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Effects of palygorskite on broiler performance, feed technological characteristics and litter quality
Applied Clay Science 49 (2010) 276–280
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Applied Clay Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l a y
Effects of palygorskite on broiler performance, feed technological characteristics and litter quality A.C. Pappas a,⁎, E. Zoidis a, N. Theophilou b, G. Zervas a, K. Fegeros a a b
Department of Nutritional Physiology and Feeding, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, 75 Iera Odos, 11855, Athens, Greece Geohellas S.A., 60 Zephyrou and Syngrou, 17564, Athens, Greece
a r t i c l e
i n f o
Article history: Received 3 March 2010 Received in revised form 31 May 2010 Accepted 2 June 2010 Available online 9 June 2010 Keywords: Attapulgite Broiler Clay Litter Palygorskite Pellet
a b s t r a c t The aim of the study was to evaluate how palygorskite supplemented in broiler diets affects growth performance, mortality, litter quality and feed technological characteristics. Day-old broilers (n = 192) were raised for 6 weeks and fed either a corn–soybean base diet with no added palygorskite, or the same diet with 1% m/m, or 10% m/m added palygorskite. Body mass, feed conversion ratio, water intake, litter score and excreta dry matter, total and non-protein nitrogen content were assessed. To test for any negative health effects of the additive, mortality was assessed and standard hematological analyses were performed. Hardness and durability of pellets were measured to determine pellet quality. The study revealed that hardness of pellets was improved by palygorskite suggesting that it may be used as an agglomerant. Data on mortality and hematology indicated that palygorskite was safe and tolerable. The luck of statistically significant differences on improvement of litter quality with palygorskite addition could be attributed to the small scale of the study and the low stress conditions. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Palygorskite, formerly called attapulgite (Bergaya et al., 2006), is found as white, gray, or transparent, dull, elongated, lath-shaped crystals in bundles that comprise thin sheets of minute interlaced fibers. Deposits with more than 75% palygorskite are seldom found (López-Galindo et al., 2007). The major chemical components of palygorskite are: SiO2 55.03%, Al2O3 10.24%, MgO 10.44%, Fe2O3 3.53%, H2O+ 8.24% and H2O− 7.29% (López-Galindo et al., 2007). Palygorskite has several physical properties such as high specific surface area, high viscosity and cation exchange capacity that favor its use in the industry. The palygorskite's cation exchange capacity ranges between 0.04 and 0.4 meq/g (Galan, 1996) usually between 0.3 and 0.4 meq/g (Murray, 2000). These properties are the reason for the palygorskite's high absorption and adsorption capacity (Carretero, 2002; Carretero and Lagaly, 2007; de Sousa Figueiredo Gomes and Pereira Silva, 2007; Viseras et al., 2007), which is the basis for many technological applications (Galan, 1996; Liu, 2007). One of the most important markets for industrial clay absorbents is as litter for companion animals, usually cats (Galan, 1996). Clay minerals, such as sepiolite, can control the concentration of ammonia in farm environments (Sugiura et al., 1991). Furthermore, palygorskite is used as an antidiarrhoea drug (WHO, 1995; Zaid et al., 1995), as a toxin binder in pigs and broilers (Bailey et al., 2006; Kubena et al., 1998; Phillips, 1999) and as an active ingredient in cosmetics (CIREP, 2003).
⁎ Corresponding author. Tel.: + 30 210 5294415; fax: + 30 210 5294413. E-mail address: [email protected] (A.C. Pappas). 0169-1317/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2010.06.003
The main objective of the present study was to investigate the effect of palygorskite on broiler performance since this clay mineral may reduce wet droppings, improve litter quality and reduce ammonia produced. Furthermore, tolerance of broilers on excess of palygorskite was assessed. Finally, pellet quality was measured to examine if palygorskite can be used as a binging agent during pellet manufacture. 2. Materials and methods 2.1. Animals, diets and design Housing and care of the birds conformed to the Faculty of Animal Science and Aquaculture of the Agricultural University of Athens guidelines. There were 4 replicates of 2 main dietary treatments namely, A and B (Table 1). The first one (A) was the control and the broilers were fed a corn–soybean base diet without addition of palygorskite. In treatment (B), broilers were fed the same corn–soybean base diet with 1% m/m added palygorskite. This level of inclusion was similar to that used in practice. To examine tolerance on excess of palygorskite, broilers were fed the same corn–soybean base diet with 10% m/m added palygorskite (treatment C) since European Food Safety Authority recommended that the inclusion level for tolerance studies should be ten times higher than that used in practice (EFSA, 2008). Treatment C was solely used to determine the mortality rate of broilers and was not compared to the other treatments. Palygorskite was provided as a light gray powder (AFG-60 mesh, Geohellas S.A., Greece). The supplemented palygorskite was mined in Greece and comprised of a
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Table 1 The diet formulation of the experimental broiler diets. Ingredients (g kg− 1)
Diet A
Diet B
Starter
Grower
Finisher
Starter
Grower
Finisher
(0–2 weeks)
(2–4 weeks)
(4–6 weeks)
(0–2 weeks)
(2–4 weeks)
(4–6 weeks)
Corn Soybean meal Herring meal Soybean oil Dicalcium phosphate Limestone NaCl Methionine Lysine Soda (NaHCO3) Choline Coccidiostat Premix Palygorskite
590.00 291.90 62.00 25.00 10.00 10.00 2.50 2.50 – 1.00 0.50 0.60 4.00a –
605.00 300.00 20.00 37.00 13.00 11.50 2.50 2.50 2.00 2.00 0.50 – 4.00a –
648.00 285.00 – 30.00 12.50 13.00 4.00 3.00 2.00 – – – 2.50b –
590.00 291.90 62.00 25.00 10.00 10.00 2.50 2.50 – 1.00 0.50 0.60 4.00a 10.00
605.00 300.00 20.00 37.00 13.00 11.50 2.50 2.50 2.00 2.00 0.50 – 4.00a 10.00
648.00 285.00 – 30.00 12.50 13.00 4.00 3.00 2.00 – – – 2.50b 10.00
Calculated analysis ME (MJ kg− 1) CP (g kg− 1) Sodium (g kg− 1) Ca (g kg− 1) Available P (g kg− 1) Methionine Methionine + cysteine (g kg− 1) Lysine (g kg− 1)
12.94 221.35 1.46 8.16 4.42 6.74 10.05 13.89
13.12 200.26 1.62 8.57 4.18 5.95 9.00 13.61
12.92 176.58 1.77 8.75 3.48 5.80 8.94 9.50
12.81 219.14 1.45 8.08 4.38 6.67 9.95 13.75
12.99 198.26 1.60 8.48 4.14 5.89 8.91 13.47
12.79 174.81 1.75 8.66 3.45 5.74 8.85 9.41
a Premix supplied per kg of diet: 12,000 IU vitamin A (retinyl acetate), 4000 IU vitamin D3 (cholecalciferol), 80 mg vitamin E (DL-α-tocopheryl acetate), 9 mg vitamin K3, 3 mg thiamin, 7 mg riboflavin, 6 mg vitamin B6, 25 μg vitamin B12, 15 mg pantothenic acid, 1.5 mg folic acid, 0.15 mg biotin, 400 mg choline, 1.5 mg iodine, 50 mg iron, 20 mg copper, 100 mg zinc. b Premix supplied per kg of diet: 9000 IU vitamin A (retinyl acetate), 3000 IU vitamin D3 (cholecalciferol), 30 mg vitamin E (DL-α-tocopheryl acetate), 2 mg vitamin K3, 1 mg thiamin, 6 mg riboflavin, 2 mg vitamin B6, 12.5 μg vitamin B12, 10.3 mg pantothenic acid, 0.4 mg folic acid, 0.05 mg biotin, 350 mg choline, 1 mg iodine, 55 mg iron, 8 mg copper, 50 mg zinc.
