Effect of clinoptilolite on performance of broiler chickens during experimental aflatoxicosis
Published: August 12, 2016
By: H. OGUZ* AND V. KURTOGLU#
Departments of *Pharmacology and Toxicology and #Animal Nutrition and Nutritional Diseases, University of Selcuk, Konya, Turkey
Summary
1. The amelioration of aflatoxicosis in broiler chickens was examined by feeding 2 concentrations of natural zeolite (clinoptilolite). Clinoptilolite (CLI), incorporated into the diet at 15 and 25 g/kg, was evaluated for its ability to reduce the deleterious effects of 2·5 mg total aflatoxin (AF; 76·40% AFB1 , 16·12% AFB2 , 6·01% AFG1 and 1·47% AFG in diet on growing broiler chicks from 1 to 21 d of age. A total of 360 broiler chicks were divided into 6 treatment groups (6 replicates of 10 broilers each): control, AF, CLI (15 g/kg), AF plus CLI (15 g/kg), CLI (25 g/kg), and AF plus CLI (25 g/kg).
2. Compared to controls, the AF treatment had significantly decreased body weight gain from week 1 onwards. The adverse effect of AF on food consumption (8·0%) and food conversion ratio (8·3%) was also shown over the entire 21-d feeding period.
3. The addition of CLI (15 g/kg) to an AF-containing diet significantly reduced the deleterious effects of AF on food consumption and body weight gain. Food conversion ratio was also slightly improved by adding CLI (15 g/kg) to AF-containing diets. Food consumption, body weight gain and food conversion ratio values were rendered numerically intermediate between AF and control groups by the addition of CLI (25 g/kg) to the AF-containing diet.
4. The addition of CLI (both 15 and 25 g/kg) to the AF-free diet did not produce any significant changes compared with the controls, except for decreased total food consumption in the CLI (25 g/kg)-alone group.
5. These results suggest that CLI (15 g/kg) addition effectively diminished the detrimental effects of AF on the values investigated. Also, the lower dietary concentration of CLI (15 g/kg) was more effective than the greater concentration against the adverse effects of AF on the variables investigated in this study.
INTRODUCTION
Aflatoxins (AF), a group of closely related, extremely toxic chemicals, are produced by Aspergillus flavus and Aspergillus parasiticus and can occur as natural contaminants of poultry foods (Edds and Bortell, 1983; Leeson et al., 1995). Aflatoxicosis is an important disease of livestock and poultry. AF may be produced in food and foodstuffs before harvest, between harvest and drying, in storage and after processing in manufacturing under certain conditions of humidity and temperature (Acet et al., 1989; Kubena et al., 1998). The frequent AF contamination of agricultural commodities and the chronic exposure of poultry to these toxins can mean the difference between profit and loss to the poultry industry (Hamilton, 1983; Kaya et al., 1990).
Aflatoxicosis in poultry is characterised by listlessness, anorexia with lowered growth rate, poor food utilisation, decreased weight gain, decreased egg production, increased susceptibility to environmental and microbial stresses and increased mortality (Bailey et al., 1998; Parlat et al., 1999). Signs of aflatoxicosis also include anaemia (Huff et al., 1988; Kececi et al., 1998), inhibition of immune function (Celik et al., 1996; Gabal and Azzam, 1998), hepatotoxicosis (Edrington et al., 1997; Kiran et al., 1998), mutagenesis, teratogenesis, carcinogenesis and haemorrhage (Edds and Bortell, 1983).
Removing AF from contaminated food and foodstuffs remains a major problem and there is a great demand for effective decontamination technology. Decontamination procedures have focused on degrading, destroying, inactivating or removing AF by physical, chemical or biological methods. A successful detoxification process must be economical, must be capable of eliminating all traces of toxin without leaving harmful residues and must not impair the nutritional quality of the commodity (Leeson et al., 1995; Kubena et al., 1998; Parlat et al., 1999). Large-scale, practical and cost- effective methods for detoxifying AF-containing foodstuffs are currently not available, although a variety of methods for detoxifying AF have been employed with limited success. Therefore, the use of AF-contaminated food remains a significant problem and one with serious economic implications (Bailey et al., 1998; Ledoux et al., 1999; Parlat et al., 1999).
