Radioimmunoassay of the anabolic agent zeranol. III. Zeranol concentrations in the faeces of steers implanted with zeranol (Ralgro)

Using a monoclonal antibody raised against zeranol, a radioimmunoassay has been validated for the determination of zeranol residues in the faeces of treated steers. The limit of decision defined as the mean apparent concentration of zeranol in the faeces of untreated cattle + 3 SD was 1 ng/g faeces. In a trial in which 27 steers were implanted with zeranol (36 mg) at the base of the ear and six steers were sham implanted, the mean maximum concentration of zeranol in faeces was 5.8 ng/g on Day 15 following implanting, declining to 1.67 ng/g on Day 34 following implanting. During this period there was a marked variation between animals sampled on the same day following implanting. At no time during the trial did the apparent concentration of zeranol in the faeces of untreated animals rise above 0.91 ng/g, which is below the limit of decision for this assay.     

Radioimmunoassay of the anabolic agent zeranol. IV. The determination of zeranol concentrations in the edible tissues of cattle implanted with zeranol (Ralgro)

Rapid solvent extraction combined with a radioimmunoassay using a monoclonal antibody raised against a derivative of zeranol has been used to measure the residues of the anabolic agent zeranol in the edible tissues (muscle, liver, kidney and fat) of cattle treated with Ralgro. Calibration curves, both with and without, tissue extracts exhibit good parallelism. Regression analysis for the extraction of zeranol from tissues dosed with standard amounts of zeranol have correlation coefficients of 0.979, 0.991, 0.986 and 0.985 for muscle, liver, kidney and fat, respectively. The limits of decision defined as the mean value + 3 SD for the concentrations apparently observed (noise) in tissues from animals not treated with Ralgro were 278, 121, 373 and 110 ng/kg for muscle, fat, liver and kidney, respectively. In the tissues of 4 cows implanted with Ralgro (36 mg), and sampled 70 days after implanting, the highest concentration of zeranol in each tissue was 232 ng/kg (muscle), 391 ng/kg (liver), 287 ng/kg (kidney) and 293 ng/kg (fat), and residues were detected in all samples of fat (4), 3 kidney samples and 1 liver sample.     

Radioimmunoassay of the anabolic agent zeranol. II. Zeranol concentrations in urine of sheep and cattle implanted with zeranol (Ralgro)

A radioimmunoassay for zeranol has been validated and used to measure the concentration of zeranol in the urine of sheep and cattle treated with zeranol (Ralgro). The assay uses an antibody raised against zeranol-16-carboxy-propyl ether conjugated to human serum albumin. In sheep and cattle urine the limits of detection were approximately 2 ng/ml and 2.5 ng/ml, respectively. In two trials 13 sheep were implanted with 12 mg zeranol at the base of the ear. The mean maximum concentrations of zeranol observed in urine were 45 ng/ml (Trial I) on day 35 and 90 ng/ml (Trial II) on day 56, and had declined to 26 ng/ml 42 days after implantation (Trial I) and 11.7 ng/ml 70 days after implantation (Trial II). In four cattle implanted with 36 mg zeranol the concentrations of zeranol in urine reached a mean maximum concentration of 13.5 ng/ml 22 days after implantation and had declined to 2.9 ng/ml 69 days after implantation.     

Radioimmunoassay of the anabolic agent zeranol I. Preparation and properties of a specific antibody to zeranol

A highly specific antibody has been raised in sheep to an antigen prepared by coupling zeranol hemisuccinate to bovine serum albumin. An assay procedure is described and has been used to measure the cross-reactivity of the antiserum, and evaluate the sensitivity of the assay method. The only compounds which reacted with the antibody were zeranol 100%, zearalanone 100%, zearalenone 19% and zearalenol 19%. The sensitivity of the assay procedure allows the determination of zeranol and/or its main metabolite zearalanone between 100 and 1000 pg.     