minimum 75% palygorskite and 25% bentonite–saponite. The main chemical components of the supplement were: SiO2 56.00%, MgO 15.05%, Fe2O3 10.60%, Al2O3 4.97%, and Lost on Ignition (water content) 10.95%. One hundred ninety two (192) Cobb broilers were used in total. There were 4 replicate pens (2 m length × 1 m width) per treatment with 8 male and 8 female broilers in each pen (16 broilers per pen, 64 per treatment). The duration of the trial was 6 weeks. The broilers were raised, according to Cobb's management manual, in a broiler house where light and ventilation were controlled. Temperature declined from 32 °C at hatching to 25 °C at 42 days of age. The lighting program was 23 h of light and 1 h of dark. Broilers were raised on a concrete floor with straw as litter. The initial quantity of straw was maintained for the entire growing period. Stocking density was according to EU legislation. The birds were fed a starter diet to the 2nd wk of their life, a grower diet to the 4th wk and a finisher diet thereafter (Table 1). Feed and water was provided ad libitum. The feed was in the form of mash. Pellets were formulated only to examine their technological characteristics (durability and hardness). Manufacture was done in a feed industry under the following pelleting conditions: steam pressure at 1 bar, maximum pressure of pelleting press at 65 bar and temperature of the homogeneously mixed meal at the exit of conditioner at 80 °C. The diameter of pellets was 3 mm. At weekly intervals, broilers were weighted and the body mass was recorded. Furthermore, feed and water consumption was recorded. A 13 L tough plastic chicken drinker was used in each pen and was weighted every day before and after fresh water addition. Every week, the litter of each pen was scored in order to assess the degree of wet droppings. Furthermore, at weekly intervals, from 6 sites in each pen fresh excreta samples were collected in sealable plastic tubes to assess dry matter, total nitrogen content (as well as its fraction of non-protein source). In detail, 3 excreta collections per day
were performed (morning, midday, evening) and the material was pooled. Excreta collection was done to test possible effects of palygorskite on the ammonia levels which are reflected in the content of non-protein fraction of the total nitrogen present in the feces. To test for any negative effects of the additive on health, blood was collected from one broiler per pen (4 per treatment) at days 0, 21 and 42 for standard hematological analyses as EFSA recommends for tolerance studies. Broilers were inspected daily and mortality was recorded on the appropriate data capture form. Total mortality was calculated as the number of broilers that died throughout the study compared to the initial number of broilers placed corrected for broilers removed for blood collection. 2.2. Analytical procedures Standard hematological analysis included determination of: hematocrit, albumin, globulin and total protein concentration. Furthermore, the different white blood cells (lymphocytes, heterophiles, monocytes, eosinophiles and basophiles) were counted. Hematocrit, albumin, globulin and total protein were determined using an ABX Pentra 400 bench top analyzer (Horiba-ABX, Montpellier, France). Leukocyte type (% of different white blood cells) was determined manually by light microscopy using a Neubauer chamber following a 1:20 dilution with the diluting solution (Turk's solution; 2% acetic acid v/v with a few drops of gentian violet) (WHO, 2000). The counting was performed by one hematologist who was blinded to the blood probes examined. Lymphocytes, heterophiles, monocytes, eosinophiles and basophiles were counted and expressed as percentage of total white blood cells. The litter of the pen was scored following a method by Murakami et al. (2000). The procedure was followed by a single operator. A visual assessment of the entire pen was undertaken using the following guide: 0=normal droppings with white caps in definition,
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1=moderately wet droppings with white caps in definition, 2=moderately wet droppings with some definition and no white caps, 3=completely liquid droppings. Additionally, from the second week onwards, and at weekly intervals fresh excreta samples were collected and dry matter was assessed in a forced-air electric oven. On fresh excreta samples, total nitrogen content (as well as its fraction of nonprotein source) was determined using AOAC 2.055–2.079 Kjeldahl method (Tecator 1015 Digester, Digestion System 20 and Tecator 2300, Kjeltec, Analyzer Unit, Sweden) (AOAC, 1984). To determine durability, pellets (500 g) were placed on a vibratory shaker with sieve diameter of 2 mm (Endecots, London, England). Pellets were shaken for 10 min and the amount of pellets that forced through the apertures was collected in a pan located on the base. Durability was expressed as the percentage of pellets that remained intact compared to the initial number of pellets placed. Hardness of pellets was measured with a Kahl hardness tester (Amandus-KAHL, Reinbeck, Germany) which is the first and common device used in industry to measure pellet hardness (Thomas and van der Poel, 1996). Pellets, one at a time, were placed in the hardness tester and the force needed to brake them was measured in kg.
3.2. Water intake, litter score, excreta dry matter and excreta nitrogen content Palygorskite addition significantly reduced water intake of broilers fed diet B compared to broilers fed diet A during the 3rd and 5th week of the study. The data on water intake are presented on Table 3. For the 6-week growing period, there was a tendency (P = 0.08) for birds fed diet B to have lower water intake (289 ± 4 ml bird− 1) compared to that of birds fed diet A (308 ± 8 ml bird− 1). For both treatments, there was a gradual increase of litter score values from about 0 at the end of first week to over 2 at the end of the 6th week (Table 3). For the 6-week growing period, there was a tendency (P = 0.10) for birds fed diet B to have drier litter conditions (1.88 ± 0.17) compared to that of birds fed diet A (1.46 ± 0.14). The excreta dry matter of birds fed diet B did not differ statistically from the excreta dry matter of birds fed diet A (Table 3). For the 6week growing period, the excreta dry matter of treatments A and B was 175 ± 6 and 175 ± 7 g kg− 1 respectively. Analysis of fecal total nitrogen content (Table 3) revealed that there were no significant differences between treatments A and B. Similarly, no differences were noted regarding the non-protein nitrogen content.
2.3. Statistical analyses
3.3. Hematology
The data were analyzed statistically using SPSS for Windows (Version 8, Chicago, USA). Pen average was the observational unit. All performance variates (body mass, feed intake, body mass gain, feed conversion ratio, mortality, litter score and water consumption) for each growth interval were analyzed by ANOVA using treatment as the main factor. Percentage data such as mortality data were angular transformed prior to analysis. The data are presented as mean ± SEM. Statements of significance were based on P ≤ 0.05 unless otherwise stated.
The age significantly affected total protein and albumin concentration (Table 4). At 21 days, the total protein and albumin concentration of treatment A was higher than that at 0 or 42 days. At 21 days, total protein and albumin concentration was higher in treatment A compared to that of treatment B. However, if the data of the 3 collection points (0, 21, 42 days) were pooled, there was no significant effect of palygorskite on total protein concentration nor on albumin or globulin concentration (pooled data not shown). Palygorskite added to the diet did not affect the hematocrit, which was about 30%, similar for all treatments (Table 4). The white blood cell type representation was unaffected by palygorskite supplementation. For both treatments, lymphocytes were the predominant white cell type in the blood of broilers (Table 4). The overall percentage representation of each white cell type was in the following order: lymphocytesN heterophilesN monocytesN eosinophilesN basophiles. The examined hematological parameters of treatment C ranged within physiological values (data not shown).
3. Results 3.1. Broiler performance Broilers receiving the diet with 1% palygorskite performed as well as broilers receiving the control diet throughout the study (Table 2). At 42 days, there was no significant difference between the mean body mass of treatments A and B which was 2648 ± 30 g and 2600 ± 76 g respectively. There was no statistical difference on FCR between broilers fed diets A and B throughout the study (Table 3). For the 6-week growing period, the FCR of broilers receiving diets A and B was 1.85 ± 0.04 and 1.85 ± 0.03 respectively. In this study, there were no statistical differences on mortality between broilers fed A or B diet (Table 3). Regarding total mortality of treatment C, not a single bird died throughout the study.
