstenone Skatole Fat quality Meat quality Castration Lipid metabolism

a b s t r a c t This exploratory study investigated the variability of fatty acid composition in entire male pigs with extremely divergent levels of boar taint compounds. Fatty acids were quantified in back fat samples from 20 selected carcasses of Pietrain*F1 sired boars (average carcass weight 84 kg) with extremely low (LL) or extremely high (HH) levels of androstenone, skatole, and indole. Concentrations of polyunsaturated fatty acids (PUFA) were significantly (p b 0.05) increased in LL boars (23.4%) compared to HH boars (19.7%). This was mainly due to increased levels of linoleic acid (C18:2 n−6) and α-linolenic acid (C18:3 n−3). Correspondingly, unsaturated fatty acids (SFA) were significantly lower (p b 0.05) in LL boars (35.2%) compared to HH boars (37.7%). The findings are discussed with respect to potential effects on flavor formation in boar fat and meat. Further research is needed to study the gender specificity and the interplay of the synthesis and the metabolism of steroids, lipids, and the clearance of skatole in pigs. © 2014 Elsevier Ltd. All rights reserved.

1. Introduction Back fat (subcutaneous adipose tissue) in pigs is mainly composed of water, collagen and lipids (which consist mainly of triacylglycerols). The concentrations of fatty acids determine the firmness of back fat and its technological as well as its nutritional quality (Wood, Enser, Whittington, & Moncrieff, 1989). Surgical castration of male piglets is routinely applied in most countries to prevent off- odors and because it reduces sex-specific behavior as the animals mature. The EU has imposed a voluntary ban on this practice. This is the background for the present study of the consequences for meat and fat quality of boars. Numerous studies have indicated that the accumulation of androstenone, skatole, and indole are the main cause for decreased consumer acceptance of boar meat as reviewed by Lundström, Matthews, and Haugen (2009) and Xue and Dial (1997). Previous studies have also identified various other volatiles such as aldehydes and short chain fatty acids as potential causes for off-odors in boar fat samples that contain very low levels of androstenone, skatole and indole (Rius, Hortos, & Garcia-Regueiro, 2005). Recently, the skatole metabolite 2-aminoacetophenone was ⁎ Corresponding author at: University of Göttingen, Department of Animal Sciences, Albrecht-Thaer-Weg 3, D-37075 Göttingen, Germany. Tel.: +49 551 39 5611; fax: +49 551 39 5587. E-mail address: [email protected] (D. Mörlein).

http://dx.doi.org/10.1016/j.meatsci.2014.08.002 0309-1740/© 2014 Elsevier Ltd. All rights reserved.

suggested as another candidate for contributing to off-flavors in boar meat (Fischer et al., 2014). It has also been suggested that the back fat composition itself may affect the release (volatilization) of androstenone and skatole and, subsequently, their impact on olfactory perception (Rius et al., 2005). Besides such effects of matrix composition on flavor release (Chevance & Farmer, 1999), fatty acids themselves directly contribute to flavor formation in pork, e.g. during oxidative processes, see for example Larick, Turner, Schoenherr, Coffey, and Pilkington (1992). Aldehydes resulting from fatty acid oxidation such as hexanal were shown to have very low detection thresholds (Abraham, Sánchez-Moreno, ComettoMuñiz, & Cain, 2012) and are described as off-flavor in foods (Brunton, Cronin, Monahan, & Durcan, 2000). In general, the susceptibility of fats to oxidative deterioration increases with the degree of unsaturation, i.e., their oxidative stability is impaired with increasing levels of MUFA and, especially, PUFA (Shahidi & Zhong, 2010). Pig genetics and diet have been shown to affect the degree unsaturation in fat tissues; and carcass fatness was shown to be inversely related to unsaturation (Wood et al., 2008). With respect to gender, sex specific differences in fat composition have been reported before (Barton-Gade, 1987; Wood et al., 1989). The carcass weight in those studies was, however, much lower than today. A recent meta-analysis using studies from 1990 until 2010 confirmed previous findings that boars have a higher average PUFA content than castrates while no difference between entire males and female pigs was found (Pauly,

