Efficacy of DL-methionine in amelioration of aflatoxicosis in coloured broiler chicken

Poultry industry suffers greatly from the economic losses due to the adverse effects of mycotoxicosis attributed to reduced feed intake, poor feed conversion and nutrient utilization, growth and production performance, immunosuppression and subsequently increased susceptibility to various infections (Xue et al. 2010). Aflatoxin is the most commonly occurring mycotoxin in India. Major forms of AF include B₁, B₂, G₁, and G₂, while AFB₁ is the most common and biologically active component (Busby and Wogan 1981). Several approaches were developed for decontamination of mycotoxins in foods (Shetty and Jespersen 2006, Diaz et al. 2004). Though many approaches are available for mycotoxin decontamination, most of them are not widely available due to high cost or practical difficulties involved in detoxification process (Shetty and Jespersen 2006). Now, the most promising and user-friendly approach is dietary manipulation, addition of adsorbents and detoxifying agent in feed. Selected adsorbents added to AF-contaminated feeds can sequester AF during the digestive process, allowing the mycotoxin to pass harmlessly through the animal (Davidson et al. 1987). However, not all of adsorbents are equally effective in protecting poultry against the toxic effects of AF and several adsorbents showed to impair nutrient utilization (Chung et al. 1990). Moreover, due to differences in binding capacity, it is obvious that some portion of the toxins gets absorbed and causes deleterious effect. Addition of sulphur amino acids to diets containing AF improved performance in chickens (Veltmann et al. 1981, 1983). Dietary methionine in excess amount (30–40% more) is reported to ameliorate the ill effects of aflatoxicosis in broiler chickens (Devegowda et al. 2000). However, such a level of methionine was very high and it may not be economical to add that amount. Moreover, methionine is considered to be a toxic amino acid. Therefore, the present study was conducted to determine the efficacy of additional methionine at lower level (excess by 5% and 10%) to ameliorate the toxic effects of aflatoxins.

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 Shotwell et al. (1966). The fermented maize was steamed to kill the fungus spores, dried and ground to a fine powder. The aflatoxin concentration from maize powder was extracted as per Pons et al. (1966) and measured using thin layer chromatography (TLC). The total AF concentration in maize powder 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 1ppm total AF.

Birds and diets:Day-old broiler (IR-3) chicks (180) obtained from CARI hatchery were used in this study. These chicks were wing banded, weighed individually and divided into 6 treatment groups (T₁ to T₆), each replicated thrice and each replication consisted of 10 birds (each group containing 30 chicks). The chicks were housed in electrically heated compartments with continuous lighting and given broiler starter feed up to 21 days and broiler finisher feed from 22 to 42 days of age. The composition of basal diets is given in Table 1. There were 6 experimental diets viz. T₁, basal diet (0.5 and 0.44% Met in starting and finishing ration) + 0 ppb aflatoxin (control); T₂, basal diet + 1 ppm aflatoxin; T₃, basal diet + 0.025% Met; T₄, basal diet +0.05% Met; T₅, basal diet + 0.025% Met + 1ppm aflatoxin, and T₆, basal diet + 0.05% Met + 1ppm aflatoxin.

Efficacy of DL-methionine in amelioration of aflatoxicosis in coloured broiler chicken - Image 1

Birds were inspected daily and their body weight and feed consumption were recorded weekly. Feed intake, body weight gain and feed conversion ratio (feed intake/ weight gain) were calculated. After completion of feeding trial at the age of 42 days, 9 birds from each treatment (3 from each replicate) were selected randomly for recording organ weights and to collect blood samples. Serum was separated and used for biochemical assays. Serum concentrations of total protein, total cholesterol, uric acid and the activities of ASAT and ALAT were determined spectrophotometrically using commercially available test kits. The data were analysed statistically using Duncan’s multiple range test (SPSS 16.0).

