INTRODUCTION

Aflatoxin causes severe economic losses to the poultry industry. Aflatoxicosis that occurs from ingesting aflatoxins is characterized in broiler chickens by decreased feed intake and growth rate, poor feed utilization and mortality (Tedesco et al., 2004; Bailey et al., 2006; Shi et al., 2006). Aflatoxicosis is also associated with changes in biochemical and haematological parameters (Denli et al., 2004; Basmacioglu et al., 2005; Bintvihok and Kositcharoenkul, 2006), liver and kidney abnormalities, and impaired immunity rendering birds more susceptible to infectious diseases (Shashidhara and Devegowda, 2003). Their contamination in feed is also practically unavoidable (Coulombe et al., 2005).

Among the many compounds examined so far for the prevention of aflatoxicosis in poultry, antioxidants are being considered due to their ability to reduce disease formation by either induction or inhibition of key enzyme systems. The synthetic feed antioxidant butylated hydroxytoluene (BHT) has been shown to be a versatile chemopreventive agent against liver cancer induced by AFB1 in rodents (Hocman, 1988). The mechanism of such protection is through inhibition of cytochrome P450s and/ or induction of enzymes such as glutathione S-transferases (Hocman, 1988; Singletary, 1990; Ulland et al., 1973). BHT was also found protective against AFB1-induced hepatocarcinogenicity in rats (Williams and Iatropoulos, 1996), and against AFB1-induced weight loss in chickens (Larsen et al., 1985). The present investigation is, therefore, an attempt to examine the effects of incorporating BHT in diet on the performance of broiler chickens during aflatoxicosis.

MATERIALS AND METHODS

Aflatoxin production

Lyophilized preparation of Aspergillus parasiticus NRRL 2999 was obtained from U.S. Department of Agriculture, Peoria, Illinois (USA). The lyophilized preparation was revived on Potato Dextrose Agar (PDA) medium and used for AF production. AF was produced from Aspergillus parasiticus NRRL 2999 by fermentation of cracked maize as per the method described by Shotwell et al. (1966). The fermented maize was then steamed to kill the fungus spores, dried and then ground to a fine powder. The aflatoxin from maize powder was extracted as per the method of Pons et al. (1966) and measured using Thin Layer Chromatography (TLC). The total AF concentration in maize powder was 980 ppm, consisted of 76.45% AFB₁, 10.52% AFB₂, and 9.89% AFG₁ and 3.14% AFG₂. The maize powder containing known concentration of AF was incorporated into the basal diets of certain dietary treatments to get the desired amount of 1 ppm total AF.

Birds, diets and experimental protocol

Day-old coloured commercial broiler chicks (n=144) were wing banded, weighed individually and divided into six (T₁ to T₆) treatment groups, each replicated four times and each replication consisted of 8 birds. The chicks were housed in electrically heated compartments with continuous lighting and were given a starter feed from 1 to 21 days and finisher feed from 22 to 42 days. The basal diet devoid of AF and BHT served as control (T₁) and was common to all groups (Table 1). The other dietary treatments were T₂ (1.0 ppm AF), T₃ (1000 ppm BHT), T₄ (2000 ppm BHT), T₅ (1.0 ppm AF+1000 ppm BHT) and T₆(1.0 ppm AF+2000 ppm BHT). Each diet was fed to four replicated groups of eight birds each from day 1 to 42 days of age. Birds were inspected daily and feed and water were offered ad libitum.

Amelioration of Aflatoxicosis in Coloured Broiler Chickens by Dietary Butylated Hydroxytoluene - Image 1

The body weight and feed consumption were recorded weekly to evaluate the BW gain and FCR. After completion of feeding trial at the age of 42 days, eight birds from each treatment group (two from each replicate) were selected randomly to collect blood samples. Serum was then separated and used for biochemical assays. Serum concentrations of total protein, total cholesterol, uric acid and the activities of AST and ALT were determined spectrophotometrically using commercially available test kits (M/s. Span Diagnostics Ltd., Surat, India). The data obtained were analysed statistically (Snedecor and Cochran, 1980.) and differences in means were tested using Duncan’s multiple range test (SPSS 12.0). Statements of statistical significance were based on P<0.05.

RESULTS AND DISCUSSION

The effect of dietary BHT on BW gain, feed consumption and feed conversion ratio of broiler chicks fed on diet containing 1.0 ppm total AF from 1 to 42 days of age is given in Table 2. During starter (0-3 week) and finisher (4-6 week) growth phases, the BW gain in T₁ was higher (P<0.05) compared to aflatoxin fed group (T₂). The BW gain of groups T₅ and T₆ was lower (P<0.05) than that of T₁ but higher (P<0.05) than that of T₂. The BW gain of groups T₃ and T₄ was statistically similar to that of control. When overall BW gain (0-6 weeks) was considered, it was significantly (P<0.05) higher in control group (T₁) than those of T₂, T₅ and T₆. However, BW gain in T₁ was statistically comparable to those of T₃ (control+1000 ppm BHT) and T₄(control+2000 ppm BHT). Therefore, the BW gain in non-AF fed treatment groups, either supplemented with BHT or without its supplementation, was significantly (P<0.05) higher than those fed diets with AF. A decrease in body weight of broiler chicks was also reported earlier (Maurice et al., 1983) on daily dose of AFB₁ at the rate of 100 µg/kg live body weight during first three weeks of age. Also, Abo-Norag et al. (1995) observed a significant reduction in BW gain (814 to 731 g) of male broiler chicks during 1-28 days of age on diets with 3.5 ppm AF. Rosa et al. (2001) also reported a decrease in BW gain when the birds were fed on diet containing 5 ppm AFB₁.

