Assessment of Aflatoxin B1 Contamination Level in Dairy Animal Feeds from Selected Towns of North Shoa Zone, Oromia, Ethiopia

Assessment of Aflatoxin B1 Contamination Level in Dairy Animal Feeds from Selected Towns of North Shoa Zone, Oromia, Ethiopia | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessment of Aflatoxin B1 Contamination Level in Dairy Animal Feeds from Selected Towns of North Shoa Zone, Oromia, Ethiopia Kasaye Bahiru Tola, Girma Salale Geleta, Argachew Nugussa This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6540017/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aflatoxins are secondary metabolites produced by fungal species in moldy animal feeds and can be transferred into the milk of lactating cows that consume aflatoxin-contaminated feeds. Among several types of aflatoxins, aflatoxin B1 (AFB1) is highly toxic and causes acute or chronic toxic effects to both humans and animals. This study, therefore, was conducted to determine the levels of AFB1 in dairy cow feed samples collected from Fiche and Degam towns of North Shoa Zone. A total of 50 feed samples comprising 41 composited and 9 individual of each composited were collected randomly from smallholder dairy farmers, and the levels of AFB1 were determined via High-Performance Liquid Chromatography coupled with Fluorescence Detector (HPLC-FLD) after being cleaned with immune affinity columns. The findings revealed that the highest AFB1 content in composited feed samples was 30.8 µg/kg and the lowest was 0.0100 µg/kg. Out of 41 analyzed composited feed samples, 35 (85.4%) samples were positive for AFB1, and 6 (14.6%) were found to be below the limit of detection. Furthermore, of the positive feed samples for AFB1, 16 (39.0%) contained AFB1 that exceeded the European Commission maximum permissible level (5 µg/kg) for dairy feed samples. Thus, the finding of this study highlights the importance of rigorous regulatory monitoring and improved feed handling techniques to safeguard consumers of animal-derived products from the serious health consequences linked to aflatoxins. AFB1 Toxic Animal feeds Smallholder dairy farmers Ethiopia Figures Figure 1 Figure 2 Figure 3 Introduction Animal feed is a balanced feed given to domestic animals, especially livestock, to promote overall health and milk production [ 43 ]. However, it is vulnerable to a variety of naturally occurring microorganisms that can cause contamination and deterioration on farms before harvest, during storage, and at storage locations [ 41 , 5 , 28 , 17 ]. Farm forage, hay, cereals, commercial concentrates (noug seed cake, peacake, maize and mix, frushka, and feed additives), and atella are the most popular dairy animal feeds required for health and increased milk production; however, they are susceptible to various chemical contaminants [ 38 ]. Among the various contaminants, fungal contamination is particularly alarming due to the potential production of aflatoxins, such as aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2), in dairy animal feed [ 32 ]. For example, toxigenic fungi such as Aspergillus flavus , Aspergillus parasiticus , and Aspergillus nomius produce aflatoxins as secondary metabolites in animal feeds and foods, leading to health risks in both humans and animals [ 7 , 8 , 18 ]. Among the most common types of aflatoxins in dairy animal feeds, AFB1 is highly toxic and causes acute or chronic toxic effects, including teratogenic, mutagenic, carcinogenic, immune toxic, or hepatotoxic effects in humans [ 30 , 13 , 27 ]. It can be metabolized hydroxylatively to AFM1 by mammalian bioconversion [ 2 , 26 , 39 ]. According to the International Agency for Research on Cancer (IARC), AFB1 is classified as a group I carcinogen as well as a genotoxin for humans and animals [ 34 , 1 , 21 , 44 ]. Thus, to ensure feed quality and public health, several countries and international organizations have set a maximum permissible limit for AFB1 in various dairy animal feeds. These standards vary depending on the economic status of the countries and the global guidelines provided by organizations such as the Food and Agricultural Organization/World Health Organization (FAO/WHO), European Union (EU), and Food and Drug Administration (FDA). For example, EU/FAO/WHO set the maximum limit of AFB1 in feed to 5 µg/kg; FDA has set the action levels for total aflatoxins in animal feed to 20 µg/kg [ 14 , 8 , 9 ]. Therefore, it is important to assess the contamination levels of AFB1 in animal feeds in order to minimize the adverse effect of aflatoxins on human and animal health. Several studies have reported AFB1 contamination of dairy animal feeds from various locations in Ethiopia. For example, recent research conducted by Tadele et al. in northwest Ethiopia found that the level of AFB1 ranged from 1.75 to 306.9 µg/kg and 96% of feed samples were contaminated with AFB1 [ 35 ]. Similarly, a study by Fikadu et al. in the Oromia Special Zone surrounding Finfinne with a range from 12.67 to 45.67 µg/kg and 26.7% of feed samples were contaminated with AFB1 (16). Additionally, a study by Mesfin, Besufekad and coworkers, Gizachew et al. in (Bishoftu, Holetta, and Hawassa), in the Gurage Zone, in Greater Addis Ababa reported the levels of AFB1 with a range between 0 and 20 µg/kg, 4.22 and 10.54 µg/kg, 7 and 419 µg/kg, respectively, which exceeded the EU’s maximum tolerance limit (5 µg/kg) [ 25 , 8 , 14 ]. Despite several studies conducted in Ethiopia, there are areas within the country that require thoroughout investigation. To our knowledge so far there is no coordinated investigation of the levels of AFB1 in various types of dairy cow feed within the selected towns of North Shoa Zone, where a high amount of utilization of feed is available by individual dairy farmers to increase milk production. Therefore, the objective of the current study was to determine the levels of AFB1 in dairy cow feed samples collected from two selected towns from North Shoa Zone, Oromia, Ethiopia, using HPLC-FLD technique. It is anticipated that this investigation would provide insightful information about the present level of AFB1 contamination in dairy cow feed. Materials and methods Study areas and sample collection The study was conducted between February and May 2024 in the North Shoa Zone, Oromia, Ethiopia, in two towns called Fiche and Degam, mainly by considering the presence of a large number of smallholder dairy farmers. The geographic location of the North Shoa Zone lies between 2738 and 2782 m above sea level at 90 N, 380 E and is characterized agro-ecologically as upper tropical to highland. The administrative center of this zone, Fitche Town, is located approximately 114 km from Addis Ababa, the capital city of Ethiopia, with a latitude of 9° 48' 0.00 N and a longitude of 38° 43' 59.99" E. Degam (Hanbiso) town is one of the woreda towns in the North Shoa Zone of Oromia, which is approximately 127 km away from Addis Ababa, with latitude 9° 39' 59.99" N and longitude 38° 39' 59.99" E. A total of 50 feed samples consisting of 41 different composite feed samples and 9 most used individual feed of the composited feed samples were collected randomly from smallholder dairy farmers of Fiche and Degam towns. 100 g of each size- reduced dairy cow feed sample was collected in a polyethylene bag, kept in ice packs during transportation, and stored at 2–8°C in a refrigerator in the laboratory to avoid any fungal growth acceleration in the packed feed until analysis [ 14 , 36 – 37 ] Methods of extracting AFB1 from dairy cow feed The procedure used for AFB1 extraction and clean-up was based on the instructions of the Ethiopian Conformity Assessment Enterprise (ECAE) for assessment of AFB1 in animal feed. Concisely, a 25g representative homogenized feed sample was weighed in a 250 mL beaker, and 125 mL of 70% methanol and 30% deionized water was added and homogenized for 10 minutes via a polytron homogenizer at 4188 x g and the extract was subsequently filtered with fast-flow filter paper in a 250 mL conical flask. 