Vol.:(0123456789) Mycotoxin Research (2025) 41:63–75 https://doi.org/10.1007/s12550-024-00566-x RESEARCH Aflatoxins and fumonisins co‑contamination effects on laying hens and use of mycotoxin detoxifiers as a mitigation strategy Phillis E. Ochieng1,2 · David C. Kemboi2,3 · Sheila Okoth4 · Siegrid De Baere2 · Etienne Cavalier5 · Erastus Kang’ethe6 · Barbara Doupovec7 · James Gathumbi8 · Marie‑Louise Scippo1 · Gunther Antonissen2,9 · Johanna F. Lindahl10,11,12 · Siska Croubels2 Received: 10 July 2024 / Revised: 20 September 2024 / Accepted: 25 September 2024 / Published online: 15 October 2024 © The Author(s) 2024 Abstract This study examined the effects of fumonisins (FBs) and aflatoxin B1 (AFB1), alone or in combination, on the productivity and health of laying hens, as well as the transfer of aflatoxins (AFs) to chicken food products. The efficacy and safety of mycotoxin detoxifiers (bentonite and fumonisin esterase) to mitigate these effects were also assessed. Laying hens (400) were divided into 20 groups and fed a control, moderate (54.6 µg/kg feed) or high (546 µg/kg feed) AFB1 or FBs (7.9 mg/kg feed) added diets, either alone or in combination, with the mycotoxin detoxifiers added in selected diets. Productivity was evaluated by feed intake, egg weight, egg production, and feed conversion ratio whereas health was assessed by organ weights, blood biochemistry, and mortality. Aflatoxins residues in plasma, liver, muscle, and eggs were determined using UHPLC-MS/MS methods. A diet with AFB1 at a concentration of 546 µg/kg feed decreased egg production and various AFB1-contaminated diets increased serum uric acid levels and weights of liver, spleen, heart, and gizzard. Interactions between AFB1 and FBs significantly impacted spleen, heart, and gizzard weights as well as AFB1 residues in eggs. Maximum AFB1 residues of 0.64 µg/kg and aflatoxin M1 (below limits of quantification) were observed in liver, plasma, and eggs of layers fed diets with AFB1. The mycotoxin detoxi- fiers reduced effects of AFB1 and FBs on egg production, organ weights, blood biochemistry, and AFB1 residues in tissues. This study highlights the importance of mycotoxin detoxifiers as a mitigation strategy against mycotoxins in poultry production. Keywords  Aflatoxins · Bentonite · Egg · Food and feed safety · Fumonisins · Kenya · Laying hen * Johanna F. Lindahl johanna.lindahl@imbim.uu.se * Siska Croubels Siska.Croubels@ugent.be 1 Department of Food Sciences, Laboratory of Food Analysis, Faculty of Veterinary Medicine, University of Liège, 4000 Liège, Belgium 2 Department of Pathobiology, Pharmacology and Zoological Medicine, Laboratory of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium 3 Department of Animal Science, Chuka University, P.O. Box 109‑60400, 00625 Chuka, Kenya 4 Department of Biology, Faculty of Science and Technology, University of Nairobi, P.O. Box 30197‑00100, Nairobi, Kenya 5 Department of Clinical Chemistry, Center for Interdisciplinary Research On Medicines (CIRM), University of Liège, University Hospital of Liège, 4000 Liège, Belgium 6 Consultant, 00100 Nairobi, Kenya 7 dsm-firmenich Animal Nutrition and Health R&D Center Tulln, 3430 Tulln, Austria 8 Department of Veterinary Pathology, Microbiology, and Parasitology, Faculty of Veterinary Medicine, University of Nairobi, P.O. Box 29053‑00100, Nairobi, Kenya 9 Chair Poultry Health Sciences, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium 10 International Livestock Research Institute (ILRI), P.O. Box 30709‑00100, Nairobi, Kenya 11 Department of Medical Biochemistry and Microbiology, Uppsala University, 751 05 Uppsala, Sweden 12 Department of Clinical Sciences, Swedish University of Agricultural Sciences, 750 07 Uppsala, Sweden http://crossmark.crossref.org/dialog/?doi=10.1007/s12550-024-00566-x&domain=pdf 64 Mycotoxin Research (2025) 41:63–75 Introduction Poultry requires less space than other livestock like cattle and is a major source of income for low-income populations in sub-Saharan Africa (SSA). The majority of the flock is made up of chickens, but pigeons, ducks, ostriches, turkeys, quails, and guinea fowls are also becoming more and more significant (Magothe et al. 2012). Commercial poultry farm- ing in SSA remains unable to meet the region’s need for this protein source (Akinola & Essien 2011). A major obstacle facing commercial chicken rearing in SSA is the scarcity of reasonably priced and high-quality feed. Furthermore, the majority of small-scale farmers in the SSA nations are igno- rant that animal health and productivity can be negatively impacted by poor quality feeds that contain mycotoxins, among other contaminants (FAO 2022). Mycotoxins are secondary metabolites produced in storage by certain fungi such as Aspergillus and Penicillium or in the fields by Fusarium fungi. Although more than 400 myco- toxins have been found, aflatoxin B1 (AFB1), fumonisin B1 (FB1), zearalenone, deoxynivalenol (DON), T-2 toxin, and ochratoxin A (OTA) are the most significant mycotoxins in animal production and human health because of their wide- spread incidence and toxicities (Kemboi et al. 2020). Aflatoxin B1-contaminated feeds have been associated with immunosuppression, stunted growth, impaired reproduc- tive function that results in delayed age of maturity, decreased egg production and hatchability, and poor egg quality in layer chickens (Fernandez et al. 1994; Lee et al. 2012). Moreover, consumers of poultry products may be in danger due to the transferability of mycotoxins from feed to these products. Studies have reported the presence of AFB1 in liver, kidney, muscle, and eggs of chickens from markets or slaughterhouses (Iqbal et al. 2014; Sineque et al. 2017). Aflatoxins (AFs) were also found in tissues from chickens fed diets contaminated with AFs (Magnoli et al. 2017; Ochieng et al. 2023; Trucksess et al. 1983). Poultry is comparatively resistant to the toxicities of FBs although damages to the kidney, liver, and gastroin- testinal tract have been documented (Antonissen et al. 2014; Chen et al. 2021). Fumonisins are not often found in poultry products and trace levels were detected in chicken tissues, blood, and eggs in recent investigations (Antonissen et al. 2020; Tangni et al. 2020; Tardieu et al. 2021). Multiple mycotoxins can contaminate feeds either because several toxigenic fungi contaminate the same feed or because the same fungi produce multiple mycotoxins (Njobeh et al. 2012). According to a survey of mycotoxin contamination of chicken feeds and feed ingredients from SSA, co-occurrence of AFs and FBs was the most common type of multiple contamination (Ochieng et al. 2021). When compared to their individual effects, interactions between mycotoxins can make their effects more severe, even at low concentrations (Huff et al. 1986). Few studies have evaluated the effects of co-contamination with AFB1 and FB1 on the immune system, blood biochemistry, and organs of broiler chickens (Ochieng et al. 2023; Tessari et al. 2006, 2010). Mycotoxin detoxifiers such as mycotoxin binders that bind to mycotoxins and prevent bloodstream absorption or mycotoxin modifiers that convert mycotoxins into less toxic compounds are considered sustainable post-harvest methods of protecting animals from the harmful effects of mycotoxins that are already in feed and being ingested by the animals. Popularly used mycotoxin binders are clay minerals and phys- icochemical qualities of these clays are influenced by various aspects, including their source and spacing within the layers, which ultimately determine their ability to adsorb mycotox- ins (Rosa et al. 2001). Compared to natural clay, artificially modified clays exhibit greater interlayer spacing and therefore increased mycotoxin-sequestering potential (Laurain et al. 2021). Bentonite (BENT) is one of the clay minerals that has been used as a mycotoxin binder to reduce AFB1 toxicities (Pappas et al. 2016; Saminathan et al. 2018). In addition to bentonite clay, biological elements, including plant extracts, algae, and Trichosporon mycotoxinivorans, which also func- tion as mycotoxin modifiers, were included in the mycotoxin binder utilized in this study (Mesgar et al. 2022). The myco- toxin modifier employed in this study is known as fumonisin esterase (FZYM) and it functions by cleaving the ester linkages in FB1 side chains, producing either fully or partially hydro- lyzed FB1 and tricarballylic acid(s) (Heinl et al. 2010). The European Food Safety Authority (EFSA) assessed BENT and FZYM and the European Commission approved both of them for use in ruminants, pigs, and poultry to mitigate the harmful effects of AFs and FBs, respectively (EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2016). The BENT and FZYM are marketed by Biomin® GmbH, a division of dsm-firmenich, as Mycofix® Secure and FUMzyme®, respectively. These mycotoxin detoxifiers are frequently tested outside of SSA in experimental condi- tions that, among other things, do not reflect the majority of SSA’s farming practices. These conditions include tempera- ture, mycotoxin contamination levels, feed composition and management, and vaccination schedules. Furthermore, there has never been a report on the use of BENT and FZYM in feed ingredients contaminated with one or more mycotoxins. The objective of the present study was to evaluate the safety and efficacy of the mycotoxin detoxifiers FZYM and BENT to reduce the negative effects of AFs and FBs, either separately or in combination, on laying hens, in experimental conditions comparable to those of small-scale commercial farming in the majority of SSA countries. Production per- formance of the layers was determined by feed intake, egg weight, feed conversion ratio, and egg production, whereas the health of the hens was evaluated by mortality rate, and changes in blood biochemistry and organ weights. 