Aflatoxins, the natural contaminants of feedstuffs, are toxic metabolites produced primarily by Aspergillus flavus and Aspergillus parasiticus. Aflatoxins may cause serious economic losses in the poultry industry because they prevent birds from achieving optimum body weight gains (Khatke et al., 2012; Oguz and Kurtoglu, 2000) Small amounts of aflatoxin B1 may cause reduction in economic parameters, hatchability and also cause increased susceptibility to disease (Sharma et al., 2014; Coulombe, 1993; Denli et al., 2004). Aflatoxicosis in poultry also causes listlessness, anorexia with lowered performance and increased mortality (Singh et al., 2011; Miazzo et al., 2000), anemia (Oguz et al., 2000), reduction of immune function (Sharma et al., 2014; Oguzet al., 2003), hepatotoxicosis and haemorrhage (Ortatatli and Oguz, 2001). Several studies were conducted using inorganic adsorbents to test their ability to detoxify feed contaminated with aflatoxin and minimize aflatoxicosis in poultry (Silambarasan et al., 2013). However, high inclusion rate and potential interactions with feed nutrients are a cause of concern (Parlat et al., 1999; Miazzo et al., 2000).
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 phenolates 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 turn helps 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. Methioninehydroxy analogue is also used as source of methionine. The objective of this study was to determine the efficacy of DL-methionine and methionine hydroxy analogue to ameliorate the toxic effects of aflatoxin present in poultry rations. The supplementation levels of MHA were selected on the basis of 100, 80 and 65% activity equivalent of 500 ppm DLM.
MATERIALS AND METHODS
Production of Aflatoxin
Aflatoxin was produced using the fungal strain Aspergillusflavus NRRL 6513 that was obtained from U.S Department of Agriculture, Illinois, U.S.A. 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 AFB1 concentration in fermented maize was 980 mg/kg of maize and the aflatoxin thus produced was very stable under normal conditions.
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.
Experimental design was completely randomized design (CRD). There were 10 dietary treatments. Each dietary treatment had 4 replicates and each replicate had 15 chicks. The experiment was conducted in quails from day old to 5 weeks of age. The various dietary treatments were prepared by mixing the required quantity of DLMethionine (DLM), methionine hydroxy analogue (MHA) 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. At the end of the experiment, gross lesions in livers of birds were recorded.
Ingredients composition of basal feed (%)
A basal diet with maize 54.2, rice bran (deoiled) 2, soybean meal (solv-extracted) 31.145, 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 vitA 8250 IU, vit. D3 1200 ICU,vit. K 1mg, vit. B1 2 mg, vit. B2 4mg, vit. B12 10mcg, niacin 60mg, pantothenic acid 10mg, choline 500mg, 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 DLM and MHA over and above the control diet considering the efficacy of MHA as 100,80 and 65% activity equivalent of 500 ppm DLM. The 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 Snedechor and Cochran (1980). The statistical analysis was done using SPSS 16.0 version.
RESULTS AND DISCUSSION
Body Weight gain
The 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 that of control. However, there was no difference in weight gain among T1 , T3 , T4 , T5 and T6 . The BWG of groups T7 and T10 did not differ (P<0.05) from that of control, however, the BWG of groups T8 and T9 was lower (P<0.05) than that of control and statistically comparable to that of aflatoxin fed group (T2 ).During second, third, fourth and fifth weeks of age, the BWG in control group was higher (P<0.05) than that of aflatoxin fed group, however, the BWG in other treatment groups was more or less comparable to that of control. Incorporation of DLmethionine (DLM) or 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 500 ppm DLM (T7 ) and 769 ppm MHA (T10) in aflatoxin contaminated diet improved the BWG of quails from first week onward. However, supplementation of MHA in aflatoxin contaminated diet at 500 and 625 ppm levels (T8 and T9 ) improved the BWG of quails from second week onward. The results indicated that depression in body weight gain of quails due to aflatoxin contamination of feed was observed from first week of age onward. Oguz and Parlat (2004) also reported growth depressing effect of aflatoxin from first week onwards in Japanese quails. Silambarsan (2011) and Abaji (2012) however, reported growth depression caused by 300 ppm aflatoxin from second week onwards in broiler chickens.
During overall growth period (0-5 wk), the weight gain in control group was 183.4g against 147.1 in aflatoxin contaminated diet (T2 ) which was significantly lower (P<0.05).The overall gain in groups T3 to T7 and T10 was statistically similar to that of control group. However, overall gain in group T8 to T9 was higher (P<0.05) than that of T2, but remained lower (P<0.05) than that of T1. The results showed that supplementation of DLM or 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). Supplementation of MHA at 500 and 625 ppm (T8 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. However, supplementation of DLM at 500 ppm (T7 ) and MHA at 769 ppm (T10) level in aflatoxin contaminated diet ameliorated 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 earlier observed when fed diets containing 2 ppm (Oguz and Parlat, 2004; Parlat et al., 1999) or 2.5 ppm (Sehuet al., 2005) or 0.5 ppm aflatoxin (Singh et al., 2015). In present study, supplementation of MHA aT769 ppm level ameliorated the adverse effects of aflatoxicosis on BWG. Naveen Kumar et al. (2007) and Sapcota et al. (2007) reported partial improvement in BWG of broiler chickens due to methionine supplementation in diet during aflatoxicosis. In present study, supplementation of DLM at 500 ppm (T7 ) and MHA at 769 ppm (T10) level in aflatoxin contaminated diet was equally efficacious in ameliorating the adverse effects of aflatoxicosis on BWG in quails.
