FULL PAPER Pathology

Pathology of the Testicle and Sex Accessory Glands Following the Administration of and Boldione as Growth Promoters in Veal Calves

Francesca T. CANNIZZO1), Gabriele ZANCANARO2), Francesca SPADA1), Chiara MULASSO1) and Bartolomeo BIOLATTI1)*

1)Università di Torino, Dipartimento di Patologia Animale, via L. da Vinci 44, 10095, Grugliasco and 2)O.R.A.P. - Regione Piemonte, Assessorato Tutela della Salute e Sanità - Direzione Sanità Pubblica- Settore Sanità Animale ed Igiene degli Allevamenti - Torino, Italy

(Received 21 February 2007/Accepted 5 July 2007)

ABSTRACT. Boldenone and its precursor Boldione are illegally used for anabolic purposes in humans, horses and cattle. To develop more effective policies and programs to maximize food security, Italian Public Health Services investigate all indicators capable of assisting the recognition of treated animals, and prioritize research and the formulation of action strategies for the promotion of healthy eating. Thus, an experimental administration of boldenone and boldione at anabolic dosages in veal calves was carried out to evaluate the changes in target organs by qualitative and semi-quantitative morphological analysis. The lesions resembled the effects already observed after the administration of hormones to cattle. Main findings were represented by prostate hypersecretion, increased rate of apoptotic cells and decreased rate of Ki67 positive cells in the germ cell line of treated animals, particularly in boldione group and finally some new features like hypertrophy of the prostate urothelial cells. KEY WORDS: boldenone, boldione, prostate, testis, veal calves. J. Vet. Med. Sci. 69(11): 1109–1116, 2007