65Mycotoxin Research (2025) 41:63–75 Material and methods Ethical statement The animals were housed at the International Livestock Research Institute (ILRI), located in Nairobi, Kenya. All methods related to maintaining, euthanizing, and sampling the animals followed the ILRI Animal Care and Use Ethics Committee approval number IACUC-RC2019-03. Preparation of experimental AFB1‑ and FBs‑contaminated diets Maize culture materials containing AFB1 or FBs were obtained according to the methods described by Ochieng et al. (2022). Aspergillus flavus and Fusarium verticillioides fungal isolates to produce AFs and FBs, respectively, were from the Mycology and Mycotoxin Laboratory, University of Nairobi, Kenya. Using LC-MS/MS methods (Monbaliu et al. 2010), the maize culture materials were examined for major AFs and FBs. Levels up to 88,174 µg AFB1/kg substrate and 1709 µg aflatoxin B2 (AFB2)/kg substrate were measured in maize cultures inoculated with A. flavus and levels up to 440,668 µg FB1/kg and 449,056 µg fumonisin B2 (FB2)/kg were found in maize cultures inoculated with F. verticillioides. The control diet (with no additional mycotoxins or detoxi- fiers) was a commercially supplied basal feed free of growth promoters, antibiotics, and coccidiostats and matched the nutritional requirements for laying hens (Nutrient Require- ments of Poultry 1994) as shown in Supplementary Table S1. Using the LC-MS/MS method developed and vali- dated by Sulyok et al. (2006), the control diet’s mycotoxin levels were determined. Supplementary Table S1 shows the concentrations of the major mycotoxins in the diet. All the tested mycotoxins were at trace levels and have been shown in previous studies to be non-toxic to poultry. The diet had AFB1 at a level of 2.26 µg/kg, FB1 at a level of 274.10 µg/ kg, and FB2 at a level of 94.98 µg/kg (all below the EU legal or recommended levels in poultry feeds, European Commis- sion 2002, 2006). The maize culture materials containing AFB1, or FBs, were mixed with 5000 g of the control diet to make a pre- mix, which was then used to formulate the experimental treatment diets contaminated with AFB1 at levels of 54.6 or 546 µg/kg feed and FBs (FB1 + FB2) at a level of 7.9 mg/ kg feed. Fumonisin B1 was at a level of 6.08 mg/kg feed whereas FB2 was at a level of 1.80 mg/kg feed in the FBs- contaminated diets. Bentonite and FZYM at doses of 2 g/kg feed and 0.012 g/kg feed, respectively, were added to certain diets. Table 1 shows the 20 dietary treatments. Table 1   The different treatment diets fed to laying hens for 28 days M AFB1 moderate aflatoxin B1, H AFB1 high aflatoxin B1, BENT bentonite, FZYM fumonisin esterase, FBs fumonisins Treatment N° AFB1 concentra- tion (µg/kg feed) FBs concentra- tion (mg/kg feed) BENT dose (g/kg feed) FZYM dose (g/kg feed) T1: Control / / / / T2: FBs / 7.9 / / T3: FBs + FZYM / 7.9 / 0.012 T4: FBs + FZYM + BENT / 7.9 2 0.012 T5: H AFB1 546 / / / T6: H AFB1 + BENT 546 / 2 / T7: H AFB1 + BENT + FZYM 546 / 2 0.012 T8: H AFB1 + FBs 546 7.9 / / T9: H AFB1 + FBs + BENT 546 7.9 2 / T10: H AFB1 + FBs + FZYM 546 7.9 / 0.012 T11: H AFB1 + FBs + BENT + FZYM 546 7.9 2 0.012 T12: M AFB1 54.6 / / / T13: M AFB1 + BENT 54.6 / 2 / T14: M AFB1 + BENT + FZYM 54.6 / 2 0.012 T15: M AFB1 + FBs 54.6 7.9 / / T16: M AFB1 + FBs + BENT 54.6 7.9 2 / T17: M AFB1 + FBs + FZYM 54.6 7.9 / 0.012 T18: M AFB1 + FBs + BENT + FZYM 54.6 7.9 2 0.012 T19: FZYM / / / 0.012 T20: BENT / / 2 / 66 Mycotoxin Research (2025) 41:63–75 Experimental birds and housing Four hundred Isa Brown laying hens, aged 19 weeks (body weight (BW) ± standard deviation = 1.7 ± 0.2 kg), were pur- chased from a small-scale commercial farm in Kenya. Two weeks were given to the chickens to adapt to their new envi- ronment before the feeding trial began. During the adaption period, all the hens were fed the control diet. At the begin- ning of the 28-day feeding trial, the birds were 21 weeks old, weighed 1.8 ± 0.1 kg, and had laying capacities of above 80%. Twenty birds (four replicates of 5 birds each) were assigned to each of the 20 treatment groups after the birds had their wings banded. Each pen measured approximately 2 m2 and was filled with sterilized pine wood shavings on concrete flooring. The pens were naturally lit and had tem- peratures of between 22 and 25 °C, emulating Kenyan small- scale commercial farming practices. Before placing the hens, the pens were thoroughly cleaned with Hy-Protectol® dis- infectant (HighChem, Nairobi, Kenya) and allowed to dry for 3 days. The birds were fed the various treatment diets and water ad libitum for the duration of the 28-day feeding period. The general flock conditions were checked twice a day and in the event that a mortality was recorded, a post- mortem examination was done immediately. Production performance; collection of blood, organs, and eggs; and blood biochemistry Production performance parameters  Every day, feed intake (FI) was calculated by deducting the amount of feed left over from the amount of feed that was supplied and corrected for mortalities. Eggs were collected every day, marked with the day they were collected and pen number before being weighed, and stored at 4 °C. Egg production was computed using the number of eggs laid by each hen each day, taking into account the production from all surviving hens. The feed conversion ratio (FCR) was determined as the weight (g) of feed used per weight (g) of egg produced (Zhu et al. 2023). Body weight gain (BWG) was computed by deduct- ing the starting BW from the final BW. Collection of blood, organs, and eggs  At the end of the feed- ing trial, blood (about 2 mL) was aseptically collected from four birds in each pen via the wing vein using a sterile 23G needle (0.65 mm × 30 mm) and a 2-mL syringe. Blood from two birds/pens was put in 10-mL plain tubes to obtain serum, while the remaining blood from the other two birds was put in sample tubes containing EDTA to obtain plasma. For the latter, after being allowed to stand at room temperature (22– 25 °C) for 2 h, each blood sample was centrifuged for 10 min at 4 °C and 3000 rpm. The collected sera and plasma sam- ples were stored at − 20 °C in vials until blood biochemistry analysis and analysis for residues of AFs, respectively. The birds were then weighed and sedated with an intramuscular injection of 3.1 mg/kg BW ketamine hydrochloride (Rotex- medica GmbH, Trittau, Germany) and 0.2 mg/kg BW mida- zolam (Troikaa, Gujarat, India), followed by euthanasia using an intravenous injection of 86 mg/kg BW pentobarbital (Bayer, Johannesburg, South Africa). Whole liver, spleen, heart, and gizzard were obtained from the same two birds/ pens from which plasma samples were taken. The organs were weighed, and the relative organ weight was computed as a proportion of the BW (Saminathan et al. 2018). About 100 g of breast muscle and the entire liver was collected from the same birds from which plasma was obtained and the samples were stored at − 20 °C until they were shipped frozen to be examined for AFs residues. Eggs collected on the final feeding trial day were weighed, shelled, centrifuged, and individually stored in 50-mL Eppendorf tubes at − 20 °C until they were delivered frozen for AFs residue analysis. Blood biochemistry analysis  An automated Cobas C600 bio- chemical analyzer (Roche Ltd, Horiba-ABX, Montpellier, France) was used to measure the levels of total protein (TP), albumin (ALB), gamma-glutamyl transferase (GGT), and uric acid (UA) in the sera samples according to the manufac- turer’s recommended protocols. By deducting the ALB from the TP, the serum globulin (GLB) levels were determined (Sakamoto et al. 2018). Analysis of residues of aflatoxins in plasma, liver, muscle tissue, and eggs using UPLC‑MS/MS Using a Moulinette 320 meat grinder (Moulinex, Barcelona, Spain), the liver and breast muscle samples were ground and homogenized. The UPLC-MS/MS methods reported by De Baere et al. (2023) for the determination of AFB1, AFB2, aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), AFM1, and afla- toxin M2 (AFM2) in