MYCOTOX Õ and aflatoxicosis in quails

British Poultry Science Volume 46, Number 4 (August 2005), pp. 520 524 MYCOTOX Õ and aflatoxicosis in quails A. S EHU, S. ÇAKIR 1,Ö. CENG_IZ 2 AND D. ES S_IZ 3 Veterinary Faculty, Department of Animal Nutrition and Nutritional Disease, University of Ankara, Ankara, 1 University of Abant _Izzet Baysal, Mudurnu Vocational Higher School, Bolu, Turkey, 2 Veterinary Faculty, Department of Animal Nutrition and Nutritional Disease, University of Adnan Menderes, Ayd n and 3 Veterinary Faculty, Department of Pharmacology and Toxicology, University of Kafkas, Kars, Turkey Abstract 1. This study was to evaluate the toxic effects of aflatoxin (AF) on growth performance of quail, and to determine the preventive efficacy of MYCOTOX Õ (oxicinol, tymol, micronised yeast). 2. One hundred and eighty 1-d-old quail (Coturnix coturnix japonica) of both sexes were weighed and randomly divided into 4 experimental groups each with 5 replicates of 9 birds. 3. There were 4 dietary treatments: (1) control with 0 mg AF/kg diet and 0% MYCOTOX Õ ; (2) 0 mg AF/kg diet and 0.5% MYCOTOX Õ ; (3) 2.5 mg AF/kg diet and 0% MYCOTOX Õ ; (4) 2.5 mg AF/kg diet plus 0.5% MYCOTOX Õ. The chicks were maintained on these treatments to 3 weeks of age. Quail consumed the diets and water ad libitum. 4. Body weight (BW) gains in groups receiving AF alone were the lowest at all periods. Feed intake was lowest in the group consuming the AF diet. The addition of MYCOTOX Õ to the AF diet did not prevent or reduce the toxic effects of AF on feed intake at any time period. Feeding diets containing MYCOTOX Õ alone did not change feed intake significantly. With the exception of the 1 to 7 d period, feed conversion of chicks fed the AF diet was similar to those of the other experimental groups. 5. Bursa of Fabricius weight decreased, whereas the relative weights of liver, kidney and spleen increased in quail consuming diets containing AF and AF plus MYCOTOX Õ. Liver colour was normal in the control and MYCOTOX Õ alone group, but was lighter in groups fed AF. 6. The results indicated that MYCOTOX Õ was not effective in preventing the deleterious effects of AF. INTRODUCTION Aflatoxins (AF) are a group of extremely toxic chemicals produced by some species of fungi in the genus Aspergillus and occur as natural contaminants of poultry and livestock feeds (Edds and Bortell, 1983). AF cause severe economic losses in the poultry and livestock industries and cause a variety of effects in poultry, including anorexia, with lowered growth rate, poor feed utilisation, decreased weight gain and egg production, increased susceptibility to environmental and microbial stress, and increased mortality (Kubena et al., 1998). Aflatoxicosis is also associated with anaemia (Huff et al., 1986; Keçeci et al., 1998), inhibition of immune function (Gabal and Azzam, 1998; Çelik et al., 2000), harmful effects in liver and kidney (Harvey et al., 1993; Jindal et al., 1994), mutagenesis, teratogenesis, carcinogenesis and haemorrhage (Edds and Bortell, 1983). Measures used by the livestock industry to protect animals from the toxic effects of AF include grain testing, use of mould inhibitors, fermentation, microbial inactivation, physical separation, thermal inactivation, irradiation, ammoniation and ozone degradation (Phillips et al., 1988). Unfortunately, most of these techniques are impractical, ineffective or potentially unsafe. At the present time, one of the more promising and practical approaches is the use of adsorbents. 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). The major advantages of these adsorbents include safety and ease of administration through addition to animal feeds. However, not all adsorbents are equally effective in protecting poultry against the toxic effects of AF and several adsorbents have been shown to impair nutrient utilisation (Scheideler, 1993). Correspondence to: Professor Dr Adnan S ehu, Ankara Üniversitesi, Veteriner Fakültesi, Hayvan Besleme ve Beslenme Hastal klar Anabilim Dal, 06110 D s kap, Ankara, Türkiye. E-mail: sehu@veterinary.ankara.edu.tr Accepted for publication 22nd March 2005. ISSN 0007 1668(print)/ISSN 1466 1799(online) ß 2005 British Poultry Science Ltd DOI: 10.1080/00071660500181529

