2.1. Ethical approval of laboratory animals The experimental broilers were used ethically and at the end of the experiment sacrificed humanely following the ethical and welfare guidelines set by the Animal Welfare and Experimental Ethics Committee of Bangladesh Agricultural University [approval number: AWEEC/ BAU/2021(07)]. 2.2. Study design A preliminary survey on poultry farming was made in the north area (Dinajpur District) of Bangladesh and five commercial farms were selected for the study after making a contract with the farmers. A meeting was held in each farm to obtain data of rearing system, treatment records, name & type of drugs used, withdrawal period maintenance etc. Awareness campaign also held in each farms to teach the poultry farmers on residual effects of antibiotics hazards for human health. After successful discussion, necessary samples (liver, thigh muscles and breast muscles) were collected for laboratory investigation. On the other hand, indoor experiment was also done on discriminate and indiscriminate use of antibiotics in broiler birds. At the end of experiments, liver, thigh muscles and breast muscles were collected & preserved under -20°C until TLC evaluations. 2.3. Indoor antibiotics experiments on broiler birds 35 apparently healthy day-old chicks (Cobb-500) were collected for indoor experiments. Chicks were reared for up to 30 days. On day 14th, the chicks were randomly divided into five groups namely control group (group A, n=6), discriminate ciprofloxacin antibiotic group (group B, n=6), indiscriminate ciprofloxacin antibiotic group (group C, n=6), discriminate oxytetracycline antibiotic group (group D, n=6) and indiscriminate oxytetracycline antibiotic group (group E, n=6). Group A was kept as untreated control and received no medicated water. Group B & C were administered ciprofloxacin and group D and E were administered oxytetracycline antibiotics. After 7 days, at the age of day 23; antibiotic supply was stopped in the group-B and D followed by withdrawal period. In group C and E, antibiotics were continued till the day of sacrifice.
2.4. Sacrificing & sample collection At the end of experiment, birds were sacrificed and liver, breast muscles and thigh muscles were collected and preserved at -20° C until examination.
2.5. Screening of antibiotic residues The experiment was conducted in the Department of Pharmacology, Bangladesh Agricultural University (BAU), Mymensingh. Collected samples were brought to the Postgraduate Laboratory for investigation of antibiotic residues by Thin Layer Chromatography (TLC) technique. All chemicals used were analytical grade.
2.6. Thin Layer Chromatography (TLC) Thin layer chromatography (TLC) is a sensitive and exact-reliable method for monitoring low amount of different biological and chemicals. Thin layer chromatography (TLC) is among the most helpful instruments for following the advancement of organic chemical reactions and for measuring the pureness of organic compounds in phytochemistry and Biotechnology. Like every single chromatographic methods, TLC exploits the distinctive affinity of the analyte with the mobile and stationary phases to accomplish the partition of complex mixtures of organic molecules [8]. Illumination of antibiotics against UV light helps as a simple detector for this mean. Employment of thin-layer chromatography (TLC) to pharmaceutical and medical or clinical or biological research comprises more than 50% of the technique’s total application. An important application of TLC is determination of drug residues in food.
1. TLC plate was cut into appropriate size (5x4 cm) 2.A straight line was drawn across the plate approximately 2cm from the bottom by a pencil. Another straight line was drawn across the plate below 1cm from the upper edge of the plate. 3. Desired spots marking were marked on the bottom line where analytes were dropped. 4. Spots were applied to the plate using thin capillary glass pipettes. A volume of 10μl was used for spotting. 5. Plate was placed in TLC tank (contained mobile phase: Acetonitrile and Methanol; 1:1) and covered by lid and it was left until the mobile phase reached the upper line. 6. Spots were visualized in UV detection box at 256 nm 7. Spots marking were done by pencil for calculation of retention factor (Rf)
2.8. Interpretation of results In order to interpret the results, first setting of standard with reference/pure substances was determined with three repeated times of examination by standard solution. A substance was positively identified in the unknown solution when it behaved identically as the reference substance. That is, after comparison of two substances (standard & unknown) based on following criteria a sample was positively identified such as:
- Same color under UV light
- Same Rf value as those of the reference sample.