Feedlot performance and biological responses to coated and non-coated steroidal implants containing trenbolone acetate and estradiol benzoate in finishing beef steers1,2,3.

Predominately Angus steers (n = 24; initial BW = 435 ± 28.3 kg) were used to evaluate non-coated (NC) and coated implants (CI) containing equal amounts of trenbolone acetate (TBA; 200 mg) and estradiol benzoate (EB; 28 mg) in finishing steers on sera metabolite responses, gene expression, and immunohistochemical analyses of the Longissimus muscle (LM). Performance data were analyzed as a randomized complete block design, and all other data were analyzed as repeated measures for a completely randomized design. Treatments were no implant (NI), NC (Synovex-PLUS; Zoetis, Parsippany, NJ), and CI (Synovex-One Feedlot) implant. There were 2 pen replicates per treatment (n = 4 steers/pen). LM biopsies, blood, and BW were collected before feeding on days 0, 14, 28, 56, 84, 112, and 133, with final BW being captured on day 140. Genes of interest were determined by RT-qPCR using two housekeeping genes. Sera was analyzed for estradiol-17ß (E2),17ß-trenbolone (TbOH), insulin-like growth factor 1 (IGF-I), NEFA, and urea-N (SUN). An α of 0.10 determined significance for performance and sera data; α of 0.05 was used for gene and histology data. No performance differences (P ≥ 0.10) were detected. An implant × day interaction (P ≤ 0.10) for E2, IGF-I, and SUN was detected; implants elevated (P ≤ 0.10) E2, 17ß-TbOH, and IGF-I; and decreased SUN across day of the study, meaning sera metabolites are not altered with time on feed. An implant × day interaction was detected for myogenic factor 5 (MYF-5) positive cells and proportions of MHCIIX. In LM, CI had greater (P < 0.10) IGF-I in LM over NI. CI increased (P < 0.05) G protein-coupled estrogen receptor 1 (GPER1) expression, as well as, GPER1 semi-quantitative scores over NI and NC. An implant × day interaction (P ≤ 0.05) for estrogen and androgen receptor-positive nuclei was detected; implants had increased (P ≤ 0.05) estrogen and androgen receptor-positive nuclei compared to NI. CIs increase genes associated with muscle tissue growth.

Death after misuse of anabolic substances (clenbuterol, stanozolol and metandienone).

A 34-year old male was found breathless and panting at home by his girlfriend three hours after a gym workout. Minutes later, he collapsed and died. Autopsy, histological and chemical analyses were conducted. The examination of the heart showed left ventricular hypertrophy, while the right coronary artery showed only a small vascular lumen (3 mm in diameter), due to its anatomical structure. In femoral blood concentrations of approx. 1 µg/L clenbuterol, approx. 56 µg/L stanozolol and approx. 8 µg/L metandienone, with trenbolone (

Pharmacokinetics of altrenogest in gilts.

Altrenogest, a synthetic progestogen, is characterized by its estrus synchronization in mares, ewes, sows, and gilts. To investigate the pharmacokinetic profile and evaluate its accumulation in gilts, 18 oral doses of 20 mg altrenogest/gilt/day were given to eight healthy gilts at an interval of 24 hr. Plasma samples were collected, and altrenogest was determined by ultra-high-performance liquid chromatography with mass spectrometry. WinNonlin 6.4 software was used to calculate the pharmacokinetic parameters through noncompartmental model analysis. After the first administration (D 1), the pharmacokinetic parameters, including Tmax , Cmax , and the elimination half-life (T1/2λz ), were similar to those observed after the final administration (D 18). However, the mean residence time at D 1 was significantly lower than D 18. As a whole, the mean steady-state plasma concentration (Css ), degree fluctuation (DF), accumulation factor (Rac ), and area under the plasma concentration-time curve in steady state (AUCss ) were 22.69 ± 6.15 ng/ml, 270.64 ± 42.51%, 1.53 ± 0.23, and 544.63 ± 147.49 ng hr/ml, respectively. These results showed that after 18 consecutive days of oral administration of altrenogest, plasma concentrations of altrenogest had a certain degree of fluctuation, without significant accumulations.

Alteration of the mare's immune system by the synthetic progestin, altrenogest.

