Aflatoxins belong to a group of mycotoxins produced as secondary metabolites by fungi of the Aspergillus genus, especially A. flavus, A. parasiticus and A. nomius (Kurtzman et al., 1987). No region of world escapes the problem of mycotoxins and according to Lawler and Lynch (2005) mycotoxin are estimated to affect as much as 20% of world’s crop each year. Crops grown under warm and moist weather in tropical or subtropical countries are especially more prone to aflatoxin contamination. Aflatoxin deteriorates the quality of the feed; therefore it has tremendous economic impact on the poultry industry as they prevent birds from achieving optimum growth rate, hatchability, feed efficiency and immunity towards diseases (Richard et al., 1986, Oguz and Kurtoglu, 2000). The contamination of aflatoxins in feed is practically unavoidable (Coulombe et al., 2005). When contamination cannot be prevented, detoxification of aflatoxins from contaminated feed is required. Methionine is an essential amino acid that contains sulfur, a substance required for the production of the body’s most abundant natural antioxidant glutathione. Aflatoxins are metabolized to highly reactive epoxides and phenolats that can bind and interfere with nucleic acid and proteins (Ciegler, 1975). The epoxides and phenolates are normally conjugated with glutathione that serves to protect vital macromolecules from these toxin intermediates. Hepatic necrosis is thought to result when glutathione reserves have been drastically depleted by conjugation with toxin intermediates so that the toxin intermediates are free to bind covalently to vital cellular macromolecules. Therefore, supplementing methionine, which in turns help to increase hepatic GSH concentration may aid to protect liver against aflatoxicosis. Moreover, methionine is the first limiting amino acid in maize-soybean meal based conventional diets of majority of poultry species under intensive feeding system. Methionine hydroxy analogue is also used as source of methionine. Considering the above facts, an investigation was undertaken to study the effects of supplementation of methionine hydroxy analogue on production parameters during aflatoxicosis in Japanese quails.
MATERIALS AND METHODS
Production and analysis of aflatoxin
Aflatoxin was produced using the fungal strain Aspergillus flavus NRRL 6513 that was obtained from US Department of Agriculture, Illinois, USA. To get the fresh spores, the culture was regularly sub-cultured on Potato Dextrose Agar (PDA) medium slants and stored at 5ºC. Aflatoxin was produced on maize substrate. Fermentations were carried out in batches as per the method described by Shotwell et al. (1966). The extraction and estimation of aflatoxin was done as per the procedure of Pons et al. (1966). Aqueous acetone was used for extraction of the toxin. Aflatoxin contents were finally quantified using a spectrophotometer.
A total of 600, day-old quail chicks were divided into ten treatment groups viz. T1 : control, T2 : T1 +500 ppb AFB1 , T3 : T1 +400 ppm MHA, T4 : T1 +500 ppm MHA, T5 : T1 +625 MHA, T6 : T1 +769 ppm MHA, T7 : T2 +400 ppm MHA, T8 : T2 +500 ppm MHA, T9 : T2 +625 MHA and T10: T2 +769 ppm MHA in a completely randomized design. Each diet was fed to four replicated groups of 15 birds each from day-old to 35 days of age. The various dietary treatments were prepared by mixing the required quantity of DL-Methionine, and mouldy maize to get the desired concentration of 500 ppb AFB1.
Biological experiment and analysis
The day-old Japanese quail chicks (six hundred) were distributed randomly into 10 treatment groups on equal weight basis. All the birds were reared under standard management conditions from 0-5 weeks of age and fed with quail starter ration from 1-35 days. Weekly body weight and feed consumption of each group were recorded.
Ingredients composition of basal feed (%)
A basal diet with maize 54.2, rice bran (deoiled) 2, soybean meal (solv-extracted) 31.15, sunflower meal 2, rapeseed meal 4, fish meal 4, limestone 0.75, dicalcium phosphate 1.4, salt, 0.15, DL-methionine 0.06, trace mineral (TM) premix 0.1, vitamin premix 0.165, and choline chloride 0.03% was formulated. The TM premix supplied Mg 300, Mn 55, I 0.4, Fe 56, Zn 30 and Cu 4 mg/kg diet. The vitamin premix supplied vit A 8250 IU, vit. D3 1200 ICU, vit. K 1mg, vit. B1 2mg, vit. B2 4 mg, vit. B12 10 mcg, niacin 60 mg, pantothenic acid 10 mg, choline 500 mg, vit. E 40 IU per kg diet. The control diet so formulated contained crude protein 23.95%, metabolisable energy 2795 kcal/kg, calcium 1.05%, available phosphorus 0.47%, lysine 1.2% and methionine 0.50%. The experimental diets were prepared by supplementing MHA over and above the control diet considering its efficacy as 65 (equivalent to 0.025, 0.03, 0.04 and 0.05% methionine) or 80% (equivalent to 0.03, 0.04, 0.05 and 0.06% methionine). The crude protein (AOAC, 1990) and calcium (Talapatra et al., 1940) contents were estimated, while the concentrations of lysine, methionine, available P and metabolizable energy values were calculated. Data were analyzed following completely randomized design (CRD) as per Snedecor and Cochran (1980). The statistical analysis was done using SPSS 16.0 version.
