Boldenone Undecylenate Research Articles

Biochemical and oxidative stress markers in the liver and kidneys of rats submitted to different protocols of anabolic steroids.

The objective of this study was to evaluate the effects of different protocols (P1, P2, and P3) of boldenone undecylenate (BU) and stanozolol (ST) on markers of liver and kidney function and variables of oxidative stress in these organs. For this, 54 male Wistar rats were divided into nine groups of six animals each. Each animal received intramuscularly 5.0mgkg-1 of BU or ST once a week for 4weeks (P1); 2.5mgkg-1 of BU or ST once a week for 8weeks (P2); and 1.25mgkg-1 of BU or ST once a week for 12weeks (P3). For each protocol, a control group was used, and they received 0.1ml of olive oil intramuscularly. Blood and fragments of liver and kidney were collected for alanine aminotransferase activity (ALT), alkaline phosphatase, albumin, creatinine, cholesterol, total protein, triglycerides, urea, reactive oxygen species, thiobarbituric acid reactive substances, total thiols, and glutathione evaluation. The results show that the BU in doses of 5 (day 30) and 2.5mgkg-1 (day 60) changes the ALT seric activity, possibly showing a hepatotoxic effect. High doses of BU may lead to increased levels of cholesterol (protocol P1) possibly due to inhibition of the normal steroid biosynthesis process. All protocols used caused changes in the redox balance of the organs studied (except in the liver, protocol P2), which indicates that these drugs might be harmful even at low doses.

Modulation of boldenone induced hepatic and renal toxicity by Moringa oleiferaas in albino rats.

Boldenone is an anabolic androgenic steroid and synthetic derivative of testosterone that was originally developed for veterinary use. Its use is very spread on veterinary medicine because its ability to increase protein synthesis. The aim of this study is to show the toxic effect in liver and kidney caused after the intramuscular injection of boldenone and focus on the role of Moringa oleifera as co-trateated substance in improving hepatic and renal toxicity of boldenone. 40 adult rats were equally divided into four main groups. Group A injected intramuscularly with olive oil, group B treated only with Moringa oleifera 200 mg/Kg body weight, group C injected with boldenone undecylenate only once every three weeks, and co-treated group D which received both intramuscular boldenone undecylenate once every three weeks beside intragastrically dose of of Moringa leaf extract twic-=0lie/week. The results showed that all the animals in the control groups (A and B) appeared healthy till the end of the experiment. The groups treated with boldenone showed a significant elevation in the levels of serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), total protein, urea, and creatinine compared to the control group. While the oxidative stress in the groups treated with boldenone showed a significant increase in the level of Malondialdehyde (MDA), nitric oxide (NO), total protein, and total thiol and marked reduction in the level of Glutathione (GSH), Catalase activity (CAT), superoxide dismutase activity (SOD). On the other hand the groups treated with Moringa olifera showed a marked reduction in the level of ALT, AST, urea, creatinine, MDA, and NO. While the level of GSH, CAT, and SOD showed a significant increase comparing with the control group. These results explain the side effect of boldenone undecylenate on the liver and kidney which may cause hepatic and renal diseases and also the role of Moringa olifera in improving these results.

Generation of phase II in vitro metabolites using homogenized horse liver.

The successful use of homogenized horse liver for the generation of phase I in vitro metabolites has been previously reported by the authors' laboratory. Prior to the use of homogenized liver, the authors' laboratory had been using mainly horse liver microsomes for carrying out equine in vitro metabolism studies. Homogenized horse liver has shown significant advantages over liver microsomes for in vitro metabolism studies as the procedures are much quicker and have higher capability for generating more in vitro metabolites. In this study, the use of homogenized liver has been extended to the generation of phase II in vitro metabolites (glucuronide and/or sulfate conjugates) using 17β-estradiol, morphine, and boldenone undecylenate as model substrates. It was observed that phase II metabolites could also be generated even without the addition of cofactors. To the authors' knowledge, this is the first report of the successful use of homogenized horse liver for the generation of phase II metabolites. It also demonstrates the ease with which both phase I and phase II metabolites can now be generated in vitro simply by using homogenized liver without the need for ultracentrifuges or tedious preparation steps.

