Livestock Research for Rural Development 28 (12) 2016 Guide for preparation of papers LRRD Newsletter

Citation of this paper

Efficacy of Thai bentonite to ameliorate the adverse effects of aflatoxin and fumonisin contaminated diets in Cherry Valley ducks

B Tengjaroenkul, C Saksangawong, S Wongtangtintan, U Tengjaroenkul1, K Ratanasinthuphong2, N Srikacha3 and L Neeratanaphan

Khon Kaen University, Khon Kaen 40002, Thailand
1 Chiang Mai University, Chiang Mai 50200, Thailand
2 Rajamangala University of Technology Isan, Nakhon Rajasima 30000, Thailand
3 Kalasin University, Kalasin 46000, Thailand


The objective of this study was to determine the efficacy of Thai bentonite clay (TB) to ameliorate the adverse effects of mixed aflatoxin (AF) and fumonisin (FMN) contaminated diets (MD) in Cherry Valley ducks. A total of 210, 7-day-old ducks were randomly allocated to 4 treatments: CTL: control diet with uncontaminated maize; ; MCT: diet with contaminated maize (total AF = 70.4 ppb and total FMN = 580 ppb);  HSCAS: MCT + 0.1% HSCAS (hydrated sodium calcium aluminosilicate);  and TB: MCT + 0.2% TB. Each treatment had three replication pens, with 14 birds per pen.

At the end of the 6 week experiment, the ducks fed MCT  had decreased weight gain, feed intake, poorer feed conversion and survival, and several adverse physiological effects, including packed cell volume, hemoglobin level and serum enzyme values. Supplementation of MCT with 0.1% HSCAS or with 0.2% TB reduced the adverse effects on all parameters measured. The results support the conclusion that 0.2% TB can alleviate mycotixicoses induced by AF and FMN in ducks.

Keywords: binder, clay, fungus, poultry


Mycotoxins are metabolic substances produced by fungi that can cause diseases and death in humans and animals. A previous survey suggested that more than one-third of grains is contaminated with mycotoxin in the Asia-Pacific region (Binder et al 2007). Aflatoxins (AF) are the most common toxin produced byAspergillus flavus and A. parasiticus. Aflatoxin B1 (AFB1) is the most hepato-toxic among AF (Hueber et al 2004, WHO 2008, Godfrey et al 2013). The symptoms of aflatoxicoses are loss of appetite, reduction of growth performance, feed efficiency and production and causes immunosuppression, liver damage and high mortality, particularly in young poultry (Bintvihok 2001, Poirier 2004, Bennett et al 2007, Okiki et al 2010, Wild and Gong 2010). Fumonisins (FMN) are a group of toxins produced by fungi in the genus Fusarium that can cause toxicities in liver and kidney of all animal species. The toxins can cause alterations in the sphingolipid metabolism and cause cardiovascular dysfunction, increment oxidative stress, the induction of lipid peroxidation, DNA fragmentation, apoptosis and impaired immunity (European Commission 2000, 2003, Marasas 2001, Yiannikouris and Jouany 2002, Stockmann and Savolainen 2008).

Physical, chemical and biological approaches are available to counteract the toxic effects of the mycotoxins. One approach to detoxify the toxin is to combine a toxin binder or adsorbent into animal feed to inhibit the bioavailability of the toxin absorbed through the alimentary tract (Basalan et al 2006). Several reports of in vivo and in vitro studies demonstrated that toxin binders including hydrate sodium calcium aluminosilicate (HSCAS), bentonite, charcoal and zeolite show considerable promise in preventing myxotoxicoses (Huebner et al 2004, Girish and Devegowda, 2006, Kossolova et al 2009, Zhao et al 2010, Bočarov-Stančić 2011, Neeff et al 2013, Neef et al 2013,Tengjaroenkul et al 2013, Wongtangtintan 2014). Thai bentonite (TB) clay from Lopburi province is capable of binding AFB1 in vitro (Tengjaroenkul et al 2013). However, in vivo reports on the efficacy of TB to reduce the toxic effects of mycotoxins are limited. Thus, the purpose of the present study was to determine the efficacy of TB to ameliorate the adverse effects of two natural mycotoxins (AF and FMN) in contaminated diets in the Cherry Valley ducks and to compare its efficacy with HSCAS.

