High levels of aflatoxin have been recorded in ingredients of poultry feed viz., soyabean, sunflower, rice polish, cotton seed etc, It accounts for around 25% of poultry feed samples and ingredients contamination¹. Chronic aflatoxicosis retards growth, increases FCR and susceptibility of chicks to infectious diseases.

Among the several methods adopted, the most effective is the neutralization of mycotoxins present in feed by inclusion of inert adsorbents or toxin binders like Zeolites², Clinoptilolite³ and Bentonites⁴,⁵ that prevent absorption of the toxin from the intestine⁵. Extract of artichoke and Curcuma longa reduced the toxic effects of aflatoxin B1 in chicks⁶,⁷. Citrus fruit oil (active ingredient D limonene) has got detoxification and antioxidant property by increasing the level of Glutathione -s- transferase⁸. The protective role of citrus fruit oil during aflatoxicosis has not been studied. In present study, protective role of herbal oil and toxin binder and their combination during induced aflatoxicosis in broilers has been investigated.

A total of 320 broilers procured from commercial hatchery at day old age. On Day 7, chicks were randomly divided into 8 groups each with 2 replicates of 20 chicks. Group wise treatment schedule of birds is presented in Table 1. Birds were maintained on deep litter system under standard managemental conditions from day old to 42^nd day of age. All chicks were vaccinated on 7^th and 11^th day of age with Lasota strain of Newcastle disease virus and Infectious Bursal Disease (Intermediate Strain) respectively.

Herbal oil containing volatile oils of citrus fruits and toxin binder containing oxime copper (0.5 per cent), Ferric acid (4.5 per cent), activated charcoal (1 per cent), mannonoligosaccharide (10 per cent) and other carrier substances like HSCAS were obtained from M/s. Tetragon Chemie Pvt. Ltd., Bangalore, India for the present study.

Aflatoxin was produced on broken rice using Aspergillus parasiticus, NRRL 2999 culture as suggested by Shotwell et al.⁹, quantified by thin layer chromatography (AOAC)¹⁰.

Broilers were given standard broiler feed and water ad libitum.To achieve required toxin levels in feed, required quantity of toxic contaminated rice was mixed in feed. Herbal oil and toxin binder was added in feed wherever required and the feed was given to birds from day 7 to 42 of age.

All birds from each group were weighed individually at weekly intervals. Also feed consumed by birds of each group was recorded to calculate feed conversion ratio (FCR). The sera collected at weekly intervals were subjected to total protein, albumin, alkaline Phosphatase (ALP), Alanine amino transferase (ALT) and Aspartate amino transferase (AST) estimations using commercially available kits on semi auto analyzer. Liver of all the birds from different groups were weighed individually on an electronic weighing balance after sacrifice. The relative weight of the organ was expressed as gram of organ per 100 g of body weight.

Relative Weight of organ = [Wt of the organ (g)/body Wt (g)] × 100

Statistical analysis of the experimental data generated was done as per Snedecor and Cochran¹¹ using one way ANOVA test.

Reduction in the body weight was observed in the broiler chicks fed with aflatoxin as well as in the birds fed with the toxin and supplemented with herbal oil or toxin binder alone or in combination as compared to control groups from 21^st day of age. The feed conversion ratio (FCR) of aflatoxin fed birds was higher in comparison with untreated control. In general herbal oil and toxin binder supplementation either alone or in combination improved body weight gain and FCR of broilers marginally than toxin alone fed birds. There was significant (P<0.05) increase in relative weight of liver in toxin alone fed group as compared to control birds (Group A), whereas in groups fed with toxin binder and herbal oil along with toxin showed no significant (P ≤ 0.05) increase in relative liver weight except on day 21 of post treatment (Table 2).

Mean serum biochemical values of birds observed at weekly intervals are presented in Table 3 and 4.

Aflatoxin fed birds showed significant (P<0.05) increase in serum ALT activity as compared to control chickens on Day 14, 21 and 28 and maximum increase on Day 21 of post treatment. A significant (P<0.05) decrease in ALT levels towards normal levels in birds fed with toxin and supplemented with herbal oil (Group F), with both herbal oil and toxin binder (Group H) was observed. Mean serum AST and ALP levels in AF alone fed group E were increased significantly (P<0.05) as compared to group A throughout the study. The birds supplemented with herbal oil and toxin binder either alone or in combination along with toxin showed significant (P<0.05) decrease and ranged in control group values from Day 14 post treatment. Mean serum total protein and albumin values were significantly (P<0.05) decreased in toxin fed birds as compared to control (Group A). Supplementation of herbal oil, toxin binder either alone or in combination along with toxin showed marginal increase in comparison to AF alone fed birds.