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Luginbühl, Ampuero, & Bee, 2012). At higher weights and with higher physical maturity, however, gender differences are likely to be stronger. Until the work of Wood et al. (1989) it had not been possible to say whether the differences in fatty acid composition were explained by differences in fatness between the sexes. The authors concluded that “the difference in sex hormone metabolism between males and females may be responsible for the effects on composition. This causes the skin to be thicker in entire males, testosterone possibly having a primary effect on the synthesis of collagen in skin and back fat”. The biosynthesis of androstenone in the testes is closely linked to the synthesis of anabolic testicular hormones such as testosterone (Claus, Weiler, & Herzog, 1994). To the best of our knowledge, no study has reported relationships between key boar taint compounds and fatty acid composition. This exploratory study thus was aimed specifically at (i) investigating the variability of fatty acid composition of entire male pigs and (ii) comparing entire males with extremely divergent levels of boar taint compounds.

2.3. Data analysis Analysis of variance (ANOVA) was used to study differences between selected groups using SAS software (The SAS Institute Inc.; Cary, NC, USA). The experimental unit was the individual pig, and the fixed effect included in the model was boar taint compound level group (LL, HH). One-way ANOVA was performed using the GLM procedure in SAS 9.3; normality was checked using the UNIVARIATE procedure. Differences between LS-means were tested for statistical significance using the PDIFF option. Multivariate analyses were performed using The Unscrambler 10.3 (CAMO Software AS; Oslo, Norway). Principal component analysis (PCA) was performed to elucidate correlations between fatty acid composition, carcass data and boar taint compound levels. For PCA, data were standardized (1/standard deviation) and mean centered (mean = 0); full cross validation was performed. 3. Results

2. Animals, material and methods

3.1. Carcass characteristics and boar taint compounds

No animal care approval was required from the University of Göttingen for these experiments because only samples of subcutaneous adipose tissue from carcasses were used.

Carcass characteristics, intramuscular fat, and boar taint compound levels of the selected animals are shown in Table 1. Hot carcass weight, age at slaughter, and back fat thickness were not significantly different (p N 0.05) between LL and HH boars. Lean meat yield was significantly higher (p b 0.05) in LL (62.9%) compared to HH boars (59.2%). Due to the selection, androstenone, skatole and indole were significantly (p b 0.01) higher in HH than in LL boars.

2.1. Samples Back fat samples for this study were taken from boar carcasses from a project which had been constructed to evaluate boar taint levels and performance data in approximately 1000 Pietrain sired crossbred males in Germany. The paternal line was purebred Pietrain, the maternal line was German Large White × German Landrace. Pigs were raised in a performance testing station (Haus Düsse) using standardized feeding (16.0% crude protein, 1.0% lysine; 13.4 MJ/kg) according to the requirements for pig performance testing in Germany (ZDS, 2007), and were slaughtered in a commercial abattoir. After chilling at slaughter, samples of subcutaneous fat were taken from the neck, individually packed in polyethylene bags under vacuum, and stored at − 20 °C until analysis. Twenty samples for fatty acid analysis were selected from the samples in storage, based on their levels of androstenone and skatole. The animals studied here originated from seven sires and 14 dams. Ten samples had very low (LL) and ten had very high (HH) levels of androstenone, skatole and indole. Of the pigs selected for the present study, the average hot carcass weight yielded about 85.6 kg (range: 77.0 to 95.5 kg); average age at slaughter was 180 days (range: 153 to 212 days). Average back fat thickness was 18 mm at 13/14th rib (range: 12 to 28 mm). Average lean meat yield was 61% (range: 53.8 to 64.7%) as estimated using the ‘Bonner Formel’ according to the procedures described in ZDS (2007). Care was taken to have a balanced variation of back fat thickness within groups.

2.2. Laboratory analyses Androstenone was quantified using gas chromatography mass spectrometry (GC–MS), skatole and indole via liquid chromatography with fluorescence detection (HPLC) as described previously (Mörlein, Grave, Sharifi, Bücking, & Wicke, 2012). Results are given in ng per g melted back fat. Fatty acid analysis was performed in duplicate using gas chromatography with flame ionization detection as described previously (Koch et al., 2011). Individual fatty acids are given as percent of total detected fatty acids, and summarized as saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). All laboratory analyses were completed within six months after sampling.