Materials and methods


The concentration of AF in contaminated maize and in diets was determined using HPLC (in-house method based on AOAC 2005), and for the FMN level using ELISA test kit (Ridascreen®, R-Biopharm AG, Germany). In this study, the control maize sample contained a total AF of 2.20ppb and a total FMN of 91 ppb. The contaminated maize contained total AF and total FMN of 70.4 ppb and 580 ppb, respectively and the contaminated diet (MCT) contained total AF and total FMN of 35.8 - 39.6 and 286 - 326 ppb, respectively.

Animals, diets and experimental design

A total of 210, 7-d-old Cherry Valley ducks were weighed and randomly allocated into 4 treatments for a 6 weeks feeding trial. The basal diet was formulated to meet nutritional requirements of ducks as recommended by NRC (1994) (Table 1). Experimental diets were prepared as follows: (1) CTL: Uncontaminated maize;  (2) MCT with contaminated maize (total AF (AFB1, AFB 2) = 70.4 ppb and total FMN (FMNB1, FMNB2) = 580 ppb);  (3) HSCAS: MCT + 0.1% commercial HSCAS (ALCA Co., LTD., Bangkok, Thailand);  and (4) TB: MCT + 0.2% TB (collected from Lopburi Province). Each treatment had three replication pens with 14 birds per pen. Feed and water were provided ad libitum, and lighting was set continuously throughout the experiment.

Table 1. Ingredient and nutrient composition (%) of experimental diets1 for Cherry Valley ducks.


Starter phase (1-3wk)

Grower phase (3-6wk)

Ingredients, %




Soybean meal



Rice bran oil



Rice bran



Fish meal






Dicalcium phosphate



Choline chloride












Vitamin-mineral premix2






Chemical composition 3

CP, %



Total phosphorus, %



Calcium, %



ME, kcal/kg4



1 The diet CTL was formulated with uncontaminated maize; the diets MCT, HSCAS and TB were formulated by replacing normal maize with
mycotoxin-contaminated maize

Vitamin-mineral premixes supplied the following per kilogram of diet: vitamin A 9,500 IU; vitamin D, 3,600 IU; vitamin E, 40 IU; vitamin K3, 5.5 mg;
thiamine, 3.0 mg; riboflavin, 12.6 mg; pyridoxine, 5.0 mg; vitamin B12, 0.025 mg; pantothenic acid, 12 mg; niacin, 75 mg; choline, 1,000 mg;
folic acid, 2.1 mg; biotin, 0.25 mg; Mn, 90 mg; Zn, 86 mg; Fe, 80 mg; Cu, 8.6 mg; Co, 1.5 mg; I, 0.35 mg; Se, 0.31 mg; thoxyquin, 125 mg

The chemical composition of the diets was determined according to AOAC (1995)
ME = Metabolizable energy calculated (NRC, 1994)

Productive performance

The ducks were monitored daily for clinical signs and mortality. At the end of the experiment, ducks were weighed individually after 12 hours of starvation and feed intakes for the ducks in each replication and treatment were analyzed to determine body weight gain (BWG), average daily gain (ADG), daily feed intake (ADFI), the feed conversion ratio (FCR) and the coefficient of variation of body weight (CVBW).

Hematology and serum enzymes

At the end of the experiment, blood samples (4 ml each) from wing veins of 9 birds (3 birds per replication) from each treatment were collected to determine hematological parameters and serum enzyme values. For the hematological study, packed cell volume (PCV) and hemoglobin concentration (Hb) were analyzed. For the enzyme study, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) were analyzed.

Organ weight

At the end of the experiment, 6 birds (2 birds per replication) of each treatment were humanely euthanized. Weights of liver, kidney, spleen, and heart were recorded and the weights were adjusted to 100 g of live body weight (g/ 100 g of BW) and the mean values were calculated.

Statistical analyses

Statistical analyses were performed using Proc. GLM (SAS, 1998). The differences in means were compared using Duncan’s Multiple Range Test (DMRT) and statistical significance was based on p<0.05.


Productive performance

Productive performance data are presented in Table 2. The mycotoxin-contaminated diet (MCT) had an adverse effect on ADG, ADFI, FCR, CVBW and SVR, when compared with data for ducks fed the control diet (CTL). The diets with toxin binder supplements (HSCAS and TB) had better ADG, FCR, ADFI, CVBW and SVR compared twith the contaminated diet (MCT).