3.4. Feed technological characteristics The kg of force needed to break pellets of treatment B was 6.68 ± 0.49 kg while that need to break pellets of treatment A was 5.06 ± 0.61 kg, indicating that the hardness of pellets from treatment B were harder by 32% than pellets from treatment A (P = 0.051). Durability of pellets was not statistically affected by palygorskite supplementation and the percentage of unaffected pellets was 98% similar for both treatments (Table 5). 4. Discussion
Table 2 The effect of palygorskite supplementation on broiler body mass (g). Age (days)
0 7 14 21 28 35 42
Body mass (g)
P value
A
B
45 ± 0 170 ± 2 464 ± 4 878 ± 9 1402 ± 18 1977 ± 8 2648 ± 30
45 ± 0 174 ± 3 470 ± 8 890 ± 10 1403 ± 30 1926 ± 49 2600 ± 76
Values are means ± SEM (n = 4).
NS NS NS NS NS NS NS
Proper environmental conditions in the house are crucial for the survival of newly hatched chicks. The pH, temperature and moisture of litter create an environment that supports bacterial growth. The decomposition of uric acid releases ammonia to the environment (Atapattu et al., 2008; McWard and Taylor, 2000; Shah, et al., 2007) and if not controlled can harm the chicken. Clays and clay products, like clay with sulphuric acid, used as bedding material or acidifiers provide control over ammonia generation (McWard and Taylor, 2000; Nakaue et al., 1981; Tasistro et al., 2007). Since decomposition of feces releases ammonia in the environment, the determination of nitrogen in the feces was preferred in the present study, rather than direct measurement of ammonia with a toxic gas detector. In the present
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Table 3 The effect of palygorskite supplementation on feed conversion ratio, mortality (%), water intake (ml bird− 1 day− 1), litter score, excreta dry matter (g kg− 1), total nitrogen content (%) and non-protein nitrogen content (% of total). Trt
FCR
1
2
3
4
5
6
1–6
A B
1.40 ± 0.04 1.44 ± 0.04 NS
1.31 ± 0.01 1.30 ± 0.03 NS
1.53 ± 0.03 1.46 ± 0.02 NS
1.59 ± 0.03 1.62 ± 0.08 NS
2.12 ± 0.01 2.14 ± 0.09 NS
2.50 ± 0.02 2.52 ± 0.02 NS
1.85 ± 0.04 1.85 ± 0.03 NS
A B
0
1.67 ± 1.67 0 NS
1.67 ± 1.67 0 NS
0 0 NS
1.78 ± 1.78 0 NS
1.92 ± 1.92 1.78 ± 1.78 NS
7.00 ± 2.85 3.50 ± 2.02 NS
NS
132 ± 4 130 ± 5 NS
213a ± 3 200b ± 3 b 0.05
311 ± 7 293 ± 7 NS
442 a ± 8 411 b ± 2 b 0.05
678 ± 35 631 ± 22 NS
308 ± 8 289 ± 4 NS (P = 0.08)
P value Mortality (%)
Age of broilers (wk)
P value
1.67 ± 1.67 NS
Water intake (ml bird− 1 day− 1) P value
A B
Litter score (arbitrary units) P value
A B
0.50 ± 0.29 0.50 ± 0.29 NS
1.25 ± 0.25 1.00 ± 0.41 NS
2.25 ± 0.48 1.25 ± 0.25 NS
2.50 ± 0.29 2.00 ± 0.41 NS
2.25 ± 0.25 2.00 ± 0 NS
2.50 ± 0.29 2.00 ± 0 NS
1.88 ± 0.17 1.46 ± 0.14 NS (P = 0.10)
Excreta dry matter (g kg− 1)
A B
177 ± 16 178 ± 13 NS
148 ± 6 156 ± 13 NS
199 ± 17 197 ± 6 NS
140 ± 24 168 ± 11 NS
198 ± 3 197 ± 9 NS
188 ± 11 155 ± 33 NS
175 ± 6 175 ± 7 NS
Total N content (%) P value
A B
4.38 ± 0.34 4.03 ± 0.51 NS
4.39 ± 1.24 5.48 ± 0.66 NS
4.23 ± 1.18 3.52 ± 1.20 NS
4.12 ± 0.36 3.83 ± 0.75 NS
4.25 ± 0.23 4.78 ± 0.75 NS
3.98 ± 0.28 3.97 ± 0.47 NS
4.22 ± 0.37 4.24 ± 0.05 NS
Non-protein N content (% of total) P value
A B
17.39 ± 3.04 19.72 ± 4.69 NS
17.66 ± 4.71 17.59 ± 4.20 NS
15.15 ± 2.72 20.31 ± 4.42 NS
11.17 ± 0.16 15.02 ± 4.69 NS
9.49 ± 0.93 11.71 ± 1.88 NS
9.05 ± 0.40 9.00 ± 0.47 NS
13.32 ± 1.39 15.56 ± 2.15 NS
P value
72 ± 3 70 ± 5
Values are means ± SEM (n = 4). Percentage data are untransformed means and SEM. Statistical differences indicated in the table and P values refer to transformed data. Table 4 The effect of palygorskite supplementation on blood protein concentration (g dl− 1), hematocrit (%) and leukocyte type (% representation of each cell type). Age Treatment (days) A Total protein (g dl− 1)
0 21 42 Albumin 0 −1 (g dl ) 21 42 Globulin 0 (g dl− 1) 21 42 Hematocrit (%) 0 21 42 Lymphocyte (%) 0 21 42 Heterophil (%) 0 21 42 Monocyte (%) 0 21 42 Eosinophil (%) 0 21 42 Basophil (%) 0 21 42
Source of variation Trt B
Age
Age × Trt
P value
4.65 ax ± 0.93 4.13 ax ± 0.46 0.057 8.45 ay ± 1.30 5.10 bx ± 0.60 4.25 ax ± 0.50 4.58 ax ± 0.38 1.90 ax ± 0.42 1.60 ax ± 0.21 NSi 3.58 ay ± 0.44 2.30 bx ± 0.23 1.35 ax ± 0.09 1.70ax ± 0.14 2.75 ± 0.51 2.53 ± 0.26 NS 4.88 ± 0.86 2.80 ± 0.37 2.90 ± 0.45 2.88 ± 0.24 29.15 ± 2.43 30.63 ± 1.65 NS 32.10 ± 0.65 34.03 ± 1.58 29.25 ± 1.18 30.00 ± 0.91 48.25 ± 8.55 51.75 ± 4.70 NS 55.25 ± 2.10 56.00 ± 2.74 42.50 ± 7.96 49.25 ± 2.87 43.25 ± 8.63 40.25 ± 4.89 NS 32.25 ± 2.39 31.50 ± 1.94 50.00 ± 8.77 42.75 ± 2.63 8.50 x ± 1.89 5.75 x ± 1.70 NS x 11.00 ± 0.41 10.00 x ± 1.22 5.50 y ± 1.26 6.25 x ± 1.93 0 1.50 ± 0.50 NS 1.50 ± 0.87 2.50 ± 0.29 2.00 ± 0.71 1.50 ± 0.65 0 0.75 ± 0.75 NS 0 0 0 0.25 ±0.25
b 0.05 NS
b 0.05 b 0.05
NS
NS
NS
NS
NS
NS
NS
NS
0.026
NS
NS
NS
NS
NS
Values are means ± SEM (n = 4). Percentage data are untransformed means and SEM. Statistical differences indicated in the table and P values refer to transformed data. Within each row, means between columns with different superscripts (a, b, c) are different at P b 0.05 unless otherwise stated (treatment effects). Within each column, means between rows with different superscripts (x, y, z) are different at P b 0.05 unless otherwise stated (age effects).