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 similar to that of control. The FC in group T4 and T6 was higher (P<0.05) than that of T2. During 2ndto5th weeks of age, the FC of control group did not differ (P<0.05) from other treatment groups. During second week of age, the FC of group T8 was higher (P<0.05) than that of T2. During fourth week, the FC in groups T3 and T6 was higher (P<0.05) than that of 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 ). In general, the feed consumption in present study remained uninfluenced due to various dietary treatments. Significant reduction in feed consumption of quails was 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. From this present study, it was revealed that at this concentration (500 ppb AFB1) did not had any adverse effect on feed consumption in growing Japanese quails.
Feed Conversion Ratio
The data pertaining to weekly and overall feed conversion ratio (FCR) as influenced by various dietary treatments are given in Table 3. At first week of age, the FCR remained uninfluenced among various dietary treatments. The deterioration of feed conversion efficiency due to aflatoxin contamination was observed from second week of age onward. During second week of age, the FCR of control group was significantly (P<0.05) lower than that of aflatoxin fed group (T2 ).The FCR of groups T3 to T7 was statistically similar to that of control, however, the FCR of group T8 was statistically similar to that of aflatoxin fed group (T2 ). The FCR in groups T9 and T10 was lower (P<0.05) than that of aflatoxin fed group but could not match with that of control diet. At third and fifth weeks of age, the FCR in control group was statistically similar to other treatment groups barring treatment T2 where in significantly (P<0.05) higher FCR compared to control was observed. At fourth week of age, the FCR in groups T3 to T7 and T10 was statistically similar to that of control, however, the FCR of groups T8 and T9 was higher (P<0.05) than that of control and statistically similar to that of aflatoxin fed group.
The overall FCR in control group (2.84 against 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, suggesting that supplementation of DLM or MHA to the basal diet did not produce any positive effect on FCR of quails. The FCR of groups T8 and T9 was lower (P<0.05) than that of aflatoxin fed group but significantly (P<0.05) higher than that of control. The FCR of groups T7 and T10 was significantly (P<0.05) lower than that of aflatoxin fed group and statistically similar to that of control. The present study indicated that contamination of feed with 500 ppb AFB1 resulted in significant increase in FCR of quails. Significant increase in FCR of broilers fed diets with 300 ppb level of dietary AFB1 was also observed by several researchers (Raju and Devegowda,2000; Silambarsan, 2011; Sapcota et al., 2007 and 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). Sehuet 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 showed 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 of feed efficiency, comparable to that of control. Supplementation 500 ppm DLM to the aflatoxin contaminated diet also resulted in significant improvement of feed efficiency that was 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 et al., 2010). However, when supplemented in aflatoxin contaminated diet (T7 to T10) there was improvement in gain and feed conversion, attributed to added methionine level. These reports are in agreement with literature (Singh et al., 2015; Sapcota et al., 2007; Naveenkumar et al., 2007), wherein methionine supplementation to the aflatoxin contaminated feed resulted in improved feed efficiency in broiler chickens. In present study, supplementation of DLM at 500 ppm (T7 ) and MHA at 769 ppm (T10) level in aflatoxin contaminated diet was equally efficacious in ameliorating the adverse effects of aflatoxicosis on feed efficiency in quails.
Gross lesions of liver
Gross pathological lesions in livers of quails kept on various dietary treatments were recorded at the end of the experiment. No lesions were seen in the liver of birds fed with toxin free diet. 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. Supplementation of MHA at 500 ppm level (T8 ) to the aflatoxin contaminated feed did not reduce any lesions caused by aflatoxicosis. However, supplementation of MHA at 625 ppm level to the aflatoxin contaminated diet (T9 ) partially ameliorated these signs of aflatoxicosis. Supplementation of 500 ppm DLM and 769 ppm MHA (T7 and T10) to the aflatoxin contaminated diet (T9 ) ameliorated these signs of aflatoxicosis, comparable to that of control.
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. In present study, incorporation of DLM (500 ppm) or MHA (769 ppm) in AF contaminated diet significantly reduced the effects of aflatoxin on. Supplementation of DLM at 500 ppm (T7 ) and MHA at 769 ppm (T10) level in aflatoxin contaminated diet was equally efficacious in ameliorating the adverse effects of aflatoxicosis on in quails.
It was concluded that dietary addition of aflatoxin B1 at the rate of 500ppb resulted in depressed body weight gain, poor feed conversion efficiency and lesions in liver morphology. Supplementation of DLM at 500 ppm or MHA at 769 ppm level in 500 ppb aflatoxin contaminated diet ameliorated those adverse effects in Japanese quails. Both supplementation of DLM at 500 ppm and MHA at 769 ppm level in aflatoxin contaminated diet was equally efficacious in ameliorating the adverse effects of aflatoxicosis in growing Japanese quails.
This article was originally published in Indian Journal of Poultry Science (2016) 51(2): 168-173; DOI: 10.5958/0974-8180.2016.00027.1.