plasma, muscle, liver, and eggs were used. The methods were in-house validated for freeze–thaw stability, matrix effect, linearity, precision, accuracy, speci- ficity, limit of detection (LOD), limit of quantification (LOQ), and extraction recovery (RE). Egg, muscle, liver, and plasma samples from healthy, untreated chickens were used to prepare matrix-matched blank and spiked samples for method validation. Details of the method validation are provided by De Baere et al. (2023). The LOQ of AFB1, AFB2, AFG1, AFG2, and AFM1 in plasma was 0.05 ng/mL, while the LOQ of AFM2 in the same matrix was 0.10 ng/ mL. In chicken liver, the LOQ was 0.05 µg/kg for AFB1 and 0.10 µg/kg for AFB2 and AFM1, whereas it was 0.25 µg/kg for AFG1 and AFG2 and 0.5 µg/kg for AFM2. For chicken muscle, the LOQ for AFB1 was 0.05 µg/kg, 0.10 µg/kg for AFM1, and 0.25 µg/kg for AFB2, AFG1, and AFG2. In 67Mycotoxin Research (2025) 41:63–75 chicken egg, the LOQ for AFB1, AFB2, and AFM1 was 0.025 µg/kg, while it was 0.050 µg/kg for AFG1 and AFG2 and 0.50 µg/kg for AFM2. For chicken plasma, the calcu- lated LOD values were from 0.0029 to 0.0300 ng/mL; for chicken liver and muscle, the LODs were between 0.006 and 0.040 µg/kg and between 0.002 and 0.097 µg/kg for egg. Statistical analysis and carry‑over factors R (R Core Team 2020) was used to analyze all the data, which are presented as least squares means and standard error of the mean. Prior to analysis, non-linear data accord- ing to the Kolmogorov–Smirnov test were first square root converted. While individual birds were employed for other analyses, the pen served as the experimental unit for the FI analysis. Linear mixed effects modelling from the R pack- age was used with the pen serving as the random variable (Tsiouris et al. 2021). The means of the various treatment groups were compared using predefined contrast analy- sis (Chowdhury et al. 2005). The threshold for statisti- cal significance in the Tukey post hoc analysis was set at p < 0.05. Aflatoxin residues in plasma, muscle, liver, and eggs were deemed positive if their concentration was higher than the limit of detection (LOD), and half of the LOQ value was applied to samples that were above LOD but below LOQ (Kemboi et al. 2023). The carry-over factors of AFs from feed into plasma, liver, muscle, and egg were expressed as the ratio of the mycotoxin concentration (µg/kg) in each tissue relative to the mycotoxin concentration (µg/kg) in feed × 100. Results Production performance In groups fed the diet with high AFB1 alone (T5) and the diet with both high AFB1 and FBs and supplemented with FZYM (T10), two mortalities were observed that were unrelated to the treatment diets as indicated in the post- mortem reports (not shown). Table 2 shows the average daily FI, FCR, egg weight, and egg production during the 28-day feeding trial. Compared to the control diet (T1), Table 2   Average daily feed intake, egg weight, feed conversion ratio, and egg production of laying hens fed different diets. Each treatment diet included 20 birds Data are presented as least square means (LSM) and standard error of the mean (SEM) for 20 birds per treatment. Values within the same column not sharing a common superscript differ significantly (p < 0.05) following a Tukey post hoc test. The feed conversion ratio was calculated by dividing the sum of feed con- sumed per hen per day by the weight of the egg produced. FBs fumonisins, H AFB1 high aflatoxin B1, M AFB1 moderate aflatoxin B1, FZYM fumonisin esterase, BENT bentonite Treatment N° Feed intake (g/ bird per day) Egg weight (g/egg) Feed conversion ratio (g of feed/g of egg) Egg pro- duction (%) T1: Control 133.7 60.0ac 2.23 92.9bcd T2: FBs 133.6 59.5ab 2.24 91.4ad T3: FBs + FZYM 133.6 60.8ac 2.20 93.2 cd T4: FBs + FZYM + BENT 133.6 60.0ac 2.23 92.9bcd T5: H AFB1 133.4 59.7ac 2.24 88.2a T6: H AFB1 + BENT 133.8 60.9bc 2.20 91.8ad T7: H AFB1 + BENT + FZYM 133.4 59.4a 2.25 94.1d T8: H AFB1 + FBs 133.5 59.8ac 2.24 90.2ad T9: H AFB1 + FBs + BENT 133.4 60.2ac 2.22 89.1ac T10: H AFB1 + FBs + FZYM 133.6 61.0c 2.21 91.6ad T11: H AFB1 + FBs + BENT + FZYM 133.4 60.1ac 2.22 89.1ac T12: M AFB1 133.7 59.8ac 2.24 91.8ad T13: M AFB1 + BENT 133.6 60.3ac 2.22 90.7ad T14: M AFB1 + BENT + FZYM 133.6 60.3ac 2.22 88.9ab T15: M AFB1 + FBs 133.8 59.4a 2.26 89.6ac T16: M AFB1 + FBs + BENT 133.8 60.2ac 2.23 92.5bcd T17: M AFB1 + FBs + FZYM 133.4 59.8ac 2.24 91.6ad T18: M AFB1 + FBs + BENT + FZYM 133.7 60.9bc 2.20 93.2 cd T19: FZYM 133.6 60.3ac 2.22 92.5bcd T20: BENT 133.6 60.0ac 2.23 92.1ad SEM 0.2 0.5 0.02 1.48 68 Mycotoxin Research (2025) 41:63–75 the production of eggs dropped by 5% (p = 0.0302) in birds fed high AFB1 alone (T5). When BENT and FZYM were added to the diets with high AFB1 (T7), egg production increased by 7% (p = 0.0066) compared to diets with high AFB1 without the detoxifiers (T5). Dietary AFB1, FBs, or both, did not significantly alter egg weights (p > 0.05). However, compared to a diet contaminated with moder- ate AFB1 and FBs and no detoxifiers (T15), egg weight increased by 3% (p = 0.0416) when both FZYM and BENT were supplemented into the moderate AFB1 and FBs diet (T18). The laying hens’ FCR and FI were unaffected by the various treatments. Moreover, neither BW nor BWG was changed by the treatments (results not shown). Relative weight of organs Table 3 shows the relative organ weights of the layers from the various treatments as a proportion of BW. In compari- son to the control diet (T1), the relative liver weight of the layers increased considerably due to diets with high AFB1 only (T5) or high AFB1 and FBs (T8) by 9% (p = 0.0148) and 8% (p = 0.0378), respectively. Additionally, compared to the control diet (T1), the diet with moderate AFB1 and FBs (T15) increased the layers’ relative liver weight by 8% (p = 0.0323). The relative liver weights were decreased when BENT was added to the AFB1-contaminated diets, although the decreases were not statistically significant (p > 0.05). In layers fed high AFB1 alone diet (T5), the relative spleen weight increased by 14% (p = 0.0158) and 12% (p = 0.0386) as opposed to the diets containing both AFB1 and FBs (T8) and the control diet (T1), respectively. When compared to a diet with high AFB1 alone and no detoxifiers (T5), adding both BENT and FZYM to the diet with high AFB1 (T7) significantly decreased the relative spleen weights by 16% (p = 0.0289). The laying hens fed high AFB1 alone (T5) or both moderate AFB1 and FBs (T15) had considerably greater relative gizzard weights by 10% (p = 0.0141) and 13% (p = 0.0356), respectively, than those fed the control diet (T1). Furthermore, hens fed diets containing both mod- erate AFB1 and FBs (T15) had relative gizzard and heart weights that were 9% (p = 0.0269) and 10% (p = 0.0205) greater, respectively, than chickens fed diets containing only moderate AFB1 (T12). When BENT was added to a diet with moderate levels of AFB1 and FBs (T16) or FZYM was added to the same diet (T17), the relative weights of the heart and gizzard were reduced (p < 0.05) compared to the Table 3   Relative weights of liver, spleen, gizzard, and heart (% body weight) and blood biochemical parameters of the laying hens at the end of the feeding trial (28 days) Data are presented as least square means (LSM) and standard error of the mean (SEM) for 8 birds per treatment. Values within the same column not sharing a common superscript differ significantly (p < 0.05) according to a Tukey post hoc test. FBs fumonisins, H AFB1 high aflatoxin B1, M AFB1 moderate aflatoxin B1, FZYM fumonisin esterase, BENT bentonite Treatment N° Relative liver weight (%) Relative spleen weight (%) Relative gizzard weight (%) Relative heart weight (%) Total protein (g/L) Albumin (g/L) Globulin (g/L) Uric acid (mg/dL) T1: Control 1.47ab 0.37acd 1.25a 0.63ad 6.22ab 4.06ac 4.71a 1.48a T2: FBs 1.52ad 0.34a 1.29abc 0.58a 5.85a 3.62a 4.58a 1.54ab T3: FBs + FZYM 1.44a 0.35ac 1.26ab 0.62abc 6.64ac 4.29bc 5.06ac 2.18 cd T4: FBs + FZYM + BENT 1.54ad 0.38ae 1.24a 0.62abc 7.00bc 4.55c 5.32ac 1.99 cd T5: H AFB1 1.60d 0.42e 1.38 cd 0.64bd 7.02bc 4.49bc 5.40ac 2.00 cd T6: H AFB1 + BENT 1.56bcd 0.38ae 1.29abc 0.63ad 7.21c 4.59c 5.95c 1.80c T7: H AFB1 + BENT + FZYM 1.57bcd 0.36acd 1.36bd 0.65 cd 6.91bc 4.47bc 5.26ac 2.16 cd T8: H AFB1 + FBs 1.58 cd 0.36acd 1.33ad 0.64bd 6.66ac 4.35bc 5.03ac 1.97d T9: H AFB1 + FBs + BENT 1.58 cd 0.36acd 1.30abc 0.63ad 7.09bc 4.56c 5.42ac 2.01bcd T10: H AFB1 + FBs + FZYM 1.57bcd 0.39bce 1.28abc 0.63ad 6.73bc 4.33bc 5.16ac 1.87ac T11: H AFB1 + FBs + BENT + FZYM 1.54ad 0.37e 1.31ad 0.63ad 6.57ac 4.18bc 5.07ac 2.12 cd T12: M AFB1 1.57bcd 0.36acd 1.29abc 0.61abc 6.44ac 4.22bc 4.87ab 2.09 cd T13: M AFB1 + BENT 1.48ac 0.36acd 1.29abc 0.62ad 6.92bc 4.44bc 5.30ac 1.95ac T14: M AFB1 + BENT + FZYM 1.54ad 0.36acd 1.34ad 0.61abc 6.80bc 4.31bc 5.25ac 2.24 cd T15: M AFB1 + FBs 1.59 cd 0.37acd 1.41d 0.67d 6.80bc 4.10ac 5.41ac 1.93ac T16: M AFB1 + FBs + BENT 1.51ad 0.34ac 1.30abc 0.60abc 6.76bc 