RESULTS AND DISCUSSION
Body weight gain
The data pertaining to effect of various dietary treatments on average body weight gain (BWG) of quails at different weeks of age and overall BWG from 1 to 5 weeks of age is presented in Table 1. At first week of age, the BWG of aflatoxin fed groups was lower (P<0.05) than control. However, there was no difference in weight gain among control, T3, T4, T5 and T6. During second, third, fourth and fifth weeks of age, the BWG in control group was higher (P<0.05) than aflatoxin fed group, however, the BWG in other treatment groups was comparable to control. Addition of methionine hydroxyl analogue (MHA) in basal diet at any level (T3 to T6 ) did not improve BWG at any stage of the experimental period. Supplementation of MHA in aflatoxin contaminated diet at various levels (T7 to T10) improved the BWG of quails from second week onward. The results showed that depression in body weight gain of quails due to aflatoxin contamination of feed was recorded from first week of age onward. Oguz and Parlat (2004) also reported growth depressing effect of aflatoxin from first week onwards in Japanese quails. However, Silambarsan (2011) and Abaji (2012) reported growth depression caused by 300 ppm aflatoxin from second week onwards in broiler chickens.
During overall growth period (0-5 wk), the gain in control group (183.4g) was significantly higher (P<0.05) than aflatoxin contaminated diet (T2 147.1 g). The overall gain in groups T3 to T6 was statistically similar to control group. However, overall gain in group T7 to T9 was higher (P<0.05) than T2, but remained lower (P<0.05) than T1 except in T10, which was comparable to T1 . The results indicated that supplementation of MHA to the basal diet did not produce any positive effect on weight gain of quails, which validated its requirement of 0.50% as suggested by NRC (1994). Moreover, supplementation of MHA at 400 to 625 ppm (T7 to T9 ) level in aflatoxin contaminated diet significantly (P<0.05) improved the weight gain but the weight gain could not match with the control diet, while MHA at 769 ppm level in aflatoxin contaminated diet ameliorated completely the adverse effects of aflatoxin on BWG in quails. Growth depression is a common feature of aflatoxicosis. The results of present study revealed that contamination of aflatoxin (500 ppb) in the diet of Japanese quails caused decrease (P<0.05) in body weight gain. Significant decrease in BWG of Japanese quails were observed when fed diets containing 2 ppm (Oguz and Parlat, 2004; Parlat et al., 1999) or 2.5 ppm aflatoxin (Sehu et al., 2005). In present study, supplementation of MHA at 769 ppm level ameliorated the adverse effects of aflatoxicosis on BWG. Naveenkumar et al. (2007) and Sapocota et al. (2007) reported partial improvement in BWG of broiler chickens due to methionine supplementation in diet during aflatoxicosis.
The effect of different dietary treatments on weekly and cumulative (0-5 weeks of age) feed consumption is presented in Table 2. At first week of age, the feed consumption (FC) in all the treatment groups was statistically similar to that of control however, FC in group T4 and T6 was higher (P<0.05) of T2 . During 2nd to 5th weeks of age, the FC of control group did not differ (P<0.05) from other treatment groups. During fourth week, the FC in group T6 was higher (P<0.05) than aflatoxin fed group. During overall growth period (0-5 week), there was no significant difference in feed consumption of various dietary treatments, however, apparently lowest feed consumption was reported in aflatoxin fed group (T2 ).
By and large, the feed consumption in present study remained uninfluenced due to various dietary treatments. Significant reduction in feed consumption of quails was also observed earlier (Parlat et al., 1999; Oguz and Parlat, 2004) when the diet containing 2 ppm aflatoxin was fed to them. Sehu et al. (2005) also reported decreased feed consumption of quails consuming the diet with 2.5 ppm of aflatoxin. The results revealed that 500 ppb AFB1 concentration did not had any adverse effect on feed consumption in growing Japanese quails.