An encouraging approach to the problem has been to use non-nutritive and inert absorbents in the diet to bind AF and reduce the absorption of AF from the gastrointestinal tract. These compounds must not be absorbed from the gastrointestinal tract and must have the ability to bind physically with chemical substances, precluding their absorption. Some aluminosilicates, clays and zeolites have been used for this purpose. These are generally inert and non-toxic to animals (Olver, 1997) and have a capacity to bind AF (Phillips et al., 1988). The dietary addition of zeolites (Harvey et al., 1993; Scheideler, 1993; Kec¸eci et al., 1998). bentonite (OgÆuz, 1997; Santurio et al., 1999) and hydrated sodium calcium aluminosilicate, a natural phyllosilicate (Kubena et al., 1993,1998; Ledoux et al., 1999), activated charcoal (Jindal et al., 1994; Edrington et al., 1997) have been used for the reduction of AF toxicity in chickens. A protective effect against AF was observed in most of the experiments.
Zeolites are crystalline, volcanic, hydrated aluminosilicates of alkali and alkaline earth cations, with infinite, 3-dimensional structures (Ming and Mumpton, 1989); one of those is clinoptilolite (CLI). Harvey et al. (1993) investigated the effects of CLI (5 g/kg) on aflatoxicosis (3·5 mg/kg diet) in broiler chicks. Results of the study demonstrated that the addition of CLI to an AF-contaminated diet did not significantly diminish the toxicity of high dietary concentrations of AF (3·5 mg/kg diet) to growing broiler chicks. However, another natural zeolite (mordenite) significantly diminished the toxicity of AF. Dwyer et al. (1997) tested CLI (10 g/kg diet) for reduction of the toxicity of cyclopiazonic acid (45 mg/kg diet) in broiler chicks. The treatment did not effectively diminish cyclopiazonic acid toxicity. Parlat et al. (1999) observed a significant improve- ment in the adverse effect of AF (2 mg/kg food) on performance (FC, BWG and FCR) of growing Japanese quail by dietary addition of CLI (50 g/ kg).
Most research has used greater concentrations of AF than naturally occur in field conditions. The AF concentrations in these experiments ranged from 2 to 5 mg/kg diet (Huff et al., 1986; 1988; Kubena et al., 1990,1993,1998; Scheideler, 1993; Kececi et al., 1998; Kiran et al., 1998) because these high concentrations may help to elicit the toxic effects of AF and also any effects of adsorbents would be seen in a shorter experimental period. Different adsorbent concentrations ranging from 0 to 50 g/kg were used in the studies above. These studies determined the adverse effect of AF on food consumption (FC), body weight gain (BWG) and food conversion (FCR). When broiler chicks were fed on a diet containing 2·5 mg AF/kg diet from 1 to 21 d of age, FC and BWG were significantly reduced by 17% to 24% in the chicks consuming the AF-containing diet (Huff et al., 1986; OgÆuz, 1997). BWG was also reduced by 10% to 19% in the birds receiving 2·5 mg/kg of AF (Huff et al., 1986,1988; Kubena et al., 1993; Scheideler, 1993; OgÆ uz, 1997). AF also increased FCR by 3% to 10% in these studies.
The purpose of the following research using growing broiler chicks was: to evaluate the toxic effects of AF on growth performance of broiler chicks; to determine the preventive efficacy of different dietary concentrations of CLI (15 and 25 g/kg); and to compare the efficacy of these 2 different dietary concentrations.
