Introduction

According to official data from global institutions such as the FAO, at least 99 countries in the world, which contain approximately 87% of the world population, have some kind of regulation for the content of mycotoxins in food for humans and/or animals. Worldwide interest in these problems is reflected in the vast number of scientific and professional papers published on mycotoxins since the discovery of aflatoxin B1 in the early 1960's. Given the ubiquitous nature of moulds, as well as the general impossibility to prevent mycotoxin contamination in foods feed materials, this topic is an integral part of modern livestock production. The increase in irregularity of meteorological conditions associated with global climate change has increased the problems of fungal contamination at harvest and the production of toxins. Although it is almost impossible to examine and measure the true impact of moulds and mycotoxins, the risks associated with the presence of mycotoxins is well researched and proven.

Mycotoxins are toxic secondary metabolites of a large number of saprophytic organisms (moulds), which, in animals and humans, proliferate in contaminated food infested with spores, conidia and/or fragments of mycelium. Fungal toxins entering the body of animals and humans via the gut cause intoxication, generally called mycotoxicosis. Damage to livestock due mycotoxicosis may be significant, and are manifested in the form of direct losses due to mortality animals or, more commonly, caused indirectly due to falling production and reproductive performance (Ožegović, Reference OŽEGOVIĆ1983). A specific problem is the mycotoxin residues found in different quantities in edible organs and animal products, which enter the foodchain and may cause adverse effects in humans.

In chemical terms, mycotoxins represent a wide range of complex compounds with different functional groups and therefore different properties. Mycotoxins do not have sufficiently large molecules, and human and animal organisms do not produce antibodies against them and so remain permanently unprotected from their action, and in addition there is no simple method for their identification. Common mycotoxins in foods include aflatoxins, ochratoxin, fusarium toxins (zearalenone, trichothecenes), ergot alkaloids, sterigmatocistin, patulin, penicillanic acid, and rubratoksins and sporodezmins.

Semmens (Reference SEMMENS1983) state that zeolites, as mineral adsorbents, in accordance of their structure, are part of the alumina silicates' group (SiO₄ and AlO₄), where the tetrahedral structure (TO₄) is the basic unit. Tetrahedrons may contain various bonds, giving the zeolite structure many channels and cavities. The channel system may be one-dimensional (unconnected parallel channels), two-dimensional (channels connected in one plane) and three-dimensional. The diffusion rate of adsorption and ion exchange are functions of the spatial channels distribution. Every mineral is characterised by the channel opening size, so zeolites selectively retain or lose molecules depending on their dimensions.

The negative charge of the three-dimensional network, which is conditioned with tetrahedral coordination, is characteristic of all the zeolites, with respective iso-structural substitution of Si⁴⁺ ions with Al³⁺ ions. The surplus negative charge is compensated for by univalent and/or divalent cations which are located in the channels and cavities. The presented ions do not enter the zeolite tetrahedron network and, with the ion exchange reaction, they can be replaced by cations of other metals. The cation exchange capacity of zeolites is a function of silicon substitution with aluminium in the tetrahedron network. More alkali cations are required to compensate for the negative charge of elementary cells if the degree of substitution is higher. The cation exchange capacity for natural zeolite is dependent on the dimensions of the channels, ionic shape and size, the charge density and ionic valency from the electrolyte composition and is in the range of 200-300 mEq/100g in zeolites (Dixon and Weed, Reference DIXON and WEED1989).

Boyer (Reference BOYER2000) states that clinoptilolite supplementation in animal feed leds to a significant increase in feed efficiency as well as reducing the potential of intestinal diseases (diarrhoea), and the toxic effects of surplus ammonium ions. Some researchers explain this as the clinoptilolite preference for large ions, such as NH₄⁺, so it serves as a reservoir for ammonia. Some natural zeolites adsorb mycotoxins from feedstuffs and offer some protection from their toxic affects during digestion.

