Administration of Clinoptilolite to Broiler Chickens During Growth and Its Effect on the Growth Rate and Bone Metabolism Indicators

Straková E., R. Pospíšil, P. Suchý, L. Steinhauser, I. Herzig: Administration of Clinoptilolite to Broiler Chickens During Growth and Its Effect on the Growth Rate and Bone Metabolism Indicators. Acta Vet Brno 2008, 77: 199-207. The growth rate and bone metabolism indicators were monitored in broiler chickens receiving the feed supplemented with clinoptilolite. One-day-old broiler chickens ROSS 308 were divided into control (C) and experimental (E) groups with 100 males and 100 females per group. The chickens received the complete feed mixture BR1 from 1 to 10 days of age, followed by the feed mixture BR2 until the age of 30 days, and the feed mixture BR3 until the end of the experiment (40 days). The feed mixtures of the experimental group were supplemented with clinoptilolite (commercial additive ZeoFeed) at a level of 0.5% (BR1), 1.5% (BR2) and 2.5% (BR3), replacing the corresponding portion of wheat. Feed mixtures and drinking water were provided ad libitum. The live weight of broiler chickens in both the control and experimental group increased steadily during the experiment. At the end of the experiment, live weights of experimental females (2,416 g) and males (2,829 g) were higher than those of control females (2,345 g) and males (2,694 g) by 3% and 5%, respectively. Significant differences in the live weight between groups were found from the age of 30 days (P  0.05 and P  0.01). At the age of 40 days, the chickens were slaughtered and the femur and tibiotarsus of the right leg were analysed for the content of dry matter, ash, calcium, phosphorus and magnesium. The ash content in dry matter ranged from 53.0 to 54.1% in group C and from 51.7 to 53.2% in group E. The Ca and P contents in dry matter in group E were lower than those in group C, except for Ca and P in the male tibiotarsus. In both groups, regardless of sex, the ash content was higher in the tibiotarsus than in the femur. Since fat levels in bones of the experimental group were increased (females by 19.5% in the femur and 21.3% in the tibiotarsus; males by 22.0% in the femur and 26.3% in the tibiotarsus), which could affect the values obtained, ash, calcium, phosphorus and magnesium were determined in the fat-free dry matter. The trends found in the originally determined dry matter were maintained, i.e. lower levels of ash, Ca, and P in the experimental group, except for male Ca in both kinds of bones. The corresponding levels in the fat-free dry matter were relatively higher as compared to the original dry matter. The results of the study showed that up to 2.5% of clinoptilolite in the diet had no adverse effect on performance and bone metabolism indicators of growing broiler chickens. Thus, it can be used as a suitable feed additive to broiler diets due to its positive effect on nutrient utilization, mechanism of digestion and pollutant elimination in connection with food safety issues. Zeofeed, weight of chickens, bone tissue, ash, calcium, phosphorus, magnesium Zeolites represent a large and very diverse group of minerals such as water-silicates that are characterized by three-dimensional structure and belong to the class of aluminosilicates from a chemical point of view. Their structure is based on a three-dimensional skeleton consisting of SiO4 and AlO4 tetrahedrons that form interconnected channels and cavities containing weakly bound (quite mobile) water molecules and cations of alkali metals (Na, K, Li, Cs) and alkaline earth metals (Ca, Mg, Ba, Sr), which compensate for the unsaturated negative valence of AlO4. Phosphorus substituting silicon and beryllium or ACTA VET. BRNO 2008, 77: 199-207; doi:10.2754/avb200877020199 Address for correspondence: Prof. Ing. Eva Straková, Ph.D. Department of Nutrition, Animal Husbandry and Animal Hygiene, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, 612 42 Brno, Czech Republic Phone: +420 541 562 672 Fax: +420 541 562 675 E-mail: strakovae@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm iron substituting aluminium may contribute to the structure of the skeleton. Zeolites have a characteristic crystal structure. Thanks to their specific physicochemical properties, they have a considerable potential to be used in the area of animal production. More than 40 kinds of zeolites have been described so far. Natural as well as synthetic zeolites are being used successfully as feed supplements, with clinoptilolite [(NaK)6 (Al6Si30O72)·20H2O] and mordenite [(CaNa2K2) (Al2Si10O24)·7H2O] that are used most frequently. Zeolites received with feed are involved in a number of biochemical processes through ion exchange, adsorption, catalysis, and dehydration-rehydration properties (Mumpton 1999). The  effectiveness  of  zeolites  was  verified  and  confirmed  in  a  number  of  areas,  for example improved performance indicators. The administration of clinoptilolite as a feed supplement enhanced feed utilization (Olver 1997). Furthermore, zeolites have a beneficial effect on the mechanism of digestion, participate in the elimination of heavy metals (Tepe et al. 2004) and radioactive elements, and repair the metabolic defects of the skeleton (Papaioannou et al. 2005). Over the last ten years, clinoptilolites have been used as adsorbents to bind a wide range of mycotoxins, thus reducing their toxic effects (Harvey et al. 1993; Skalická and Makoová 1999; Skalická et al. 2000; Parlat et al. 1999; Ortatatli and Oguz 2001; Rizzi et al. 2003). In in vitro experiments, sodium bentonite showed a high sorption capacity for aflatoxin B1 and decreased the overall content of aflatoxins in growing broilers (Rosa et al. 2001). The lower mortality and improved weight gains in piglets have been reported by Papaioannou et al. (2002; 2004). Clinoptilolites were shown to be highly effective with regard to the metabolic utilization of nitrogen in poultry and pigs. This may indicate that one may decrease the concentration of nitrogen-containing substances in a feeding dose without affecting performance of animals. One of  the  significant  features  of  clinoptilolites  is  that  they  are  able  to  bind  ammonia, which reduces the release of nitrogen via excrements and thus limits the concentration of nitrogen in animal houses and the environment (Amon et al. 1997; Meisinger et al. 2001; Melenova et al. 2003). Data on how to affect bone tissue composition in birds (deposition of Ca, P and Mg) after the administration of clinoptilolites is published only sporadically in literature (Leach et al. 1990; Papaioannou et al. 2005). Grosicki and Kowalski (2003), Grosicki et al. (2004), and Grosicki and Rachubik (2005) studied the effect of bentonite on the kinetics of iron, calcium, and selenium in rats. The absorption of selenium and calcium in organs was decreased while the absorption of iron increased. The relationship between natural/ synthetic zeolites and tibial dyschondroplasia in chickens was investigated by Ledwaba and Roberson (2003), Edwards (1988), Ballard and Edwards (1988), and Watkins and Southern (1991). The main aim of the presented work was to investigate the effect of clinoptilotite as a feed supplement on the selected production and bone metabolism indicators in broilers during their growth at defined experimental conditions. Materials and Methods The experiment was performed in the accredited experimental enclosure of the Department of Nutrition, Animal Husbandry and Animal Hygiene, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Brno. A total of 400 sexed one-day-old chickens (hybrid ROSS 308) were selected and divided into two groups: control group (C) and experimental group (E), each consisting of 100 male chickens and 100 female chickens. Chickens were reared individually in aviaries equipped with an automatic system of watering and manual feeding, in a setting with controlled light and temperature regimen. The experiment was finished when the chickens reached the age of 40 days. Chickens received the complete feed mixture BR1 from the start of the experiment until Day 10, followed by the feed mixture BR2 from Day 11 to Day 30 and the feed mixture BR3 from Day 31 to Day 40. All feed mixtures contained the same components; the only difference was that the mixtures designed for the experimental group 200