MYCOTOX Õ AND AFLATOXICOSIS IN QUAILS 521 Recent approaches adopted to deal with the presence of aflatoxin in feedstuffs have been directed towards the prevention of their absorption from the gastrointestinal tract by the use of zeolites (Phillips et al., 1988; Kubena et al., 1991; Eraslan et al., 2003), bentonite (NRC, 1994), hydrated sodium calcium aluminosilicate (Bailey et al., 1998; Kubena et al., 1998; Ledoux et al., 1999) and activated charcoal (Jindal et al., 1994; Edrington et al., 1997). Saccharomyces cerevisae, another non-toxic adsorbent, binds toxic molecules and eliminates them from the gastrointestinal tract (Stanley et al., 1993; Devegowda, 1999). MYCOTOX Õ is a new approach to prevent the deleterious effects of AF. The objective of the present experiment using quail chicks was to evaluate the toxic effects of AF on growth performance and to determine the preventive efficacy of MYCOTOX Õ. MATERIALS AND METHODS Animals and rearing conditions One hundred and eighty 1-d-old quail of both sexes were obtained from a commercial hatchery (Lezzet Bildircin, Akyurt, Ankara, Turkey). Individually weighed quail were randomly divided into 4 experimental groups. There were 5 replicates of 9 birds per dietary treatment. Newly hatched quail were housed in environmentally controlled cages with paper on wire floors and fed a commercial quail chick starter for 3 weeks. Lighting was continuous in an enclosed building. Diets and experimental design Quails were fed a commercial non-medicated, maize and soybean meal diet that met the nutrient recommendations of the National Research Council (1994), with no added antibiotics, coccidiostats or growth promoters, up to 21 d. The diets of the experimental groups are presented in (Table 1). There were 4 treatments: (1) control with 0 mg AF/kg of diet and 0% MYCOTOX Õ (oxicinol, tymol, micronised yeast); (2) 0.5% MYCOTOX Õ ; (3) 2.5 mg AF/kg of diet; (4) 2.5 mg AF/kg of diet plus 0.5% MYCOTOX Õ. AF and MYCOTOX Õ concentrations were chosen on the basis of information from the literature and distributor company, respectively. Quails were on these treatments from one day old to 3 weeks of age; feed and water were provided ad libitum. Group feeding was applied in all replications. Birds were weighed as groups once a week. Feed was weighed weekly to evaluate intake and feed conversion ratio per cage. Mortality was recorded. Birds were inspected daily and any health-related problems Table 1. Chemical composition of experimental diets Ingredients Control MYCOTOX Õ (g/kg) Maize 494.9 494.9 Soybean meal 254.2 254.2 Full-fat soybean Rice 1 179. 5 33.8 179. 5 33.8 CaCO 3 9.6 9.6 Dicalcium phosphate Salt 19.1 2.5 19.1 2.5 Methionine 2.9 2.9 Vitamin premix 2 2.5 2.5 Mineral premix 3 1. 0 MYCOTOX Õ (toxin binder) 1. 0 5.0 Silica 4 5.0 Values analysed, g/kg Metabolisable energy, MJ/kg 13.17 13.17 Crude protein 219.5 219.5 Crude fibre Ether extract 31.0 63.0 31.0 63.0 Ash 67.8 67.8 Dry matter 920.0 920.0 1 Rice included mycotoxin at 73. 96 mg/kg. 2 Each 1 kg of vitamin premix contained: 15 000 000 IU vit. A, 2 500 000 IU vit. D 3, 80 000 mg vit. E, 5000 mg K 3, 300 mg B 1, 600 mg B 2, 5000 mg B 6, 30 mg B 12, 50 000 mg vit. C, nicotinamide 50 000 mg, 12 000 mg panthotenic acid, 1500 mg folic acid, 100 mg D-biotin. 3 Each 0. 5 kg mineral premix contained: 80 000 mg Mn, 60 000 mg Fe, 60 000 mg Zn, 5000 mg Cu, 200 mg Co, 1000 mg I, 150 mg Se. 4 Groups without toxin binder included silica instead of MYCOTOX Õ. recorded. At 3 weeks of age, 25 birds per group (5 birds per replicate) were killed by cervical dislocation and liver, spleen, kidney and bursa of Fabricius were removed and weighed. Preparation of toxin and inclusion of toxin and toxin binder to diet Aflatoxin was produced, with minor modifications (Demet et al., 1995), by the method described by Shotwell et al. (1966) by growing Aspergillus parasiticus NRLL 2999 (National Center for Agricultural Utilization Research, Peoria, IL, USA) on rice. The rice culture was autoclaved, dried and ground to a fine powder. The aflatoxin content of the culture material was analysed using immunoaffinity columns (Vicam AflaTest Õ Affinity Column) and quantified by high performance liquid chromatography (HPLC) (Agillent 1100 Series) using a Hypersil BDS C-18 column (25 cm 4.6mm5mm) with post column derivatisation with a KOBRA electrochemical cell, fluorescent detection (Agillent FLD) at 360 nm excitation and 440 nm emission wavelengths. A water:methanol:acetonitrile solution (6:2:3, v:v:v; flow rate 1 ml/min) containing 120 mg KBr and 350 ml HNO 3 at 4 moles/l of mobile phase was used (Stroka et al., 2000). The standards of AFs B 1,B 2,G 1 and G 2 were purchased from Sigma-Aldrich (Taufkirchen, Germany) (ref. A-6636, A-9887, A-0138 and A-0263, respectively). The stock solutions,