Boldenone-induced apoptotic, structural, and functional alterations in the liver of rabbits

Boldenone undecylenate (BOL) is an anabolic androgenic steroid used in livestock to improve growth and food conversion. This study investigated the actions of BOL on structure and functions of rabbit liver as well as the effects of its withdrawal. Eighteen mature male New Zealand rabbits were divided into 2 groups: Control group (n=6) were injected with 0.25 mL corn oil/kg body weight (BW), while BOL group (n=12) received 3 intramuscular injections, 2 wk apart, of BOL (4.5 mg/kg BW). Animals were scarified 1 d after last injection except for 6 rabbits from BOL group that served as the BOL-withdrawal group (4 wk after the 3rd injection). Intramuscular injection of BOL increased ( P <0.05) malondialdehyde (MDA) level, but markedly lowered activities of superoxide dismutase (SOD) and catalase (CAT) and reduced glutathione (GSH) concentration compared to both control and BOL-withdrawal groups. Treatment with BOL significantly ( P <0.05) increased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) compared to the control group. BOL injection caused different histopathological alterations and apoptosis in liver, but these changes were less evident in the BOL-withdrawal group. Expression of p53 and tumour necrosis factor-α (TNF-α) genes was up regulated in BOL compared to control group, while the expressions of p53 and TNF-α were down regulated in BOL-withdrawal group in comparison with BOL group. In conclusion, BOL injection induced structural and functional changes in the liver of rabbits, increasing oxidative stress and mediators of apoptosis such as ROS, p53 and TNF-α. All these parameters returned to near the control values after withdrawal.

Carcass traits and some blood stress parameters of summer stressed growing male rabbits of different breeds in response to boldenone undecylenate

The present study was conducted to investigate the effects of boldenone undecylenate (BUL) on carcass traits and blood stress parameters in growing male rabbits. A total of 170 male rabbits comprising of three breeds namely New Zealand White (NZW; n=55), Californian (CAL; n=50), and Rex (RX; n=65) were taken for this study. The rabbits of each breed were divided into three groups viz ., D0 (control), D1 provided with normal dose of BUL (at 4.4 mg/kg body weight), and D2 provided with BUL (at 8.8 mg/kg bwt). The rabbits were kept under temperature ranged from 28-32°C during the experimental period. Carcass traits of the rabbits were studied, and the blood parameters were measured by radioimmunoassay. Most of carcass traits, globulin and cholesterol levels in the serum were significantly improved for the injection of BUL at normal dose. Plasma corticosterone levels in normal dose injected rabbits were lowered by 27.21 and 15.25% as compared to controls and double dose, respectively. The effect of interaction between dose and breed was non-significant ( P >0.05) on almost all carcass traits and blood parameters. In conclusion, BUL improves all carcass traits in male growing rabbits when injected with normal dose of BUL, with the exception of dressing-out%, and has a significant lowering effect on stress hormone ( i.e ., corticosterone), and increasing effect on serum total protein, globulin and cholesterol. DOI: http://dx.doi.org/10.5455/javar.2015.b64

Effects of Short and Long Term Growth Promoter Boldenone Undecylenate Treatments on Antioxidant Enzyme Activities and Oxidative Stress Markers in Rabbit Muscles

Effects of Short and Long Term Growth Promoter Boldenone Undecylenate Treatments on Antioxidant Enzyme Activities and Oxidative Stress Markers in Rabbit Muscles

Investigations into the feasibility of routine ultra high performance liquid chromatography–tandem mass spectrometry analysis of equine hair samples for detecting the misuse of anabolic steroids, anabolic steroid esters and related compounds