Zeolites represent a large and very diverse group of minerals such as water-silicates that are characterized by three-dimensional structure and belong to the class of aluminosilicates from a chemical point of view.Their structure is based on a three-dimensional skeleton consisting of SiO 4 and AlO 4 tetrahedrons that form interconnected channels and cavities containing weakly bound (quite mobile) water molecules and cations of alkali metals (Na, K, Li, Cs) and alkaline earth metals (Ca, Mg, Ba, Sr), which compensate for the unsaturated negative valence of AlO 4 .Phosphorus substituting silicon and beryllium or iron substituting aluminium may contribute to the structure of the skeleton.Zeolites have a characteristic crystal structure.Thanks to their specific physicochemical properties, they have a considerable potential to be used in the area of animal production.
More than 40 kinds of zeolites have been described so far.Natural as well as synthetic zeolites are being used successfully as feed supplements, with clinoptilolite [(NaK) 6 (Al 6 Si 30 O 72 )•20H 2 O] and mordenite [(CaNa 2 K 2 ) (Al 2 Si 10 O 24 )•7H 2 O] that are used most frequently.Zeolites received with feed are involved in a number of biochemical processes through ion exchange, adsorption, catalysis, and dehydration-rehydration properties (Mumpton 1999).
The effectiveness of zeolites was verified and confirmed in a number of areas, for example improved performance indicators.The administration of clinoptilolite as a feed supplement enhanced feed utilization (Olver 1997).Furthermore, zeolites have a beneficial effect on the mechanism of digestion, participate in the elimination of heavy metals (Tepe et al. 2004) and radioactive elements, and repair the metabolic defects of the skeleton (Papaioannou et al. 2005).
Over the last ten years, clinoptilolites have been used as adsorbents to bind a wide range of mycotoxins, thus reducing their toxic effects (Harvey et al. 1993;Skalická and Makoová 1999;Skalická et al. 2000;Parlat et al. 1999;Ortatatli and Oguz 2001;Rizzi et al. 2003).In in vitro experiments, sodium bentonite showed a high sorption capacity for aflatoxin B1 and decreased the overall content of aflatoxins in growing broilers (Rosa et al. 2001).The lower mortality and improved weight gains in piglets have been reported by Papaioannou et al. (2002;2004).
Clinoptilolites were shown to be highly effective with regard to the metabolic utilization of nitrogen in poultry and pigs.This may indicate that one may decrease the concentration of nitrogen-containing substances in a feeding dose without affecting performance of animals.One of the significant features of clinoptilolites is that they are able to bind ammonia, which reduces the release of nitrogen via excrements and thus limits the concentration of nitrogen in animal houses and the environment (Amon et al. 1997;Meisinger et al. 2001;Melenova et al. 2003).
Data on how to affect bone tissue composition in birds (deposition of Ca, P and Mg) after the administration of clinoptilolites is published only sporadically in literature (Leach et al. 1990;Papaioannou et al. 2005).Grosicki and Kowalski (2003), Grosicki et al. (2004), and Grosicki and Rachubik (2005) studied the effect of bentonite on the kinetics of iron, calcium, and selenium in rats.The absorption of selenium and calcium in organs was decreased while the absorption of iron increased.The relationship between natural/ synthetic zeolites and tibial dyschondroplasia in chickens was investigated by Ledwaba and Roberson (2003), Edwards (1988), Ballard and Edwards (1988), and Watkins and Southern (1991).
The main aim of the presented work was to investigate the effect of clinoptilotite as a feed supplement on the selected production and bone metabolism indicators in broilers during their growth at defined experimental conditions.

