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In this work, localized surface plasmon resonance (LSPR) sensing as applicable in the detection of Trenbolone acetate dopant is demonstrated. We show that the LSPR of the Trenbolone acetate/silver nanoparticle (Tren Ac/AgNPs) complex is sensitive to changes in the adsorbent concentration. The results show an average redshift of + 18 nm in the LSPR peak with variations in intensity and broadening behavior of the LSPR band of the Tren Ac/AgNPs complex. AgNPs were synthesized using laser ablation in liquid (LAL) technique with water as the solvent. UV-Vis spectroscopy was used for absorbance measurements and particle size and morphology were monitored using scanning electron microscopy (SEM). The aggregation behavior of the Tren Ac/AgNPs complex was monitored using energy-dispersive X-ray spectroscopy (EDS). Molecular Electrostatic Potential (MEP) and the HOMO-LUMO orbitals of the optimized Trenbolone acetate structure were obtained using Density Function Theory (DFT). The molecule was optimized at the B3LYP level of theory using the 6-311 basis set carried out using the Gaussian 09 software package. The results showed that O2- is Trenbolone acetate's active site that would interact with Ag+ to form a complex that would influence the plasmon behavior. The results presented in this work demonstrate the feasibility of LSPR for anabolic androgenic steroid detection.

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Long-term use of anabolic androgenic steroids (AAS) in supratherapeutic doses is associated with severe adverse effects, including physical, mental, and behavioral alterations. When used for recreational purposes several AAS are often combined, and in scientific studies of the physiological impact of AAS either a single compound or a cocktail of several steroids is often used. Because of this, steroid-specific effects have been difficult to define and are not fully elucidated. The present study used male Wistar rats to evaluate potential somatic and behavioral effects of three different AAS; the decanoate esters of nandrolone, testosterone, and trenbolone. The rats were exposed to 15 mg/kg of nandrolone decanoate, testosterone decanoate, or trenbolone decanoate every third day for 24 days. Body weight gain and organ weights (thymus, liver, kidney, testis, and heart) were measured together with the corticosterone plasma levels. Behavioral effects were studied in the novel object recognition-test (NOR-test) and the multivariate concentric square field-test (MCSF-test). The results conclude that nandrolone decanoate, but neither testosterone decanoate nor trenbolone decanoate, caused impaired recognition memory in the NOR-test, indicating an altered cognitive function. The behavioral profile and stress hormone level of the rats were not affected by the AAS treatments. Furthermore, the study revealed diverse AAS-induced somatic effects i.e., reduced body weight development and changes in organ weights. Of the three AAS included in the study, nandrolone decanoate was identified to cause the most prominent impact on the male rat, as it affected body weight development, the weights of multiple organs, and caused an impaired memory function.

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The objective of this study was to evaluate potency and timing of trenbolone acetate (TBA) administration on live performance and carcass characteristics of beef × dairy steers. A total of 6,895 beef × dairy steers [initial body weight (BW) = 157 ±â€…5.2 kg] were allotted into 30 pens, with pen as the experimental unit. Each pen was randomly assigned one of three implant treatments: 1) Revalor-IS (IS) at d 0, IS at d 80, and Revalor-XS (XS) at d 160 (IS/IS/XS); 2) Ralgro at d 0, IS at d 80, and XS at d 160 (Ral/IS/XS); or 3) Encore at d 0 and XS at d 160 (Enc/XS). Steers were blocked by arrival date, each pen was terminally sorted in three ways at 257 ±â€…22 days on feed and harvested at 329 ±â€…25 days on feed. For live and carcass outcomes, fixed effect of implant treatment and random effect of block was evaluated. Data are reported on a deads and removals out basis. Removals, morbidity, and mortality were similar (P ≥ 0.45). Steers administered TBA prior to d 160 were 5.8 kg heavier (P = 0.03) than Enc/XS steers at d 160. Final BW was not different (P = 0.78). Early administration of a TBA-containing implant resulted in an increased prevalence of bullers [2.40%, 5.18%, 6.86% (for Enc/XS, Ral/IS/XS, and IS/IS/XS) respectively; P < 0.01]. Dry matter intake (DMI) was 2.3% greater (P < 0.01) in steers administered Enc/XS compared to IS/IS/XS; however, DMI as a percentage of BW, average daily gain, and feed efficiency were not different (P ≥ 0.12). Dressing percentage, hot carcass weight, heavy carcass occurrence, Longissimus muscle area, and 12th rib fat thickness were similar among all steers (P ≥ 0.28). Marbling score tended to be greatest for Enc/XS and Ral/IS/XS (P = 0.09). Enc/XS graded a greater proportion of USDA Prime and fewer USDA Select carcasses than IS/IS/XS (P < 0.05). Enc/XS and Ral/IS/XS tended (P = 0.09) to have more USDA Yield Grade (YG) 1 carcasses. While delayed administration or decreased total potency of TBA-containing implants may decrease buller incidence and improve Quality Grade, few differences were observed in live or carcass outcomes.