The use of boldenone (1,4-androstadiene-17beta-ol-3- development of human prostate carcinomas one; BOL) and its precursor boldione (1,4-androstadiene-3- xenotransplanted into nude mice [3]. To prevent the abuse 17dione; ADD) as anabolic in livestock, of anabolics in animal production, biological samples, particularly in veal calves production, has been recognized including urine, blood and hair, are periodically taken from in several EU States and the legislation against its use selected farms. Existing literature relates the presence in the (Council Directive 96/22/EC; Council Directive 96/23/EC) urine samples of BOL and ADD derivatives, like 17α-BOL have been used for prosecutions of those using it illegally. and 17β-BOL conjugates, to proof of illegal treatment [7], BOL is an anabolic very similar to , with while the presence of 17α-BOL free in urine and faeces of dehydrogenation at the C-1,2 position. This drug has been animal species can come from resources other than illegal developed for veterinary use: with a low androgenic potency treatment [18], like phytosterols in food [19]. Since no clear and a very long half-life, trace amounts can easily be correlation has yet been found between different detected for months after discontinued use (http:// observations, studies are being carried out in order to better en.wikipedia.org/wiki/Boldenone). BOL causes increases understand the significance of these metabolites in urine and of muscle size due to promotion of positive nitrogen balance faeces of cattle [7]. On the other hand, few morphological by stimulating protein production and reducing protein studies are available on the target organs of treated animals. destruction, moreover it produces a retention of body water, Groot and Biolatti [14] investigated the prostate, bulbo- nitrogen, sodium, potassium and [11, 16]. urethral gland and testis of veal calves from the field found BOL improves growth and feed conversion in veal calves positive for 17β-BOL residues in urine. Findings showed and therefore might be used illegally to achieve more hypersecretion, cyst formation in prostate and bulbo- efficient meat production [1, 2, 7, 25]. ADD is used by urethral gland, and hyperplasia of urethral epithelium. In bodybuilders as a product with an even greater anabolic the testis, reduced development and degeneration of the potency than BOL itself [7, 9]. Conversely, like the other germinal epithelium was also found. androgenic steroids, 17β-BOL is classified by the It is recognized that analytical methods are often International Agency for Research on Cancer (IARC) in ineffective to determine residues of drugs in biological class 2A (growth promoters - steroids), as a probable human matrices, and the illegal use of a large variety of growth carcinogen (e.g. prostate and liver tumors), with a promoters is still actual. Their detection is far from evident carcinogenicity index higher than that of other , by, e.g. extensive metabolism of the compounds and/or the such as , and testosterone [7]. A low dosages used. Furthermore, structural changes of recent study has demonstrated the role of 17α-BOL in the known representative can elude screening, while new substances and even new classes of compounds are *CORRESPONDENCE TO: BIOLATTI, B., Dipartimento di Patologia Ani- male, Sez. Anatomia Patologica,Via L. da Vinci 44, 10095 introduced unknown to the laboratories. The increased use Grugliasco (TO) - Italy. for screening purposes of very specific methodology, such e-mail: [email protected] as mass spectrometry (MS) with single monitoring and 1110 F. T. CANNIZZO ET AL. even tandem MS, is a handicap in detecting new molecules. Committee of the University of Turin. Carcasses of treated The application of screening methods based on different animals were destroyed (2003/74/CE - DL 16 march 2006, approaches, including group specific methods and methods n. 158). Bulbo-urethral gland, testis and prostate of each based on the measurement of indirect parameters also animal were collected after slaughter. Tissue samples were should be encouraged. The use of histological and fixed in 10% neutral buffered formalin overnight at room physiological indicators should be worked out [6]. Since temperature and paraffin embedded according to routine veal calves category is largely bred in north of Italy, an histological procedures. Representative sections of each experimental administration of BOL and ADD in veal sample were stained with hematoxylin-eosin (HE). calves was carried out in order to establish new, validated Immunohistochemistry: Sections of testis were parameters to recognize treated animals. Purpose of the immunohistochemically stained with monoclonal Ki-67 work was to evaluate the changes in target organs, both with antibodies (DakoCytomation, CA, U.S.A.) as follows: qualitative and semi-quantitative morphological analysis. sections (3 µm) were deparaffinized and re-hydrated. Endogenous peroxidase activity was blocked by incubating MATERIALS AND METHODS sections in 3% H2O2 for 15 min. After repeated rinsing with phosphate-buffered saline solution (PBS), sections were Animals: The study was carried out on 14 Friesian male immersed in citrate buffer (0.005 M, pH 6.0) and veal calves, about 25 days old, housed in a farm situated in microwaved three times for 5 min each, then were Piedmont (North West Italy). The animals were housed for incubated with a primary antibody for 30 min (1:100). After 6 months in individual boxes (0.80 × 1.80 m) with concrete primary antibody incubation, the sections were washed in floor without litter or lateral partitions. The calves were PBS. A streptavidin-biotin multilink method (StrAviGen tethered and fed with liquid milk replacer, twice a day [dry Multilink kit; Biogenex, San Ramon, CA) was used for matter 0.95, crude protein 0.23, ether extract 0.21, ash 0.6, detection of reactivity. Diaminobenzidine-H2O2 solution cellulose 0.001; vit. A 25.000 IU/kg, vit. C 50 mg/kg, Cu 5 (DakoCytomation, CA, U.S.A.) was used as chromogen mg/kg, vit. D3 5.000 IU/kg, vit. E 80 mg/kg]. The amount and applied for 5 min. The slides were then rinsed in of the fodder was increased gradually up to 16 l/calf/day; distilled water to stop the reaction. After washing, the slides after one month 0.5 kg of barley straw (D.M. 0.90, C.P. were counterstained with hematoxylin, dehydrated and 0.02, E.E. 0.01, ash 0.06, crude fibre 0.41) was added to the mounted with a cover-slip. diet according to the indications suggested by the European In situ detection of apoptosis: In situ detection of Commission (97/182/EC). Calves were weighed at monthly apoptosis was performed using the TUNEL technique. This intervals and slaughtered at 210 days of age. method is based on the addition of labelled deoxynucleotide Treatment: Calves were randomly allotted to 3 groups. triphosphate to the 3’-OH ends of DNA fragments catalyzed Table 1 reports the average body weight of each group at the by deoxynucleotidyl transferase (TdT). This enzyme start of the experiment. Each animal in Group A (5 animals) selectively detects apoptotic rather than necrotic cells [12]. was administered 1 mg/Kg BOL undecylenate (1,4- The reaction was revealed immunohistochemically by androstadiene-17beta-ol-3-one) (Novartis Consumer Health means of an anti-digoxigenin antibody peroxidase