The results of present study demonstrated that dietary aflatoxin adversely affected the body weights and performance of broilers. The reason for reduced body weight gain and increased FCR can be attributed to anorexia, inhibitory effect of aflatoxin on protein synthesis and lipogenesis¹². Similar observations have also been made earlier workers⁴,¹³,¹⁴,¹⁵. There was significant improvement in the body weight of herbal oil and toxin binder treated chicks both individually and in combination. Significant improvement in the body weight of Toxiroak treated birds was also reported by Kalorey et al¹⁶.This could be attributed to the possible partial amelioration of aflatoxin by the herbal oil and toxin binder in the study. The increase in the relative weight of liver in Aflatoxin alone treated birds could be attributed to the AFB1 induced impaired fat metabolism in the liver with an increase in the fat content of the hepatocytes. These findings have been well documented by earlier workers⁴,¹²,¹⁷.The birds fed with toxin and simultaneously treated with herbal oil and toxin binder alone or in combination showed reduction in relative weight of liver as compared to aflatoxin alone fed birds. These findings could be related to the incorporation of toxin binder and herbal oil alone or in combination in the diet and their possible role in marginally ameliorating the aflatoxin and accordance of mild degree of histological lesions in these groups provided adequate support to this observation.

The reduction in total protein and albumin level in toxin fed birds could be due to degeneration of endoplasmic reticulum in hepatocytes and covalent binding of AF metabolites to template RNA. This process causes inhibition of protein synthesis¹⁸,¹⁹. Supplementation of herbal oil, toxin binder either alone or in combination along with toxin showed marginal protein increase in comparison to AF alone fed birds. This could be attributed to partial alleviation of toxic effects. Similarly Kurkure et al.⁷, reported that treatment of chicks with Curcuma longa during aflatoxicosis helped to maintain normal serum protein levels. Aflatoxin fed birds showed significant (P<0.05) increase in serum ALT activity as compared to control chickens. Higher levels of ALT in younger birds could be attributed to microsomal AFB1 activation to the reactive AFB1-8-9 epoxide (AFBO) which is most efficient in younger birds and they are also deficient in cytosolic Glutathione -S- Transferase (GST) which protects from free radical injury²⁰. Mean serum AST and ALP levels in AF alone fed group E were increased significantly (P<0.05) as compared to group A. The increased enzyme levels were attributed to hepatic degeneration and subsequent leakage of enzymes in AF treated birds ²¹.

A significant (P<0.05) decrease in ALT levels towards normal levels in birds fed with toxin and supplemented with herbal oil (Group F), with both herbal oil and toxin binder (Group H) whereas, ALT values of birds fed with toxin, supplemented with toxin binder showed significant (P<0.05) decrease only towards the end of the experiment. This clearly indicates both herbal oil and toxin binder together have better protective effects as compared to individual supplementation and herbal oil is superior to toxin binder. The birds supplemented with herbal oil and toxin binder in combination along with toxin showed significant (P<0.05) decrease in mean AST and ALP enzyme levels towards control group from Day 14 post treatment indicating the beneficial effect of both herbal oil and toxin binder than individually. This could be due to synergistic effect of both during aflatoxicosis.

From the results, it was suggested that feeding of herbal oil and toxin binder in combination was more effective in reducing the toxicity of aflatoxin than individually; further herbal oil supplementation is marginally superior to toxin binder alone. So, it can be concluded that herbal oil and toxin binder can be used in combination for partial amelioration of aflatoxin.

Authors thank Indian Council of Agricultural Research (ICAR) for providing junior research fellowship during the study. Also thanks are due to M/S Tetragon Chemie, Bangalore, India for providing necessary chemicals to carry out the study.

Mean ±SEM with same superscripts within a row do not differ significantly, P<0.05

>Means ±SEM with same superscripts within a row do not differ significantly, P<0.05

>Means ±SEM with same superscripts within a row do not differ significantly, P<0.05.

REFERENCES