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Sexually mature female guppies (Poecilia reticulata) were exposed to environmentally relevant concentrations (20, 200, and 2000 ng/L) of 17ß-trenbolone for four weeks. As evidenced by the increased caudal fin index and anal fins developing into gonopodium-like structures, exposed females displayed masculinized secondary sexual characteristics. Differential gene expression and subsequent pathway analysis of mRNA sequencing data revealed that the transcription of transforming growth factor beta/bone morphogenetic protein signaling pathway and Wnt signaling pathway were upregulated following 17ß-trenbolone exposure. Enzyme-linked immunosorbent assays showed that the bone morphogenetic protein 7 protein content was elevated after 17ß-trenbolone exposure. Finally, real-time PCR revealed that 17ß-trenbolone treatment significantly increased androgen receptor mRNA levels, and molecular docking showed potent interaction between 17ß-trenbolone and guppy androgen receptor. Furthermore, 17ß-trenbolone-induced masculinization of caudal and anal fins in female guppies, concomitant to the upregulated expression of differentially expressed genes involved in the above-mentioned two signaling pathways, was significantly inhibited by flutamide (androgen receptor antagonist). These findings demonstrated that 17ß-trenbolone masculinized fins of female guppies by activating the androgen receptor. This study revealed that 17ß-trenbolone could upregulate signaling pathways related to fin growth and differentiation, and eventually cause caudal and anal fin masculinization in female guppies.

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The development of new electrochemiluminescence (ECL) luminophores has become a hot research topic in the field of ECL. Metal-organic frameworks (MOFs) are widely used in ECL sensors due to their excellent ECL performance, high porosity, and abundant surface functional groups. In the work, we developed a cerium-based organic backbone as an ECL luminophor using 5,10,15,20-tetrakis(4-carboxyphenyl)-porphyrin (TCPP) as organic ligand for the detection of trenbolone (TRE). Importantly, the Ce-MOFs can effectively prevent the self-aggregation and self-quenching of TCPP, so that the ECL signal is amplified. To further improve the conductivity and antigen loading capacity of the Ce-MOFs, platinum nanosheets (Pt NPs) were modified at its surface (Pt NPs@MOFs). The Au-ZnO with good biocompatibility is used as the substrate material to load that antibody through the Au-NH2. Based on the above strategy, we constructed a competitive immunosensor to achieve a highly sensitive detection of TRE. Under suitable circumstances, it was discovered that the ECL sensor had a linear relationship with the logarithm of the TRE concentration, with a limit of detection (LOD) of 3.61 fg/mL (S/N = 3). This work provides direction for the application of organic luminescent porphyrins and their derivatives in ECL sensors.