PROBLEM: Progestins are immunomodulatory in a variety of species. In the horse, the most commonly administered synthetic progestin is altrenogest (ALT), but its effect on the immune system of the non-pregnant mare is unknown. METHODS: Peripheral blood mononuclear cells (PBMCs) from diestrous mares were incubated with varying concentrations of progesterone (P4) or ALT to assess intracellular production of IFNγ and the expression of select cytokines. Additionally, ten mares received either ALT or VEH daily utilizing a switchback design beginning on the day of ovulation and continuing for 7 days. Circulating PBMCs and endometrial biopsies were obtained to assess the production and expression of the same cytokines. RESULTS: In vitro, both P4 and ALT caused a dose-dependent decrease in intracellular IFNγ in PBMCs. P4 caused a dose-dependent decrease in the expression of IFNγ, IL-10 and IL-4, while ALT caused an increase in the expression of IL-6 and IL-1ß in PBMCs. In vivo, ALT suppressed the intracellular levels of IFNγ in PBMCs on d6. While control mares experienced a decrease in IL-1ß expression from d0 to d6, ALT-treated mares did not. In the endometrium, ALT increased the expression of IL-1RN and IFNγ in comparison with VEH-treated mares. CONCLUSION: P4 and ALT appear to alter the immune system of the non-pregnant mare both systemically in addition to locally within the endometrium. Further research is necessary to determine the pathways through which this synthetic progestin functions on the immune system of the horse, and the consequences it may have.

Characterization of trenbolone acetate and estradiol metabolite excretion profiles in implanted steers.

Exogenous growth promoters have been used in US beef cattle production for over 50 yr. The environmental fate and transport of steroid growth promoters suggest potential for endocrine-disrupting effects among ecological receptors; however, the initial excretion of steroid metabolites from cattle administered growth promoters has not been well characterized. To better characterize excretion of trenbolone acetate and estrogen metabolites, steers were assigned to 1 of the following treatment groups: control, given no implant, or treatment, administered a combination implant (200 mg trenbolone acetate, 40 mg estradiol). Blood, urine, and fecal samples were collected over the course of 112 d following implantation. Samples were extracted and analyzed by liquid chromatography tandem mass spectrometry for trenbolone acetate and estrogen metabolites. In both urine and feces, 17α-trenbolone and 17α-estradiol were the predominant metabolites following implantation. Mean concentrations of 17α-trenbolone and 17α-estradiol in feces of implanted steers were 5.9 ± 0.37 ng/g and 2.7 ± 0.22 ng/g, respectively. A best-fit model is presented to predict 17α-trenbolone and 17α-estradiol excretion from steers receiving implants. The present study provides the first characterization of both trenbolone and estrogen metabolites in excreta from implanted cattle and will help provide estimates of steroid production from feedyards in the United States.

Mass balance approaches to characterizing the leaching potential of trenbolone acetate metabolites in agro-ecosystems.

Several studies have documented the occurrence and fate of trenbolone acetate (TBA) metabolites in soil and water. However, considerable uncertainty still exists with respect to TBA risk in agro-ecosystems because limited data are available to quantify excretion, transformation, and leaching processes. To address these uncertainties, we used experimental mesocosms and a mass balance approach to estimate the TBA metabolite leaching potential from manure excreted by implanted (40 mg TBA, 8 mg 17ß-estradiol) beef cattle. Manure sample analysis indicates that over 113 days, a maximum of 9.3% (3,200 µg/animal unit [AU]) of the implant dose was excreted as 17α-trenbolone (17α-TBOH), and <1% was excreted as 17ß-trenbolone (65 µg/AU) or trendione (3 µg/AU). While most (>97%) of the total excreted mass of 17α-TBOH transforms to uncharacterized products, 0.3-0.6% (100-220 µg/AU) of the implant dose accumulates on land surfaces and is available for subsequent transport. During rainfall or irrigation events, a maximum of 0.005-0.06% (1.6-22 µg/AU 17α-TBOH) or 0.005-0.012% (1.8-4 µg/AU 17α-TBOH) of the dose leached into runoff, respectively. Leaching potentials peak at 5-30 days postimplantation, suggesting that targeted timing of implantation and irrigation could minimize steroid leaching during rainfall and irrigation events.

Concentrations of altrenogest in plasma of mares and foals and in allantoic and amniotic fluid at parturition.

