The mycoestrogen zeranol at high dosage antagonizes transient receptor potential channel activities in 3T3 L1 cells.

Recent findings have revealed that exposure to environmental contaminants may result in obesity and pose a health threat to the general public. As the activity of transient receptor potential channels (TRPs) plays a permissive role in adipogenesis, the interactions between TRPs and some food pollutants, i.e. bisphenol A, di (2-ethylhexyl) phthalate, zearalenone, and zeranol at 10 µM were investigated in the present study. TRP-V1,-V3, -C4 and -C6 are reported to be differentially expressed in the adipocyte differentiation, and immunoblotting was performed to quantify changes in these TRPs affected by the pollutants. Our result indicated that the mycoestrogen zeranol or α-zearalanol suppressed the expression of the V1 and C6 isoforms. Subsequently, confocal microscopy was used to measure the calcium inflow repressed by zeranol from 0.1 µM to 10 µM. Oil Red O staining was used to determine the differentiation of 3T3 L1 preadipocytes. Zeranol could suppress the expression of TRP-V1 and -C6 protein and inhibit the associated flow of calcium into the cytosol of 3T3 L1 cells. Its IC50 value for inhibiting calcium inflow stimulated by 40 µM capsaicin or 10 µM GSK1702934A was estimated to be around 6 µM. Reduced TRP-V1 or -C6 activity might result in promoting adipogenesis. In conclusion, this study demonstrated that zeranol could potentiate fat cell differentiation through antagonizing TRP-V1 and -C6 activities.

Resorcylic acid lactones in urine samples of slaughtered animals resulting from potential feed contamination with zearalenone.

Resorcylic Acid Lactones, including zeranol, anabolics listed in the group A4 of Directive 96/23/EC, are banned in Europe for use in animals since 1985. Zeranol, after administration to animals, is metabolized to taleranol and zearalanone. It can also naturally occur in the urine due to conversion of zearalenone that may be present in animal feed. In 2010-2017, in Poland, 7746 animal samples were tested for zeranol residues within the official monitoring program. In 13, zeranol was detected after screening. Re-examinations confirmed resorcylic acid lactones in six samples. The recommendations state that only the presence of zeranol and/or taleranol gives the basis for non-compliance. Confirmation should cover the entire profile of six resorcylic acid lactones. In case of detection, the relationship ratio should be verified. Following the proposed criteria, it could be concluded that zeranol detected in urine samples in Poland originated from contamination of feed with mycotoxin, not from illegal use.

Synergistic estrogenic effects of Fusarium and Alternaria mycotoxins in vitro.

Mycotoxins are toxic secondary metabolites formed by various fungal species that are found as natural contaminants in food. This very heterogeneous group of compounds triggers multiple toxic mechanisms, including endocrine disruptive potential. Current risk assessment of mycotoxins, as for most chemical substances, is based on the effects of single compounds. However, concern on a potential enhancement of risks by interactions of single substances in naturally occurring mixtures has greatly increased recently. In this study, the combinatory effects of three mycoestrogens were investigated in detail. This includes the endocrine disruptors zearalenone (ZEN) and α-zearalenol (α-ZEL) produced by Fusarium fungi and alternariol (AOH), a cytotoxic and estrogenic mycotoxin formed by Alternaria species. For evaluation of effects, estrogen-dependent activation of alkaline phosphatase (AlP) and cell proliferation were tested in the adenocarcinoma cell line Ishikawa. The estrogenic potential varied among the single substances. Half maximum effect concentrations (EC50) for AlP activation were evaluated for α-ZEL, ZEN and AOH as 37 pM, 562 pM and 995 nM, respectively. All three mycotoxins were found to act as partial agonists. The majority of binary combinations, even at very low concentrations in the case of α-ZEL, showed strong synergism in the AlP assay. These potentiating phenomena of mycotoxin mixtures highlight the urgent need to incorporate combinatory effects into future risk assessment, especially when endocrine disruptors are involved. To the best of our knowledge, this study presents the first investigation on synergistic effects of mycoestrogens.