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Introduction: Holstein steers (n = 32) were used to determine if the ergot analog, bromocriptine decreases muscle protein synthesis through inhibitory action on the mTOR pathway via a direct effect on signal proteins, and if these negative effects can be alleviated with anabolic agents. Methods: Steers were treated with intramuscular administration of bromocriptine (vehicle or 0.1 mg/kg BW) and a subdermal commercial steroidal implant containing trenbolone acetate (TBA) and estradiol 17ß (with or without), in a 2×2 factorial design. During the 35 day experiment, intake was restricted to 1.5 times maintenance energy requirement. On days 27 through 32, steers were moved to metabolism stalls for urine collection, and whole-body protein turnover was determined using a single pulse dose of [15N] glycine into the jugular vein on day 28. On day 35, skeletal muscle samples were collected before (basal state) and 60 min after (stimulated state) an i.v. glucose challenge (0.25 g glucose/kg). Blood samples were collected at regular intervals before and after glucose infusion for determination of circulating concentrations of glucose and insulin. Results: Bromocriptine reduced insulin and glucose clearance following the glucose challenge, indicating decreased insulin sensitivity and possible disruption of glucose uptake and metabolism in the skeletal muscle. Conversely, analysis of whole-body protein turnover demonstrated that bromocriptine does not appear to affect protein synthesis or urea excretion. Western immunoblot analysis of skeletal muscle showed that it did not affect abundance of S6K1 or 4E-BP1, so bromocriptine does not appear to inhibit activation of the mTOR pathway or protein synthesis. Estradiol/TBA implant decreased urea excretion and protein turnover but had no effect on protein synthesis, suggesting that steroidal implants promote protein accretion through unchanged rates of synthesis and decreased degradation, even in the presence of bromocriptine, resulting in improved daily gains. Implanted steers likely experienced increased IGF-1 signaling, but downstream activation of mTOR, S6K and 4E-BP1, and thus increased protein synthesis did not occur as expected. Conclusions: Overall, this data suggests that bromocriptine does not have a negative impact on muscle protein synthetic pathways independent of DMI.

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Endocrine-disrupting chemicals-compounds that directly interfere with the endocrine system of exposed animals-are insidious environmental pollutants that can disrupt hormone function, even at very low concentrations. The dramatic impacts that some endocrine-disrupting chemicals can have on the reproductive development of wildlife are well documented. However, the potential of endocrine-disrupting chemicals to disrupt animal behaviour has received far less attention, despite the important links between behavioural processes and population-level fitness. Accordingly, we investigated the impacts of 14 and 21-day exposure to two environmentally realistic levels of 17ß-trenbolone (4.6 and 11.2 ng/L), a potent endocrine-disrupting steroid and agricultural pollutant, on growth and behaviour in tadpoles of an anuran amphibian, the southern brown tree frog (Litoria ewingii). We found that 17ß-trenbolone altered morphology, baseline activity and responses to a predatory threat, but did not affect anxiety-like behaviours in a scototaxis assay. Specifically, we found that tadpoles exposed to our high-17ß-trenbolone treatment were significantly longer and heavier at 14 and 21 days. We also found that tadpoles exposed to 17ß-trenbolone showed higher levels of baseline activity, and significantly reduced their activity following a simulated predator strike. These results provide insights into the wider repercussions of agricultural pollutants on key developmental and behavioural traits in aquatic species, and demonstrate the importance of behavioural studies in the ecotoxicological field.

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INTRODUCTION: There are a variety of harms associated with anabolic-androgenic steroids (AAS), with some AAS associated with an increased risk profile for users. Despite potentially different risk profiles, these harms are seldom discussed with respect to specific compounds although recent ethnographic research has identified a need to do so. Specifically, myth has developed among users with trenbolone reportedly having more dramatic effects on individuals, with reports of aggression, violent behaviour and extreme mood disturbances, and this is reflected in extant literature. This paper aims to report on the narrative surrounding the use of trenbolone among AAS users. METHOD: As part of a larger qualitative study, a number of AAS users were interviewed regarding their usage practices. A narrative emerged regarding the physical and psychological harms which accompanied their AAS use of which trenbolone played a central role (N = 16). RESULTS: Of all the AAS, trenbolone was viewed as having the most deleterious consequences for those who used it. Users reported an extreme shift in risk profile for psychosocial harms, particularly increased aggression and violent behaviour, as well as impulsivity regulation issues. AAS-using peers and family members of users reported the readily observable effect of trenbolone. DISCUSSION AND CONCLUSIONS: Users should be cognisant of the potential for significant harms and health-care providers working with this group may consider more focused screening strategies. Future policy decisions regarding AAS may wish to consider the pivotal role trenbolone plays in adverse outcomes for this unique group of substance users.

