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[A simple semiquantitative determination of trenbolone acetate and trenbolone in biological material from farm animals].

A technique of high-performance thin-layer chromatography (HPTLC plates) by which to identify the anabolic substance of trenbolone acetate (TBA) and its metabolite trenbolone is described in this paper. Plasma, bile, urine, faeces, liver, and meat can be used for testing. The sensitivity of the method is 1 ng. Additional reliability criteria are enumerated. Semiquantitative TBA determination has proved to be suitable in random sampling for residual analysis.

Anabolic Agents↗

Trenbolone acetate and trenbolone: trace analysis in animal chow, wastewater and human urine by high pressure liquid chromatography and electron capture gas chromatography.

Analytical methodology is described for determining residues of the synthetic anabolic steroid trenbolone acetate (TBA) and its hydrolysis product trenbolone (TBOH) in admixture in animal chow, human urine, and wastewater. Benzene extracts of the substrates are subjected to liquid-liquid partitioning, further cleanup on a column of silica gel, and direct analysis by high pressure liquid chromotography or derivatization with pentafluoropropionic anhydride and analysis by electron capture gas chromatography. Satisfactory recoveries were obtained with both compounds from all three substrates. Residue levels of TBA and TBOH as low as 0.32 and 0.04 ppm, respectively, could be detected in chow; about 0.6 ppb of each compound could be detected in urine and wastewater. Thin layer chromatographic behavior of the two compounds in 7 solvent systems and other ancillary analytical data are also presented.

Acetates↗

Skeletal muscle protein metabolism and serum growth hormone, insulin, and cortisol concentrations in growing steers implanted with estradiol-17 beta, trenbolone acetate, or estradiol-17 beta plus trenbolone acetate.

Skeletal muscle protein degradation, measured by urinary N tau-methylhistidine excretion, and circulating concentrations of growth hormone (GH), insulin (INS), and cortisol (CT) were monitored in steers before and after implantation with estradiol-17 beta (E2; 24 mg) and trenbolone acetate (TBA; 300 mg). Yearling crossbred steers (n = 43) were randomly assigned to four treatment groups in a 2 x 2 factorial arrangement: nonimplanted controls (C); TBA; E2; and TBA plus E2 (TBA+E2). A subgroup (Block 1) of 16 steers was bled on d -12, 31, and 72 after implanting. Deposition of skeletal muscle protein was markedly increased (P less than .001) by E2 and TBA+E2 treatment. This response occurred mainly within the first 40 d after implantation and declined (P less than .001) in concert with decreasing (P less than .01) concentration of serum E2. Anabolic steroid treatment did not affect the rate of skeletal muscle protein breakdown. There was no apparent relationship between reduced serum CT concentration (linear effect; P less than .01) in TBA-treated steers and skeletal muscle protein degradation rate. Blood concentration and pulse activity of INS were not affected by anabolic steroid administration. Both TBA- and TBA+E2-implanted steers displayed a linear decrease (P less than .05) in serum GH concentration over time, which was similar to C. Lowered mean GH concentration resulted from a reduction (TBA main effect; P less than .05) in pulse amplitude of GH. Unlike TBA, TBA+E2, and C, only E2 maintained serum GH concentrations over time. Although increased muscle protein deposition was evident in TBA+E2-treated steers, an obvious causal relationship between this response and circulating GH, INS, and CT was not revealed. These results do not support the concept that combined androgenic agent and estrogen administration effectively reduce bovine muscle protein degradation by static modulation of circulating endogenous anabolic and antianabolic hormones.

Anabolic Agents↗

Fate and residues of trenbolone acetate in edible tissues from sheep amd calves implanted with tritium-labeled trenbolone acetate.

In order to study the fate and residues of trenbolone acetate in edible tissues, two groups of six animals from two ruminant species (ewes and calves) were implanted with [3H]trenbolone acetate. The distribution of extractable radioactive residues was measured in liver, kidney and muscle. We found that the largest proportion of residues was not extractable and thus was considered as covalently bound residues. The proportion of the main extractable metabolites (17 alpha-trenbolone, trendione, 17 beta-trenbolone) was measured. The evaluation of the distribution of trenbolone acetate metabolites directly soluble in water showed that unknown metabolite(s) were predominant. The covalent binding to nucleic acids was measured. It was so low that it was not detectable. The results are discussed in light of the data presented in the scientific report on anabolic agents in animal production from the European scientific working group.

