Effects of aflatoxin-detoxifizyme on growth performance and liver biochemical indices of broilers fed with aflatoxin B₁

Abstract

Aflatoxin-detoxifizyme (ADTZ) was evaluated for its ability to reduce the deleterious effects of aflatoxin B₁ (AFB₁) in broiler diets. A total of 624 one-day-old AA broilers were randomly divided into 3 treatment groups with 8 replicates each. The control, AFB₁ and ADTZ treatments were fed with basal diet, basal diet+ 0.1 mg/kg AFB₁ and basal diet+ 0.1 mg/kg AFB₁ + 0.3% ADTZ, respectively. The trials lasted 42 days. The results showed that: (1) AFB₁ diminished the growth performance of broilers significantly (P<0.01). (2) The AFB₁ supplement caused significant alteration of related liver enzyme activities and significantly decreased the body’s antioxidant capacity. (3) Related parameters were returned to normal levels by a 0.3% ADTZ addition to the AFB₁-contaminated diet. All the results in the trials indicate that ADTZ has the ability to detoxify AFB₁ and recover the negative effects of AFB₁.

Keywords

aflatoxin-detoxifizyme (ADTZ) / aflatoxin B₁(AFB₁) / broilers / growth performance / liver biochemical indices

Introduction

It is reported that as much as 25% of crop products in the world are mildewed annually, completely losing about 2%-3% of food (fed) values (Richard, 1989). The aflatoxin B₁ (AFB₁) is very toxic and exists in a wide range of mycotoxins. Its high toxicity to both animals and humans makes AFB₁ the most dangerous mycotoxin (Wilson et al., 1994). Liver is the target organ of AFB₁, long-term intake of diets containing AFB₁ can cause a reduction of broiler growth performance and antiviral ability, liver dysfunction, and even cancer (Quezada et al., 2000). In the feed industry, the most common method to control AFB₁ is the application of nonnutritive adsorptive materials so as to reduce the concentration of mycotoxin in the gastrointestinal tract of animals. But these adsorbents may also adsorb other feed nutrients, resulting in the decrease in their nutritional value. Reports show that hydrated aluminosilicate and yeast cell wall polysaccharide have non-selective adsorption to vitamin C and vitamin E, in which hydrated aluminosilicate has stronger adsorption to both of them (Chen et al., 2008). Therefore, more advanced and efficient methods of detoxifying AFB₁-containing feedstuffs are in great demand. Aflatoxin-detoxifizyme (ADTZ) is a protease from edible fungus A. tabescens, it can specifically act on the 8, 9 double bond, the toxic part of AFB₁, to open the ring structure and degrade AFB₁ into a nontoxic substance. This study was conducted to examine the influence of AFB₁ on the growth performance and related liver biochemical indices of broilers and the antitoxic effect of ADTZ.

Materials and methods

Chemicals

Aflatoxin-detoxifizyme (7000 U/g) was provided by Guangzhou Co-win Bioengineering Co., Ltd.

AFB₁ (purity>99%) was purchased from Beijing Rapid-bio Co., Ltd.

Animals and experimental design

624 one-day-old AA broilers were randomly divided into 3 treatment groups with 8 replicates each. The birds were given 24 h illumination and access to the assigned diet and water ad libitum. Trials were conducted in two phases: 1-21 d and 21-42 d. All birds were assigned to one of the three treatments: the control, AFB₁ and ADTZ treatment, which were fed with basal diet (Table 1), basal diet+ 0.1 mg/kg AFB₁ and basal diet+ 0.1 mg/kg AFB₁ + 0.3% ADTZ, respectively.

Collection of hepatic samples

At the end of this trial, 12 birds were randomly selected from each treatment, and then killed to obtain liver samples for subsequent determination of enzymes activities. All samples were flash frozen by liquid nitrogen and stored at -70°C.

Measurements of growth performance and liver biochemical indices

On the 1st, 21st and 42nd day of the experiment, birds were weighed by replicates for the measurement of growth performance. Average daily gain (ADG), average daily feed intake (ADFI) and feed efficiency (F/G) were also measured.