The detection of the abuse of anabolic steroids in equine sport is complicated by the endogenous nature of some of the abused steroids, such as testosterone and nandrolone. These steroids are commonly administered as intramuscular injections of esterified forms of the steroid, which prolongs their effects and improves bioavailability over oral dosing. The successful detection of an intact anabolic steroid ester therefore provides unequivocal proof of an illegal administration, as esterified forms are not found endogenously. Detection of intact anabolic steroid esters is possible in plasma samples but not, to date, in the traditional doping control matrix of urine. The analysis of equine mane hair for the detection of anabolic steroid esters has the potential to greatly extend the time period over which detection of abuse can be monitored.Equine mane hair samples were incubated in 0.1M phosphate buffer (pH 9.5) before anabolic steroids (testosterone, nandrolone, boldenone, trenbolone and stanozolol), anabolic steroid esters (esters of testosterone, nandrolone, boldenone and trenbolone) and associated compounds (fluticasone propionate and esters of hydroxyprogesterone) were extracted by liquid–liquid extraction with a mix of hexane and ethyl acetate (7:3, v:v). Further sample clean up by solid phase extraction was followed by derivatisation with methoxylamine HCL and analysis by UHPLC–MS/MS. Initial method development was performed on a representative suite of four testosterone esters (propionate, phenylpropionate, isocaproate and decanoate) and the method was later extended to include a further 18 compounds.The applicability of the method was demonstrated by the analysis of mane hair samples collected following the intramuscular administration of 500mg of Durateston® (mixed testosterone esters) to a Thoroughbred mare (560kg). The method was subsequently used to successfully detect boldenone undecylenate and stanozolol in hair samples collected following suspicious screening findings from post-race urine samples. The use of segmental analysis to potentially provide additional information on the timing of administration was also investigated.

Efficacy Of Boldenone Undecylenate And Probiotic As Growth Promotors On Male Buffalo Calves

This study was planned to investigate the effect of probiotic and Boldenone undecylenate on body weight gain, feed conversion rate, blood picture and some biochemical alterations of male buffalo calves. This study was done on twenty male healthy buffalo calves 7-8 months old and weight ranges from 164-170 Kg. They were divided into 4 equal groups each of 5 calves, 1st group healthy buffalo calves (control), 2nd group healthy buffalo calves treated with probiotic (Biogen), 3rd group, healthy buffalo calves treated with half therapeutic dose of Boldenone undecylenate and 4th group healthy buffalo calves treated with the therapeutic dose of Boldenone undecylenate. Two blood samples were collected at 1st, 2nd, 3rd, 4th and 5th injection at 4 weeks and 4 weeks post last injection. 1st Sample used for hematological studies. 2nd sample used for biochemical analysis. Oral dosing of probiotic and I/M injection of full dose of boldenone undecylenate induced a significant increase in weight gain, erythrocytic count, hemoglobin% content, packed cell volume %, total leukocytic count, total proteins, , globulin, calcium and phosphorus and significant decrease in feed conversion rate beside insignificant effect on albumin compared with control buffalo calves. Half therapeutic dose of boldenone undecylenate induce insignificant effect in all above parameters. Probiotic and Boldenone undecylenate in therapeutic dose showed non significant effect on serum ALT, AST, ALP, urea and creatinine and significant decrease in blood glucose level. It could be concluded that the Probiotic and boldenone undecylenate induce good improvement in body weight and feed conversion rate but boldenone undecylenate has some adverse effects on some biochemical parameters than probiotic, so probiotics can be used as safe growth promoter.

Adverse effects of the anabolic steroid, boldenone undecylenate, on reproductive functions of male rabbits

This study was conducted to evaluate the adverse effects of the anabolic steroid, boldenone undecylenate (BOL) on reproductive functions of male rabbits. Thirty white New Zealand mature male rabbits were divided into three groups (10 rabbits each). Group A rabbits served as a control group. Group B rabbits received 4.4 mg/kg body weight (bwt) BOL 5% oily solution. Group C rabbits received 8.8 mg/kg bwt BOL. Rabbits were injected intramuscularly twice weekly for two months. BOL had no significant effect on the bwt and bwt gain. Testes and epididymis weights were decreased significantly in the BOL-treated groups. BOL caused significant reduction in serum testosterone level, seminal volume, sperm motility, and sperm count. No abnormalities were detected in the sperm morphology of the BOL-treated groups. Histopathological alterations in the testes and epididymis were marked in the group C rabbits. These results indicate that administration of BOL exerts a significant harmful effect on the reproductive functions of male rabbits.

