Pathology and residues in veal calves treated experimentally with clenbuterol

Researchin VeterinaryScience1994,57, 365-371

Pathology and residues in veal calves treated experimentally with clenbuterol B. BIOLATTI, Universith degli Studi di Padova, Facoltgt di Medicina Veterinaria, Istituto di Patologia e Igiene Veterinaria, via Gradenigo 6, 35131 Padova, Italy, E. BOLLO, G. RE, Universitg~ degli Studi di Torino, Facoltit di Medicina Veterinaria, Dipartimento di Patologia Animale, via Nizza 52, 10126 Torino, Italy, S. APPINO, Universitit degli Studi di Padova, Faeoltgt di Medicina Veterinaria, Istituto di Patologia e Igiene Veterinaria, via Gradenigo 6, 35131 Padova, Italy, E. TARTARI, G. BENATTI, Universitit degli Studi di Torino, Facoltit di Agraria, Dipartimento di Scienze Zootecniche, via Geneva 6, 10126 Torino, Italy, C. T. ELLIOTT, W. J. McCAUGHEY, Veterinary Sciences Division, Stormont, Belfast, Northern Ireland

Six veal calves were medicated with clenbuterol at 20 gg kg bodyweight -1 day -1 for 42 days before they were slaughtered, to evaluate the lesions and residues in target organs. Compared with six unmedicated calves the most noticeable changes were tracheal dilatation, decreased uterine weight, slight mucous hypersecretion in the uterus and vagina and depletion of liver glycogen. The highest concentrations of clenbuterol (62 to 128 ng/g -1) were recorded in the choroid/retina, and the aqueous humonr had the lowest concentration (0.5 to 2.4 ng ml-1). The residue concentrations were higher than the maximum residue level set for clenhuterol (0.5 ng g-l) THE [3-agonist clenbuterol is not licensed for use as a growth promoting agent. However, its persistent and widespread illegal use as a repartitioning agent in farm animals (banned by the EC Directives 466/86 and 88/146) has demanded better and cheaper methods for detecting its residues and the lesions which might lead to the identification of medicated animals. There have been several studies of the detection of residues (Meyer and Rinke 1991, Degand et al 1992, Elliott et al 1993a, b, c, Sauer and Limer 1993), but less is known about the morphological changes induced by the chronic administration of clenbuterol (Cantoni et al 1988, Borghetti et al 1989, Corradi et al 1989, Cantoni 1990, Re et al 1991, 1993, Biolatti et al 1992, 1994a, b). The present study combined analytical and morphological techniques to improve

the ability to detect the abuse of clenbuterol by providing veterinary inspectors with more information on the recognition of illegally treated calves at the slaughterhouse.

Materials and methods Twelve female Friesian veal calves were fed from the age of 20 to 141 days exclusively with milk replacer (Table 1). Six of the animals were kept as a control group, while the ration of the other six was medicated with 20 gg kg bodyweight-1 day-1 from day 99 until they were TABLE 1: Total feed consumed (as fed basis) of control and treated calves Number of the animals and mean value (x)

Whole period (kg) 141 days

First period (kg) 99 days

Period of treatment (kg) 42 days

288.9 288.9 288-9 291.4 291.4 291-2 290.1 (1.33)

174-7 174.7 174-7 174.4 174-4 174.7 174.6 (0.15)

114-2 114.2 114-2 117.0 117.0 116.5 115.5 (1.45)

288.6 282-2 289.5 290.8 289.5 292.5 288.8 (3.53)

175.4 169-1 175.4 174.1 172.8 174.7 173.5 (2.40)

113.2 113.1 114.1 116.7 116.7 117.8 115.2 (2.04)

Controls 1

2 3 4 5 6 (x) Treated

365

7 8 9 10 11 12 (x)

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B. BioIattg E. Bollo, G. Re, S. Appino, E. Tartari, G. Benatti, C. T. Elliott, W. J. McCaughey

TABLE 2: Initial and final bodyweights (BW), the average daily gains (ADG), food conversion ratios (FOR) and dressing percentages (DP) of six control calves and six calves treated with clenbuterol

Calves Controls 1 2 3 4 5 6 Mean Treated 7 8 9 10 11 12 Mean

Initial BW (kg)