Radioimmunoassay of hexoestrol residues in faeces, tissues and body fluids of bulls and steers

A fast, reliable and sensitive radioimmunoassay method using either 3H-hexoestrol or a 125I-hexoestrol derivative as the competitive radioligand has been developed for the measurement of residues of hexoestrol in faeces. The blank value for untreated cattle was 113 +/- 98 pg/g faeces, the intra-assay coefficient of variation 10.8 per cent, interassay coefficient of variation 11.1 per cent and recovery of 3H-hexoestrol added to faeces was 58.7 per cent. The concentration of hexoestrol in faeces was measured in 18 steers and 14 bulls at regular intervals after implantation of 36, 45 and 60 mg of hexoestrol. Residues were detected in all samples up to 104 days after implantation and in nine out of 13 samples taken 111 to 153 days after implantation. Residues of hexoestrol were also measured in edible tissues and body fluids of 14 bulls and eight steers slaughtered between 90 and 153 days after implantation of 45 mg hexoestrol. The percentages of samples containing residues were 82, 73, 64, 82 and 27 per cent for bile, urine, liver, kidney and muscle, respectively.     

Preparation and properties of monoclonal antibodies to the anabolic agent zeranol

Monoclonal antibodies have been produced using two haptens, zeranol-7-hemisuccinate coupled to bovine serum albumin and zeranol-16-carboxypropyl ether coupled to human serum albumin. An assessment of cross-reactivity demonstrated that the monoclonal antibody raised against the 7-hemisuccinate derivative reacted with zeranol (100%), talernol (12%), zearalenone (17%), zearalanone (100%) and α-and ß-zearalenol (17% and <0.01%, respectively). In contrast the antibodies to the 16-carboxypropyl ether derivative reacted only with zeranol (100%) and also with a α-zearalenol (13-16%). All monoclonal antibodies were more specific than the polyclonal antibodies raised to the same haptens by conventional methods using sheep.     

The effects of zeranol,an anabolic agent,on the serum immunoglobulins of rabbits immunized with B.19 brucellosis vaccine

Rabbits were immunized with strain 19 antibrucella vaccine and repeatedly administered zeranol subcutaneously (total dose 10 mg in 28 days). The antibody response was followed up for 14 weeks. The following effects were observed: a) an increase in serum proteins during the period of zeranol treatment; b) increases of the agglutinating and, later, of the complement fixing activity, probably caused by an enhanced antibody avidity; c) an increased production and persistence of IgM (19 S).     

Female sex steroid residues in the tissues of steers treated with progesterone and oestradiol-17 beta.

Progesterone, oestrone and oestradiol-17 beta levels in the muscle, fat, liver, kidney and plasma of steers treated with Synovex S, untreated steers, and cows during the normal oestrous cycle, were examined. Progesterone levels in female tissues reached a maximum in the luteal phase and fell to a minimum in the follicular phase. Oestrogen levels (oestrone and oestradiol-17 beta) did not change during the cycle. Progesterone and oestrogen levels in the tissues of steers treated with Synovex S and untreated steers were not statistically different. Tissue progesterone levels in steers were much lower than those in cows during the luteal phase. The highest oestrogen levels were found in the fat of female animals. These results indicate that residue levels of progesterone and oestrogen in the muscle and fat of steers treated with Synovex S were within the physiological range, and lower than those of cows.     

Elimination of sulfamethazine from edible tissues, blood, urine, and feces of turkey poults.

Sulfamethazine was administered to 8- to 10-week-old turkey poults intravenously (IV) at the dose level of 71.5 mg/kg of body weight, orally at the dose level of 143 mg/kg of body weight, or in the drinking water at the concentration of 0.1% over a 6-day period. The concentrations of free sulfamethazine in blood, muscle, skin, kidney, and liver were determined and semilogarithmic plots of concentration vs time for the various tissues indicated that the curve had a linear portion within the first 72-hour period of drug withdrawal. The rates of disappearance of sulfamethazine from the various tissues were proportional to the concentration in the tissues. After 72 hours of withdrawal and for as long as 14 days, sulfamethazine concentrations in kidney, liver, and skin of turkeys given the drug in the drinking water fluctuated between 0.1 and 0.4 ppm. Only 8.6% of the oral dose (143 mg/kg) and 16.5 to 17% of the IV dose (71.5 mg/kg) were recovered in urine and feces as the parent compound during the initial 72-hour period.     