4.23bc 5.27ac 2.23 cd T17: M AFB1 + FBs + FZYM 1.52ad 0.37acd 1.30abc 0.61abc 6.74bc 4.07ac 5.37ac 1.85ac T18: M AFB1 + FBs + BENT + FZYM 1.53ad 0.34ab 1.31ad 0.60abc 7.12c 4.31bc 5.66bc 2.45d T19: FZYM 1.50ac 0.40de 1.29abc 0.63ad 6.70ac 3.93ab 5.71bc 2.10 cd T20: BENT 1.51ad 0.39ce 1.31ad 0.59ab 6.38ac 4.04ac 4.94ab 2.04 cd SEM 0.04 0.02 0.04 0.02 0.32 0.20 0.32 0.18 69Mycotoxin Research (2025) 41:63–75 contaminated diet without the detoxifiers (T15). None of the organs under investigation was affected by diets containing only FZYM (T19) or BENT (T20) (p > 0.05). Biochemical parameters Table 3 also shows the alterations in serum TP, ALB, GLB, and UA brought on by the various experimental diets. When the diet contaminated with FBs was supplemented with both FZYM and BENT (T4), the concentrations of TP and ALB were considerably increased by 20% (p = 0.0289) and 26% (p = 0.0011), respectively, compared to the diet with FBs only and no detoxifiers (T2). Additionally, compared to a diet with FBs only (T2), the addition of FZYM to a diet with FBs only (T3) raised the serum ALB by 19% (p = 0.0305). When BENT was added to a high AFB1 only diet (T6), serum TP and GLB levels were elevated by 16% (p = 0.0297) and 26% (p = 0.0069), respectively, in comparison to the control diet (T1). When compared to the control diet (T1), the addition of FZYM and BENT to a diet with moderate AFB1 and FBs (T18) increased the GLB by 21% (p = 0.0289), while the diet with FZYM alone (T19) increased the serum GLB by 20% (p = 0.0372). The various treatments significantly affected serum UA concentrations. Serum UA concentrations in the layers fed diets with high AFB1 only (T5), high AFB1 and FBs (T8), or moderate AFB1 (T12) were higher than those on the control diet (T1). In comparison to the control diet (T1), the addition of FZYM, BENT, or both to contaminated diets (T3, T4, T6, T7, T9, T11, T14, T16, and T18) also elevated the UA concentrations of the layers (p < 0.05). The serum UA of layers fed diets containing FZYM alone (T19) or BENT alone (T20) were greater by 42% (p = 0.0111) and 38% (p = 0.0238), respectively, than those fed the control diet (T1). The various treatments had no effect on serum GGT (results not shown). Aflatoxins residues in plasma, liver, muscle, and egg Aflatoxin B1 residues were found in plasma, liver, and egg samples (Table 4). In breast muscle samples from all experimental groups, all of the tested AFs were below detectable levels (results not shown). Levels of AFB1 were in the range of LOQ to 0.639 µg/kg in liver, LOQ to 0.063 ng/mL in plasma, and LOQ to 0.040 µg/kg in eggs. The livers of layers fed both high AFB1 and FBs (T8) had the highest level of AFB1 residues (0.64 µg/kg), but this value did not differ statistically from AFB1 residues found in the livers of layers fed a diet with high AFB1 only (T5) (p > 0.05). Layers fed moderate AFB1 alone (T12) or with FBs (T15) had levels of liver AFB1 residues of 0.22 µg/ kg and 0.10 µg/kg, respectively, and these values did not differ statistically from each other. When compared to a diet with high AFB1 (T5), the addition of both BENT and FZYM to the diet (T7) significantly reduced the AFB1 residues in the liver by 82% (p = 0.0044). Similarly, the liver samples from birds fed high AFB1 and FBs and sup- plemented with BENT (T9) or FZYM (T10) showed a significant reduction in AFB1 residues of 71% and 81%, respectively, when compared to the same diet without the detoxifiers (T8) (p < 0.001). Aflatoxin M1 was present in plasma and liver of birds given high or moderate AFB1 alone or with FBs or the detoxifiers, although below the LOQ of 0.050 ng/mL or 0.10 µg/kg, respectively. Trace levels (below LOQs) of AFG1, AFG2, AFB2, and AFM2 were found in the liver and plasma samples of birds that were given high AFB1- contaminated diets (results not shown). Table 4 shows that AFB1 was only found in eggs above the LOQ of 0.025 µg/kg in birds fed diets containing high AFB1 alone (T5), or in combination with FBs (T8), or in diets containing both high AFB1, FBs, and supplemented with BENT (T9). When comparing the transfer of AFB1 Table 4   Aflatoxin B1 (AFB1) concentrations in laying hens’ plasma (ng/mL), liver, and eggs (µg/kg) from the different treatments at the end of the feeding period (28 days) LOQ limit of quantification (0.050  µg/kg or ng/mL for AFB1 resi- dues in liver and plasma samples and 0.025 µg/kg for AFB1 residues in egg samples). ND not detected. Data are presented as least square means (LSM) and standard error of the mean (SEM) for 8 birds per treatment. FBs fumonisins, H AFB1 high aflatoxin B1, M AFB1 mod- erate aflatoxin B1, FZYM fumonisin esterase, BENT bentonite Treatment No AFB1 concentration (ng/mL or µg/kg) Plasma Liver Eggs T1: Control  < LOQa NDa NDa T2: FBs  < LOQa  < LOQa NDa T3: FBs + FZYM  < LOQa NDa NDa T4: FBs + FZYM + BENT  < LOQa NDa NDa T5: H AFB1 0.063b 0.439cde 0.040 g T6: H AFB1 + BENT 0.059b 0.327bd  < LOQde T7: H AFB1 + BENT + FZYM  < LOQa 0.080ab  < LOQc T8: H AFB1 + FBs  < LOQa 0.639e 0.028f T9: H AFB1 + FBs + BENT  < LOQa 0.187abc 0.025ef T10: H AFB1 + FBs + FZYM  < LOQa 0.124ab  < LOQcd T11: H AFB1 + FBs + BENT + FZYM  < LOQa 0.457de  < LOQe T12: M AFB1 NDa 0.215ad  < LOQab T13: M AFB1 + BENT NDa  < LOQa NDa T14: M AFB1 + BENT + FZYM  < LOQa  < LOQa  < LOQab T15: M AFB1 + FBs 0.025a 0. 101ab  < LOQab T16: M AFB1 + FBs + BENT NDa  < LOQa  < LOQbc T17: M AFB1 + FBs + FZYM  < LOQa  < LOQa  < LOQab T18: M AFB1 + FBs + BENT + FZYM NDa NDa  < LOQab T19: FZYM NDa  < LOQa NDa T20: BENT NDa NDa NDa SEM 0.012 0.105 0.002 70 Mycotoxin Research (2025) 41:63–75 from feeds into eggs between layers fed a diet with high AFB1 only (T5) and a diet with both high AFB1 and FBs (T8), the results showed a significant difference (p < 0.001). The addition of BENT (T6) or both BENT and FZYM (T7) significantly reduced the carry-over of AFB1 into the eggs when compared to a diet with high AFB1 without the detoxifiers (T5) (p < 0.001). Similarly, supplementing FZYM (T10) or both FZYM and BENT (T11) into a diet containing high AFB1 and FBs (T8) statistically reduced the concentration of AFB1 transferred into eggs (p < 0.0010 and p = 0.0183, respectively). Eggs from layers fed diets high in AFB1 (T5-T11) showed detectable levels of AFM1, albeit below the LOQ of 0.025 µg/kg. Other tested AFs including AFB2, AFM2, AFG1, and AFG2 were not found in eggs from all treatment groups (data not shown). Table 5 shows the AFB1 carry-over factors from feed into plasma, liver, and eggs. Birds fed diets containing high levels of AFB1 and FBs (T8) had liver samples with the greatest carry-over factor (0.12%) overall. When comparing AFB1 carry-over factors from feed to eggs with carry-over factors to liver and plasma from the same laying hens, the former showed lower carry-over factors. Discussion The results of this study demonstrated that some of the det- rimental effects of FBs and AFB1 were mitigated by FZYM and BENT, respectively. Overall, the different treatments had no effect on FI and FCR. Zaghini et al. (2005) also found no changes in the FI of laying hens fed diets with AFB1 up to levels of 2500 µg/kg feed for 4 weeks. However, when AFB1 and DON (both at 2000 µg/kg feed) were fed to com- mercial-strain hens during their peak production, reduced FI and high FCR were observed (Lee et al. 2012). In the latter study, usage of higher concentrations of AFB1 might explain the observed differences. The highest dosage of AFB1 used in the present study was shown to lower egg production. Fernandez et al. (1994) also observed a reduction in egg production in laying hens fed AFB1 at a concentration of 5000 µg/kg feed for 32 days. However, when AFB1 was fed to laying hens at doses nearly similar to the current study (500 µg/kg) for 8 weeks, no effect on egg production was noted (Oliveira et al. 2000). The differences in the breed sensitivity of the Babcock hens in the latter study versus Isa Brown in the current study could be the cause of the discrepancy in the results. In the current study, the addition of both FZYM and BENT to diets containing high or moderate AFB1 and FBs improved the egg production and egg weights, respectively. These results suggest the need for multi-component detoxifiers since poul- try diets are often contaminated with multiple mycotoxins. The liver is the main target organ for AFs and FBs tox- icities and an increase in the relative weight of the livers from layers fed AFB1 alone or in combination with FBs was expected. Laying hens fed dietary AFB1 at levels of 150–5000 µg/kg feed showed increased liver weights as a result of lipid accumulation (Fernandez et al. 1994; Zhao et al. 2021). According to Lee et al. (2012), laying hens fed dietary AFB1 and DON, both at levels of 1500 or 2000 µg/ kg feed, also had increased liver weights. The increased liver weights in the latter study were associated with AFB1. The present study showed that lower liver weights were observed when BENT was