Pretreatment BW (kg)

Final BW (kg)

ADG whole period (g) (141 days)

ADG ADG 1st period of period of treatment (99 days) (g) (42 days)

42.5 39.5 39.0 40.0 41.0 41.5 40.6 (1.3)

158 147 147 145 139 137 145.5 (7.4)

213 208 198 197 192 186 199 (10.0)

1201 1195 1128 1106 1063 1018 1119 (72.2)

1167 1086 1091 1061 990 965 1060 (73)

44.0 34.0 37.0 38.0 39.0 40-0 38.66 (3.33)

143 123 146 148 153 155 144.7 (11.5)

212 186 218 218 222 224 213.3 (14.0)

1191 1078 1284 1268 1289 1296 1234 (85.7)

1000 899 1101 1111 1152 1162 1071 (102)

FCR whole period

FCR 1st period

FCR period of treatment

DP

1310 1452 1214 1209 1236 1140 1260 (109)

1.69 1.71 1.82 1.86 1.93 2.01 1.84 (0.12)

1.51 1.63 1.62 1.71 1.78 1.83 1.68 (0.12)

2.08 1.87 2.24 2.25 2.21 2.39 2.17 (0.18)

58.2 61.1 61-1 57.9 59-4 58-1 53-9 (1.5)

1643 1500 1714 1628 1605 1605 1616 (69)**

1.73 1-86 1.61 1.63 1-58 1-59 1-67 (0-11)*

1-79 1.90 1.63 1-60 1.52 1-52 1.66 (0-15)

1.64 1.79 1.59 1.68 1.69 1.70 1-68 (0.07)**

64.5 70.0 63.8 63.3 66.2 67.4 65.9 (2-6)**

* P<0.05,** P<0.01

slaughtered on day 141. The animals were killed by exsanguination after they had been stunned with a captive bolt. The initial, pre-treatment and final bodyweights of the animals, the weight of the reproductive organs, the daily liveweight gain (ADG), the food conversion ratio (FCR) and the dressing percentage were recorded. For the morphological investigations the following samples were collected: genitalia, respiratory tract, liver, heart, thyroid, mammary gland, kidney, adrenal glands, central nervous system (CNS) and thymus. The samples were fixed in 10 per cent buffered formalin, paraffin-embedded and cut in 4 gm thick sections, stained with haematoxylin and eosin or periodic acid-Schiff (PAS), and PAS diastase for glycogen, and Sudan black for lipids. The concentration of clenbuterol in the tissues was measured by the enzyme-immunoassay (EIA) method of Elliott et al (1993a). The samples of retina, liver, kidney, brain, muscle and uterus were initially digested with a non-specific proteolytic enzyme (protease type XXiii, Sigma Chemical). The digest supernatants were then adjusted to pH 10 by the addition of 5N sodium hydroxide and extracted with diethyl ether. The ether extracts were evaporated to dryness and the dry residue resuspended in a 10 mM sodium acetate assay buffer pH 7.0 containing 2 mg m1-1 bovine serum albumin (BSA). Fluid samples (urine and aqueous humour) were extracted in a similar way except that the pro-

teolytic digest stage of the protocol was omitted. The extracted samples were transferred into the wells of a microtitre plate coated with an immobilised clenbuterol polyclonal antibody. To each of the wells was added 50 ~tl of clenbuterol-horseradish peroxidase conjugate diluted 1/10,000 in assay buffer. After an overnight incubation at 4°C the plate was washed with a salt Tween buffer, and a chromagen (3,3',5,5'-tetramethyl-benzidine and hydrogen peroxidase) was added to each well. After a 10-minute incubation at 37°C the reaction was stopped by the addition of 25 gl of 2.5 M hydrogen sulphate. The concentrations of clenbuterol in the samples were determined by reference to standards incorporated into the assay. The assay was validated by the analysis of fluids and tissues taken from 20 calves known not to have been treated with clenbuterol. A range of these samples was spiked with clenbuterol and the coefficients of variation (cv) of the assays and the recoveries were assessed. Positive samples of liver, retina and urine were assayed to determine between assay variations. The data were analysed satistically by analysis of variance and Student's t test. Results