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Research suggests that androgens increase skeletal muscle growth by modulating polyamine biosynthesis. As such, the objective of this study was to investigate effects of anabolic hormones, polyamine precursors, and polyamines relative to proliferation, protein synthesis, and the abundance of mRNA involved in polyamine biosynthesis, proliferation, and protein synthesis in C2C12 and Sol8 cells. Cultures were treated with anabolic hormones (trenbolone acetate and/or estradiol), polyamine precursors (methionine or ornithine), or polyamines (putrescine, spermidine, or spermine). Messenger RNA was isolated 0.5 or 1, 12, or 24 h post-treatment. The cell type had no effect (p > 0.10) on proliferation, protein synthesis, or mRNA abundance at any time point. Each treatment increased (p < 0.01) proliferation, and anabolic hormones increased (p = 0.04) protein synthesis. Polyamines increased (p < 0.05) the abundance of mRNA involved in polyamine biosynthesis, proliferation, and protein synthesis. Treatment with polyamine precursors decreased (p < 0.05) the abundance of mRNA involved in proliferation and protein synthesis. Overall, C2C12 and Sol8 myoblasts do not differ (p > 0.10) in proliferation, protein synthesis, or mRNA abundance at the time points assessed. Furthermore, anabolic hormones, polyamines, and polyamine precursors increase proliferation and protein synthesis, and polyamines and their precursors alter the abundance of mRNA involved in growth.

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Research objectives were to evaluate effects of two implant programs for beef heifers fed three different durations (days-on-feed; DOF) on carcass weight and composition (primary outcomes) and feedlot performance (secondary outcomes) at commercial feedlots. Data from a randomized trial in Kansas were analyzed separately and also pooled with data from two previously published trials conducted in Texas. Heifers were randomly allocated to pens within a block, and pens were randomized to treatments in a 2 × 3 factorial randomized complete block design. Implant programs were IH + 200 - an initial Revalor-IH implant [80 mg trenbolone acetate (TBA) and 8 mg estradiol (E2)] and a re-implant after a mean of 98-d (± 10.8 SD) with Revalor-200 (200 mg TBA and 20 mg E2), or XH - Revalor-XH, a single extended-release implant (200 mg TBA and 20 mg E2). Heifers were fed to a baseline endpoint (BASE; pooled mean 166-d ± 11.9 SD), +21, or +42 additional DOF. A total of 10,583 crossbred heifers with mean initial body weight (BW) 315 kg (± 20.1 SD) were enrolled in 144 pens in 24 blocks (treatment replications) across the three trials. General and generalized linear mixed models accounting for clustering of trials, blocks, and pens were used to test for effects of treatments, with significance set at α = 0.05. The only implant program × DOF interaction in pooled analyses was for dry matter intake (DMI; P < 0.01); IH + 200 heifers had lower mean DMI than XH when fed +42 DOF. Gain:feed was higher for IH + 200 compared to XH with dead and removed animals excluded (P < 0.01) or included (P = 0.03). For IH + 200, hot carcass weight (HCW) increased (P < 0.01), USDA Yield Grade (YG) distributions shifted towards lower numerical categories (P < 0.01), and Prime carcasses decreased while Select increased compared to XH (P < 0.01). For each incremental increase in DOF, final BW (P < 0.01) and HCW increased (P < 0.01), while daily gain (P < 0.01) and gain:feed (P < 0.01) decreased. Categories of YG were affected by DOF (P < 0.01); there were fewer YG 1 and 2 and more YG 4 and 5 carcasses for +42 compared to BASE and +21. USDA Quality Grade (QG) distributions differed by DOF (P < 0.01); each incremental increase in DOF resulted in more Prime and fewer Select carcasses. Without meaningful interactions, tested implant programs likely have a consistent effect when heifers are fed to similar DOF, while changes in HCW, QG, and YG may influence marketing decisions when extending DOF.