Histopathological and Immunohistochemical Changes in the Testes of Rabbits After Injection With the Growth Promoter Boldenone

Recently, boldenone (androgenic steroid) is used in improvement of the growth and food conversion in food-producing animals. In addition, it is used by bodybuilders during both off-season and precontest, where it is well known for increasing vascularity while preparing for a bodybuilding contest. The present study was designed to investigate the possible effect of growth promoter boldenone undecylenate on the structure and functions of rabbit testes. A total of 32 adult New Zealand rabbits were divided into 4 groups. The first group in the control group includes animals that were intramuscularly injected with olive oil and dissected after 3 weeks. Three experimental groups include animals that receive 1, 2, and 3 intramuscular injections of 5mg/kg body weight boldenone, and dissected after 3, 6, and 9 weeks, respectively. Treating rabbits with boldenone increased the testosterone levels compared to the control group. Seminiferous tubules of the rabbit testis treated with boldenone showed reduced development and degeneration of the germinal epithelium, leading to debris and syncytial cell formation in the lumina of seminiferous tubules. Our immunohistochemical results indicated severe reduction in proliferating cell nuclear antigen-positive spermatogonia in boldenone-treated animals as compared to the control group. These findings explain the common phenomena among athletics and bodybuilders who suffer from infertility as they were injected with some drugs such as steroids (boldenone) to build muscles.

Changes in the hepatic and renal structure and function after a growth promoter boldenone injection in rabbits

Boldenone is an androgenic steroid that improves the growth and food conversion in food producing animals. In most countries worldwide, this anabolic steroid is forbidden for meat production. Recently, it is used by bodybuilders in both off-season and pre-contest, where it is well known for increasing vascularity while preparing for a bodybuilding contest. Therefore, our study was designed to investigate the possible effect of using growth promoter boldenone undecylenate on the rabbit liver and kidney structure and functions. Thirty-two adult New Zealand rabbits were divided into four groups. Control group includes animals that injected intramuscularly with olive oil and dissected after 3 weeks. Three experimental groups include animals that receive one, two and three intramuscular injections of 5 mg/kg body weight boldenone, respectively and dissected after 3, 6 and 9 weeks respectively, where the interval of each dose of boldenone was three weeks. Intramuscular injection of rabbits with boldenone increased the serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), urea and creatinine compared with the control group. We also found significant increases in the total protein, total lipid, nitric oxide (NO), superoxide dismutase activity (SOD), glutathione (GSH) and malondialdehyde (MDA) in liver and kidney tissues compared with the control group. Intramuscular injection of rabbits with boldenone exhibited mild to severe histopathological lesions in liver tissue as hepatocellular vacuolation in the centrilobular region and sinusoids and in the kidney, the renal glomerulei had completely lost their typical shape with the appearance of some vacuoles of different shapes and sizes with markedly congested sinusoidal and dilated blood vessels. These findings suggest that misuse of growth promoter boldenone undecylenate may contribute to continuous damage of the hepatic and renal function and structure that may lead to a progressive hepatic and renal diseases, so people should be careful if they want to use such steroids to enhance their strength and endurance.

Deterioration of glomerular endothelial surface layer and the alteration in the renal function after a growth promoter boldenone injection in rabbits

Boldenone is an anabolic steroid developed for veterinary use. Recently, it is used by bodybuilders in both off-season and precontest, where it is well known for increasing vascularity while preparing for a bodybuilding contest. However, the side effect of this steroid on the human health is still unclear. Therefore, the present study was designed to investigate the possible effect of the growth promoter, boldenone undecylenate, on the function and structure of the rabbit's kidneys. A total of 36 adult New Zealand rabbits were divided into 4 groups. Control group includes animals that were injected intramuscularly with olive oil and dissected after 3 weeks. Three experimental groups include animals that receive one, two and three intramuscular injections of 5 mg/kg body weight boldenone, and dissected after 3, 6, and 9 weeks, respectively, and the interval of each dose of boldenone was 3 weeks. The biochemical analysis of the blood serum of treated rabbit showed a significant increase in the total protein, urea and creatinine concentrations, with a significant decrease in albumin/globulin (A/G) ratio. At the same time, a significant glomerulus mass reduction that accompanied with the expression of CD34, a marker for endothelial cells deterioration, was also determined. The incidence of the glomerulosclerosis was significantly increased compared with the control group (0.46 ± 0.05, p < 0.05). The glomerulosclerosis scores were 1.32 ± 0.10, 2.14 ± 0.11 and 3.02 ± 0.09 in groups 2, 3 and 4, respectively. These findings suggest that misuse of the boldenone undecylenate may contribute to the occurrence of a chronic renal injury that may lead to a progressive renal failure.

