1. Introduction
Animal welfare has become a very important factor in livestock breeding, including that of pigs. A general ban on surgical castration of entire male pigs was expected in all EU member states by the end of 2018. Due to various circumstances, it was postponed until after 2021. One feasible alternative to painful surgical castration is to fatten all males. It is well known that boars grow faster and more efficiently, and they have higher lean meat content in carcass than surgical castrates. On the other hand, fattening boars increases the risk of and results in a higher incidence of boar taint and thus the dissatisfaction of consumers with such meat.
The incidence of boar taint is mainly attributed to two substances, androstenone [
1] and skatole [
2], especially after heat treatment of pork. Androstenone (α-androst-16-en-3-one) is a steroid hormone synthesized in the Leydig cells of the testis of uncastrated boars. This compound has an odour similar to urine or sweat, is perceived by approximately two-thirds of the human population and has been shown to be different according to country/region. Deposition of androstenone in fat tissue has high heritability estimates (0.55–0.88), which indicates that it is affected mainly by genetics [
3,
4]. Skatole (3-methyl-indole) is a metabolite derived from microbial catabolism of the amino acid tryptophan in the hindgut of pigs. Its deposition is influenced mainly by environmental factors (
h2 = 0.23–0.55) [
5], especially nutrition, the system of feeding and the housing conditions [
6,
7,
8,
9,
10]. Since both of these compounds are lipophilic, they can accumulate in adipose tissue and therefore may have a negative effect on sensory attributes and result in the rejection of such meat by consumers.
Since the odour of skatole is negatively perceived by practically the entire human population, reduction efforts have focused mainly on various feeding strategies in fattening all males. Promising results have been achieved by supplementation of diets with a variety of feed additives, such as chicory root or inulin [
11,
12,
13,
14,
15,
16,
17,
18,
19,
20], raw potato starch [
21,
22,
23,
24], sugar beet pulp [
25], Jerusalem artichoke [
26], oligofructose and fructooligosaccharides [
15,
27] and tannins [
28,
29,
30].
Tannins are plant metabolites with great diversity, resulting in different physiological effects according to their form, animal species and amount of supplementation [
29,
31]. Sweet chestnut (
Castanea sativa Mill.) wood extract, consisting mainly of hydrolysable tannins, has antimicrobial and antiviral properties. Therefore, products containing this substance are used in animal nutrition, especially in piglets, as supportive treatment for diarrhoea after weaning [
32,
33,
34,
35,
36,
37,
38].
Several studies have demonstrated a reduction in total protein digestibility, as well as inhibition of microbial activity, in the colons of pigs after supplementation of the pig diet with hydrolysable tannins [
39,
40,
41]. Lower disponibility of tryptophan and cell debris in the hindgut may lead to reduced intestinal production of skatole [
28,
42]. These findings are interesting from the entire male production point of view.
Apart from a few studies dealing with Iberian pigs fed natural sources [
43,
44,
45,
46], other research on the effects of hydrolysable tannins (HTs) has been mainly aimed at growth and carcass parameters, meat quality and oxidative stability or the intestinal skatole production and microbiota composition in the large intestines of boars [
28,
29,
30,
31,
32,
37,
41,
47], but almost no research has addressed the effect of HTs on the sensory traits of entire male meat.
The main objective of the present study was to assess the impact of hydrolysable tannins on parameters of eating quality, considering the possible effect of the sex of consumers, as well as to investigate the relationships between sensory evaluation and the content of skatole and androstenone in adipose tissue.
2. Materials and Methods
2.1. Animals and Diet
Eighty young boars were used in the experiment. They were the progeny of Landrace sows and Yorkshire × Pietrain boars. Two weeks before the experiment, the pigs were housed in pairs/pens at the experimental test station of the Research Institute for Animal Production (RIAP) Nitra. Subsequently, the boars were randomly distributed within litters to one control and four experimental groups (each containing 16 animals). Control pigs (T0) received a diet without any supplementation. Experimental groups received the same diet as the control group but supplemented with 10 (T1), 20 (T2), 30 (T3) or 40 (T4) g/kg sweet chestnut wood extract (SCWE) rich in hydrolysable tannins (
Table 1). The producer of the Farmatan product is Tanin Sevnica d.d., Sevnica, Slovenia, and the supplier was Product Feed a.s., Luzianky, Slovakia. The content of tannins in this product is 73 ± 2% (the value declared by the producer). The analysis of feed was performed according to the Folin–Ciocalteu method [
48]. The total phenolic content is expressed as gallic acid equivalents and is 45.1%.