Anabolic Agents↗

Immunotoxicity of trenbolone acetate in Japanese quail.

Trenbolone acetate is a synthetic androgen that is currently used as a growth promoter in many meat-exporting countries. Despite industry laboratories classifying trenbolone as nonteratogenic, data showed that embryonic exposure to this androgenic chemical altered development of the immune system in Japanese quail. Trenbolone is lipophilic, persistent, and released into the environment in manure used as soil fertilizer. This is the first study to date to assess this chemical's immunotoxic effects in an avian species. A one-time injection of trenbolone into yolks was administered to mimic maternal deposition, and subsequent effects on the development and function of the immune system were determined in chicks and adults. Development of the bursa of Fabricius, an organ responsible for development of the humoral arm of the immune system, was disrupted, as indicated by lower masse, and smaller and fewer follicles at day 1 of hatch. Morphological differences in the bursas persisted in adults, although no differences in either two measures of immune function were observed. Total numbers of circulating leukocytes were reduced and heterophil-lymphocyte ratios were elevated in chicks but not adults. This study shows that trenbolone acetate is teratogenic and immunotoxic in Japanese quail, and provides evidence that the quail immune system may be fairly resilient to embryonic endocrine-disrupting chemical-induced alterations following no further exposure posthatch.

Anabolic Agents↗

Effect of hexoestrol on the response of finishing steers to treatment with trenbolone acetate.

The relative effectiveness of implanting 300 mg trenbolone acetate alone or in combination with either 15, 30 or 45 mg hexoestrol was studied in three 90-day experiments using 64 Friesian steers. In experiment 1 hexoestrol was shown to improve live-weight gain and efficiency of feed conversion in steers implanted with trenbolone acetate. In experiments 2 and 3 trenbolone acetate in combination with 30 mg hexoestrol gave a better growth response than when combined with either 15 or 45 mg. However, in experiment 3 trenbolone acetate plus 15 mg hexoestrol was shown to improve live-weight gain by about 36 per cent compared with untreated controls. In experiment 2 small differences between treated groups in mean values for plasma urea, serum albumin, plasma glucose and free fatty acids were recorded.

Animals↗

Lipogenesis in adipose tissue from ovariectomized and intact heifers immunized against estradiol and (or) implanted with trenbolone acetate.

Forty-two heifers were allotted randomly to six treatment groups: intact controls, intact heifers implanted with trenbolone acetate, ovariectomized heifers, ovariectomized heifers implanted with trenbolone acetate, intact heifers immunized against estradiol and intact heifers immunized against estradiol and implanted with trenbolone acetate. Blood titers of estradiol-17 beta were increased over 100-fold in heifers immunized against estradiol in Freund's complete adjuvant or saline:squalene/arlacel containing Mycobacterium. Lipogenic enzyme activities and acetate incorporation into fatty acids were increased in subcutaneous adipose tissue obtained at slaughter from heifers receiving immunization or the combination of immunization and trenbolone acetate. The increased lipogenic capacity was not reflected in either cell diameter or cells per gram adipose tissue. Ovariectomy in combination with trenbolone acetate caused the lowest activities for all enzymes measured. This treatments also caused the greatest decrease in cell diameter, which resulted in the largest number of cells per gram of adipose tissue. Trenbolone acetate alone had no detectable effect on lipogenesis in the intact heifer, but the combination of ovariectomy and trenbolone acetate caused substantial decreases in enzyme activities, in most cases a significant decrease as compared with ovariectomized heifers. The data suggest that trenbolone acetate is able to depress lipogenesis only when not competing with the effects of circulating estradiol.

Adipose Tissue↗

Growth hormone, insulin, prolactin and total thyroxine in the plasma of sheep implanted with the anabolic steroid trenbolone acetate alone or with oestradiol.

The mode of action of the anabolic steroid trenbolone acetate (19-norandrost-4,9,11-trien-3-one-17-acetate) was studied through the endogenous hormonal response of castrated male sheep to subcutaneous implantation of 140 mg of trenbolone acetate and 20 mg of oestradiol both separately and in combination. Radioimmunoassay of delta-4,9,11-trienic steroids and oestradiol-17 beta in plasma confirmed that simultaneous administration of trenbolone acetate with oestradiol led to a significantly greater persistence of oestradiol-17 beta residues in plasma (P less than 0.05) than with implantation of oestradiol alone. Oestradiol treatment increased concentrations of growth hormone and insulin (P less than 0.05; P less than 0.001 respectively) in plasma samples collected weekly. Trenbolone acetate by itself had no significant effect and the oestrogenic response was blocked on the simultaneous implantation of trenbolone acetate and oestradiol (despite higher plasma levels of oestradiol-17 beta with this treatment). Plasma total thyroxine was markedly depressed to 45 per cent of its basal level by trenbolone acetate, alone or with oestradiol (P less than 0.001) and depressed to 80 per cent of basal by oestradiol treatment alone (P less than 0.001). Plasma prolactin was unaltered by the above treatments.