15 mL of the filtrate was taken into a 100 mL Erlenmeyer flask and 30 mL deionized water was added in to IAC obtained from Libios (Pontcharra sur-Turdine, France) for the purification of the filtrate. Subsequently, 15 mL of the filtrate was loaded into the conditioned immune-affinity column at a rate of 1 drop per second. It was then washed with 10 mL of deionized water and allowed to air dry. The AFB1 was then eluted with 3 mL of methanol. The eluted mixture was evaporated to dryness in a 40°C nitrogen gas evaporator. Finally, and before HPLC-FLD analysis, the final residue was reconstituted with 1 mL of a mobile phase consisting of a deionized water: acetonitrile: methanol mixture (60:25:15) and a volume of 10 µL was injected into the injection loop for both the standard and sample solutions into the HPLC coupled with a fluorescence detector. The proposed schematic representation for the determination of AFB1 via HPLC-FLD is shown in Fig. 2 Analysis method using HPLC-FLD A HPLC (water alliance e2695) system equipped with FLD (2475) was employed for the quantification of AFB1. The chromatographic separation was achieved on a reversed-phase chromatographic C18 column (3.50mm column; 4.60 × 150mm) (HS, Bellefonte, USA). The mobile phase consisted of 60% deionized water, 25% acetonitrile, and 15% methanol (v/v/v), which was filtered through a micro-syringe filter for use. The following instrument parameters were set: injection volume of 10.00µl, flow rate of 1mL min − 1 , run time of 10 minutes, temperature set to 35°C, excitation wavelength of 360nm, and emission wavelength of 440nm. AFB1 standard was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and was used to prepare a series of AFB1 standard solutions (0.5, 1, 2, 4, 6, 8 and 10 µg/L) using the deionized water:acetonitrile:methanol mixture (60:25:15 v/v) through dilution. Calibration curves of peak areas versus AFB1 concentrations were then plotted and used for AFB1 determination in samples. Determination of AFB1 in dairy cow feed samples The concentrations (µg/kg) of AFB1 in cow feed samples were determined via HPLC‒FLD. The actual amount of AFB1 in µg/kg was calculated via the following Eq. ( 1 ) below: $$\:\text{m}\text{t}=\frac{\text{V}2.\text{V}4}{\text{v}1.\text{v}3}\text{*}\text{m}\text{o}$$ 1 ……………… Where: mt is the mass of the test sample present in the fraction of the second filtrate taken for the immune affinity column in grams (g) mo is the mass of the test portion in grams (mo = 25 g). V 1 is the total volume of the first filtrate in milliliters (V 1 = 125 mL). V 2 is the fraction volume of the first filtrate taken for dilution in milliliters (V 2 = 15 mL). V 3 is the total volume of the second filtrate in milliliters (V 3 = 45 mL) V 4 is the fraction volume of the second filtrate in milliliters (V 4 = 15 mL). The mass fraction of each aflatoxin ( wi ) in µg/kg of sample was calculated via Eq. ( 2 ) below. $$\:\text{w}i=\frac{v5.mi}{v6.mt}$$ 2 ……………………. Where: V 5 is the volume of the eluate in microliters (V 5 = 1000 µL). V 6 is the volume of the purified and injected sample extract in microliters (V 6 = 10 µL). mi is the mass of each aflatoxin present in the injection volume, corresponding to the measured peak area read off the calibration graph in nanograms (ng). mt is the mass of the test sample present in the fraction of the second filtrate taken for the immune affinity column in grams (g) [ 24 ]. Statistical analysis All statistical analyses i.e., mean, standard deviations, %RSD, coefficient of determination (r 2 ), maximum and minimum concentration was carried out using SPSS (Statistical Package for the Social Sciences). An independent t-test was applied at 95% mean confidence interval to determine mean differences of AFB1 concentrations in dairy cow feed samples between the two towns. The concentrations of AFB1 are expressed as the means ± standard deviation, minimum and maximum values. Results and discussions Method of Validation Limit of detection (LOD), limit of quantification (LOQ), accuracy (recovery), and precisions (repeatability) were determined to test the validity of the HPLC-FLD method for AFB1 determination in dairy cow feed. Linearity was determined by constructing seven-point calibration curves for AFB1 standard solutions across concentration ranges of 0.500, 1.00, 2.00, 4.00, 6.00, 8.00 and 10.0 µg/kg. The calibration curves were constructed by plotting the peak area against the concentration of AFB1, with linearity determined through linear regression analysis, indicated by the coefficient of determination (r²). Hence, LOD was found to be 0.0100 µg/kg, calculated as 3 times the standard deviation (SD) of the blank sample relative to the analytical curve slope (m), 3SD/m. Similarly, LOQ was found to be 0.0500 µg/kg, calculated as 10 times the standard deviation (SD) of the blank sample relative to the analytical curve slope (m) (10SD/m). Furthermore, the accuracy and precision evaluation results of all sample types are expressed in percent of recoveries and percent of relative standard deviation, respectively. Thus, the calculated percentage recovery of AFB1 for triplicate measurements (n = 3) in spiked feed samples ranged between 70.0% and 95.5%, and the %RSD for triplicate measurements (n = 3) ranged between 1.63% and 2.23%. This demonstrates the accuracy (recoveries) and precision (%RSD) of the method were found within the range of European Commission limits of 70.0–120% and < 20.0% respectively [ 31 , 24 ]. Therefore, the optimized and validated HPLC-FLD method used in this study was considered to be both accurate and precise because all the determined method performance characteristics were found to be consistent with EC regulative requirements [ 12 ]. Applied to this method of validation, the mean value of the all analysed composite dairy cow feed samples for AFB1 level was summarized in the Fig. 3 . AFB1 contamination levels in dairy cow feed samples A total of 50 feed samples were collected and analyzed for AFB1, comprising 41 different composited feed samples (20 samples from Fiche town, 21 samples from Degam town) and 9 individual feed sample of most used in composited feed sample (4 samples from Fiche town, 5 samples from Degam town). AFB1 was identified in 35 feed samples, representing 85.4% of the 41 analyzed different composited feed samples, with concentrations from 0.0100 to 30.8 µg/kg. Notably, only 6 samples, representing 14.6% of the total, were found to be below the limit of detection (LOD) of 0.0100 µg/kg. Variation in the level of AFB1 in dairy cow feed between two towns AFB1 was found in 35 (85.4%) dairy cow feed samples, among these, 16 samples (39.0%) had levels of AFB1 above the European Commission's maximum allowable limit (5 µg/kg) and 19 samples (46.4%) had levels below the European Commission's maximum allowable limit (5 µg/kg). The analysis of dairy cow feed samples from 2 towns revealed varying concentrations of AFB1; with statistically significant differences among the samples from these towns at the 95% confidence level, as determined by independent t-test (P < 0.05). The Fiche sample exhibited the higher mean concentration at 13.4 µg/kg compared to Degam town at 3.14 µg/kg, as shown in Table 1 as well as the highest individual measurement of AFB1 concentration levels, as shown in Fig. 3 . Table 1 Summary of AFB1 contamination level in most used composite dairy feeds Location N Contaminated sample Range(µg/kg) Mean ± STD Above EU (5 µg/kg) N % N % Degam 21.0 17.0 