CHAPTER 8 Efficacy of adsorbents (bentonite and diatomaceous earth) and turmeric
8.2 Material and methods
8.2.1 Experimental design, birds and diets
Two hundred and fifty Ross 308 day-old male broiler chicks were purchased from a commercial hatchery, weighed, wing banded, and assigned to stainless steel chick batteries so that initial body weights (41 g/bird) were equal among treatments. A completely randomized design was used with five replicate pens of five chicks assigned to each of 10 dietary treatments. The chicks were maintained on a 24 h continuous light schedule and allowed ad libitum access to feed and water from hatch to day 21. The temperature of the room ranged from 35 to 36.1oC at the beginning of the experiment to between 30 and 31.1oC towards the end of the experiment.
The protocol of the experiment was approved by the University of Missouri-Columbia Animal Care and Use Committee. Mortality was recorded as it occurred, and the birds were inspected daily for any health related problems.
8.2.2 Diets
A maize-soybean meal-based basal diet (Table 8.1), in mash form formulated to meet the nutritional requirements of young broilers (1-21d post-hatch) as recommended by the National Research Council (NRC, 1994), was used. Aflatoxin (700 mg/kg culture material) from ground
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Aspergillus parasiticus strain NRRL 2999 culture material was incorporated into the basal diet to achieve the required dietary aflatoxin B1 concentration of 2 mg/kg diet. Commercially available food grade turmeric powder (Curcuma longa), containing 24,700 mg/kg of curcuminoids was used as a source of antioxidants. Turmeric powder (0.81 %) was incorporated into the basal diet to produce dietary treatments containing 200 mg/kg curcuminoids. Dietary treatments included: 1) a positive control basal diet (BD) containing no aflatoxin (AFB1), no bentonite (BC), no diatomaceous(DE) or no turmeric (TUM); 2) BD + 0.75 % BC, but with no AF, DE or TUM; 3) BD + 0.75 % DE but with no AFB1, BC or TUM; 4) BD+ TUM but with no BC, DE or AFB1; 5) BD + 2 mg AFB1/kg diet but with no BC, DE or TUM; 6) BD + 0.75 % BC + 2 mg AFB1/kg diet but with no DE or TUM; 7) BD + 0.75 % DE + 2 mg AFB1/kg diet but with no BC or TUM; 8) BD+ 2 mg AFB1/kg diet + TUM but with no BC or DE; 9) BD+ 2 mg AFB1/kg diet + 0.75 % BC + TUM, but with no DE; 10) BD+ 2 mg AFB1/kg diet + 0.75 % DE + TUM, but with no BC. Dietary AF (B1, B2, G1, and G2) concentrations were confirmed by analyses. In brief, feed samples were extracted with acetonitrile and water (86:14), and an aliquot of the extract was passed through a puriTox TC-M 160 cleanup column and suitably diluted with water before analysis using HPLC with cobra cell post column derivatization with fluorescence detection at 365 nm excitation and 440nm emissions. All diets were screened by the method of Rottinghaus et al. (1982) for the presence of citrinin, T-2 toxin, vomitoxin, zearalenone, fumonisins and ochratoxin A, before the start of the experiment and were below detection limits for these mycotoxins. Total curcuminoid content, including curcumin, bisdemethoxycurcumin, and demethoxycurcumin, were determined by the method described by Gowda et al. (2008).
8.2.3 Measurements
Chicks were weighed by pen at the beginning (day 1), day 7 and at day 21 of the experiment.
Feed was weighed per pen at day 21, and feed conversion ratio was calculated from average feed consumption per pen (g) divided by average body weight gain per pen (g). Mortalities were recorded as they occurred and the dead birds were necropsied. On day 21, three birds per pen were anaesthetized with carbon dioxide and blood samples collected via cardiac puncture for determination of serum chemistries. Livers and kidneys were removed from the same three birds, and weighed.
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Table 8.1. Composition and nutrient value of the basal diet (g/kg as-is)
Ingredient g/kg
Corn 506.4
Soybean Meal 383.2
Soybean oil 52.4
Dicalcium 17.2
Limestone 12.4
Salt 4.6
Methionine 1.9
Vitamin Mix
22.5
Sand 19.4
TOTAL 1000.0
Calculated nutrient composition
3Crude Protein (g/kg) 230.0
Metabolizable Energy (MJ/kg) 12.9
Lysine (%) 12.7
Methionine (%) 5.3
Methionine + Cysteine (%) 9.0
Threonine (%) 8.6
Tryptophan 3.1
Calcium (%) 10.0
Phosphorus (% Av.) 4.5
2Supplied per kilogram of feed: manganese, 100 mg; zinc, 100 mg; iron, 50 mg; copper, 11.25 mg; iodine, 1.5 mg; selenium, 0.15 mg; vitamin A, 2.31 mg ; vitamin D3, 68.75 µg ; vitamin E, 16.5 mg; vitamin B12, 11 µg; vitamin K, 0.83 mg; riboflavin, 6.6 mg; thiamin, 1.1 mg;
pantothenic acid, 6.6 mg; niacin, 27.5 mg; pyridoxine, 1.37 mg; folic acid, 0.69 mg; biotin, 33 µg; choline, 385 mg.
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The liver weight of each bird was recorded and a piece of liver tissue (2 to 3 g) was collected and rinsed with ice-cold phosphate buffered saline (pH 7.4) containing 0.16 mg/ml heparin to prevent blood clot formation. Liver samples were also harvested from 6 birds per treatment, and fixed in 10 % neutral buffered formalin for gross and histopathology evaluation. Fixed liver tissues were trimmed, embedded in paraffin, sectioned at 4 µm, and stained with hematoxylin and eosin for microscopic examination. Liver lesions were scored using a score system of 1 to 4 (1= liver unremarkable; 2= mild aflatoxin lesions; 3= moderate aflatoxin lesions; and 4= severe aflatoxin lesions.
Blood was centrifuged at 1400g at 8oC for 30 min (Sorval, RC 3 B plus) and the serum was collected and preserved at -20oC until submitted for biochemical analyses. Serum samples were analyzed for total proteins, albumin, globulin, glucose, uric acid, γ-glutamyltransferase (EC 2.3.2.2), aspartate aminotransferase, and calcium using an auto analyzer (Kodak Ekatachem Analyzer, Eastman Kodak Co, Rochester, NY).
The shank colour of all surviving birds was determined using the DSM colour fan for broilers.
Right tibiae were collected from three birds per pen for determination of tibia ash. Tibiae were stripped of adhering tissue, dried at 100oC, and fat was extracted with a mixture of ether and methanol (90 and 10 %, respectively). Fat-extracted tibiae were than dried at 100oC for 24 h and ashed in a muffle furnace at 600oC overnight.
8.2.4 Statistical analyses
Data on growth performance, liver and kidney weights, gross pathology, histopathology (liver), serum chemistry, tibia ash and shank colour were analyzed using the GLM procedure of SAS software (SAS Institute Inc., 2011). Treatment means were compared by the Tukey’s test and statistical significance was accepted based on the 0.05 level of probability. Data on mortality and liver lesion scores were analyzed using the Chisquare test Proc FEQ in SAS.