Effect of the Feed Additive Clinoptilolite ( ZeoFeed ) on Nutrient Metabolism and Production Performance of Laying Hens

The aim of this study was to assess the effects of two levels of clinoptilolite administered in feed (2% and 4%) on some selected performance indicators, metabolic utilization of basic nutrients and the health status of laying hens. The selected 24 Bovans Goldline hybrid laying hens were divided into three equal groups, two experimental groups (E1 and E2) and one control group (C). The laying hens were housed individually in cages with an automatic supply of drinking water, manual feeding, in a setting with controlled light and temperature regimens. Hens from individual groups were all fed a complete feed mix of the same composition and the only difference was in clinoptilolite supplementation: feed mixes for E1 and E2 groups contained 2% and 4% of clinoptilolite (commercial additive ZeoFeed) respectively, replacing the same amounts of wheat. The hens received feed mixes and drinking water ad libitum. During this 28-day experiment, feed consumption and the number and weight of eggs laid were monitored individually for each hen. At the end of the experiment, the balance test using the indicator method (Cr2O3) was performed in all eight hens in each of the groups. The results of balance tests were then used to calculate the metabolic utilization of selected nutrients (nitrogen, fat, ash, nitrogen-free extracts, starch, gross energy, Ca, P). After the balance tests, blood samples for haematological and biochemical examinations were collected via puncture of the vena basilica. The addition of 2% clinoptilolite to feed mix resulted in a highly significant (P ≤ 0.01) increase in mean egg weight to 64.69 g, but the addition of 4% clinoptilolite in group E2 resulted in a highly significant (P ≤ 0.01) decrease in mean egg weight to 62.20 g compared to the control (63.73 g). Moreover, daily feed mix consumption in group E1 decreased to 114 g per one laying hen/day compared to the controls (118 g per one laying hen/day). In group E2 (4% clinoptilolite), daily consumption of feed mix increased compared with the controls to 124 g. The 2% clinoptilolite supplementation of E1 group feed slightly increased metabolic utilization of fat, nitrogen-free extracts, starch and gross energy compared to group C. Results of haematological test of the hens’ blood showed significant changes in haemoglobin, whose values in groups C and E1 were significantly higher (P ≤ 0.05) than in group E2. Differences in the values of the biochemical indicators monitored (total protein, glucose, cholesterol, triacylglycerols, calcium, phosphorus, magnesium and the AST enzyme) between group C and experimental groups E1 and E2 were not significant and remained within the range of reference values. ZeoFeed, nutrient balance, biochemical profile, mineral profile, haematological profile Feeds may be carriers of a number of contaminating exogenous substances that spread to food via food-producing animals and their products. For that reason, suitable approaches are being sought to prevent such substances from penetrating the animal organism and, subsequently, the food chain. There is a group of sorbents among feed additives that might be used to help resolve these problems; they are clinoptilolite-based natural zeolites. The crystal lattice of clinoptilolite offers a large internal sorption surface area. The size of its pores allows for selective ion exchange (Boranić 2000; Melenova et al. 2003), which is limited by the pore size. In terms of chemical composition, clinoptilolite is a hydrated aluminosilicate of alkaline metals and alkaline earth metals. ACTA VET. BRNO 2010, 79: S29–S34; doi:10.2754/avb201079S9S029 Address for correspondence: MVDr. Miroslav Macháček Department of Nutrition, Animal Husbandry and Animal Hygiene Faculty of Veterinary Hygiene and Ecology University of Veterinary and Pharmaceutical Sciences Brno Palackého 1-3, 612 42 Brno, Czech Republic Phone: +420 541 562 683 Fax: +420 541 562 675 E-mail: machacekm@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm In animal nutrition, clinoptilolites are used mainly to protect animals against mycotoxin intoxications (Skalicka and Makoova 1999; Skalicka et al. 2000; Rizzi et al. 2003; Modirsanei et al. 2004; Desheng et al. 2005), to eliminate heavy metals (Boranić 2000) and radionuclides (Poschl and Balas 1999; Vitorovic et al. 2002; Branislava and Gordana 2004), as well as to prevent digestive tract diseases, to improve utilization of feed nutrients, and to improve hygienic and sanitary conditions in animal housing facilities (Meisinger et al. 2001; Melenova et al. 2003). They have a positive effect on the production performance in pigs (Veldman and Aar 1997; Papaioannou et al. 2004) and in poultry (Amon et al. 1997; Olver 1997). Vogt (1991) found no significant improvement in production performance of laying hens associated with clinoptilolite supplementation. There was a slight improvement in the egg shell strength, reduction of the egg white height and improvement in yolk colour. Elliot and Edwards (1991) reported a positive effect of dietary natural clinoptilolite on feed utilization of broiler chickens. In theory, sorbents may be assumed to have a negative effect on the digestibility and utilization of important nutrients such as minerals and vitamins. The majority of published papers, however, seem to testify to a positive effect of clinoptilolite-based sorbents on the mineral metabolism of animals, e.g. in pigs (Papaioannou et al. 2002) or in poultry (Elliot and Edwards 1991; Watkins and Southern 1991; Gezen et al. 2004; Desheng et al. 2005). The use of feed additives had no negative effect on biological efficiency of certain vitamins as demonstrated by Edwards et al. (1992) for vitamin D3 and by Gomonaj et al. (2000) for vitamin B1. Data on the effect of various clinoptilolite levels on the metabolic utilization of basic nutrients are missing in the literature. Therefore, our aim was to investigate the effect of two different clinoptilolite levels (2% and 4%) administered in the feed on the metabolic utilization of basic nutrients, laying hens’ production performance and their health status assessed on the basis of haematological and biochemical profiles of their blood. Materials and Methods Animals and procedures The experiments were conducted in accredited experimental barns of the Department of Nutrition, Animal Husbandry and Animal Hygiene at the Faculty of Veterinary Hygiene and Ecology of the University of Veterinary and Pharmaceutical Sciences in Brno and approved by the local Ethics Committee. The selected 24 Bovans Goldline hybrid laying hens were divided into three equal groups of 8 hens, i.e. two experimental groups (E1 and E2) and one control group (C). The laying hens were housed individually in cages with automatic watering, manual feeding, in a setting with controlled light and temperature regimens. The hens were all fed complete feed mixes with the same component composition and the only difference was their clinoptilolite supplementation, when feed mixes for E1 and E2 groups contained 2% and 4% of clinoptilolite (commercial additive ZeoFeed), respectively. The feed mixes and drinking water were provided ad libitum. The feed additive ZeoFeed contains at least 80% clinoptilolite as the active substance, and its moisture content is 6% maximum. Its other components include 62% SiO2, 14% Al2O3, 2.3% Fe2O3 and 5. % CaO. ZeoFeed grain size is within the range of 0.2 to 0.5 mm. During the 28-day experiment, feed consumption and the number and weight of laid eggs were monitored individually for each hen. At the end of the experimental period, individual balance tests using the Cr2O3 indicator method were performed twice for 5 days on each of the eight hens in all three groups. The results of balance tests were then used to calculate metabolic utilization of selected nutrients (nitrogen, fat, ash, nitrogen-free extracts, starch, gross energy, Ca, P). After the balance tests, blood samples for haematological and biochemical examinations of the laying hens in the study were collected via puncture of the vena basilica. Haematological indicators, i.e. erythrocytes, haematocrit values, haemoglobin and leukocytes, were calculated in whole blood. Haematological examinations were performed using classical methods (Doubek et al. 2003). Biochemical indicators, such as total protein, glucose, cholesterol, triacylglycerols, aspartate aminotransferase (AST), Ca, P, Mg, were determined in blood plasma after blood centrifugation. Biochemical indicators were determined using the biochemical analyzer Cobas EMira and commercial diagnostic kits, and mineral substances were determined using AAS. During the experiment, the health status of the laying hens was monitored daily. No clinical symptoms of disease or death were observed. The results obtained were processed by mathematical-statistical methods using the statistical programme Unistat 5.6, mean values and their differences were assessed by multiple comparisons using Tukey-HSD test at levels of significance P ≤ 0.01 and P ≤ 0.05. S30