Hepatic metabolic enzyme activities of succinic dehydrogenase (SDH), choline esterase (CHE), alkaline phosphatase (AKP) activities in the liver were measured using a 752 spectrophotometer, with kits purchased from Nanjing Jiancheng Bioengineering Institute, China.

Hepatic antioxidant indices were determined by measuring superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione peroxidase (GSH-Px) activity, and glutathione reductase (GR) activity, using the methods of xanthine oxidase, ammonium molybdate, dithio-bis-nitrobenzoic acid, and colorimetry, respectively. The concentration of malonaldehyde (MDA) in the liver was measured using the method of barbituric acid by a 752 spectrophotometer and kits purchased from Nanjing Jiancheng Bioengineering Institute, China.

Data statistical analysis

One-way ANOVA was performed using the GLM procedure of the SPSS 13.0 software. Significant differences among treatment means were determined at P<0.05 by the LSD multiple comparison test. Values were expressed as the mean±SD.

Results

Growth performance

The growth performance of broilers was significantly affected by the 0.1 mg/kg AFB₁-containing diet (Table 2). In the first phase (from 1 to 21d) of the experiment, the ADG and ADFI of the AFB₁ treatment were decreased by 3.45% (P<0.05) and 0.74% (P>0.05), respectively, compared with the control; however, the F/G was increased by 2.72% (P>0.05). The ADG and ADFI of the ADTZ treatment were raised by 3.47% (P<0.05) and 2.77% (P<0.05), respectively, compared with the AFB₁ treatment; however, the F/G was decreased by 0.71% (P = 0.615). In the second phase (from 21 to 42 d) of the experiment, the ADG and ADFI of the AFB₁ treatments were decreased by 5.62% (P<0.05) and 0.88% (P>0.05), respectively, compared with the control, but the F/G was increased by 4.93% (P<0.05). The ADG and ADFI of the ADTZ treatment were raised by 5.58% (P<0.05) and 3.40% (P<0.05), respectively, compared with the AFB₁ treatment, but the F/G was decreased by 2.05% (P>0.1). In the entire experiment (from 1 to 42 d), compared with control, the ADG and ADFI of the AFB₁ treatment were decreased by 5.09% (P<0.05) and 0.85% (P>0.05), respectively, and the F/G was increased by 4.42% (P<0.05). As compared with AFB₁ treatment, the ADG and ADFI of the ADTZ treatment were raised by 5.06% (P<0.05) and 3.27% (P<0.05), respectively, and the F/G was decreased by 1.7% (P>0.1).

Compared with the control, the ADFI of the ADTZ treatment in the second phase and the entire treatment course was raised significantly, and the feed efficiency of the ADTZ treatment did not reach the same level of the control.

Hepatic metabolic enzyme activities

The changes in the hepatic metabolic enzyme activities are shown in Table 3. Compared with the control, the SDH and CHE activities of the AFB₁ treatment were decreased by 14.52% (P<0.05) and 12.56% (P = 0.072), respectively, whereas the AKP activity was increased by 13.57% (P = 0.065). In the ADTZ treatment, SDH and CHE activities were raised by 17.98% (P<0.05) and 14.83% (P = 0.06), and AKP activity was decreased by 12.68% (P = 0.052), compared with that of the AFB₁ treatment, respectively.

The hepatic metabolic enzyme activities showed no significant difference between the ADTZ treatment and the control.

Hepatic anti-oxidation

The antioxidant indices in liver are shown in Table 4. A significant effect of 0.1 mg/kg AFB₁ on the hepatic anti-oxidation of broilers was observed. In the liver, the GSH-Px, SOD, GR and CAT activities of the AFB₁ treatment were decreased by 10.14% (P<0.05), 5.5% (P<0.05), 12.08% (P = 0.071) and 10.07% (P>0.1), respectively, compared with control, but the MDA content there was increased by 9.22% (P = 0.061). In the ADTZ treatment, SOD activity was raised by 5.58% (P = 0.056), and the GSH-Px, GR and CAT activities were raised by 8.29% (P>0.1), 10.48% (P>0.1) and 10.08% (P>0.1), respectively, whereas MDA content in liver was decreased by 7.80% (P = 0.082), compared with that of the AFB₁ treatment.

