Description of the study area
The study was conducted in Bishoftu, Oromia Regional State which is found about 47 km south east of Addis Ababa, the capital city of Ethiopia. Bishoftu is located at 8º 43’- 8º 45’ N latitude and 38º 56’E longitudes at an altitude of 1,850 meters above sea level in the central high land of Ethiopia. It has an annual rainfall of 866 mm of which 84% is in the long rainy season (June to September). The dry season extends from October to February. The mean annual maximum and minimum temperatures are 26 and 14°C respectively, with mean relative humidity of 61.3% (ADARDO, 2007).
Study design, Sample size determination
Across sectional study designs was used to determine the level of aflatoxin in poultry feed during December, 2018 to May, 2019. Large commercial poultry farmers were unwilling to provide samples because of the fear of risks of contamination of their farm and the regulatory actions that may be taken by the government authorities if the publication of the research result is un rewarding as a result small to medium scale poultry farms organized in micro-enterprises in Bishoftu town were used as a target of the study in collaboration with the Bishoftu town municipal office by advising the microenterprise members about the importance of the study.
Based on the secondary data obtained from Bishoftu municipal office there were a total of 36 Small to medium scale poultry farm micro-enterprises actively functioning in 2019. Therefore, this number is so relatively small and the sampling fraction exceeds 10%, the finite population correction factor (FPC) was used to calculate the required sample size (n’) as follow: (Daniel, 1999)
n’ = 1/(1/n+1/N)
Where, n= the original estimate of the required sample size in an infinite population and it is calculated as follow:
Where, Zα=1.96,p = expected contamination (p=50%=0.5), q=1-p=0.5, L2=5% = 0.05, 1.96 (CI = 95%) hence, n=384 and N=36= number of poultry farm micro-enterprises actively functioning in 2019 in Bishoftu town. Thus, the desired sample size was n’=33
Sample collection and sampling
Poultry feed samples were collected randomly from five vilages based on the accessibility of small scale poultry farms and sampling. They include Air force (n=3), Kajima (n=7), Hora (n=8), Cheleleka (n=8) and Babogaya (n=8). Since aflatoxins occur in heterogeneous fashion in feed, stratified random sampling was used to make a composite sample consisting of subsamples from every part of a store, sack, or unit of feeds. During sampling the feeds were grouped into 4 parts depending on the layers and height of the bags. Then choose a simple random sample from each group and 100g of sample was weighed and zipped with plastic bag.
Analytical Procedure
The aflatoxin content in the feeds and feed ingredients was determined at the veterinary drug and animal feed administration and control authority-Ethiopia, following the manufacturer’s instructions. All chemicals and reagents were of analytical grade and HPLC standard. (Sigma-aldrich, St. Louis, MO, USA). The working solutions of aflatoxins B1, B2, G1 and G2 standards (50 µg/g of each aflatoxin in capped amber bottles) were prepared according to the AOAC procedure (AOAC, 2000)
Instrumentation: Quantitative analyses of the aflatoxins were carried out using a HPLC unit that consisted of a pump and quaternary gradient system. Fluorescence detector was used for the quantitation under the following conditions: 360 nm excitation and 440 nm emission. The analytical column was a ZORBAX SB-C18, 150 x 4.6, 3.5μm particle size. All HPLC analysis were carried out under isocratic conditions using a mobile phase of water: acetonitrile: methanol (60:25:15) and the flow rate was fixed at 1.0 ml/ min. The injection volume was 20 μl and it was standard injection with needle wash. Stop time for quaternary pump was 10 min and the temperature of the column was 35°C on both sides (Shimadzu, USA)
Sample Preparation: Sample preparation was conducted using the method of AOAC Official method 950.02 for animal feed. To achieve the maximum particle size reduction and thoroughness of the mix the entire lots of samples were ground through hammer mill and passed through number 14 sieve split sample sequentially in sample splitter. The coarse portions were regrind and Weigh 50gm from each sample for aflatoxin estimation.
Extraction: About 20 g of grounded sample was added into a beaker with 2.0 g of NaCl. Then extraction was done with 100 ml methanol/water (4/1, v/v) and 50 mL of n-hexane in a blender jar at high speed for five minutes. The extract was then passed through a plaited filter. Fourteen milliliter of the purified extract was added to 86 mL PBS buffer (pH 7.2). Finally the evenly mixed sample was filtered using a Buchner funnel and a filter paper.
Clean-up: The AflaCLEANTM immunoaffinity column was used for cleanup purposes. After opening the column, the storage buffer was drained until the level reaches the upper frit. The sample was passed through a 0.2 µm syringe filter to remove residual turbidity. Then 25 mL of the diluted extract was taken and passed through the AflaCLEANTM column with a flow rate of 2 mL/min (1 drop/sec). Then the sample was allowed to drain through the column until there is no more sample in the column. The column was then washed with 10 mL of distilled water and the residual water removed from the column by applying a gentle pressure of vacuum pump. Finally the elution was done using 1ml of methanol at least 2 times. The first addition of methanol was let for 5 minutes in order the methanol act on the column to break the aflatoxin-antibody bond.
Calibration: Calibration curve was prepared using the working calibration solutions prior to analysis according to the method and check the plot for linearity. These solutions cover various ranges of aflatoxin concentrations. To establish a calibration curve, 8 calibration points of a mixed aflatoxin standard containing various concentrations per injection solvent (0.5, 1.25, 2.5, 5, 7.5, 10, 15, 20 µg/g) prepared. Linear regression was performed using statistical program. The correlation coefficient of calibration curve is an indicator of method quality. The correlation coefficient should be greater or equal to 0.995 to run the samples with it. Accordingly it was found that 0.99978, secured to run analysis.
Determination of moisture content:
Moisture content in poultry feed samples were determined using official methods of AOAC. The samples were dried at 105°C to constant weight, and the average content was calculated as percentage on wet basis (AOAC International, 2011).
Statistical analysis
For data analysis, Microsoft Excel 2013 and IBM SPSS Statistics version 20 software were used. One-way analysis of variance (ANOVA) was performed to evaluate the levels of total aflatoxin mean comparison between the study villages. A P-value of less than 0.05 (P < 0.05) was considered as statistical significance.