Feeds may be carriers of a number of contaminating exogenous substances that spread to food via food-producing animals and their products.For that reason, suitable approaches are being sought to prevent such substances from penetrating the animal organism and, subsequently, the food chain.There is a group of sorbents among feed additives that might be used to help resolve these problems; they are clinoptilolite-based natural zeolites.The crystal lattice of clinoptilolite offers a large internal sorption surface area.The size of its pores allows for selective ion exchange (Boranić 2000;Melenova et al. 2003), which is limited by the pore size.In terms of chemical composition, clinoptilolite is a hydrated aluminosilicate of alkaline metals and alkaline earth metals.
In animal nutrition, clinoptilolites are used mainly to protect animals against mycotoxin intoxications (Skalicka and Makoova 1999;Skalicka et al. 2000;Rizzi et al. 2003;Modirsanei et al. 2004;Desheng et al. 2005), to eliminate heavy metals (Boranić 2000) and radionuclides (Poschl and Balas 1999;Vitorovic et al. 2002;Branislava and Gordana 2004), as well as to prevent digestive tract diseases, to improve utilization of feed nutrients, and to improve hygienic and sanitary conditions in animal housing facilities (Meisinger et al. 2001;Melenova et al. 2003).They have a positive effect on the production performance in pigs (Veldman and Aar 1997;Papaioannou et al. 2004) and in poultry (Amon et al. 1997;Olver 1997).Vogt (1991) found no significant improvement in production performance of laying hens associated with clinoptilolite supplementation.There was a slight improvement in the egg shell strength, reduction of the egg white height and improvement in yolk colour.Elliot and Edwards (1991) reported a positive effect of dietary natural clinoptilolite on feed utilization of broiler chickens.
In theory, sorbents may be assumed to have a negative effect on the digestibility and utilization of important nutrients such as minerals and vitamins.The majority of published papers, however, seem to testify to a positive effect of clinoptilolite-based sorbents on the mineral metabolism of animals, e.g. in pigs (Papaioannou et al. 2002) or in poultry (Elliot and Edwards 1991;Watkins and Southern 1991;Gezen et al. 2004;Desheng et al. 2005).The use of feed additives had no negative effect on biological efficiency of certain vitamins as demonstrated by Edwards et al. (1992) for vitamin D3 and by Gomonaj et al. (2000) for vitamin B1.
Data on the effect of various clinoptilolite levels on the metabolic utilization of basic nutrients are missing in the literature.Therefore, our aim was to investigate the effect of two different clinoptilolite levels (2% and 4%) administered in the feed on the metabolic utilization of basic nutrients, laying hens' production performance and their health status assessed on the basis of haematological and biochemical profiles of their blood.