Effect of nursing-calf implant timing on growth performance and carcass characteristics.

The objective of this study was to compare pre- and postweaning growth performance, carcass characteristics, and meat quality attributes of calves that did not receive an implant or were implanted early or late in the nursing period. Crossbred steer calves ( = 135) were stratified by birth date and birth weight and randomly assigned to the following implant treatments: control (CON; no preweaning implant), 58 d (EARLY; 36 mg zeranol, administered at an average of 58 ± 13 d of age), and 121 d (LATE; 36 mg zeranol, administered at an average 121 ± 13 d of age). After weaning, steers were blocked by initial feed yard BW to 15 pens (5 pens/treatment and 9 steers/pen). All steers were implanted on d 21 after arrival at the feed yard and again on d 108 of finishing. Steer BW and ultrasound assessment of rib eye area (uREA), rib fat thickness (uRFT), and percent intramuscular fat (uIMF) were collected when implants were administered, at weaning, and on harvest day. Carcass measurements included HCW, rib eye area (REA), 12th-rib fat thickness (FT), and marbling score. Objective color (L*, a*, and b*) was recorded, and a 3.8-cm strip loin section was removed from both sides of each carcass and portioned into 2.54-cm steaks that were aged for 3 or 14 d for analysis of cook loss and Warner-Bratzler shear force (WBSF). The remaining portion of each sample was used for analysis of moisture and crude fat. Steer BW, ADG, and G:F did not differ among treatments ( > 0.05). Steers implanted in the EARLY treatment had a greater ( < 0.05) cumulative DMI than CON but were not different from steers implanted in the LATE treatment. Ultrasound REA and uRFT (averaged across all collection days) did not differ ( > 0.05); however, steers on the CON treatment had a greater ( ≤ 0.05) percent uIMF than EARLY implanted steers, whereas steers receiving the LATE implant were intermediate and not different from the other treatments. Hot carcass weight, REA, FT, USDA yield grade, marbling score, and objective color did not differ ( > 0.05) among treatments. The proportion of steers in each USDA yield and quality grade was similar ( > 0.05) among treatments, and no differences were detected for total carcass value or price per 45.4 kg (hundredweight; > 0.05). Treatment did not influence ( > 0.05) percent cook loss, crude fat, moisture, or WBSF. In conclusion, administering a nursing implant, regardless of timing, did not influence live performance, carcass characteristics, or meat quality of steers fed in this study.

Effects of timing of anabolic implant insertion on growth and immunity of recently weaned beef steers.

We evaluated the effects of timing of estrogenic implant insertion, relative to weaning, on growth performance and measurements of innate and humoral immunity of beef calves. On d -14, Angus × Simmental crossbred steers ( = 48; BW = 217 ± 5 kg; age = 191 ± 3 d) were stratified by BW, age, and cow parity and randomly assigned to receive no implant (NOIP) or 36 mg of zeranol on d -14, 0, or 14, relative to weaning (IP-14, IP0, and IP+14, respectively; 12 steers/treatment). From d -14 to 0, cow-calf pairs remained on a single, tall-fescue pasture with no access to concentrate supplementation. Steers were weaned on d 0, stratified by treatment and BW, and then allocated into 1 of 16 drylot pens to receive daily free-choice access to a corn silage-based diet during the preconditioning phase (d 0 to 56). Steers were vaccinated against infectious bovine rhinotracheitis (IBRV), bovine viral diarrhea virus (BVDV), and on d -27 and 0. From d 56 to 252 (postpreconditioning phase), steers remained in their respective feedlot pens and were provided free-choice access to corn silage-based growing (d 56 to 167) and finishing total mixed rations (d 168 to 252). Body weight on d 0 did not differ among treatments ( ≥ 0.29) but was greater for IP-14 and IP0 than NOIP and IP+14 steers on d 14, 42, and 56 ( ≤ 0.05). Treatment effects were not detected for G:F and DMI from d 0 to 56 ( ≥ 0.34), but ADG from d -14 to 56 was greater for IP-14 compared to NOIP ( ≤ 0.05) and intermediate for IP0 and IP+14 steers. Plasma IGF-1 concentrations were greater for IP-14 than NOIP ( ≤ 0.05) and intermediate for IP0 and IP+14 steers on d -7, 0, 14, and 21. Plasma concentrations of cortisol and haptoglobin and serum titers against BVDV types 1a and 2 did not differ among treatments from d 0 to 56 ( ≥ 0.37). However, serum IBRV titers were greater for IP+14 than NOIP, IP-14, and IP0 steers ( ≤ 0.02). On d 252, BW was greater for IP-14 and IP0 than NOIP steers ( ≤ 0.05) and intermediate for IP+14 steers, but ADG and G:F from d 57 to 252 and carcass characteristics at slaughter did not differ among treatments ( ≥ 0.16). Thus, the 36-mg zeranol implant did not elicit an inflammatory response or affect the overall vaccine response of steers (except for IBRV titers). However, growth of steers during a 56-d preconditioning period was enhanced by administering 36-mg zeranol implant 14 d before weaning, without affecting subsequent postpreconditioning growth and carcass characteristics at slaughter.