3.2. Fatty acid composition Results of fatty acid analysis with respect to boar taint levels are shown in Table 2. Saturated fatty acids were significantly (p b 0.05) increased in entire males with high levels of androstenone and skatole (= HH boars). This was mainly due to significantly higher levels of myristic acid (C14:0), palmitic acid (C16:0), and arachidic acid (C20:0) in HH animals. The level of monounsaturated fatty acids (MUFA), especially oleic acid (18:1 n−9 cis), did not (p N 0.05) differ between LL and HH boars. Polyunsaturated fatty acids (PUFA) were significantly (p b 0.05) increased in boars with low levels of androstenone and skatole (= LL boars). This was mainly due to increased levels of linoleic acid (C18:2 n−6) and α-linolenic acid (C18:3 n−3). 3.3. Relationship of fatty acid composition, carcass characteristics and boar taint compounds (PCA) Fig. 1 shows the results of the principal component analysis using the individual fatty acid levels as input variables; carcass data, summed fatty acids (SFA, MUFA, PUFA) and boar taint compounds were used with very low weights (‘passified’) such as they were not contributing to the model but used for graphing only. As to be seen from the score plot that shows the distribution of samples in the space of the input Table 1 Carcass characteristics (LS-means) of entire males selected for very low levels of androstenone and skatole (LL, n = 10) compared to very high levels (HH, n = 10).

Hot carcass weight, kg Age, d Back fat thickness, mm Lean meat yielda, % Intramuscular fat, % Androstenone, ng/gb Skatole, ng/gb Indole, ng/gb a b

LL

HH

s.e.

F-value

p-Value

86.6 177.7 17.7 62.9 1.04 97.7 37.5 33.9

84.7 182.2 18.5 59.2 1.19 2983.7 464.2 331.9

2.00 5.41 0.13 0.62 0.13 305.53 38.41 68.67

0.47 0.35 0.19 17.99 0.69 44.61 61.71 9.41

0.4998 0.5641 0.6688 0.0005 0.4164 b.0001 b.0001 0.0066

Estimated (according to ZDS, 2007). Given in ng/g melted fat.

D. Mörlein, E. Tholen / Meat Science 99 (2015) 1–7 Table 2 Fatty acid composition (LS-means) in subcutaneous tissue of entire male pigs selected for very low (LL) compared to very high levels (HH) of androstenone and skatole, n = 10 per group.

C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C20:0 C22:0 Σ SFA C14:1 C16:1 C18:1 n−9 cis C18:1 n−9 trans C20:1 C22:1 n−9 Σ MUFA C18:2 n−6 cis C18:2 n−6 trans C18:3 n−3 C18:3 n−6 C20:2 C20:3 n−3 C20:3 n−6 C20:4 C20:5 n−3 C22:2 C22:6 n−3 Σ PUFA

LL

HH

s.e.

F-value

p-Value

0.01 0.07 0.07 1.22 22.00 11.56 0.16 0.13 35.21 0.07 2.49 37.64 0.41 0.81 0.00 41.43 18.83 0.06 1.64 0.07 1.88 0.26 0.18 0.44 0.001 0.002 0.000 23.36

0.00 0.08 0.08 1.37 23.43 12.45 0.19 0.09 37.70 0.05 2.84 38.52 0.35 0.80 0.01 42.57 15.80 0.05 1.38 0.05 1.75 0.24 0.12 0.34 0.00 0.005 0.001 19.73

0.005 0.004 0.003 0.034 0.296 0.481 0.009 0.011 0.661 0.004 0.153 0.695 0.008 0.044 0.002 0.703 0.843 0.003 0.065 0.004 0.042 0.013 0.014 0.034 0.001 0.003 0.001 0.968

0.89 1.46 2.79 9.39 11.74 1.73 8.14 7.24 7.08 12.12 2.68 0.79 25.53 0.02 0.91 1.32 6.43 9.90 7.98 9.73 4.94 2.19 8.16 4.54 0.11 0.85 1.00 7.04

0.3569 0.2420 0.1122 0.0067 0.0030 0.2049 0.0106 0.0149 0.0159 0.0027 0.1190 0.3850 b.0001 0.8936 0.3525 0.2661 0.0207 0.0056 0.0112 0.0059 0.0394 0.1563 0.0105 0.0473 0.7397 0.3682 0.3306 0.0162

s.e. = standard error of LS-means, SFA = saturated fatty acids, MUFA = monounsaturated fatty acids, and PUFA = poly-unsaturated fatty acids.