Partitioning of nutrient and trace elements in feed between body retention,faeces and urine by growing dairy-breed steers

The partitioning of nutrients and trace elements from feeds, drinking water and mineral supplements on growth, faeces and urine of growing cattle was studied with eight steers in a balanced Latin square design with two replicates, each comprising four treatments (diet compositions), four periods and four animals. The treatments were four rations with a silage proportion of 15, 30, 60 and 100% of the dry matter. Records were taken of the individual intake of silage, concentrates, mineral supplements and water, and excretions by collecting all urine and faeces. All feeds, urine, faeces and water were analysed for their content of Al, Ca, Cd, Cl, Co, Cu, Fe, K, Mg, Mn, Mo, N, Na, Ni, P, S, Se, and Zn. Elements that were mainly excreted in faeces (Ca, Cd, Cu, Fe, P, and Zn) tended to have a constant uptake in amount. As there is a fairly good correlation between DM consumption and faeces production, it is possible to estimate the amount of these elements excreted at farm level by analysing a faeces sample. Elements that are excreted in significant amounts in both faeces and urine (Cl, K, Mg, Mo, N, Na, S, and Se) tended to have a constant uptake over treatments as a fraction of the feed content. It is more difficult to estimate the amounts of these elements at farm level because it is more difficult to predict urine production than faeces production.     

Nitrogen and protein metabolism and metabolites in plasma and urine of beef steers treated with somatotropin

The objectives of this study were to determine the effects of daily injection of bovine somatotropin (bST) on the metabolism of N and 1-[14C]leucine and on hormone and metabolite concentrations in growing beef steers. Injection of bST increased N retention (P less than .05) primarily through decreased (P less than .10) urinary N excretion. Plasma concentration of somatotropin, insulin and glucose increased (P less than .01) and of urea-N (P less than .01) and alpha-amino-N (P less than .10) decreased with bST compared with excipient injection. Total leucine flux was not altered by treatment; however, the partition of flux was. Leucine oxidation decreased (P less than .05) and leucine used for protein synthesis (P less than .10) increased, with bST compared with excipient injection. During excipient injection, 10.3 g protein were synthesized for each gram crude protein deposited, whereas during bST injections only 6.4 g were required. The average maximum contribution of myofibrillar protein degradation to whole body protein degradation, calculated from excretion of 3-methylhistidine, was 16%. Although the ratio of protein deposition/protein synthesis was low for both excipient- and bST-injected steers, the incremental efficiency of protein deposition was 50%, reflecting a dilution of protein synthesis required for turnover and a proportionately greater increase in protein synthesis than protein degradation with bST injection. In growing beef steers, bST stimulated whole body protein synthesis and decreased leucine oxidation. The change in partition of leucine flux, but not of total flux (irreversible loss), demonstrates a chronic redirection in metabolism consistent with homeorhetic control. These data from steers injected with bST suggest mechanisms by which bST affects metabolism during normal growth.     

Application of diethylstilbestrol dipropionate in bulls. II. Residues in bile, liver, kidney, muscle tissues and application site

The second part of an experiment is described in which 20 one year old bulls were injected with diethylstilbestrol (DES) dipropionate containing preparations. Analysis of DES content was performed in several tissues, such as the injection site, diaphragm muscle, psoas muscle, liver, kidney and bile. In the injection site appreciable amounts of DES were found. Measurable amounts of DES were also found in liver and kidney until 4 weeks after injection. In bile, DES concentrations were even higher than those in urine, and were well correlated with DES concentrations in urine. Implications for screening purposes are discussed.     

A radioimmunoassay method for the measurement of residues of the anabolic agent, hexoestrol, in tissues of cattle and sheep