Individual and combined effects of deoxynivalenol and α-zearalenol on cell proliferation and steroidogenesis of granulosa cells in cattle.

This study was conducted to evaluate the impact of deoxynivalenol (DON) and zearalenone (ZEA) metabolite, α-zearalenol (α-Zol), on cell proliferation and steroidogenesis of bovine large (LG) follicle granulosa cells (GC). LGGC were obtained from bovine ovarian follicles (8-22 mm) and were cultured for 2 days in medium containing 10% fetal bovine serum followed by 1 or 2 days in serum-free medium without (control) or with treatments. Three different experiments were performed using different dosages of DON and α-Zol and in different combinations and a fourth experiment evaluated estradiol effects on granulosa cell proliferation. DON inhibited progesterone (P4) and estradiol (E2) production at high dose. α-Zol alone and in combination with DON increased cell growth. Estradiol inhibited cell growth indicating α-Zol is not acting as an estrogen agonist. This study demonstrates that α-Zol and DON can impact in vitro GC function, however further studies will be required to better understand the mechanism of action and reproductive effects of Fusarium mycotoxins.

Cytotoxicity induced by ochratoxin A, zearalenone, and α-zearalenol: effects of individual and combined treatment.

This study investigated the cytotoxicity of combined mycotoxins of ochratoxin A (OTA), zearalenone (ZEA), and/or α-zearalenol (α-ZOL). The cytotoxicity of two mycotoxin combinations (two two-toxin combinations and one three-toxin combination) on human Hep G2 cells was evaluated using a tetrazolium salt (MTT) assay and isobologram analysis. Our results demonstrated significant cytotoxic effects of the two-toxin combination and the three-toxin combination on Hep G2 cells in a time- and concentration-dependent manner. The combination indexes (CI) were 2.73-7.67 for the OTA+ZEA combination and 1.23-17.82 for the OTA+α-ZOL combination after 24 h, 48 h, and 72 h of exposure at all inhibit concentration (IC) levels (IC10-IC90), indicating an antagonism. The CIs of the ZEA+α-ZOL combination were 1.29-2.55 after 24 h and 72 h of exposure (IC10-IC90), indicating an antagonism. The CIs of the ZEA+α-ZOL combination were 0.74-1.68 after 48 h of exposure, indicating synergism (IC80-IC90), additive effects (IC50-IC70), or antagonism (IC10-IC40). For the OTA+ZEA+α-ZOL combination, the CIs were 1.41-14.65 after 24 h, 48 h, and 72 h of exposure (IC10-IC90), indicating an antagonism.

Alleviation of plasma homocysteine level by phytoestrogen α-zearalanol might be related to the reduction of cystathionine ß-synthase nitration.