Materials and Methods
The experiment was performed in the accredited experimental enclosure of the Department of Nutrition, Animal Husbandry and Animal Hygiene, Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences, Brno.A total of 400 sexed one-day-old chickens (hybrid ROSS 308) were selected and divided into two groups: control group (C) and experimental group (E), each consisting of 100 male chickens and 100 female chickens.Chickens were reared individually in aviaries equipped with an automatic system of watering and manual feeding, in a setting with controlled light and temperature regimen.The experiment was finished when the chickens reached the age of 40 days.
Chickens received the complete feed mixture BR1 from the start of the experiment until Day 10, followed by the feed mixture BR2 from Day 11 to Day 30 and the feed mixture BR3 from Day 31 to Day 40.All feed mixtures contained the same components; the only difference was that the mixtures designed for the experimental group were supplemented with 0.5% (BR 1), 1.5% (BR 2), and 2.5% (BR 3) of clinoptilolite (commercial additive ZeoFeed) to replace respective portions of wheat.Feed mixtures and drinking water were provided ad libitum.The levels of basic components and nutrient composition of feed mixtures are provided in Table 1.

Active substance specification
The feed additive ZeoFeed which was used in this experiment contains at least 80% of clinoptilolite as an active substance, at a moisture level of max.6%.It also contains 62% of SiO 2 , 14% of Al 2 O 3 , 2.3% of Fe 2 O 3 and 5.5% of CaO.Particle size varies in a range of 0.2 -0.5 mm.

Live weight determination
The weight of the chickens was determined on Days 1, 11, 20, 30, and 40 of the chickens' age.The weight was determined at the accuracy of one tenth of a gram, at the same time of day and at the same order of groups.Together with live weight determination, the consumption of feed mixtures was also estimated.

