Mycotoxins are secondary toxic metabolites that are produced by fungi growing on food products, such as corn, peanut, and wheat, among others. ¹ Exposure to mycotoxins occurs predominantly by the ingestion of contaminated cereals such as corn, wheat, peanuts and sorghum, as well as other raw materials used in the preparation of animal feed. ² Aflatoxicosis in poultry can cause disease and increased mortality. ³,⁴ Aflatoxins (AFs) are produced by fungi of the genus Aspergillus, particularly A. flavus, A. parasiticus, and A. nomius.⁵ Seventeen metabolites have been identified as aflatoxins, among them AFB ₁ exhibits the highest toxigenic effects and being the most commonly found metabolite in cereals. ⁶ Biochemically, aflatoxins affect energy, carbohydrates lipids, nucleic acids, and protein metabolism. ⁷ Their biological effects include carcinogenicity, mutagenicity, teratogenicity, and hepatotoxicity caused by oxidative damage. ⁸

Determination of biochemical toxic effects of AFs is important for diagnosis of toxicosis in broilers. ⁹ AF toxicity in broilers has been manifested by decreased serum concentrations of total protein, albumin, total cholesterol, ⁴ uric acid, ¹⁰ and increased hepatic enzyme activities such as Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT). ¹¹,¹² In Japanese quail, reduction has been seen in the levels of serum total protein, albumin, globulin, glucose, cholesterol, ALT and elevation of AST and Gamma-glutamyltransferase (GGT) and variable Alkaline phosphatase (ALP) levels observed in toxin treated groups. ¹² Also, the activity of serum enzymes such as AST has been extensively used as a measure of aflatoxin toxicity in chickens. ¹³

Silymarin, an extract from the seeds of milk thistle (Silybum marianum), is a complex of four flavonolignans that have strong antioxidant and free radical scavenging activity. ¹⁴ It has been shown that milk thistle seed (MTS) protects birds against adverse effects of Aflatoxin B ₁ (AFB ₁ ). ¹⁴ Milk thistle extracts have been used to treat diseases. ¹⁵ These extracts are also used to successfully treat hepatitis patients. ¹⁶ They have hepatoprotective, antiinflammatory, cytoprotective, and anticarcinogenic effects. ¹⁶ Keeping in view the medicinal value of milk thistle, this study was conducted to investigate the efficacy of MTS on serum biochemistry, lipid profile, and liver enzymes in broiler chickens contaminated with AFB ₁ during an early stage of exposure (21 days). In our knowledge, this is the first work on the effect of AF in 21-day-old broilers and assessment of MTS on its adverse effects.

Production of aflatoxin on rice

To produce AF, A. flavus obtained from fungi collection of the Centre of Scientific and Industrial Research Organization in Iran, PTCC NO:5004 (IR111). AFB ₁ production performed via fermentation of rice by the method of Shotwell et al., (1966) with minor modifications by Oguz. ⁴,¹⁷ Successfully fermented rice was then steamed to kill the fungus, dried and ground to a fine powder. The AF content in rice powder was analyzed by the method of Shotwell et al., (1966) and measured on a thin layer chromatography (TLC) fluorometric densitometer (Camag-III, Basel, Switzer-land) on the TLC spots. ¹⁷ The AF within the rice powder consisted of 60 ppm AFB ₁ .

Experimental design and diets

The individual and combined effects of dietary AFB ₁ and MTS on serum biochemistry factors, fatty acid and liver enzymes of broilers were evaluated at 21 days of age. Dietary treatments included a 3 × 3 factorial arrangement with three levels of AFB ₁ (0, 250, and 500 ppb), and three levels of MTS (0%, 0.5%, and 1.0%) were incorporated into the basal diet (corn, wheat, and soybean meal). The effect of nine experimental treatments was assessed using 216 one-d-old Ross 308 male broiler chicks in a randomized complete design with four replicates of six birds for each dietary treatments: Control (T1), 250 ppb AFB ₁ (T2), 500 ppb AFB ₁ (T3), 0.5% MTS (T4), 0.5% MTS Plus 250 ppb AFB ₁ (T5), 0.5% MTS Plus 500 ppb AFB ₁ (T6), 1.0% MTS (T7), 1.0% MTS Plus 250 ppb AFB ₁ (T8), 1.0% MTS Plus 500 ppb AFB ₁ (T9). Basal diet was formulated and compounded to meet the nutrient requirements of broiler chicks based on Ross recommendation strain during the whole period of experiment without inclusion of either aflatoxin or binder Table 1.{Table 1}

Biochemical parameters, lipid profile, and liver enzymes

When the chicks reached to three weeks of age, blood samples from two chicks of each treatment was collected by puncturing the brachial vein. The blood sample was allowed to stand for one hour and centrifuged at a speed of 3,000 rpm for 10 minutes. The clear serum was collected in sterilized disposable plastic tubes and stored at −20°C. The serum samples were analyzed for serum biochemistry factors (Glucose, Calcium (Ca), Phosphorus, Iron, Creatinine, and Uric acid), lipid profile (Triglyceride, Cholesterol, Low-density lipoprotein (LDL), and High-density lipoprotein (HDL)) and liver enzymes (AST and ALT), were measured by biochemical test.