81.0 0.0100–12.3 3.14 ± 3.25 3.00 14.3 Fiche 20.0 18.0 90.00 0.0100–30.8 13.4 ± 10.4 13.0 65.0 Total 41.0 35.0 85.4 0.0100–30.8 16.0 39.0 N:-represents number of sample Upon comparing the two towns, 7.30% (n = 3) and 31.7% (n = 13) of the feed samples collected from Degam and Fiche towns were found to exceed the maximum permissible level (5µg/kg) of AFB1 established by the European Commission [ 11 ], respectively. Furthermore, to determine how much each individual contributed to the overall contamination of the composite feed samples with AFB1, the most often used individual components of the composite feed samples, such as hay, mixin, frushka, maize grain, and pea cake, were also analyzed, as results shown in Table 2 . Thus, the lowest level of AFB1 contamination was detected in the hay collected from the two towns. This indicates that the sample's contribution to the composite feed sample that was collected from two towns was negligible. Table 2 Levels of AFB1 in most commonly used individual feed samples taken from two towns Location Feed type Level of AFB1 (µg/kg) Fiche Frushka ND Mixin 19.8 Pea cake 0.10 Hay ND Max - min 0.01–19.75 Degam Frushka 23.8 Mixin 20.2 Pea cake 8.30 Hay ND Maize grain 12.1 Max - min 0.01–23.80 Hint: - ND-represents less than limit of detection To the opposite, the highest degree of contamination of AFB1 level (19.8 µg/kg) was detected in the mixin feed sample collected from Fiche town. Similarly, 23.8 µg/kg and 20.2 µg/kg degrees of contamination were detected in frushka and mixin feed samples collected from Degam town, respectively. These clearly demonstrated that these two types of individual feed samples contributed more contamination than did other individuals in a composite feed. Thus, factors such as the local environment, high humidity, and the absence of storage animal feed in small, confined places may be responsible for the apparent variation in AFB1 levels in dairy cow feed from the study location [ 19 ]. Comparison of the results of this study with those of other studies The presence of AFB1 contamination in dairy feed samples has been reported in Ethiopia as well as worldwide, as shown in Table 3 below. The results of this study indicated lower levels of AFB1 in dairy feed samples compared to those reported in Ethiopia by Gizachew et al. , which ranged from 7 to 419 µg/kg [ 14 ] Similarly, the research conducted by Fikadu et al. reported higher concentrations of AFB1, ranging from 12.67 to 45.67 µg/kg [ 16 ]. Additionally, Tadele et al. reported AFB1 concentration ranging from 1.75 to 306.9 µg/kg, which is higher than those found in the current study [ 35 ]. Furthermore, several countries, such as Pakistan (10, 42), Egypt [ 15 , 3 ], India [ 20 ], Kenya [ 22 ], and Malawi [ 29 ], have reported higher AFB1 levels than those observed in the current study. On the other hand, the results of this finding revealed a greater range of AFB1 than those values reported in Ethiopia by Tefera and Vegarud, which ranged from 0.067 to 29.69 µg/kg [ 37 ]; Yegrem, which ranged from 0.164 to 0.490 µg/kg [45]; India, which ranged from 0.9 to 15 µg/kg [ 31 ]; Morocco, which ranged from 1.02 to 13.59 µg/kg [ 4 ]; and Spain, which ranged from 0.07 to 5.17 µg/kg [ 9 ], whereas this finding ranged from 0.0100 to 30.8 µg/kg, and 85.4% of feed samples were contaminated with AFB1. However, it was in line with the values reported by Tefera & Vegarud in the selected rural area of the Sidama Zone (Ethiopia) that ranged from 0.067 to 29.69 µg/kg, and 82.9% of feed samples were contaminated with AFB1 [ 37 ]. In general, the disparity of those results reported in literature and this study might be due to sampling periods, different quantification techniques, geographical conditions, methods of harvesting and storing animal feed, type of feed samples, and sampling techniques [ 28 , 33 , 23 , 6 ]. Table 3 Comparison of AFB1 levels in dairy feeds with various location of Ethiopia and those reported in literatures. Location Methods of Detection Type of feed N o of sample +ve sample(%) Range(µg/kg) References Ethiopia ELISA wheat bran and noug cake 156 26.2 7-419 14 Ethiopia HPLC wheat bran, noug seed cake, 90 26.7 12–46 16 Ethiopia ELISA hay, grain byproducts, and maize stove 240 82.9 0.067–29.69 37 Ethiopia ELISA atella, hay, commercial concentrates 100 96 1.75–306.9 35 Spain HPLC Compound feed 22 86 0.07–5.17 9 Egypt HPLC Grain and fourages 20 55 7.16–69.81 15 Morocco ELISA Dairy feed 44 40.91 1.02–13.59 4 Pakistan ELISA Dairy concentrate 50 64 5.31–42.39 10 India HPTLC Cattle feed 24 26 20–60 20 India HPLC Concentrate feeds 189 59 0.9–15 31 Kenya ELISA Fourage, concentrates 74 56 0–147.86 22 Malawi ELISA Maize bran, Dairy mash and Hay 51 62.7 2.36–60.29 29 Egypt UPLC Complete feed samples 83 30.12 3-300 3 Ethiopia HPLC-FLD -composite of concentrate feed -Individual of composited feed 41 9 83.36 66.67 0.0100–30.8 0.0100–23.8 This study Conclusion In this study, different concentrations of AFB1 were detected in dairy feed samples collected from 2 towns within the North Shoa Zone, exhibiting considerable levels of contamination. Overall, of 41 different composited dairy feed samples collected from two towns, 85.4% (n = 35) were contaminated with AFB1, and 39.0% of contaminated dairy cow feed samples had AFB1 levels exceeding limits set by the European Commission (5 µg/kg). Furthermore, significant levels of AFB1, which can hydroxylate to AFM1, were found in most dairy feed samples that were collected from Fiche town. In light of this result and based on the analyses of the most used individual of the composited feed samples, the major contributors to AFB1 contamination level in composite feed samples were found to be mixin and frushka. Therefore, on the basis of individual sample feed results, risk mitigation should focus on mixin and frushka. Finally, owing to financial constraints, the designed seasonal variation and location of the studied area may not be sufficient; therefore, it is highly recommended that additional research be conducted on the seasonal variation of AFB1 contamination levels in dairy animal feeds and at different areas with a representative number of samples since this study is the baseline for further studies. Declarations Conflict of Interest All authors declare no competing interests. Funding: The author (s) disclosed receipt of the following financial support for the research, authorship, and /or publication of this article: This study was financially supported by Salale University. Author Contribution The study was carried out by Kasaye Bahiru Tola, Girma Selale Geleta, and Argachew Nugussa. Kasaye Bahiru Tola wrote the manuscript and created the figures and tables, while Girma Selale Geleta and Argachew Nugussa offered additional insights and discussions regarding the article. The final version of manuscript has been approved for publication by all authors after review. Acknowledgement The authors would like to acknowledge the Department of Chemistry, Salale University, for the funds provided for this study. Additionally, we would like to extend our thanks to the Ethiopian Confirmative Assessment Enterprise (ECAE), Addis Ababa, for providing laboratory facilities during the study period. 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Patyal A, Gill JP, Bedi JS.