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Evaluation of boldenone as a growth promoter in broilers: safety and meat quality aspects
The object of this study was to evaluate the safety and meat quality criteria in broilers following intramuscular injection of boldenone. Twenty-four broiler chicks, divided into two groups, were used in the present study. Boldenone was injected intramuscularly at a single-dose level of 5 mg/kg body weight into 12 broiler chicks at 2 weeks old; the other 12 chicks were injected with sesame oil and kept as controls. Blood samples were collected from the wing and metatarsal veins after 1, 2, and 3 weeks through the experimental course for hematological and clinic-chemical safety parameters. On the last day, chicks were humanely sacrificed and livers and kidneys were removed for histopathological examination.
Breast muscles were also removed to assess meat-quality parameters. Boldenone significantly (p < 0.05) increased total erythrocytic count and hemoglobin and hematocrit values, while mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration indices decreased. Leukogram showed leukopenia, lymphopenia, and granulocytosis (p < 0.05) as compared to control. Hepatorenal biomarkers, including alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, urea, and creatinine were significantly (p < 0.05) higher than the corresponding control values.
Additionally, boldenone significantly (p < 0.05) increased metabolic markers, including total protein, globulins, cholesterol, triacylglycerols, and glucose, with parallel decreases in albumin and albumin/globulin ratio. Degenerative changes were recorded in liver and kidney tissues from chicks treated with boldenone. Muscle samples exhibited raised pH values and higher microbial counts as compared to the corresponding control. These data may discourage the use of boldenone as a growth promoter in broilers due to safety and meat quality reasons.
1. Boldenone Introduction
Anabolic androgenic steroids are synthetic derivatives structurally related to androgens (the male sex hormones). These agents promote development of male sexual characteristics (androgenic effects) and, thus, are used in treating delayed puberty, impotence, and related conditions. They also promote growth of skeletal muscle (anabolic effects) and can be used in repairing wasting of the body caused by some emaciating diseases . However, these anabolic steroids can be abused in human and animal sports for better performance . Also, they can be used for increasing body weight in livestock as growth-promoting agents either legally or illegally.
Growth-promoting implants have been used in the production of cattle and sheep for >40 years. Such implants may improve growth rates (approx. 10–30%), feed efficiency (approx. 5–15%), and carcass leanness (approx. 5–8%). Historically, the earliest use of hormones as growth enhancers in farm animal production included iodinated proteins in dairy cows for increased milk production and estrogen implants (diethylstilbestrol and dienestrol) in broilers for enhanced fat deposition .
The first hormone used in beef cattle and sheep for growth, efficiency, and lean meat promotion was diethylstilbestrol in 1954 . However, because of potential carcinogenicity from the use of diethylstilbestrol in humans, this compound was banned for use in 1979 by the Food and Drug Administration (FDA). Parallel to this trend, most hormonal growth promoters have been banned, and the restricted use of only a few agents have been permitted. Many countries permit the use of a short list of growth-promoting steroid hormones to bulk up animals and increase the yield of meat. The USA and Canada, in addition to all developing countries, follow this practice; however, the EU has banned the use of hormones in food-producing animals due to health concerns .
Boldenone is an anabolic steroid that differs from testosterone by its high anabolic and low androgenic activities. Therefore, it has been produced as a veterinary product as boldenone undecylenate under various trade names for improving meat mass in cattle and veal calves. Boldenone is restricted to veterinary purposes only in some countries; nonetheless, sports competitors and bodybuilders have been known to administer this anabolic steroid.
It was approved by the U.S. FDA for use in horses; however, in most countries worldwide, it is forbidden for meat production and human use . The control of boldenone for illegal uses was based on the identification of either 17β-boldenone or 17α-boldenone (the main metabolite in cattle) in edible tissues, hair, feces, or urine . Boldenone has been increasingly detected in a number of biological samples in different EU member states. The question arose concerning whether this increased number of boldenone findings was due to the illegal treatment of animals or whether, in some circumstances, boldenone could be of endogenous origin. For instance, it was demonstrated that boldenone could be formed from phytosterols present in vegetable fat , . Previous studies were conducted to study the effects and metabolism of boldenone in various species, including horses , cattle , veal calves , weaned lambs , rabbits , , and human volunteers .
