research methodology

CHAPTER THREE: MATERIALS AND METHODS

3.1 Data Collection Procedure

In order to achieve the results of the first objective of the study which was to investigate the common antibiotics used in Nakawa division, the researcher used a questionnaire design to collect data from 50 employees of the 5 selected poultry farms in Nakawa division where each farm had 10 employees purposely selected.

Sample of Questionnaire

Dear Respondents,

I am MUGARURA GASTON, a student of Kyambogo University pursuing A master’s degree in chemistry. Am conducting a study on “on determination of antibiotics residues in chicken meat”. The research study is a partial requirement leading to the award of a master degree. Therefore, the information provided therein is for only academic purposes and will be highly treated with utmost confidentiality.   

Part I: BIO DATA

Please tick your appropriate option

What is the name of this farm…………………………………………………………………?

PART II: THE COMMON ANTIBIOTICS USED AT THIS FARM

In a bid to achieve the results of the second objective of the study which is to investigate the common antibiotics used in Uganda, the researcher poses different questions with the alternatives to the poultry farmers in Nakawa division and the respondents are expected to tick their appropriate option

Which of the following antibiotics do you use here?

Please tick your appropriate option

If any other antibiotic used, specify………………………………………

Who administers these antibiotics?

Where do you get these antibiotics?

Any other source, specify………………………………………………

Why do you use these antibiotics?

3.2: Sample Collection

The fresh samples of Breast Muscle, Liver and Gizzard of broilers fifty (50) in number respectively were randomly collected from difference markets of Nakawa, Bugolobi, Luzira, Ntinda and Banda in Nakawa Division, located in Kampala City Council Authority. The coordinates of the division are 0° 20’00.0″N, 32° 37’00.0″E (Latitude: 0.333333; Longitude: 32.616667 (UBOS, 2014).

The collected samples, were packed in properly labelled sterile polyethylene bags at room temperature and transported to the Directorate of Government Analytical Laboratory (DGAL) in Wandegeya, Kampala and kept under refrigeration at 4^oC.

3.3: Equipment

Laboratory blender (Thomas Scientific, USA), Vortex mixer-VM18 (Schiltern Scientific, Beds, UK), Centrifuge (Hettich D-78532, Germany), Centrifuge tubes, Pasteur pipettes and Rubber bulbs, Nitrogen evaporator – 6 Position N-Evap (Thomas Scientific, USA), Mas Hunter work station software LC/MS Data Acquisition for 6400 series triple quadrupole, version B.08.00 and Quantitative Analysis version B.07.01/Build 7.1.524.0 for QQQ.

3.4: Sample Preparation

The 150 whole tissue samples of liver, breast muscle and gizzard of broilers were blended using a laboratory blender (Thomas Scientific, USA) each sample separately to make minced samples that could easily be analysed. 4 g of the minced samples were measured and properly stored at -18 ^OC in preparation for samples analysis.

3.5: Chemicals

The purity of all standard chemicals and reagents were at least 99% HPLC grade, methanol (Merck-Germany), Trichloroacetic acid (TCA), diethyl ether and acetone were used.

3.6: Preparation of standards

Enrofloxacin (ENR), Oxytetracycline (OTC), Tetracycline (TC), Penicillin G and Chloramphenicol (CAP) are USP reference standard (USP pharmacopeial convention), USA. The standards for the selected antibiotics were prepared by dissolving 0.1 gm of powder in 4 mL solution of methanol and standard solutions were stored in – 4°C.

3.7: Sample analysis

The minced samples that were analysed were stored in a refrigerator to temperatures of -4 ⁰ C for an hour.  However, where the analysis could not be performed within 2 days, the samples were stored to a temperature of -18 ⁰ C and to further ensure that the samples remained in the same state after two days, 50 µL of work solution of internal standards (multresidue) were added to 4 g of minced samples in their storage facilities.

