Abstract: In an in vivo study, Sodium Bentonite (SB) was evaluated for its ability to reduce the deleterious effects of Aflatoxin B₁ (AFB₁) in broiler chickens. In this study, 288 days old Ross male broiler chickens were used and randomly assigned to 9 treatment groups, with 4 replicates of 8 birds each. Three levels of Aflatoxin B₁, AFB₁ (0, 500 and 1000 ppb) and 3 levels of SB (0.0, 0.5 and 1.0%) were fed to chickens from 0-42 days of age. Feeding levels of AFB₁ alone significantly decreased (p<0.05) feed intake, body weight and body weight gain, while, SB levels alone or in combination with AFB₁, had no effect on the mentioned traits. No significant difference was seen for feed conversion ratio during periods of 0-42 days of age. In the diet containing AFB₁, relative weight of the carcass, thigh and breast were significantly decreased (p<0.05). Adding AFB₁ alone into the diets significantly increased (p<0.05) serum ALT, AST and LDH enzymes and decreased serum GGT enzyme (p<0.05). However, SB alone or in combination with AFB₁ had no effect on the above mentioned enzymes. It is suggested that sodium bentonite at the levels of 0.5-1.0% might be used for reducing the adverse effects of aflatoxins in broiler chickens.

INTRODUCTION

Mycotoxins are secondary toxic metabolites formed by certain fungi growing on Food and animal feeds usually belonging to the genus of Aspergillus flavus and Aspergillus parasiticus (Tedesco et al., 2004) and can happen as natural contaminants of poultry feed (Leeson et al., 1995). Contamination of cereals and grains and their related products with mycotoxins causes food and feed-born intoxicants in human and animals (Meltor, 2001). Aflatoxin B₁ (AFB₁) is the most potent mycotoxins causes a wide range of clinical and sub-clinical problems in poultry (Kermanshahi et al., 2007; Hussein and Brasel, 2001). Many different toxic effects for aflatoxins, including reduced performance, hepatic intoxication, adverse effects on carcass as well as egg shell quality, immunosuppression and carcinogenicity have been reported in poultry (Charmley et al., 1995). Increase in relative weights of liver, kidney, heart, proventriculus, gizzard, spleen and pancreas in broilers consuming aflatoxin contaminated diets, have been reported (Kubena et al., 1990). Liver enlargement and discoloration in broilers have been reported by Kermanshahi et al. (2007). Also, it has been demonstrated that aflatoxins are able to alter concentrations of some blood enzymes (Kubena et al., 1990). Negative effects of aflatoxins in poultry production are both dose and time dependent (Leeson et al., 1995). At present, aflatoxins are considered unavoidable contaminants of feed and food. To combat animal aflatoxicosis and detoxification of mycotoxin-contaminated feeds, different physical methods are recommended but since mycotoxins are heat stable and resistance to heat, current agricultural practices and routine feed processing procedures can not reduce their toxicity (Bassapa and Shanta, 1996; Doyle et al., 1982). In animal nutrition, the use of mycotoxin adsorbents as feed additives is one of the most promising and widely used approaches to reduce the deleterious effect of aflatoxicosis in farm animals. It is good method to reduce carry-over of mycotoxins from the contaminated feeds into animal-derived products (Ramos et al., 1996). Phillips et al. (1987, 1988) in in vitro and in vivo studies reported that Hydrated Sodium Calcium Aluminosilicate (HSCAS) have the high capacity to adsorb aflatoxins in poultry HSCAS is a generic description and it does not uniquely define the material of use. Most of the classified HSCAS are as natural montmorillonits and bentonits (Ramos and Fernadez, 1997). In many reports, different adsorbents including zeolites, acticated charcoal and phylosilicate minerals have been used to decrease the negative effect of aflatoxins (Devegowda et al., 1996; Rosa et al., 2001). There are many adsorbents in the markets and their capacity to adsorb specific mycotoxins are not clear. Although, from nutritional point of view, bentonite type of binders would supply little nutritive value but, it is the most potent mineral substances to reduce the deleterious effect of aflatoxins in poultry (Miazzo et al., 2000, 2005; Rosa et al., 2001). Therefore, the purpose of this research was to evaluate the capacity of locally available bentonits in male broiler chickens fed diets contaminated with aflatoxin B₁.