study, no significant differences were noted between treatments regarding the nitrogen content. However, there was a tendency to have drier litter conditions in the treatment with 1% m/m added palygorskite compared to the control diet. Previous work on sepiolite by Ouhida et al. (2000) reported no significant effect of sepiolite supplementation on the live mass and FCR of broilers. In the present study, at 42 days of age, broilers fed both diets A and B exceeded Cobb body mass target of 2345 g indicating that broilers fed on 1% m/m added palygorskite converted feed equally well as broilers on the control diet. Palygorskite supplementation at 10% m/m did not cause any mortality indicating that from this point of view, palygorskite was safe and tolerable by the broilers. Under commercial conditions where stress factors, like disease prevalence and aflatoxin-contaminated diets exist, palygorskite may reduce mortality rates and benefit broiler growth and performance. In addition, standard hematological analysis did not reveal any negative effect of palygorskite on health. It is well known that broiler hematology has several distinctive characteristics such as red blood cells with nucleus and short life span. Furthermore, heterophil function parallels mammalian neutrophil function. In the present study, hematocrit ranged between 27 and 34%, values that are within the physiological values (22–35%) (Bounous and Stedman, 2000). The same applied for lymphocytes and heterophiles that accounted for the 80% of all white cells (Bounous and Stedman, 2000). Low water intake can affect the body fluids and in turn protein concentration in blood. Total protein of blood is a test often used to determine dehydration. In the present study, palygorskite added to the diet was accompanied by the tendency for lower water intake compared to the control treatment. However, it should be mentioned that water was provided ad libitum and total protein values were about 4 g dl− 1 in line with previously reported total protein values for healthy, non-dehydrated avian species like chickens (Filipović et al., 2007), pigeons (Gayathri et al., 2004) and quails (El-Hommosany, 2008). Clays are incorporated as agglomerant in animal diets in order to improve feed manufacture (Angulo et al., 1995; Xia et al., 2004). The
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Table 5 The effect of palygorskite supplementation on feed technological characteristics (hardness and durability of pellets).
Hardness (kg) Durability (%)
A
B
n
P value
5.06a ± 0.61 98.68 ± 0.04
6.68b ± 0.49 98.69 ± 0.52
11 4
P = 0.051 NS
Values are means ± SEM (n = 11 for hardness data; n = 4 for durability data). Means within a column with different superscripts are different at P b 0.05 unless otherwise stated.
pelleting process is influenced by several factors including but not limited to: content and level of different ingredients, fat and fiber content, particle size, inclusion of binders and factors related to the process of pelleting per se such as conditioning, distance between roller and die and hole compression (Angulo et al., 1995, Angulo et al., 1996). Earlier work by Angulo et al. (1995) reported that sepiolite, a clay with similar physical properties to palygorskite (Galan, 1996), improved the performance of pelleted diets. Furthermore, the same authors reported that sepiolite was more effective when fat and fiber levels were high (Angulo et al., 1995). In the present study, pellets manufactured with 1% m/m added palygorskite showed better pellet hardness than pellets manufactured without palygorskite. It is also possible that under different circumstances i.e. diets with higher fiber or fat content, the noted difference will be more pronounced. Improvement of pellet hardness could be attributed to palygorskite's special sorptive and colloidal-reological properties, which are the basis for its most technological applications (Galan, 1996, Liu, 2007). In detail, the presence of micropores and channels in palygorskite together with the fine particle size and fibrous habit account for its very high surface area (IARC, 1997). Palygorskite absorbs polar liquids and forms gels which in turn may improve pellet hardness possibly by increasing solid–solid bonding interactions. In conclusion, the hardness of pellets was improved by palygorskite addition suggesting that it may be used as an agglomerant (binder). Data on mortality and hematology indicated that palygorskite was also safe and tolerable. This trial was a small scale one with low levels of stress however, under commercial conditions, tendencies of litter quality improvement as those noted in the present study could be more pronounced. Future studies on palygorskite may reveal the optimum inclusion level in broiler diets where the quality of litter is maintained without compromising growth. Acknowledgements We would like to thank A. Zervou for her assistance during the conduct of the trial and A. Arapis for excellent technical support. References Angulo, E., Brufau, J., Esteve-Garcia, E., 1995. Effect of sepiolite on pellet durability in feeds differing in fat and fibre content. Anim. Feed Sci. Technol. 53, 233–241. Angulo, E., Brufau, J., Esteve-Garcia, E., 1996. Effect of a sepiolite product on pellet durability in pig diets differing in particle size and in broiler starter and finisher diets. Anim. Feed Sci. Technol. 63, 25–34. AOAC, 1984. Official Methods of Analysis of the Association of Official Analytical Chemists. Kjeldahl method, 2.055–2.079, 14th. AOAC, Virginia, USA. Atapattu, N.S.B., Senaratna, M.D., Belpagodagamage, U.D., 2008. Comparison of ammonia emission rates from three types of broiler litters. Poult. Sci. 87, 2436–2440. Bailey, C.A., Latimer, G.W., Barr, A.C., Wigle, W.L., Haq, A.U., Balthrop, J.E., Kubena, L.F., 2006. Efficacy of montmorillonite clay (NovaSil Plus) for protecting full-term broilers from aflatoxicosis. J. Appl. Poult. Res. 15, 198–206.
Bergaya, F., Theng, B.K.G., Lagaly, G., 2006. History of clay science: a young discipline, In: Bergaya, F., Theng, B.K.G., Lagaly, G. (Eds.), Handbook of Clay Science, 1st ed. Elsevier, Amsterdam, The Netherlands. Bounous, D.I., Stedman, N.L., 2000. Normal avian hematology: chicken and turkey, In: Feldman, B.F., Zinkl, J.G., Jain, N.C., Schalm, O.W. (Eds.), Schalm's Veterinary Hematology, 5th ed. Blackwell Publishing, USA. Carretero, M.I., 2002. Clay minerals and their beneficial effects upon human health. A review. Appl. Clay Sci. 21, 155–163. Carretero, M.I., Lagaly, G., 2007. Clays and health: an introduction. Appl. Clay Sci. 36, 1–3. CIREP (Cosmetic Ingredient Review Expert Panel), 2003. Final report on the safety assessment of aluminum silicate, calcium silicate, magnesium aluminum silicate, magnesium silicate, magnesium trisilicate, sodium magnesium silicate, zirconium silicate, attapulgite, bentonite, fuller's earth, hectorite, kaolin, lithium magnesium silicate, lithium magnesium sodium silicate, montmorillonite, pyrophyllite, and zeolite. Int. J. Toxicol. 22 (Suppl. 