2.3. Sample collection
Five samples of each chicken feed, different tissues of broiler chicken and their manure were simultaneously collected from five different poultry farms on weekly basis. Feed samples were collected directly from the bags and feeder duct. About two hundred healthy mature broiler chickens, weight and age ranged, 50–1500 g and 1–3 to 4–6 week, respectively, were brought from poultry farms of Hyderabad, Pakistan (25 22
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E). Manure samples were collected from different spots of the poultry farms, mixed thoroughly and made five sub-samples/week from each poultry farm and transferred to polythene containers. Each collected chicken was dissected in the laboratory and different muscles were separated (leg, breast, liver and heart). Then each of the muscle tissues was washed separately with deionized water. While feed and manure were treated directly. All samples (feed, different tissues of chicken and manure) were freeze-dried in freeze-drying system (Make) up to a constant weight. The dried samples were ground in vibrational agate ball mill for 5 min using a power of 60%. The powdered samples were sieved through nylon sieve to obtain particle size [ø] 125 lm and were stored in closed polyethylene bottles and kept in a refrigerator at 18 C until analysis.
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