MATERIALS AND METHODS Three hundred and sixty 1-d-old unvaccinated broiler chicks (Avian) of both sexes were obtained from a commercial hatchery. Individually weighed chicks were divided at random into 6 groups. There were 6 replicates of 10 broiler chicks for each dietary treatment, totalling 360 chicks. The birds were housed in electrically heated batteries under fluorescent lighting. The chicks were fed on a commercial diet (a maize and soyabean meal providing 230 g protein and 13·26 MJ ME/kg) formulated to contain National Research Council (1994) requirements of all nutrients, without added antibiotics, coccidiostats or growth promoters. Food and water were always available and lighting was continuous. Composition of the basal diet is shown in Table 1. The basal diet was tested for possible residual AF before feeding (Howel and Taylor, 1981) and was found to be negative (detection limit 1 µg/kg
1Batches of diet were prepared weekly and stored in a dry and cool place. 2Each 2´5 kg of vitamin premix contained: 3600 mg Vit A; 50´0 mg Vit D3 ; 3000 mg Vit K3 ; 3000 mg Vit B1 ; 6000 mg Vit B2 ; 5000 mg Vit B6 ; 15´0 mg Vit B12 ; 25 000 mg niacin; 4000 mg biotin; 8000 mg carotenoid 1000 mg folic acid; 30 000 mg choline chloride; 50 000 mg Vit C. 3Each 1 kg of mineral premix contained: 8000 mg Mn; 35 000 mg Fe; 50 000 mg Zn; 5000 mg Cu; 2000 mg I; 440´0 mg Co; 150´0 mg Se. diet). The chicks were weighed weekly to determine body weight gain. Food consumption was also determined weekly. Mortality was recorded as it occurred. The trial period was 3 weeks.
The experimental design consisted of 6 dietary treatments. 1) CONT: basal diet; 2) AF, basal diet plus 2·5 mg AF (Total aflatoxin; the composition given below)/kg diet; 3) CLI (15 g/kg), basal diet plus 15 g clinoptilolite/kg diet; 4) AF+CLI (15 g/ kg), basal diet plus 2·5 mg AF plus 15 g clinoptilolite/kg diet; 5) CLI (25 g/kg), basal diet plus 25 g clinoptilolite; 6) AF+CLI (25 g/kg), Basal diet plus 2·5 mg AF plus 25 g clinoptilolite/kg diet. Clinoptilolite (CLI/NUT-1000), which is a member of the heulandite-stilbite group, was provided by Incal Biotechnology and Mining Ltd., Izmir, Turkey; its chemical formula is KNa2Ca2 (Si29AL7 ) O72.32H2O.
The AF was produced by the method of Shot- well et al. (1966) with minor modifications by Oguz (1997). Briefly, 100 g of sterile polished rice was inoculated with 1 ml of resuspended spores (1·5×106 spores/ml) of Aspergillus parasiticus NRRL 2999 (USDA, Agricultural Research Service, Peoria IL), placed an incubator at 28°C and fermented for 5 d. Successfully fermented rice was then steamed to kill the fungus, dried and ground to a fine powder. The rice powder was then analysed for AF content by the method of Shotwell et al. (1966) and measured following TLC (Thin Layer Chromatography) using a densitometer (Perkin Elmer MPF 43-A) at 365 nm excitation and 425 nm emission wavelengths (TLC Plates from Merck; other equipment from Desega; the AF standards from Makor Chemical Ltd. Jerusalem, Israel). The total amount of aflatoxin (AFB1 , B2 , G1 and G2 ) in the fermented rice was measured. The AF within the rice powder consisted of 76·40% AFB1 , 16·12% AFB2 , 6·01% AFG1 and 1·47% AFG2 based on total AF in the rice powder (detection limit: 1 µg AF/kg rice powder: recovery of the extraction method: 92%). The rice powder was incorporated into the basal diet to provide the desired amount of 2·5 mg AF/kg food.