added to the AFB1-contaminated diets; however, these differences were not statistically significant, and this could be because the diets contained high levels of AFB1. Shannon et al. (2017) also reported that BENT could not fully prevent the increase in liver weights in broiler hens fed a very high dietary AFB1 dose (2000 µg/kg) from day 1 to day 21. Thus, there is a need to determine the dosage of a detoxifier based on the level of mycotoxin in a feed. In the present study, laying hens given high AFB1 alone showed increased spleen weights. On the other hand, Zhao et al. (2021) found that layers fed AFB1 (150 µg/kg feed) along with DON (1500 µg/kg feed) and OTA (120 µg/kg feed) had reduced spleen weights. The additional negative effects of DON and OTA in the latter study may be the cause of the observed discrepancies in the results. The spleen is an organ of the immune system, and any impairments therein suggest interferences with the immune system. The laying hens’ gizzard weights in the current study increased when they were fed diets contaminated with AFB1. However, in studies conducted with broiler chickens, feeding AFB1 in the range of 20 to 500 µg/kg for 35 days had no effect on the weight of the gizzard (Mesgar et al. 2022; Ochieng Table 5   Carry-over factors (%) of AFB1 from feed to plasma, liver, and eggs of laying hens fed diets contaminated with the high AFB1 level (546 µg/kg feed), alone or in combination with FBs, or BENT, and/or FZYM for 28 days Carry-over factors (%) from feed into plasma, liver, and eggs expressed as a percentage of the concentration of AFB1 in tissues (µg/kg) compared to the concentration of AFB1 in feed (µg/kg). NA not applicable, FBs fumonisins, H AFB1 high aflatoxin B1, M AFB1 moderate aflatoxin B1, FZYM fumonisin esterase, BENT bentonite Treatment N° Carry-over factors (%) Plasma Liver Eggs T5: H AFB1 0.012 0.080 0.007 T6: H AFB1 + BENT 0.010 0.060 NA T7: H AFB1 + BENT + FZYM NA 0.015 NA T8: H AFB1 + FBs NA 0.117 0.005 T9: H AFB1 + FBs + BENT NA 0.034 0.005 T10: H AFB1 + FBs + FZYM NA 0.023 NA T11: H AFB1 + FBs + BENT + FZYM NA 0.084 NA 71Mycotoxin Research (2025) 41:63–75 et al. 2023; Saminathan et al. 2018). When compared to the effects of AFB1-contaminated diets alone, the combination of AFB1 and FBs caused more severe effects on the weights of the spleen, gizzard, and heart, suggesting interactions between the two mycotoxins. A study by Huff et al. (1988) revealed that interactions between mycotoxins can result in enhanced harmful effects on the health and production of chickens. Pappas et al. (2016) observed that broiler hens fed diets containing 100 µg/kg feed of both OTA and AFB1 for 42 days showed increased heart weight. In the present study, the detrimental effects of the myco- toxins on the weights of the chickens’ hearts and spleens were reduced when BENT, FZYM, or both were added to the contaminated diets. This result supports the reports on the efficacy of BENT and FZYM for usage in poultry to reduce effects of AFB1 and FBs as previously documented in other studies (EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), 2020; Shannon et al. 2017). The FZYM only at levels of 0.012 g/kg feed or the BENT only at levels of 2 g/kg feed were safe and had no effect on any of the organs examined, indicating the safety of these detoxifiers. Serum TP, ALB, and GLB concentrations were higher in birds given contaminated diets but supplemented with FZYM, BENT, or both, showing the mycotoxin mitigation effects of the detoxifiers. Grenier et al. (2017)) and Shannon et al. (2017) also observed that FZYM and BENT reduced the effects of FBs and AFB1, respectively, on the blood bio- chemistry of broiler chickens. The present study showed an increase in serum UA concentrations of hens fed AFB1- contaminated diets or contaminated diets supplemented with FZYM, BENT, or both. In contrast, Swamy et al. (2002) reported that broiler hens exposed to AFs have lower serum UA levels which could indicate changes in renal filtra- tion and reabsorption rates. Studies with broiler chickens revealed no changes in the serum UA levels due to dietary AFB1 at dosages of 100 to 220 µg/kg feed (Ochieng et al. 2023; Oğuz et al. 2002). The various treatment diets in the current study had no effect on the serum GGT concentra- tion. However, Fernandez et al. (1994) observed elevated serum GGT levels in laying hens fed AFB1 at concentra- tions nearly five times greater (2500 µg/kg feed) than the present study. An increase in blood enzymes, such as GGT, can signify damage of hepatocytes and can be used to diag- nose for mycotoxin exposure long before significant clinical symptoms manifest (Tessari et al. 2010). In the present study, AFB1 was found above LOQ in plasma, liver, and egg samples from layers fed AFB1-con- taminated diets. Breast muscle samples from all experimen- tal groups did not contain any of the AFs that were tested. The maximum AFB1 residual concentration of 0.64 µg/kg corresponding to a carry-over factor of 0.12% was found in liver samples of layers fed a diet with both FBs and AFB1. The present study’s carry-over was slightly lower than in another study with laying hens where diets contaminated with AFB1 at levels of 894 µg/kg feed resulted in liver AFB1 residues of 1.59 µg/kg and thus a calculated carry-over fac- tor of 0.18% (Herzallah 2013). Other researchers found that feeding 2500 µg/kg feed of AFB1 to laying hens produced up to 4.13 µg/kg of liver AFB1 residues in one trial and 2.21 µg/ kg in another, and corresponding calculated carry-over fac- tors of 0.17% and 0.09%, respectively (Rizzi et al. 2003; Zaghini et al. 2005). For a short-term 7-day feeding trial, Trucksess et al. (1983) found that laying hens fed diets with extremely high AFB1 levels of 8000 µg/kg feed had a calcu- lated lower carry-over factor of 0.01% when compared to the present study. In our previous work, the highest AFB1 resi- due level of 0.12 µg/kg corresponding to a calculated carry- over factor of 0.06% was found in liver samples of broiler chickens fed dietary AFB1 at concentrations of 220 µg/kg feed and 17,430 µg FB1 + FB2/kg feed (Ochieng et al. 2023). The differences in the mycotoxin concentrations in feeds, the length of exposure time, and the sensitivity of the breed of hens used in the trials can contribute to the discrepancies in the carry-over factors seen in the various research works. Residues of AFB1 of up to 16.36 µg/kg were found in field surveys of chicken liver samples taken from markets and slaughterhouses, suggesting that the hens were exposed to AFB1, particularly through contaminated diets (Amirkhizi et al. 2015; Iqbal et al. 2014; Sineque et al. 2017). In comparison to plasma, eggs, and muscle samples, the current study’s findings showed that liver samples had the highest levels of AFB1, and these findings are consistent with those of previous studies (Bintvihok & Kositcharoenkul 2006; Trucksess et al. 1983). Aflatoxin B1 is metabolized in the liver into AFB1-8,9-epoxide, which can then bind to DNA, RNA, or other macromolecules, including proteins in the liver. Additionally, the epoxide can cause malignant growths by deactivating antioxidant enzymes (Yunus et al. 2011). The BENT utilized in this study decreased the concen- tration of AFB1 that accumulated in the liver of layers fed diets contaminated with AFB1, supporting the findings of Bhatti et al. (2018) that BENT can bind to AFB1 and the binder-AFB1 complex is excreted through feces, thereby reducing the level of AFB1 that bioaccumulates in organs. Both plasma and liver samples of layers fed diets con- taining high levels of AFB1 or a diet consisting only of BENT showed traces of AFM1 (below the LOQ). One of the hydroxylated metabolites of AFB1 is AFM1 and it is frequently found in the tissues, milk, or eggs of animals that have been exposed to AFB1 (Kemboi et al. 2023). Trucksess et al. (1983) reported that AFM1 in the con- centrations ranges of 0.04 to 0.10 µg/kg was present in kidney samples from laying hens fed AFB1 at extremely high concentrations of 8000 µg/kg feed for a short period of 7 days. 72 Mycotoxin Research (2025) 41:63–75 Other AFs examined in this study such as AFB2, AFG1, AFG2, and AFM2 were also found (below the LOQ values) in plasma and liver samples of the layers that were fed diets contaminated with AFB1. These AFs make up a very minor portion of all naturally produced AFs and are rarely found in chicken tissues (Bintvihok & Kositcharoenkul 2006; Okoth et al. 2018). In the present study, only egg samples taken from layers fed diets with the highest dosage of AFB1 contained residues of AFB1. The highest concentration of AFB1 residues of 0.040 µg/kg (carry-over factor of 0.007%) found in layers fed AFB1 only was significantly higher than the concentration of 0.028 µg/kg found in the egg samples of layers fed both AFB1 and FBs. Future research should look into the effects of mycotoxins' interactions on the transmission of AFB1 