P53 and Bcl-2expression in response to boldenone induced liver cells injury

Boldenone is an anabolic steroid developed for veterinary use. Recently, it is used by bodybuilders in both off-season and pre-contest, where it is well known for increasing vascularity while preparing for a bodybuilding contest. So, the present study was designed to investigate the possible effect of using growth promoter boldenone undecylenate on the rabbit liver tissue. Thirty-two adult New Zealand rabbits were divided into four groups (8 animals each). Control group includes animals that injected intramuscularly with olive oil and dissected after 3 weeks. The experimental groups include animals that receive one, two and three intramuscular injections of 5 mg/kg body weight boldenone, respectively. The animals were dissected after 3, 6 and 9 weeks respectively, where the interval of each dose of boldenon was 3 weeks. Small pieces of the liver tissues were sent for the histopathological examination. Apoptotic p53 and antiapoptotic Bc1-2 proteins were localized immunohistochemically. Histological observations of the liver tissue showed that the sinusoidal congestion was the most prominent feature that extended from the centrilobular to the periportal regions. Hepatocellular vacuolation in the centrilobular region was also detected. Liver immunohistochemical observation showed a significant increase of the apoptotic protein p53 and a significant decrease in the antiapoptotic Bc1-2 proteins. The highest frequency of p53 positive cells was observed in the liver sections of three dose of boldenone injections, while the lowest in control group, also the highest frequency of Bcl-2 positive cells was observed in the liver sections of control group while the lowest in three dose of boldenone injections. The present results investigate that people should be careful if they want to use such steroids to enhance their strength and endurance.

76 DOES MALE'S AGE AFFECT ART RESULTS (FERTILIZATION, DEVELOPMENT AND EMBRYO QUALITY)?

of those substances may provoke adverse effects on several organs and systems. Even though the use of AAS is worldwide extended, this matter and the possible adverse effects have not been studied in our country. The aim of this study was to evaluate the effects of the use of the AAS on the reproductive function and the lipidic metabolism in Venezuelan bodybuilders. Twenty two bodybuilders (age range: 18 47; mean: 28.2; 7.9 years-old) gave their consent to participate in the study and were classified as intermediate (36%) and advanced level of training (63%). Information about AAS consumption, training performance and reproductive data was collected. Clinical examination and seminal analysis, according to WHO guidelines, were also done. Serum hormonal and lipidic pattern were assessed. All of the participants admitted to be using AAS continually for at least 12 weeks. The more frequent parenterally administered AAS were Testosterona (100%), Boldenone undecylenate (73%), Stanozolol (41%), Nandrolone (14%), methenolone enanthate (9%), drostanolone propionate (5%), and the orally AAS: Methandrostenolone (27%) and Oximetolone (5%). Dosage of AAS was over the therapeutical level. Seminal analysis revealed oligozoospermia (45%), azoospermia (32%) and normozoospermia (23%); 86% of the subjects presented asthenozoospermia. LH and FSH levels were low, eventhough free testosterone level was normal. Hypercholesterolemia was detected in 55% of the athletes; LDL was increased in 77% and HDL was low in 55% of them. Venezuelan bodybuilders show a long and intense AAS pattern of use associated with damage to the spermatogenical function and an increased risk of adverse cardiovascular events.

