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Suppression of androstenone in entire male pigs by anabolic preparations
Livestock Production Science 69 (2001) 139–144 www.elsevier.com / locate / livprodsci
Suppression of androstenone in entire male pigs by anabolic preparations a,
a
a
a
Andreas Daxenberger *, Melanie Hageleit , Wolf-Dieter Kraetzl , Iris G. Lange , Rolf Claus b , Bruno Le Bizec c , Heinrich H.D. Meyer a a
¨ Physiologie, Forschungszentrum f ur ¨ Milch und Lebensmittel, TU Munchen-Weihenstephan ¨ , Weihenstephaner Berg 3, Institut f ur D-85354 Freising-Weihenstephan, Germany b ¨ Tierhaltung und Tierzuchtung ¨ ¨ Hohenheim, (470), Fachgebiet Tierhaltung und Leistungsphysiologie, Universitat Institut f ur Postfach 70 05 62, D-70539 Stuttgart, Germany c ´ ´ ´´ , Ecole Nationale Veterinaire des Nantes, B.P. 50707, Laboratoire des Dosages Hormonaux, Laboratoire National Reference F-44307 Nantes Cedex 3, France Received 8 November 1999; received in revised form 20 July 2000; accepted 22 November 2000
Abstract At an age of 19 weeks and an average weight of 80.5 kg, four boars each were implanted i.m. with two doses of either Synovex-H (SH; total dose 400 mg testosterone propionate 1 40 mg oestradiol benzoate) or Synovex Plus (SP; total dose 400 mg trenbolone acetate 1 56 mg oestradiol benzoate), respectively. A third group of four boars served as control. The animals were slaughtered 5 weeks after implantation and the testes weights recorded. Hormone (androgen and oestradiol benzoate) residues in implantation sites were quantified by high-performance liquid chromatography with UV detection, and the boar taint steroid androstenone was determined in fat by enzyme-immunoassay (EIA). Gas chromatography–mass spectrometry analysis additionally confirmed the identity. Plasma testosterone and trenbolone concentrations were measured by EIA. Plasma testosterone and fat androstenone concentrations were reduced to basal levels by the SP treatment. With SH the decrease in fat androstenone concentrations was inconsistent between animals. Androgen and estrogen residues in implantation sites were higher in SH boars than in SP boars (179 mg 1 19 mg versus 85 mg 1 15 mg, respectively). Besides the resulting higher resorbed doses, the higher SP efficiency was attributed to greater hypothalamic negative feedback activity of trenbolone compared with testosterone. Including the implantation site, whole batches of processed meat products would be contaminated above the international threshold levels for these hormones in meat. 2001 Elsevier Science B.V. All rights reserved. Keywords: Boar taint; Androstenone; Testes; Residues; Anabolic
1. Introduction *Corresponding author. Tel.: 1 49-8161-714-202; fax: 1 498161-714-204. E-mail address: [email protected] (A. Daxenberger).
Compared with castrates, the portion of lean meat in boars is increased and the feed conversion is more efficient (Hansson, 1974; Hansson et al., 1975;
0301-6226 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 00 )00259-1
140
A. Daxenberger et al. / Livestock Production Science 69 (2001) 139 – 144
Dobrowolski et al., 1995) due to the anabolic action of testicular steroids. After implantation of an anabolic preparation (20 mg oestradiol 1 140 mg trenbolone acetate) into growing castrated male pigs, nitrogen retention increased by 23 to 88%, and was paralleled by a 56% increasing protein and a 15% decreasing fat deposition (Van Weerden and Grandadam, 1976). Higher nitrogen retention of boars is also desirable with respect to environmental aspects. Meanwhile, rearing of boars is favoured mainly by the animal welfare aspect because castration is avoided. The main argument against rearing of boars is an unpleasant odour which occurs regularly in carcasses of postpuberal male pigs. On the one hand, the odour is caused by the steroid 5a-androst-16-en-3-one (androstenone), the main representative of D-16 steroids in the boar. Androstenone has no hormonal activity but works as a sex pheromone (Claus, 1979). The D-16 steroids are produced parallel to testicular hormones in the Leydig cells from common precursors, pregnenolone and progesterone, and their synthesis is stimulated by luteinizing hormone (LH) which is controlled by gonadotropin-releasing hormone from the hypothalamus. This system is balanced by the feedback function of testicular hormones on the hypothalamus (Claus et al., 1994). Due to its lipophilic properties, androstenone is accumulated in the adipose tissue and eliminated within 3–6 weeks after castration, depending on the age at castration (Claus, 1976). On the other hand, skatole, which is formed in the intestinal tract by microbial degradation of the amino acid tryptophan, has been identified as another substance responsible for the off-flavour of boar meat. However, the production of skatole is dependent on the presence of tryptophan under a simultaneous deficit of energy in the gut, and therefore can be influenced by feeding (Claus et al., 1994). The intention of our study was to determine the effect of the anabolic implant preparations SynovexH (testosterone propionate 1 oestradiol benzoate) and Synovex Plus (trenbolone acetate 1 oestradiol benzoate) on fat androstenone concentration of intact boars. The use of certain anabolic sex hormones is licensed in many countries (except the European Union) when implanted into the ears of cattle. Thus, the availability of such substances may lead to
misuse in other species. Furthermore, we examined the hormone residues at implantation sites with respect to consumer safety.