into eggs, as this study showed. The majority of research works have assessed the transmission of mycotoxins from feed to eggs in the presence of a single mycotoxin contamination. Oliveira et al. (2000) reported that eggs from laying hens that consumed 500 µg/kg of dietary AFB1 had AFB1 residues of 0.16 µg/kg, which translates to a computed carry-over factor of 0.032%. There may have been a greater accumulation and transfer of AFB1 from feeds into eggs in the latter trial since the feeding time was double (8 weeks) compared to the pre- sent study (4 weeks). According to Trucksess et al. (1983), residues of AFB1 of 0.2 µg/kg resulting in a calculated carry- over factor of 0.04% were observed in egg samples of laying hens fed very high concentrations of AFB1 (8000 µg/kg feed) for just 7 days. The latter study’s shorter feeding duration combined with likely low analysis accuracy at low AFB1 levels may have led to the low carry-over observed. In field surveys conducted in SSA, levels of AFB1 residues of up to 7.6 µg/kg were found in egg samples from farms and markets (Tatfo Keutchatang et al. 2022; Tchana et al. 2010). Research done outside SSA showed that egg samples taken from mar- kets and slaughterhouses had AFB1 levels ranging from 0.3 to 5.8 µg/kg (Herzallah 2009; Iqbal et al. 2014). Wang et al. (2018) found up to 168 µg/kg of AFB1 residues in a single egg sample taken from a market. In the present study, AFB1 carry-over factor into eggs was decreased when BENT was added to diets contami- nated with AFB1, suggesting that BENT could bind to AFB1 and decrease its gastrointestinal absorption and subsequent transfer into eggs. In conclusion, this study demonstrated that feeding lay- ing hens either high (546 µg/kg) or moderate (54.6 µg/kg) AFB1 or FBs (7.9 mg/kg) alone or in combination had no influence on the hens’ FCR and FI. On the other hand, the contaminated diets decreased egg production, increased gizzard, liver, and spleen weights, and elevated serum uric acid levels. When compared to the effects of a single mycotoxin, the interactions between AFB1 and FBs had a significant effect on the weights of the spleen, heart, and gizzard as well as the concentration of AFB1 residues in eggs. Residues of AFB1 (maximum 0.64 µg/kg) and trace levels of AFM1 (below LOQ values) were found in plasma, liver, and egg samples of laying hens that were fed AFB1- contaminated diets. The addition of BENT and/or FZYM to contaminated diets reduced the individual or combined effects of AFB1 and FBs on changes in blood biochemis- try, organ weights, egg production, and egg weight. The detoxifiers also decreased the level of AFB1 that accu- mulated in the liver and eggs. Therefore, the mycotoxin detoxifiers provided an appropriate way to mitigate the detrimental effects of AFB1 and FBs on the productivity and health of laying hens as well as decreased the concen- tration of AFB1 that was transferred into chicken products, guaranteeing the safety of these food items, especially in SSA where mycotoxin monitoring along the food chain is not consistently conducted. Supplementary Information  The online version contains supplemen- tary material available at https://​doi.​org/​10.​1007/​s12550-​024-​00566-x. Acknowledgements  The authors would like to acknowledge personnel of the Mycology and Mycotoxin Laboratory, University of Nairobi, Kenya, for their help with the laboratory production of mycotoxins. The management and staff of ILRI, Nairobi, Kenya, for their coopera- tion and help with the animal experiments, are also acknowledged by the authors. Many thanks to Nicholas Ndiwa (ICRAF) and Jane Poole (ILRI) for helping with the data analysis and animal trial design. Author contributions  P.E. Ochieng: Methodology, investigation, for- mal analysis, writing—original draft preparation. D.C. Kemboi: Inves- tigation, writing—review and editing. S. Okoth: Supervision, concep- tualization, writing—review and editing. S. De Baere: Methodology, investigation, writing—review and editing. E. Cavalier: Methodology, investigation, writing—review and editing. E. Kang’ethe: Supervision, writing—review and editing. B. Doupevec: Methodology, investiga- tion, writing—review and editing. J. Gathumbi: Funding acquisition, conceptualization, supervision, writing—review and editing. M-L. Scippo: Funding acquisition, supervision, methodology, writing— review and editing. G. Antonissen: Funding acquisition, project coordi- nation, supervision, conceptualization, methodology, writing—review and editing. J.F. Lindahl: Supervision, conceptualization, methodol- ogy, formal analysis, writing—review and editing. S. Croubels: Fund- ing acquisition, supervision, conceptualization, project coordination, methodology, writing—review and editing. All authors reviewed the manuscript. Funding  Open access funding provided by Uppsala University. This research was conducted within the ERA-NET LEAP-Agri MycoSafe- South project and was funded by the Kenyan Ministry of Education Science and Technology (MOEST), the Belgian National Fund for Scientific Research (FNRS), the Belgian Science Policy Office (BEL- SPO), Research Council of Norway (RCN), South Africa’s National Research Foundation (NRF), BIOMIN Holding GmbH (dsm-firmen- ich), and Harbro Ltd. The mycotoxin detoxifiers (Mycofix® Secure and FUMzyme®) were kindly provided by Biomin® GmbH (Getzersdorf, Austria, part of dsm-firmenich). The AFs levels in biological samples were determined using a UPLC-MS/MS instrument part of the Ghent University MSsmall Expertise Centre for advanced mass spectrometry analysis of small organic molecules and partly funded by a Research Foundation Flanders (FWO) grant FWO.HMZ.2022.0008.01. https://doi.org/10.1007/s12550-024-00566-x 73Mycotoxin Research (2025) 41:63–75 Data Availability  No datasets were generated or analyzed during the current study. Declarations  Competing interests  The authors declare no competing interests. Open Access  This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. References Akinola LAF, Essien A (2011) Relevance of rural poultry production in developing countries with special reference to Africa. World’s Poult Sci J 67(4):697–705. https://​doi.​org/​10.​1017/​S0043​93391​ 10007​78 Amirkhizi B, Arefhosseini SR, Ansarin M, Nemati M (2015) Aflatoxin B1 in eggs and chicken livers by dispersive liquid–liquid micro- extraction and HPLC. Food Addit Contam: Part B 8(4):245–249. https://​doi.​org/​10.​1080/​19393​210.​2015.​10676​49 Antonissen G, Martel A, Pasmans F, Ducatelle R, Verbrugghe E, Van- denbroucke V, Li S, Haesebrouck F, Van Immerseel F, Croubels S (2014) The impact of Fusarium mycotoxins on human and animal host susceptibility to infectious diseases. Toxins 6(2):430–452. https://​doi.​org/​10.​3390/​toxin​s6020​430 Antonissen G, De Baere S, Novak B, Schatzmayr D, den Hollander D, Devreese M, Croubels S (2020) Toxicokinetics of hydrolyzed fumonisin B1 after single oral or intravenous bolus to broiler chickens fed a control or a fumonisins-contaminated diet. Toxins 12(6):413. https://​doi.​org/​10.​3390/​toxin​s1206​0413 Bhatti SA, Khan MZ, Hassan ZU, Saleemi MK, Saqib M, Khatoon A, Akhter M (2018) Comparative efficacy of bentonite clay, acti- vated charcoal and Trichosporon mycotoxinivorans in regulating the feed-to-tissue transfer of mycotoxins: regulating mycotoxins deposition in liver. J Sci Food Agric 98(3):884–890. https://​doi.​ org/​10.​1002/​jsfa.​8533 Bintvihok A, Kositcharoenkul S (2006) Effect of dietary calcium pro- pionate on performance, hepatic enzyme activities and aflatoxin residues in broilers fed a diet containing low levels of aflatoxin B1. Toxicon 47(1):41–46. https://​doi.​org/​10.​1016/j.​toxic​on.​2005.​ 09.​009 Chen J, Wei Z, Wang Y, Long M, Wu W, Kuca K (2021) Fumonisin B1: mechanisms of toxicity and biological detoxification progress in animals. Food Chem Toxicol 149:111977. https://​doi.​org/​10.​ 1016/j.​fct.​2021.​111977 Chowdhury SR, Smith TK, Boermans HJ, Woodward B (2005) Effects of feed-borne Fusarium mycotoxins on hematology and immunol- ogy of laying hens. Poult Sci 84(12):1841–1850. https://​doi.​org/​ 10.​1093/​ps/​84.​12.​1841 De Baere S, Ochieng PE, Kemboi DC, Scippo M-L, Okoth S, Lindahl JF, Gathumbi JK, Antonissen G, Croubels S (2023) Develop- ment of high-throughput sample preparation procedures for the quantitative determination of aflatoxins in biological matrices of chickens and cattle using UHPLC-MS/MS. Toxins 15(1):37. https://​doi.​org/​10.​3390/​toxin​s1501​0037 EFSA Panel on Additives and Products or Substances used in Ani- mal Feed (FEEDAP), Rychen G, Aquilina G, Azimonti G, Bam- pidis V, de Lourdes Bastos M, Bories G, Chesson A, Cocconcelli PS, Flachowsky G, Gropp J, Kolar B, Kouba M, López‐Alonso M, Mantovani A, Mayo B, Ramos F, Saarela M, Villa RE, …, López Puente S (2016) Safety and efficacy of fumonisin ester- ase (FUMzyme®) as a technological feed additive for all avian species. EFSA J 14(11). https://​doi.​org/​10.