Statistical analysis

The data was statistically analyzed with the standard procedures of analysis of variance (ANOVA), using on 3 × 3 factorial with completely randomized design as suggested by Andrea Onofri (2010). ¹⁸ The statistical packages Macros-1.002 (2010) were used to perform the above analysis on computer. ¹⁸

The effects of AFB ₁ and MTS on serum biochemistry (Phosphorus, Iron and Uric Acid), liver profile (triglyceride, cholesterol, LDL, and ALT) are shown in Table 2 and Table 3. Feeding AF caused significant decrease in serum glucose, calcium and HDL after 21 days compared with the control group (T1) Table 2. AFB ₁ (500 ppb) had an adverse effect on liver enzyme (AST and ALT) enhancing the values (244.51 ± 4.91 and 36.22 ± 3.93), respectively, compared to control group (196.11 ± 5.45 and 20.11 ± 3.21) (P < 0.05). Furthermore, addition of different levels of AFB ₁ alone (250 ppb and 500 ppb), into the diet significantly decreased creatinine (1.81 ± 0.024 and 2.25 ± 0.011, respectively) compared to the control group (0.58 ± 0.027), (P < 0.05). Interestingly, MTS supplementation to AF-contaminated feed (500 ppb) significantly increased serum glucose and decreased creatinine and AST levels (P < 0.05) compare to AFB ₁ (500 ppb). Although, MST declined ALT levels compare to AFB ₁ groups (T ₂ and T ₃ ) but it was not significant (P = 0.51). AF (500 ppb) caused a marked drop in HDL and MTS at 1% alone increased HDL (91.25 ± 2.71) concentration in comparison to control group (85.12 ± 1.95), and MTS supplementation to AF-contaminated feed also enhanced HDL but it was not significant.{Table 2}{Table 3}

Our results showed that serum total glucose level was significantly different between groups contaminated with different levels of AFB ₁ . Previous studies performed with high levels of AF (2.5-5 mg/kg diet) showed significant decreases in serum total glucose, protein, albumin, total cholesterol and uric acid levels. ⁴,¹⁹ The differences between previous studies and our current findings can be related to the AF doses in the diet. In the present study, the use of low AF levels (250 ppb) did not cause any significant changes in the calcium, glucose, HDL, and AST compared to control group. ¹⁹ Protein synthesis inhibition in the liver together with other damage to the liver and kidney could induce the biochemical changes during aflatoxicosis. ²⁰ The results also revealed that treatment with MTS prevented changes in AST activity, supporting the idea that these MTS may provide protection against toxic effects of AFB ₁ . Other authors reported no significant changes in serum biochemistry for the lower dietary AF, such as 100 ppb ²¹ and 200 ppb ²² in broiler feed. However, when AF levels increased in food up to 300 ppb and more the serum biochemistry was significantly affected and total protein, albumin, and cholesterol levels were decreased. ²² These results agree with data reported by Henry et al., who fed broilers 80 ppm AFB ₁ for three weeks and observed an increase in the levels of ALT and AST liver enzymes. ²³ Similar results were also observed by Ledoux et al., in experiments with both turkeys and broilers. ²⁴ The liver is the principal organ for AF toxicity where most aflatoxins are bioactivated to the reactive form, which is known to bind deoxyribonucleic acid (DNA) and proteins, damaging the liver structures. ¹⁹,²⁵ Liver function tests, including serum levels of liver enzymes, are used to assess injury to liver cells, the liver′s ability to synthesize proteins, and the excretory functions of the liver. ²⁶,²⁷ Elevated serum enzyme test results usually indicate liver injury earlier than other indicators of liver function. The key enzymes are ALT and AST, which are present in liver cells. ALT is liver specific, whereas AST is derived from organs other than the liver. In most cases of liver damage, there are parallel rises in ALT and AST like what has been shown in this research. In the present study, serum AST and ALT activity was significantly increased in birds consuming 500 ppb of AFB ₁ Table 3. The elevation of AST and ALT may be due to disruption of hepatic cell as a result of necrosis or a consequence of altered membrane permeability. ²⁸ Supplementation with low (0.5) and high (1.0) percent of MTS to the diet resulted normal serum biochemistry on glucose and AST levels. Tedesco et al., (2004) found similar results on serum biochemistry in broilers consumed with milk thistle diets alone. ¹⁴ In addition, ALT activity in the blood plasma of aflatoxin-treated group supplied with silymarin did not differ from the non-treated control group in their experiment. ¹⁴,²⁰ It has been demonstrated that the activation of AFB ₁ in human and rat liver is a complex process controlled by multiple cytochrome P450 enzymes. ¹,¹⁹ Silymarin can inhibit the cytochrome P450 system, and consequently inhibit AFB ₁ activation. ¹⁴ However, Gordon et al., (2006) did not prove the significant decrease of ALT activity in patients suffering from chronic hepatitis when they were given formulas with thistle seeds. ²⁹ Presence of AFB ₁ in food chain is associated with decrease in quality and quantity of food and feed materials. ¹⁹,²⁰ Almost all of the biochemical changes due to AF presented in this research is in accordance with rat and human. ¹,³⁰,³¹ The hazard of AF to human health may result from direct ingestion of mycotoxins contaminated cereals with fungi as well as secondary contamination through products of animals and poultry with remains of mycotoxins or their metabolites in the animal products. In conclusion, our results suggest that MTS may provide protection against the negative effects of AFB ₁ on broiler chicks and indirectly reduce development of liver and kidney disease in human.

Results showed that treatment with MTS can be effective in counteracting the negative effects of AFB ₁ intoxication on AST, HDL, and calcium in broilers. The results showed that consumption of different levels of MTS from 0.5% to 1.0% in poultry diets may alleviate some of the adverse effects of aflatoxin in growing broilers and more importantly reduce the aflatoxin-contaminated meat used in human food.

This study was conducted at the University of Birjand and was supported by the Office of Graduate Studies of University of Birjand.