variables, PCs 1 and 2 yield no perfect discrimination with respect to boar taint. By trend, however, samples of LL and HH boars are grouped together. From the correlation loading plot it is then to be seen, which of the input variables contain enough structured variation to be discriminating for the samples, i.e., for the boar taint levels. Variables in the radius between the ellipses (50% explained variance, inner ellipse; 100% explained variance, outer ellipse) are more discriminating for the samples being analyzed, e.g. C18:2c and C16:0. By contrast, variables close to origin in the 2-D plot, e.g., C8:0 are neither well explained by PCs 1 and 2 nor do they contribute to discriminating between the boar taint levels. The correlation loading plot further illustrates positive correlations of skatole, androstenone and indole with saturated fatty acids (SFA), mainly palmitic acid (C16:0) and stearic acid (C18:0), and mono-unsaturated fatty acids (MUFA), mainly oleic acid (C18:1). On the contrary, low levels of androstenone, skatole and indole are positively correlated with polyunsaturated fatty acids (PUFA) and with low levels of lean meat yield. Fig. 2 shows the results of another PCA using only aggregated fatty acid data (SFA, MUFA, PUFA), carcass characteristics, and boar taint compounds (passified) as input variables. Using PCs 1 and 2, the score plot again indicates a grouping of LL and HH animals although there is some overlap. Higher levels of androstenone, indole and skatole are positively correlated with the amount of saturated fatty acids (SFA) in subcutaneous tissue of boars; PCs 1 and 2, however, explain less than 50% of the variation of boar taint compounds. Furthermore, the concentration of PUFA is correlated to lean meat percentage while the fat thickness is positively correlated to concentration of MUFA.

4. Discussion To the best of our knowledge, this study is the first to explore the variability of subcutaneous fatty acid composition in boars with divergent levels of androstenone and skatole in back fat. Being exploratory,

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it was beyond the scope of the present communication to study the functional mechanisms in depth. We will, however, provide suggestions for physiological relationships and sensory consequences below. Hence, potential effects of carcass weight, back fat thickness, breed type, diet, and lipid metabolism are discussed. Finally, potential impact of back fat composition on eating quality is addressed. 4.1. PUFA differences and carcass weight Boars selected for extremely low levels of boar taint compounds (LL) in this study had, on average, 18% higher polyunsaturated fatty acid (PUFA) levels than boars with extremely high levels of boar taint compounds (HH). It has been shown previously that PUFA levels of boars were, on average, 30% higher compared to surgically castrated males (Pauly, Spring, Doherty, Kragten, & Bee, 2009). In line with that, the level of n− 6 PUFA was found to be 21% lower (p = 0.001) in barrows compared with entire males (Mackay, Pearce, Thevasagayam, & Doran, 2013). A recent meta analysis confirmed the higher abundance of PUFA in entire males (Pauly et al., 2012). The values for androstenone (mean: 0.7 μg/g, range: 0.2 to 1.9 μg/g) and skatole (average: 0.19 μg/g, range: 0.03 to 1.23 μg/g) observed in Pauly et al. (2009) were low in comparison to the present study, while boar taint compound levels were not reported in Mackay et al. (2013). In those studies, animals were slaughtered at comparable weights (84.0 kg carcass weight (Pauly et al., 2009); 102.2 kg live weight (Mackay et al., 2013)) to those in the present study (85.6 kg carcass weight). Wood et al. (2008) found that, at the same fat thickness as females, subcutaneous adipose tissue from entire males contained a higher proportion of water and a lower proportion of lipid, and concluded that entire males have less mature fat tissue (Wood et al., 2008). Possibly lower values of androstenone in LL boars in this study indicate lesser physiological maturity and in turn might be the reason for the increased degree of unsaturation as C18:2 cis decreases with age (Wood et al., 2008). 4.2. Back fat thickness Various other effects on fatty acid composition of subcutaneous tissue have been reported previously. Generally, carcass leanness has been found to be strongly related to fatty acid composition. Increased back fat thickness is inversely related to the degree of unsaturation (Wood et al., 2008). In the present study, however, samples were selected such as the back fat thickness was not significantly different between LL and HH boars. For gender effects see, e.g., the latest meta-analysis (Pauly et al., 2012). 4.3. Breed Although mechanisms regulating fat deposition are not yet fully understood – it has been suggested that they may be breed-specific – significant differences between breed types have been reported previously (Lopes et al., 2014; Marriott, Chevillon, Spencer-Phillips, & Doran, 2013). In the present study, all boars resulted from crossbreeding using Pietrain sires, so that breed effects can be ruled out. 4.4. Diet As for dietary effects, monogastric species such as pigs are responsive to composition of the diet and the subsequent incorporation of these fatty acids in fat tissues (Wood et al., 2008). Including sources of highly unsaturated fatty acids such as linseed in the diet increases the degree of unsaturation in porcine subcutaneous tissue (Kouba, Enser, Whittington, Nute, & Wood, 2003), and this increase is linear with dietary intake (Wood et al., 2008). In the present study, all pigs were fed the same diet as required for pig performance testing. Therefore, dietary effects between LL and HH groups can be ruled out. This might be