Amelioration of Aflatoxicosis in Coloured Broiler Chickens by Dietary Butylated Hydroxytoluene - Image 2

In the present study, addition of 1.0 ppm total aflatoxins resulted in significant reductions in BW gain but this reduction improved partially (P<0.05) by the addition of 1000 or 2000 ppm BHT in AF diets. The addition of BHT in diet at these levels (1000 or 2000 ppm) could not bring reductions in BW gain equivalent to that of control. Klein et al. (2002) also reported that the BW gain in the AFB₁+ BHT fed groups were significantly higher than that in the AFB₁-only group and the BW gain in the AFB₁+ high BHT (4000 ppm) fed group was not statistically different from control. They further reported that the BWG in birds given BHT alone was not statistically different from control. In our study also the BW gain in birds given either level of BHT alone did not differ statistically, however, the values were numerically higher than that of control. Similar protective effects of BHT on decreases in BW gain due to AFB1 have also been noted in chickens in earlier studies (Larsen et al., 1985; Dalvi, 1986, Ehrich et al., 1988).

Amelioration of Aflatoxicosis in Coloured Broiler Chickens by Dietary Butylated Hydroxytoluene - Image 3

With regard to feed consumption (FC) of broilers in starter phase (0-3 week), it was significantly (P<0.05) lower in group T₂ compared to that of control (T₁). The FC in other treatment groups was statistically similar to that of control. In case of finisher phase (4-6 week), the FC of groups T₂ and T₅ was significantly (P<0.05) lower than that of control (T₁). The FC in other treatment groups was statistically similar to that of control. In case of cumulative FC (0-6 week), the FC in T₁ was significantly (P<0.05) higher than those of T₂ and T₅ whereas, it was almost similar in rest of the treatment groups. Thus, the FC in non-AF fed treatment groups was higher than those of AF fed treatment groups. Miazzo et al. (2000) reported that FC decreased significantly in broiler chicks given AF (2.5 ppm) for 3 weeks. However, Basmacioglu et al. (2005) reported that decreasing effect of AF (2.0 ppm) was not statistically different though it was about 8 per cent lower. In this study the FC in T₆ was numerically less but did not differ significantly compared to control, indicating that the BHT supplementation along with AF fed groups tended to improve the FC.

With regard to FCR, during starter growth phase (0-3 week), the FCR in T₁ was lower (P<0.05) than that of T₂. The FCR of groups T₅ and T₆ was lower (P<0.05) than that of T₂ but higher (P<0.05) than that of T₁. The FCR of groups T₃ and T₄ was statistically similar to that of control. During finisher phase (4-6 week), the FCR did not differ significantly among various dietary treatments. In case of cumulative FCR (0-6 week), the FCR in T₁ differed significantly (P<0.05) from that of T₂ and T₃ whereas, it remained comparable to those of T₄, T₅ and T₆. Thus, AF contamination deteriorated the feed efficiency and these adverse effects on feed efficiency were reversed by BHT supplementation. Oguz et al. (2000) also observed adverse effect of 100 ppb AF on the cumulative FC and FCR values by 5.64 and 4.89 per cent respectively, but they did not reach statistical significance during the experiment. Azzam and Gabal (1997) found significant adverse effects of AF on BWG, FC and FCR in broiler chickens fed on a 100 ppb AF- containing diet for two months. Raju and Devegowda (2000) also reported significant adverse effects of AFB1 on BWG, FC and FCR when the broilers were fed on 0.3 ppm AFB1 containing diet. The improvement in feed efficiency was also reported by Ehrich et al. (1986) where BHT at 1000 and 3000 ppm counteracted many of the adverse effects on BWG and feed efficiency in chickens.

With regard to biochemical parameters (Table 3), the serum total protein concentration in T₁ was significantly (P<0.05) higher than those in T₂ and T₅ and remained comparable to those in T₃, T₄ and T₆. The protein concentration in T₂ was the lowest as compared to other treatment groups. Cholesterol content in T₁ were significantly (P<0.05) higher than that of T₂ and comparable to rest of the treatment groups. Uric acid content did not differ significantly among various treatment groups. In earlier studies, dietary AF contamination (2.5-5 ppm) showed significant decreases in serum total protein, cholesterol and uric acid levels (Kubena et al., 1998; Oguz et al., 2000a; Rosa et al., 2001). Basmacioglu et al. (2005) also reported that feeding AF (2.0 ppm) caused significant decreases in serum total protein and cholesterol in broiler chickens. BHT supplementation in AF contaminated groups reversed these parameters significantly and the values were almost comparable to that of control.

The AST and ALT activities in T₁ were statistically lower than that of T₂ and remained almost comparable to T₃, T₄, T₅ and T₆. Significantly highest activities of these enzymes were recorded in T2. The principal target for AF is the liver. Serum AST and ALT activities are considered sensitive markers of hepatocellular damage/dysfunction, indicating liver inflammation, lesions or obstruction of the biliary tract (Kubena et al., 1998). In our study, serum AST and ALT activities were significantly elevated by feeding 1 ppm AF. These findings are in agreement with the earlier reports where 500 to 750 ppb dietary AF was fed to broiler chickens (Jindal et al., 1994; Amer et al., 1998). BHT supplementations in AF contaminated groups reversed these parameters significantly and were comparable to that of control group. These findings are in agreement with the earlier report (Klein et al., 2002).

It is thus concluded that dietary supplementation of BHT at 1000 and 2000 ppm levels provided moderate alleviation against the adverse effects of 1.0 ppm total aflatoxin in terms of investigated parameters in broiler chickens.

This article was originally published in Animal Nutrition and Feed Technology (2013) 13: 235-242.

Amelioration of Aflatoxicosis in Coloured Broiler Chickens by Dietary Butylated Hydroxytoluene

INTRODUCTION

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