​2903/j.​efsa.​2016.​4617 EFSA Panel on Additives and Products or Substances used in Animal Feed (FEEDAP), Bampidis V, Azimonti G, de Bastos ML, Chris- tensen H, Dusemund B, Kos Durjava M, Kouba M, López‐Alonso M, López Puente S, Marcon F, Mayo B, Pechová A, Petkova M, Ramos F, Sanz Y, Villa RE, Woutersen R, Brozzi R, …, López‐ Gálvez G (2020) Safety and efficacy of fumonisin esterase from Komagataella phaffii DSM 32159 as a feed additive for all animal species. EFSA J 18(7). https://​doi.​org/​10.​2903/j.​efsa.​2020.​6207 European Commission (2002) Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable sub- stances in animal feed—Council statement. L 140:10–22 European Commission (2006) Commission Recommendation 2006/576/EC of 17 August 2006 on the presence of deoxynivale- nol, zearalenone, ochratoxin A, T-2 and HT-2 toxin and fumoni- sins in products intended for animal feeding. L 229:7–9 FAO (2022) Africa sustainable livestock 2050: business models along the poultry value chain in Kenya Evidence from Kiambu and Nai- robi City Counties. FAO. https://​doi.​org/​10.​4060/​cb819​0en Fernandez A, Verde MT, Gascon M, Ramos J, Gomez J, Luco DF, Chavez G (1994) Variations of clinical biochemical parameters of laying hens and broiler chickens fed aflatoxin-containing feed. Avian Pathol 23(1):37–47. https://​doi.​org/​10.​1080/​03079​45940​84189​73 Grenier B, Schwartz-Zimmermann HE, Gruber-Dorninger C, Dohnal I, Aleschko M, Schatzmayr G, Moll WD, Applegate TJ (2017) Enzymatic hydrolysis of fumonisins in the gastrointestinal tract of broiler chickens. Poult Sci 96(12):4342–4351. https://​doi.​org/​ 10.​3382/​ps/​pex280 Heinl S, Hartinger D, Thamhesl M, Vekiru E, Krska R, Schatzmayr G, Moll W-D, Grabherr R (2010) Degradation of fumonisin B1 by the consecutive action of two bacterial enzymes. J Biotechnol 145(2):120–129. https://​doi.​org/​10.​1016/j.​jbiot​ec.​2009.​11.​004 Herzallah SM (2009) Determination of aflatoxins in eggs, milk, meat and meat products using HPLC fluorescent and UV detectors. Food Chem 114(3):1141–1146. https://​doi.​org/​10.​1016/j.​foodc​ hem.​2008.​10.​077 Herzallah SM (2013) Aflatoxin B1 residues in eggs and flesh of lay- ing hens fed aflatoxin B1 contaminated diet. Am J Agric Biol Sci 8(2):156–161. https://​doi.​org/​10.​3844/​ajabs​sp.​2013.​156.​161 Huff WE, Kubena LF, Harvey RB, Hagler WM, Swanson SP, Phillips TD, Creger CR (1986) Individual and combined effects of afla- toxin and deoxynivalenol (DON, Vomitoxin) in broiler chickens. Poult Sci 65(7):1291–1298. https://​doi.​org/​10.​3382/​ps.​06512​91 Huff WE, Harvey RB, Kubena LF, Rottinghaus GE (1988) Toxic syn- ergism between aflatoxin and T-2 toxin in broiler chickens. Poult Sci 67(10):1418–1423. https://​doi.​org/​10.​3382/​ps.​06714​18 Iqbal SZ, Nisar S, Asi MR, Jinap S (2014) Natural incidence of aflatox- ins, ochratoxin A and zearalenone in chicken meat and eggs. Food Control 43:98–103. https://​doi.​org/​10.​1016/j.​foodc​ont.​2014.​02.​046 Kemboi DC, Ochieng PE, Antonissen G, Croubels S, Scippo M-L, Okoth S, Kangethe EK, Faas J, Doupovec B, Lindahl JF, Gath- umbi JK (2020) Multi-mycotoxin occurrence in dairy cattle and poultry feeds and feed ingredients from Machakos Town Kenya. Toxins 12(12):762. https://​doi.​org/​10.​3390/​toxin​s1212​0762 Kemboi D, Antonissen G, Ochieng P, Croubels S, De Baere S, Scippo M-L, Okoth S, Kangethe E, Faas J, Doupovec B, Lindahl J, http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.1017/S0043933911000778 https://doi.org/10.1017/S0043933911000778 https://doi.org/10.1080/19393210.2015.1067649 https://doi.org/10.3390/toxins6020430 https://doi.org/10.3390/toxins12060413 https://doi.org/10.1002/jsfa.8533 https://doi.org/10.1002/jsfa.8533 https://doi.org/10.1016/j.toxicon.2005.09.009 https://doi.org/10.1016/j.toxicon.2005.09.009 https://doi.org/10.1016/j.fct.2021.111977 https://doi.org/10.1016/j.fct.2021.111977 https://doi.org/10.1093/ps/84.12.1841 https://doi.org/10.1093/ps/84.12.1841 https://doi.org/10.3390/toxins15010037 https://doi.org/10.2903/j.efsa.2016.4617 https://doi.org/10.2903/j.efsa.2020.6207 https://doi.org/10.4060/cb8190en https://doi.org/10.1080/03079459408418973 https://doi.org/10.3382/ps/pex280 https://doi.org/10.3382/ps/pex280 https://doi.org/10.1016/j.jbiotec.2009.11.004 https://doi.org/10.1016/j.foodchem.2008.10.077 https://doi.org/10.1016/j.foodchem.2008.10.077 https://doi.org/10.3844/ajabssp.2013.156.161 https://doi.org/10.3382/ps.0651291 https://doi.org/10.3382/ps.0671418 https://doi.org/10.1016/j.foodcont.2014.02.046 https://doi.org/10.3390/toxins12120762 74 Mycotoxin Research (2025) 41:63–75 Gathumbi J (2023) Efficacy of bentonite and fumonisin esterase in mitigating the effects of aflatoxins and fumonisins in two Ken- yan cattle breeds. J Agric Food Chem 71(4):2143–2151. https://​ doi.​org/​10.​1021/​acs.​jafc.​2c082​17 Laurain J, Tardieu D, Matard-Mann M, Rodriguez MA, Guerre P (2021) Fumonisin B1 accumulates in chicken tissues over time and this accumulation was reduced by feeding algo-clay. Toxins 13(10):701. https://​doi.​org/​10.​3390/​toxin​s1310​0701 Lee JT, Jessen KA, Beltran R, Starkl V, Schatzmayr G, Borutova R, Caldwell DJ (2012) Mycotoxin-contaminated diets and deactivat- ing compound in laying hens: 1. effects on performance charac- teristics and relative organ weight. Poult Sci 91(9):2089–2095. https://​doi.​org/​10.​3382/​ps.​2012-​02136 Magnoli AP, Rodriguez MC, González Pereyra ML, Poloni VL, Peralta MF, Nilson AJ, Miazzo RD, Bagnis G, Chiacchiera SM, Cava- glieri LR (2017) Use of yeast (Pichia kudriavzevii) as a novel feed additive to ameliorate the effects of aflatoxin B1 on broiler chicken performance. Mycotoxin Res 33(4):273–283. https://​doi.​org/​10.​ 1007/​s12550-​017-​0285-y Magothe TM, Okeno TO, Muhuyi WB, Kahi AK (2012) Indigenous chicken production in Kenya: I. Current Status. World’s Poult Sci J 68(1):119–132. https://​doi.​org/​10.​1017/​S0043​93391​20001​28 Mesgar A, Aghdam Shahryar H, Bailey CA, Ebrahimnezhad Y, Mohan A (2022) Effect of dietary L-threonine and toxin binder on per- formance, blood parameters, and immune response of broilers exposed to aflatoxin B1. Toxins 14(3):192. https://​doi.​org/​10.​ 3390/​toxin​s1403​0192 Monbaliu S, Van Poucke C, Detavernier C, Dumoulin F, Van De Velde M, Schoeters E, Van Dyck S, Averkieva O, Van Peteghem C, De Saeger S (2010) Occurrence of mycotoxins in feed as analyzed by a multi-mycotoxin LC-MS/MS method. J Agric Food Chem 58(1):66–71. https://​doi.​org/​10.​1021/​jf903​859z Njobeh PB, Dutton MF, Åberg AT, Haggblom P (2012) Estimation of multi-mycotoxin contamination in South African compound feeds. Toxins 4(10):836–848. https://​doi.​org/​10.​3390/​toxin​s4100​836 Nutrient Requirements of Poultry (1994) Ninth Revised Edition. National Academies Press, p 2114. https://​doi.​org/​10.​17226/​2114 Ochieng PE, Scippo M-L, Kemboi DC, Croubels S, Okoth S, Kang’ethe EK, Doupovec B, Gathumbi JK, Lindahl JF, Antonissen G (2021) Mycotoxins in poultry feed and feed ingredients from Sub-saharan Africa and their impact on the production of broiler and layer chickens: a review. Toxins 13(9):633. https://​doi.​org/​10.​3390/​ toxin​s1309​0633 Ochieng PE, Kemboi DC, Scippo M-L, Gathumbi JK, Kangethe E, Doupovec B, Croubels S, Lindahl JF, Antonissen G, Okoth S (2022) Maximizing laboratory production of aflatoxins and fumonisins for use in experimental animal feeds. Microorganisms 10(12):2385. https://​doi.​org/​10.​3390/​micro​organ​isms1​01223​85 Ochieng PE, Croubels S, Kemboi D, Okoth S, De Baere S, Cavalier E, Kang’ethe E, Faas J, Doupovec B, Gathumbi J, Douny C, Scippo M-L, Lindahl JF, Antonissen G (2023) Effects of aflatoxins and fumonisins, alone or in combination, on performance, health, and safety of food products of broiler chickens, and mitigation efficacy of bentonite and fumonisin esterase. J Agric Food Chem 71:13462–13473. https://​doi.​org/​10.​1021/​acs.​jafc.​3c017​33 Oğuz H, Kurtoğlu F, Kurtoğlu V, Bırdane YO (2002) Evaluation of biochemical characters of broiler chickens during dietary afla- toxin (50 and 100 ppb) and clinoptilolite exposure. Res Vet Sci 73(1):101–103. https://​doi.​org/​10.​1016/​S0034-​5288(02)​00040-1 Okoth S, De Boevre M, Vidal A, Diana Di Mavungu J, Landschoot S, Kyallo M, Njuguna J, Harvey J, De Saeger S (2018) Genetic and toxigenic variability within Aspergillus flavus population isolated from maize in two diverse environments in Kenya. Front Microbiol 9:57. https://​doi.​org/​10.​3389/​fmicb.​2018.​00057 Oliveira CAF, Kobashigawa E, Reis TA, Mestieri L, Albuquerque R, Correa LMB (2000) Aflatoxin B1 residues in eggs of laying hens fed a diet containing different levels of the mycotoxin. Food Addit Contam 17(6):459–462. https://​doi.​org/​10.​1080/​ 02652​03005​00340​37 Pappas AC, Tsiplakou E, Tsitsigiannis DI, Georgiadou M, Iliadi MK, Sotirakoglou K, Zervas G (2016) The role of bentonite binders in single or concomitant mycotoxin contamination of chicken diets. Br Poult Sci 57(4):551–558. https://​doi.