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A)

B)

Fig. 1. Graphical representation of the first and second principal components (PC-1 and PC-2) summarizing the variation of individual fatty acids in subcutaneous tissue of boars with low levels of boar taint compounds (LL) compared to high levels (HH). A) Score plot showing the distribution of samples, and B) correlation loading plot showing the variables; the inner and the outer circles indicate the proportion of variance of these variables as explained by PC-1 and 2 50% and 100% explained variance, respectively. Abbreviations are defined in Tables 1 and 2.

relevant, however, for boars with increased muscle growth potential but with limitations in diets. 4.5. Lipid synthesis and metabolism Expression of lipogenic enzymes such as stearoyl-CoA desaturase (SCD), fatty acid synthase (FAS), and delta-6 desaturase (Δ6D) is associated with fatty acid composition of pig tissues, as reviewed by Wood et al. (2008). SCD catalyzes the desaturation of saturated fatty acids, preferably palmitoyl-CoA (C16:0) and stearoyl-CoA (C18:0) into palmitoleoyl-CoA (C16:1) and oleoyl-CoA (18:1) (Miyazaki & Ntambi, 2003). Significantly higher SCD protein expression in subcutaneous adipose tissue of entire males compared to barrows was recently shown; this was associated with significantly higher levels of mono- and polyunsaturated fatty acids in boars compared to surgically castrated pigs (Mackay et al., 2013). Most recently, a functional variant in the SCD gene promoter was identified as causative for increased fatty acid

desaturation in pork (Estany, Ros-Freixedes, Tor, & Pena, 2014). FAS catalyzes the fatty acid synthesis. Increased SFA deposition has been shown to be related to increased abundance of FAS protein expression (Marriott et al., 2013). Furthermore, expression of Δ6D, which modifies, for example linoleic acid (C18:2 n− 6) into linolenic acid (C18:3 n − 6), tended to be higher in boar fat tissue compared to barrows which had higher back fat thickness, SFA and MUFA (Mackay et al., 2013). Significantly lower expression of SCD proteins has been shown in subcutaneous adipose tissue of pigs selected for decreased back fat thickness compared with a control group. This was, however, not accompanied by significant differences of MUFA and PUFA in subcutaneous tissue between treatments (Cánovas, Estany, Tor, Pena, & Doran, 2009). In line with that, a subsequent experiment also revealed decreased FAS and Δ6D protein expression in liver tissue of pigs selected for decreased back fat thickness. No significant changes were found, however, for fatty acid composition between treatments (Muñoz, Estany, Tor, & Doran, 2013).

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A)

B)

Fig. 2. Graphical representation of the first and second principal components (PC-1 and PC-2) summarizing the variation of aggregated fatty acids in subcutaneous tissue of boars and carcass characteristics of boars and their relationship to boar taint compounds. A) Score plot showing the distribution of samples with low (LL) or high levels (HH) of boar taint compounds; and B) loading plot showing the variables; the inner and the outer circles indicate 50% and 100% explained variance, respectively. Abbreviations are defined in Tables 1 and 2.