The process of mixing is an important phase in the production of premixes and feeds, in order to attain uniformity in the basic matrix (carrier) distribution of the components so that each consumed amount of feed contains the required amount of nutrients (Filipović et al., Reference FILIPOVIĆ, FILIPOVIĆ and IVIĆ1993), including toxin binders. Spoilage of animal feed means deviation from the normal quality in terms of changes of organoleptic characteristics, nutritional value and/or hygiene of foodstuffs (Šefer et al., Reference ŠEFER, NEDELJKOVIĆ-TRAILOVIĆ, JOVANOVIĆ and SINOVEC Z.1997). Feed contamination with fungi causes poorer productive performance, increased metabolic disorders and diseases, and can eventually cause death. As a result, the presence of moulds in animal feed is an important criterion in evaluation of the quality of an animal feed.

In Serbian conditions, feed samples commonly contain zearalenone, ochratoxin A, trichothecenes (T-2, DAS) and aflatoxins. Research by Bočarov-Stančić et al. (Reference BOČAROV-STANČIĆ, MILOVAC and GOLOŠIN2000) and Jajić et al. (Reference JAJIĆ, JURIĆ, GLAMOČIĆ and KRSTOVIĆ2010) showed that in years with good climatic conditions, between 20 and 25% of cereals were contaminated with mould and mycotoxins. Rajić and Ožegović (Reference RAJIĆ and OŽEGOVIĆ1990) suggested a set of measures for the prevention and control of moulds in corn, namely: the selection of corn resistant to mould, seeding early varieties, prevention of grain breakage, artificial grain drying, hygienic cleaning of the grain hoppers, grain aeration, tighter control measures for moisture, and treating grain with propionic acid or a mixture of organic acids.

In intensive poultry production adsorbents most often used to neutralise the harmful effects of mycotoxins achieve this by bonding. Adsorption therapy is one of the most important methods in preventing mycotoxicosis. Some of the adsorbents used include activated charcoal, hydrated sodium-aluminosilicates calcium (HSCAS), bentonite, clay soil and various aluminosilicates or zeolites (Adamović et al., Reference ADAMOVIĆ, TOMAŠEVIĆ-ČANOVIĆ, MILOŠEVIĆ, DAKOVIĆ and LEMIĆ2003; Resanović et al., Reference RESANOVIĆ, SINOVEC and SINOVEC2004). Zeolites are classified as safe or GRAS (Generally Regarded As Safe) compounds as per EU 21 CFR Part 582.2729.

Supplementing zeolites at a rate of 0.5% in poultry feed under field conditions has achieved good results in the prevention of mycotoxins. Further research on the different categories of poultry showed that zeolites can be used in amounts of 0.2% of the feed to effectively prevent the harmful effects of mycotoxicosis (Radović et al., Reference RADOVIĆ, RAJIĆ and RADOVANOVIĆ2000), which confirmed the previous results obtained in vitro. Adding 0.5% of the commercial zeolite ‘Min-a-Zel’(MZ) in feed for laying hens contaminated with T-2 toxin (2 mg/kg in corn and 1.2 mg/kg in sunflower pellet) had a positive impact on increasing laying capacity and mitigated the negative effects of T-2 mycotoxicosis. The research using MZ in hens diets, supplemented at levels of 0, 0.2, 0.3 and 0.5%, confirmed that the best performance was with 0.5% MZ, giving increased laying, higher egg weight, better feed intakes, less feed consumption per egg, and improved FCR (Radović, Reference RADOVIĆ1997; Radović et al., Reference RADOVIĆ, RAJIĆ and RADOVANOVIĆ2000). The use of MZ at a level of 0.2% in broilers diets had a positive impact on reducing mortality, achieving higher body weights and improving FCR (Palić et al., Reference PALIĆ, VUKIĆEVIĆ and RAJIĆ1990; Reference PALIĆ, PETROVIĆ, STANOJLOVIĆ, JOVANOVIĆ and Dj.1991; Radović et al., Reference RADOVIĆ, RAJIĆ and RADOVANOVIĆ1999; Reference RADOVIĆ, RAJIĆ, STANIĆ and NADAŽDIN2003; Reference RADOVIĆ, RAJIĆ and BOGOSAVLJEVIĆ-BOŠKOVIĆ2004). Bočarov-Stančić et al. (Reference BOČAROV-STANČIĆ, MILOVAC and GOLOŠIN2000) added zeolites into meal feeds for broilers and found enhanced growth of 1.7-2.6%, and better FCR by 5.9-6.4% in the supplemented compared to the control group. Zeolite (clinoptilolite) supplemented at levels of 1.2% and 4% in feed had a positive effect on weight gain, final body weight and FCR in broilers, with the best performances seen in those receiving the 2% zeolite feed. Adding MZ at a level of 0.5% had a positive impact on body weight and growth of broilers and prevented the deposition of aflatoxin B1 in organs (Sinovec and Resanovic, 2005; Resanović and Sinovec, Reference RESANOVIĆ and SINOVEC2006).