Animals↗

Performance, blood and carcase characteristics of finishing steers treated with trenbolone acetate and hexoestrol.

Twenty British Friesian steers were divided into four uniform groups and either not treated or implanted with hexoestrol, trenbolone acetate, or hexoestrol plus trenbolone acetate. Hexoestrol was given 90 days and trenbolone acetate 70 days, before slaughter. Animals in the treatment groups grew significantly faster, converted food to live-weight gain more effciently faster, converted food to live-weight gain more efficiently and had lower levels of plasma urea and to a lesser extent serum albumin than untreated controls for the final 70 days before slaughter. The combined treatment of hexoestrol plus trenbolone acetate produced more pronounced effects than either compound given alone. Steers treated with hexoestrol had significantly greater levels of serum growth hormone than steers implanted with trenbolone acetate alone or untreated controls, but the treatments had no significant effect on levels of plasma glucose, free fatty acids or serum insulin. Carcase conformation and fat cover assessed subjectively did not differ between treated and control animals but killing out percentage was generally higher in all treatment groups.

Anabolic Agents↗

Effects of zeranol and trenbolone acetate on testis function, live weight gain and carcass traits of bulls.

The ability of zeranol and trenbolone acetate (trenbolone) to alter testis function, weight gain and carcass traits of young bulls was studied. In Exp. 1, the effects of age at initial zeranol implantation was determined. After a 235-d experimental period, sequential implantation (56-d intervals) beginning at 100 or 150 d of age had reduced testis growth (P less than .01), sperm production (P less than .01) and serum testosterone concentration in response to gonadotropin releasing hormone (GnRH; P less than .01). The 200-d age group was partially suppressed, while the 250-d age group was not affected. Body weights were similar to controls in all groups. In Exp. 2, bulls previously implanted with zeranol at 175 and 231 d of age received single implants of zeranol, trenbolone or trenbolone plus zeranol at approximately 300 d of age. At slaughter (135 d later), body weight and carcass characteristics in all treatments were similar to controls. However, trenbolone reduced sperm production (P less than .05), zeranol reduced sperm production and testes weight (P less than .05), but trenbolone plus zeranol was similar to controls. Mean testosterone response to GnRH was suppressed in all implant groups on d 65 (P less than .01), but only in trenbolone or trenbolone plus zeranol groups on d 112 (P less than .05). Results indicate that zeranol suppresses spermatogenesis and testosterone production if implanted before approximately 200 d of age. Reduction of endogenous testosterone without alteration of weight gain or carcass characteristics may be of benefit if behavioral or masculinity traits of bulls are altered. Also, it appears that no benefit is derived from implanting bulls with both trenbolone and zeranol.

Animals↗

Late-gestation treatment of pregnant cows with trenbolone acetate does not increase subsequent growth of heifer calves.

Fifty crossbred cows (38 multiparous and 12 nulliparous) were used to evaluate in utero androgenization of heifer calves with trenbolone acetate. Three 200-mg trenbolone acetate (Finaplix-H) implants were implanted in the ear of treated cows (n = 24) on d 214 +/- 11 of gestation; the remaining animals (n = 26) were used as controls. Cows' rate of gain, serum levels of trenbolone acetate, gestation length, degree of dystocia, percentage bred back, days until conception, and 24-h milk production were evaluated. Fourteen-day weigh periods until parturition indicated that trenbolone acetate-treated dams had an increased (P < .05) average daily gain (1.05 +/- .1 kg) compared with control cows (.55 +/- .1 kg). Serum concentrations of trenbolone acetate were higher (P < .01) in treated cows with a peak at 9 d after implantation and returned to basal concentrations by d 77. Treatment did not affect degree of dystocia among all cows (P > .05) but seemed to increase (P < .01) the incidence of dystocia in nulliparous cows compared with nulliparous control cows. Gestation length and subsequent fertility were not affected by treatment (P > .05). Similarly, there was no difference in 24-h milk production (P > .05) between treated and control cows. Calf birth weight, phenotypic measurements at birth, vigor, average daily gain, carcass characteristics, and heifer reproductive tract and ovarian weights did not differ with treatment (P > .05). These data showed that late-gestation treatment with 600 mg of trenbolone acetate significantly increased weight gain of dams without demonstrating any androgenizing effects on the growth or physical characteristics of heifer calves.