Weight

The average final bodyweight of the treated animals was 14.3 kg higher than that of the controls;

367

Pathology and residues of clenbuterol in calves

During the whole period of the experiment the ADG was 10 per cent higher and the FCR 10 per cent lower in the treated animals than in the controls (P<0.05) (Table 2). Morphology Respiratory tract. All the treated animals showed a marked dilatation of the trachea and the tracheal crest had disappeared; as a result the dorsal side of the trachea was completely flat (Figs 1 and 2). FIG 1 : Control calves; normal appearance of tracheas

FIG 2: Treated calves; the tracheas are dilated and the tracheal crest has disappeared

the increase was not statistically significant. The animals treated with clenbuterol had a higher ADG during the period of treatment (1616 g vs 1260 g, P<0.01) and a better FCR (1'68 vs 2-17, P<0.01).

Genital tract. No differences were detected either in morphology or in the absolute and relative weights of the ovaries between the control and the treated calves (Table 3). Macroscopically there was a small volume of clear mucus in the uterine lumen of the treated calves. No other differences were detected (Figs 3 and 4). There was a significant decrease in the absolute (P<0.05) and relative (P<0-01) weights of the uterus, including the cervix, of the treated animals (Table 3). There were no morphological differences between the cervices of the two groups. The vaginal mucosa of the treated calves was covered by small to large amounts of clear mucus, whereas the vaginal mucosa of the controls was usually dry (Figs 3 and 4). Liver. Macroscopically the livers of all the treated calves were darker than those of the controls (Fig 5). Histologically, no differences were detected in the sections stained with haematoxylin and eosin and Sudan black, but there was a marked differ-

TABLE 3: Absolute and relative weights of the uteri and ovaries of six control calves and six calves treated with clenbuterol

Calves

Weight of the uterus (g)

Controls 1 2 3 4 5 6 Mean

139-8 106.6 95-0 88.7 53.3 87-0 95-1 (28-2)

Treated 7 8 9 10 11 12 Mean

53.0 52.5 62-2 94-0 76-0 52.8 65.1" (16.85)

* P<0.05, ** P<0.01

Weight of the ovaries (g)

Relative weight of the uterus (%)

left

right

Mean weight of the ovaries (g)

Relative weight o1 the ovaries (%)

0.07 0.05 0.05 0-04 0.03 0.05 0.05 (0-01)

3.58 4.76 6.69 3-75 2.76 2.68 4.04 (1.50)

0.89 4.12 9.90 2.61 3.02 1.98 3,75 (3.20)

2.24 4.44 8.30 3.23 2.89 2-33 3-91 (2.30)

0.0010 0.0021 0.0042 0.0016 0.0015 0.0012 0.0019 (0.001)

0.02 0-03 0.03 0.04 0.03 0.02 0.03** (0.01)

2-50 0-95 4-00 3-11 4-70 3.60 3.14 (1.31)

2.23 1.93 3-80 4-26 4.85 5.00 3.68 (1.31)

2.36 1.44 3.90 3-69 4.78 4-30 3.41 (1.26)

0.0022 0.0008 0.0018 0.0017 0.0021 0-0019 0.0018 (0.0005)

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B. Biolatti, E. Bollo, G. Re, S. Appino, E. Tartari, G. Benatti, C. T. Elliott, W. J. McCaughey

FIG 3: Control calf; the mucosa of the genital tract appears dry

FIG 4: Treated calf; abundant mucus in the vagina and small amounts in the uterus

ence between the sections stained with PAS and PAS diastase. The livers of the control calves had large amounts of glycogen stored throughout the lobule (Figs 6 to 9) whereas in the livers of the treated calves the glycogen was concentrated mainly around the central vein.

0.31 ng m1-1 for the tissues, the retina and the fluids, respectively. The recoveries of clenbuterol from all the types of sample ranged between 81 and 85 per cent. There was no evidence of residues of clenbuteroi in any of the tissue or fluid samples taken from the six control animals, but residues were widely distributed in the medicated animals and all the samples analysed were positive for the drug. However, there was considerable variation in the concentrations. The highest concentrations (62 to 128 ng g-l) were recorded in the choroid/ retina samples. Intermediate concentrations were found in the urine, kidney and liver specimens, with the brain and uterine tissues having lower concentrations (Table 4). The aqueous humour had the lowest concentration (0.5 to 2-4 ng ml-1).