Sample extraction was performed according to Poppelka (2005) where to the 4 g of each minced samples, 10 mL Phosphate Buffer Saline (pH-6.5) was then added and mixed by vortexing using a Centrifuge (Hettich D-78532, Germany) @ 4000 rpm for 20 min after mixing with 2 mL 30% TCA. The supernatant was removed using Pasteur pipette and filtered by Whatman filter paper of 50 µm in thickness. The Filtrate was collected in another falcon tube and 2 mL of diethyl ether was added and left for 10 min in room temperature. The bottom layer was collected and extraction was repeated twice using diethyl ether. Final volume of the filtrate was pooled carefully into screw cap vial and kept into refrigerator for future analysis.

The tube containing the filtrate was put into a water bath at 40^oC under nitrogen flow and ether was evaporated to dryness.

3.8: Sample analysis

The remains of the filtrate were reconstituted in 1 ml water/methanol/acetonitrile (50/25/25) +/- 0.05% acetic acid and vortexed.

The filtrate was transferred in a LC-MS vail and capped with a capper. Five (5µL) of the filtrate was injected in heated LC-MS system where the mobile phase entered the column from the left, passed through the particle bed, and exited at the right.  The flow occurred in the column at time zero (the moment of injection), when the sample extracts entered the column and begun to form a band, a mixture of dyes, appeared at the inlet of the column as a single black band where a detector was used to monitor the separated compounds as they eluted. After a few minutes during which mobile phase had flown continuously and steadily past the packing material particles, the individual dyes moved in separate bands at different speeds. Since each dye band moved at different speed, separation of residues chromatographically was achieved and the data collected by the computer was used to analyse the levels of antibiotics in the samples.

The analysis of the antibiotic residues Oxytetracycline (OTC), Tetracycline, Enrofloxacin, Penicillin G and Chloramphenicol were done using analytical column C18 Eclipse Plus, 95, 1.8µm, 21 x 100 mm, manufactured by Agilent Technologies, Column Description: ZORBAX RRHD Eclipse C18, Pore Size: 95, Particle Size: 1.8µ, Inner Diameter: 2.1mm, Length: 100mm, Carbon Load: 9%, USP Number: L1, Category: Reversed Phase (RP) part number 959758-902.

The separation of antibiotics was accomplished at 30 °C, the flow rate and injection volume were 0.5 mL/min and 10 μL, respectively. The mobile phases used were (A) water and (B) acetonitrile. The gradient elution program started with 10% B for 1 min, increased to 65% for 6 min, then increased to 95% for 1 min and returned to the initial conditions in 1 min. The final run time of the method was 12 min. The separation of sulfamides, tetracyclines, and quinolones was accomplished at 40 °C. The flow rate and injection volume were 0.3 mL/min and 10 μL, respectively. The mobile phases used were (A) TFA (0.1%) in water and (B) acetonitrile. The gradient elution program was as follow: A (90%) (3min), A (25%) (5 min), and A (90%) (1min); the final run time of the method was 15 min.

3.9 Data analysis

Data analysis on the concentration of antibiotics was analysed using SPSS statistical program (SPSS Inc., Chicago, IL, USA). Analysis of variance, ANOVA, was performed to compare levels of antibiotic residues in chicken breast muscle, liver and gizzard.

CHAPTER FOUR

RESULTS AND DISCUSSION

4.1. Response Rate

The study sought to gather information from staff of different poultry farms in Nakawa division. A total of 50 questionnaires were distributed to 50 employees of the 5 selected poultry farms in Nakawa division and only 45 were collected having been filled completely with useful information. This made a response rate of 90% which was sufficient for data analysis.

4.2 Bio Data of the Respondents

Results of the bio-data of respondents including gender, age, marital status, education level, and period spent at the farm are presented below:

Table 4.1: Showing the Bio Data of the Respondents

Source: Primary Data 2022

The table above show that 60% (27) of the respondents were male and 40% (18) were female. This implies that the information provided was from both genders.

The table above also show that 51.1% (23) of the respondents were in the age group of 30-39years, 22.2% (10) in the age bracket of 20-29year, 15.6% in the age bracket of 40-49years and 11.1% (05) in the age bracket 50-59 years. This implies that information in the study is the representative of all age bracket of poultry farm staff.

