According to this act, chicken embryo is not definite as the animal. Fertilized eggs

(n = 150; 56 ± 2.2 g) from hens of the Ross line were obtained from a commercial hatchery and stored at 12°C for 4 days. After 4 days, the eggs were weighed and randomly divided into six groups (n = 25 eggs per group). The control group was not treated, while the other groups were treated www.selleckchem.com/products/Rapamycin.html with 1, 5, 10, 15, or 20 μg/ml of NP-Pt solutions. The experimental solutions were given in ovo by injection into the albumen (at two-thirds of the egg’s height from the blunt end) using a sterile 1-ml insulin syringe. Injection consisted of 0.3-ml NP-Pt hydrocolloid. The injection holes were sterilized, and the eggs were then incubated at 37.5°C and 60% humidity and were turned once per hour for 19 days. At day 20 of incubation, the embryos were sacrificed by decapitation. Embryos and organs (brain, heart, liver, spleen, bursa of Fabricius) were weighed and evaluated by Hamburger LY2606368 clinical trial and Hamilton [18] (HH) standards. Biochemical indices Blood serum samples were collected from the jugular vein on

the 20th day of incubation. The samples were centrifuged at 3,000 rpm for 15 min (Sorvall ST 16, Thermo Fisher Scientific, Waltham, MA, USA), and concentrations of alanine aminotransferase (ALT), asparagine aminotransferase, lactate dehydrogenase, alkaline phosphatase (ALP), glucose level, and blood urea nitrogen were measured in the blood serum. Biochemistry markers were examined using a dry chemistry equipment Vitros DT 60 II (Johnston and Johnston, New Brunswick, NJ, USA). Brain morphology: examination of brain tissue microstructure Chicken brains (n = 12), three from the control group and nine from groups treated with 1, 10, and 20 μg/ml of NP-Pt solutions, were sampled

and fixed in 10% buffered formalin (pH 7.2). Fixed samples were dehydrated in a graded series of ethanols, embedded in Paraplast, and cut into 5-μm sections using a microtome (Leica RM 2265, Leica, Nussloch, Germany). The morphology of the chicken brains was examined using hematoxylin-eosin staining. Proliferating cells were identified via immunohistochemistry using antibodies directed against Elongation factor 2 kinase proliferating cell nuclear antigen (PCNA) [19]. Apoptotic cells were detected using rabbit polyclonal anti-caspase-3 antibody (C8487, Sigma-Aldrich Corporation, St. Louis, MO, USA). Sections for this purpose were incubated for 1 h with the rabbit polyclonal anti-caspase-3 antibody at room temperature and were visualized with Dako EnVision+System-HRP (Dako K 4010, Dako A/S, Glostrup, Denmark), while further procedures were identical as for PCNA detection. The proliferation and apoptosis levels were expressed as the number of PCNA-positive cells and caspase-3-positive cells in the chicken brain cortex, respectively (the area counted was 3,500 μm2).