B.V.) suspended in sesame oil by intramuscular injection conjugate. Representative 4-µm-thick sections of each once weekly for five weeks, starting from the seventh week sample were stained by means of ApopTag® In Situ before slaughter. Each animal in Group B (5 animals) was Apoptosis Detection Kit (Intergen Company, Purchase, NY, administered 1 mg/Kg ADD (1,4-androstadiene-3,17-dione) U.S.A.) for identification of apoptotic nuclei. Briefly, (Sigma-Aldrich, Saint Louis, MO, U.S.A.) suspended in sections were deparaffinized, re-hydrated and treated with sesame oil by intramuscular injection once weekly for 4 proteinase K (Sigma, Saint Louis, MO, U.S.A.) 20 µg/ml for weeks, starting from the sixth week before slaughter. Four 15 min at room temperature. The slides were then washed calves received 5 ml of sesame oil by intramuscular in distilled water and endogenous peroxidases were blocked injection (Group C), once a week for 5 times as group A. with 3% H2O2 in PBS for 5 min at room temperature. Dosages were chosen according to literature [23]. Calves Subsequently, the sections were allowed to react with TdT- were slaughtered at 210 days of age, 15 days after the last Enzyme for 1 hr at 37°C and then incubated with the anti- treatment. digoxigenin-peroxidate antibody for 30 min in a humidified Samples and processing of tissue: The experiment was chamber at room temperature. The reaction was developed authorized by the Italian Ministry of Health and the Ethic with diaminobenzidine and H2 O 2 solution (DakoCytomation, CA, U.S.A.) for 5 min, counterstained Table 1. Body weight of animals when settled in the farm with 0.5% (W:V) Methyl Green for 10 min and destaining in n-butanol. A negative control was obtained by omitting the Groups N. animals Body weight TdT in the reaction mixture during the labelling steps. (range; media ± SD) Image analysis: Digital images were obtained with A 5 61–77 Kg; 70 ± 6.3 Coolscope (Nikon Instruments s.p.a., Calenzano, Italy). B 5 64–73 Kg; 67 ± 3.7 Image enhancement and image measurements were made C 4 65–75 Kg; 72 ± 4.1 using software Image-Pro Plus. A sample of prostrate was BOLDENONE AND BOLDIONE AS ANABOLICS IN CATTLE 1111 obtained from every subject and 12 different fields of cells, both in treated and control animals. No hyperplasia or urethral epithelium from each sample of prostate were metaplasia were found in treated groups (Fig. 2). randomly chosen for magnification at 200 ×. For each field, Concerning the prostate urothelium, the thickness of the 20 thickness measurements were made. Also relative area epithelial layer did not show significant differences between of nuclei in urethral epithelium was calculated (nuclear area the experimental groups (Fig. 3) and no metaplasia or (N) / total epithelium area (N+C)) on a single representative epithelial folding were detected. However, the size of the picture captured from each sample. Concerning testis, a epithelial cells was increased in group A and B when sample was obtained from each animal and stained with Ki- compared with C. In the treated animals, the superficial and 67. Five different fields of seminiferous tubules from each intermediate epithelial cells of the urothelium were larger sample were randomly chosen for taking pictures at × 200 than those in the controls while there were no changes in the magnification [10]. For each picture, the area positive for basal layer. Cytoplasm was swollen and paler than in the Ki-67 stain was measured and calculated using software. controls in which there was less cytoplasm that stained more Also, the frequency of apoptotic cells was evaluated by intensely, while the nuclei were normal (Fig. 4). The ratio using the software Image-Pro Plus on samples of testis from between the area of nuclei and the total area of the urethra each animal. The analysis was done by scanning, at × 200 (N/N+C) was calculated (Fig. 5): the highest values were in magnification, microscopic fields picked at random in the control group, followed by group A and group B. seminiferous epithelium. For each animal, the number of Differences between groups were not significant although a apoptotic cells in germinal tissue was expressed as a mean ± clear tendency has been revealed (Kruskal-Wallis test: SEM. p=0.08). Statistical analyses: Statistical analyses were carried out Testis: Administration of BOL and ADD to veal calves using the GraphPad Software, version 3.05 (GraphPad induced a slight reduction of testis weight, although no Software, Inc., San Diego, California, U.S.A.) and R statistically relevant differences were detected between software, version 2.2.0 ((C) R Foundation, from http:// groups (Fig. 6). The bilateral average weight of testis was www.r-project.org). To compare data the Kruskal Wallis 136.6 g, 130.6 g and 149.5 g in animals of group A, B and C test (Non-Parametric ANOVA) with the Dunn’s Multiple respectively. The weight difference was ~8.5% less in comparison post test were used. Differences between group A and ~12.5% less in group B when compared with groups were considered significant at p<0.05. the control group. HE stained sections of testis tissue showed a reduced number of germinal cells and no RESULTS spermatids when compared to controls. Leydig cells did not show relevant differences between groups. Size and pattern Body weight: Table 2 shows the monthly average body of cytoplasm was comparable. Apoptotic figures were weight of experimental groups. From the beginning to the present, although rare, in subjects of all groups. Ki-67 end of the treatment, the animals of groups C increased from immunohistochemistry showed a significant reduction of 197 to 225 kg; the animals of group A from 201 to 249 kg cell proliferation in both BOL and ADD groups, along with and the animals of group B from 201 to 239 kg. No a restriction of germ cells to spermatogonia and a few statistically relevant difference was detected between spermatocytes in extensive areas of testicular parenchyma groups. (Fig. 7). When relative positive areas to Ki-67 were Muscle: No muscular lesion were detected in the site of calculated in germ cell epithelium, and median values were injection of the drugs or placebo in treated and control compared (Fig. 8), group C showed the highest median animals. value, followed by group B, and then group A. The Student- Prostate, Urethra and Bulbo-urethral gland: The Newman-Keuls test confirmed a significant difference prostates of treated animals were macroscopically normal, between groups A and C (p<0.05). Furthermore, the in situ but histologically showed various degrees of hypersecretion apoptosis stain showed a significant (P<0.05) increased and cyst formation, characterized by diffuse and moderate number of positive cells in the seminiferous tubules of the tubular dilation, as well as accumulation of secretion. In the group B animals compared to controls (Fig. 9). Although in same groups, among the undifferentiated glandular group A the number of positive cells was higher than epithelial cells, small areas of well-differentiated mucous controls, the difference from apoptotic cells between group secreting cells were present, compared with none in group C A and C was not significant (Fig. 10). (Fig. 1). The same findings were detected in bulbo-urethral glands, except for a more diffuse differentiation of secreting