Chemical composition of bones
At the end of the experiment, the chickens were slaughtered.The femur and the tibiotarsus of the right leg in 60 control and 60 experimental chickens were collected and analysed for dry matter, fat, ash, calcium in ash, phosphorus, and magnesium in the original mass, absolute matter, and fatfree dry matter (Anonymous -AOAC 2001).

Health
The health condition of broiler chickens was monitored throughout the experiment, with no clinical symptoms of any disease being recorded.In the course of the experiment, 5 female and 8 male chickens died in the control group, and 4 female and 7 male chickens (yolk peritonitis, cardiac failure) died in the experimental group.

Statistical evaluation
ANOVA was used to process the data with homogeneous dispersion followed by Tukey-HSD test to perform multiple comparisons in order to find the pairs of groups with significant differences.The Kruskal-Wallis ANOVA method was used for the data with non-homogeneous dispersion, followed by non-parametric rank test (Tukeytype multiple comparisons).

Results
The administration of clinoptilolite (0.5; 1.5, and 2.5% of the commercial additive ZeoFeed) in the feed mixtures for the chickens in the experimental group (Group E) was positively reflected in performance indicators.Chickens in both groups showed steady weight gains throughout the whole experiment but male and female chickens in the experimental group exhibited higher weights from Day 20 or Day 30, respectively.The corresponding differences were significant on Day 30 of the chickens' age (P  0.05), the weight of chickens in the experimental group at the end of the experiment was significantly higher (P  0.01) (♂ 2,829 g and ♀ 2,416 g), as compared to that in the control group (♂ 2,694 g and ♀ 2,345 g) (Table 2).
Chickens were slaughtered at the end of the experiment, i.e. on Day 40.The femur and the tibiotarsus were collected and analysed for the contents of dry matter, fat, ash, Ca, P and Mg ( tibiotarsus varied in the range of 53.0 -54.1% in the control group and 51.7 -53.2% in the experimental group, which was non-significantly less as compared to the control group. The level of ash in dry matter was higher in the tibiotarsus as compared to that in the femur, in both groups, irrespective of sex.The level of Ca and P in dry matter in the experimental group was lower than in the control group, the only exception were the levels of Ca in the tibiotarsus in male chickens.The level of Ca in ash in the control group was 20.6% in the femur and 21.5% in the tibiotarsus in female chickens, while in male chickens lower values of 19.9% and 19.7%, respectively, were found.Similar levels were found in the experimental group of female (19.7% and 19.6%) and male chickens (19.8% and 20.0%).
The levels of phosphorus were similar, ranging from 9.06% to 10.4% in the control group, and from 7.92% to 9.55% in the experimental group, which is slightly less, as compared to the control group.The levels of magnesium did not differ between both groups and sexes.No significant differences were found.As the content of fat in bones increased by 19.5% in the femur and by 21.3% in the tibiotarsus in experimental female chickens, and by 22.0% in the femur and 26.3% in the tibiotarsus in experimental male chickens (Table 4), which was supposed to affect the corresponding indicators of bone tissue, the variables monitored (ash, calcium, phosphorus and magnesium) were determined in the fat-free dry matter (Table 5), all of them showing relatively higher levels, as compared to those in the original dry matter (Table 5).However, the trends found in the original dry matter (Table 3) were the same, i.e. the lower levels of ash, Ca, and P in the experimental group except for Ca in male chickens, in both kinds of bones.
Table 6 provides an overview of the monitored values in the absolute dry matter.Even in this presentation of the results, the trends in individual values were maintained and did not practically differ from the values found in the fat-free dry matter.