Hyperhomocysteinemia is strongly associated with cardiovascular diseases. Previous studies have shown that phytoestrogen α-zearalanol can protect cardiovascular system from hyperhomocysteinemia and ameliorate the level of plasma total homocysteine; however, the underlying mechanisms remain to be clarified. The aim of this research is to investigate the possible molecular mechanisms involved in ameliorating the level of plasma homocysteine by α-zearalanol. By the successfully established diet-induced hyperhomocysteinemia rat models, we found that, after α-zearalanol treatment, the activity of cystathionine ß-synthase, the key enzyme in homocysteine metabolism, was significantly elevated and level of nitrative stress in liver was significantly reduced. In correlation with this, results also showed a decreased nitration level of cystathionine ß-synthase in liver. Together data implied that alleviation of plasma homocysteine level by phytoestrogen α-zearalanol might be related to the reduction of cystathionine ß-synthase nitration.

Interactive effects of zearalenone and its metabolites on cytotoxicity and metabolization in ovarian CHO-K1 cells.

Zearalenone (ZEA) is a non-steroidal estrogen mycotoxin with high binding affinity to estrogen receptors. ZEA is rapidly absorbed and metabolized in vivo to α-zearalenol (α-ZOL) and ß-zearalenol (ß-ZOL). So, mixtures of them may be present in biological systems and suppose a hazard to animals and human health. The aims of this study were to determine the cytotoxic effects of ZEA and its metabolites, alone and in combination in ovarian (CHO-K1) cells during 24, 48 and 72h by the MTT assay; and to investigate the metabolism of the CHO-K1 cells on ZEA, and its conversion into α-ZOL and ß-ZOL by CHO-K1 cell after 24 and 48h of exposure. The IC50 value obtained for individual mycotoxins range from 60.3 to >100.0µM, from 30.0 to 33.0µM and from 55.0 to >75.0µM for ZEA, α-ZOL and ß-ZOL, respectively. Cytotoxic interactions were assayed by the isobologram method, which provides a combination index (CI) value as a quantitative measure of the degree of the three mycotoxin interaction. The CI values for binary combinations ranged from 0.56±0.15 (synergism at low concentrations) to 5.25±5.10 (addition at high concentrations) and tertiary combinations from 2.95±0.75 (antagonism at low concentrations) to 0.41±0.23 (synergism at high concentrations). The concentration of ZEA and its metabolites was determined with liquid chromatography coupled to the mass spectrometer detector-linear ion trap (LC-MS-LIT). The percentage of ZEA degradation ranged from 4% (24h) to 81% (48h). In the same conditions, α-ZOL and ß-ZOL concentration decreased from 8% to 85%. No conversion of ZEA in α-ZOL and ß-ZOL was detected. However, at 24h of exposure other degradation products of ZEA and its derived were detected.

Effects of increasing dosages of zeranol implants on lamb growth, carcass characteristics, blood hormones, and nitrogen metabolism.

The objective of this research was to compare the growth performance, incidence of prolapse and mortality, carcass characteristics, blood hormone concentration, and N balance of lambs implanted with increasing dosages of zeranol. One hundred forty-four crossbred lambs (29.6 ± 4.9 kg) were used in a completely random design and placed into 16 feedlot pens (4 pens/treatment) for a 116 d finishing study. Lambs were fed an 84.7% corn and 15.3% market lamb pellet (DM basis) diet ad libitum. Treatments were 0, 12, 24, and 36 mg zeranol (Ralgro; Schering-Plough), and lambs were implanted in the ear according to treatment on d 0. Lambs were weighed. Thirty lambs (67.6 ± 3.4 kg) and 96 lambs (65.8 ± 5.1 kg) were harvested on d 84 and d 118, respectively. Carcass data were collected 24 h after chill. Blood samples were collected on d 0, 28, 56, 70, 82, 99, and 116 from 64 lambs (29.6 ± 2.1 kg) in the feedlot study (subsample of 4 lambs per pen) and analyzed for thyroxine, triiodothyronine, and IGF-I. A second study was conducted to compare effects of 0, 12, 24, or 36 mg zeranol on N balance in 16 crossbred lambs (34.8 ± 2.1 kg). There were no differences among treatments for BW, ADG, DMI, and G:F (P > 0.05) in the feedlot study. However, there was a linear increase for incidence of prolapse (P = 0.006; 2.78, 5.55, 24.98, and 27.75%, respectively) and mortality (P = 0.005; 0.00, 5.55, 11.10, and 13.88%, respectively) as zeranol dosage increased. Carcass characteristics, blood hormone concentrations, and N balance were not affected by treatment (P > 0.05). These results indicate zeranol increases incidence of prolapse and mortality without increasing growth performance.