2. Materials and methods
2.1. Animal experiments Twelve boars (Landrace 3 Pietrain crossbreds) were weaned at an age of 5 weeks and fed a commercial diet (12.8 MJ / kg, 17.7% crude protein) ad libitum until an age of 12 weeks and a mean weight of 35 kg. Thereafter, they were fed ad libitum with a finishing feed (12.9 MJ / kg, 18.0% crude protein) until the end of the experiment. During the whole period an artificial light programme with 10 h light per day was applied. At an age of 19 weeks and an average weight of 80.5 kg, the animals were allocated to three groups of four animals. One group served as control (C; no treatment). The treatment groups were either implanted with two doses of Synovex-H (SH; total dose 400 mg testosterone propionate 1 40 mg oestradiol benzoate) or two doses of Synovex Plus (SP; total dose 400 mg trenbolone acetate 1 56 mg oestradiol benzoate; both preparations from Fort Dodge Laboratories, IA, USA). In order to simulate off-label use in pigs, the preparations were injected into the necks of the animals, because hormone depots would have been visible after application into the pinna of the ear. The implantation was performed with the original implanter pistol by injecting two doses of each preparation 4–5 cm deep into the neck muscles of each animal. Five weeks after implantation the animals were slaughtered, some tissues sampled and the carcasses of the treated animals removed from the food chain.
2.2. Sample collection Blood samples for plasma were taken directly before treatment (day 0) and 7, 21 and 31 days after implantation by venipuncture of the V. jugularis. Immediately after slaughter, the complete implantation sites, the testes and perirenal fat were collected. The testes were cut from the scrotum, weighed and measured with a slide gauge. The implantation sites
A. Daxenberger et al. / Livestock Production Science 69 (2001) 139 – 144
141
were prepared further in the laboratory. The whole tissue area containing the implants was cut into layers of 5 mm. All visible residual implants were taken out from the embedding tissue. Furthermore, the tissue that contained the implants was dissected (up to a distance of 3–5 cm from the recovered implants).
2.3. Steroid determinations The content of testosterone and trenbolone-17b in plasma was analysed by enzyme-immunoassay (EIA) using standardized kits (R-Biopharm, Darmstadt, Germany) as published previously (Meyer and Hoffmann, 1987; Blottner et al., 1996). Hormone residues in recovered implants and the tissues of the injection sites were quantified by high-performance liquid chromatography (HPLC) with diode array detection (DAD) (Daxenberger et al., 2000). Boar taint steroids in adipose tissue were quantified by EIA (Claus et al., 1997). Androstenone identity was additionally confirmed by gas chromatography–mass spectrometry (Le Bizec et al., 1996).
3. Results The mean feed consumption was 2.2 kg per day between the beginning of the finishing period and the time of the hormone treatments. During the 5-week treatment period, the average food consumption was 2.8 kg / day. Average daily weight gain was 804 g from week 13 to 19 and 824 g during the treatment period (weeks 20–24). There was no significant difference between groups. At slaughter, the mean weight of the boars was 109 kg, corresponding to a mean carcass weight of 91.1 kg.
3.1. Testosterone and trenbolone in plasma, androstenone in fat and testis size A clear decrease of plasma testosterone concentrations (Fig. 1 and Table 1) was observed in SP boars. The concentrations dropped from more than 1 ng / ml before treatment down to , 0.15 ng / ml on day 7. In SH boars, there was no reduction in testosterone concentrations. After SP treatment only traces of androstenone were detected in adipose
Fig. 1. Effect of anabolic preparations on testosterone and trenbolone concentrations in plasma of individual boars (day 0 5 treatment).
tissue (Table 1). In SH boars, the situation was less clear: Animal no. 500 revealed a considerable concentration (1291 ng / g) whereas, in two other boars, low androstenone concentrations were found. In animal no. 502 androstenone concentrations were above base levels observed in sows ( , 50 ng / g), but with 190 ng / g did not exceed the level of acceptance in Germany (500 ng / g; BGBl, 1996). The testes in the SH group and the SP group were 15% and 21% lighter, respectively, than in the C group (Table 1). The differences were not significant (t-test; P 5 0.275 and 0.065, respectively). Across all 12 individuals, there was a slight (r 5 0.679) but significant (P 5 0.015) linear regression
A. Daxenberger et al. / Livestock Production Science 69 (2001) 139 – 144
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Table 1 Effect of Synovex-H and Synovex Plus on testosterone in plasma and androstenone in adipose tissue, and on testis weight of boars a Group
Animal No.