The effects of different types and levels of mineral adsorbents (MZ and ‘Min-a-Zel Plus’ (MZ+)) on production and slaughter characteristics of broilers at 42 days were studied by Karović (Reference KAROVIĆ2009), Karović et al. (Reference KAROVIĆ, RADOVIĆ, OKANOVIĆ, Đ., DŽINIĆ, IKONIĆ, TASIĆ and GUBIĆ2010) and Radovic et al. (2010a; 2010c). During the fattening stage, different zeolites at different levels were used and compared to a negative control; MZ at 0.5%; MZ+ at 0.2% and MZ+ at 0.3% inclusion. The authors concluded that the maximum weight gain was seen at 0.5% inclusion, and the percentage of inedible products was highest in the 0.2% group. Radović et al. (Reference RADOVIĆ, FILIPOVIĆ, OKANOVIĆ, Đ., KAROVIĆ, KORMANJOŠ and Š.2010a) examined the impact of different levels of mineral adsorbents (MZ and MZ’) added to broiler feed on production. A trial was conducted with 400 broilers from one day old, of Cobb 500 breed, divided into four experimental groups. The control group (K) and experimental groups (I, II and III) were fed a complete feed of the same composition, with different levels of added mineral adsorbent (0.5, 0.2 and 0.3% respectively). The authors showed that 0.5% supplementation gave the best feed conversion (1.716 g/g), followed by 0.3% (1.891 g/g) and 0.2% (1.978 g/g), with the poorest seen for the control group (2.161 g/g). In addition, the highest average body weights, average daily gains, and best FCR was observed for the 0.5% group. The authors concluded that broilers fed complete feeds with 0.5% clinoptilolite gave better results, and that the differences between the mid values were statistically significant (P<0.05).

Similar studies were conducted by Radović et al. (Reference RADOVIĆ, KAROVIĆ, OKANOVIĆ, Đ., FILIPOVIĆ, KORMANJOŠ and Š.2009; Reference RADOVIĆ, FILIPOVIĆ, OKANOVIĆ, Đ., RISTIĆ, KORMANJOŠ, Š. and KAROVIĆ2010b) who explored the influence of the addition of the mineral adsorbents MZ and MZ+ to feed on the yield of broiler chickens separated by gender. The research was conducted on 400 Cobb 500 broilers, divided into four treatment groups, until they were 42 days of age. Dietary treatments included a control (0%), 0.5% MZ, 0.2% MZ+ and 0.3% MZ+. The highest mortality was seen in the control group, and the lowest in the experimental group with the addition of 0.3% ‘MZ+. The highest average body weight of chickens were in the 0.5% group (♂ 2179.8 g, ♀ 2086.3 g). Weight gain of chickens fed 0.3% MZ+ ranged between 1976.5 g (♂) and 1899.3 g (♀). The average weight of chickens fed 0.2% MZ+ was 1874.8 g (♂) and 1836.0 g (♀). The chickens in the control group had the lowest weights (♂ 1754.5 g, ♀ 1649.0). Best total weight of chilled carcasses was found in the 0.5% supplemented group of chickens (♂ 1570.0 g, ♀ 1510.0 g), followed by 0.3% (♂ 1428.8 g, ♀ 1371.3 g), 0.2% (♂ - 1358.8 g, ♀ - 1303.8 g) and control (♂ - 1265.0 g, ♀ - 1176.3). The lowest yield (71%) was found in female broilers in the 0.2% group, while the highest value of 72.5% was in the same treatment of male broilers.