The table above shows that 57.8% of the respondents were married, 24.4% were single and 17.8% were widowed.

The table above shows that 37.8% of the respondents were degree holders, 28.9% were certificate holders, 26.7% were degree holders and 06.7% were master’s degree holders. This implies that the information provided is from informed point of view.

The table above shows that 51.1% of the respondents had above 5 years’ experience in poultry farming, 26.7% had experience of 2-4 years and only 22.2% had experience in poultry farming for less than one years.

4.3 The types of antibiotics used by poultry farmers in Nakawa Division

In a bid to achieve the results of the first objective of the study which was to investigate the common antibiotics used by poultry farmers in Nakawa Division.

Table 4.2: Showing the Common Antibiotics Used

Source: Primary Data

Tetracycline shows a high usage rate with 82% responding positively. Only 18% reported not using it. This suggests that Tetracycline is a commonly employed antibiotic in the dataset. Oxytetracycline has a lower usage rate compared to Tetracycline, with 35.6% responding positively. The majority (64.4%) indicated not using Oxytetracycline, Enrofloxacin has a moderate usage rate, with 57.8% responding positively. Around 42.2% reported not using Enrofloxacin. Penicillin is widely used, with 68.9% responding positively. However, there is a significant portion (31.1%) that does not use Penicillin.

Chloramphenicol has a relatively high usage rate, with 62.2% responding positively. About 37.8% reported not using Chloramphenicol, these findings provide insights into the prevalence of antibiotic usage in the dataset. Further analysis may be needed to understand the reasons behind the choices and to assess the effectiveness of these antibiotics in the given context. It is also essential to consider factors such as dosage, duration of use, and potential implications for antibiotic resistance.

Source: Primary Data 2022

The figure above shows that 82.2% (37) of the respondents use Tetracycline at the poultry farm, 68.8% (31) use Penicillin,62.2% (28) use Chloramphenicol, 57.8% (26) use Enrofloxacin while only 35.6% (16) use Oxytetracycline. This implies that the commonly used antibiotics in poultry are Tetracycline, Penicillin, Chloramphenicol and Enrofloxacin respectively.

4.4 The administering of antibiotics

The study sought to investigate who administers the antibiotics used by poultry farmers in Nakawa Division. The results from the study that were collected are illustrated below,

Table 4.3: Showing the administering of antibiotics

Source: Primary Data 2022

The table 4.3 above show that 24.4 % (11) of the respondents accepted that antibiotics used by poultry farmers in Nakawa Division are administered by farmers themselves, 84.4 % (38) by Veterinary doctors, 95.6 %(43) use because the antibiotics are effective on the birds, 42.2 %( 19) use purposely to boost fast growth of their birds and 46.7 %( 21) by Farm employees. This means antibiotics are administered by qualified personnel with relevant knowledge in poultry farming.

4.5 The sources of antibiotics

The study also sought to identify the sources of antibiotics and the following results were obtained

Table 4.4: Showing the sources of antibiotics

Source: Primary Data 2022

The table 4.4 above show that 88.9 % (40) of the respondents accepted that antibiotics are brought by veterinary doctors, 60 % (27) from veterinary drug shops, and 04.4 % (02) from hawkers. This implies that most poultry farmers get antibiotics mainly to treat their birds from recommendable sources hence administering right drugs.

4.6 Reasons for the use of Antibiotics

The study also sought to identify the reasons for the usage of antibiotics and the following results were obtained.

Table 4.5: Reasons for the use of antibiotics

Source: Primary Data 2022

The table 4.5 above show that 46.7 % (21) of the respondents use antibiotics because they can easily have accessed, 100 % (45) use antibiotics to treat their birds when they get sick, 95.6 %(43) use because the antibiotics are effective on the birds, 42.2 %( 19) use purposely to boost fast growth of their birds and 17.8 %( 08) use because they are cheap. This implies that most poultry farmers use antibiotics mainly to treat my birds when they get sick, antibiotics are effective, and some to boost fast growth of the birds.