Animals and procedures
The experiments were conducted in accredited experimental barns of the Department of Nutrition, Animal Husbandry and Animal Hygiene at the Faculty of Veterinary Hygiene and Ecology of the University of Veterinary and Pharmaceutical Sciences in Brno and approved by the local Ethics Committee.The selected 24 Bovans Goldline hybrid laying hens were divided into three equal groups of 8 hens, i.e. two experimental groups (E1 and E2) and one control group (C).The laying hens were housed individually in cages with automatic watering, manual feeding, in a setting with controlled light and temperature regimens.The hens were all fed complete feed mixes with the same component composition and the only difference was their clinoptilolite supplementation, when feed mixes for E1 and E2 groups contained 2% and 4% of clinoptilolite (commercial additive ZeoFeed), respectively.The feed mixes and drinking water were provided ad libitum.The feed additive ZeoFeed contains at least 80% clinoptilolite as the active substance, and its moisture content is 6% maximum.Its other components include 62% SiO 2 , 14% Al 2 O 3 , 2.3% Fe 2 O 3 and 5. % CaO.ZeoFeed grain size is within the range of 0.2 to 0.5 mm.
During the 28-day experiment, feed consumption and the number and weight of laid eggs were monitored individually for each hen.At the end of the experimental period, individual balance tests using the Cr 2 O 3 indicator method were performed twice for 5 days on each of the eight hens in all three groups.The results of balance tests were then used to calculate metabolic utilization of selected nutrients (nitrogen, fat, ash, nitrogen-free extracts, starch, gross energy, Ca, P).After the balance tests, blood samples for haematological and biochemical examinations of the laying hens in the study were collected via puncture of the vena basilica.Haematological indicators, i.e. erythrocytes, haematocrit values, haemoglobin and leukocytes, were calculated in whole blood.Haematological examinations were performed using classical methods (Doubek et al. 2003).Biochemical indicators, such as total protein, glucose, cholesterol, triacylglycerols, aspartate aminotransferase (AST), Ca, P, Mg, were determined in blood plasma after blood centrifugation.Biochemical indicators were determined using the biochemical analyzer Cobas EMira and commercial diagnostic kits, and mineral substances were determined using AAS.During the experiment, the health status of the laying hens was monitored daily.No clinical symptoms of disease or death were observed.
The results obtained were processed by mathematical-statistical methods using the statistical programme Unistat 5.6, mean values and their differences were assessed by multiple comparisons using Tukey-HSD test at levels of significance P ≤ 0.01 and P ≤ 0.05.

Production performance
The addition of 2% clinoptilolite to the feed mix resulted in a highly significant (P ≤ 0.01) increase in the mean egg weight to 64.69 g in group E1.The addition of 4% clinoptilolite in group E2, on the other hand, resulted in a highly significant (P ≤ 0.01) decrease in the mean egg weight to 62.20 g compared to the control (63.73 g).Moreover, daily feed mix consumption in group E1 decreased to 114 g per one laying hen/day compared to the controls (118 g per one laying hen/day).In group E2 (4% clinoptilolite), the daily consumption of feed mix increased compared to the controls to 124 g.The numbers of eggs laid were 197 (group C), 190 (group E1) and 177 (group E2).