Palić et al. (Reference PALIĆ, VUKIĆEVIĆ and RAJIĆ1990) conducted a trial using one-day old chickens, divided into three groups of 50 animals. One experimental group received T-2 toxin at a level of 1 mg/kg body weight directly into the crop, and the other groups received the same treatment, but with the addition of 0.2% MZ into the feed. The group that received the MZ achieved the highest average body weight (899 g) compared to the chicks that did not receive the mycotoxin adsorbent (805 g). In a further experiment with broilers, (Palić et al., Reference PALIĆ, PETROVIĆ, STANOJLOVIĆ, JOVANOVIĆ and Dj.1991) zeolite supplement in doses of 0, 0.2, 0.5, and 1.0% in feed contaminated with 5.0 and 5.4 mg/kg zearalenone. It was determined that 0.2% MZ gave the best results, showing the lowest mortality, highest weight and lowest average feed consumption per kg gain.

In a study with 100 broilers divided into four groups of 25 animals, a complete diet either without zeolite (control) or with the addition of 0.2%, 0.5% and 1% zeolite, Rajić et al. (Reference RAJIĆ, KONJEVIĆ and VLAHOVIĆ1992) found that the average weight of the experimental groups, compared to the control, on the 35th and 40th day of age were significantly higher (P<0.001). FCR, relative to the control group, was 3.4% lower for the 0.2% group, 3.66% for the 0.5% group and 3.70% for the 0.1% group. Based on these results, the authors concluded that the addition of zeolite to feed for chickens had a positive impact on body weight and feed conversion.

In a dose response trial for zeolite (0, 2.5 and 5%) Rajić et al. (Reference RAJIĆ, KONJEVIĆ and VLAHOVIĆ1992) found that chickens fed 2.5% supplemented feed had 9% higher body weights and 4-8% improved feed conversion. Kelemen and Bogaromy (Reference KELEMEN and BOGAROMY1983) fed broiler chickens with a feed containing 3% and 10% zeolite. The 3% diet had less influence on body weight than that supplemented with 10% in the final (grower) stage of feeding. Average chicken body weight on day 49 was 3.5% to 4% less in the control chickens group. In addition, Duff et al. (Reference DUFF, BURNS and DWIEVEDI1987) found that mycotoxins that cause inhibition of growth can also cause a reduction in bone strength. It is considered that the presence of even minimal amounts of OTA (> 0.5 ppm) can cause a negative effect on the broiler performance. These negative effects are already evident at the end of the first week of age, when fed contaminated feed, and chicks then lag behind in growth, which is dose dependent, but which is more pronounced in males. Exposure to contaminated feed without supplementation with a mineral adsorbent for longer periods causes a significant decrease in body weight (~ 12%) and increased feed conversion ratio. The results for average daily gains in broilers show that mycotoxins cause significantly negative effects on this parameter. Ayed et al. (Reference AYED, DAFALLA, YAGI and SEI1991) found significantly lower daily weight gains in broilers fed contaminated feeds without adsorbant supplementation.

Valdivia et al. (1993) found that the addition of 5% zeolite stimulated the growth of broilers fattened to 49 days. Similarly, Talanov et al. (Reference TALANOV, CHUPALKHINA, BRICHKO, USTENKO and SKVORTESOV1994) noted that 3% of zeolite or 3% zeolite plus 0.04% methionine, significantly (P<0.05) affected daily gain compared to the control group. Similar studies with 5% zeolite supplementation in broiler feed was carried out by Karelin (1985) who found that the average chicken gains were 1593 g for the supplemented group versus 1528 g for the control group. The percent of live chickens was 91.1 and 96.5 respectively. Sukan et al. (Reference SUKAN, ERKEK and ONGEN-BAYASAL1994) added 1% of zeolite to broiler feed which was not contaminated with mycotoxins. Results showed that body weight of chicks and daily gain were increased with zeolite, but also caused higher (poorer) feed conversion. Similar tests were conducted by Dobeic and Amon (Reference DOBEIC and AMON1994), where clinoptilolite was added in quantities of 1, 2, 4 and 6% to feed. Clinoptilolite at 1, 2 and 4% resulted in increasing gain, higher final body weights and better feed conversion. Clinoptilolite increased the number of erythrocytes in the blood, while the 6% inclusion level led to decreased moisture in the faeces. Furthermore, the addition of 1% zeolite in feed reduced aflatoxin B1 residues by 72%, although 2% zeolite gave the same levels of residues as the control group (no aflatoxins), with gain improvements of 1.7-2.6%, and better feed utilisation of 5.9-6.4%. The use of zeolites (clinoptilolite) in various amounts (1, 2, 4 and 6%) showed that feeding 1, 2 and 4% had a positive effect on body weight gain and final weight of broiler chickens. The highest body weight and feed conversion efficiency were in the group of broilers fed 2% zeolite in the ration.