1), 37–102. de Sousa Figueiredo Gomes, C., Pereira Silva, J.B., 2007. Minerals and clay minerals in medical geology. Appl. Clay Sci. 36, 4–21. El-Hommosany, Y.M., 2008. Study of the physiological changes in blood chemistry, humoral immune response and performance of quail chicks fed supplemental chromium. Int. J. Poult. Sci. 7, 40–44. European Food Safety Authority (EFSA), 2008. Technical guidance: tolerance and efficacy studies in target animal. EFSA J. 778, 1–14. Filipović, N., Stojević, Z., Milinković-Tur, S., Beer Ljubić, B., Zdelar-Tuk, M., 2007. Changes in concentration and fractions of blood serum proteins of chickens during fattening. Veterinarski Arhiv. 77, 319–326. Galan, E., 1996. Properties and applications of palygorskite–sepiolite clays. Clay Miner. 31, 443–453. Gayathri, K.C., Shenay, K.B., Hegde, S.N., 2004. Blood profile of pigeons (Columba livia) during growth and breeding. Comp. Biochem. Physiol.-Part A. 138, 187–193. IARC, 1997. Palygorskite (attapulgite). Silica, Some Silicates, Coal Dust and Para-Aramid Fibrils. : IARC Monographs on the Evaluation of Carcinogenic Risks to Humans, 68. International Agency for Research on Cancer, Lyon, pp. 245–255. Kubena, L.F., Harvey, R.B., Bailey, R.H., Buckley, S.A., Rottinghaus, G.E., 1998. Effects of a hydrated sodium calcium aluminosilicate (T-Bind) on mycotoxicosis in young broiler chickens. Poult. Sci. 77, 1502–1509. Liu, P., 2007. Polymer modified clay minerals: a review. Appl. Clay Sci. 38, 64–76. López-Galindo, A., Viseras, C., Cerezo, P., 2007. Compositional, technical and safety specifications of clays to be used as pharmaceutical and cosmetic products. Appl. Clay Sci. 36, 51–63. McWard, G.W., Taylor, D.R., 2000. Acidified clay litter amendment. J. Appl. Poult. Res. 9, 518–529. Murakami, A.E., Saleh, E.A., Watkins, S.E., Waldroup, P.W., 2000. Sodium source and level in broiler diets with and without high levels of animal protein. J. Appl. Poult. Res. 9, 53–61. Murray, H.H., 2000. Traditional and new applications for kaolin, smectite, and palygorskite: a general overview. Appl. Clay Sci. 17, 207–221. Nakaue, H.S., Koelliker, J.K., Pierson, M.L., 1981. Studies with clinoptilolite in poultry. II. Effect of feeding broilers and the direct application of clinoptilolite (zeolite) on clean and reused broiler litter on broiler performance and house environment. Poult. Sci. 60, 1221–1228. Ouhida, I., Pérez, J.F., Piedrafita, J., Gasa, J., 2000. The effects of sepiolite in broiler chicken diets of high, medium and low viscosity. Productive performance and nutritive value. Anim. Feed Sci. Technol. 85, 183–194. Phillips, T.D., 1999. Dietary clay in the chemoprevention of aflatoxin-induced disease. Toxicol. Sci. 52, 118–126. Shah, S.B., Baird, C.L., Rice, J.M., 2007. Effect of a metabolic stimulant on ammonia volatilization from broiler litter. J. Appl. Poult. Res. 16, 240–247. Sugiura, M., Hayashi, H., Suzuki, T., 1991. Adsorption of ammonia by sepiolite in ambient air. Clay Sci. 8, 87–100. Tasistro, A.S., Ritz, C.W., Kissel, D.E., 2007. Ammonia emissions from broiler litter: response to bedding materials and acidifiers. Br. Poult. Sci. 48, 399–405. Thomas, M., van der Poel, A.F.B., 1996. Physical quality of pelleted animal feed 1. Criteria for pellet quality. Anim. Feed Sci. Technol. 61, 89–112. Viseras, C., Aguzzi, C., Cerezo, P., Lopez-Galindo, A., 2007. Uses of clay minerals in semisolid health care and therapeutic products. Appl. Clay Sci. 36, 37–50. WHO., 1995. The Treatment of Diarrhoea: A Manual for Physicians and other Senior Health Workers. World Health Organization, Geneva. WHO., 2000. Recommended Methods for the Visual Determination of White Blood Cell Count and Platelet Count. The Expert Panel on Cytometry of the International Council for Standardization in Heamatology. World Health Organization, Geneva. Xia, M.S., Hu, C.H., Xu, Z.R., 2004. Effects of copper-bearing montmorillonite on growth performance, digestive enzyme activities, and intestinal microflora and morphology of male broilers. Poult. Sci. 83, 1868–1875. Zaid, M.R., Hasan, M., Khan, A.A., 1995. Attapulgite in the treatment of acute diarrhoea: a double-blind placebo-controlled study. J. Diarrhoeal Dis. Res. 13, 44–46.
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Applied Clay Science j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l a y
Effects of palygorskite on broiler performance, feed technological characteristics and litter quality A.C. Pappas a,⁎, E. Zoidis a, N. Theophilou b, G. Zervas a, K. Fegeros a a b
Department of Nutritional Physiology and Feeding, Faculty of Animal Science and Aquaculture, Agricultural University of Athens, 75 Iera Odos, 11855, Athens, Greece Geohellas S.A., 60 Zephyrou and Syngrou, 17564, Athens, Greece
a r t i c l e
i n f o
Article history: Received 3 March 2010 Received in revised form 31 May 2010 Accepted 2 June 2010 Available online 9 June 2010 Keywords: Attapulgite Broiler Clay Litter Palygorskite Pellet
a b s t r a c t The aim of the study was to evaluate how palygorskite supplemented in broiler diets affects growth performance, mortality, litter quality and feed technological characteristics. Day-old broilers (n = 192) were raised for 6 weeks and fed either a corn–soybean base diet with no added palygorskite, or the same diet with 1% m/m, or 10% m/m added palygorskite. Body mass, feed conversion ratio, water intake, litter score and excreta dry matter, total and non-protein nitrogen content were assessed. To test for any negative health effects of the additive, mortality was assessed and standard hematological analyses were performed. Hardness and durability of pellets were measured to determine pellet quality. The study revealed that hardness of pellets was improved by palygorskite suggesting that it may be used as an agglomerant. Data on mortality and hematology indicated that palygorskite was safe and tolerable. The luck of statistically significant differences on improvement of litter quality with palygorskite addition could be attributed to the small scale of the study and the low stress conditions. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Palygorskite, formerly called attapulgite (Bergaya et al., 2006), is found as white, gray, or transparent, dull, elongated, lath-shaped crystals in bundles that comprise thin sheets of minute interlaced fibers. Deposits with more than 75% palygorskite are seldom found (López-Galindo et al., 2007). The major chemical components of palygorskite are: SiO2 55.03%, Al2O3 10.24%, MgO 10.44%, Fe2O3 3.53%, H2O+ 8.24% and H2O− 7.29% (López-Galindo et al., 2007). Palygorskite has several physical properties such as high specific surface area, high viscosity and cation exchange capacity that favor its use in the industry. The palygorskite's cation exchange capacity ranges between 0.04 and 0.4 meq/g (Galan, 1996) usually between 0.3 and 0.4 meq/g (Murray, 2000). These properties are the reason for the palygorskite's high absorption and adsorption capacity (Carretero, 2002; Carretero and Lagaly, 2007; de Sousa Figueiredo Gomes and Pereira Silva, 2007; Viseras et al., 2007), which is the basis for many technological applications (Galan, 1996; Liu, 2007). One of the most important markets for industrial clay absorbents is as litter for companion animals, usually cats (Galan, 1996). Clay minerals, such as sepiolite, can control the concentration of ammonia in farm environments (Sugiura et al., 1991). Furthermore, palygorskite is used as an antidiarrhoea drug (WHO, 1995; Zaid et al., 1995), as a toxin binder in pigs and broilers (Bailey et al., 2006; Kubena et al., 1998; Phillips, 1999) and as an active ingredient in cosmetics (CIREP, 2003).