No data are available concerning the altering effects of boldenone administration to broilers as a popular farm animal species. Therefore, this study was designed to investigate the possible effects of boldenone on broiler chicks from safety and meat quality aspects as a trial to evaluate its possible use as a growth promoter in the broiler industry.
2. Boldenone Methods
Chemically, 17β-boldenone, also called 1-dehydrotestosterone, androsta-1,4-diene-17β-ol-3-1, is a steroid with androgenic activity that differs from 17β-testosterone by only one double bond at the 1 position. Esters of 17β-boldenone (e.g., undecylenate ester) are pharmaceutically produced as anabolic preparations for veterinary use.
In the present study, boldenone was obtained as the patent preparation Equigan (Laboratorios Tornel S.A., México) that is an intramuscular therapy for muscle building in equines. This preparation is formulated as 10 mL glass vials containing 50 mg boldenone undecylenate/mL sesame oil. The drug was administered intramuscularly in the thigh as single doses of 5 mg/kg body weight of broiler chick.
2.2. Experimental animals
Twenty-four Ross broiler chicks, aged 2 weeks and weighing approximately 600 g, were used for the present study. Animals were purchased from a local farm at 1 week old and kept in our controlled environment for a further week for acclimatization before use. All animals were maintained on standard growing rations and water ad libitum. Animal care and experimentation were according to guidelines of the European Commission Directive 86/609/EEC for animal experiments and were approved by our local institutional committee.
2.3. Experimental design
A parallel design is followed in this study, where animals were randomly divided into two groups (n = 12 for each) and labeled appropriately. The first group received 0.1 mL of sesame oil (solvent of boldenone) intramuscularly in the thigh muscle and kept as the control group. The second group received a single dose of boldenone undecylenate (5 mg/kg) as 0.1 mL of sesame oil-diluted Equigan injectable solution in the same manner and kept as the treated group. After different treatments, all animals were observed daily for extraordinary symptoms throughout the period of study.
Blood samples were collected from the wing and metatarsal veins using syringes with 22-gauge needles 1 week, 2 weeks, and 3 weeks post-injection. The samples were received into centrifuge tubes containing lithium heparin. Each sample was divided into two parts, with the first kept as whole blood for hematological analysis and the second centrifuged for 5 min at 12,000g to separate plasma that was used for the clinicochemical study. At the end of experiment, all animals were humanely sacrificed, and the liver and kidneys were removed for histopathological examination. Also, pectoral muscles on the keel bone were picked for meat-quality examination.
2.4. Hematological assays
To assess the blood-safety profile of boldenone, the following hematological parameters were automatically evaluated by auto-hematology analyzer (Mindray, Model BC-2800Vet, Shenzhen, China). Erythrocytic parameters included red blood cell (RBC) count, hematocrit value (Hct), mean corpuscular volume (MCV), hemoglobin concentration (Hgb), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).
Leukocytic parameters included white blood cell (WBC) count, differential leukocyte count (neutrophils, lymphocytes, monocytes, eosinophils, and basophils), platelet (PLT) concentration, and mean platelet volume (MPV). The analyzer adopted the Coulter Principle to count RBC, WBC, and PLT cells and to draw their corresponding histograms. The Hgb concentration was obtained by the colorimetric method and the MCV was calculated electronically. The rest of the indices were mathematically derived.
2.5. Clinicochemical assays
Estimating plasma clinicochemical parameters and evaluating hepatotoxicity, nephrotoxicity and/or metabotoxicity was carried out spectrophotometrically (Model 6500, Jenway, Germany) using diagnostic kits purchased from Analyticon Biotechnologies AG (Lichtenfels, Germany). The parameters were alkaline phosphatase , aspartate aminotransferase , alanine aminotransferase , total protein , creatine kinase , albumin , urea , creatinine , glucose , and total cholesterol .