Anabolic steroids have long-lasting effects on male social behaviors

Anabolic androgenic steroids (AAS) use by adolescents is steadily increasing. Adolescence involves remodeling of steroid-sensitive neural circuits that mediate social behaviors, and previous studies using animal models document effects of AAS on male social behaviors. The present experiments tested whether AAS have persistent and more pronounced behavioral consequences when drug exposure occurs during adolescence as compared to exposure in adulthood. Male Syrian hamsters were injected daily for 14 days with either vehicle or an AAS cocktail containing testosterone cypionate (2 mg/kg), nandrolone decanoate (2 mg/kg), and boldenone undecylenate (1 mg/kg), either during adolescence (27–41 days of age) or adulthood (63–77 days of age). As adults, subjects were tested two or four weeks after the last injection for either sexual behavior with a receptive female or male-male agonistic behavior in a resident-intruder test. Compared with vehicle-treated males, AAS-treated males, regardless of age of treatment, displayed fewer long intromissions and a significant increase in latency to the first long intromission, indicative of reduced potential to reach sexual satiety. Increased aggression was observed in males exposed to AAS compared with males treated with vehicle, independently of age of AAS treatment. However, unlike hamsters exposed to AAS in adulthood, hamsters exposed to AAS during adolescence did not display any submissive or risk-assessment behaviors up to 4 weeks after discontinuation of AAS treatment. Thus, AAS have long-lasting effects on male sexual and agonistic behaviors, with AAS exposure during adolescence resulting in a more pronounced reduction in submissive behavior compared to AAS exposure in adulthood.

Anabolic androgens restore mating after sexual satiety in male rats

Androgen receptors and estrogen receptors importantly participate in the neuroendocrine control of masculine mating behavior. Sexual satiety is the long term inhibition of masculine mating behavior after repeated ejaculations and is associated to changes in both androgen receptor and estrogen receptor-α expression. Androgen receptor expression is up-regulated by systemic chronic administration of anabolic androgens, 5α-dihydrotestosterone or estradiol benzoate. This study was carried out to investigate the effect of these treatments on sexual satiety development and recovery; additionally flutamide or tamoxifen treatments – alone or together with anabolic androgens – were also included. Chronic 15-day treatment with 5α-dihydrotestosterone (5 mg/kg) or tamoxifen (15 mg/kg) inhibited, whereas estradiol benzoate treatment (5 mg/kg) facilitated, mating behavior during sexual satiety development. The proportion of animals that ejaculated 48 h after sexual satiety was increased after 17-day treatment with a mixture of anabolic androgens containing 2 mg/kg testosterone propionate, 2 mg/kg nandrolone decanoate and 1 mg/kg boldenone undecylenate. This effect was only blocked by the combined administration of flutamide plus tamoxifen. The data suggest that anabolic androgens metabolites synergize to restore mating behavior after sexual satiety.

Anabolic androgenic steroids differentially affect social behaviors in adolescent and adult male Syrian hamsters

Anabolic androgenic steroids (AAS) are synthetic derivatives of testosterone used by over half a million adolescents in the United States for their tissue-building potency and performance-enhancing effects. AAS also affect behavior, including reports of heightened aggression and changes in sexual libido. The expression of sexual and aggressive behaviors is a function of complex interactions among hormones, social context, and the brain, which is extensively remodeled during adolescence. Thus, AAS may have different consequences on behavior during adolescence and adulthood. Using a rodent model, these studies directly compared the effects of AAS on the expression of male sexual and aggressive behaviors in adolescents and adults. Male Syrian hamsters were injected daily for 14 days with either vehicle or an AAS cocktail containing testosterone cypionate (2 mg/kg), nandrolone decanoate (2 mg/kg), and boldenone undecylenate (1 mg/kg), either during adolescence (27–41 days of age) or in adulthood (63–77 days of age). The day after the last injection, males were tested for either sexual behavior with a receptive female or agonistic behavior with a male intruder. Adolescent males treated with AAS showed significant increases in sexual and aggressive behaviors relative to vehicle-treated adolescents. In contrast, AAS-treated adults showed significantly lower levels of sexual behavior compared with vehicle-treated adults and did not show heightened aggression. Thus, adolescents, but not adults, displayed significantly higher behavioral responses to AAS, suggesting that the still-developing adolescent brain is more vulnerable than the adult brain to the adverse consequences of AAS on the nervous system and behavior.