Anabolic Agents↗

Androgen receptors in skeletal muscle cytosol from sheep treated with trenbolone acetate.

Female sheep were implanted with the anabolic agent trenbolone acetate and the effects of the treatment on the skeletal muscle androgen receptor studied. In two separate trials the binding capacity/mg skeletal muscle protein was reduced. In trial 1 the animals were implanted (80 mg trenbolone acetate) at the start and after 5 weeks of the trial and samples taken one week later while in the second trial samples were taken 15 days after a single implant (80 mg trenbolone acetate). The ability of a variety of steroids and steroid-like compounds to displace [3H] testosterone and [3H] methyltrienolone was also studied. The androgen receptor was also partially purified using heparin-sepharose chromatography The involvement of the anddrogen receptor in the mode of action of trenbolone is discussed.

Animals↗

The action of trenbolone acetate, a synthetic anabolic steroid, on ovarian function in the guinea pig.

The action of trenbolone acetate, a synthetic anabolic steroid, on ovarian function was investigated in the guinea pig. Certain comparisons were made with testosterone, the naturally occurring androgen, administered as the phenylpropionate ester. Two milligrams trenbolone acetate per kg given subcutaneously on alternate days for 20 days blocked oestrous cyclicity and ovulation in 9 of 10 animals. A similar effect was shown by 2.2 mg of testosterone phenylpropionate. Treatment of trenbolone acetate-treated animals with exogenous gonadotrophins suggested that the production of follicle-stimulating hormone had been suppressed. Signs of abnormality were seen in the livers of animals receiving 2 mg trenbolone acetate and 2.2 mg testosterone phenylpropionate.

Anabolic Agents↗

Effect of prenatal trenbolone acetate treatment on lamb performance and carcass characteristics.

Forty-three pregnant Dorset and Dorset crossbred ewes were assigned randomly to a control group or implanted with either 300 mg trenbolone acetate (Low TBA) or 1,200 mg trenbolone acetate (High TBA) between d 40 and 60 of gestation. Adjusted weaning weights for ewe lambs were 23.3% less (P less than .10) with vs without TBA treatments. Postweaning ADG of ewe lambs was lower (P less than .05) but ADG of ram lambs was greater (P less than .05) for high TBA vs low TBA. Ewe lambs receiving high TBA had 19% less (P less than .05) gain per unit of feed than those receiving low TBA. Days on test for ewe lambs was greater (P less than .05) due to TBA treatment and for high TBA vs low TBA. Days on test for ram lambs was decreased (P less than .05) due to high TBA compared to low TBA. Subcutaneous fat over the ribeye and lower rib were greater (P less than .05) for high-TBA ewe lambs vs low-TBA ewe lambs. Percentage kidney and pelvic fat of ewe lambs was lower (P less than .05) due to TBA treatments. Ribeye area per unit of carcass weight was lower (P less than .05) in high-TBA ewe lambs vs low-TBA ewe lambs. Yield grade of ewe lambs was lower (P less than .05) for low TBA vs high TBA. Prenatal trenbolone acetate treatment of ewe lambs did not improve their subsequent postnatal growth performance and carcass traits. In addition, TBA implantation of the pregnant ewe produced dystocia and less milk production, as evidenced by the need for more lambs to be grafted.

Anabolic Agents↗

Reduction of energy requirements of steers fed on low-quality-roughage diets using trenbolone acetate.