Testosterone (ng / ml)
Androstenone (ng / g)
Control
450 504 562 565
1.93 2.35 0.68 0.79
1312 319 312 807
Mean S.E.M.
1.4 0.42
688 238
451 500 502 563
0.60 4.82 1.60 1.90
Mean S.E.M.
Synovex-H
Synovex Plus
a
Testis (g) Left
Right
Total
233 211 229 234
263 208 244 259
496 419 473 493
227 5.4
244 13
470 18
38 1291 190 84
121 317 213 158
89 327 207 166
210 644 420 324
2.2 0.91
401 298
202 43
197 50
400 92
452 453 503 564
0.13 0.10 0.87 0.08
39 16 72 73
247 116 243 146
230 114 267 123
477 230 510 269
Mean S.E.M.
0.30 0.19
50 14
188 33
184 38
372 71
Plasma testosterone levels are given as the mean during the implantation period (days 7, 21 and 31).
between the testes weights (sum of both testis) and fat androstenone concentrations. All boars with testis weights below 200 g had low levels of androstenone.
3.2. Hormone residues in implantation sites During the 5-week implantation period the implants were not completely degraded. The recovered implant remnants and the surrounding tissue contained considerable amounts of hormones after slaughter (Table 2). Additionally, the surrounding tissues contained traces of hydrolysed androgens (i.e., trenbolone-17b or testosterone), but hydrolysis of oestradiol benzoate was not observed. A mean total of 179 mg testosterone propionate (range 138– 208 mg), 85 mg trenbolone acetate (range 69–108 mg) and 17 mg oestradiol benzoate (range 12–21 mg) remained in the respective injection sites. Consequently, the total applied doses did not enter the circulation completely. The actual effective dose was the difference of the applied dose minus the residual hormones. Thus, the relative effective doses per kg
live weight, based on the average live weights at implantation and slaughter, were 2.32 mg testosterone propionate 1 0.22 mg oestradiol benzoate (SH) and 3.33 mg trenbolone acetate 1 0.43 mg oestradiol benzoate (SP).
4. Discussion
4.1. Effect of anabolic steroids on fat androstenone and testicular function The possibility of a ‘‘hormonal castration’’ and thus the control of boar taint by exogenous application of gonadal hormones were studied previously and included the use of several substances. For example, diethylstilbestrol proved to be effective to depress testicular development but the loss of growth potential was not compensated by the application for this oestrogen alone (Echternkamp et al., 1969; Palmer et al., 1971). Application of SP 5 weeks prior to slaughter resulted in a reduced androstenone
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Table 2 Hormone residues in implants and at implantation sites at the time of slaughter, and the dose of hormone actually released (T, testosterone propionate or trenbolone acetate; OE, oestradiol benzoate; SH, Synovex-H ; SP, Synovex Plus ) Group
Animal No.
Hormone residues (mg) in implants and at implantation sites Pellets
SH
SP
Hydrolysed a
Total
Implantation
T
OE
T
OE
T
T
OE
451 500 502 563
136 164 206 194
14.6 19.9 20.9 19.7
2.1 12.1 2.2 2.0
0.3 1.2 0.4 0.4
0.0 0.1 0.1 0.1
138.1 175.8 208.2 195.7
14.9 21.1 21.3 20.1
79 90 81 73
Mean S.E.M.
175 16
18.8 1.4
4.6 2.5
0.58 0.21
0.08 0.03
179 15.3
19.4 1.5
452 453 503 564
47.7 66.4 31.9 106
6.7 9.8 4.8 16.4
18.6 14.2 48.6 1.4
5.4 4.5 9.6 2.5
2.6 1.1 1.4 0.5
68.9 81.7 81.9 107.7
63.0 16.0
9.4 2.5
20.7 10.0
5.5 1.5
1.4 0.4
85.1 8.1
Mean S.E.M. a
Tissue
Live weight (kg)
Released doses Slaughter
mg
mg/kg
T
OE
T
OE
109 120 105 105
262 224 192 204
25.1 18.9 18.7 19.9
2.79 2.14 2.06 2.30
0.27 0.18 0.20 0.22
81 3.5
110 3.5
221 15.3
20.7 1.5
2.32 0.16
0.22 0.02
12.1 14.3 14.4 18.9
81 84 85 72
113 115 106 100
331 318 318 292
43.9 41.7 41.6 37.1
3.45 3.11 3.40 3.38
0.46 0.41 0.44 0.43
14.9 1.4
81 3.0
109 3.4
315 8.2
41.1 1.4
3.33 0.08
0.43 0.01
Refers to free testosterone or trenbolone in tissue.