⁎ Corresponding author. Tel.: + 30 210 5294415; fax: + 30 210 5294413. E-mail address: [email protected] (A.C. Pappas). 0169-1317/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.clay.2010.06.003
The main objective of the present study was to investigate the effect of palygorskite on broiler performance since this clay mineral may reduce wet droppings, improve litter quality and reduce ammonia produced. Furthermore, tolerance of broilers on excess of palygorskite was assessed. Finally, pellet quality was measured to examine if palygorskite can be used as a binging agent during pellet manufacture. 2. Materials and methods 2.1. Animals, diets and design Housing and care of the birds conformed to the Faculty of Animal Science and Aquaculture of the Agricultural University of Athens guidelines. There were 4 replicates of 2 main dietary treatments namely, A and B (Table 1). The first one (A) was the control and the broilers were fed a corn–soybean base diet without addition of palygorskite. In treatment (B), broilers were fed the same corn–soybean base diet with 1% m/m added palygorskite. This level of inclusion was similar to that used in practice. To examine tolerance on excess of palygorskite, broilers were fed the same corn–soybean base diet with 10% m/m added palygorskite (treatment C) since European Food Safety Authority recommended that the inclusion level for tolerance studies should be ten times higher than that used in practice (EFSA, 2008). Treatment C was solely used to determine the mortality rate of broilers and was not compared to the other treatments. Palygorskite was provided as a light gray powder (AFG-60 mesh, Geohellas S.A., Greece). The supplemented palygorskite was mined in Greece and comprised of a
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Table 1 The diet formulation of the experimental broiler diets. Ingredients (g kg− 1)
Diet A
Diet B
Starter
Grower
Finisher
Starter
Grower
Finisher
(0–2 weeks)
(2–4 weeks)
(4–6 weeks)
(0–2 weeks)
(2–4 weeks)
(4–6 weeks)
Corn Soybean meal Herring meal Soybean oil Dicalcium phosphate Limestone NaCl Methionine Lysine Soda (NaHCO3) Choline Coccidiostat Premix Palygorskite
590.00 291.90 62.00 25.00 10.00 10.00 2.50 2.50 – 1.00 0.50 0.60 4.00a –
605.00 300.00 20.00 37.00 13.00 11.50 2.50 2.50 2.00 2.00 0.50 – 4.00a –
648.00 285.00 – 30.00 12.50 13.00 4.00 3.00 2.00 – – – 2.50b –
590.00 291.90 62.00 25.00 10.00 10.00 2.50 2.50 – 1.00 0.50 0.60 4.00a 10.00
605.00 300.00 20.00 37.00 13.00 11.50 2.50 2.50 2.00 2.00 0.50 – 4.00a 10.00
648.00 285.00 – 30.00 12.50 13.00 4.00 3.00 2.00 – – – 2.50b 10.00
Calculated analysis ME (MJ kg− 1) CP (g kg− 1) Sodium (g kg− 1) Ca (g kg− 1) Available P (g kg− 1) Methionine Methionine + cysteine (g kg− 1) Lysine (g kg− 1)
12.94 221.35 1.46 8.16 4.42 6.74 10.05 13.89
13.12 200.26 1.62 8.57 4.18 5.95 9.00 13.61
12.92 176.58 1.77 8.75 3.48 5.80 8.94 9.50
12.81 219.14 1.45 8.08 4.38 6.67 9.95 13.75
12.99 198.26 1.60 8.48 4.14 5.89 8.91 13.47
12.79 174.81 1.75 8.66 3.45 5.74 8.85 9.41
a Premix supplied per kg of diet: 12,000 IU vitamin A (retinyl acetate), 4000 IU vitamin D3 (cholecalciferol), 80 mg vitamin E (DL-α-tocopheryl acetate), 9 mg vitamin K3, 3 mg thiamin, 7 mg riboflavin, 6 mg vitamin B6, 25 μg vitamin B12, 15 mg pantothenic acid, 1.5 mg folic acid, 0.15 mg biotin, 400 mg choline, 1.5 mg iodine, 50 mg iron, 20 mg copper, 100 mg zinc. b Premix supplied per kg of diet: 9000 IU vitamin A (retinyl acetate), 3000 IU vitamin D3 (cholecalciferol), 30 mg vitamin E (DL-α-tocopheryl acetate), 2 mg vitamin K3, 1 mg thiamin, 6 mg riboflavin, 2 mg vitamin B6, 12.5 μg vitamin B12, 10.3 mg pantothenic acid, 0.4 mg folic acid, 0.05 mg biotin, 350 mg choline, 1 mg iodine, 55 mg iron, 8 mg copper, 50 mg zinc.
minimum 75% palygorskite and 25% bentonite–saponite. The main chemical components of the supplement were: SiO2 56.00%, MgO 15.05%, Fe2O3 10.60%, Al2O3 4.97%, and Lost on Ignition (water content) 10.95%. One hundred ninety two (192) Cobb broilers were used in total. There were 4 replicate pens (2 m length × 1 m width) per treatment with 8 male and 8 female broilers in each pen (16 broilers per pen, 64 per treatment). The duration of the trial was 6 weeks. The broilers were raised, according to Cobb's management manual, in a broiler house where light and ventilation were controlled. Temperature declined from 32 °C at hatching to 25 °C at 42 days of age. The lighting program was 23 h of light and 1 h of dark. Broilers were raised on a concrete floor with straw as litter. The initial quantity of straw was maintained for the entire growing period. Stocking density was according to EU legislation. The birds were fed a starter diet to the 2nd wk of their life, a grower diet to the 4th wk and a finisher diet thereafter (Table 1). Feed and water was provided ad libitum. The feed was in the form of mash. Pellets were formulated only to examine their technological characteristics (durability and hardness). Manufacture was done in a feed industry under the following pelleting conditions: steam pressure at 1 bar, maximum pressure of pelleting press at 65 bar and temperature of the homogeneously mixed meal at the exit of conditioner at 80 °C. The diameter of pellets was 3 mm. At weekly intervals, broilers were weighted and the body mass was recorded. Furthermore, feed and water consumption was recorded. A 13 L tough plastic chicken drinker was used in each pen and was weighted every day before and after fresh water addition. Every week, the litter of each pen was scored in order to assess the degree of wet droppings. Furthermore, at weekly intervals, from 6 sites in each pen fresh excreta samples were collected in sealable plastic tubes to assess dry matter, total nitrogen content (as well as its fraction of non-protein source). In detail, 3 excreta collections per day
were performed (morning, midday, evening) and the material was pooled. Excreta collection was done to test possible effects of palygorskite on the ammonia levels which are reflected in the content of non-protein fraction of the total nitrogen present in the feces. To test for any negative effects of the additive on health, blood was collected from one broiler per pen (4 per treatment) at days 0, 21 and 42 for standard hematological analyses as EFSA recommends for tolerance studies. Broilers were inspected daily and mortality was recorded on the appropriate data capture form. Total mortality was calculated as the number of broilers that died throughout the study compared to the initial number of broilers placed corrected for broilers removed for blood collection. 2.2. Analytical procedures Standard hematological analysis included determination of: hematocrit, albumin, globulin and total protein concentration. Furthermore, the different white blood cells (lymphocytes, heterophiles, monocytes, eosinophiles and basophiles) were counted. Hematocrit, albumin, globulin and total protein were determined using an ABX Pentra 400 bench top analyzer (Horiba-ABX, Montpellier, France). Leukocyte type (% of different white blood cells) was determined manually by light microscopy using a Neubauer chamber following a 1:20 dilution with the diluting solution (Turk's solution; 2% acetic acid v/v with a few drops of gentian violet) (WHO, 2000). The counting was performed by one hematologist who was blinded to the blood probes examined. Lymphocytes, heterophiles, monocytes, eosinophiles and basophiles were counted and expressed as percentage of total white blood cells. The litter of the pen was scored following a method by Murakami et al. (2000). The procedure was followed by a single operator. A visual assessment of the entire pen was undertaken using the following guide: 0=normal droppings with white caps in definition,
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1=moderately wet droppings with white caps in definition, 2=moderately wet droppings with some definition and no white caps, 3=completely liquid droppings. Additionally, from the second week onwards, and at weekly intervals fresh excreta samples were collected and dry matter was assessed in a forced-air electric oven. On fresh excreta samples, total nitrogen content (as well as its fraction of nonprotein source) was determined using AOAC 2.055–2.079 Kjeldahl method (Tecator 1015 Digester, Digestion System 20 and Tecator 2300, Kjeltec, Analyzer Unit, Sweden) (AOAC, 1984). To determine durability, pellets (500 g) were placed on a vibratory shaker with sieve diameter of 2 mm (Endecots, London, England). Pellets were shaken for 10 min and the amount of pellets that forced through the apertures was collected in a pan located on the base. Durability was expressed as the percentage of pellets that remained intact compared to the initial number of pellets placed. Hardness of pellets was measured with a Kahl hardness tester (Amandus-KAHL, Reinbeck, Germany) which is the first and common device used in industry to measure pellet hardness (Thomas and van der Poel, 1996). Pellets, one at a time, were placed in the hardness tester and the force needed to brake them was measured in kg.