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The wide and even whole pH range electrochemiluminescence (ECL) is attractive for steroid estrogens detection under harsh conditions (such as strong acid and alkali). Herein, we presented an efficient europium-based metal-organic framework (Eu-MOF) as ECL luminophore, which has been synthesized via the specific 2, 4-bis(3, 5-dicarboxyphenylamino)-6-oltriazine (H4BDPO) ligand with acid-base buffering effect. The functional groups with weak acid and base endowed the H4BDPO with eight ionogenic group states, thereout different total charges of H4BDPO were derived, thus high and steady ECL signals of Eu-MOF were acquired under different environments with pH = 1.0-14.0. Most notably, combined with the means of UV-vis, fluorescence spectra, cyclic voltammetry (CV) and density functional theory (DFT) calculations, the Eu-MOF has been explored different luminescence mechanisms with variational total charges. The constructed ECL biosensor based on the Eu-MOF realized sensitive detection of trenbolone under wide pH range (In order to maintain the biological activity of antigen and antibody, the studied pH value is 5-8.5), in which the limits of detection were 3.95 fg/mL (pH = 5.0), 2.36 fg/mL (pH = 7.4) and 5.48 fg/mL (pH = 8.5) respectively. This work provides a considerable method to realize efficient trace detection of steroid estrogens under the wide or even whole pH conditions.

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The majority of beef cattle in the United States often receive at least one anabolic implant resulting in improved growth, feed efficiency, and environmental and economic sustainability. However, the physiological and molecular mechanisms through which anabolic implants increase skeletal muscle growth of beef cattle remain elusive. The objective of this study was to identify transcriptional changes occurring in skeletal muscle of steers receiving anabolic implants containing different steroid hormones. Forty-eight steers were stratified by weight into 1 of 4 (n = 12/treatment) implant treatment groups: (1) estradiol (ImpE2; 25.7 mg E2; Compudose, Elanco Animal Health, Greenfield, IN), (2) trenbolone acetate (ImpTBA; 200 mg TBA; Finaplix-H, Merck Animal Health, Madison, NJ), (3) combination (ImpETBA; 120 mg TBA + 24 mg E2; Revalor-S, Merck Animal Health), or (4) no implant (CON). Skeletal muscle biopsies were taken from the longissimus 2 and 10 d post-implantation. The mRNA abundance of 94 genes associated with skeletal muscle growth was examined. At 10 d post-implantation, steers receiving ImpETBA had greater (P = 0.02) myoblast differentiation factor 1 transcript abundance than CON. Citrate synthase abundance was increased (P = 0.04) in ImpETBA steers compared to CON steers. In ImpE2 steers 10 d post-implantation, muscle RING finger protein 1 decreased (P = 0.05) compared to CON steers, and forkhead box protein O4 decreased (P = 0.05) in ImpETBA steers compared to CON steers. Interleukin-6 abundance tended to be increased (P = 0.09) in ImpE2 steers compared to both ImpETBA and CON steers. Furthermore, interleukin-10 mRNA abundance tended to be increased (P = 0.06) in ImpTBA steers compared to ImpETBA steers. Leptin receptor abundance was reduced (P = 0.01) in both ImpE2 and ImpTBA steers when compared to CON steers. Abundance of phosphodiesterase 4B was increased (P = 0.04) in ImpTBA steers compared to CON steers 2 d post-implantation. Taken together, the results of this research demonstrate that estradiol increases skeletal muscle growth via pathways related to nutrient partitioning and mitochondria function, while trenbolone acetate improves steer skeletal muscle growth via pathways related to muscle growth.