522 A. S EHU ET AL. working standards and the calibration curve were prepared as described by Stroka et al. (2000). The total amount of AF (AF B 1,B 2,G 1 and G 2 ) was 73.96 mg/kg in rice powder, and consisted of 83.87% AF B 1,9.51% AF G 1,6.18% AF B 2 and 0.43% AF G 2, based on the total AF in the rice powder. The rice powder was incorporated into the basal diet in order to provide the desired amount of 2.5 mg AF/kg feed. The toxin binder was added to the diet at 5 g/kg. Statistical analysis Data for all response variables were subjected to ANOVA (Snedecor and Cochran, 1967) using an SPSS program (SPSS Inc., Chicago, IL, USA). Significant differences among treatment means were determined by multiple range test with a 5% level of probability. RESULTS Performance data are given in Table 2 body weight (BW), BW gain, feed intake and feed conversion ratio (FCR) were not significantly influenced by addition of MYCOTOX Õ to diet without AF. No significant differences were observed for BW at the start of the trial. Feeding aflatoxin alone caused a significant decrease in BW. Table 2. Body weight (BW), body weight gain (BW gain), feed intake and feed conversion ratio (FCR) of experimental groups (n ¼ 5) MYCOTOX Õ (%) 0 0.5 0 0.5 SEM AF (mg/kg) 0 0 2.5 2.5 BW (g) 1d 7.98 7.93 7.97 7.84 0.062 7d 32.76 a 34.13 a 28.99 b 29.16 b 0.716** 14 d 21 d 71. 37 a 110.67 a 71. 91 a 112.84 a 63. 52 b 99.04 b 64. 04 b 101.29 b 1. 109*** 1.552*** BW gain (g) 1to7d 24.78 a 26.20 a 21.02 b 21.32 b 0.693* 8to14d 38.62 a 37.78 a 34.53 b 34.88 b 0.566** 15 to 21 d 39.30 40.93 35.52 37.25 0.599** 1 to 21 d 102. 70 104. 91 91. 07 93. 45 1. 598*** Feed intake (g) 1to7d 5.43 a 5.69 a 4.29 b 4.35 b 0.176*** 8to14d 15 to 21 d 11. 96 a 15.05 a 11. 34 ab 15.40 a 9. 11 c 13.46 b 10. 36 bc 14.19 b 0. 324** 0.210*** 1to21d 32.44 a 32.43 a 26.87 b 28.90 b 0652*** FCR (kg/kg) 1to7d 0.22 a 0.22 a 0.21 ab 0.20 b 0.003* 7to14d 0.31 0.30 0.27 0.30 0.007 15 to 21 d 1to21d 0.32 0.31 0.30 0.30 0. 004 0.004 a,b,c : Means on the same row followed by different letters differ significantly. MYCOTOX Õ ¼ toxin binder (oxicinol, tymol, micronised yeast); AF ¼ aflatoxin. *P <0. 05; **P <0. 01; ***P <0. 001. Quail consuming the diet with 2.5 mg/kg of AF had lower BW (P <0.001) than the MYCOTOX Õ and control groups. The inclusion of 5 g/kg MYCOTOX Õ to diet containing AF had no effect on BW at any period. BW gains of the groups given AF were the lowest at all periods. The reduction in BW gain caused by 2.5 mg AF/kg of diet was not prevented or reduced by the addition of 0.5% MYCOTOX Õ to the diet. No significant interactions occurred between AF and MYCOTOX Õ during the experiment. Feed intake was lowest in the groups consuming the AF diet. The addition of MYCOTOX Õ to the AF diet did not protect against the decrease in feed intake at any time period. Quail fed MYCOTOX Õ alone had similar feed intake to those fed the control diet. FCR (kg feed/kg BW gain) values of quail fed AF diet were similar to those of the other experimental groups. None of the treatments altered the FCR significantly. Although a difference (P <0.05) was observed between groups at the end of the first period, the addition of MYCOTOX Õ to the AF diet had no effect on the FCR of the groups. Data in (Table 3) show the effects of dietary treatments on relative organ weights and some blood variables. With the exception of kidney weight, quail fed the MYCOTOX Õ alone diet had organ weights similar to those of controls. Relative kidney weight decreased in the AF alone group but increased (P <0.001) in the quails consuming the AF plus MYCOTOX Õ diet. When compared to the control, kidney weights increased (P <0.001) in birds fed AF only, MYCOTOX Õ only, and the AF plus MYCOTOX Õ treatments. The increase in kidney weight in quail fed AF plus MYCOTOX Õ was significantly greater than observed in quail fed AF or MYCOTOX Õ alone. No significant effect of MYCOTOX Õ supplementation to AF diet was Table 3. The effect of toxin binder on relative organ weights and some blood variables (n ¼ 25) MYCOTOX Õ (%) 0 0.5 0 0.5 SEM AF (mg/kg) 0 0 2.5 2.5 Liver (g/100 g BW) 2.14 b 2.08 b 2.76 a 2.92 a 0.053*** Kidney (g/100 g BW) 0.57 c 0.86 b 0.82 b 1.05 a 0.029*** Spleen (g/100 g BW) Bursa of Fabricius 0.05 b 0.14 a 0.05 b 0.13 a 0.09 a 0.10 b 0.08 a 0.10 b 0.004*** 0.006** (g/100 g BW) Protein (g/dl) 3.85 3.56 2.90 3.20 0.178 Cholesterol (mg/dl) Glucose (mg/dl) 135. 