& Aulakh RS (2021) Assessment of aflatoxin contamination in dairy animal concentrate feed from Punjab, Indian Journal of Chelonian Conservation And Biology, 28: 37705–37715, Popescu RG, Luminit A, & Radulescu SEG (2022) Aflatoxins in Feed: Types, Metabolism, Health Consequences in Swine and Mitigation Strategies, Journal of Toxins, 14: 853. Seetha A, Munthali W, Msere H W, Swai E, Muzanila Y, Sichone E, Tsusaka T W, Rathore A & Okori P (2017) Occurrence of aflatoxins and its management in diverse crop ping systems of central Tanzania. Mycotoxin Research, 33(4): 323–331. Senerwa DM, Sirma AJ, Mtimet N, Kang’ethe EK, G.D. and J.L. (2016) Prevalence of aflatoxin in feeds and cow milk from five counties in kenya, Food, Agriculture,Nutrition and Development, 16(3): 11004–11021. Tadele F, Demissie B, Amsalu A, Demelash H, Mengist Z, Ambelu A & Yenew C (2023) Aflatoxin contamination of animal feeds and its predictors among dairy farms in Northwest Ethiopia: One Health approach implications,Journal Front. [Preprint]. https://doi.org/10.3389/fvets.2023.1123573 Tefera AK (2017) Determination of Aflatoxins in Maize (Zea Mays L.) samples from Dera and Fogera Districts of South Gondar Using LC- MS / MS, Journal of Agricultural and Food Sciences. Tefera W, Vegarud GE, Taye M & Taye T (2022) Aflatoxin contamination in cow milk during wet and dry season in selected rural areas of Sidama zone Southern Ethiopia, International Journal of Food Properties, 25(1): 2425–2434. Tesfaye A, Kurtu M.Y, Mummed YY, Mohammed A (2024) Aflatoxins Levels in Concentrate Feeds Collected from Specialized Dairy Farms and Local Markets in Selected Urban Centers of Eastern Ethiopia. Toxins 2024, 16: 418. https://doi.org/10.3390/toxins1610041 . Torres B, Salazar D, Cachiguango M (2020) Determination of Aflatoxin M1 in Raw Milk from Different Provinces of Ecuador’, Journal of Toxins, 12(8): 498. Visiol F. & Strata A (2014) Milk, dairy products, and their functional effects in humans: A narrative review of recent evidence, Journal of Advances in Nutrition, 5(2): 131–143. Waqas M, Pervaiz W, Zia KM & Iqba SZ (2021) Assessment of aflatoxin B 1 in animal feed and aflatoxin M 1 in raw milk samples of different species of milking animals from Punjab, Pakistan, Journal of Food Safety. https://doi.org/10.1111/jfs.12893 Worku B (2016) Indigenous Processing Methods of Cheka: A Traditional Fermented Beverage in Southwestern Ethiopia, Journal of Food Processing & Technology, 07(01). doi.10.4172/2157-7110.1000540 Yang X, Liu B, Zhang L, Wang X, Xie J, Liang J (2025) Spatial Distribution and Dietary Risk Assessment of Aflatoxins in Raw Milk and Dairy Feedstuff Samples from Different Climate Zones in China, Toxins, 17: 41. Yegrem L (2020) Aflatoxin Contaminations Levels in Animal Feeds and, Austin Journal of Veterinary Science & Animal Husbandry, 7(2): 1072. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6540017","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":448594846,"identity":"3128a962-9649-40cb-b065-26e7743957fb","order_by":0,"name":"Kasaye Bahiru Tola","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+UlEQVRIiWNgGAWjYBACCQh1gMEARH0AYjZ2UrQwzgBpYSZFCzMPiE1Ii2T78YefeSruyJuzn30mbfNrmzwfMwPjh485uLVI8+QYS/OceWa4syfdTDq377ZhGzMDs+TMbbi1yDHkMEjzth1m3HAgjU06t+c2I1ALGzMvPi38zx//5v132H7D+Wds0pY9t+0JapGWSDCT5m04nLjhBtAWhh+3EwlqkZzxxsxyzrFnyRtuPGO27G24ndzGzNiM1y8S59Mf33hTc8d2w/k0xhs//ty2nd/efPDDRzxaQICJB8ZibAOTDfjVg5T8gDP/EFQ8CkbBKBgFIxAAAGMxUkZPju4uAAAAAElFTkSuQmCC","orcid":"","institution":"Salale University","correspondingAuthor":true,"prefix":"","firstName":"Kasaye","middleName":"Bahiru","lastName":"Tola","suffix":""},{"id":448594847,"identity":"4c2b361f-c825-441f-9fd0-95546051e9c8","order_by":1,"name":"Girma Salale Geleta","email":"","orcid":"","institution":"Salale University","correspondingAuthor":false,"prefix":"","firstName":"Girma","middleName":"Salale","lastName":"Geleta","suffix":""},{"id":448594848,"identity":"c98f85fa-c591-4b75-992f-a8a0b26ac789","order_by":2,"name":"Argachew Nugussa","email":"","orcid":"","institution":"Salale University","correspondingAuthor":false,"prefix":"","firstName":"Argachew","middleName":"","lastName":"Nugussa","suffix":""}],"badges":[],"createdAt":"2025-04-27 11:23:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6540017/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6540017/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81615818,"identity":"00b2c88b-971d-4898-a629-c7d82fd4b3c7","added_by":"auto","created_at":"2025-04-29 08:09:23","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":122430,"visible":true,"origin":"","legend":"\u003cp\u003eMap of the North Shoa Zone, Oromia, Ethiopia\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6540017/v1/bc1fbc0dbe96980bb6c1dc9b.jpeg"},{"id":81614978,"identity":"3d0dfdf9-bece-4367-b65d-218ebfab50bf","added_by":"auto","created_at":"2025-04-29 08:01:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":180706,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of AFB1 determination in feed samples by HPLC-FLD\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6540017/v1/7c1143eb16ac436275c19007.png"},{"id":81614983,"identity":"fa4eb361-e729-49a3-bb9b-f83b64cbc535","added_by":"auto","created_at":"2025-04-29 08:01:23","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":291355,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of all total analysed composited dairy feed samples collected from two towns (FF: Fiche Feed, DF: Degam Feed. Number: - represent code of the sample)\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6540017/v1/4a298a65578a88361d19ce9e.jpeg"},{"id":86269831,"identity":"551c2990-9048-408a-bd6b-80f741458426","added_by":"auto","created_at":"2025-07-08 16:53:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1342146,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6540017/v1/f5b1b186-f5b1-4d2a-a7a9-d15d0e2d66c4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Assessment of Aflatoxin B1 Contamination Level in Dairy Animal Feeds from Selected Towns of North Shoa Zone, Oromia, Ethiopia","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAnimal feed is a balanced feed given to domestic animals, especially livestock, to promote overall health and milk production [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, it is vulnerable to a variety of naturally occurring microorganisms that can cause contamination and deterioration on farms before harvest, during storage, and at storage locations [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Farm forage, hay, cereals, commercial concentrates (noug seed cake, peacake, maize and mix, frushka, and feed additives), and atella are the most popular dairy animal feeds required for health and increased milk production; however, they are susceptible to various chemical contaminants [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Among the various contaminants, fungal contamination is particularly alarming due to the potential production of aflatoxins, such as aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2), in dairy animal feed [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor example, toxigenic fungi such as \u003cem\u003eAspergillus flavus\u003c/em\u003e, \u003cem\u003eAspergillus parasiticus\u003c/em\u003e, and \u003cem\u003eAspergillus nomius\u003c/em\u003e produce aflatoxins as secondary metabolites in animal feeds and foods, leading to health risks in both humans and animals [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Among the most common types of aflatoxins in dairy animal feeds, AFB1 is highly toxic and causes acute or chronic toxic effects, including teratogenic, mutagenic, carcinogenic, immune toxic, or hepatotoxic effects in humans [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It can be metabolized hydroxylatively to AFM1 by mammalian bioconversion [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to the International Agency for Research on Cancer (IARC), AFB1 is classified as a group I carcinogen as well as a genotoxin for humans and animals [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Thus, to ensure feed quality and public health, several countries and international organizations have set a maximum permissible limit for AFB1 in various dairy animal feeds. These standards vary depending on the economic status of the countries and the global guidelines provided by organizations such as the Food and Agricultural Organization/World Health Organization (FAO/WHO), European Union (EU), and Food and Drug Administration (FDA). For example, EU/FAO/WHO set the maximum limit of AFB1 in feed to 5 \u0026micro;g/kg; FDA has set the action levels for total aflatoxins in animal feed to 20 \u0026micro;g/kg [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Therefore, it is important to assess the contamination levels of AFB1 in animal feeds in order to minimize the adverse effect of aflatoxins on human and animal health.