As compared with the control, no significant difference was found in the hepatic antioxidant indices of the ADTZ treatment.

Discussion

AFB₁ is a highly toxic substance and chronic poisoning caused by long-term ingestion has become most common in livestock and poultry production, resulting in the reduction of animal growth performance including feed efficiency, litter size, poultry egg production and hatching rate (Lindemann et al., 1993; Verma et al., 2002). In this trial, results showed that the performance of broilers was significantly reduced by AFB₁, in which ADG and ADFI were significantly decreased, while F/G was significantly increased. This result is consistent with that of Hou et al. (2008a), who reported that feeding AFB₁-contained diets led to a significant decrease in BW, ADG, and ADFI (P<0.05) and an increase in F/G.

In the ADTZ treatment, ADG and ADFI were significantly raised, while F/G decreased compared with the AFB₁ treatment. Broiler growth performance indices were basically returned to normal levels, but the feed efficiency did not reach the control’s. It indicates that ADTZ can diminish the inhibitive effects of AFB₁ on broiler growth performance by decomposing AFB₁.

Liver is the target organ of AFB₁, aflatoxicosis can cause abnormal liver enzyme activities. SDH, as the first enzyme of the mitochondrial respiratory chain, is also the key enzyme of the citric acid cycle. The changes in SDH activity could indicate whether the liver function is normal or not. CHE is a sensitive parameter, reflecting the synthesis of liver cells and positive correlation between its decrease of activity and liver damage (Long et al., 2007). AKP are a group of enzymes that can hydrolyze phosphate ester under alkaline conditions, and it is widespread in tissues and body fluids. AKP activity may abnormally increase because of hepatobiliary disease. Hou et al. (2008b) reported that SDH and CHE activities in broiler liver were significantly decreased, whereas AKP activity was significantly increased after the ingestion of AFB₁. We found similar results, wherein compared with the control, liver SDH activity was significantly decreased and CHE activity decreased, whereas AKP activity increased in the AFB₁ treatment. After adding ADTZ, SDH and CHE activities were significantly raised, whereas AKP activity decreased, compared with the AFB₁ treatment, and there was no significant difference between them and the control. All data indicate that AFB₁ can damage the liver, thereby affect liver function, however, the damages can be reduced by adding ADTZ.

There is an antioxidant system in aerobiont cells, mainly including SOD, CAT, GSH-Px, GR, etc., which can be induced and synthesized by the increasing number of free radical compounds. Their content and activities affect the levels of active oxygen radicals in vivo and also the concentration of MDA as the terminal metabolites of lipid peroxidation (Wills, 1966). The decline of these series of enzyme activities suggests the reduction of free radicals’ scavenging ability in the liver. Shi et al. (2007) reported that with the addition of 0.1 mg/kg AFB₁ into growing-finishing pig diets lead to a significant reduction of liver antioxidant capacity, GSH-Px, GR, and CAT activities were significantly decreased. The results in this study also showed a significant reduction in liver antioxidant capacity in those birds fed with the AFB₁ diet, with significant decreases in the GSH-Px and SOD activities, with GR and CAT activities also decreased, whereas the content of MDA in the liver was increased. The above-mentioned liver antioxidant indices were basically returned to normal levels without significant differences between them and the control by the addition of 0.3% ADTZ. It indicates that AFB₁ can cause a significant decline in liver antioxidant indices and ADTZ has the ability to recover this negative effect of AFB₁.

Overall, the results demonstrate that exposing broilers to 0.1 mg/kg AFB₁ in their diet is associated with a number of negative effects manifested by reductions in ADG and ADFI and a change in several liver parameters. Moreover, we found that ADTZ is safe for broilers and its use to detoxify AFB₁-contaminated feed is possible because it can degrade AFB₁ into a nontoxic substance and helps birds recover from the negative effects of AFB₁.

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