4.6. Effects of high androstenone and skatole It remains open, whether increased levels of androstenone and skatole affect lipid synthesis and lipid metabolism related enzyme activity in subcutaneous or other tissues, e.g., in the liver which is also involved in lipogenesis (Cánovas et al., 2009). It has been shown previously that high levels of androstenone effectively inhibited cytochrome P450 (CYP) activity (Doran, Whittington, Wood, & McGivan, 2002; Rasmussen, Zamaratskaia, & Ekstrand, 2011). On the other hand, it has been suggested that CYP2E1 gene expression is involved in lipid synthesis and metabolism of entire males with divergent fatty acid composition (Corominas et al., 2013). Recent studies suggested differential gene expression in the liver indicative for several metabolic processes including lipid and amino acid metabolism in boars with divergent skatole levels (Gunawan, Sahadevan, Cinar, et al., 2013) and with divergent androstenone

levels (Gunawan, Sahadevan, Neuhoff, et al., 2013). Among others, genes for energy metabolism were identified such as IDH1 (isocitrate dehydrogenase) which is involved in the citrate and fatty acid synthesis. This was supposed to be in line with earlier findings that pigs with a higher metabolic rate (= fatter pigs, e.g., Large White and Duroc) have higher androstenone and skatole levels than lean breeds with lower energy metabolism such as Pietrain. This is confirmed by the present results: Pietrain × F1 crossbred boars with very high levels of androstenone and skatole (HH) tended to be fatter compared to boars with very low levels of androstenone and skatole (LL). Recently, another study found differentially-expressed genes in female pigs with divergent fatty acid composition (Ramayo-Caldas et al., 2012). Those genes were related to the lipid and fatty acid metabolism, especially the linolenic and arachidonic acid metabolism. Furthermore, high concentrations of skatole are suggested to be toxic while detoxification involves enzymes such as the cytochrome

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P450 family which are also involved in lipid metabolism; gender differences of hepatic enzyme activity related to detoxification are well known in rodents, but also pigs (Black, 1994; Waxman & Holloway, 2009). Toxic effects of skatole have been shown for example in the bovine lung, where skatole from rumen degradation causes acute pulmonary edema and interstitial emphysema (ABPE) accompanied by abnormalities in phospholipid synthesis and glycogen metabolism (Bray & Kirkland, 1990). The authors reported that the metabolism of skatole by cytochrome P-450-dependent mixed function oxidases (MFO) or prostaglandin H synthase (PHS) caused free radicals. It is well established that lipid peroxidation, especially of highly unsaturated fatty acids, is promoted by free radicals. The question thus arises whether decreased PUFA levels in HH animals could (partly) be due to oxidative processes because of radical formation as induced by high skatole content. Furthermore, changes in phospholipid synthesis were shown to be associated with increased neutral lipid storage (Igal & Coleman, 1998). This study showed that HH pigs had significantly higher SFA levels in subcutaneous adipose tissue and tended to have higher IMF compared to LL pigs. It has to be noted though that skatole formation in pigs is under genetic control such as toxic effects of endogenous skatole appear to be unlikely. Feeding a low fiber diet to commercial, fast growing breed types may, however, increase the probability that high skatole levels occur. By contrast, in wild boars very low (if any) skatole levels have been reported despite very high androstenone concentrations (Fischer & Wüst, 2012). All in all, the results of the present explorative study suggest an interaction of increased levels of androstenone and skatole with lipid metabolism. As it was beyond the scope of the present study to define functional relationships, further research needs to study the role of enzymes involved in the synthesis and metabolism of steroids and lipids as affected by the boar taint compounds in entire males as well as the clearance of skatole and androstenone. As regards a potential toxicity of very high skatole in pigs, the tissue specificity of radical formation and its effect on lipid oxidation in LL vs. HH needs to be validated. Using divergent boars having, e.g. low androstenone but high skatole (LH) versus high androstenone but low skatole (HL) might allow a more detailed study of the effects of androstenone and skatole individually as they were not differentiated here.