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Pollutants, such as endocrine disrupting chemicals (EDCs), are increasingly being detected in organisms and ecosystems globally. Agricultural activities, including the use of hormonal growth promotants (HGPs), are a major source of EDC contamination. One potent EDC that enters into the environment through the use of HGPs is 17ß-trenbolone. Despite EDCs being repeatedly shown to affect reproduction and development, comparatively little is known regarding their effects on behaviour. Amphibians, one of the most imperilled vertebrate taxa globally, are at particular risk of exposure to such pollutants as they often live and breed near agricultural operations. Yet, no previous research on amphibians has explored the effects of 17ß-trenbolone exposure on foraging or antipredator behaviour, both of which are key fitness-related behavioural traits. Accordingly, we investigated the impacts of 28-day exposure to two environmentally realistic concentrations of 17ß-trenbolone (average measured concentrations: 10 and 66 ng/L) on the behaviour and growth of spotted marsh frog tadpoles (Limnodynastes tasmaniensis). Contrary to our predictions, there was no significant effect of 17ß-trenbolone exposure on tadpole growth, antipredator response, anxiety-like behaviour, or foraging. We hypothesise that the differences in effects found between this study and those conducted on fish may be due to taxonomic differences and/or the life stage of the animals used, and suggest further research is needed to investigate the potential for delayed manifestation of the effects of 17ß-trenbolone exposure.

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Two methods that the beef cattle industry can use to improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive an anabolic implant at some point during production resulting in an overall increase in skeletal muscle growth. Recent research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to anabolic implants. The objective of this study was to characterize changes that occur in skeletal muscle following implanting in Bos indicus influenced steers or Bos taurus steers. Twenty steers were stratified by initial weight in a 2 × 2 factorial design examining two different breeds: Angus (AN; n = 10) or Santa Gertrudis influenced (SG; n = 10), and two implant strategies: no implant (CON; n = 10) or a combined implant containing 120 mg TBA and 24 mg E2 (IMP; n = 10; Revalor-S, Merck Animal Health). Skeletal muscle biopsies were taken from the longissimus thoracis (LT) 2 and 10 d post-implantation. The mRNA abundance of 24 genes associated with skeletal muscle growth were examined, as well as the protein expression of µ-calpain and calpastatin. Succinate dehydrogenase mRNA abundance was impacted (P = 0.05) by a breed × treatment interaction 2 d post-implanting, with SG-CON having a greater increased abundance than all other steers. A tendency for a breed × treatment interaction was observed for calpain-6 mRNA (P = 0.07), with SG-CON having greater abundance than AN-CON and SG-IMP. Additionally, calpastatin protein expression was altered (P = 0.01) by a breed × treatment interaction, with SG-CON and SG-IMP steers having increased expression (P = 0.01) compared with AN-CON steers. At 2 d post-implanting, a breed × treatment interaction was observed with SG-CON steers having greater (P = 0.05) mRNA abundance of mitogen-activated protein kinase compared with AN-CON steers. Furthermore, breed affected (P = 0.05) calpastatin abundance with AN steers having increased (P = 0.05) abundance 2 d post-implanting compared with SG steers. Meanwhile, implants tended to affect (P = 0.09) muscle RING finger protein-1 mRNA abundance, with CON steers having increased (P = 0.09) abundance compared with that of IMP steers. These findings suggest that cattle breed type and anabolic implants impact calpastatin expression and mRNA abundance associated with protein turnover in the LT of feedlot steers 2 and 10 d post-implantation.

Two methods that the beef cattle industry can use to potentially improve efficiency, sustainability, and economic viability are growth promotants and crossbreeding cattle of different breed types. In the United States, over 90% of cattle receive at least one anabolic implant during the production cycle resulting in improvements in production and overall economic and environmental sustainability. Research suggests that the two main cattle breed types, Bos indicus and Bos taurus, respond differently to different anabolic implant strategies. The objective of this study was to characterize changes that occur in the skeletal muscle following implanting in Bos indicus influenced animals and Bos taurus animals. This research measured mRNA abundance of 24 genes associated with skeletal muscle growth, and protein expression of calpain-1 and calpastatin. The findings of this research suggest that anabolic implants and cattle breed type interact to cause changes in mRNA abundance in the longissimus thoracis that are related to protein turnover of skeletal muscle. Furthermore, calpastatin protein abundance was also altered by this breed × treatment interaction. This research demonstrates that anabolic implants cause molecular changes in skeletal muscle of feedlot steers, with some of these changes being breed dependent.