storage in fat, which was paralleled by a distinctive reduction of plasma testosterone concentrations. SH seemed to be less effective, because one animal still had androstenone concentrations typical for boars. Plasma testosterone remained unaffected or even tended to increase in SH, indicating a substitution of endogenous testosterone by the testosterone released from the preparation. The difference between preparations might be due to faster release of hormones from SP pellets, compared with SH. An additional explanation for the more pronounced feedback function of trenbolone compared to testosterone is offered by its higher activity on the androgen receptor and its strong binding to the gestagen receptor (Meyer and Rapp, 1985). The reduced androstenone and testosterone concentrations support the assumption by Sheridan et al. (1990) that exogenous application of sex hormones retards the pulsatile secretion of LH, thereby causing atrophy of the Leydig cells and decreased testicular activity even if the effects on testes size were not significant in our study. From a practical point of view, our study demonstrated that application of anabolic preparations basically could reduce or block fat androstenone accumulation, but the efficacy of the treatment depends of the dose and the kind of preparation.
4.2. Residues of anabolic steroids in implantation sites The potential of anabolic preparations (designed for cattle) to reduce boar taint together with the greater food conversion and weight gain of boars in contrast to castrates indicate a clear potential for misuse. Misuse of veterinary drugs has always been observed as long as the economic benefit is big enough. It has to be pointed out that the scenario presented herein disregards application as prescribed for cattle. Injection into the ear followed by discarding of the ear after slaughter might also reduce androstenone accumulation in boar fat effectively, but will leave hormone residues in edible tissues that are similar to cattle. However, as the use in pigs is not licensed, it can be assumed that potential application is performed at locations where the implantation site is not obviously visible. Therefore, injection into the neck muscle instead of the ear might be a realistic scenario in boars. Total implantation sites in our studies contained a mean of 179 mg testosterone propionate or 85 mg trenbolone acetate combined with 17 mg of oestradiol benzoate. In meat, a maximum residue limit (MRL) for trenbolone of 2 mg / kg was adopted by the Commission of Codex Alimentarius (1995). For oestradiol-17b, acceptable
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daily intake (ADI) was suggested to be 0.05 mg per kg body weight, corresponding to 3 mg for a 60-kg adult (JECFA, 1999). Assuming a maximum consumption of 500 g meat per day, the threshold value would be 3 mg / 500 g or 6 mg / kg in meat. For example, after industrial processing of a double injection site (like that used in our experiment) in a batch size of 300 kg of meat, the MRL for trenbolone is exceeded by a factor of 142 and the (hypothetical) threshold for oestradiol-17b by a factor of 9. Thus, our exemplary injection site would be able to contaminate 42.5 tons of meat products above the MRL for trenbolone. As a conclusion, suppression of androstenone accumulation in fat by implantation of anabolic sex hormones might be effective, but it is not consistent with consumer protection, at least when implantation is given into the usable (carcass) portion of the pig.
Acknowledgements This study was financially supported by the European Commission. The boars were slaughtered at the ¨ slaughterhouse of the Bayerische Landesanstalt fur Tierzucht, Grub, Germany.
References ¨ BGBl, 1996. In: Anderungsverordung zur Fleischhygieneverordnung, p. S2120. Commission of Codex Alimentarius, 1995. ALINORM 95 / 21 Part I. Joint FAO / WHO Expert Committee on Food Additives (JECFA), 1999. 52nd Meeting, Rome, 2–11 February 1999. Blottner, S., Hingst, O., Meyer, H.H.D., 1996. Seasonal spermatogenesis and testosterone production in roe deer (Capreolus capreolus). J. Reprod. Fertil. 108, 299–305. Claus, R., 1976. Duration of the clearance of boar taint stores in ¨ fatty tissue after castration of boars. Z. Tierzuchtg. ¨ Zuchtgsbiol. 93, 38–47. Claus, R., 1979. Mammalian pheromones with special reference to the boar taint steroid and its relationship to other testicular steroids. Fortschr. Tierphysiol. Tierernahr. 10, 1–136.