RESULTS AND DISCUSSION

Body weight gain: Significant (P<0.05) differences in body weight gain (BWG) among various dietary treatments were recorded from third week of age onward (Table 2). At third week of age, the BWG was lower (P<0.05) in T₂ than in T₁, while BWG remained statistically similar in other treatments to that of control. At fourth, fifth and sixth weeks of age also the birds under T₂ had lower body weight than control. Moreover, the BWG in T₁, T₃ and T₄ were statistically similar. Dietary supplementation of methionine at both the levels (T₅ and T₆) to aflatoxin-contaminated diet improved (P<0.05) the BWG, even then the values were lower (P<0.05) than that of control barring T₆ at sixth week of age wherein the BWG was statistically similar to that of control. During overall growth phase (0 to 6 weeks), the BWG decreased significantly on inclusion of 1 ppm of total aflatoxin in the diet. These results were in agreement with earlier workers who reported reduction in BWG at 0.2 to 0.5 ppm level of dietary aflatoxin (Azzam and Gopal 1997, Raju and Devegowda 2000, Sapocota et al. 2007, Silambarsan et al. 2013, Miazzo et al. 2000, Ledoux et al. 1999, Kubena et al. 1998, Santurio et al. 1999, and Rosa et al. 2001). In the present study, the results showed that supplementation of methionine to the basal diet did not produce any positive effect on weight gain of broilers, which validated its requirement of 0.50% as suggested by NRC (1994). Methionine supplementation in the aflatoxin contaminated diet resulted in significant improvement in the BWG. These findings are in agreement with earlier report (Sapocota et al. 2007) where partial improvement in BWG was reported when aflatoxin contaminated diet was supplemented with methionine. Devegowda et al. (2000) also reported that excess dietary methionine can ameliorate the adverse effects of aflatoxin in broiler chickens.

Efficacy of DL-methionine in amelioration of aflatoxicosis in coloured broiler chicken - Image 2

Efficacy of DL-methionine in amelioration of aflatoxicosis in coloured broiler chicken - Image 3

Efficacy of DL-methionine in amelioration of aflatoxicosis in coloured broiler chicken - Image 4

Feed consumption: Feed intake (FI) pattern differed significantly (P<0.05) among various dietary treatments from third week of age onward (Table 3). FI in T₂ was lower (P<0.05) than that of T₁ from third week of age onward. The FI in T₃, T₄ and T₆ was statistically similar to that of control but was statistically lower in T₅ during third and fourth week of age. During overall growth phase (0–6 wk), the FI in aflatoxin fed group (T₂) was lower (P<0.05) than control group (T₁). These results were in conformity with earlier reports where aflatoxin contaminated feed (0.3 ppm or higher) resulted in significant decrease in FI of broiler chickens (Kubena et al. 1990, Kubena et al. 1998, Ledoux et al. 1999, Santurio et al. 1999, Raju and Devegowda 2000). The overall FI in T₃ and T₄ was statistically similar to that of control indicating that supplementation of methionine to basal diet did not produce any positive effect on weight gain of broilers. Dietary addition of methionine at both levels (0.025 and 0.05%) to the aflatoxin contaminated diet (T₅ and T₆) significantly (P<0.05) improved the FI, however the values were significantly (P<0.05) lower than that of control. Significant difference (P<0.05) in values of T₅ and T₆ was observed, being higher in T₆.

Feed conversion ratio: Feed conversion ratio (FCR) did not differ (P<0.05) among various dietary treatments during first, second, third and sixth weeks of age (Table 4). During overall growth phase (0 to 6 wk), FCR of toxin fed group (T₂) was poorer (P<0.05) than of control group (T₁). Raju and Devegowda (2000) also reported poor feed efficiency in broilers fed diets containing 0.3 ppm aflatoxin. Several others have also reported a decrease in feed efficiency due to presence of aflatoxin in diet (Scheideler 1993, Kubena et al. 1998, Rosa et al. 2001). In present study, the inclusion of methionine in basal diet (T₃ and T₄) did not bring any positive effect on FCR. However, addition of DL-methionine at both the levels (0.025 and 0.05%) to the aflatoxin contaminated diet (T₅ and T₆) resulted in significant (P<0.05) improvement in FCR, statistically comparable to control. These reports were in agreement with other workers (Sapocota et al. 2007, Naveenkumar et al. 2007), wherein methionine supplementation to the aflatoxin contaminated feed resulted in improvement of feed conversion efficiency in broiler chickens.