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Globally, amphibian species are experiencing dramatic population declines, and many face the risk of imminent extinction. Endocrine-disrupting chemicals (EDCs) have been recognised as an underappreciated factor contributing to global amphibian declines. In this regard, the use of hormonal growth promotants in the livestock industry provides a direct pathway for EDCs to enter the environment-including the potent anabolic steroid 17ß-trenbolone. Emerging evidence suggests that 17ß-trenbolone can impact traits related to metabolism, somatic growth, and behaviour in non-target species. However, far less is known about possible effects of 17ß-trenbolone on anuran species, particularly during early life stages. Accordingly, in the present study we investigated the effects of 28-day exposure to 17ß-trenbolone (mean measured concentrations: 10 and 66 ng/L) on body size, body condition, metabolic rate, and anxiety-related behaviour of tadpoles (Limnodynastes tasmaniensis). Specifically, we measured rates of O2 consumption of individual tadpoles as a proxy for metabolic rate and quantified their swimming activity and their time spent in the upper half of the water column as indicators of anxiety-related behaviour. Counter to our predictions based on effects observed in other taxa, we detected no effect of 17ß-trenbolone on body size, metabolic rate, or behaviour of tadpoles; although, we did detect a subtle, but statistically significant decrease in body condition at the highest 17ß-trenbolone concentration. We hypothesise that 17ß-trenbolone may induce taxa-specific effects on metabolic function, growth, and anxiety-related behaviour, with anurans being less sensitive to disruption than fish, and encourage further cross-taxa investigation to test this hypothesis.

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The suprachiasmatic nucleus (SCN) is the main control area of the clock rhythm in the mammalian brain. It drives daily behaviours and rhythms by synchronizing or suppressing the oscillations of clock genes in peripheral tissue. It is an important brain tissue structure that affects rhythm stability. SCN has high plasticity and is easily affected by the external environment. In this experiment, we found that exposure to the endocrine disruptor 17ß-trenbolone (17ß-TBOH) affects the rhythmic function of SCN in the brains of adolescent male balb/c mice. Behavioural results showed that exposure to 17ß-TBOH disrupted daily activity-rest rhythms, reduced the robustness of endogenous rhythms, altered sleep-wake-related behaviours, and increased the stress to light stimulation. At the cellular level, exposure to 17ß-TBOH decreased the c-fos immune response of SCN neurons to the large phase shift, indicating that it affected the coupling ability of SCN neurons. At the molecular level, exposure to 17ß-TBOH interfered with the daily expression of hormones, changed the expression levels of the core clock genes and cell communication genes in the SCN, and affected the expression of wake-up genes in the hypothalamus. Finally, we observed the effect of exposure to 17ß-TBOH on energy metabolism. The results showed that 17ß-TBOH reduced the metabolic response and affected the metabolic function of the liver. This study revealed the influence of environmental endocrine disrupting chemicals (EDCs) on rhythms and metabolic disorders, and provides references for follow-up research.