Table 2. Efficacy of adsorbents to ameliorate the toxin effects of AF and FMN on productive performance and survival of Cherry Valley ducks.





ADG, (g)






ADFI, (g)












CVBW, (%)






SVR, (%)






a-d Values within a row without common letters differ at p<0.05
ADG = average daily gain; ADFI = average daily feed intake; FCR = feed consumption ratio; CVBW = coefficient of variation of body weight;
SVR = survival rate; CTL = control diet with uncontaminated maiz, MCT = diet with mycotoxin-contaminated maize; HSCAS MCTn+ 0.1% HSCAS
(hydrated sodium calcium aluminosilicate), and TB: MCT + 0.2% Thai bentonite clay (TB) (collected from Lopburi Province)

Hematology and serum enzymes

Blood and serum enzyme data are presented in Table 3. The MCCT diet significantly decreased PCV and Hb concentrations, when compared to the control diet (CLO). HSCAS and TB supplementation to MCT significantly enhanced levels of PCV and Hb, when compared with the MCT diet .

Table 3. Efficacy of adsorbents to ameliorate the toxin effects of AF and FMN on hematology and serum enzymes of Cherry Valley ducks.





Blood hematology

PCV, (%)







Hb, (g/dl)





Serum enzyme

ALT, (U/L)






AST, (U/L)






ALP, (U/L)






a-d Values within a row with the difference superscripts significantly different (p<0.05)
PCV = pack cell volume; Hb = hemoglobin; ALT = alanine aminotransferase; AST = aspartate aminotransferase; ALP = alkaline phosphatase;
SEM (standard error of mean) = dividing the standard deviation by the square root of sample size

The MCT treatment resulted in a significant increase in ALT, AST and ALP levels, when compared with the control diet treatment. HSCAS and TB supplementation of MCT diet reduced ALT, AST and ALP levels, when compared with the MCT treatment.

Organ weight

Table 4. Efficacy of adsorbents to ameliorate the toxin effects of AF and FMN on organ weight (% body weight) of Cherry Valley ducks







Relative organ weight2 (% body weight)














0.068 ab

0.072 a

0.067 ab

0.064 b



0.578 b





Bursa of Fabricius






a-d Values within a row without common letters are different (p<0.05);

Organ weights are presented in Table 4. The MCT treatment showed a significant increase in the weights of the liver, kidney, heart and bursa of fabricius. It was found that 0.1% HSCAS and 0.2% TB supplementation of the MCT diet caused a significant reduction in the weights of liver, kidney, heart and bursa of fabricius, when compared with the MCT diet. Supplementation of the MCT diet with HSCAS or TB of the binders had no effect on spleen weight.


Productive performance

Our results indicated that the mycotoxins  in the contaminated diet affected production performance by decreasing ADG and ADFI, and increasing FCR and CVBW in Cherry Valley ducks. These observations are similar to several previous studies. Wan et al (2013) reported that natural AFB1 less than 100 ppb linearly decreased ADG and linearly increased mortality  between 1 to 3 weeks. Han et al (2008) demonstrated that duck diets containing 20-40 ppb AFB1 decreased BWG and feed intake and increased FCR during a 1 to 6 week period.  In addition, Yang et al (2014) demonstrated that ducks fed mycotoxin-contaminated maize had reduced nutrient digestibility.

Hematology and serum enzymes

Hematology and serum enzyme data are helpful tools to determine health status, metabolic diseases, nutritional deficiencies and the welfare of animals. The MCT treatment in this study had adverse effects by decreasing PCV and Hb values, when compared with the control diet. Similar results were also mentioned in several studies of poultry species. Li et al (2012) reported that Cherry Valley ducks fed high AFB1 (170 ppb) diets had decreased Hb, platelets and mean corpuscular hemoglobin concentrations. Khajarern (2003)  found that AFB1 (60 and 120 ppb) contaminated diets decreased Hb and PCV concentrations. Chowdhury et al (2005) demonstrated that Fusarium mycotoxins can induce small decreases in hematocrit values and the total number of white blood cells in laying hens. Andretta et al (2012) used meta-analysis of 98 articles published between 1980 and 2009 to study the association of mycotoxins with hematological and biochemical profiles and found that mycotoxins reduced the hematocrit and hemoglobin levels in broilers by 5% and 15%, respectivly. The decreases in PCV and Hb may occur due to mycotoxins causing necrosis or altering hemopoietic processes (formation of blood cellular components by stem cells) in the bone marrow (Birbrair and Frenette 2016).