Claus, R., Weiler, U., Herzog, A., 1994. Physiological aspects of androstenone and skatol formation in the boar – a review with experimental data. Meat Sci. 38, 289–305. Claus, R., Herbert, E., Dehnhard, M., 1997. Comparative determination of the boar taint steroid androstenone in pig adipose tissue by a rapid enzyme immunoassay and a HPLCmethod. Arch. Lebensmittelhyg. 2, 27–30. Daxenberger, A., Lange, I.G., Meyer, K., Meyer, H.H.D., 2000. Residues of anabolic preparations in misplaced implantations sites. J. AOAC Int. 83, 809–819. ¨ Dobrowolski, A., Horeth, R., Branscheid, W., 1995. In: Der ¨ ¨ Schlachtkorper von Ebern und Borgen und Probleme der ¨ Klassifizierung. Schriftenreihe des Bundesministeriums fur ¨ Ernahrung, Landwirschaft und Forsten /A, Angewandte Wis¨ senschaft, 449. Landwirschaftsverlag, Munster, pp. 6–13. Echternkamp, S.E., Teague, H.S., Plimpton, Jr. R.F., Grifo, Jr. A.P., 1969. Glandular development, hormonal response and boar odour and flavour intensity of untreated and diethylstilbestrol-implanted boars. J. Anim. Sci. 28, 653–658. Hansson, I., 1974. Effect of sex and weight on growth, feed efficiency and carcass characteristics of pigs. 1. Growth rate and feed efficiency of boars, barrows and gilts. Swedish J. Agric. Res. 4, 209–218. Hansson, I., Lundstrom, K., Malmfors, B., 1975. Effect of sex and weight on growth, feed efficiency and carcass characteristics of pigs. 2. Carcass characteristics of boars, barrows and gilts, slaughtered at four different weights. Swedish J. Agric. Res. 5, 69–80. ´ F., 1996. Le Bizec, B., Montrade, M.B., Monteau, F., Andre, ´ ´ Graisse de Porc: Detection et Quantification de l’Androstenone ´ a` la Specpar Chromatographie en Phase Gazeuse Couplee ´ ´ trometrie de Masse en Mode Impact Electronique. DGAL / SDSPA / N96 / 8134. Agriculture Ministry, Paris. Meyer, H.H.D., Rapp, M., 1985. Reversible binding of the anabolic steroid trenbolone to steroid receptors. Acta Endocrinol. 108 (Suppl. 267), 129. Meyer, H.H.D., Hoffmann, S., 1987. Development of a sensitive microtitration plate enzyme immunoassay for the anabolic steroid trenbolone. Food Addit. Contam. 4, 149–160. Palmer, W.M., Teague, H.S., Grifo, Jr. A.P., 1971. Effect of diethylstilbestrol implantation on testicular development in the boar. J. Anim. Sci. 32, 525–530. Sheridan, P.J., Austin, F.H., Bourke, S., Roche, J.F., 1990. The effect of anabolic agents on growth rate and reproductive organs of pigs. Livest. Prod. Sci. 26, 263–275. Van Weerden, E.J., Grandadam, J.A., 1976. The effect of an anabolic agent on N deposition, growth, and slaughter quality in growing castrated male pigs. In: Lu, C.F., Rendel, J. (Eds.), Anabolics in Animal Production. Georg Thieme, Stuttgart, pp. 115–122.
Suppression of androstenone in entire male pigs by anabolic preparations a,
a
a
a
Andreas Daxenberger *, Melanie Hageleit , Wolf-Dieter Kraetzl , Iris G. Lange , Rolf Claus b , Bruno Le Bizec c , Heinrich H.D. Meyer a a
¨ Physiologie, Forschungszentrum f ur ¨ Milch und Lebensmittel, TU Munchen-Weihenstephan ¨ , Weihenstephaner Berg 3, Institut f ur D-85354 Freising-Weihenstephan, Germany b ¨ Tierhaltung und Tierzuchtung ¨ ¨ Hohenheim, (470), Fachgebiet Tierhaltung und Leistungsphysiologie, Universitat Institut f ur Postfach 70 05 62, D-70539 Stuttgart, Germany c ´ ´ ´´ , Ecole Nationale Veterinaire des Nantes, B.P. 50707, Laboratoire des Dosages Hormonaux, Laboratoire National Reference F-44307 Nantes Cedex 3, France Received 8 November 1999; received in revised form 20 July 2000; accepted 22 November 2000
Abstract At an age of 19 weeks and an average weight of 80.5 kg, four boars each were implanted i.m. with two doses of either Synovex-H (SH; total dose 400 mg testosterone propionate 1 40 mg oestradiol benzoate) or Synovex Plus (SP; total dose 400 mg trenbolone acetate 1 56 mg oestradiol benzoate), respectively. A third group of four boars served as control. The animals were slaughtered 5 weeks after implantation and the testes weights recorded. Hormone (androgen and oestradiol benzoate) residues in implantation sites were quantified by high-performance liquid chromatography with UV detection, and the boar taint steroid androstenone was determined in fat by enzyme-immunoassay (EIA). Gas chromatography–mass spectrometry analysis additionally confirmed the identity. Plasma testosterone and trenbolone concentrations were measured by EIA. Plasma testosterone and fat androstenone concentrations were reduced to basal levels by the SP treatment. With SH the decrease in fat androstenone concentrations was inconsistent between animals. Androgen and estrogen residues in implantation sites were higher in SH boars than in SP boars (179 mg 1 19 mg versus 85 mg 1 15 mg, respectively). Besides the resulting higher resorbed doses, the higher SP efficiency was attributed to greater hypothalamic negative feedback activity of trenbolone compared with testosterone. Including the implantation site, whole batches of processed meat products would be contaminated above the international threshold levels for these hormones in meat. 2001 Elsevier Science B.V. All rights reserved. Keywords: Boar taint; Androstenone; Testes; Residues; Anabolic
1. Introduction *Corresponding author. Tel.: 1 49-8161-714-202; fax: 1 498161-714-204. E-mail address: [email protected] (A. Daxenberger).