Treatment with the progestin altrenogest is widely used in pregnant mares. The fact that foals born from healthy mares treated with altrenogest until term suffered from neonatal problems raises the question of direct effects of altrenogest on vital functions in the neonate. We have therefore investigated altrenogest concentrations in maternal and neonatal blood plasma and in fetal fluids. Pregnant mares were treated with altrenogest orally once daily (0,088 mg/kg bodyweight, n = 7) or left untreated (n = 8) from 280 d of gestation until foaling. Altrenogest concentration was determined in plasma of the mares, their foals and in amniotic and allantoic fluid. The concentration of altrenogest in plasma from treated mares (2.6 +/- 1.0 ng/mL) was significantly lower than in plasma from their foals immediately after birth (5.6 +/- 1.9 ng/mL; p < 0.05), but was significantly higher than in their fetal fluids (amniotic fluid: 0.4 +/- 0.1 ng/mL; p < 0.05; allantoic fluid: 3.0 +/- 1.5 ng/mL). Altrenogest was undetectable in maternal and fetal plasma and fetal fluids of control pregnancies at all times. Altrenogest concentration in plasma of foals from treated mares was strongly correlated to the altrenogest concentration in plasma of their dams (r = 0.938, p < 0.001) and in amniotic (r = 0.886, p < 0.001) and allantoic fluid (r = 0.562, p < 0.05). A significant decrease in altrenogest concentration between the time periods 0-15 min, 30-120 min, and 180-360 min after parturition was seen in the plasma from foals born to altrenogest-treated mares. In conclusion, our data demonstrate that altrenogest reaches the equine fetus at high concentrations.

Pharmacokinetics of altrenogest in horses.

The Federation Equestre Internationale has permitted the use of altrenogest in mares for the control of oestrus. However, altrenogest is also suspicious to misuse in competition horses for its potential anabolic effects and suppression of typical male behaviour, and thus is a controlled drug. To investigate the pharmacokinetics of altrenogest in horses we conducted an elimination study. Five oral doses of 44 mug/kg altrenogest were administered to 10 horses at a dose interval of 24 h. Following administration blood and urine samples were collected at appropriate intervals. Altrenogest concentrations were measured by liquid chromatography-tandem mass spectrometry. The plasma levels of altrenogest reached maximal concentrations of 23-75 ng/mL. Baseline values were achieved within 3 days after the final administration. Urine peak concentrations of total altrenogest ranged from 823 to 3895 ng/mL. Twelve days after the final administration concentrations were below the limit of detection (ca 2 ng/mL).

Validation of a gas chromatography-mass spectrometry method for the determination of pg/ml levels of 17beta-estradiol and 17beta-trenbolone in bovine serum.

A method for the quantitation of pg/ml levels of 17beta-estradiol and 17beta-trenbolone in bovine serum by gas chromatography/electron-capture mass spectrometry has been developed and validated. Using the area ratios of the integrated molecular-ion peaks of the analytes to their corresponding deuterated internal standards, [2,4,16,16-2H4] 17beta-estradiol (17beta-estradiol-d(4)) and [16,16-2H2] 17beta-trenbolone (17beta-trenbolone-d(2)), and non-weighted linear regression, two calibration curves per analyte; 5-50 and 50-500 pg/ml for 17beta-estradiol in sera, and 25-250 and 250-2500 pg/ml for 17beta-trenbolone in sera, respectively, were constructed. Splitless injection of 200 fg 17beta-estradiol and 1000 fg 17beta-trenbolone could be detected and quantified. Tested batches of control bovine sera did not exhibit interference for 17beta-trenbolone, and showed expected background presence of endogenous 17beta-estradiol. Intra-day residual errors did not exceed 20%, and regression correlations were greater than 0.99. Intra-day precision data was similar to inter-day precision data. Using this method, 16 samples can be processed within one working day.

Determination of trenbolone and its metabolite in bovine fluids by liquid chromatography-tandem mass spectrometry.

A liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed for the determination of trenbolone in bovine urine and serum. The aim was a control of the misuse of trenbolone in food-producing animals. The procedure involved, in both cases, a preliminary solid-phase clean-up followed by a liquid-liquid extraction for urine samples after a preliminary enzymatic hydrolysis. The extracts have been directly analysed by reversed-phase LC-MS-MS in selected reaction monitoring (SRM), acquiring two diagnostic product ions from the chosen precursor [M+H](+). The procedures were validated across the concentration range of 1-1500 ng/ml. The linearity, the inter- and intra-day accuracy and precision have been determined. The procedure was specific and the accuracy values were better than 20% at the limit of quantitation of spiked samples. The limit of quantification (LOQ) and the limit of detection (LOD) were, respectively, 1 ng/ml and 350 pg/ml for urine and serum. According to the draft, SANCO/1805/2000, we determined the decision limit CCalpha and the detection capability CCbeta. The recovery values for urine ranged from 87 to 128%, and for plasma the recovery was 70+/-4%. The procedure proved to be simple and suitable for routine and confirmatory purposes such as those developed for residue studies.