Changes in intramuscular fat, fatty acid profile and cholesterol content induced by zeranol implantation strategy in hair lambs.

BACKGROUND: The effect of zeranol implantation strategy on intramuscular fat, fatty acid profile and cholesterol content of the longissimus dorsi muscle of hair lambs was studied. Four treatments were tested: C, control group; Z12, 12 mg zeranol; Z24, 24 mg zeranol in a single application; and RZ12, 12 mg zeranol given twice. One-way analysis of variance was employed to estimate the effect of treatments (P < 0.05). To separate the effect of the mean, orthogonal contrasts were tested: C1, C versus Z12 + Z24 + RZ12; C2, Z12 versus Z24 + RZ12; and C3, Z24 versus RZ12. RESULTS: A decrease (P < 0.05) in intramuscular fat content was observed from implanting (C1 effect) and zeranol reimplantation (C3 effect). Implanted lambs exhibited an increase (P < 0.05) in monounsaturated fatty acids compared with control group (40.60% versus 35.35%). All contrasts were significant for the sum of n-6 and n-3, with values lower (P < 0.05) in the control (n-6: 0.84% and n-3: 1.38%) and higher in the RZ12 treatment (n-6: 7.55% and n-3: 14.9%). Cholesterol decreased by 78% with implantation and increasing the dose. CONCLUSION: The results indicate that it is possible to induce favorable changes in the fatty acid profile and cholesterol content using a zeranol implantation strategy on hair lambs.

Zeranol enhances the proliferation of pre-adipocytes in beef heifers.

BACKGROUND: The high morbidity and mortality of breast cancer among women is a serious problem. The adverse effects of the consumption of beef with zeranol (Z, a growth promoter widely used in beef industry in North American) residue on human health are still unknown. MATERIALS AND METHODS: The effects of Z implantation on the growth of heifer pre-adipocytes were evaluated. The stimulatory effects of Z and estradiol-17beta (E(2)) on the proliferation of pre-adipocytes isolated from control heifers and Z-implanted heifers were measured. Real-time PCR and Western-blotting analysis were performed to evaluate the expression of cyclin D1 and p53 at both mRNA and protein levels. RESULTS: The growth of pre-adipocytes from heifers bearing for 2 months of Z-implants was about 12-fold faster than that observed in control heifers. The pre-adipocytes isolated from Z-implanted heifers were more sensitive to treatment with Z and E(2). Z up-regulated the expression of cyclin D1 and down-regulated p53 in pre-adipocytes isolated from Z-implanted heifers. CONCLUSION: The implantation of Z increases body weight gain by enhancing growth of pre-adipocytes. The stimulation of pre-adipocytes division by Z and E(2) might be partially mediated by up-regulation of cyclin D1 and down-regulation of p53 at mRNA and protein levels.

Effects of Zeranol upon luteal maintenance and fetal development in peripubertal gilts.