Supplementation of the diet with tannins started when the boars reached an average live weight of 80 kg (after a 2-week transitional period) and lasted for 40 days prior to slaughter. Access of the animals to drinking water and feed (automatic feeders—Schauer s.r.o., Nitra, Slovakia) was ad libitum.
2.2. Slaughter and Sample Collection
Entire males were slaughtered in one batch at the experimental slaughterhouse of RIAP Nitra. The average live weight of the pigs was 122.28 kg ± 5.63 kg. Standard slaughter conditions were used: electrical stunning (90–100 V, 0.9–1.0 A, 50 Hz) followed by exsanguination. Evisceration was completed approximately 20 min post mortem. Chilling of the carcasses (air temperature 2–4 °C, velocity 0.5–1.0 m.s−) started approximately 60 min after slaughter and was continued overnight. After 24 h of chilling of the carcasses, musculus longissimus thoracis (LT) samples (1.0 cm thick) with subcutaneous fat were removed from the right side of the carcass (at the level of the last rib) and stored at −20 °C until sensory evaluation.
2.3. Sample Preparation
One day before sensory evaluation, the raw LT sample was thawed overnight at 4 °C. Subsequently, each LT slice was trimmed to have 5 mm of subcutaneous fat. Each LT slice was placed in a grill and cooked for 4 min at 180 °C. The average measured internal temperature of the samples was 80 °C. After grilling, each steak was cut into four strips and immediately served to different panellists.
2.4. Sensory Evaluation
Panellists were recruited from the staff of RIAP Nitra. All of them were consumers who liked and ate pork regularly (2–3 times weekly). Before the sensory evaluation of samples, consumers were tested for their sensitivity to androstenone according to the modified method of Weiler et al. [
49]. On the basis of this method, 20 panellists were selected (12 men and 8 women aged 32 to 60 years old).
In total, 320 samples were evaluated in eight sessions (each of 40). In each session, eight panellists evaluated five samples (one from each dietary group). Each panellist participated in several sessions. Meat samples were randomly served to the consumers, and the attributes classified were odour, flavour, tenderness and juiciness. The scale applied in the sensory test was structured into 5 points, with 1 being the worst and 5 the best evaluation (
Table 2). The panellists were asked to score for odour first, followed in order by flavour, tenderness and juiciness.
2.5. Skatole and Androstenone Determination
Fat samples (100 g from a part of the belly) from entire males were removed 24 h after slaughter. One part of each sample was individually packed in a microtene bag, marked and frozen (−20 °C) until panel testing. The second part of each fat sample was transported to the authorized private laboratory of EKOLAB, s.r.o. (Košice, Slovakia), to analyse the androstenone and skatole concentrations according to the methods of Ampuero Kragten et al. [
50] and Bekaert et al. [
51]. Briefly, adipose tissue samples were melted in a microwave for 4 min. Liquid fat was transferred to centrifuge tubes (2 mL), and 1.75 mL of extraction solvent methanol:hexane (9:1) were added. The extract was ultrasonically cleaned in a bath at 50 °C for 5 min and centrifuged for 5 min at 10,000×
g. After cooling, approximately 2.0 mL of extract were then injected into a gas chromatograph equipped with a mass spectrometry (MS) detector (Shimadzu GCMS-TQ8030, Shimadzu Corp., Kyoto, Japan) at an injection temperature of 260 °C.
The limits for detection were 0.02 µg/g for androstenone and 0.01 µg/g for skatole.
2.6. Statistical Analysis
The observed data were evaluated by 1-way analysis of variance (ANOVA) with fixed effects [
52] using the following model:
yij= μ + αi + eij with N (0,σ2) for androstenone and skatole
For multiple comparisons of treatment means, Bonferroni’s test was used [
53]. Dunnett’s test was not used to compare the control and treatment groups since, from the analyses of variance of both traits, it could be concluded that differences between all groups by the F test were nonsignificant. The observations of sensory traits were evaluated by 2-way ANOVA with fixed effects using the following statistical model:
yijk = μ + αi + (αβ)jj + eijk with N (0,σ2)
Pearson’s correlation coefficients of androstenone and skatole with sensory traits were calculated.