1. Six steers implanted with 300 mg trenbolone acetate and six steers not implanted were fed on low protein, low-quality-roughage diets ad lib. in two experiments. The steers were Hereford (Bos taurus) x Brahman (Bos indicus) crossbreds (50:50), initially of about 400 kg mean live weight (LW). In the first experiment of 8 weeks duration roughage was given alone. In the second experiment of 6 weeks duration the diet was supplemented with 100 g urea and 4.6 g sulphur daily. The same steers were implanted in each experiment. At the conclusion of each experiment metabolic rate was measured after a 72 h fast. 2. In the first experiment control and implanted steers had similar rates of LW loss (0.57 and 0.59 kg/d respectively). Implanted steers had significantly (P less than 0.01) lower feed intakes (12.8 v. 10.9 g dry matter (DM)/kg LW), significantly (P less than 0.01) lower fasting metabolic rates even after adjustment for intake (83.3 v. 74.5 kJ/kg per d) and significantly (P less than 0.01) lower plasma insulin concentrations (24 v. 19 mu units/ml). Differences in plasma concentrations of free 3,5,3'-triiodothyronine (T3), non-esterified fatty acids and urea-nitrogen were not significant. 3. In the second experiment intake of the supplemented diet was similar in both control and trenbolone acetate-treated steers (19.5 and 20.0 g DM/kg LW respectively). LW gains were 0.23 and 0.41 kg/d for control and implanted steers respectively, the difference being significant (P less than 0.05). Fasting metabolic rate (76.9 v. 70.7 kJ/kg per d) was significantly (P less than 0.05) lower in implanted steers.

Animals↗

Effects of the anabolic steroid trenbolone acetate, implanted alone or in combination with oestradiol-17 beta, on liver cell ultrastructure in steers.

The ultrastructural changes in hepatocytes of steers were studied in animals implanted with 140 mg trenbolone acetate, alone or in combination with 20 mg oestradiol. The mean volume of the nucleus in the hepatocytes of the animals implanted with trenbolone acetate alone was significantly greater than that in control animals (P less than 0.001) and in animals implanted with the combined preparation (P less than 0.05). It is suggested that the increase indicates an androgen-mediated response in the nuclei which is not seen when oestradiol is included in the implant. Similarly, the peroxisome volume density was elevated significantly above that in the controls by trenbolone acetate (P less than 0.001); although the inclusion of oestradiol in the implant significantly reduced this level (P less than 0.001) it was still greater than the control values (P less than 0.05). No mitochondrial abnormalities were observed.

Anabolic Agents↗

Reproductive toxicity of trenbolone acetate in embryonically exposed Japanese quail.

This study was conducted to assess the effects of a one time embryonic exposure to trenbolone acetate on reproductive development and function in Japanese quail (Coturnix japonica). Embryos were exposed to either 0.05, 0.5, 5, or 50microg trenbolone or a sesame oil vehicle control at embryonic day 4. Onset of puberty, gonadal histopathology, sperm motility, cloacal gland size, and male copulatory behavior were assessed in adults. Trenbolone delayed onset of puberty in males, inhibited cloacal gland development, and reduced male reproductive behaviors. Industry laboratories have shown trenbolone acetate to be non-teratogenic in mammalian studies. Our study, however, shows that this one time in ovo exposure delayed onset of puberty in and suppressed adult copulatory behavior in quail males. These results suggest that this one time embryonic exposure to trenbolone may have disrupted development of either the central nervous system or the hypothalamic-pituitary-gonadal axis. This is the first study to demonstrate a demasculinizing effect on copulatory behavior in Japanese quail from embryonic exposure to a non-aromatizable androgenic chemical. More studies are needed to determine the mechanisms behind the observed effects.

Animals↗

Identification of metabolites of trenbolone acetate in androgenic runoff from a beef feedlot.

Little is known concerning the potential ecological effects of hormonally active substances associated with discharges from animal feeding operations. Trenbolone acetate is a synthetic anabolic steroid that is widely used in the United States to promote growth of beef cattle. Metabolites of trenbolone acetate include the stereoisomers 17alpha- and 17beta-trenbolone, both of which are stable in animal wastes and are relatively potent androgens in fish and mammals. Our purpose in this study was to evaluate the occurrence of 17alpha- and 17beta-trenbolone in a beef cattle feedlot discharge and in river water upstream and downstream from the discharge. In conjunction with that effort, we measured in vitro androgenic activity of the discharge using CV-1 cells that had been transiently cotransfected with human androgen receptor and reporter gene constructs. Samples were collected on nine different occasions during 2002 and 2003. Whole-water samples from the discharge caused a significant androgenic response in the CV-1 cells and contained detectable concentrations of 17alpha- and 17beta-trenbolone. Further work is needed to ascertain the degree to which synthetic androgens such as trenbolone contribute to androgenic activity of feedlot discharges.

Anabolic Agents↗