Other tissues. There were no significant morphological differences, either macroscopically or microscopically, in the heart, thymus, muscle, kidney, adrenal glands, CNS, lung and thyroid gland between the treated and control calves. Analytical observations

The limits of determination was calculated as the mean value recorded from the 20 negative conrols plus three standard deviations, and the limit of decision as the mean value plus six standard deviations. These calculations gave values of 0.13 and 0.20 ng g-l, 1.44 and 2.28 ng g-1 and 0-17 and

Discussion The administration of clenbuterol increased the ADG and FCR of the calves during the period of

Pathology and residues of clenbuterol in calves

369

FIG 7: Liver of control calf; vacuolisation of the cytoplasm of the hepatocytes due to the loss of glycogen after histochemical treatment Periodic acid-Schiff-diastase (Bar = 25 ILtm)

FIG 5: Control (above) and treated (below) calves; the liver appears darker in the treated than in the control calves

FIG 8: Liver of treated calf; the hepatocytes are Periodic acidSchiff-negative owing to the depletion of glycogen (Bar = 25 gin)

FIG 6: Liver of control calf; the hepatocytes appear Periodic acidSchiff-positive owing to the storage of glycogen (Bar = 25 ,am)

FIG 9: Liver of treated calf; vacuoles are almost absent from the cytoplasm of the hepatocytes, owing to the lack of glycogen. Periodic acid-Schiff-diastase (Bar = 25 gm)

treatment (P<0.01), which resulted in an increase in their final bodyweight and a significant increase in the dressing percentage (P<0.01) of the treated animals. The post mortem observations suggested that there were major effects on the trachea which resulted in a marked dilatation of its lumen owing

to the relaxation of the tracheal smooth musculature and the disappearance of the tracheal crest; similar changes were observed in an experiment in which different doses of clenbuterol were administered to veal calves (Biolatti et al 1994a).

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B. Biolatti, E. Bollo, G. Re, S. Appino, E. Tartari, G. Benatti, C. T. Elliott, W. J. McCaughey

TABLE 4: Concentrations of clenbuterol (ng g-l) in the tissues of six control calves and six calves treated with clenbuterol Calves

Retina/choroid

Liver

Urine

Kidney

Brain

Muscle

Uterus

Aqueous humour

Control 1 2 3 4 5 6

<0.2 <0.2 <0.2 <0.2 <0.2 <0.2

<0.2 <0.2 <0.2 <0.2 <0.2 <0.2

<0.2 <0.2 <0.2 <0-2 <0.2 <0-2

<0.2 <0.2 <0.2 <0.2 <0.2 <0.2

<0.2 <0-2 <0.2 <0-2 <0-2 <0-2

<0.2 <0.2 <0.2 NA NA NA

<0-2 <0.2 NA <0.2 <0-2 <0.2

<0.2 <0.2 <0.2 <0.2 <0.2 <0.2

Treated 7 8 9 10 11 12

73 NA 87 62 98 128

17.0 29.2 20.9 20.9 21.8 34.3

47.3 55-5 15-1 19-8 35-1 32-2

12.5 11.1 15.4 9.8 19.9 25.0

7.1 13-9 9-9 11-1 4-2 18-8

5.6 12.3 10.3 NA NA NA

3.6 6.9 NA 4.8 7.1 8.8

1-4 1.5 0.7 0-5 2-4 1-5

NA Not assayed

This finding suggests that a simple examination of the cross-sectional area and outline of this organ during veterinary inspections may provide evidence of treatment with clenbuterol. Changes in the genital tract were also observed, similar to those described in previous experiments in rats and pigs (Re et al 1993, Biolatti et al 1994b). However, the ovaries did not appear to be affected, probably owing to their prepubertal condition, that is the absence of ovulatory follicles and corpora lutea. This finding was in contrast with the findings in the ovaries of pubertal pigs, the function of which appears to be impaired by clenbuterol (Biolatti et al 1994b). Medication with clenbuterol caused significant decreases in the weight of the uterus, and some secretion of mucus into the uterus and vagina. However, the changes were minor and unlikely to assist in the identification of medicated veal calves at slaughter. The darker appearance of the liver could be the result of the depletion of glycogen and may aid in the recognition of treated animals; this evidence can be supported by the histological examination of PAS-stained sections. These observations are in good agreement with the enhanced glycogenolysis induced by clenbuterol (Moloney and Allen 1992). The distribution of clenbuterol in the tissues was similar to that previously reported. Elliott et al (1993b, c) found that the tissue with the highest concentration of clenbuterol was the choroid/retina. Saner and Limer (1993) also found that this layer was the site with the highest concentration of clenbuterol within the eye; as in the present study, they also recorded low concentrations of clenbuterol in the aqueous humour of the eye. The