Compared with castrates, the portion of lean meat in boars is increased and the feed conversion is more efficient (Hansson, 1974; Hansson et al., 1975;
0301-6226 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0301-6226( 00 )00259-1
140
A. Daxenberger et al. / Livestock Production Science 69 (2001) 139 – 144
Dobrowolski et al., 1995) due to the anabolic action of testicular steroids. After implantation of an anabolic preparation (20 mg oestradiol 1 140 mg trenbolone acetate) into growing castrated male pigs, nitrogen retention increased by 23 to 88%, and was paralleled by a 56% increasing protein and a 15% decreasing fat deposition (Van Weerden and Grandadam, 1976). Higher nitrogen retention of boars is also desirable with respect to environmental aspects. Meanwhile, rearing of boars is favoured mainly by the animal welfare aspect because castration is avoided. The main argument against rearing of boars is an unpleasant odour which occurs regularly in carcasses of postpuberal male pigs. On the one hand, the odour is caused by the steroid 5a-androst-16-en-3-one (androstenone), the main representative of D-16 steroids in the boar. Androstenone has no hormonal activity but works as a sex pheromone (Claus, 1979). The D-16 steroids are produced parallel to testicular hormones in the Leydig cells from common precursors, pregnenolone and progesterone, and their synthesis is stimulated by luteinizing hormone (LH) which is controlled by gonadotropin-releasing hormone from the hypothalamus. This system is balanced by the feedback function of testicular hormones on the hypothalamus (Claus et al., 1994). Due to its lipophilic properties, androstenone is accumulated in the adipose tissue and eliminated within 3–6 weeks after castration, depending on the age at castration (Claus, 1976). On the other hand, skatole, which is formed in the intestinal tract by microbial degradation of the amino acid tryptophan, has been identified as another substance responsible for the off-flavour of boar meat. However, the production of skatole is dependent on the presence of tryptophan under a simultaneous deficit of energy in the gut, and therefore can be influenced by feeding (Claus et al., 1994). The intention of our study was to determine the effect of the anabolic implant preparations SynovexH (testosterone propionate 1 oestradiol benzoate) and Synovex Plus (trenbolone acetate 1 oestradiol benzoate) on fat androstenone concentration of intact boars. The use of certain anabolic sex hormones is licensed in many countries (except the European Union) when implanted into the ears of cattle. Thus, the availability of such substances may lead to
misuse in other species. Furthermore, we examined the hormone residues at implantation sites with respect to consumer safety.
2. Materials and methods
2.1. Animal experiments Twelve boars (Landrace 3 Pietrain crossbreds) were weaned at an age of 5 weeks and fed a commercial diet (12.8 MJ / kg, 17.7% crude protein) ad libitum until an age of 12 weeks and a mean weight of 35 kg. Thereafter, they were fed ad libitum with a finishing feed (12.9 MJ / kg, 18.0% crude protein) until the end of the experiment. During the whole period an artificial light programme with 10 h light per day was applied. At an age of 19 weeks and an average weight of 80.5 kg, the animals were allocated to three groups of four animals. One group served as control (C; no treatment). The treatment groups were either implanted with two doses of Synovex-H (SH; total dose 400 mg testosterone propionate 1 40 mg oestradiol benzoate) or two doses of Synovex Plus (SP; total dose 400 mg trenbolone acetate 1 56 mg oestradiol benzoate; both preparations from Fort Dodge Laboratories, IA, USA). In order to simulate off-label use in pigs, the preparations were injected into the necks of the animals, because hormone depots would have been visible after application into the pinna of the ear. The implantation was performed with the original implanter pistol by injecting two doses of each preparation 4–5 cm deep into the neck muscles of each animal. Five weeks after implantation the animals were slaughtered, some tissues sampled and the carcasses of the treated animals removed from the food chain.
2.2. Sample collection Blood samples for plasma were taken directly before treatment (day 0) and 7, 21 and 31 days after implantation by venipuncture of the V. jugularis. Immediately after slaughter, the complete implantation sites, the testes and perirenal fat were collected. The testes were cut from the scrotum, weighed and measured with a slide gauge. The implantation sites
A. Daxenberger et al. / Livestock Production Science 69 (2001) 139 – 144
141
were prepared further in the laboratory. The whole tissue area containing the implants was cut into layers of 5 mm. All visible residual implants were taken out from the embedding tissue. Furthermore, the tissue that contained the implants was dissected (up to a distance of 3–5 cm from the recovered implants).
2.3. Steroid determinations The content of testosterone and trenbolone-17b in plasma was analysed by enzyme-immunoassay (EIA) using standardized kits (R-Biopharm, Darmstadt, Germany) as published previously (Meyer and Hoffmann, 1987; Blottner et al., 1996). Hormone residues in recovered implants and the tissues of the injection sites were quantified by high-performance liquid chromatography (HPLC) with diode array detection (DAD) (Daxenberger et al., 2000). Boar taint steroids in adipose tissue were quantified by EIA (Claus et al., 1997). Androstenone identity was additionally confirmed by gas chromatography–mass spectrometry (Le Bizec et al., 1996).