Discussion
The application of clinoptilolite (0.5; 1.5 and 2.5% of the commercial additive ZeoFeed) in feed mixtures designed for chickens in the experimental group was reflected positively in performance parameters, which is in good agreement with the findings made by the above-mentioned authors.Male and female chickens in the experimental group showed higher weights from Day 20 or Day 30 of the chickens' age, respectively.The differences on Day 30 were significant (P  0.05) and the weight of experimental chickens at the end of the experiment was significantly higher (P  0.01).The improved performance is likely to be associated with the improved utilization of nutrients (Olver 1997) and detoxifying effects of zeolites (Harvey et al. 1993;Skalická and Makoová 1999;Skalická et al. 2000;Parlat et al. 1999;Ortatatli and Oquz 2001;Rizzi at al. 2003), which has been documented repeatedly.The efficacy can be explained by slower passage of pre-digested food through the intestine which leads to the increased utilization of nutrients from the feeding dose, particularly nitrogen (Mumpton and Fishman 1977;Meisinger et al. 2001;Melenova et al. 2003).Similarly, the adsorption of mycotoxins as common components of feed mixtures could also be reflected in the performance indicators and the health condition of broiler chickens (Rosa et al. 2001;Papaioannou et al. 2005).Zeolites also participate in the elimination of heavy metals (Tepe et al. 2004) and radioactive elements, and play an important role in the repair of the metabolic defects of the skeleton (Papaioannou et al. 2005).
The components received with the feed and harmful substances formed by intestinal microorganisms (e.g.NH 4 , enterotoxins) undergo adsorption and elimination which prevents their penetration into the blood circulation, thus decreasing the load of the liver with toxic substances.The energy necessary for detoxification is used to increase performance.Another favourable aspect of zeolites is that they can contribute to the metabolic utilization of nitrogen and decrease its excretion in faeces.Apart from the beneficial effect of zeolites on performance indicators in animals, the supplementation of diets with zeolites helps to prevent some diseases and improves the general health of animals (Papaioannou et al. 2005).
The effect of the addition of zeolite on performance indicators and bone metabolism indicators in broiler chickens was studied by Leach et al. (1990) who reported weight gains in broilers and the enhanced deposition of calcium in bones in the case of deficient or boundary level of calcium, thus relieving the symptoms of rickets.The level of ash in bones was also higher.The results of our experiment showed that the level of ash in the dry matter of bone tissue varied in a range of 53.0 -54.1% in the control group and in a range of 51.7 -53.2% in the experimental group which was non-significantly lower.The amount of ash in the tibiotarsus was higher than that in the femur in both groups, regardless of sex.The levels of calcium in the diets of chickens corresponded to the respective demand.In comparison with the results reported by Leach et al. (1990), the levels of Ca and P in dry matter in the experimental group were lower than those in the control group.The only exception was the level of Ca in the tibiotarsus in male chickens.The level of Ca in ash was about 20% in the control group, being lower in male chickens, while in the experimental group it was below 20%.Similar results were found in the case of phosphorus where the levels of phosphorus in the control group varied in a range of 9.06 -10.4%, the lower level of phosphorus was found in the experimental group, ranging between 7.92 and 9.55%.The levels of magnesium between both groups and sexes did not differ.Venalainen et al. (2006) reported that the level of ash, calcium and phosphorus in the tibiotarsus in broilers receiving a diet with a lower content of ME (11.0 MJ/kg) was higher 204 than that in broilers receiving a diet with a higher content of ME (12.0 MJ/kg).The levels of ash, calcium, and phosphorus increased, depending on the content of usable phosphorus in a diet.The feed mixtures used in our experiment (BR1, BR2, BR3) contained significantly more ME, as compared to those reported by Venalainen et al. (2006), ranging from 12.6 to 13.5 MJ/kg.One may assume that the reason for different results lies in the level of ash, calcium, and phosphorus in bones.Grosicki and Kowalski (2003), Grosicki et al. (2004), and Grosicki and Rachubik (2005) reported the slightly lowered absorption of calcium and selenium in organs and increased absorption of iron after the 28-day administration of a diet supplemented with 2% of bentonite in rats, which is in good agreement with our results with respect to calcium.
As reported in literature (Papaioannou et al. 2005), zeolites received by the body participate in a number of biochemical processes through ion exchange, adsorption, and catalysis.Further investigations will allow a wider use of zeolites in the prevention and therapy of some of the diseases of farm animals.

Table 3 )
. The level of ash in dry matter in bone tissue from the femur and the

Table 2 .
Live weight of broiler chickens in the course of fattening (g) n -number of chickens, x -arithmetic means, SD -standard deviation, P - level of significance, ab P ≤ 0.05,

Table 3
. Levels of ash, Ca, P, and Mg in Groups C and E in original dry matter (g)

Table 5 .
Levels of ash, Ca, P, and Mg in Groups C and E in fat-free dry matter (g)

Table 6 .
Levels of ash, Ca, P, and Mg in C and E groups, per absolute dry matter (g)