Závodský et al. (1985) fed males and females with 1% and 2% of zeolite in feed. On the 50th day of feeding, the body weight was 1803 g and 1819 g (with 1% or 2% of zeolite respectively). The body weight of chickens fed the negative control was 1873 g. Feed conversion in which the zeolite was added was 2.36 and 2.184, and without zeolite it was 2.00. Lon-Wo et al. (Reference LON-WO, PEREZ and GONZALES1987) fattened chickens with feed containing 5% of zeolite for a period of eight weeks. They determined that the fattening production results at the beginning of the trial in both groups of chickens were very similar, but at the end of trial period, feed efficiency was better and mortality lower for the broilers which received zeolite (P<0.05). Addition of 5% zeolite in the finishing feed for chickens gave significant improvements in feed conversion (P<0.05). In a second experiment, 20 geese aged 7-21 days old were fed a ration contaminated with aflatoxin B1, B2 and G1 concentrate, with and without zeolite. Zeolite addition improved conversion and carcass yield.

Application of zeolite in feed to prevent poultry mycotoxicosis has been shown to be effective (Sinovec et al., Reference SINOVEC, RESANOVIĆ, NEDELJKOVIĆ-TRAILOVIĆ, ŠEFER and SINOVEC2002; Radović and Bogosavljević-Bosković, 2006) both in experimental (Resanović et al., Reference RESANOVIĆ, SINOVEC and SINOVEC2004), and practical conditions (Palić et al., Reference PALIĆ, PETROVIĆ, STANOJLOVIĆ, JOVANOVIĆ and Dj.1991). Adding zeolite in feed contaminated with different mycotoxins reduces patho-morphological alterations in target organs (Nedeljković-Trailović et al., Reference NEDELJKOVIĆ-TRAILOVIĆ, ŠEFER, SINOVEC, ZUROVAC-KUZMAN and SINOVEC2001; Reference NEDELJKOVIĆ-TRAILOVIĆ, JOVANOVIĆ and SINOVEC2004). In addition, significant reductions in the presence of residues in edible tissues of broilers have been noted (Sinovec et al., Reference SINOVEC, RESANOVIĆ, NEDELJKOVIĆ-TRAILOVIĆ, ŠEFER and SINOVEC2002), but it should be stated that the efficiency of zeolite depends on the application and form in which it is used (Radović et al., Reference RADOVIĆ, RAJIĆ and RADOVANOVIĆ2000; Resanović et al., Reference RESANOVIĆ, SINOVEC and SINOVEC2004).

It is assumed that different levels of mineral adsorbents added to the feed of broiler chickens have different and unequal effects on consumption and feed conversion, body weight gain, final body weights, carcass traits and meat quality as well as the health status of chickens in accordance with duration and economic performance. Tica et al. (Reference TICA, OKANOVIĆ, Đ., ZEKIĆ, KAROVIĆ and MILIĆ2010) also demonstrated the impact of zeolites on the economic performance of growing broiler chickens exposed to mycotoxins. The use of mineral adsorbents in feeds and premixes can provide a solution to mycotoxicosis, showing better productive performance and economic returns to growers. However, in Serbia, the full benefits of the use of mineral absorbents can only be seen with the readiness of customers to purchase poultry meat products according to their quality.