\u003c/p\u003e \u003cp\u003eSeveral studies have reported AFB1 contamination of dairy animal feeds from various locations in Ethiopia. For example, recent research conducted by Tadele \u003cem\u003eet al.\u003c/em\u003e in northwest Ethiopia found that the level of AFB1 ranged from 1.75 to 306.9 \u0026micro;g/kg and 96% of feed samples were contaminated with AFB1 [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Similarly, a study by Fikadu \u003cem\u003eet al.\u003c/em\u003e in the Oromia Special Zone surrounding Finfinne with a range from 12.67 to 45.67 \u0026micro;g/kg and 26.7% of feed samples were contaminated with AFB1 (16). Additionally, a study by Mesfin, Besufekad and coworkers, Gizachew \u003cem\u003eet al.\u003c/em\u003e in (Bishoftu, Holetta, and Hawassa), in the Gurage Zone, in Greater Addis Ababa reported the levels of AFB1 with a range between 0 and 20 \u0026micro;g/kg, 4.22 and 10.54 \u0026micro;g/kg, 7 and 419 \u0026micro;g/kg, respectively, which exceeded the EU\u0026rsquo;s maximum tolerance limit (5 \u0026micro;g/kg) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite several studies conducted in Ethiopia, there are areas within the country that require thoroughout investigation. To our knowledge so far there is no coordinated investigation of the levels of AFB1 in various types of dairy cow feed within the selected towns of North Shoa Zone, where a high amount of utilization of feed is available by individual dairy farmers to increase milk production. Therefore, the objective of the current study was to determine the levels of AFB1 in dairy cow feed samples collected from two selected towns from North Shoa Zone, Oromia, Ethiopia, using HPLC-FLD technique. It is anticipated that this investigation would provide insightful information about the present level of AFB1 contamination in dairy cow feed.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy areas and sample collection\u003c/h2\u003e \u003cp\u003eThe study was conducted between February and May 2024 in the North Shoa Zone, Oromia, Ethiopia, in two towns called Fiche and Degam, mainly by considering the presence of a large number of smallholder dairy farmers. The geographic location of the North Shoa Zone lies between 2738 and 2782 m above sea level at 90 N, 380 E and is characterized agro-ecologically as upper tropical to highland. The administrative center of this zone, Fitche Town, is located approximately 114 km from Addis Ababa, the capital city of Ethiopia, with a latitude of 9\u0026deg; 48' 0.00 N and a longitude of 38\u0026deg; 43' 59.99\" E. Degam (Hanbiso) town is one of the woreda towns in the North Shoa Zone of Oromia, which is approximately 127 km away from Addis Ababa, with latitude 9\u0026deg; 39' 59.99\" N and longitude 38\u0026deg; 39' 59.99\" E. A total of 50 feed samples consisting of 41 different composite feed samples and 9 most used individual feed of the composited feed samples were collected randomly from smallholder dairy farmers of Fiche and Degam towns. 100 g of each size- reduced dairy cow feed sample was collected in a polyethylene bag, kept in ice packs during transportation, and stored at 2\u0026ndash;8\u0026deg;C in a refrigerator in the laboratory to avoid any fungal growth acceleration in the packed feed until analysis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMethods of extracting AFB1 from dairy cow feed\u003c/h3\u003e\n\u003cp\u003eThe procedure used for AFB1 extraction and clean-up was based on the instructions of the Ethiopian Conformity Assessment Enterprise (ECAE) for assessment of AFB1 in animal feed. Concisely, a 25g representative homogenized feed sample was weighed in a 250 mL beaker, and 125 mL of 70% methanol and 30% deionized water was added and homogenized for 10 minutes via a polytron homogenizer at 4188 x g and the extract was subsequently filtered with fast-flow filter paper in a 250 mL conical flask. 15 mL of the filtrate was taken into a 100 mL Erlenmeyer flask and 30 mL deionized water was added in to IAC obtained from Libios (Pontcharra sur-Turdine, France) for the purification of the filtrate. Subsequently, 15 mL of the filtrate was loaded into the conditioned immune-affinity column at a rate of 1 drop per second. It was then washed with 10 mL of deionized water and allowed to air dry. The AFB1 was then eluted with 3 mL of methanol. The eluted mixture was evaporated to dryness in a 40\u0026deg;C nitrogen gas evaporator. Finally, and before HPLC-FLD analysis, the final residue was reconstituted with 1 mL of a mobile phase consisting of a deionized water: acetonitrile: methanol mixture (60:25:15) and a volume of 10 \u0026micro;L was injected into the injection loop for both the standard and sample solutions into the HPLC coupled with a fluorescence detector. The proposed schematic representation for the determination of AFB1 via HPLC-FLD is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAnalysis method using HPLC-FLD\u003c/h3\u003e\n\u003cp\u003eA HPLC (water alliance e2695) system equipped with FLD (2475) was employed for the quantification of AFB1. The chromatographic separation was achieved on a reversed-phase chromatographic C18 column (3.50mm column; 4.60 \u0026times; 150mm) (HS, Bellefonte, USA). The mobile phase consisted of 60% deionized water, 25% acetonitrile, and 15% methanol (v/v/v), which was filtered through a micro-syringe filter for use. The following instrument parameters were set: injection volume of 10.00\u0026micro;l, flow rate of 1mL min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, run time of 10 minutes, temperature set to 35\u0026deg;C, excitation wavelength of 360nm, and emission wavelength of 440nm. AFB1 standard was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and was used to prepare a series of AFB1 standard solutions (0.5, 1, 2, 4, 6, 8 and 10 \u0026micro;g/L) using the deionized water:acetonitrile:methanol mixture (60:25:15 v/v) through dilution. Calibration curves of peak areas versus AFB1 concentrations were then plotted and used for AFB1 determination in samples.\u003c/p\u003e\n\u003ch3\u003eDetermination of AFB1 in dairy cow feed samples\u003c/h3\u003e\n\u003cp\u003eThe concentrations (\u0026micro;g/kg) of AFB1 in cow feed samples were determined via HPLC‒FLD. The actual amount of AFB1 in \u0026micro;g/kg was calculated via the following Eq.\u0026nbsp;(\u003cspan refid=\"Equ1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) below:\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{m}\\text{t}=\\frac{\\text{V}2.\\text{V}4}{\\text{v}1.\\text{v}3}\\text{*}\\text{m}\\text{o}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u003c/p\u003e \u003cp\u003eWhere:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003emt\u003c/b\u003e is the mass of the test sample present in the fraction of the second filtrate taken for the immune affinity column in grams (g)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003emo is the mass of the test portion in grams (mo\u0026thinsp;=\u0026thinsp;25 g).