3. Results The mean feed consumption was 2.2 kg per day between the beginning of the finishing period and the time of the hormone treatments. During the 5-week treatment period, the average food consumption was 2.8 kg / day. Average daily weight gain was 804 g from week 13 to 19 and 824 g during the treatment period (weeks 20–24). There was no significant difference between groups. At slaughter, the mean weight of the boars was 109 kg, corresponding to a mean carcass weight of 91.1 kg.
3.1. Testosterone and trenbolone in plasma, androstenone in fat and testis size A clear decrease of plasma testosterone concentrations (Fig. 1 and Table 1) was observed in SP boars. The concentrations dropped from more than 1 ng / ml before treatment down to , 0.15 ng / ml on day 7. In SH boars, there was no reduction in testosterone concentrations. After SP treatment only traces of androstenone were detected in adipose
Fig. 1. Effect of anabolic preparations on testosterone and trenbolone concentrations in plasma of individual boars (day 0 5 treatment).
tissue (Table 1). In SH boars, the situation was less clear: Animal no. 500 revealed a considerable concentration (1291 ng / g) whereas, in two other boars, low androstenone concentrations were found. In animal no. 502 androstenone concentrations were above base levels observed in sows ( , 50 ng / g), but with 190 ng / g did not exceed the level of acceptance in Germany (500 ng / g; BGBl, 1996). The testes in the SH group and the SP group were 15% and 21% lighter, respectively, than in the C group (Table 1). The differences were not significant (t-test; P 5 0.275 and 0.065, respectively). Across all 12 individuals, there was a slight (r 5 0.679) but significant (P 5 0.015) linear regression
A. Daxenberger et al. / Livestock Production Science 69 (2001) 139 – 144
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Table 1 Effect of Synovex-H and Synovex Plus on testosterone in plasma and androstenone in adipose tissue, and on testis weight of boars a Group
Animal No.
Testosterone (ng / ml)
Androstenone (ng / g)
Control
450 504 562 565
1.93 2.35 0.68 0.79
1312 319 312 807
Mean S.E.M.
1.4 0.42
688 238
451 500 502 563
0.60 4.82 1.60 1.90
Mean S.E.M.
Synovex-H
Synovex Plus
a
Testis (g) Left
Right
Total
233 211 229 234
263 208 244 259
496 419 473 493
227 5.4
244 13
470 18
38 1291 190 84
121 317 213 158
89 327 207 166
210 644 420 324
2.2 0.91
401 298
202 43
197 50
400 92
452 453 503 564
0.13 0.10 0.87 0.08
39 16 72 73
247 116 243 146
230 114 267 123
477 230 510 269
Mean S.E.M.
0.30 0.19
50 14
188 33
184 38
372 71
Plasma testosterone levels are given as the mean during the implantation period (days 7, 21 and 31).
between the testes weights (sum of both testis) and fat androstenone concentrations. All boars with testis weights below 200 g had low levels of androstenone.
3.2. Hormone residues in implantation sites During the 5-week implantation period the implants were not completely degraded. The recovered implant remnants and the surrounding tissue contained considerable amounts of hormones after slaughter (Table 2). Additionally, the surrounding tissues contained traces of hydrolysed androgens (i.e., trenbolone-17b or testosterone), but hydrolysis of oestradiol benzoate was not observed. A mean total of 179 mg testosterone propionate (range 138– 208 mg), 85 mg trenbolone acetate (range 69–108 mg) and 17 mg oestradiol benzoate (range 12–21 mg) remained in the respective injection sites. Consequently, the total applied doses did not enter the circulation completely. The actual effective dose was the difference of the applied dose minus the residual hormones. Thus, the relative effective doses per kg
live weight, based on the average live weights at implantation and slaughter, were 2.32 mg testosterone propionate 1 0.22 mg oestradiol benzoate (SH) and 3.33 mg trenbolone acetate 1 0.43 mg oestradiol benzoate (SP).
4. Discussion
4.1. Effect of anabolic steroids on fat androstenone and testicular function The possibility of a ‘‘hormonal castration’’ and thus the control of boar taint by exogenous application of gonadal hormones were studied previously and included the use of several substances. For example, diethylstilbestrol proved to be effective to depress testicular development but the loss of growth potential was not compensated by the application for this oestrogen alone (Echternkamp et al., 1969; Palmer et al., 1971). Application of SP 5 weeks prior to slaughter resulted in a reduced androstenone
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Table 2 Hormone residues in implants and at implantation sites at the time of slaughter, and the dose of hormone actually released (T, testosterone propionate or trenbolone acetate; OE, oestradiol benzoate; SH, Synovex-H ; SP, Synovex Plus ) Group
Animal No.