Table 2. Monthly average weight of veal calves for each group Groups N. animals Month 1 Month 2 Month 3 Month 4 Month 5 A 5 118 Kg 158 Kg 201 Kg 232 Kg 249 Kg B 5 116 Kg 157 Kg 201 Kg 229 Kg 239 Kg C 4 120 Kg 158 Kg 197 Kg 222 Kg 225 Kg 1112 F. T. CANNIZZO ET AL.

Fig. 1. Prostate glandular tissue of a) boldenone treated animal, Fig. 2. Bulbo-urethral gland of a) boldenone treated animal, b) b) boldione treated animal and c) control animal. Note hyperse- boldione treated animal and c) control animal. Note hyperse- cretion, cyst formation and moderate tubular dilation in treated cretion, small systs and moderate tubular dilation in treated animals compared to control (HE, × 200 magnification). animals compared to control (HE, × 200 magnification).

DISCUSSION decreased weight of the testicles, decreased expression of Ki-67 immunostaining, and increased apoptosis rate in Bulbo-urethral and secreting epithelial cells of the seminiferous tubules were induced by BOL and ADD. The prostate were in an advanced stage of differentiation, of effects of exogenous high doses of androgens on bovine gland secretion and the size of the urothelial cells was prostate morphology are known [5, 15]: treated animals moderately increased in treated animals. In addition, show an active gland with increased PAS positive secretion, BOLDENONE AND BOLDIONE AS ANABOLICS IN CATTLE 1113

Fig. 3. Thickness of the urethral epithelial layer. No significant differences are detected between groups.

Fig. 5. N/N+C ratio shows that the cytoplasm volume of the urethral epithelial cells in prostates of treated animals is increased compared with controls.

Fig. 4. Prostatic urothelial cells of a) boldenone treated animal, b) boldione treated animal and c) control animal. Note increased cytoplasm volume, like hypertrophy, in urothelial cells of the treated animals (HE, × 600 magnification).

which sometimes leads to cyst formation and acid-basic modification of the secretion. Similar findings were confirmed in our experiment, although not as conspicuously as observed by other authors using different androgens [13]. Fig. 6. Testis weight, was slightly decreased in In addition, N/N+C ratio was lower in treated groups than in treated animals but not significantly compared to the control group. The evident tendency of the value may control. Data are expressed as mean ± SEM. suggest that, following the administration of androgens, 1114 F. T. CANNIZZO ET AL.