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eV\u003csub\u003e1\u003c/sub\u003e is the total volume of the first filtrate in milliliters (V\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;125 mL).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eV\u003csub\u003e2\u003c/sub\u003e is the fraction volume of the first filtrate taken for dilution in milliliters (V\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;15 mL).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eV\u003csub\u003e3\u003c/sub\u003e is the total volume of the second filtrate in milliliters (V\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;45 mL)\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eV\u003csub\u003e4\u003c/sub\u003e is the fraction volume of the second filtrate in milliliters (V\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;15 mL).\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eThe mass fraction of each aflatoxin (\u003cb\u003ewi\u003c/b\u003e) in \u0026micro;g/kg of sample was calculated via Eq.\u0026nbsp;(\u003cspan refid=\"Equ2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) below.\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\text{w}i=\\frac{v5.mi}{v6.mt}$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;\u0026hellip;.\u003c/p\u003e \u003cp\u003eWhere:\u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003eV\u003csub\u003e5\u003c/sub\u003e is the volume of the eluate in microliters (V\u003csub\u003e5\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1000 \u0026micro;L).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003eV\u003csub\u003e6\u003c/sub\u003e is the volume of the purified and injected sample extract in microliters (V\u003csub\u003e6\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10 \u0026micro;L).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003emi is the mass of each aflatoxin present in the injection volume, corresponding to the measured peak area read off the calibration graph in nanograms (ng).\u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003emt is the mass of the test sample present in the fraction of the second filtrate taken for the immune affinity column in grams (g) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses i.e., mean, standard deviations, %RSD, coefficient of determination (r\u003csup\u003e2\u003c/sup\u003e), maximum and minimum concentration was carried out using SPSS (Statistical Package for the Social Sciences). An independent t-test was applied at 95% mean confidence interval to determine mean differences of AFB1 concentrations in dairy cow feed samples between the two towns. The concentrations of AFB1 are expressed as the means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation, minimum and maximum values.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results and discussions","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eMethod of Validation\u003c/h2\u003e \u003cp\u003eLimit of detection (LOD), limit of quantification (LOQ), accuracy (recovery), and precisions (repeatability) were determined to test the validity of the HPLC-FLD method for AFB1 determination in dairy cow feed. Linearity was determined by constructing seven-point calibration curves for AFB1 standard solutions across concentration ranges of 0.500, 1.00, 2.00, 4.00, 6.00, 8.00 and 10.0 \u0026micro;g/kg. The calibration curves were constructed by plotting the peak area against the concentration of AFB1, with linearity determined through linear regression analysis, indicated by the coefficient of determination (r\u0026sup2;). Hence, LOD was found to be 0.0100 \u0026micro;g/kg, calculated as 3 times the standard deviation (SD) of the blank sample relative to the analytical curve slope (m), 3SD/m. Similarly, LOQ was found to be 0.0500 \u0026micro;g/kg, calculated as 10 times the standard deviation (SD) of the blank sample relative to the analytical curve slope (m) (10SD/m). Furthermore, the accuracy and precision evaluation results of all sample types are expressed in percent of recoveries and percent of relative standard deviation, respectively. Thus, the calculated percentage recovery of AFB1 for triplicate measurements (n\u0026thinsp;=\u0026thinsp;3) in spiked feed samples ranged between 70.0% and 95.5%, and the %RSD for triplicate measurements (n\u0026thinsp;=\u0026thinsp;3) ranged between 1.63% and 2.23%. This demonstrates the accuracy (recoveries) and precision (%RSD) of the method were found within the range of European Commission limits of 70.0\u0026ndash;120% and \u0026lt;\u0026thinsp;20.0% respectively [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, the optimized and validated HPLC-FLD method used in this study was considered to be both accurate and precise because all the determined method performance characteristics were found to be consistent with EC regulative requirements [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Applied to this method of validation, the mean value of the all analysed composite dairy cow feed samples for AFB1 level was summarized in the Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eAFB1 contamination levels in dairy cow feed samples\u003c/h3\u003e\n\u003cp\u003eA total of 50 feed samples were collected and analyzed for AFB1, comprising 41 different composited feed samples (20 samples from Fiche town, 21 samples from Degam town) and 9 individual feed sample of most used in composited feed sample (4 samples from Fiche town, 5 samples from Degam town). AFB1 was identified in 35 feed samples, representing 85.4% of the 41 analyzed different composited feed samples, with concentrations from 0.0100 to 30.8 \u0026micro;g/kg. Notably, only 6 samples, representing 14.6% of the total, were found to be below the limit of detection (LOD) of 0.0100 \u0026micro;g/kg.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVariation in the level of AFB1 in dairy cow feed between two towns\u003c/h2\u003e \u003cp\u003eAFB1 was found in 35 (85.4%) dairy cow feed samples, among these, 16 samples (39.0%) had levels of AFB1 above the European Commission's maximum allowable limit (5 \u0026micro;g/kg) and 19 samples (46.4%) had levels below the European Commission's maximum allowable limit (5 \u0026micro;g/kg). The analysis of dairy cow feed samples from 2 towns revealed varying concentrations of AFB1; with statistically significant differences among the samples from these towns at the 95% confidence level, as determined by independent t-test (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The Fiche sample exhibited the higher mean concentration at 13.4 \u0026micro;g/kg compared to Degam town at 3.14 \u0026micro;g/kg, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e as well as the highest individual measurement of AFB1 concentration levels, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of AFB1 contamination level in most used composite dairy feeds\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eContaminated sample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eRange(\u0026micro;g/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;STD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eAbove EU (5 \u0026micro;g/kg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e%\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDegam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e21.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e81.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0100\u0026ndash;12.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3.14\u0026thinsp;\u0026plusmn;\u0026thinsp;3.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e14.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFiche\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.0100\u0026ndash;30.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.4\u0026thinsp;\u0026plusmn;\u0026thinsp;10.