sensory evaluation, i.e., they might be judged as deviant from ordinary back fat samples because of off-odors such as rancidity (Rossi et al., 2013) although they are low in boar taint compounds. The relevance of lipid content and lipid composition in flavor formation during heating of meat has been shown previously (Mottram & Edwards, 1983). Those experiments indicated that phospholipids or their degradation products inhibit the formation of alkyl pyrazines from the Maillard reaction responsible for roast, biscuit-like flavor. This quenching effect of lipids was later confirmed in model systems, where the addition of lipids also reduced resulting sulfurous notes (Farmer & Mottram, 1990). Though they are likely to be of minor importance compared to muscle tissue, these interactions of lipids (and lipid degradation products, e.g., aldehydes) and Maillard reactions also need to be considered in fat tissues. As both the lipid content and the fatty acid composition in subcutaneous tissue of boars varies remarkably depending on carcass fatness and age (Wood et al., 2008), the question arises whether and to what extend this could affect lipid derived flavor formation and flavor release including the release of the boar taint compounds themselves; this would be highly relevant for sensory evaluation in abattoirs for sensory quality control or in research units aiming to decrease boar taint by means of breeding for example. To our knowledge, flavor formation in porcine fat tissues varying in fat content, fat composition and boar taint compounds has yet not been extensively studied. Further investigation is also needed to quantify the increase of free fatty acids that might occur due to enzymolysis (Song et al., 2014) during longer storage before sensory evaluation. Finally, in regard to flavor release, volatile compounds held responsible for boar taint (here: androstenone, skatole and indole) are likely to interact with proteins from the connective tissue matrix in back fat as suggested earlier (Larick et al., 1992). Also, varying water and fat content might affect flavor release as has been shown for example in sausages (Chevance & Farmer, 1999). All told, the flavor formation and flavor release in boar meat and fat tissue needs to be studied beyond the abundance of key odorants, i.e., androstenone and skatole, in order to better understand why consumer acceptance may be affected.

4.7. Implications for eating quality: flavor formation and release

Being exploratory, the present study cannot posit any functional relationship. It does, however, for the first time report divergent fatty acid composition in subcutaneous tissue of boars selected for high or low levels of boar taint compounds. The entire male pigs with low androstenone, skatole, and indole levels (ASI) show significantly increased polyunsaturated (PUFA) levels and less saturated fatty acids (SFA). Literature suggests an involvement of cytochromes both in the lipid and steroid synthesis and metabolism as well as the clearance of skatole which need further investigation. The results give rise to the intriguing possibility that higher levels of androstenone and/or skatole might affect the lipid metabolism in pigs. However, this remains to be verified. Further research also needs to show the extent to which a varying lipid content and fatty acid composition influence flavor formation and the release of boar taint compounds from the subcutaneous tissue matrix. The question remains whether the increased susceptibility of boar fat to oxidation promotes the development of off-flavor in boars despite low ASI levels. In general, flavor formation in boar meat and fat needs to be studied beyond these key volatiles in order to better understand why consumer acceptance is often impaired.

Finally, potential impact of adipose tissue composition on sensory quality should be addressed. This includes PUFA levels, susceptibility to oxidation, and effects of heating. In LL boars, the increased levels of PUFA (+18% on average) could be responsible for increased levels of volatiles as shown earlier in lean meat from pigs fed different proportions of linoleic acid (Larick et al., 1992). That effect is very likely to be even more pronounced in fat tissue as the absolute amount of fat is higher compared to lean meat. As regards flavor formation, PUFA have been shown to be precursors for aroma active aldehydes, e.g., hexanal due to oxidative processes (Shahidi & Zhong, 2010). In general, these aldehydes have very low olfactory thresholds that depend on their respective chain length; the potency of these odorants is, among other reasons, due to a strong response of human odorant receptors to these substances (Abraham et al., 2012; Cometto-Muñiz & Abraham, 2010). The increased susceptibility to fat oxidation is especially relevant when samples are stored before sensory evaluation, e.g., in research projects. Furthermore, for sensory evaluation the release of androstenone and skatole needs to be facilitated by microwave heating or other means. Thus, the flavor formation due to oxidation of PUFA is also increased. Thermal oxidation, though for a longer period of time than typically applied for boar taint evaluation, was shown to be most effective for flavor development in beef (Song et al., 2014). Here, LL samples appear to be more susceptible to fat oxidation due to their elevated levels of PUFA. They are, thus, at risk of being false positives in

5. Conclusion and implication

Conflict of interest None of the authors has a financial or personal relationship with other individuals or organizations that could inappropriately influence or bias the contents of this paper.

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