When the chicks reached 21 d of age, the feeding trial was terminated. Data for food consumption, body weight gains and food conversion ratio were grouped and expressed as mean±pooled standard errors of means. The results obtained were analysed statistically using Duncan’s multiple range test (SPSS, 1988). Differences were considered to be significant at the 5% level of probability
RESULTS
The results presented in Table 2, 3 and 4 show the effect of dietary treatments on food consumption (FC), body weight gain (BWG) and food conversion ratio (FCR) respectively. Feeding AF alone caused significant decreases in BWG from week 1 onwards compared with the controls. The decreasing effect of AF on FC was not statistically significant but it was about 8% to 10% during the experiment. The deleterious effect of AF on FCR was 8·3%. The addition of CLI (15 g/kg diet) to an AF containing diet significantly reduced the adverse effect of AF on BWG and FC. However, the ameliorating effectof CLI (25 g/kg) on BWG was not as great as CLI (15 g/kg) except for week 1. The FCR values were intermediate between those of the AF and CONT groups. The overall BWG was reduced by 10·9% among the broiler chicks consuming the AF diet without CLI but by only 3·7% for the birds fed AF plus CLI (25 g/kg) diet. There was no difference between control and CLI (15 g/kg)-alone group on FC, BWG and FCR. However, the addition of CLI (25 g/kg) to the AF-free diet significantly decreased overall FC compared with the controls. In the CLI (25 g/kg)-alone group FCR was positively different from CLI (15 g/kg)-alone group. No significant difference was found in the mortality values of the groups (Table 4).
DISCUSSION
AF are important to the poultry industry because of their frequent occurrence in foodstuffs which produces severe economic losses and health problems in the poultry industry. The most prevalent symptom of chronic aflatoxicosis in poultry and livestock is reduced growth rate and poor performance (Abo- Norag et al., 1995). Diagnosis is rather difficult and medical treatment may be almost impossible. Chronic, subclinical and acute aflatoxicosis can affect profit in the poultry industry (Sanli, Y., 1995; Bailey et al., 1998). In this study, experimental aflatoxicosis was induced in 1-d-old broiler chicks by feeding 2·5 mg total AF/kg diet for 3 weeks.
Producers and scientists aim to develop an effective preventive method dealing with this food- borne toxin. The use of agents which act as antidotes or antagonise the effects of toxic substances such as AF have therapeutic and economic importance. The major advantages of these adsorb- ents include expense, safety and easy administration through addition to animal foods (Ledoux et al., 1999). In the present study, the performance of chicks were not negatively affected by the addition of CLI (both 15 and 25 g/kg) alone to the AF-free diet, except for FC values in the CLI (25 g/kg) alone group. These data show that the CLI used in this study was inert and non-toxic, as indicated by Harvey et al. (1993), Olver (1997), Balevi et al. (1998) and Parlat et al. (1999).
BWG was significantly reduced by AF treat- ment compared with the control. The growth inhibiting effect of AF was seen from week 1 onwards. The effect of AF on FC was not statisti- cally significant. These findings agree with the results of Huff et al. (1986, 1988), Kubena et al. (1993,1998 ), Scheideler (1993) and OgÆuz (1997) who reported that FC and BWG decreased significantly (10% to 20%) in broiler chicks given 2·5 mg AF/kg diet for 3 weeks (P<0·05). 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 mechanisms may also have affected growth and general health (Kececi et al., 1998; Kiran et al., 1998).