Fig. 8. Ki-67 immunohistochemistry. Treated animals show a decreased expression of Ki-67 in testis which is accentuated in group A. Data are expressed as mean ± SEM. * P<0.05 (Dunn’s Multiple comparison test).

metaplasia of the whole epithelial layer as a consequence of estrogens treatment has been shown [15,20]. As testosterone is the primary substrate for the synthesis of in the male, the aromatization of BOL and ADD could produce sufficient estradiol to induce the urothelial cell swelling. The findings confirm what has already been observed in the field by Groot and Biolatti [14] in samples from positive animals subjected to analytical testing. Considering testis morphology, by the time of slaughter all the animals were pre-pubertal [4]. BOL and ADD had a detrimental effect on spermatogenesis and testis size, causing a reduction in testis weight and the number of developing germ cells, as previously reported in horses [14, 17, 22]. Although it is well known that these drugs induce changes in human and animal sexual behaviour and sperm quality [14, 17, 24], biological changes of the testicular structures have received less attention. The evaluation of proliferation and apoptosis in germ cell lines by means of image analysis, immunohistochemistry and TUNEL methods, showed that BOL played a role mostly by depressing germinal cell proliferation, while ADD seemed to act both by depressing germ cell proliferation and by Fig. 7. Ki-67 immunohistochemistry in testis from representa- inducing apoptosis. Although the mechanism of action has tive sections of group A, B and C animals. The positive cells still to be clarified, and hormonal regulation of show darker nuclei (arrow). Note the lower expression of ki-67 spermatogenesis is still not completely understood, it is well and a considerable regression of germinal cell line in treated known that testosterone and its analogues play an essential animals (× 400 magnification). role in spermatogenesis, particularly in the initiation and maintenance of the spermatogenic process and inhibition of prostatic urothelial cells become swollen due to the germ cell apoptosis [21]. Exogenous treatment with increased cytoplasm volume as a probable consequence of testosterone or anabolic androgenic steroids such as BOL, cell hypertrophy. The increased size of the urothelial are followed by suppression of both Gonadotropin intermediate and superficial cells has not yet been described Releasing Hormone production by the hypothalamus and following administration of androgens, though squamous Luteinising hormone production by the pituitary gland and BOLDENONE AND BOLDIONE AS ANABOLICS IN CATTLE 1115

Fig. 10. Apoptosis expression of germ cell lines of groups A, B and C. Data are expressed as mean ± SEM. Apoptosis is more expressed in treated groups and it is significantly higher in group B compared with group C. * P<0.05 (Dunn’s Multi- ple comparison test).

Therefore the suppression of testosterone production by the Leydig cells, induced by exogenous androgens, will result in a deficient spermatogenesis, despite the high circulating levels of administered hormone, as can also be seen in men taking anabolic-androgenic steroids [8]. An analogous pathogenetic mechanism seems to be responsible for testicular findings detected in treated animals recruited in this study. In conclusion, the described lesions and effects, although less severe, were similar to those already observed after androgen administration, both in cattle and other species, and included prostate hypersecretion and hypogonadism, they also included hypertrophy of the urothelium. The exact meaning of the latter is still to be clarified and requires more investigation. By contrast, the morphological findings caused by BOL and ADD administration in veal calves are too slight to be adopted as reliable positive histological markers to detect treated animals at the abattoir. Nevertheless, from the public health point of view, the histological study of anabolic target organs must be further investigated since it may provide additional information to the official methods adopted by Fig. 9. Immunocytochemical demonstration of apoptosis in tes- the European National Residues Programs. On the other tis of a) boldenone treated animal. Several apoptotic cells are hand, difficulties of analytical methods to detect residues of present (arrows), b) boldione treated animal. Increased number modified or unknown new molecules, result in an incorrect of apoptotic cells are present in the germ cell line (arrows), and quantification of the risk for the consumer health. c) control animal. Few apoptotic cells are detectable (arrows) Histology, is a fast and cheap screening test which is able to (× 400 magnification). show morphological changes in target organs, lasting several weeks, following treatment with growth promoters subsequently suppression of testicular testosterone [5]. Thus, histological test can provide more information production. It is acknowledged that, in order to allow about the misuse of hormones in live stock production and, regular spermatogenesis, high levels of testosterone are finally, a better quantification of the risk for consumer needed inside the testis, and this can never be accomplished health. by oral or parenteral administration of androgens. 1116 F. T. CANNIZZO ET AL.

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