Serum enzyme activities of ALT, AST and ALP can be used as evaluation criteria for the grading of severity of AFB1 and FMN toxicities or specific measurement of liver function or hepatic injuries in poultry (Quist et al 2000). In this study, ducks fed the MCT diet had an increase of AST, ALT and ALP levels compared with the control diet. Similarly, previous studies reported that ducks fed 100 ppb AFB1 showed increases in AST and ALT activity as well as AST:ALT ratios (Méndez-Albores et al 2007). Chen et al (2014) found that Pekin ducks fed 110-210 ppb AFB1 had linear increases in ALP and AST activity with an increase in the AFB1 levels. Ducklings fed AFB1 contaminated maize had increased ALP activity. The ducks fed the MCT diet in this study demonstrated these adverse effects by increases in AST, ALT and ALP activity. This occurs when liver tissue is injured and the enzymes contained in liver cells are released into the blood stream, resulting i increased levels of these enzymes in the serum.

Organ weight

Measuring weights of immune-regulating organs is a common method for evaluation of immune status in poultry. As ducks may be similar, when exposed to the AF, organ weights were tested in this study. The MCT treatment increased weights of liver, kidney, heart and bursa of fabricius (p<0.05), when compare with control diet treatment. These results are similar to several studies including Wan et al (2013), who demonstrated that increasing natural AFB 1 concentrations from 25-100 ppb in diets with or without toxin binder supplementation in ducklings (during 1-3 wk) caused linearly decreases in the weights of the liver, spleen, thymus and bursa of fabricius. Khajarern and Khajarern (2003) reported that 60 and 120 ppb AFB1 increased the organ weights in Cherry Valley ducks (at 4 wk). He et al (2013) found that increases in liver and spleen weights were observed when ducks were fed contaminated maize diets (196.8 ppb AFB1). Increases in internal organ weights (liver, heart, kidney, spleen and pancreas) of the poultry is due to congestion of blood during the inflammatory response and tissue hypertrophy after expose to the toxin according to Bondy and Pestka (2000), Khajarern and Khajarern (2003) and Oswald et al (2005).

Toxin binders

In the present study, both the HSCAS and TB supplements ameliorated the adverse effects of mycotoxins on productive performance as well as on blood hematology, and serum enzymes when compared with the MCT  treatment.  HSCAS demonstratedSF contaminated broiler diets ((5 ppm) b, and the HSCAS significantly prevented the negative effects on performance and serum biochemistry in chicks. Ledoux et al (1999) mixed 1% HSCAS to AF (4 ppm) broiler diets, and demonstrated that the adsorbent improved performance, organ weights, serum biochemistry, and gross pathology in chicks. HSCAS also effectively reduced the incidence and severity of the hepatic and renal histopathological changes associated with aflatoxicosis. Eraslan et al (2004) added 0.5 and 1% HSCAS to AF (2.5 ppm) in quail diets, and the HSCAS provided a moderate amelioration the negative effects of AF on performance and biochemistry. Abousadi et al (2007) incorporated 0.5% SB and 0.5% HSCAS into AF contaminated (125 ppb) broiler diets, and demonstrated a reduction of negative effects of AF on performance and biochemistry. Gowda et al (2008) incorporated 0.5% HSCAS and 0.5% turmeric powder to AF contaminated (1 ppm) broiler diets, and found that the clay detoxified the toxin in the diet. Diaz et al (2009) infused mixed aluminosilicate and phytogenic substances to AF contaminated (up to 500 ppb) turkey diets, and showed that the binder partially reduced the negative effects of AF on performance and immunity. Wan et al (2013) put HSCAS at 0.1% in AF contaminated (up to 100 ppb) diets, which was effective in preventing the toxic effects of AF on weight gain, organ weight, serum immunoglobulin and protein concentrations, intestinal morphology and mortality in ducklings.



We thank Dr. Frank F. Mallory, Laurentian University, Canada for reviewing the manuscript and financial support was provided by the Research Group on Toxic Substances in Livestock and Aquatic Animals, and the Faculty of Veterinary Medicine, Khon Kaen University.


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Received 11 May 2016; Accepted 1 November 2016; Published 1 December 2016

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