The most important effect of aflatoxicosis is poor body weight gain since this directly affects profit in the poultry industry. In the present study, CLI (15 g/kg) addition to the AF-containing diet significantly reduced the adverse effect of AF on FC and BWG in broiler chickens (P<0·05). These improvements agree with previous results on the protective effects of CLI (50 g/kg) against an AF-containing (2 mg/kg) diet in growing Japanese quail (Parlat et al., 1999) and those showing that adding natural zeolites such as phillosilicate (Kubena et al., 1990,1993,1998; Huff et al., 1992; Abo-Norag et al., 1995; Ledoux et al., 1999 ), mordenite (Harvey et al., 1993), perlite and zeobrite (Scheideler, 1993) significantly diminished the toxicity of AF to broiler chicks (P<0·05). Harvey et al. (1993) found no beneficial effect on broiler chicks of adding CLI (5 g/kg) to an AF-containing diet (3·5 mg/kg) for 3 weeks. The reason for these conflicting results may be attributable to differences in type or physical characteristics of CLI, concentration of AF in the diet or broiler strains. The present results agree with those of of Parlat et al. (1999) who reported a significant amelioration in the adverse effects of AF on FC, BWG and FCR by CLI in the Japanese quail. The CLI used by Parlat et al. (1999) had the same chemical formula as in the present work. Addition of CLI (25 g/kg) to the AF-containing diet also provided a slight improve- ment on BWG; the BWG values were numerically intermediate between AF and CONT groups.
In the present study, the test clay had no Table 4. Effect of clinoptilolite (CLI) on food conversion ratio and mortality values for broiler chicks fed on diet containing 2´5 mg total aflatoxin (AF) kg diet at 1 to 21 days of age AF CLI addition Food conversion ratio (g food/g gain) Change from control (%) Mortality 15 g/kg 25 g/kg 1 to 7 d 8 to 14 d 15 to 21 d 1 to 21 d (dead/total) ± ± ± 1´64±0´067 1´94±0´081 1´86±0´220ab 1´81±0´102ab 0 4/60 + ± ± 1´89±0´130 2´03±0´074 1´82±0´120ab 1´96±0´110a +8´28 2/60 ± + ± 1´63±0´060 1´97±0´120 2´26±0´193a 2´04±0´086a +12´70 3/60 + + ± 1´75±0´054 1´89±0´098 1´94±0´200a 1´88±0´080ab +3´86 6/60 ± ± + 1´88±0´096 1´87±0´081 1´38±0´068b 1´67±0´049b ±7´74 1/60 + ± + 1´83±0´110 2´13±0´133 1´85±0´112ab 1´94±0´064a +7´18 6/60 a ±bValues within columns with no common superscripts are signi®cantly different (P<0´05), according to multiple range test. Values represent the mean±SEM of 6 groups of 60 broiler chicks (6 replicates of 10 broilers in each) per treatment. CLINOPTOLITE AND AFLATOXICOSIS 515 detrimental effect on broilers at a concentration of 15 g/kg in the basal diet in accordance with the studies cited above. However, FC values were altered by CLI (25 g/kg) addition to the AF-free diet from week 1 until the end of the experimental period. Such time-dependent increases and decreases have been seen in some previous work (Scheideler, 1993; Jindal et al., 1994; Oguz, 1997; Parlat et al., 1999). The reason for this unpredicted result is unknown but it may have resulted from a possible adsorbent- nutrient interaction as suggested by some previous workers. Further studies should be conducted, paying particular attention to the potential for nutrient interactions.
The results of the present study clearly demonstrate that: The growth performance (FC, BWG and FCR) of broiler chicks was significantly affected and decreased by giving an AF (2·5 mg/kg)- containing diet for 3 weeks; that CLI (15 g/kg) addition to the AF-containing (2·5 mg/kg) diet significantly ameliorated the adverse effects of AF on performance. Further the protective effect of 15 g/kg CLI used in this study against the toxic effects of AF was greater than that of 25 g/kg CLI, and the lower concentration of CLI (15 g/kg diet) was inert and non-toxic for broiler chickens. These improvements should contribute to a solution of AF problem in broiler chickens when adsorbents are used in conjunction with other AF prevention strate- gies.
ACKNOWLEDGMENTS
The authors gratefully acknowledge The Scientific and Technical Research Council of Turkey (TU¨ BI`TAK), whose Veterinary Medicine and Animal Husbandry Research Grand Committee funded this project. Project no: VHAG-1437.
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