liver and kidney have been shown to be useful tissues for detecting the administration of clenbuterol to cattle which have been subject to either no withdrawal period or a short withdrawal period (Meyer and Rinke 1991, Elliott et al 1993a, b, Saner and Limer 1993). The concentrations found in these calves were lower than those reported to have caused outbreaks of food poisoning (Martinez-Navarro 1990, Pulse et al 1991), but were above the maximum residue level set for the compound (0.5 gg kg -1) and they should not be assumed to be without a health risk to the consumer. Several morphological changes have been shown to occur in animals treated with clenbuterol, and these may prove useful to veterinary inspectors seeking to detect the abuse of the compound. Samples such as the eyes and liver can be removed from suspect carcases, for laboratory analysis, to confirm the presence of the compound. In this study the aminals were not subject to a withdrawal period, and further investigations would be required to determine for how long after the removal of the medication the gross morphological changes would persist.

Acknowledgements The research was supported by the National Research Council of Italy, Special Project ~AISA, Sub-Project Number 3, Paper Number 1658. The authors thank Mr Mario Monaco and Mrs Angela Bellonio for their excellent technical assistance. Part of this work has been communicated at the 1 lth Autumn Meeting of the European Society of

Pathology and residues of clenbuterol in calves

Veterinary Pathology and published in the proceedings (Medicina Veterinaria 9, [Supplement] 100-101). References BIOLATTI, B., BOLLO, E., APPINO, S., AMEDEO, S., GUARDA, F. & TARTARI, E. (1992) Somministrazione eroniea di clenbuterolo in vitelli a canle bianca. Aspetti anatomo-patologiei. Atti della Societ~ ltaliana di Buiatria 24, 319-325 BIOLATTI, B., BOLLO, E., APPINO, S. & DONN, A. (1994a) Patologia por anabolizantes en temeras de came bianca. Medicina Veterinaria 4, 241-248 BIOLATTI, B., CASTAGNARO, M., BOLLO, E., APPINO, S. & RE, G. (1994b) Genital lesions following long-term administration of clenbuterol in female pigs. Veterinary Pathology 31, 82-92 BORGHETTI, P., UBALDI, A., FUSARI, A., NOSARI, U. & CABASSI, E. (1989) Effetti del trattamento con clenbuterolo sul tessuto adiposo e sul metabolismo energetico dei vitelli. Atti della Societd ltaliana de lle Seienze Veterinarie 43, 1019-1023 CANTONI, A. M. (1990) Sull'impiego dei promotori di ereseita negli animali in produzione zootecnica: rilievi anatomoistopatologiei. Annali della Facoltd di Medicina Veterinaria 10, 21-33 CANTONI, A. M., CORRADI, A. & GABBI, C. (1988) Variazioni istochimiche ed istoenzimologiche sul m. longissimus dorsi di suini trattati con ~-adrenergici. Atti della Societd ltaliana delle Scienze Veterinarie 43, 521-523 CORRADI, A., CANTON[, A. M., BORGHETTI, P., GABBI, C., FUSARI, A. & BARCHI, D. (1989) L'impiego di un e.d. ripartitore di energia (GAH 034) nel suino da came: indagini istologiehe ed istochimiche sul tessuto muscolare ed adiposo. Selezione Veterinaria 30, 1657-1665 DEGAND, G., BERNES-DUYCKAERTS, A. & MAGHUIN-ROGISTER, G. (1992) Determination of clenbuterol in bovine tissues and urine by enzyme immunoassay. Journal of Agriculture and Food Chemistry 40, 70-76

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