Efficacy of DL-methionine in amelioration of aflatoxicosis in coloured broiler chicken - Image 5

Biochemical parameters: The total serum protein in T₂ was lower (P<0.05) than that of T₁ (Table 5), while it remained comparable to that of control in T₃ and T₄. The serum protein content of groups T₅ and T₆ were lower (P<0.05) than that of control but higher (P<0.05) than that of T₂. Methionine addition (0.025 and 0.05%) to aflatoxin-contaminated diet partially reduced the adverse effects of aflatoxicosis on serum protein. Decrease in total serum protein due to aflatoxin contamination in diet was also reported by earlier researchers (Kubena et al. 1998, Ledoux et al. 1999, Raju and Devegowda 2000, Ahamad 2000). With regard to serum cholesterol content, it was lower (P<0.05) in T₂ compared to T₁. Addition of methionine to the aflatoxin contaminated diet (T₅ and T₆) increased (P<0.05) the cholesterol content, but the values remained lower (P<0.05) than that of control. These results are in agreement with the other reports (Bailey et al. 1998, Kececi et al. 1998, Raju and Devegowda 2000, Ahamad 2000). The uric acid content in T1 was higher (P<0.05) than that of aflatoxin fed group (T₂). The uric acid content of groups T₅ and T₆ was statistically similar to that of T₂ but lower (P<0.05) than that of control. Decrease in uric acid due to aflatoxin contamination in diet was reported earlier (Bailey et al. 1998, Kececi et al. 1998, Ahmad 2000). The aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) activities in T1 were lower (P<0.05) than that of T₂ and remained comparable to T₃, T₄, T₅ and T₆. Significant increase in the activities of ASAT was also reported by Basmacioglu et al. (2005) where 2 ppm aflatoxin was fed to broilers, whereas, Kubena et al. (1993) reported a decrease in ASAT activities at 3.5 ppm of aflatoxin in the diet of broilers. In present study, ALAT activities were increased (P<0.05) by feeding 1 ppm aflatoxin. These results are in agreement with Santurio et al. (1999) where increase in ALAT activities was reported at 3 ppm level of aflatoxin. Methionine supplementations to toxin contaminated diet reversed these parameters significantly (P<0.05) and were comparable to that of control. The information on effect of supplementation of methionine on ASAT and ALAT activities during aflatoxicosis in poultry is lacking in literature.

Organ weights:The relative weights of liver and gizzard in control group was lower (P<0.05) than that of aflatoxin fed group (T₂) (Table 6), while in other treatments (T₃ to T₆), the values were statistically similar to that of control. Significant increase in the relative weights of liver and gizzard due to aflatoxin feeding (0.3 to 5 ppm) was also reported earlier (Kubena et al. 1998, Raju and Devegowda 2000, Rosa et al. 2001, Miazzo et al. 2000, Sapocota et al. 2007). In the present study, incorporation of methionine at 0.025 and 0.05% levels in the aflatoxin containing diet (T₅ and T₆) reversed the relative weights of liver and gizzard as the values were statistically similar to that of control. These results were in agreement with Sapocota et al. (2007). In the present study, the relative weight of heart remained unaffected due to various dietary treatments, however, several researchers (Kubena et al. 1990, Bailey et al. 1998, Kubena et al. 1998, Rosa et al. 2001) reported an increase in the relative weight of heart in aflatoxin fed groups. In the present study, the relative weight of bursa of Fabricius in T₂ was lower (P<0.05) than that of in T₁. The relative weight of bursa in T₃ to T6 was statistically similar to that of T₁. Incorporation of methionine at 0.025 and 0.05% levels in the aflatoxin contaminated diet (T5 and T6) reversed (P<0.05) the adverse effects of aflatoxicosis on relative weight of bursa of Fabricius. Kubena et al. (1990) also reported a decrease in relative weight of bursa when diet contained 3.5 ppm aflatoxin. However, Sapocota et al. (2007) observed no effect on the relative weight of bursa in broiler chickens fed diet with 0.3 ppm aflatoxin.

The results showed that incorporation of methionine at 0.025 and 0.05% levels to the basal diet (T₃ and T₄) did not produce any significant improvement on different parameters investigated. However, supplementation of methionine at 0.025 and 0.05% levels to the aflatoxin contaminated diet (T₅ and T₆) of broiler chickens provided partial protection against the ill effects of aflatoxicosis in terms of investigated parameters. Thakur et al. (2008) reported that the level of glutathione significantly decreased in aflatoxin fed group compared to that of control. Methionine acts as precursor of glutathione, which forms conjugated complexes with aflatoxin inside the animal and especially in the liver. These complexes are eliminated through faeces and urine.

It was concluded that inclusion of methionine at 0.025 and 0.05% levels in the 1 ppm total aflatoxin contaminated diet provided partial protection from the adverse effects of aflatoxicosis in broiler chickens.

This article was originally published in Indian Journal of Animal Sciences 83 (12): 1329–1334, December 2013/Article.