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e65.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e35.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.0100\u0026ndash;30.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e39.0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eN:-represents number of sample\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eUpon comparing the two towns, 7.30% (n\u0026thinsp;=\u0026thinsp;3) and 31.7% (n\u0026thinsp;=\u0026thinsp;13) of the feed samples collected from Degam and Fiche towns were found to exceed the maximum permissible level (5\u0026micro;g/kg) of AFB1 established by the European Commission [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], respectively.\u003c/p\u003e \u003cp\u003eFurthermore, to determine how much each individual contributed to the overall contamination of the composite feed samples with AFB1, the most often used individual components of the composite feed samples, such as hay, mixin, frushka, maize grain, and pea cake, were also analyzed, as results shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Thus, the lowest level of AFB1 contamination was detected in the hay collected from the two towns. This indicates that the sample's contribution to the composite feed sample that was collected from two towns was negligible.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLevels of AFB1 in most commonly used individual feed samples taken from two towns\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFeed type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eLevel of AFB1 (\u0026micro;g/kg)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eFiche\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFrushka\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMixin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e19.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePea cake\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eMax - min 0.01\u0026ndash;19.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eDegam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eFrushka\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMixin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePea cake\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.30\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eHay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eMaize grain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eMax - min 0.01\u0026ndash;23.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eHint: - ND-represents less than limit of detection\u003c/p\u003e \u003cp\u003eTo the opposite, the highest degree of contamination of AFB1 level (19.8 \u0026micro;g/kg) was detected in the mixin feed sample collected from Fiche town. Similarly, 23.8 \u0026micro;g/kg and 20.2 \u0026micro;g/kg degrees of contamination were detected in frushka and mixin feed samples collected from Degam town, respectively. These clearly demonstrated that these two types of individual feed samples contributed more contamination than did other individuals in a composite feed. Thus, factors such as the local environment, high humidity, and the absence of storage animal feed in small, confined places may be responsible for the apparent variation in AFB1 levels in dairy cow feed from the study location [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eComparison of the results of this study with those of other studies\u003c/h2\u003e \u003cp\u003eThe presence of AFB1 contamination in dairy feed samples has been reported in Ethiopia as well as worldwide, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e below. The results of this study indicated lower levels of AFB1 in dairy feed samples compared to those reported in Ethiopia by Gizachew \u003cem\u003eet al.\u003c/em\u003e, which ranged from 7 to 419 \u0026micro;g/kg [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] Similarly, the research conducted by Fikadu \u003cem\u003eet al.\u003c/em\u003e reported higher concentrations of AFB1, ranging from 12.67 to 45.67 \u0026micro;g/kg [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, Tadele \u003cem\u003eet al.\u003c/em\u003e reported AFB1 concentration ranging from 1.75 to 306.9 \u0026micro;g/kg, which is higher than those found in the current study [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Furthermore, several countries, such as Pakistan (10, 42), Egypt [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], India [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], Kenya [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and Malawi [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], have reported higher AFB1 levels than those observed in the current study.\u003c/p\u003e \u003cp\u003eOn the other hand, the results of this finding revealed a greater range of AFB1 than those values reported in Ethiopia by Tefera and Vegarud, which ranged from 0.067 to 29.69 \u0026micro;g/kg [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]; Yegrem, which ranged from 0.164 to 0.490 \u0026micro;g/kg [45]; India, which ranged from 0.9 to 15 \u0026micro;g/kg [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]; Morocco, which ranged from 1.02 to 13.59 \u0026micro;g/kg [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]; and Spain, which ranged from 0.07 to 5.17 \u0026micro;g/kg [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], whereas this finding ranged from 0.0100 to 30.8 \u0026micro;g/kg, and 85.4% of feed samples were contaminated with AFB1. However, it was in line with the values reported by Tefera \u0026amp; Vegarud in the selected rural area of the Sidama Zone (Ethiopia) that ranged from 0.067 to 29.69 \u0026micro;g/kg, and 82.9% of feed samples were contaminated with AFB1 [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In general, the disparity of those results reported in literature and this study might be due to sampling periods, different quantification techniques, geographical conditions, methods of harvesting and storing animal feed, type of feed samples, and sampling techniques [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of AFB1 levels in dairy feeds with various location of Ethiopia and those reported in literatures.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMethods of Detection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eType of feed\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eo\u003c/span\u003e of sample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e+ve sample(%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRange(\u0026micro;g/kg)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eReferences\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthiopia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ewheat bran and noug cake\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7-419\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthiopia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ewheat bran, noug seed cake,\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12\u0026ndash;46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthiopia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ehay, grain byproducts, and maize stove\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e240\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e82.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.067\u0026ndash;29.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthiopia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eatella, hay, commercial concentrates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.75\u0026ndash;306.