Hormone residues (mg) in implants and at implantation sites Pellets
SH
SP
Hydrolysed a
Total
Implantation
T
OE
T
OE
T
T
OE
451 500 502 563
136 164 206 194
14.6 19.9 20.9 19.7
2.1 12.1 2.2 2.0
0.3 1.2 0.4 0.4
0.0 0.1 0.1 0.1
138.1 175.8 208.2 195.7
14.9 21.1 21.3 20.1
79 90 81 73
Mean S.E.M.
175 16
18.8 1.4
4.6 2.5
0.58 0.21
0.08 0.03
179 15.3
19.4 1.5
452 453 503 564
47.7 66.4 31.9 106
6.7 9.8 4.8 16.4
18.6 14.2 48.6 1.4
5.4 4.5 9.6 2.5
2.6 1.1 1.4 0.5
68.9 81.7 81.9 107.7
63.0 16.0
9.4 2.5
20.7 10.0
5.5 1.5
1.4 0.4
85.1 8.1
Mean S.E.M. a
Tissue
Live weight (kg)
Released doses Slaughter
mg
mg/kg
T
OE
T
OE
109 120 105 105
262 224 192 204
25.1 18.9 18.7 19.9
2.79 2.14 2.06 2.30
0.27 0.18 0.20 0.22
81 3.5
110 3.5
221 15.3
20.7 1.5
2.32 0.16
0.22 0.02
12.1 14.3 14.4 18.9
81 84 85 72
113 115 106 100
331 318 318 292
43.9 41.7 41.6 37.1
3.45 3.11 3.40 3.38
0.46 0.41 0.44 0.43
14.9 1.4
81 3.0
109 3.4
315 8.2
41.1 1.4
3.33 0.08
0.43 0.01
Refers to free testosterone or trenbolone in tissue.
storage in fat, which was paralleled by a distinctive reduction of plasma testosterone concentrations. SH seemed to be less effective, because one animal still had androstenone concentrations typical for boars. Plasma testosterone remained unaffected or even tended to increase in SH, indicating a substitution of endogenous testosterone by the testosterone released from the preparation. The difference between preparations might be due to faster release of hormones from SP pellets, compared with SH. An additional explanation for the more pronounced feedback function of trenbolone compared to testosterone is offered by its higher activity on the androgen receptor and its strong binding to the gestagen receptor (Meyer and Rapp, 1985). The reduced androstenone and testosterone concentrations support the assumption by Sheridan et al. (1990) that exogenous application of sex hormones retards the pulsatile secretion of LH, thereby causing atrophy of the Leydig cells and decreased testicular activity even if the effects on testes size were not significant in our study. From a practical point of view, our study demonstrated that application of anabolic preparations basically could reduce or block fat androstenone accumulation, but the efficacy of the treatment depends of the dose and the kind of preparation.
4.2. Residues of anabolic steroids in implantation sites The potential of anabolic preparations (designed for cattle) to reduce boar taint together with the greater food conversion and weight gain of boars in contrast to castrates indicate a clear potential for misuse. Misuse of veterinary drugs has always been observed as long as the economic benefit is big enough. It has to be pointed out that the scenario presented herein disregards application as prescribed for cattle. Injection into the ear followed by discarding of the ear after slaughter might also reduce androstenone accumulation in boar fat effectively, but will leave hormone residues in edible tissues that are similar to cattle. However, as the use in pigs is not licensed, it can be assumed that potential application is performed at locations where the implantation site is not obviously visible. Therefore, injection into the neck muscle instead of the ear might be a realistic scenario in boars. Total implantation sites in our studies contained a mean of 179 mg testosterone propionate or 85 mg trenbolone acetate combined with 17 mg of oestradiol benzoate. In meat, a maximum residue limit (MRL) for trenbolone of 2 mg / kg was adopted by the Commission of Codex Alimentarius (1995). For oestradiol-17b, acceptable
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daily intake (ADI) was suggested to be 0.05 mg per kg body weight, corresponding to 3 mg for a 60-kg adult (JECFA, 1999). Assuming a maximum consumption of 500 g meat per day, the threshold value would be 3 mg / 500 g or 6 mg / kg in meat. For example, after industrial processing of a double injection site (like that used in our experiment) in a batch size of 300 kg of meat, the MRL for trenbolone is exceeded by a factor of 142 and the (hypothetical) threshold for oestradiol-17b by a factor of 9. Thus, our exemplary injection site would be able to contaminate 42.5 tons of meat products above the MRL for trenbolone. As a conclusion, suppression of androstenone accumulation in fat by implantation of anabolic sex hormones might be effective, but it is not consistent with consumer protection, at least when implantation is given into the usable (carcass) portion of the pig.
Acknowledgements This study was financially supported by the European Commission. The boars were slaughtered at the ¨ slaughterhouse of the Bayerische Landesanstalt fur Tierzucht, Grub, Germany.
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