The results showed that 99% of the farmers utilize at least of the antibiotics under investigation namely, Enrofloxacin, Penicillin, Oxytetracycline, Tetracycline and Chloramphenicol in treatment of poultry and almost 80% administer all the antibiotics to the poultry animals. The results continue to show that 100% of the poultry farmers use the antibiotics under investigation for the purposes of treating their poultry birds against sickness from different poultry diseases. The results further showed that 95.6% of the poultry farmers use antibiotics because they are effective on any diseases. This is in agreement with Clarke. B, (2004) & Gerber et al. (2007) who noted that the increasing market base has led to usage of antibiotics for growth promotion and fighting diseases to meet with the demand and supply locally and internationally.

4.7: The antibiotic residue in breast muscle, liver and gizzard of broilers collected from selected local markets in Nakawa Division.

In a bid to achieve the results of the second objective of the study which was to identify and quantify the antibiotic residue in breast muscle, liver and gizzard of broilers collected from selected local markets in Nakawa Division.

4.7.1: Concentration of Antibiotic Residues in Gizzard Samples from selected Markets in Nakawa Division.

Figure 10: Shows Concentration of antibiotic residues in Gizzard samples collected from selected markets

The results used to obtain the graphical presentation of Concentration of Tetracycline (TC), Oxytetracycline (OTC), Enrofloxacin, Penicillin G and Chloramphenicol (CAP) of Gizzard samples collected were obtained from table 1 in the appendix B.

4.7.2: Concentration of Antibiotic Residues in Muscles Samples from selected Markets in Nakawa Division.

Figure 11: Shows Concentration of antibiotic residues in Muscle Samples collected from 5 selected markets

The results used to obtain the graphical presentation of Concentration of Tetracycline (TC), Oxytetracycline (OTC), Enrofloxacin, Penicillin G and Chloramphenicol (CAP) of Muscle samples collected were obtained from table 2 in the appendix B.

4.7.3: Concentration of Antibiotic Residues in Liver Samples from selected Markets in Nakawa Division.

Figure 12: Shows Concentration of Antibiotics residues in Liver samples collected from selected markets

The results used to obtain the graphical presentation of Concentration of Tetracycline (TC), Oxytetracycline (OTC), Enrofloxacin, Penicillin G and Chloramphenicol (CAP) of Liver samples collected were obtained from table 3 in the appendix B.

A total of 150 random fresh poultry Liver, Muscle and Gizzard samples from different local markets (Ntinda, Bugolobi, Banda, Luzira and Nakawa) in Nakawa division, Kampala, Uganda were collected and examined using Liquid Chromatography-Mass Spectrometry (LC-MS) to evaluate the antibiotics residues present (Ahmed & Gareib, 2016).

The study revealed that of 30 samples collected from Ntinda market, 13.33% had the occurrence of antibiotic residues namely; Oxytetracycline (OTC), Tetracycline (TC) and Enrofloxacin (ENRO), with a mean residual concentration of 7.88 ppb and of the positive samples, 99.65%, 0.35% and 0.00% were found in Liver, Muscles and Gizzard respectively with 67.45%, 1.05% and 31.50% Enrofloxacin, Tetracycline and Oxytetracycline antibiotic residues respectively.  In addition, of the 30 samples collected from Bugolobi market, 30% had the occurrence of antibiotic residues namely; Oxytetracycline (OTC), Tetracycline (TC) and Penicillin G (PEN), with a mean residual concentration of 24.41 ppb and of 30% contaminated samples 51.43%, 29.04% and 19.53% were found in Liver, Muscles and Gizzard respectively with 86.16%, 11.21% and 2.63% Tetracycline, Oxytetracycline and Penicillin G antibiotic residues respectively.