​org/​10.​ 1080/​00071​668.​2016.​11877​12 R Core Team (2020) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Retrieved from https://​www.R-​proje​ct.​org/. Accessed 20 Apr 2023 Rizzi L, Simioli M, Roncada P, Zaghini A (2003) Aflatoxin B1 and clinoptilolite in feed for laying hens: effects on egg quality, myco- toxin residues in livers, and hepatic mixed-function oxygenase activities. J Food Prot 66(5):860–865. https://​doi.​org/​10.​4315/​ 0362-​028X-​66.5.​860 Rosa CAR, Miazzo R, Magnoli C, Salvano M, Chiacchiera SM, Ferrero S, Saenz M, Carvalho ECQ, Dalcero A (2001) Evaluation of the efficacy of bentonite from the South of Argentina to ameliorate the toxic effects of aflatoxin in broilers. Poult Sci 80(2):139–144. https://​doi.​org/​10.​1093/​ps/​80.2.​139 Sakamoto MI, Murakami AE, Fernandes AM, Ospina-Rojas IC, Nunes KC, Hirata AK (2018) Performance and serum biochemical pro- file of Japanese quail supplemented with silymarin and contami- nated with aflatoxin B1. Poult Sci 97(1):159–166. https://​doi.​org/​ 10.​3382/​ps/​pex277 Saminathan M, Selamat J, Abbasi Pirouz A, Abdullah N, Zulkifli I (2018) Effects of nano-composite adsorbents on the growth per- formance, serum biochemistry, and organ weights of broilers fed with aflatoxin-contaminated feed. Toxins 10(9):345. https://​doi.​ org/​10.​3390/​toxin​s1009​0345 Shannon TA, Ledoux DR, Rottinghaus GE, Shaw DP, Daković A, Marković M (2017) The efficacy of raw and concentrated benton- ite clay in reducing the toxic effects of aflatoxin in broiler chicks. Poult Sci 96(6):1651–1658. https://​doi.​org/​10.​3382/​ps/​pew408 Sineque A, Macuamule C, Dos Anjos F (2017) Aflatoxin B1 contami- nation in chicken livers and gizzards from industrial and small abattoirs, measured by ELISA technique in Maputo, Mozambique. Int J Environ Res Public Health 14(9):951. https://​doi.​org/​10.​ 3390/​ijerp​h1409​0951 Sulyok M, Berthiller F, Krska R, Schuhmacher R (2006) Development and validation of a liquid chromatography/tandem mass spectro- metric method for the determination of 39 mycotoxins in wheat and maize. Rapid Commun Mass Spectrom 20(18):2649–2659. https://​doi.​org/​10.​1002/​rcm.​2640 Swamy H, Smith T, Cotter P, Boermans H, Sefton A (2002) Effects of feeding blends of grains naturally contaminated with Fusarium mycotoxins on production and metabolism in broilers. Poult Sci 81(7):966–975. https://​doi.​org/​10.​1093/​ps/​81.7.​966 Tangni EK, Van Pamel E, Huybrechts B, Delezie E, Van Hoeck E, Daeseleire E (2020) Carry-over of some Fusarium mycotoxins in tissues and eggs of chickens fed experimentally mycotoxin- contaminated diets. Food Chem Toxicol 145:111715. https://​doi.​ org/​10.​1016/j.​fct.​2020.​111715 Tardieu D, Travel A, Le Bourhis C, Metayer J-P, Mika A, Cleva D, Boissieu C, Guerre P (2021) Fumonisins and zearalenone fed at low levels can persist several days in the liver of turkeys and broiler chickens after exposure to the contaminated diet was stopped. Food Chem Toxicol 148:111968. https://​doi.​org/​10.​ 1016/j.​fct.​2021.​111968 Tatfo Keutchatang FDP, Tchuenchieu AK, Nguegwouo E, Mouafo HT, Bouelet Ntsama IS, Kansci G, Medoua GN (2022) Occur- rence of total aflatoxins, aflatoxin B1, and ochratoxin A in chicken and eggs in some Cameroon urban areas and population dietary https://doi.org/10.1021/acs.jafc.2c08217 https://doi.org/10.1021/acs.jafc.2c08217 https://doi.org/10.3390/toxins13100701 https://doi.org/10.3382/ps.2012-02136 https://doi.org/10.1007/s12550-017-0285-y https://doi.org/10.1007/s12550-017-0285-y https://doi.org/10.1017/S0043933912000128 https://doi.org/10.3390/toxins14030192 https://doi.org/10.3390/toxins14030192 https://doi.org/10.1021/jf903859z https://doi.org/10.3390/toxins4100836 https://doi.org/10.17226/2114 https://doi.org/10.3390/toxins13090633 https://doi.org/10.3390/toxins13090633 https://doi.org/10.3390/microorganisms10122385 https://doi.org/10.1021/acs.jafc.3c01733 https://doi.org/10.1016/S0034-5288(02)00040-1 https://doi.org/10.3389/fmicb.2018.00057 https://doi.org/10.1080/02652030050034037 https://doi.org/10.1080/02652030050034037 https://doi.org/10.1080/00071668.2016.1187712 https://doi.org/10.1080/00071668.2016.1187712 https://www.R-project.org/ https://doi.org/10.4315/0362-028X-66.5.860 https://doi.org/10.4315/0362-028X-66.5.860 https://doi.org/10.1093/ps/80.2.139 https://doi.org/10.3382/ps/pex277 https://doi.org/10.3382/ps/pex277 https://doi.org/10.3390/toxins10090345 https://doi.org/10.3390/toxins10090345 https://doi.org/10.3382/ps/pew408 https://doi.org/10.3390/ijerph14090951 https://doi.org/10.3390/ijerph14090951 https://doi.org/10.1002/rcm.2640 https://doi.org/10.1093/ps/81.7.966 https://doi.org/10.1016/j.fct.2020.111715 https://doi.org/10.1016/j.fct.2020.111715 https://doi.org/10.1016/j.fct.2021.111968 https://doi.org/10.1016/j.fct.2021.111968 75Mycotoxin Research (2025) 41:63–75 exposure. J Environ Public Health 2022:1–9. https://​doi.​org/​10.​ 1155/​2022/​55410​49 Tchana A, Moundipa P, Tchouanguep F (2010) Aflatoxin contamina- tion in food and body fluids in relation to malnutrition and cancer status in Cameroon. Int J Environ Res Public Health 7(1):178– 188. https://​doi.​org/​10.​3390/​ijerp​h7010​178 Tessari ENC, Oliveira CAF, Cardoso ALSP, Ledoux DR, Rottinghaus GE (2006) Effects of aflatoxin B1 and fumonisin B1 on body weight, antibody titres and histology of broiler chicks. Br Poult Sci 47(3):357–364. https://​doi.​org/​10.​1080/​00071​66060​07560​71 Tessari ENC, Kobashigawa E, Cardoso ALSP, Ledoux DR, Rot- tinghaus GE, Oliveira CAF (2010) Effects of aflatoxin B1 and fumonisin B1 on blood biochemical parameters in broilers. Toxins 2(4):453–460. https://​doi.​org/​10.​3390/​toxin​s2040​453 Trucksess MW, Stoloff L, Young K, Wyatt RD, Miller BL (1983) Aflatoxicol and aflatoxins B1 and M1 in eggs and tissues of laying hens consuming aflatoxin-contaminated feed. Poult Sci 62(11):2176–2182 Tsiouris V, Tassis P, Raj J, Mantzios T, Kiskinis K, Vasiljević M, Delić N, Petridou E, Brellou GD, Polizopoulou Z, Mittas N, Georgopou- lou I (2021) Investigation of a novel multicomponent mycotoxin detoxifying agent in amelioration of mycotoxicosis induced by aflatoxin-B1 and ochratoxin A in broiler chicks. Toxins 13(6):367. https://​doi.​org/​10.​3390/​toxin​s1306​0367 Wang L, Zhang Q, Yan Z, Tan Y, Zhu R, Yu D, Yang H, Wu A (2018) Occurrence and quantitative risk assessment of twelve mycotoxins in eggs and chicken tissues in China. Toxins 10(11):477. https://​ doi.​org/​10.​3390/​toxin​s1011​0477 Yunus AW, Razzazi-Fazeli E, Bohm J (2011) Aflatoxin B1 in affect- ing broiler’s performance, immunity, and gastrointestinal tract: a review of history and contemporary issues. Toxins 3(6):566–590. https://​doi.​org/​10.​3390/​toxin​s3060​566 Zaghini A, Martelli G, Roncada P, Simioli M, Rizzi L (2005) Manna- noligosaccharides and aflatoxin B1 in feed for laying hens: effects on egg quality, aflatoxins B1 and M1 residues in eggs, and afla- toxin B1 levels in liver. Poult Sci 84(6):825–832. https://​doi.​org/​ 10.​1093/​ps/​84.6.​825 Zhao L, Feng Y, Wei J-T, Zhu M-X, Zhang L, Zhang J-C, Karrow NA, Han Y-M, Wu Y-Y, Guo Y-M, Sun L-H (2021) Mitigation effects of bentonite and yeast cell wall binders on AFB1, DON, and OTA induced changes in laying hen performance, egg qual- ity, and health. Toxins 13(2):156. https://​doi.​org/​10.​3390/​toxin​ s1302​0156 Zhu F, Zhu L, Xu J, Wang Y, Wang Y (2023) Effects of moldy corn on the performance, antioxidant capacity, immune function, metabo- lism and residues of mycotoxins in eggs, muscle, and edible vis- cera of laying hens. Poult Sci 102(4):102502. https://​doi.​org/​10.​ 1016/j.​psj.​2023.​102502 Publisher's Note  Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. https://doi.org/10.1155/2022/5541049 https://doi.org/10.1155/2022/5541049 https://doi.org/10.3390/ijerph7010178 https://doi.org/10.1080/00071660600756071 https://doi.org/10.3390/toxins2040453 https://doi.org/10.3390/toxins13060367 https://doi.org/10.3390/toxins10110477 https://doi.org/10.3390/toxins10110477 https://doi.org/10.3390/toxins3060566 https://doi.org/10.1093/ps/84.6.825 https://doi.org/10.1093/ps/84.6.825 https://doi.org/10.3390/toxins13020156 https://doi.org/10.3390/toxins13020156 https://doi.org/10.1016/j.psj.2023.102502 https://doi.org/10.1016/j.psj.2023.102502 Aflatoxins and fumonisins co-contamination effects on laying hens and use of mycotoxin detoxifiers as a mitigation strategy Abstract Introduction Material and methods Ethical statement Preparation of experimental AFB1- and FBs-contaminated diets Experimental birds and housing Production performance; collection of blood, organs, and eggs; and blood biochemistry Analysis of residues of aflatoxins in plasma, liver, muscle tissue, and eggs using UPLC-MSMS Statistical analysis and carry-over factors Results Production performance Relative weight of organs Biochemical parameters Aflatoxins residues in plasma, liver, muscle, and egg Discussion Acknowledgements References