Multi residue screening of intact testosterone esters and boldenone undecylenate in bovine hair using liquid chromatography electrospray tandem mass spectrometry

The abuse of esters of natural androgenic steroids in cattle fattening and sports is hard to control via routine urine testing. The esters are rapidly hydrolysed in vivo into substances which are also endogenously present in urine. In veterinary control strange findings of 17beta-testosterone and 17alpha-testosterone in urine are often ignored because of the lack of statistically sound reference data of naturally occurring levels. An interesting alternative for inconclusive urine analyses in veterinary control can be provided by the analysis of the administered steroids themselves, i.e. the analysis of intact steroid esters in hair. Unfortunately, the analysis of intact steroid esters is complicated not only by the vulnerability of the esters which precludes alkaline hydrolysis of the hair, but also by the wide polarity range of short and long-chain esters yielding very poor recoveries for either the one or the other. In this study, a multi-steroid esters LC/MS/MS screening method is presented for trace analysis of the synthetic intact esters of 17beta-testosterone and the undecylenate ester of 17beta-boldenone in bovine hair. The method, requiring only 200 mg of pulverised hair, features a mild digestion procedure using tris(2-carboxyethyl)phosphine hydrochloride (TCEP) and the use of four deuterium-labelled steroid esters as internal standards covering the wide polarity range of the analytes. In spiked hair samples for most of the analytes the limit of detection and the accuracy using isotope dilution were 2-5 ng/g and 97-105%, respectively. The applicability was demonstrated using hair samples from a controlled experiment in which six bovines were injected intramuscularly with two different doses of two commercial mixtures of testosterone esters, and with two different doses of boldenone undecylenate. Depending on the dose all administered testosterone- and boldenone esters were found to be incorporated in bovine hair following a single intramuscular injection, except testosterone propionate which dose might have been too low.

Expression analysis of androgen-responsive genes in the prostate of veal calves treated with anabolic hormones

In order to identify indirect molecular biomarkers of anabolic treatments in veal calves, an animal experiment was performed using two combinations of growth promoters (consisting of boldenone undecylenate and estradiol benzoate, and of testosterone enantate and estradiol benzoate). We selected a set of 12 genes that are known to be androgen responsive in other mammalian species. The expression profile of this set of genes was analysed on prostate samples of veal calves using a real-time RT-PCR approach. For each selected gene the corresponding bovine sequence was obtained and a gene specific real-time assay was optimised and validated. The amplification was shown to be highly specific, linear and efficient. High reproducibility (<1%) and low-test variability (<2.5%) were also been achieved. Messenger RNA levels were quantified in prostate samples, non-parametric analysis of variance showed significant up-regulation of three genes (MAF, ESR1 and AR) and significant down-regulation of four genes (HMGCS1, HPGD, DBI, and LIM) in treated samples when compared with untreated controls. To assess the possibility of identifying hormone-treated animals by molecular means we performed a discriminant analysis that was effective in classifying treated and non-treated samples with an accuracy of 93%. Our results indicate that identification of treatment with steroid hormones in veal calves by means of gene expression analysis is a feasible approach and could be improved increasing both the number of genes and the number of controls analysed.

Direct detection of boldenone sulfate and glucuronide conjugates in horse urine by ion trap liquid chromatography–mass spectrometry

A study of the equine phase II metabolism of the anabolic agent boldenone is reported. Boldenone sulfate, boldenone glucuronide and their C17-epimers were synthesised as reference standards in our lab and a method was developed for their detection in a horse urine matrix. Solid phase extraction was used to purify the analytes, which were then detected by ion trap LC/MS. Negative and positive ionisation mode MS 2 were used for the detection of sulfate and glucuronide conjugates, respectively. Boldenone sulfate and 17-epiboldenone glucuronide were detected as the major and minor phase II metabolites, respectively, in horse urine samples collected following the administration of boldenone undecylenate by intramuscular injection.