A radioimmunoassay (RIA) method for hexoestrol using an antiserum against hexoestrol-carboxypropyl ether-BSA and H³-hexoestrol was used to measure the concentrations of residues of hexoestrol in 0.1 ml biological fluids and 1 g edible tissues of implanted cattle and sheep. A preliminary ether extraction of biological fluids was necessary before RIA. The ether extract from tissues was further purified by solvent partition and silica gel column chromatography before RIA. Conjugates of hexoestrol were measured after enzymatic hydrolysis to free hexoestrol. In untreated animals residues were either not detected or very low in all tissues except urine from sheep. The method has a lower limit of detection of approximately 0–10 pg/ml for biological fluids in cattle and 20–100 pg/g for tissues in both sheep and cattle but the lower limit of detection in sheep urine was 70–294 pg/ml urine. In two heifers implanted with 60 mg hexoestrol and slaughtered 2 and 7 days after implantation, residues of hexoestrol were detected in all tissues except muscle with highest concentrations between 2 - 17 ng/g in urine, bile and kidney. The concentration of residues in steers which had been implanted with 45 mg or 60 mg hexoestrol and slaughtered at 90 days after implantation were 0, < 50, 46–96 and 200 pg/ml or g of plasma, muscle, liver and urine, respectively. The concentrations of hexoestrol in sheep implanted with 15 ml hexoestrol and slaughtered after 60 days were 70, 0, 964, 3100 and 4074 pg/g or ml of muscle, fat, liver, kidney and urine, respectively. No hexoestrol was found in control untreated cattle and sheep. It was concluded that some residues of hexoestrol were present in the excretory fluids and tissues of cattle and sheep which had been implanted with hexoestrol at the recommended dose and slaughtered after the recommended withdrawal periods. However, the concentrations of hexoestrol in muscle and fat were extremely low or not detectable. The method could be used for the routine screening of animals for treatment with hexoestrol.     

The effects of two shipping treatments on the carcass characteristics of bulls implanted with zeranol and unimplanted steers

A total of 144 male crossbred calves were allocated to four management treatments (bulls; steers; bulls implanted with zeranol at 100 d of age and re-implanted at 69, 93 and 56 d thereafter; bulls implanted with zeranol at 168 d of age and re-implanted at 93 and 56 d thereafter), and two pre-slaughter shipping treatments (minimum pre-slaughter stress with cattle shipped and slaughtered within 4 h of leaving the feedlot pen; moderate pre-slaughter stress with cattle mixed, trucked 160 km and slaughtered up to 24 h of leaving the feedlot pen) in a 4 X 2 factorial arrangement. Management treatment had no significant effect on carcass pH (45 min), carcass muscle temperature (45 min), or peak shear-force of cooked longissimus muscle. Steers had significantly lower dressing percentage, warm-carcass weight, hide weight and carcass-lean content, but higher marbling score, fat thickness and intramuscular-fat content than all treatments with bulls. Minimum pre-slaughter stress resulted in significantly lower dressing percentage, warm-carcass weight, and carcass pH (45 min), but generally had no effect on carcass tissue-yield measurements compared with the moderate stress treatment. Implanted bulls produced carcasses with significantly darker meat, higher 24-h pH and lower meat expressible juice than bulls and castrates for the moderate pre-slaughter stress treatment. These results provide evidence that zeranol implantation in bulls had a minor influence on carcass characteristics, and did not reduce the incidence of dark-cutting carcasses in young bulls subjected to moderate pre-slaughter shipping stress.     

Influence of zeranol implants on growth, behavior and carcass traits in Angus and Limousin bulls and steers

A 2(3) factorial arrangement of treatments was utilized to determine effects of postweaning zeranol implantation, breed (Angus vs Limousin) and castration (bull vs steer) on growth, behavior and carcass traits. An initial slaughter group was used to account for breed differences in composition and to determine fat and lean growth in the 9-10-11th rib section (NTE). The remaining cattle were fed a finishing diet to a fat end point of .76 cm, as determined by a backfat probe. Control bulls outgained (P less than .01) control steers both to the first kill date and over the entire test and did not require significantly more time to reach the fat end point. The implant did not influence gain in bulls but did increase gain in steers. Angus and Limousins were similar in growth rate for the first 126 d before the first slaughter date. Limousins required more (P less than .01) time to reach the fat end point. Bulls and Limousins produced heavier (P less than .01) carcasses and larger rib eyes (P less than .05; bulls; P less than .01; Limousins). Steers and Angus had higher (P less than .01) marbling scores and lower bone maturity. Implanting decreased (P less than .05) marbling and increased carcass maturity. Small but significant shifts in carcass wholesale cut weight distribution were found between breed and sex condition groups. Bulls and Limousins had greater lean growth in the NTE. Bulls and steers were similar in fat growth, but Angus exceeded Limousin in this trait. Zeranol reduced scrotal circumference (P less than .01) and testicle weight at slaughter (P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth, carcass and palatability traits of intact males and steers implanted with zeranol or estradiol early and throughout life