Metabolic utilization of some selected nutrients
Compared to the control group C, the 2% clinoptilolite supplementation of E1 group feed slightly increased metabolic utilization of fat (92.15 vs. 93.63%),nitrogen-free extracts (89.03 vs. 90.68%),starch (97.01 vs. 97.82%)and gross energy (74.32 vs. 75.66%)(Table 1).More efficient utilization of the above nutrients helped to increase gross energy utilization, which means that clinoptilolite supplementation also helped to increase the feed metabolizable energy (ME).Higher metabolic utilization was also observed in nitrogen (33.69 vs. 35.87%)(Table 1).The higher (4%) clinoptilolite content had a negative effect on most of the above indicators with the exception of starch and gross energy.Metabolic utilization of mineral substances (Table 1) characterized by concentrations of ash, calcium (Ca) and phosphorus (P) was significantly reduced by the addition of 2% clinoptilolite (group E1).Interestingly, the addition of a higher amount (4%) of clinoptilolite, on the other hand, increased calcium and phosphorus utilization.
It follows from the results of the conducted balance tests that the optimum level of clinoptilolite supplementation in feed mixes for laying hens is up to 2% clinoptilolite.

Results of haematological examinations
Compared to the control, lower mean values were found in the examined blood indicators (Table 2) particularly in group E2.The differences between mean values of the control group and the experimental groups were non-significant with the exception of the haemoglobin content, whose mean value in group E2 (4% clinoptilolite) was significantly lower (P ≤ 0.05) compared to both control and group E1.The results indicate that higher in-feed clinoptilolite concentrations (4% in our case) may have a negative effect on haematopoiesis.

Results of biochemical examinations
No significant differences between biochemical indicators of blood plasma of laying hens from S31 individual groups were found (Table 3).The experimental groups (E1 and E2) were found to have lower concentrations of total protein, cholesterol and triacylglycerol, but higher concentrations of glucose.Based on our results we assume that clinoptilolite administered at 2% and 4% levels will not significantly influence blood plasma composition.

Discussion
Zeolites (clinoptilolite) are used in animal nutrition for a number of beneficial effects they provide including better utilization of feed nutrients, positive effects on intestinal microflora and the mechanism of digestion, protection of animals against harmful effects of mycotoxins, stimulation of liver detoxification processes, elimination of heavy metals and radioactive elements.
Because of their properties, in-feed zeolites participate in many biochemical processes including exchange, adsorption, catalysis and dehydration-rehydration (Mumpton 1999).It has been demonstrated in a number of studies that the inclusion of zeolites in animal diets improves weight gains and feed conversion in broilers (Fethiere et al. 1994;Olver 1997), egg production in laying hens (Olver 1997), and has a positive effect of egg weight and their physical properties (Tserveni-Gousi et al. 1997).The extent of positive effects on performance depends on the type of zeolite used, its purity, physical and chemical characteristics and its concentration in diet.
Clinoptilolites have been demonstrated to have a positive effect on the metabolic utilization of nitrogen in poultry and pigs.This suggests that it may be possible to reduce the concentrations of nitrogenous substances in feed rations without adversely affecting production performance of animals.An important benefit of their use is the ability of clinoptilolites to bind ammonia, which reduces nitrogen excretion in faeces and thus also lowers its concentrations in livestock barns and in the environment (Amon et al. 1997;Meisinger et al. 2001;Melenova et al. 2003).
Research into application of these substances in laying hens is a specific area (Wu-Haan et al. 2007;Olver 1997;Rabon et al. 1991;Roland et al. 1993;Roland et al. 1985).Nys (1999) noted that synthetic zeolites with a high cation exchange capacity slightly improve egg shell quality because they form complexes with Ca.In terms of specific weight, a positive effect of zeolites on eggshell quality was demonstrated in a majority of studies.Positive effects were observed in particular when dietary Ca was at a marginal level of 2.75% (Roland et al. 1985) and at the time of elevated environmental temperatures (Keshavarz and McCormick 1991).In addition to their positive effects on production performance in animals, in-feed zeolites are effective in enhancing prevention of some diseases and improving the health status.Mechanisms of enhancing properties of dietary zeolite on production performance of animals were summarized by Papaioannou et al. (2005).
The results presented in this paper corroborate some literary data and complement others.The most important finding is the positive effect of lower clinoptilolite concentration on metabolic utilization of nutrients, production performance indicators (egg weight), and lowering of daily feed consumption.This may be explained by the above-mentioned range of positive effects of zeolites.Our results show that clinoptilolite is a useful feed additive.Concentrations in rations for laying hens up to 2% are considered optimal.Higher concentrations present certain risks in terms of laying hens' production performance and possible negative effects on haemopoiesis.Indirect effects of clinoptilolite should also be considered: at higher doses and long-term administration it may affect the utilization of biologically active substances such as vitamins, trace elements and other specific-action substances and thus influence nutrient metabolism indicators.To verify this hypothesis, further experiments will have to be conducted.