Plasma triacylglycerol level was estimated using a diagnostic kit purchased from Biolabo SA (Maizy, France) according to Fossati and Prencipe . Globulin level was calculated by subtracting the value of albumin from the value of total protein according to Doumas and Biggs .
2.6. Histopathological assay
The liver and the two kidneys were taken from sacrificed birds in both groups, preserved in formalin solution (10%), and subjected for histopathological examination according to Bancroft and Gamble .
2.7. Determination of pH values of chicken breasts (pectoralis) samples during cold-chilled storage
Chicken breasts (pectoralis) pH values for the boldenone and control groups were determined after 2 h, 24 h, 48 h and 72 h post mortem during cold storage at 4°C. The measurement was based on using a fiber-optic pH Meter (Jenway 3505 pH Meter, Barloworld Scientific Ltd., Dunmow, UK) after calibrating using standard buffers at pH 4.0 and pH 7.0 (Merck Millipore, Waltham, MA, USA). The glass electrodes were mechanically inserted into pre-opened pectoralis with a sterile scalpel .
2.8. Microbiological Analysis
Two groups of chicken breasts (pectoralis) from boldenone-treated and control groups were subjected for microbiological analysis after 24-h storage at 4°C. Preparation of samples, decimal dilutions, culturing, and enumeration techniques for bacteria were performed according to the methods described by the American Public Health Association , . Briefly, 25 g of the sample was removed with sterile scalpels and transferred to a sterile Stomacher bag (Stomacher 400, Seaward Medicals, UK.) under aseptic conditions.
The sample was then diluted to a 10:1 dilution with 225 mL peptone water (M0216, Park Scientific, Ltd., Northampton, UK) and stomached for 2 min using a Seward’s Stomacher 400 Circulator (Stomacher 400, Seaward Medicals). Serial dilutions were then performed using sterile 0.1% peptone water (Park Scientific, Ltd.). Determination of the aerobic plate count (APC) was performed using plate-count agar (Difco Laboratories, Detroit, MI, USA), inoculated with serial dilutions on triplicate agar plates, and incubated at 37°C for 48 h. Countable plates were those containing from 25 to 250 colonies.
Determination of coliform count was performed using Violet Red Bile Lactose agar (VRBA, Park Scientific, Ltd.). VRBA triplicate agar plates were inoculated using an overlay method and incubated at 37°C for 24 h. Purple-red colonies, 0.5 mm in diameter or larger, surrounded by a zone of precipitated bile acids were counted.
Enumeration of total Staphylococci count was performed using Baird-Parker agar (Park Scientific, Ltd.). The inoculums were surface plated using a sterile bent-glass streaking rod on triplicate agar plates. Plates were inverted and incubated at 37°C for 48 h.
2.9. Statistical analysis
Results are expressed as mean ± standard error of the mean for 12 observations (n). A factorial linear statistical model was fitted to the data, and differences between the control and treated group at different time points were tested for significance using a one-way analysis of variance, followed by least-significant difference post-hoc test. A p ≤ 0.05 was considered significant. All statistical analytical procedures were done using SPSS software version 20 (IBM Corp., Armonk, NY, USA).
The animals post-boldenone administration appeared lively with good appetites and did not show any abnormal signs. No mortalities were recorded throughout the experiment, and no significant increases in body weight gain were recorded.
3.1. Hematological study
The administration of boldenone significantly affected both the erythrocyte and leukocyte parameters as compared to the control group (p > 0.05). A single dose of boldenone (second group) significantly increased RBC count and Hct and Hgb values in the 2nd and 3rd weeks. Meanwhile, MCH and MCHC index values decreased. Boldenone significantly decreased total leukocytic count, with changes in differential count, where lymphocytes exhibited significant decreases and granulocytes exhibited significant increases as compared to control (Table 1, Table 2).