MATERIALS AND METHODS

Total 288 days old Ross male broiler chickens were divided into 9 treatments of 4 replicates each and randomly assigned to each of 36 floor pens of 1x1 m. All birds had free access to feed and water during the experiment (0-42 days). Aflatoxin B₁ (AFB₁) for this experiment was produced using Aspergillus parasiticus strain 2999 according to the method described by Shotwell et al. (1966) and West et al. (1973). More than 80% of the aflatoxin produced by this strain is B₁ type (Rosa et al., 2001). AFB₁ concentration was determined using TLC according to Afzali (1998) method. Relative weights of edible meat, thigh and breast to live body weight measured at 42 days of age by killing 2 birds from each replicate of treatments. Serum concentrations of Aspartate amino Transferase (AST), Alanine amino Transferase (ALT), Gama Glutamyl Transferase (GGT) and Lactate Dehydrogenase (LDH) were determined at 21 and 42 days of age using commercial available kits (Darman Kave Research Laboratory, Isfahan, Iran). Starter, grower and finisher diets (Table 1) were formulated for 0-14, 14-28 and 28-42 days of age, respectively, according to the Ross 308 manual recommendations. Dietary treatments were consisted of 3 levels of AFB₁ in diet (0.0, 500 and 1000 ppb) and 3 levels of a locally prepared sodium bentonite (0.0, 0.5 and 1.0% SB, Ashkaftook mine, South of Khorasan, Iran). The SB composition was SiO₂,58.80%; Al₂O₃, 18.50%; Fe₂O₃, 5.0%; CaO, 2.15%; MgO, 2.10%; Na₂O, 3.50%; K₂O, 0.45%; TiO₂, 0.15%; moisture, 9.35%. In order to reach intended AFB₁ concentrations in diets, an appropriate mixture of Aspergillus parasiticus culture on rice was added into the basal diet. AFB₁ contaminated diets were fed to broiler chickens from 0-42 days of age. Feed intake and body weight gain recorded weekly and then performance data expressed as week basis.

Table 1:	Composition of the experimental diets
1In each period, 9 treatment diets prepared as arranged in treatment arrangements, 2Each kg of vitamin premix contained: vitamin A, 3,600,000 IU; vitamin D3, 800,000 IU; vitamin E, 7,200 IU; vitamin K3, 800 mg, vitamin B1, 720 mg; vitamin B2, 2,640 mg, vitamin B3, 4,000 mg, vitamin B5, 12,000 mg, vitamin B6, 1,200 mg vitamin B9, 400 mg; vitamin B12, 6 mg, vitamin H2, 40 mg, choline chloride, 200,000 mg, 3Each kg of mineral premix contained: Mn, 40,000 mg, Fe, 20,000 mg, Zn, 40,000 mg; Cu, 4,000 mg, Se, 80 mg

Statistical analysis: The 9 treatments were allotted in a completely randomized design, with 3x3 factorial arrangements. All data were analyzed using GLM procedure of SAS and treatment means were compared by Touki test (SAS, 1990).

RESULTS AND DISCUSSION

Appearance and performance: In the 1st and 2nd week of experiment, chickens didn’t show any sign of toxicity in their appearance. Gradually, at week 3 and later on, chickens fed AFB₁ showed some sign of aggressions, pale color in their combs and skins, fluffy feathers and retarded growth rate. Chickens receiving SB or SB with AFB₁ had normal appearance.

Data of weekly Feed Intake (FI) is presented in Table 2. Treatments had no effect on Feed intake till the end of week 3. However, chickens receiving AFB₁ alone (500 and 1000 ppb) fed lower feed when compared to other treatments. At week 4-6 FI significantly decreased in birds fed AFB₁ contaminated diets (p<0.05). The lowest FI was seen in birds fed 1000 ppb AFB₁ alone. However, the difference between 500 and 1000 ppb AFB₁ was not different only at 4th week.

Table 2:	Effect of treatments on feed intake of broiler chickens fed diets containing different levels of aflatoxin B1 and sodium bentonite
1AFB1: Aflatoxin B1; SB: Sodium Bentonite; Results for each treatments are obtained from 32 chickens in each treatment with ±SEM = 11.7; a, b, cIn each column, means with different superscripts are significantly different (p<0.05)

Table 3:	Effect of treatments on body weight gain of broiler chickens fed diets containing different levels of aflatoxin B1 and sodium bentonite
1AFB1: Aflatoxin B1, SB: Sodium Bentonite, Results for each treatments are obtained from 32 chickens in each treatment with ±SEM = 14.5, a, b, cIn each column, means with different superscripts are significantly different (p<0.05)

Addition of SB into contaminated diets increased FI similar to control birds. Data indicated that by increasing levels of AFB₁, FI decreases more and SB addition alleviated this effect. These results are in agreement with those of Ledoux et al. (1999), Santin et al. (2003), Kermanshahi et al. (2007), Rosa et al. (2001) and Miazzo et al. (2005). Data of weekly Mean Body Weight (MBW) and weekly Body Weight Gain (BWG) of treatments are presented in Table 3 and 4. The results and trends were similar with those of FI. Mean body weight and body weight gain of chickens fed AFB₁ alone started to decrease from 3rd week and continued to end of the experiment. The deleterious effect of AFB₁ was also changed to normal conditions (control) when SB added into their contaminated diets. The results of MBW and BWG in this experiment are also in agreement with those of other studies (Rosa et al., 2001; Desheng et al., 2005; Miazzo et al., 2005; Kermanshahi et al., 2007). Dersjant-Li et al. (2003) reviewed the impact of low oncentrations of aflatoxins in poultry diets and suggested that the growth reduction due to aflatoxin contaminated diets can be related to reduction in both feed intake and feed efficiency. In this experiment, AFB₁ did not negatively influence feed conversion ratio (Table 5) that is in agreement with the results of Edrington et al. (1997).