Eighty gilts were utilized to determine whether zeranol implants could maintain hCG-induced corpora lutea (CL) in peripubertal gilts and to examine the effects of a Zeranol implant on fetal development. Crossbred gilts (171+/-0.3 days of age, 109.1+1.4 kg) were blocked by weight and ancestry to control (n=40) or treatment (n=40) groups. To induce ovulation and CL maintenance, treated gilts received 500 IU of hCG i.m. and a Zeranol ear implant (Ralgro, 36 mg; day 0). All gilts were checked once daily for estrus with a mature boar from days 3-58 of the experiment. On day 42, treated gilts received two 10 mg injections of Lutalyse (PGF(2)alpha) spaced 6 h apart. Treated gilts not displaying estrus within 7 days of PGF(2)alpha received two additional 10 mg of PGF(2)alpha spaced 6 h apart on day 49. On days 44-58, gilts detected in estrus were inseminated twice, 24 h apart with pooled semen via AI. Blood samples were obtained on days 0, 7, 18 and 42 and analyzed for serum progesterone (P(4)). Bred gilts were slaughtered on days 58-62 of gestation. Ovulation, as determined by serum concentrations of P(4) on day 7 of the experiment, was induced by hCG in 79.5% of treated gilts. Zeranol implants, however, failed to increase (P>0.05) the proportion of gilts available for breeding (treated, 21/39; control, 18/40). Of gilts inseminated on days 44-58, 16/21 treated gilts and 16/18 control gilts were pregnant at slaughter on days 58-62 of gestation. Number of fetuses (7.5 versus 12), fetal weight (83 versus 121 g), fetal length (117 versus 132 mm) and fetal survival (45% versus 78%) were reduced (P<0.001) by Zeranol implants. These data indicate that treatment of peripubertal gilts with a 36 mg Zeranol implant did not increase the proportion of gilts available for breeding while causing deleterious effects upon the fetuses.

Assessment of proliferative activity by AgNOR and PCNA in prostatic tissues of ram lambs implanted with zeranol.

The aim of this study was to assess cellular proliferation using silver-stained nucleolar organizer regions (AgNOR) and the proliferating cell nuclear antigen (PCNA) in various tissues in the prostate of ram lambs implanted with increasing zeranol doses and to compare the sensitivity of different tissues of lamb prostate to zeranol. Twenty-four Akkaraman lambs were implanted with increasing zeranol doses, including 12 mg (n = 8), 24 mg (n = 8) and 96 mg (n = 8), with eight lambs serving as controls. After 33 days, the prostate tissues of the lambs were stained using AgNOR and PCNA techniques. The prostate tissues were divided into two compartments--the epithelial tissues, including glandular acinus, collecting duct and penile urethra, and the non-epithelial tissues, including interstitial tissue and striated muscle. AgNOR dots and PCNA index on each prostatic tissue were counted under a light microscope and were evaluated statistically. AgNOR staining in the treatment groups showed a higher score in the non-epithelial tissues than the epithelial components, whereas the PCNA index was significant in the epithelial tissues and non-epithelial tissues had very low PCNA immunostaining. According to the PCNA index, collecting duct epithelium showed more sensitivity to increasing zeranol doses and according to AgNOR counts, there was no difference of sensitivity to zeranol among tissues of the same origin. Both AgNOR counts and PCNA indexes seem to be valuable proliferating markers for the epithelial components of ram prostate, but PCNA index had no significance in relation to the non-epithelial components in contrast to AgNOR counts. Therefore, the controversial results arising from the combined use of both techniques as proliferating markers for the ram prostate should be considered in further studies.

Transformation of MCF-10A human breast epithelial cells by zeranol and estradiol-17beta.

Among the endocrine factors associated with breast cancer, estrogens are considered to play a central role in human breast carcinogenesis. Breast cancer risks are increased by long-term exposure to estrogens. Zeranol (Ralgro) is a nonsteroidal agent with estrogenic activity that is used as a growth promoter in the U.S. beef and veal industry. To determine whether zeranol and estradiol-17beta play a role in the neoplastic transformation of human breast and to compare the estrogenic potency of zeranol to that of estradiol-17beta in human breast, we treated human breast epithelial cell MCF-10A with different doses of zeranol or estradiol-17beta for 10 repeated treatment cycles. By utilizing the doubling time assay, soft agar assay, and reverse transcriptase polymerase chain reaction (RT-PCR) assay, we showed that 10 repeated estradiol-17beta or zeranol treatment cycles to MCF-10A cells decrease the doubling time of the cells by 30 to 40% and stimulate colony formation in soft agar and induce estrogen receptor beta (ER-beta) mRNA expression, all of which are not dose related in our tested dose range. Furthermore, we show that zeranol and estradiol-17beta have a similar potency in the stimulation and inhibition of gene expressions in human breast cancer cell line MCF-7 by RT-PCR. These results indicate that both zeranol and estradiol-17beta can induce human breast epithelial cell neoplastic transformation with similar potency in the long-term exposure through the oxidation-reduction (redox) pathway and/or ER-beta-mediated pathway.