Feed conversion ratio
The data pertaining to weekly and overall FCR are given in Table 3. At first week of age, the FCR among various dietary treatments did not differ significantly. The deterioration in feed conversion efficiency due to aflatoxin contamination was observed from second week of age onward. During second T8 was statistically similar to that of aflatoxin fed group (T2 ). At third, fourth and fifth weeks of age, the FCR in control group was statistically similar to other treatment groups barring treatment T2 wherein significantly (P<0.05) higher FCR compared tond week of age, the FCR of control group was significantly (P<0.05) lower than aflatoxin fed group (T2 ). The FCR of groups T3 , T4 , T5 , T9 and T10 was statistically similar to that of control, however, the FCR of groups T7 control was observed. The overall FCR in control group (2.84 vs.3.40) was lower (P<0.05) than in aflatoxin fed group. The overall FCR in groups T3 to T6 was statistically similar to that of control, indicating that supplementation of MHA to the basal diet did not produce any positive effect on FCR of quails. The FCR of groups T7 , T8 and T9 was lower (P<0.05) than aflatoxin fed group but significantly (P<0.05) higher than control. The FCR of group T10 was significantly (P<0.05) lower than aflatoxin fed group and similar to control.
The present study revealed that contamination of feed with 500 ppb AFB1 resulted in significant poor FCR by quails. Significantly poor FCR in broilers fed diets with 300 ppb level of dietary AFB1 was also observed by several researchers (Raju and Devegowda, 2000; Silambarsan, 2011; Sapocota et al., 2007; Abaji, 2012). Impaired feed efficiency in quails due to 2 ppm aflatoxin containing diet was also reported (Parlat et al., 1999; Oguz and Parlat, 2004). Sehu et al. (2005) on the other hand, reported that FCR values of quails fed diet containing 2.5 ppm aflatoxin were similar to those of the other experimental groups. The results of the present investigation revealed that supplementation of 625 ppm or less MHA to the aflatoxin contaminated diet may not be sufficient to ameliorate the harmful effect of aflatoxin on FCR, however, supplementation at higher level (769 ppm) to the aflatoxin contaminated diet resulted in significant improvement in feed efficiency, comparable to that of control. Moreover, supplementation of MHA beyond 0.05% methionine level did not prove to be beneficial validating the optimum requirement of dietary methionine as 0.50% (0.90% TSAA-methionine+cysteine) for growing meat type quails during 0-5 weeks of age (Rana Parvin et al., 2010). However, when supplemented in aflatoxin contaminated diet (T7 to T10) there was improvement in gain and FCE, due to added methionine level. These reports are in agreement with earlier reports (Sapocota et al., 2007; Naveenkumar et al., 2007), wherein methionine supplementation to the aflatoxin contaminated feed resulted in improved FCE in broiler chickens.
Gross lesions of liver
Alteration of liver both morphologically and histopathologically is a common phenomenon of aflatoxicosis. Marked morphological changes occurred in the liver of quails fed diet with aflatoxin. Aflatoxin contamination in diet (T2 ) resulted in enlarged, pale, congested and round bordered liver (Table 4). Supplementation of MHA at 400 and 500 ppm level (T7 and T8 ) to the aflatoxin contaminated feed did not reduce any lesions caused by aflatoxicosis. Supplementation of MHA to the aflatoxin contaminated diet (T9 and T10) partially to completely ameliorated these signs of aflatoxicosis. In present study, incorporation of aflatoxin (500 ppb) to the basal diet resulted in significant gross lesions in liver of aflatoxin affected birds. Similar lesions were also reported by Silambarsan (2011), at 300 ppb aflatoxin level in broiler chickens. Manegar et al. (2010) observed these changes at 100 ppb level of aflatoxin contamination in commercial broilers. Incorporation of MHA at higher level (769 ppm) in AF contaminated diet significantly reduced the effect of aflatoxin on liver morphology.
It may be concluded that supplementation of methionine hydroxy analogue beyond 0.50% dietary methionine did not prove beneficial for growth and feed conversion efficiency in growing Japanese quails. Dietary addition of aflatoxin B1 at the rate of 500 ppb depressed body weight gain and feed conversion efficiency and supplementation of MHA at 769 ppm level in 500 ppb aflatoxin contaminated diet ameliorated those adverse effects in growing Japanese quails.
This article was originally published in Animal Nutrition and Feed Technology (2015) 15: 227-234. doi: 10.5958/0974-181X.2015.00025.6.