A possible correlation between the blood leukocyte formula and the use of glucocorticoids as growth promoters in beef cattle.

Clinical analyses designed to set welfare parameters were performed on blood drawn from the caudal vein of 14 groups of cattle (young bulls and heifers) (n = 10) from 480 to 550 kg b.w., each group representative of a different farm. The leukocyte formula exhibited a lymphocytopenia in four groups compared with the values from a control group (n = 50). This finding was related to the possible illicit use of corticoids as growth promoters in meat production. The individual plasmas tested negative by two different ELISA kits for corticosteroids, but chemical analyses by LC-MS/APCI (detection limit 0.5 ng/ml) on the pooled plasma of each of the 14 groups revealed the presence of beclomethasone and fluocinolone acetonide in 3 of the 4 suspect farms. These corticosteroids are not always efficiently screened by commercially available immunoassays. The epidemiological reliability of blood analysis as a screening test for such drugs is discussed in the light of the need for quality certification of the whole meat production processes 'from farm to fork', and for enhanced animal welfare.

Pour on application of growth promoters in veal calves: analytical and histological results.

To investigate the possibilities for screening and confirmation methods when the 'pour on' method of application is used for administration of growth promoters, an animal experiment was performed using a cocktail of a combination of growth promoters derived from (illegal) practice. Two cocktails were used, cocktail A consisting of stanozolol and estradiol benzoate and cocktail B consisting of stanozolol, estradiol benzoate and beclomethasone dipropionate. The intended dose per animal was 110 mg stanozolol, 25 mg estradiol and 10 mg beclomethasone. The experiment was performed on 20 male veal calves, 16 treated and 4 vehicle treated controls and 3 female veal calves, 2 treated and 1 vehicle treated control. Half of the animals were shaven prior to the application of the drugs. The cocktails were administered using two types of vehicles: vehicle A; Miglyol 840 with butylated hydroxytoluene and vehicle B; di(ethyleneglycol) monobutylether. During a 28 day treatment period, one group of animals was treated once a week, another group of animals was treated once every two weeks and slaughtered. Preliminary results showed that pour on application of anabolic steroids markedly increased growth performance of veal calves, the animals treated with cocktail A performed better than the animals treated with cocktail B. Macroscopically, the thymus was reduced in weight and size in the B animals. The bulbo-urethral glands were enlarged in all treated animals. Histologically all treated animals showed squamous metaplasia in the prostate, bulbo-urethral gland and Bartholins glands. Moreover, a changed secretion pattern was observed in both the prostate and the bulbo-urethral gland. Severe cortical atrophy was observed in the thymus and to a lesser extent the adrenals of the beclomethasone treated animals. The recently discovered 16 beta-hydroxy-metabolite of stanozolol was detected in urine, in relatively high concentrations. This is the first report of the excretion of this metabolite in urine after pour on administration showing the prospect for detection of dermal treatment. Estradiol levels were remarkably elevated (up to 200 micrograms l-1) exceeding the endogenous levels (< 1 microgram l-1).

Analysis of dexamethasone and betamethasone in bovine urine by purification with an "on-line" immunoaffinity chromatography-high-performance liquid chromatography system and determination by gas chromatography-mass spectrometry.

A method for the immunoaffinity extraction of dexamethasone and betamethasone in bovine urine, followed by high-pressure liquid chromatography (HPLC) fractionation and gas chromatography-mass spectrometry determination, is described. A commercial immunoaffinity gel, containing antibodies raised against dexamethasone, was used to prepare an immunoaffinity cartridge which was inserted in an automatic HPLC system for on-line extraction and purification. By injecting urine samples (spiked with flumethasone as internal standard) directly into the system, it was possible to collect purified fractions, containing the analytes of interest. The fractions were dried and derivatized to yield the tetra-trimethylsilyl derivatives of the three corticosteroids, which were analyzed by selected ion monitoring gas chromatography-mass spectrometry. The method allowed a very good purification of samples and reached a detection limit of 0.1 ng/ml for dexamethasone and 0.2 ng/ml for betamethasone. Several samples, coming from a steer treated with dexamethasone and from other bovines coming from breedings in northern Italy, were analyzed with the method described. Dexamethasone levels ranged from 0.12 to 146 ng/ml.

Experimental administration of 19-nortestosterone and dexamethasone in cattle: elimination of the two drugs in different biological matrices.

19-Nortestosterone (19-NT) is one of the mostly recurrent anabolic agents on the black market of illicit drugs. Dexamethasone (DEXA) is licensed for therapy in veterinary practice but its misuse, although often suspected, has seldom been demonstrated. The excretion of 19-NT and DEXA is well documented when the compounds are administered independently but poor information is available in literature for instances when both drugs are administered as a mixture. To evaluate a radioimmunoassay (RIA) for anabolic residues in urine, blood and faeces, the effect of the simultaneous administration and the subsequent elimination of the two drugs, two animals were injected (4 times) with 19-NT and another two (4 times) with 19-NT-DEXA. After preparation and a sequential clean-up on C18 and alumina columns, the samples were analysed by means of a specific 19-NT radioimmunoassay kit and an anti-DEXA antibody. Detection limits for both drugs were 0.5 ppb in blood samples and 2.0 ppb in urine and faecal samples. After the final treatment, positive 19-NT results were recorded at 14 and 21 d in urine and faecal samples, respectively. In the same matrices, positive DEXA values were found at 11 and 28 d, respectively. In the same matrices, positive DEXA values were found at 11 and 28 d, respectively. Urinary excretion was the main metabolic path both for 19-NT and DEXA; only 30% of the residues were excreted via the faeces. The simultaneous injection of DEXA and 19-NT increased the urinary excretion of 19-NT; however, the excretion of 19-NT via faecal matter was similar for both groups.