4. Discussion
Generally, tannins are considered to be antinutrients, as they create compounds with other nutrients, such as proteins, minerals, or digestive enzymes, and therefore are capable of reducing feed palatability, feed intake and nitrogen digestibility [
39,
54,
55]. However, pigs, wild as well as domestic, seem to be relatively resilient to the intake of feedstuffs with a high content of tannins without any negative consequences on performance or health status [
56]. This resistance is associated with elevated synthesis of proline-rich proteins (PRPs) in the saliva, which bind tannins from feedstuffs and prevent intoxication of organisms with diets rich in hydrolysable tannins [
57,
58]. Moreover, recent studies have suggested that tannins have antimicrobial, anticancer, and antioxidant properties and can improve feed efficiency and reduce bacterial proteolytic reactions in piglets, thus protecting them from severe diarrhoea during weaning [
32,
33,
34,
35,
36,
37,
59]. It is well known that wild pigs, as well as some special native breeds of domestic pigs (Iberian, Cinta, etc.), can eat foodstuffs rich in tannins (content: 4–7%); therefore, a dose of 40 g per kg of feed mixture was selected as the highest dosage in the present study.
It is well known that the level of skatole in fatty tissue is influenced by many processes, such as formation, absorption, metabolism and deposition. The main role is associated with the activities of digestive enzymes in the CYP450 family, such as 2E1, 2A19, 1A1, and 1A2, in the liver [
6,
28].
Generally, data relating the effect of tannins on skatole production and accumulation in pig adipose tissue are limited. Some studies have shown that tannins can inhibit proliferation and apoptosis in the caecum. This inhibition results in decreased skatole production in the large intestine due to the lower availability of cell debris from lower apoptosis and tryptophan [
41]. In the present study, skatole accumulation in adipose tissue tended to decrease with increasing tannin supplementation. Similar numerical decreases were observed in other studies after 2–3% [
28] or 3% [
29] tannin supplementation, but surprisingly, lower supplementation (1.0 or 1.5%) resulted in higher skatole accumulation than in the control group [
28,
30]. Čandek-Potokar et al. [
28] suggest that this result could be associated with lower activity of CYP2E1 and CYP2A19 enzymes, two major enzymes of the phase 1 metabolism of skatole in the liver. It should be mentioned that some of the above studies [
29,
30] used products with lower contents (only 50%) of hydrolysable tannins than our study.
Regarding the effect of tannins on androstenone, the present study showed that supplementation with these additives did not have any impact, even though the two highest doses had higher (although nonsignificant) androstenone concentrations in fat tissue than the control group. This result is partially in contrast with that of other studies [
28,
29,
30] in which numerical reductions were found after supplementation of diet with 1–3, 3, and 3% hydrolysable tannin extracts. However, the pigs in the latter study [
30] had low levels of androstenone overall.
Generally, the results regarding the effects of tannin supplementation on skatole and androstenone levels in back fat were not consistent and were highly dependent on the dose of tannin supplementation, often reporting curvilinear dependence. Therefore, research to determine the optimal dose of tannin supplementation for reducing boar taint is still needed. Moreover, the effects of hydrolysable tannins on androstenone are still unclear, and if any are confirmed, further studies will be needed to clarify the mechanism of action.
As previously mentioned, very few studies have been published thus far relating the effects of hydrolysable tannins on pigs. Those studies focused mainly on intestinal skatole production, growth rate, carcass and meat quality, and the intestinal microbiota [
30,
32,
37,
47,
59], but almost none focused on the sensory properties of pork. Thus, our results related to the effects of tannins on eating quality are difficult to compare with those of other studies. Only one study in pigs [
30] and one in sheep [
60] investigated the impact of tannin supplementation on the sensory traits of meat. In the present study, the odour and flavour of boar meat were not affected by tannin supplementation. Panellists scored these two parameters in the control and supplemented groups at almost the same level, and the differences were not significant. Bee et al. [
30] reported different findings. Panel members detected a stronger boar taint odour but not flavour in the meat of entire males supplemented with 1.5% tannins compared to the controls and group with 3% tannins. In lambs, Priolo et al. [
60] observed lower sheep meat odour in meat from animals supplemented with tannins compared to those not supplemented. Contrary to odour and flavour, the other two sensory traits—tenderness and juiciness—in the present study showed significant differences. Panellists, but only men, scored (
p < 0.05) tenderness better in the control group than in the two supplemented groups (T3–T4), and juiciness in the control group was evaluated better than in T4. This outcome is contrary to the results of a study [
30] that reported no significant effect of tannin supplementation on juiciness and/or tenderness. Regarding differences between the sexes of panellists in the present study, women scored tenderness and juiciness worse than men in both the control and supplemented groups.
It seems that higher supplementary tannin doses (3–4%) in our study significantly reduced juiciness (T4) and tenderness (T3–T4) compared with the control group. However, this result may be sex-dependent.