Serum concentrations of trenbolone-17 beta and estradiol-17 beta and performance of heifers treated with trenbolone acetate, melengestrol acetate, or estradiol-17 beta.

The effects of the growth-promoting steroids estradiol-17 beta (E2), trenbolone acetate (TBA), and melengestrol acetate (MGA) in heifers on serum concentrations of E2 and trenbolone-17 beta (TBOH) were examined. Feed intake and growth performance were also measured. Serum concentrations of E2 and TBOH were measured on d 0, 1, 3, 5, 7, 13, 21, 28, 42, 56, 84, 112, and 140 in finishing heifers administered the following treatments: 1) control; 2) MGA, .5 mg per heifer daily; 3) Revalor-H (140 mg TBA + 14 mg E2); 4) Revalor-H + MGA; 5) Finaplix-H (200 mg TBA); and 6) Finaplix-H + MGA. Revalor-H implantation (Treatments 3 and 4) increased (P < .05) serum E2 concentrations; peak concentrations (67.5 pg/mL) occurred between d 21 and 56. Feeding MGA (Treatment 4) had no effect (P > .05) on this increase in serum E2 concentrations (63.3 pg/mL). From d 84 until d 140, serum E2 was greater (P < .05) for the Revalor-H treatment (average of 19 pg/mL) than for the control (7 pg/mL) or Finaplix-H treatments (6.5 pg/mL). Serum E2 concentrations increased numerically two- to threefold from d 56 to 140 in controls fed MGA, compared with controls not fed MGA. There was the expected increase in serum TBOH concentrations after TBA implantation in the Revalor-H and Finaplix-H treatments; concentrations were similar (P > .05) for Revalor-H (221 pg/mL) and Finaplix-H (280 pg/mL). After d 56, serum TBOH concentrations decreased in both treatments to 10 and 20% of these concentrations, respectively. Feeding MGA increased serum TBOH (P < .05). Dry matter intake by heifers did not differ among treatments. Feeding of MGA improved gains (P = .12) and efficiencies (P < .01) in nonimplanted heifers and had no effect (P > .4) on gains or efficiencies in Finaplix-implanted heifers.

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.

Effect of a combined trenbolone acetate and estradiol implant on feedlot performance, carcass characteristics, and carcass composition of feedlot steers.

Objectives of this study were to determine the influence of trenbolone acetate (TBA) and estradiol (E2) in a combined implant on feedlot performance, carcass characteristics, and carcass composition in finishing steers. Sixty-four large-framed (394.1 kg) crossbred steers were randomly assigned to one of four pens. Subsequently, pens were randomly assigned to one of two treatments, implanted (120 mg of TBA and 24 mg of E2) and nonimplanted. Eight steers/treatment were slaughtered for initial carcass composition. Remaining steers were assigned to one of three serial slaughter dates (d 40, 115, or 143). Implantation increased circulating trenbolone (TBOH) and E2 concentrations throughout the trial. Implantation increased ADG 18% (P < .001) during d 0 to 40, 21% (P < .001) from d 0 to 115, and 16% for the entire 143 d. Implant status had no effect (P > .05) on dry matter intake. Feed efficiency was improved 13% during d 0 to 40 (P < .01) and from d 41 to 115 (P = .07). Longissimus muscle area was larger (P < .05) in implanted steers than in nonimplanted steers on d 115. Carcasses from implanted steers had a smaller (P < .05) percentage of kidney, pelvic, and heart (KPH) fat on d 143 than those from nonimplanted steers. Carcasses from implanted steers possessed more carcass protein (P < .05) on d 40. Implanted steers had an 82% increase (P < .05) in daily carcass protein accretion during the first 40 d. Implantation increased (P < .01) carcass water but did not affect carcass fat accumulation throughout the feeding period. The combined TBA+E2 implant improved feedlot performance and stimulated carcass protein accretion in feedlot steers.