Effects of implant regimens (trenbolone acetate-estradiol administered alone or in combination with zeranol) and vitamin D3 on fresh beef color and quality.

In the first oftwo experiments, 123 calf-fed steers were used over a 2-yr period to evaluate the effects of trenbolone acetate (TBA)-based implants administered alone or in combination with zeranol implants on fresh beef muscle quality, color, and physiological maturity of the carcass. Implant treatments decreased (P < 0.05) a* values (d 0 and d 3 of retail display) and b* values (d 0, d 1, and d 3 of retail display) after 14 d of aging. Carcasses from cattle initially implanted with Revalor-S and reimplanted with Revalor-S on d 60 of the finishing period showed increased lean and bone maturity scores and ash content of the 9th to 11th thoracic buttons and Warner-Bratzler shear force values (WBS) compared to those initially implanted with Ralgro and subsequently reimplanted with Revalor-S or control cattle. In addition, implants decreased (P < 0.05) marbling, percentage of the carcasses grading Choice, and kidney, pelvic, and heart fat (KPH). Implant treatments increased (P < 0.05) ADG, hot carcass weights, and longissimus muscle (LM) area. In the second experiment over a 2-yr period, 166 steers fed as yearlings were allotted to one of two implant treatments and one of two vitamin D3 preharvest supplementation treatments. Implanted steers had heavier (P < 0.05) final body weights and higher (P < 0.05) ADG, less (P < 0.05) KPH fat, and larger (P < 0.05) LM. Also, implanted steers had more (P < 0.05) advanced bone maturity scores, higher (P < 0.05) ash content of the 9th to 11th thoracic buttons, and higher (P < 0.05) WBS values on 5-d postmortem loin steaks. Vitamin D3 feeding decreased (P < 0.05) final live weight, ADG (P < 0.05), and LM (P < 0.05), but did not significantly improve WBS values. In Experiment 2, neither implant treatment nor vitamin D3 supplementation had significant effects on L*, a*, or b* values of muscles in steaks before or during simulated retail display.

Metabolic profiles of the mycotoxin zearalenone and of the growth promoter zeranol in urine, liver, and muscle of heifers.

A group of five heifers were fed for 84 days with 2 kg of zearalenone-contaminated oats (1370 microg/kg) resulting in an average daily intake of 2740 microg of zearalenone per animal. In a parallel experiment five heifers were implanted with two 25 mg zeranol pellets, one at the beginning of the study and one after 42 days, and fed with 2 kg of "blank" control oats (79 microg/kg, daily intake = 158 microg). A third group of five animals were also fed with 2 kg of "blank" oats and served as control. Urine samples of all animals were collected every 5-6 days during the whole period of the study. Animals of all three groups were killed 84 days after the beginning of the feeding study. Tissue samples (back, femoral region, liver, and residues of implanted pellets) were taken during post-mortem investigations. The content of zearalenone and zeranol and their metabolites in urine and tissue samples was established by an analytical method combining solid-phase extraction and high-performance liquid chromatography-tandem mass spectrometry. Urinary excretion rates of zeralenone and zeranol were calculated from these results.

Impact of doramectin treatment at the time of feedlot entry on the productivity of yearling steers with natural nematode infections.

