Sample Collection and Procedures

At 10 days of age, one chick was selected from each cage for blood sample collection. At the end of the experiment (13 days of age), two chickens from each replicate were selected and sacrificed. Blood samples were obtained from the wing vein of each chicken using a heparinized syringe. Plasma was obtained after centrifugation at 400 g (4°C, 10 min) and stored at −20°C for further analysis (34, 35). After obtaining blood samples, the chickens were slaughtered through the intravenous injection of pentobarbital sodium (30 mg/kg body weight) and jugular exsanguination. The hypothalamus was collected, in accordance with the method described by Liu et al. (36), via a 4–5 mm deep incision, made parallel to the base of the brain (37). Hypothalamus samples were flash-frozen in liquid nitrogen and stored at −80°C.