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Exposure to 17ß-trenbolone caused a skewed sex ratio in fish. However, the molecular initiating event and key molecular event(s) remain unknown. In this study, zebrafish were exposed to 17ß-trenbolone at nominal concentrations of 2 ng/L, 20 ng/L, 200 ng/L, and 2000 ng/L from fertilization to 60 days post fertilization (dpf). First, the sex ratio at 60 dpf was calculated to evaluate adverse outcomes on sexual differentiation. 17ß-Trenbolone caused a skewed sex ratio toward males, with intersex individuals observed in the 20 ng/L group and all-male populations found in the 200 ng/L and 2000 ng/L groups. Then, the distribution and number of primordial germ cells, the expression of sex differentiation-related genes, and plasma vitellogenin concentrations were detected in wild-type zebrafish and the EGFP-nanos-3'UTR transgenic line using whole-mount in situ hybridization, real-time PCR, EGFP fluorescence quantification, and enzyme-linked immunosorbent assay. The results indicated that 17ß-trenbolone exposure decreased the number of primordial germ cells at 1 dpf and 3 dpf, decreased expression of ovarian differentiation-related genes foxl2 and cyp19a1a at 60 dpf, increased expression of testis differentiation-related genes dmrt1, sox9a, and amh at 60 dpf, and decreased plasma vitellogenin levels at 60 dpf, revealing the key molecular events at different time points involved in affected sexual differentiation by 17ß-trenbolone. Finally, molecular docking showed that 17ß-trenbolone docked into ligand-binding domain of zebrafish androgen receptor with high binding energy (-3.72 kcal/mol), suggesting that binding to androgen receptor is the molecular initiating event affecting sexual differentiation by 17ß-trenbolone. We found that 17ß-trenbolone can bind to the zebrafish androgen receptor, decrease the number of primordial germ cells during the early embryonic stage, modulate the expression of genes related to sexual differentiation during gonadal differentiation, and eventually cause a skewed sex ratio toward males in adults.

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Forty-seven Zebu calves were used to determine the effects of class (bull or steer), supplementation (SUPPL, a poultry litter-based supplement or mineral supplementation), and implant (20 mg estradiol combined with 120 mg of trenbolone acetate or no implant) on growth and carcass performance and beef eating quality. The average daily gain (ADG) of implanted cattle significantly increased for steers, but not for bulls. The SUPPL treatment increased ADG by 8.63% from day 0 to end, and shortened in 73.3 d the time to reach 480 kg BW (p < 0.01). Compared to bulls, the steer carcasses exhibited more desirable maturity and finish scores, thicker back fat (p < 0.05), and yielded greater (p < 0.01) percentages of high-value boneless subprimals (HVBLS) (+1.64%) and total cuts (1.35%). The SUPPL bulls dressed 2.63 and 1.63% greater than non-supplemented bulls and SUPPL steers, respectively (p < 0.05). Meat sensory quality was subtly affected (p < 0.05) by sex class or supplementation. The implant did not affect (p > 0.05) shear force or sensory ratings. The supplementation improved key growth performance traits while it adversely affected tenderness-related sensory traits. The implant enhanced the rate of gain of steers only, without improving cut-out yields or inducing adverse effects on palatability traits in both steers and bulls.

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In vivo testing systems for environmental androgens are scarce. The aim of this study was to evaluate the potential of male-specific brightened body coloration in female guppies (Poecilia reticulata) to serve as an in vivo biomarker of environmental androgens using 17ß-trenbolone as an example. The high bioaccumulation of 17ß-trenbolone in the skin of female guppies suggests that it is a potential target tissue of environmental androgens. The coloration index, pigment cell ultrastructure, pigment levels, sexual attractiveness, and reproductive capability of female guppies were analyzed following 28 days of exposure to 20 ng/L, 200 ng/L, and 2000 ng/L 17ß-trenbolone. Increases in the coloration index caused by 17ß-trenbolone exposure were attributable to increased pteridine and melanin levels. Decreases in the sexual attractiveness, number of offspring, and survival rate of offspring suggested that the changes in body coloration translated into adverse outcomes. Finally, mRNA sequencing indicated that 17ß-trenbolone increased pteridine levels by activating genomic effects of androgen receptor on xanthine dehydrogenase and increased melanin levels by exerting non-genomic effects targeting microphthalmia-associated transcription factor, tyrosinase, and tyrosinase-related protein 1 that were mediated by mitogen-activated protein kinase and calcium signaling pathways. We have derived a robust adverse outcome pathway of environmental androgens, and our findings suggest that indicators at different biological levels related to brightened body coloration in female guppies can serve as less-invasive or noninvasive in vivo biomarkers of short-term exposure to environmental androgens.