Use of trenbolone acetate and estradiol in intact and castrate male cattle: effects on growth, serum hormones, and carcass characteristics.

The effects of anabolic implant on growth, carcass characteristics, and serum hormones were examined in 30 young bulls and steers fed a growing diet then a finishing diet. In a 2 X 3 factorial arrangement, steers and bulls received an implant of trenbolone acetate (TBA), TBA and estradiol-17 beta (E2), or no implant. Blood samples were taken serially (every 20 min for 6 h) at intervals during the growing and finishing phases. Percentage of DM, fat, protein, and ash and Warner-Bratzler shear test were measured and taste panel evaluations of the 9-10-11 rib section were obtained. Treatment with TBA and E2 increased weight gain in steers but not in bulls. There were no differences in feed efficiency, serum growth hormone (GH), and cortisol concentrations between bulls and steers or between treated groups and controls in bulls or steers, although during the finishing phase mean GH concentrations in treated steers were twofold higher than in controls and were similar to those in the bull groups. Serum insulin-like growth factor-I (IGF-I) increased twofold during the growing phase, then remained at that level. Steers implanted with TBA and E2, which had the highest gains among the steer groups, had the highest serum GH and IGF-I. Longissimus steaks from bulls treated with TBA alone or TBA and E2 were comparable to steaks from steers in the shear test. Taste panelists found steaks from TBA- and E2-treated bulls to be similar in tenderness and connective tissue to steaks from steers.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth and hormonal response of intact and castrate male cattle to trenbolone acetate and estradiol.

Effects of castration and anabolic implants on weight gain, rib soft tissue composition and serum hormones were studied in cattle using a completely random design with a 2 x 2 factorial arrangement. Half of 16 bulls and 16 steers (Angus or Angus x Brahman) aged 9 mo and weighing 290 kg were treated with an implant (200 mg trenbolone acetate and 24 mg estradiol). Half of each group were not treated with an implant. A growing diet was fed for 95 d and half the animals in each group were slaughtered. Animals in the treated groups were reimplanted with trenbolone acetate and fed a finishing diet for 84 d and slaughtered. Percentage dry matter, fat and protein were determined on soft tissue from the 9-10-11th rib. Two blood samples were collected from each animal every 2 wk. Serum was assayed for five hormones. During the growing phase, untreated and treated bulls and treated steers gained more weight and had leaner rib sections that untreated steers (P less than .05); after the finishing phase, there were no differences among groups. Untreated steers had lower insulin-like growth factor (IGF-I) and higher cortisol concentrations during both phases of growth than untreated bulls did (P less than .05). Treatment with implants increased IGF-I concentrations in steers during both phases and reduced cortisol during the finishing phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of LH secretion by oestradiol, dihydrotestosterone and trenbolone acetate in the acutely castrated bull.

Twenty acutely castrated bulls were used to investigate the role of androgenic and oestrogenic steroids in the feedback control of LH secretion. The effects of 5 alpha-dihydrotestosterone (DHT) or the growth stimulants trenbolone acetate (TBA) or oestradiol-17 beta (OE2) on serum LH secretory profiles were measured. In addition, pituitary LH responses to exogenous LH releasing hormone (LHRH) were determined to differentiate between hypothalamic and pituitary sites of steroid action. At the time of castration, two groups of animals were given implants of either 45 mg OE2 or 200 mg TBA. Another group received equivalent to 30 mg daily injections of DHT. Control steers showed an increase in LH from 2.4 +/- 0.5 (S.E.M.) micrograms/l to 7.0 +/- 0.5 micrograms/l during the week after castration. Treatment with DHT and TBA prevented the post-castration rise in serum LH. In contrast, steers given implants of OE2 showed a significantly greater increase in LH than controls 1 day after castration, but by day 5 LH declined in the OE2-treated group to precastration values. Five weeks after castration control steers secreted LH in pulses at intervals of 40-50 min and with an amplitude of 4.2 +/- 0.4 micrograms/l. Pulses were not detected in the LH profiles of the steroid-treated steers. Dihydrotestosterone and TBA significantly reduced pituitary LH responses to exogenous LHRH, whereas steers receiving OE2 showed LH responses to LHRH which were similar to those observed in castrated controls. These results support the hypothesis that androgenic and oestrogenic components participate separately in the feedback control of LH secretion in the bull.(ABSTRACT TRUNCATED AT 250 WORDS)