OBJECTIVE: To measure the reduction in fecal nematode egg counts and productivity impact of treatment of yearling steers with doramectin at entry into the feedlot, compared with control steers treated only with fenthion. ANIMALS: 6,096 crossbred yearling steers with a mean (+/- SD) body weight of 377.0 (+/- 37) kg. PROCEDURE: Steers were implanted with zeranol and alternately separated to fill each of 24 pens. Groups of steers within 12 matched pairs of pens were randomly allocated to treatment with doramectin or no treatment with doramectin for internal nematodes. Fecal samples were collected from approximately every twentieth steer from each pen at day 0 and at reimplant (approx day 60). Each steer was weighed on day 0 and at reimplant and then mean body weights of steers per pen were determined at 120 to 140 days after trial initiation. RESULTS: Treatment steers had a significantly lower fecal egg count at reimplant than control steers. Treatment steers had a significantly greater mean daily gain during the study, significantly greater feed consumption, significantly lower feed-to-gain ratio, and significantly better quality carcass grades at slaughter. CONCLUSIONS AND CLINICAL RELEVANCE: Under the conditions of our trial, there was a significant fecal egg count reduction response to doramectin treatment, which resulted in significantly improved productivity. Results of economic analysis of return on investment indicated that even with low egg counts in heavy body weight cattle, nematode egg count reduction with doramectin significantly improved returns.

Hormone contents in peripheral tissues after correct and off-label use of growth promoting hormones in cattle: effect of the implant preparations Filaplix-H, Raglo, Synovex-H and Synovex Plus.

Certain hormonal growth promoters are licensed in several beef producing countries outside the European Union (EU). Use in compliance with Good Veterinary Practice is mandatory. As risk assessment of hormone residues in animal tissues up to now has neglected potential off-label use, the present study dealt with two topics: 1) multiple treatment with the implant preparations Finaplix-H (200 mg trenbolone acetate), Ralgro (36 mg zeranol) and Synovex-H (200 mg testosterone propionate plus 20 mg estradiol benzoate) in heifers (1-fold, 3-fold and 10-fold dose), and 2) non-approved treatment of female veal calves (1-fold dose of Synovex-H or Synovex Plus with 200 mg trenbolone acetate plus 28 mg estradiol benzoate). Residues of estradiol-17beta, estradiol-17alpha, estrone and testosterone, trenbolone-17beta, trenbolone-17alpha and trendione or zeranol, respectively, were measured in loin, liver, kidney and peri-renal fat by high performance liquid chromatography/enzyme immunoassay (HPLC/EIA) after liquid-liquid extraction and solid-phase clean-up. The hormone residues in the multiple-dose experiments were dose-dependent and partially exceeded the threshold values: in the liver in one animal after 3-fold dose and in two animals after 10-fold dose of Finaplix-H, and in the liver and kidney after 3-fold and 10-fold dose of Synovex-H. Mean hormone residues in calves were mainly below those of heifers and did not infringe threshold values.

Quantification of rainbow trout (Oncorhynchus mykiss) zona radiata and vitellogenin mRNA levels using real-time PCR after in vivo treatment with estradiol-17 beta or alpha-zearalenol.

Estrogen receptor-mediated induction of zona radiata (ZR) and vitellogenin (VTG) mRNA and protein in rainbow trout (Oncorhynchus mykiss) was compared to assess their utility as biomarkers for exposure to estrogenic compounds. Partial sequences of rainbow trout ZR and beta-actin were cloned by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerate primers based on conserved regions across a number of species. A 549 bp fragment of the rainbow trout ZR-gene showed a high degree of amino acid sequence identity to that of salmon (77%), winter flounder (64%), carp ZP2 (63%) and medaka (61%) ZR-proteins. The 1020 bp beta-actin fragment was approximately 100% identical to sequences from several species. Real-time PCR was used to quantify the induction of ZR-gene and VTG in rainbow trout liver after in vivo exposure to estradiol-17 beta (E(2)) (0.01, 0.1, 1.0 or 10 mg/kg body weight (bw) fish) or alpha-zearalenol (alpha-ZEA) (0.1, 1.0 or 10 mg/kg bw). Real-time PCR and indirect enzyme-linked immunosorbent assay (ELISA) showed that ZR and VTG were induced in both the liver and the plasma after a single injection of E(2) or alpha-ZEA. ZR was more responsive to low levels of E(2) and alpha-ZEA than VTG, and real-time PCR was shown to be more sensitive than the ELISA. Rainbow trout ZR-gene and proteins provide a sensitive biomarker for assessing estrogenic activity.