Table 4:	Effect of treatments on mean body weight of broiler chickens fed diets containing different levels of aflatoxin B1 and sodium bentonite
1AFB1: Aflatoxin B1; SB: Sodium Bentonite; Results for each treatments are obtained from 32 chickens in each treatment with ±SEM = 24.5; a, b, cIn each column, means with different superscripts are significantly different (p<0.05)

Table 5:	Effect of treatments on Feed Conversion Ratio (FCR) of broiler chickens fed diets containing different levels of aflatoxin B1 and sodium bentonite
1AFB1: Aflatoxin B1; SB: Sodium Bentonite; Results for each treatments are obtained from 32 chickens in each treatment with ±SEM = 0.07; In each column, means with no superscripts are not significantly different (p>0.05)

Table 6:	Effect of treatments on relative organ weights (g/100 g of body weight) of broiler chickens fed diets containing different levels of aflatoxin B1 and sodium bentonite
1AFB1, aflatoxin B1; SB, Sodium Bentonite, a, b, cIn each column, means with different superscripts are significantly different (p<0.05)

Feed Conversion Ratio (FCR) is an index of feed intake and body weight gain and usually wouldn’t be considered alone.

Relative organ weights: The effect of AFB₁ alone or in combination with SB on carcass weight, thigh and breast yield relative to live body weight is presented in Table 6. The data clearly shows the negative effect of AFB₁ on the mentioned organ weights.

Table 7:	Serum ALT, AST, GGT and LDH concentrations (U/L) in broiler chickens fed diets containing different levels of aflatoxin B1 and sodium bentonite
1AFB1: Aflatoxin B1, SB: Sodium Bentonite, Results for each treatments are obtained from 32 chickens in each treatment, 2ALT: Alanine Aminotransferase, AST: Aspartate Aminotrasferase, GGT: Gamma Glutamyl Trasferase, LDH: Lactate Dehydrogenase, a, b, cIn each column, means with different superscripts are significantly different (p<0.05)

AFB₁ significantly decreased carcass weight, thigh and breast yield (p<0.05). This negative effect was cleared when SB added as feed supplement into the experimental diets. Kermanshahi et al. (2007) showed that AFB₁ significantly increases some of the organ weights. Liver is the first organ affected by AFB₁ (Leeson et al., 1995). Increase in liver relative weight has also been reported by Kubena et al. (1993) during aflatoxicosis. This enhancement in liver weight is usually due to fat deposition in the liver (Leeson et al., 1995). Increase in internal organ weights like liver may decreases relative carcass weight. As seen in this study, the lower values of edible parts of the carcass (most valued parts) brings lots of losses to poultry producers and using SB is one of the economical ways to combat with this problem.

Blood enzymes: Serum concentrations of ALT, AST, GGT and LDH for different treatment groups are shown in Table 7. AFB₁ alone in the diet at both 21 and 42 days of age caused a significant increase in serum concentrations of ALT and AST (p<0.05).

The results obtained in this study concerning increase activity of ALT and AST when chickens fed AFB₁ are in agreement with those of many studies (Devegowda et al., 1996; Celik et al., 2005; Kermanshahi et al., 2007). Dafalla et al. (1987) also reported an increase in serum concentration of AST and ALT due to AFB₁ administration into the diet. Generally, AST and ALT are intracellular enzymes that do not belong to plasma; so, their appearance in serum indicates cell injury (Coles, 1974).

Increase in serum concentrations of AST and ALT in this experiment, might be a result of hepatocytes injury. Using bentonite brought the ALT and AST enzyme activities back to normal conditions that is also showed by Eraslan et al. (2006). AFB₁ alone significantly increased GGT activity in both 21 and 42 days of age (p<0.05).

The lowest level of GGT was seen in chickens fed 1000 ppb AFB₁. Supplementation of contaminated diets with SB corrected the GGT values similar to that of control treatment in both ages. Our results concerning decrease in GGT activity and AFB₁ effect is similar to those of Jansen et al. (2006) and Ersalan et al. (2006). Eraslan et al. (2006) was also shown that using SB alleviates the effect of AFB₁ on GGT and increases its activity. AFB₁ significantly increased activity of LDH in both 21 and 42 days of age (p<0.05).

This effect was more pronounced in 1000 ppb concentration of AFB₁. Addition of SB into the contaminated diets corrected the increasing effect of AFB₁ so LDH activity gain decreased and reached to control group.

CONCLUSION

Under the conditions of this study, it was concluded that feeding aflatoxin B₁ at the used levels can impair feed intake, growth performance and efficiency of carcass yield, thigh and breast meat. Using sodium bentonite showed a positive effect to chickens and nearly cleared the negative effect of aflatoxin B₁. It seems that using 0.5-1% sodium bentonite into the diets is the acceptable range to combat the negative effects of aflatoxin B₁ in broiler chickens.

ACKNOWLEDGEMENT

The financial support of the Excellence Center of Animal Science, Ferdowsi University of Mashhad, Iran is gratefully acknowledged by the authors.