To add on, the 30 samples collected from Banda market, 73.33% had the occurrence of antibiotic residues namely; Oxytetracycline (OTC), Tetracycline (TC), Enrofloxacin (ENRO), Penicillin G (PEN) and Chloramphenicol (CAP) with a mean residual concentration of 49.37 ppb and of the contaminated samples, 23.36%, 38.91% and 37.73% were found in Liver, Muscles and Gizzard respectively with 0.02%, 6.83%, 25.84%, 35.04% and 32.28% Oxytetracycline, Tetracycline, Enrofloxacin, Penicillin G and Chloramphenicol respectively.

Furthermore, of the 30 samples collected from Nakawa market, 40% had the occurrence of antibiotic residues namely; Oxytetracycline (OTC), Tetracycline (TC), Enrofloxacin (ENRO), Penicillin G (PEN) and Chloramphenicol (CAP) with a mean residual concentration of 53.77 ppb. It continued to show that of 40% contaminated samples 71.92%, 10.84% and 17.24% were found in Liver, Muscles and Gizzard respectively with 4.13%, 54.27%, 9.72%, 0.44% and 31.44% Oxytetracycline, Tetracycline, Enrofloxacin, Penicillin G and Chloramphenicol respectively.

Lastly, of 30 samples collected from Luzira market, 6.67% had the occurrence of antibiotic residue namely; Penicillin G with a mean residual concentration 0.01 ppb. The 6.67% contaminated samples were found in Liver only. These antibiotics have the capacity to stay and be absorbed in an animal’s tissues more so if the initial dosage is high or if the withdrawal time is less that the recommended before slaughtering (Mund et al., 2017) making the easily detectable as residues and some antibiotics for example Tetracycline and Oxytetracycline have a property that enables them to adsorb onto the organic matter which could result into degradation of the antibiotics making it easy for such to be detected by LC-MS (Pugazhendhi et al., 2021).

The study indicated that, Banda market had the highest percentage of contaminated poultry samples with 54.78% followed by Nakawa with 32.55%, Luzira had the lowest percentage with 0.001%. The highest percentage of contaminated poultry carcass in Banda could be attributed to the annual presence of students of Kyambogo University most of whom reside in Banda and majority of these students buy poultry carcasses as food thereby increasing the demand which forces the breeding and brooding companies to use antibiotics as growth promoters to meet the market demand. Similarly, the high percentage of antibiotics residues in samples collected from Nakawa market could be due to the presence of several institutes of higher learning such Management Training and Advisory Center (MTAC), Makerere University Business School (MUBS) etc. However, the lowest percentage of antibiotic residues in Luzira market could be attributed to its proximity and lack market for poultry products compared to Nakawa and Banda markets.

It is evident that Liver samples had the highest percentage of antibiotics residues with 43.49% followed by Muscles and Gizzard with 28.07% and 28.45% respectively (Aslam et al., 2016; Jakubowski et al., 2010; Tajik et al., 2010; Yibar et al., 2011). The highest percentage of antibiotic residues in liver is attributed to the fact that liver is an organ responsible for protein synthesis, metabolism and detoxification of most toxic substances and residues from the body (Ramatla et al., 2017).

The mean residual levels of Oxytetracycline (OTC), Tetracycline (TC), Enrofloxacin, Penicillin G and Chloramphenicol (CAP) were within the maximum residual limits (MRLs) of antibiotics recommended for human consumption (Codex Alimentarius, 2018a) except for Penicillin G in Gizzard sample collected from Banda market which was 212.64 ppb above the MRLs recommended level for human consumption of 200 ppb. Although this was the case, it is confirmed that antibiotics usage in poultry is a common practice (Bartkiene et al., 2020). This research continue to show that given different regulations and recommendations on the application of antibiotics in poultry for treatment and growth promotion, the residues had detectable antibiotics confirming continued usage and failure to adhere to the recommended withdrawal period (Tajik et al., 2010; Ahmed & Gareib, 2016; Aslam et al., 2016b) which may present public health implications (Ben et al., 2019; Vougat Ngom et al., 2017; Kang et al., 2021; Girmatsion et al., 2021;  de Faria et al., 2021).