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpain\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCompound feed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.07\u0026ndash;5.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEgypt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrain and fourages\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.16\u0026ndash;69.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorocco\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDairy feed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.02\u0026ndash;13.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePakistan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDairy concentrate\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e5.31\u0026ndash;42.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPTLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCattle feed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20\u0026ndash;60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIndia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eConcentrate feeds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e189\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.9\u0026ndash;15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eKenya\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFourage, concentrates\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0\u0026ndash;147.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMalawi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eELISA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMaize bran,\u003c/p\u003e \u003cp\u003eDairy mash and Hay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e62.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.36\u0026ndash;60.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEgypt\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUPLC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eComplete feed samples\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3-300\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEthiopia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHPLC-FLD\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-composite of concentrate feed\u003c/p\u003e \u003cp\u003e-Individual of composited feed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41\u003c/p\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e83.36\u003c/p\u003e \u003cp\u003e66.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.0100\u0026ndash;30.8\u003c/p\u003e \u003cp\u003e0.0100\u0026ndash;23.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, different concentrations of AFB1 were detected in dairy feed samples collected from 2 towns within the North Shoa Zone, exhibiting considerable levels of contamination. Overall, of 41 different composited dairy feed samples collected from two towns, 85.4% (n\u0026thinsp;=\u0026thinsp;35) were contaminated with AFB1, and 39.0% of contaminated dairy cow feed samples had AFB1 levels exceeding limits set by the European Commission (5 \u0026micro;g/kg). Furthermore, significant levels of AFB1, which can hydroxylate to AFM1, were found in most dairy feed samples that were collected from Fiche town. In light of this result and based on the analyses of the most used individual of the composited feed samples, the major contributors to AFB1 contamination level in composite feed samples were found to be mixin and frushka. Therefore, on the basis of individual sample feed results, risk mitigation should focus on mixin and frushka. Finally, owing to financial constraints, the designed seasonal variation and location of the studied area may not be sufficient; therefore, it is highly recommended that additional research be conducted on the seasonal variation of AFB1 contamination levels in dairy animal feeds and at different areas with a representative number of samples since this study is the baseline for further studies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of Interest\u003c/h2\u003e \u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThe author (s) disclosed receipt of the following financial support for the research, authorship, and /or publication of this article: This study was financially supported by Salale University.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eThe study was carried out by Kasaye Bahiru Tola, Girma Selale Geleta, and Argachew Nugussa. Kasaye Bahiru Tola wrote the manuscript and created the figures and tables, while Girma Selale Geleta and Argachew Nugussa offered additional insights and discussions regarding the article. The final version of manuscript has been approved for publication by all authors after review.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors would like to acknowledge the Department of Chemistry, Salale University, for the funds provided for this study. Additionally, we would like to extend our thanks to the Ethiopian Confirmative Assessment Enterprise (ECAE), Addis Ababa, for providing laboratory facilities during the study period.\u003c/p\u003e\u003ch2\u003eAvailability of Data and Materials\u003c/h2\u003e\n\u003cp\u003eAll data generated in the study are incorporated in the manuscript\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdmasu FT, Melak A, Demissie B, Yenew C., Habtie ML, Bekele TT, Feyesa TO, Chanie ES, G/Medhin MT, Malik T \u0026amp; Dejenie TA (2021) Occurrence and associated factors of aflatoxin M1 in raw cow,Journal of Food Science and Nutrition, pp 6286\u0026ndash;6293. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/fsn3.2589\u003c/span\u003e\u003cspan address=\"10.1002/fsn3.2589\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhlberg S, Grace D, Kiarie G, Kirino Y \u0026amp; 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Among several types of aflatoxins, aflatoxin B1 (AFB1) is highly toxic and causes acute or chronic toxic effects to both humans and animals. This study, therefore, was conducted to determine the levels of AFB1 in dairy cow feed samples collected from Fiche and Degam towns of North Shoa Zone. A total of 50 feed samples comprising 41 composited and 9 individual of each composited were collected randomly from smallholder dairy farmers, and the levels of AFB1 were determined via High-Performance Liquid Chromatography coupled with Fluorescence Detector (HPLC-FLD) after being cleaned with immune affinity columns. The findings revealed that the highest AFB1 content in composited feed samples was 30.8 \u0026micro;g/kg and the lowest was 0.0100 \u0026micro;g/kg. Out of 41 analyzed composited feed samples, 35 (85.4%) samples were positive for AFB1, and 6 (14.6%) were found to be below the limit of detection. Furthermore, of the positive feed samples for AFB1, 16 (39.0%) contained AFB1 that exceeded the European Commission maximum permissible level (5 \u0026micro;g/kg) for dairy feed samples. Thus, the finding of this study highlights the importance of rigorous regulatory monitoring and improved feed handling techniques to safeguard consumers of animal-derived products from the serious health consequences linked to aflatoxins.\u003c/p\u003e","manuscriptTitle":"Assessment of Aflatoxin B1 Contamination Level in Dairy Animal Feeds from Selected Towns of North Shoa Zone, Oromia, Ethiopia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-29 08:01:18","doi":"10.21203/rs.3.rs-6540017/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"02fdf00a-d080-4c35-8714-9ef57d6ffd4d","owner":[],"postedDate":"April 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-08T16:53:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-29 08:01:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6540017","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6540017","identity":"rs-6540017","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}