3.2. Water intake, litter score, excreta dry matter and excreta nitrogen content Palygorskite addition significantly reduced water intake of broilers fed diet B compared to broilers fed diet A during the 3rd and 5th week of the study. The data on water intake are presented on Table 3. For the 6-week growing period, there was a tendency (P = 0.08) for birds fed diet B to have lower water intake (289 ± 4 ml bird− 1) compared to that of birds fed diet A (308 ± 8 ml bird− 1). For both treatments, there was a gradual increase of litter score values from about 0 at the end of first week to over 2 at the end of the 6th week (Table 3). For the 6-week growing period, there was a tendency (P = 0.10) for birds fed diet B to have drier litter conditions (1.88 ± 0.17) compared to that of birds fed diet A (1.46 ± 0.14). The excreta dry matter of birds fed diet B did not differ statistically from the excreta dry matter of birds fed diet A (Table 3). For the 6week growing period, the excreta dry matter of treatments A and B was 175 ± 6 and 175 ± 7 g kg− 1 respectively. Analysis of fecal total nitrogen content (Table 3) revealed that there were no significant differences between treatments A and B. Similarly, no differences were noted regarding the non-protein nitrogen content.
2.3. Statistical analyses
3.3. Hematology
The data were analyzed statistically using SPSS for Windows (Version 8, Chicago, USA). Pen average was the observational unit. All performance variates (body mass, feed intake, body mass gain, feed conversion ratio, mortality, litter score and water consumption) for each growth interval were analyzed by ANOVA using treatment as the main factor. Percentage data such as mortality data were angular transformed prior to analysis. The data are presented as mean ± SEM. Statements of significance were based on P ≤ 0.05 unless otherwise stated.
The age significantly affected total protein and albumin concentration (Table 4). At 21 days, the total protein and albumin concentration of treatment A was higher than that at 0 or 42 days. At 21 days, total protein and albumin concentration was higher in treatment A compared to that of treatment B. However, if the data of the 3 collection points (0, 21, 42 days) were pooled, there was no significant effect of palygorskite on total protein concentration nor on albumin or globulin concentration (pooled data not shown). Palygorskite added to the diet did not affect the hematocrit, which was about 30%, similar for all treatments (Table 4). The white blood cell type representation was unaffected by palygorskite supplementation. For both treatments, lymphocytes were the predominant white cell type in the blood of broilers (Table 4). The overall percentage representation of each white cell type was in the following order: lymphocytesN heterophilesN monocytesN eosinophilesN basophiles. The examined hematological parameters of treatment C ranged within physiological values (data not shown).
3. Results 3.1. Broiler performance Broilers receiving the diet with 1% palygorskite performed as well as broilers receiving the control diet throughout the study (Table 2). At 42 days, there was no significant difference between the mean body mass of treatments A and B which was 2648 ± 30 g and 2600 ± 76 g respectively. There was no statistical difference on FCR between broilers fed diets A and B throughout the study (Table 3). For the 6-week growing period, the FCR of broilers receiving diets A and B was 1.85 ± 0.04 and 1.85 ± 0.03 respectively. In this study, there were no statistical differences on mortality between broilers fed A or B diet (Table 3). Regarding total mortality of treatment C, not a single bird died throughout the study.
3.4. Feed technological characteristics The kg of force needed to break pellets of treatment B was 6.68 ± 0.49 kg while that need to break pellets of treatment A was 5.06 ± 0.61 kg, indicating that the hardness of pellets from treatment B were harder by 32% than pellets from treatment A (P = 0.051). Durability of pellets was not statistically affected by palygorskite supplementation and the percentage of unaffected pellets was 98% similar for both treatments (Table 5). 4. Discussion
Table 2 The effect of palygorskite supplementation on broiler body mass (g). Age (days)
0 7 14 21 28 35 42
Body mass (g)
P value
A
B
45 ± 0 170 ± 2 464 ± 4 878 ± 9 1402 ± 18 1977 ± 8 2648 ± 30
45 ± 0 174 ± 3 470 ± 8 890 ± 10 1403 ± 30 1926 ± 49 2600 ± 76
Values are means ± SEM (n = 4).
NS NS NS NS NS NS NS
Proper environmental conditions in the house are crucial for the survival of newly hatched chicks. The pH, temperature and moisture of litter create an environment that supports bacterial growth. The decomposition of uric acid releases ammonia to the environment (Atapattu et al., 2008; McWard and Taylor, 2000; Shah, et al., 2007) and if not controlled can harm the chicken. Clays and clay products, like clay with sulphuric acid, used as bedding material or acidifiers provide control over ammonia generation (McWard and Taylor, 2000; Nakaue et al., 1981; Tasistro et al., 2007). Since decomposition of feces releases ammonia in the environment, the determination of nitrogen in the feces was preferred in the present study, rather than direct measurement of ammonia with a toxic gas detector. In the present
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Table 3 The effect of palygorskite supplementation on feed conversion ratio, mortality (%), water intake (ml bird− 1 day− 1), litter score, excreta dry matter (g kg− 1), total nitrogen content (%) and non-protein nitrogen content (% of total). Trt
FCR
1
2
3
4
5
6
1–6
A B
1.40 ± 0.04 1.44 ± 0.04 NS
1.31 ± 0.01 1.30 ± 0.03 NS
1.53 ± 0.03 1.46 ± 0.02 NS
1.59 ± 0.03 1.62 ± 0.08 NS
2.12 ± 0.01 2.14 ± 0.09 NS
2.50 ± 0.02 2.52 ± 0.02 NS
1.85 ± 0.04 1.85 ± 0.03 NS
A B
0
1.67 ± 1.67 0 NS
1.67 ± 1.67 0 NS
0 0 NS
1.78 ± 1.78 0 NS
1.92 ± 1.92 1.78 ± 1.78 NS
7.00 ± 2.85 3.50 ± 2.02 NS
NS
132 ± 4 130 ± 5 NS
213a ± 3 200b ± 3 b 0.05
311 ± 7 293 ± 7 NS
442 a ± 8 411 b ± 2 b 0.05
678 ± 35 631 ± 22 NS
308 ± 8 289 ± 4 NS (P = 0.08)
P value Mortality (%)
Age of broilers (wk)
P value
1.67 ± 1.67 NS
Water intake (ml bird− 1 day− 1) P value
A B
Litter score (arbitrary units) P value
A B
0.50 ± 0.29 0.50 ± 0.29 NS
1.25 ± 0.25 1.00 ± 0.41 NS
2.25 ± 0.48 1.25 ± 0.25 NS
2.50 ± 0.29 2.00 ± 0.41 NS
2.25 ± 0.25 2.00 ± 0 NS
2.50 ± 0.29 2.00 ± 0 NS
1.88 ± 0.17 1.46 ± 0.14 NS (P = 0.10)
Excreta dry matter (g kg− 1)
A B
177 ± 16 178 ± 13 NS
148 ± 6 156 ± 13 NS
199 ± 17 197 ± 6 NS
140 ± 24 168 ± 11 NS
198 ± 3 197 ± 9 NS
188 ± 11 155 ± 33 NS
175 ± 6 175 ± 7 NS
Total N content (%) P value
A B
4.38 ± 0.34 4.03 ± 0.51 NS
4.39 ± 1.24 5.48 ± 0.66 NS
4.23 ± 1.18 3.52 ± 1.20 NS
4.12 ± 0.36 3.83 ± 0.75 NS
4.25 ± 0.23 4.78 ± 0.75 NS
3.98 ± 0.28 3.97 ± 0.47 NS
4.22 ± 0.37 4.24 ± 0.05 NS
Non-protein N content (% of total) P value
A B
17.39 ± 3.04 19.72 ± 4.69 NS
17.66 ± 4.71 17.59 ± 4.20 NS
15.15 ± 2.72 20.31 ± 4.42 NS
11.17 ± 0.16 15.02 ± 4.69 NS
9.49 ± 0.93 11.71 ± 1.88 NS
9.05 ± 0.40 9.00 ± 0.47 NS
13.32 ± 1.39 15.56 ± 2.15 NS
P value
72 ± 3 70 ± 5
Values are means ± SEM (n = 4). Percentage data are untransformed means and SEM. Statistical differences indicated in the table and P values refer to transformed data. Table 4 The effect of palygorskite supplementation on blood protein concentration (g dl− 1), hematocrit (%) and leukocyte type (% representation of each cell type). Age Treatment (days) A Total protein (g dl− 1)
