In vitro and in vivo assessment of humic acid as an aflatoxin binder in broiler chickens

Poster presentation at World Nutrition Forum 2006

Aflatoxins are often encountered in food and animal feeds at alarming concentrations around the world. One of the most practical approaches is the use of non-nutritive adsorbents, which bind the mycotoxins and inhibit their absorption from the gastrointestinal tract. Aluminosilicates, activated charcoal, polymers such as cholestyramine and polyvinylpyrrolidones, and yeast and yeast products, have been extensively studied with promising, but varying results. Humic acids are ubiquitous and are found wherever matter is being decomposed, or has been transposed as in the case of sediments.

Humic substances have demonstrated a strong affinity to bind various substances, such as heavy metals, herbicides, different mutagens, monoaromatic and polycyclic aromatic compounds, minerals and Bacillus subtilis bacteria. In spite of its known binding characteristics, humic acids have never been evaluated previously as a mycotoxin adsorbent. A new large-scale process was developed to regenerate pure, high quality humic acids from bituminous coal by reversing the process whereby the coal was formed. Humic acids produced in this manner, called oxihumate, differ only slightly chemically from humic acids obtained from other sources.

The first objective of this study was to evaluate the efficacy of oxihumate as an aflatoxin binder, both in vitro and in vivo. The binding capability of oxihumate for aflatoxin B1 (AFB1) was tested and the Langmuir and Freundlich adsorption isotherms of oxihumate were determined for AFB1 under different pH conditions to describe surface adsorption. Oxihumate showed a high in vitro affinity for AFB1. The Freundlich isotherm fitted the data better than the Langmuir isotherm and binding capacities of 10.3, 7.4 and 11.9 mg AFB1/g oxihumate at pH 3, 5 and 7, respectively, were calculated.

The second objective of this study was to evaluate the effects of oxihumate on growth performance, liver morphology and serum biochemical and haematological variables in male broiler chickens exposed to aflatoxincontaminated feed from 7 to 42 days of age. 420 chicks of similar weight were randomly assigned to 28 clean pens in an environmentally controlled broiler house. The birds were divided into 7 treatment groups, with 4 replicates per treatment and 15 birds per replicate. Three levels of AFB1 were used (0, 1 and 2 mg AFB1/kg feed). The AFB1-contaminated diets contained either no additive, 3.5 g oxihumate/kg feed or 3.5 g of a wellknown commercially available adsorbent containing esterified glucomannan (EGM). The oxihumate, EGM and aflatoxin-contaminated rice powder were mixed into the different treatment diets to required concentrations.

A concentration of 1 mg AFB1/kg feed did not affect the body weight and thus growth of the broilers up to 42 days of age. However, the 2 mg AFB1/kg feed level depressed growth with significantly lower body weights evident from an age of 21 days onwards, two weeks after intake of the contaminated feed commenced. The reduction in weight gain continued throughout the study. The decrease in body weight gain caused by the addition of 2 mg AFB1/kg feed was diminished at 42 days of age by the addition of 3.5 g oxihumate/kg to the diet. In the present study, no indication of any improvement in weight gain was observed when the chicks consumed diets containing aflatoxin plus EGM.

The relative weights for the livers were significantly increased by almost 75% for the chicks consuming diets contaminated with 2 mg AFB1/kg. The livers of these chicks also appeared to be friable and pale yellow as a result of fat accumulation in the cytoplasm of the hepatocytes. Oxihumate showed significant protective effects with respect to liver damage, as indicated by an inhibition of liver enlargement.

Microscopically, aflatoxin ingestion had a pronounced dose-response effect on the quantity and severity of hepatic lesions with a lesion value of 1.75 for the controls, 4.13 for the broilers that received 1 mg AFB1/kg feed and 9.60 for the 2 mg AFB1/kg feed treatment group. Supplementation of oxihumate improved the liver lesion value of the latter significantly to 6.30. EGM treatment of the contaminated diets did not show any positive effect on liver enlargement or liver lesions values.

The feed contaminated with 1 mg AFB1/kg had no effect on the relative weight of the liver and heart but it did cause a significant increase in stomach weight, indicating possibly a higher sensitivity of the stomach to aflatoxin contamination than the other organs. The additions of oxihumate, and EGM to a lesser extent inhibited this effect at the contamination level of 1 mg AFB1/kg and prevented the enlargement of the heart at 2 mg AFB1/kg feed.

1 mg AFB1/kg feed did not affect hematocrit levels of the broilers. Hematocrit was, however, significantly reduced by the 2 mg AFB1/kg feed treatment. Oxihumate supplementation to this diet significantly improved hematocrit values, probably as a result of effective adsorption in the gut to reduce the amount of aflatoxin absorption by the body. EGM did not improve the hematocrit of the contaminated birds.

The observed reduction in serum concentration of total protein and albumin in all groups fed aflatoxin indicates impaired protein synthesis in the liver caused by the blockage of RNA synthesis, resulting from the hepatotoxicity seen in aflatoxicosis. Albumin and total protein levels in the serum proved to be sensitive indicators of aflatoxicosis in broilers, as significant decreases were observed at 1 mg AFB1/kg feed, a level which did not affect body weight gain. The decrease in serum albumin levels in broilers that consumed the 2 mg AFB1/kg diet with added oxihumate was less profound when compared to the group that received aflatoxin alone. The inclusion of EGM with 2 mg AFB1/kg in the diet resulted in a significant lower total serum protein level compared with birds that consumed the 2 mg AFB1/kg diet without EGM.

The mortality rate did not differ significantly among treatments. The negative control group had the lowest mortality rate with a mean of 0.25 birds per pen (1.67%). The highest mortality rate observed was for the 2 mg AFB1/kg group with a mean of 1.75 birds per pen (13.3%).

Results demonstrated that oxihumate was able to effectively bind AFB1 in vitro and was also effective in diminishing the growth inhibitory effects of aflatoxin in vivo. There was apparent protection noted for some of the organ, haematological and serum biochemical changes associated with aflatoxin toxicity. In this study, oxihumate proved to be much more effective in the amelioration of aflatoxicosis in broilers than the commercially available mycotoxin binder containing EGM. The data suggest that oxihumate may alleviate some of the toxic effects of aflatoxin in growing broilers, and when used in combination with other mycotoxin management practices might prove to be beneficial in the management of aflatoxin-contaminated feedstuffs for poultry.

Authors:C. JANSEN VAN RENSBURG*1, C. E. J. VAN RENSBURG†, J. B. J. VAN RYSSEN*, N. H. CASEY* AND G. E. ROTTINGHAUS‡

*Department of Animal and Wildlife Sciences, University of Pretoria, Pretoria, 0002, South Africa; †Department of Pharmacology, University of Pretoria, Pretoria, 0002, South Africa; ‡Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, P.O. Box 6023, Columbia, MO 65205, USA; *Corresponding author at: Department of Animal and Wildlife Sciences, University of Pretoria, Pretoria, 0002, South Africa.

In vitro and in vivo assessment of humic acid as an aflatoxin binder in broiler chickens

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