This study was conducted to assess the impact of implanting intact beef males with protein anabolic agents at varying intervals throughout life. Ninety-six intact males were assigned to three implant treatments: 1) not implanted, 2) implanted at 9 wk of age, weaning and at 56-d intervals thereafter with a 36-mg zeranol implant or 3) estradiol implant at 9 wk of age and 68 d post-weaning. During the 118-d, post-weaning growing period, eight animals per treatment (one replication) were castrated. After a 114-d finishing period, cattle were slaughtered (average age of 13 to 14 mo). Feedlot performance, carcass and palatability data were obtained. Average daily gains and feed efficiency did not differ (P greater than .05) between zeranol and estradiol-implanted intact males. Regardless of implant treatment, steers had lighter carcass weights (P less than .05) and higher (P less than .01) quality grades than intact males. Implanting either intact males or steers with zeranol or estradiol resulted in higher (P less than .05) numerical yield grades. Quality grades were higher in zeranol-implanted cattle than the non-implanted or estradiol-implanted cattle. Intact males implanted with zeranol were similar in carcass fatness to zeranol-implanted steers. No differences (P greater than .05) in tenderness or connective tissue were detected. Implanting intact males early and throughout life with zeranol made them similar to steers in fatness, while estradiol implantation had few effects on carcass and palatability traits of intact males or steers.     

Pathologie changes in endocrine glands and certain other tissues of lambs implanted with the synthetic growth promotant zeranol.

As doses of zeranol implants (0 (control), 12, 24, 48, and 96 mg) were increased, there were increased reaction and activity in target organs (such as urogenital tract and mammary, adrenal, hypophyseal, and thyroid glands) of castrated male sheep (wethers). Hyperplasia and transitional and squamous transformation in the prostate were mild (1+) in the wethers given 12- and 24-mg doses, moderate to marked (2.5+) in the wethers given 48-mg doses, and severe (4+) in the wethers given 96-mg doses. Papillary proliferation and fibrosis increased correspondingly in the seminal vesicle. Changes in the distal penile urethra increased from papillary hyperplasia in the wethers given a 24-mg dose to 100% squamous transformation in the wethers given a 96-mg dose. Mammary gland development was noticeable in the wethers given a 24-mg dose and increased thereafter to progressive alveolar growth and secretory activity in the wethers given 48- and 96-mg doses. Along with a progressive increase of adrenal gland weight and adrenal gland/thyroid gland ratios over the controls, the principals had hypertrophy and hyperplasia of the adrenal cortex. Mean adrenal cortex widths for control wethers and wethers given 24-, 48-, and 96-mg doses were 2,089, 2,140 (adjusted value, 2,194), 2,416, and 2,425 mum, respectively. Mean adrenal gland weights for control wethers and wethers given 12-, 24-, 48-, and 96-mg doses were 2.50, 2.61, 2.53, 2.70, and 2.78 g. respectively. Hyperplasia (nodule formation) plus exhaustive and pyknotic changes of the adrenal cortex increased similarly with increasing zeranol dose. After the thyroid gland weights decreased (2.19, 2.04, 2.00, and 1.72 g, respectively, for control wethers and wethers given 12-, 48-, and 96-mg doses), secretory activity of thyroid epithelial cells decreased. In the glandular portion of the hypophysis, secretory activity and proliferation of eosinophilic cells increased with the larger zeranol doses (48 and 96 mg). There was a corresponding decrease in the number of basophils. These changes are consistent with increased somatotropin and adrenocorticotropin secretion and decreased thyrotropin secretion. Muscle and ligament structures appeared looser and widened in the wethers given the 96-mg dose, and fat cell formation was increased in the muscles along ligament muscle junctions.     