0 21 42 Albumin 0 −1 (g dl ) 21 42 Globulin 0 (g dl− 1) 21 42 Hematocrit (%) 0 21 42 Lymphocyte (%) 0 21 42 Heterophil (%) 0 21 42 Monocyte (%) 0 21 42 Eosinophil (%) 0 21 42 Basophil (%) 0 21 42
Source of variation Trt B
Age
Age × Trt
P value
4.65 ax ± 0.93 4.13 ax ± 0.46 0.057 8.45 ay ± 1.30 5.10 bx ± 0.60 4.25 ax ± 0.50 4.58 ax ± 0.38 1.90 ax ± 0.42 1.60 ax ± 0.21 NSi 3.58 ay ± 0.44 2.30 bx ± 0.23 1.35 ax ± 0.09 1.70ax ± 0.14 2.75 ± 0.51 2.53 ± 0.26 NS 4.88 ± 0.86 2.80 ± 0.37 2.90 ± 0.45 2.88 ± 0.24 29.15 ± 2.43 30.63 ± 1.65 NS 32.10 ± 0.65 34.03 ± 1.58 29.25 ± 1.18 30.00 ± 0.91 48.25 ± 8.55 51.75 ± 4.70 NS 55.25 ± 2.10 56.00 ± 2.74 42.50 ± 7.96 49.25 ± 2.87 43.25 ± 8.63 40.25 ± 4.89 NS 32.25 ± 2.39 31.50 ± 1.94 50.00 ± 8.77 42.75 ± 2.63 8.50 x ± 1.89 5.75 x ± 1.70 NS x 11.00 ± 0.41 10.00 x ± 1.22 5.50 y ± 1.26 6.25 x ± 1.93 0 1.50 ± 0.50 NS 1.50 ± 0.87 2.50 ± 0.29 2.00 ± 0.71 1.50 ± 0.65 0 0.75 ± 0.75 NS 0 0 0 0.25 ±0.25
b 0.05 NS
b 0.05 b 0.05
NS
NS
NS
NS
NS
NS
NS
NS
0.026
NS
NS
NS
NS
NS
Values are means ± SEM (n = 4). Percentage data are untransformed means and SEM. Statistical differences indicated in the table and P values refer to transformed data. Within each row, means between columns with different superscripts (a, b, c) are different at P b 0.05 unless otherwise stated (treatment effects). Within each column, means between rows with different superscripts (x, y, z) are different at P b 0.05 unless otherwise stated (age effects).
study, no significant differences were noted between treatments regarding the nitrogen content. However, there was a tendency to have drier litter conditions in the treatment with 1% m/m added palygorskite compared to the control diet. Previous work on sepiolite by Ouhida et al. (2000) reported no significant effect of sepiolite supplementation on the live mass and FCR of broilers. In the present study, at 42 days of age, broilers fed both diets A and B exceeded Cobb body mass target of 2345 g indicating that broilers fed on 1% m/m added palygorskite converted feed equally well as broilers on the control diet. Palygorskite supplementation at 10% m/m did not cause any mortality indicating that from this point of view, palygorskite was safe and tolerable by the broilers. Under commercial conditions where stress factors, like disease prevalence and aflatoxin-contaminated diets exist, palygorskite may reduce mortality rates and benefit broiler growth and performance. In addition, standard hematological analysis did not reveal any negative effect of palygorskite on health. It is well known that broiler hematology has several distinctive characteristics such as red blood cells with nucleus and short life span. Furthermore, heterophil function parallels mammalian neutrophil function. In the present study, hematocrit ranged between 27 and 34%, values that are within the physiological values (22–35%) (Bounous and Stedman, 2000). The same applied for lymphocytes and heterophiles that accounted for the 80% of all white cells (Bounous and Stedman, 2000). Low water intake can affect the body fluids and in turn protein concentration in blood. Total protein of blood is a test often used to determine dehydration. In the present study, palygorskite added to the diet was accompanied by the tendency for lower water intake compared to the control treatment. However, it should be mentioned that water was provided ad libitum and total protein values were about 4 g dl− 1 in line with previously reported total protein values for healthy, non-dehydrated avian species like chickens (Filipović et al., 2007), pigeons (Gayathri et al., 2004) and quails (El-Hommosany, 2008). Clays are incorporated as agglomerant in animal diets in order to improve feed manufacture (Angulo et al., 1995; Xia et al., 2004). The
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Table 5 The effect of palygorskite supplementation on feed technological characteristics (hardness and durability of pellets).
Hardness (kg) Durability (%)
A
B
n
P value
5.06a ± 0.61 98.68 ± 0.04
6.68b ± 0.49 98.69 ± 0.52
11 4
P = 0.051 NS
Values are means ± SEM (n = 11 for hardness data; n = 4 for durability data). Means within a column with different superscripts are different at P b 0.05 unless otherwise stated.
pelleting process is influenced by several factors including but not limited to: content and level of different ingredients, fat and fiber content, particle size, inclusion of binders and factors related to the process of pelleting per se such as conditioning, distance between roller and die and hole compression (Angulo et al., 1995, Angulo et al., 1996). Earlier work by Angulo et al. (1995) reported that sepiolite, a clay with similar physical properties to palygorskite (Galan, 1996), improved the performance of pelleted diets. Furthermore, the same authors reported that sepiolite was more effective when fat and fiber levels were high (Angulo et al., 1995). In the present study, pellets manufactured with 1% m/m added palygorskite showed better pellet hardness than pellets manufactured without palygorskite. It is also possible that under different circumstances i.e. diets with higher fiber or fat content, the noted difference will be more pronounced. Improvement of pellet hardness could be attributed to palygorskite's special sorptive and colloidal-reological properties, which are the basis for its most technological applications (Galan, 1996, Liu, 2007). In detail, the presence of micropores and channels in palygorskite together with the fine particle size and fibrous habit account for its very high surface area (IARC, 1997). Palygorskite absorbs polar liquids and forms gels which in turn may improve pellet hardness possibly by increasing solid–solid bonding interactions. In conclusion, the hardness of pellets was improved by palygorskite addition suggesting that it may be used as an agglomerant (binder). Data on mortality and hematology indicated that palygorskite was also safe and tolerable. This trial was a small scale one with low levels of stress however, under commercial conditions, tendencies of litter quality improvement as those noted in the present study could be more pronounced. Future studies on palygorskite may reveal the optimum inclusion level in broiler diets where the quality of litter is maintained without compromising growth. Acknowledgements We would like to thank A. Zervou for her assistance during the conduct of the trial and A. Arapis for excellent technical support. References Angulo, E., Brufau, J., Esteve-Garcia, E., 1995. Effect of sepiolite on pellet durability in feeds differing in fat and fibre content. Anim. Feed Sci. Technol. 53, 233–241. Angulo, E., Brufau, J., Esteve-Garcia, E., 1996. Effect of a sepiolite product on pellet durability in pig diets differing in particle size and in broiler starter and finisher diets. Anim. Feed Sci. Technol. 63, 25–34. AOAC, 1984. Official Methods of Analysis of the Association of Official Analytical Chemists. Kjeldahl method, 2.055–2.079, 14th. AOAC, Virginia, USA. Atapattu, N.S.B., Senaratna, M.D., Belpagodagamage, U.D., 2008. Comparison of ammonia emission rates from three types of broiler litters. Poult. Sci. 87, 2436–2440. Bailey, C.A., Latimer, G.W., Barr, A.C., Wigle, W.L., Haq, A.U., Balthrop, J.E., Kubena, L.F., 2006. Efficacy of montmorillonite clay (NovaSil Plus) for protecting full-term broilers from aflatoxicosis. J. Appl. Poult. Res. 15, 198–206.
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