58 a 118. 50 ab 80. 29 c 100. 88 bc 5. 124*** 13.22 16.6 20.14 13.25 1.464 a,b,c : Means on the same column followed by different letters differ significantly. MYCOTOX Õ ¼ toxin binder (oxicinol, tymol, micronised yeast); AF ¼ aflatoxin. **P <0. 05; ***P <0. 001.

MYCOTOX Õ AND AFLATOXICOSIS IN QUAILS 523 observed in liver and spleen weights. Bursa of Fabricius weight decreased (P <0.01) in groups fed diets containing AF and AF plus MYCOTOX Õ. Liver colour was normal in the control and the group receiving MYCOTOX Õ only but groups receiving AF had a lighter colour. No significant differences were observed for total protein and glucose concentrations. There was a significant difference in cholesterol between the control and groups receiving AF. No signs of nutritional deficiencies were found in any of the organs examined in any of the treatments. A total of 14 birds (5 in MYCOTOX Õ alone group, 4 in AF alone group and 5 in AF plus MYCOTOX Õ group) died during the course of the study. There were no significant differences in mortality. Deaths mostly occurred during the first period of the experiment and did not appear to be related to treatment. DISCUSSION Aflatoxins (AF) are important to the poultry industry because of their toxicity and frequency of occurrence in feedstuffs; their toxic effects in poultry have been widely documented (Huff et al., 1986). Our study defined the toxicity of AF in quails. The results of the present study with MYCOTOX Õ agree with previous results of Kubena et al. (1990a) who showed that HSCAS diminished the toxicity of AF alone. In the absence of toxins MYCOTOX Õ did not adversely affect any of the variables measured. Quail performance was not adversely affected by the addition of 5 g MYCOTOX Õ /kg to the basal diet. The absence of any signs of nutritional deficiencies in any of the organs indicates that MYCOTOX Õ did not adversely affect dietary nutrients (such as minerals and vitamins) at this dietary inclusion rate. Body weights were lowest in groups fed diets containing AF alone and those fed AF plus MYCOTOX Õ. Such effects may have resulted from anorexia, listlessness and the inhibitory effect of AF on protein synthesis and lipogenesis. Impaired liver functions and protein/lipid utilisation mechanism may have affected growth and general health (Keçeci et al., 1998). The addition of MYCOTOX Õ to the AF diet did not reverse the growth-depressing effect of AF. When compared with the control, BW at 21 d was reduced ( 10.51%) by AF alone, showing that MYCOTOX Õ had no protective effect on BW depression. Poor BW gain during aflatoxicosis directly affects profit in the poultry industry. The BW gain response was similar to that of BW. When compared with the control, overall BW gain was reduced 11.32% by AF alone. The addition of MYCOTOX Õ to the AF diet did not improve BW gain. These findings support the results of Kubena et al. (1998). There was no difference in feed intake between the AF alone and the AF plus MYCOTOX Õ treatment at any time period. Decreased feed intake observed in birds fed AF alone was consistent with previous trials (Edds and Bortell, 1983; Kubena et al., 1990b). The liver is regarded as a principal target organ for AF. Increases in the relative liver weights of birds fed AF have been reported previously (Kubena et al., 1998; Ledoux et al., 1999). In our study, too, relative liver weight was increased in quail receiving the AF diet and addition of MYCOTOX Õ to the AF diet did not prevent the increased organ weights observed in quail fed AF alone. Normally, changes in serum albumin, globulin and total protein are indicators of impaired protein synthesis that occur in quail suffering from aflatoxicosis (Kubena et al., 1993). In our study total protein and glucose concentrations did not change significantly. Total cholesterol was lower in AF groups suggesting that lipid metabolism might to be affected. 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