Local antibody responses in the bile and faeces of sheep infected with Fasciola hepatica

The effect of infection with the liver fluke Fasciola hepatica on serum, bile and faecal immunoglobulin and antibody levels was studied in Scottish Blackface sheep. In the serum the immunoglobulins showing the most marked increase were IgG1 and IgG2 and their maximal values were reached at 16 weeks after infection. In the bile IgG2 rose to peak values at two weeks and IgG1, IgA and IgM were maximal at four weeks after infection. The levels of faecal IgG and IgA were low after primary infection but after reinfection a rapid increase in IgA concentration was observed within one to two weeks. Haemagglutinating antibody levels against egg antigens, juvenile and adult excretory-secretory antigens and adult fluke somatic antigens were evaluated. In the sera high titres were observed starting from two to four weeks after infection and persisting until 14 to 16 weeks. Bile haemagglutinating antibodies against excretory-secretory antigens showed the highest level at two and four weeks after infection while antibodies against adult somatic antigens reached maximal titres between four and eight weeks. Faecal antibody levels after primary infection were low but increased rapidly within two weeks after reinfection, coinciding with the elevation in faecal IgA concentration. However, there was no reduction in the number of flukes established in reinfected animals.     

Plasma concentrations of thyroid hormone in steers treated with Synovex-S and 3,5,3'-triiodothyronine.

This experiment examined the effect of daily administration of 3,5,3'-triiodothyronine (T3) on plasma profiles of T3, thyroxine (T4), 3,3',5'-triiodothyronine (reverse T3; rT3) and thyrotropin (TSH) in beef steers in which protein accretion was increased by using implants of Synovex-S (SYN). Twenty-four Angus-Hereford steers (302 +/- 16 kg) were individually fed a diet of a corn-based concentrate and silage mixture for 56 d at equal energy intake per steer (ME/unit BW.75). A 2 x 2 factorial arrangement of treatments was used in which treatments were SYN ear implants (200 mg of progesterone and 20 mg of estradiol benzoate) or no implants and s.c. injections of T3 in polyethylene glycol (2 micrograms of T3/kg BW every 48 h) or no injections of T3. Blood samples were collected every 2 wk. Plasma T3 concentration during the experimental period was increased in T3-treated steers (3.0 +/- .1 vs 2.2 +/- .1 ng/mL, P < .01) and was decreased in SYN-implanted steers (2.4 +/- .1 vs 2.7 +/- .1 ng/mL, P < .01). Plasma T4 and rT3 concentrations were reduced (22 +/- 4 vs 75 +/- 2 and .04 +/- .01 vs .12 +/- .01 ng/mL, respectively, P < .01) in T3-treated steers. Concurrently, plasma TSH concentration was decreased in T3-treated steers (.37 +/- .01 vs .51 +/- .02 ng/mL, P < .02). Synovex-S increased BW gain (21.0%, P < .01) and protein gain (35.6%, P < .01) compared with that of nonimplanted steers. Body weight gain and protein gain were not affected by treatment with T3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrocortisone levels in the urine and blood of horses treated with ACTH.

An investigation was undertaken to demonstrate whether therapeutic treatment with ACTH raises hydrocortisone (cortisol) levels in horse urine above the limit (1000 ng/ml) established by the International Conference of Racing Authorities with the aim of controlling the abuse of cortisol and ACTH in equine sports. ACTH (200 iu) was administered i.m. to 3 Thoroughbred horses; urine and blood samples were collected at intervals afterwards and analysed by an immunoenzymatic system (ELISA) and HPLC-MS. To ascertain post exercise cortisol levels in untreated horses, 101 urine and 103 serum samples were taken from horses immediately after racing and analysed by ELISA. The peak urine level of cortisol, detected 8 h after ACTH administration, was around 600 ng/ml using either ELISA or HPLC-MS. The peak serum cortisol concentration was found to be around 250 ng/ml by ELISA, but consistently less by HPLC-MS. Mean cortisol levels in post race horses were 135.1+/-72.1 ng/ml in urine and 90.1+/-41.7 ng/ml in serum. High levels of the metabolite 20beta-dihydrocortisol in urine and the cortisol precursor 11beta-desoxycortisol in serum were found. The latter showed high cross-reactivity with cortisol on ELISA. In our experiment, treatment with ACTH 200 iu i.m. did not raise urinary cortisol levels above the 1000 ng/ml threshold proposed by the ICRA.