A rapid decline in egg production of laying hens begins after 480 d of age. Such a rapid decrease results predominantly from the ovarian aging, accompanied by endocrine changes, decreased yolk synthesis and accumulation, and the reduction in follicles selected into the preovulatory hierarchy. In this study, hens at 90, 150, 280, and 580 d old (D90, D150, D280, and D580, respectively) were compared for yolk precursor formation in the liver to elucidate effects of aging on laying performance. The results showed that liver lipid synthesis increased remarkably in hens from D90 to D150, but decreased sharply at D580 as indicated by the changes in triglyceride (TG) levels. This result was consistent with the age-related changes of the laying performance. The levels of liver antioxidants and total antioxidant capacity decreased significantly in D580 hens and the methane dicarboxylic aldehyde in D580 hens was much higher than that at other stages. The serum 17β-estradiol level increased from D90 to D280, but decreased at D580 (P<0.05). The expression of estrogen receptor α and β mRNAs in the liver displayed similar changes to the serum 17β-estradiol in D580 hens. Expressions of the genes related to yolk precursor formation and enzymes responsible for fat acid synthesis were all decreased in D580 hens. These results indicated that decreased yolk precursor formation in the liver of the aged hens resulted from concomitant decreases of serum 17β-estradiol level, transcription levels of estrogen receptors and critical genes involved in yolk precursor synthesis, and liver antioxidant status.
Age Factors ; Animals ; Antioxidants ; metabolism ; Chickens ; Egg Yolk ; metabolism ; Estradiol ; blood ; Female ; Lipids ; biosynthesis ; Liver ; metabolism ; Oviposition ; Receptors, Estrogen ; genetics

A highly sensitive and specific method for the determination of roxithromycin in broiler tissues by LC/MS was developed and validated. A dichloromethane extract of the sample was separated on C18 reversed-phase column with acetonitrile-50 mM ammonium acetate (80:20, v/v) as the mobile phase and analyzed by LC/MS via atmospheric pressure ionization/electrospray ionization interface. The limit of detection and limit of quantitation were 1 ng/g and 5 ng/g. The method has been successfully applied to determine for roxithromycin in various tissues of broilers. Residue concentrations were associated with administered dose. At the termination of treatment, roxithromycin was found in all collected samples for both dose groups. Liver was detected to have the highest residual concentration of roxithromycin. Residue concentrations of roxithromycin were lower than its LOQ in all tissues from both dose groups 10 days after the treatment of roxithromycin mixed with drinking water at a dose rate of 15 mg/L or 60 mg/L to each broiler for 7 days.
Adipose Tissue/metabolism ; Animals ; Chickens/blood/*metabolism ; Chromatography, Liquid ; Drug Administration Schedule ; Intestine, Small/metabolism ; Kidney/metabolism ; Liver/metabolism ; Mass Spectrometry ; Molecular Structure ; Muscle, Skeletal/metabolism ; Roxithromycin/*administration&dosage/blood/chemistry/*pharmacokinetics ; Sensitivity and Specificity ; Skin/metabolism

AIMTo investigate the effect of cold stress on the energy metabolism in Yisha chickens.

METHODSMale Yisha chickens were subjected to acute (0.25, 1, 3, 6, 12 and 24 h) and chronic (5, 10 and 20 d) cold stress (12 +/- 1 degrees C). This study detected uncoupling protein (UCP) mRNA levels in gastrocnemius, glucagons (GLU) content in blood plasma and insulin (INS), blood glucose (BG) and free fatty acid (FFA) content in serum in the chicken.

RESULTSThe results were as follow: with the time lapsing during acute cold stress, UCP mRNA levels gradually increased, the content of INS and FFA showed fluctuant change, GLU content gradually increased, and BG content first increased and then decreased. During chronic cold stress, UCP mRNA levels significantly increased compared with their control group at every stress time point, and the content of INS, GLU, BG and FFA were all gradually increased with the time lapsing.

CONCLUSIONCold stress could change the energy metabolism in chickens. And the different extent cold stress would produce different effects on the energy metabolism.

Animals ; Chickens ; Cold Temperature ; Energy Metabolism ; physiology ; Fatty Acids, Nonesterified ; blood ; Insulin ; blood ; Ion Channels ; genetics ; metabolism ; Male ; Mitochondrial Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Stress, Physiological ; physiology ; Uncoupling Protein 1

Fuantai-03(FAT-03), isolated from the Dasyatis akajei, has a strong antiangiogenic activity. The recombinant Fuantai-03 (GST/rFAT-03) fusion protein can be obtained with the DNA recombination technology. In this study, expression conditions of GST/rFAT-03 were optimized by response surface experimental design method. The constructed engineering bacteria containing GST/rFAT-03 plasmid was induced by isopropy-beta-D-thiogalactosid (IPTG), the GST affinity column was used for isolation and purification, and then the effects of different culture time, IPTG concentration, induction temperature and induction time on the amount of soluble GST/rFAT-03 fusion protein were compared. The culture time for optimal expression was 6.13 h, IPTG concentration was 0.36 mmol/L, induction temperature was 19.71 degrees C, and induction time was 13.60 h. The amount of soluble GST/rFAT-03 fusion protein was 7.57 mg/L under above mentioned expression conditions. The results also showed that rFAT-03 significantly inhibited angiogenesis in chicken chorioallantoic membrane in a dose-dependent manner. Moreover, the soluble form of the target protein is useful for further work on purification and on studying its biological function.
Angiogenesis Inhibitors ; biosynthesis ; genetics ; Animals ; Chickens ; Chorioallantoic Membrane ; blood supply ; Escherichia coli ; genetics ; metabolism ; Fish Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Skates (Fish)

In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.
Animals ; Antibodies, Viral ; biosynthesis ; blood ; Chickens ; immunology ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vaccines, Subunit ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

The cDNA encoding human Vascular Endothelial Growth Factor 165 (VEGF165) was amplified using RT-PCR from human tonsil tissue and cloned into eukaryotic expression vector pcDNA3.1 (+). The recombinant plasmid pcDNA/V was transferred into 293 cells mediated by liposome and the cells stably expressing VEGF were selected under the pressure of G418. ELISA and Western blotting demonstrated that the eukaryotic expression vector pcDNA/V was successfully constructed and its corresponding protein could be expressed efficiently in vitro. Chick Charioallantoic Membrane (CAM) bioassay showed that recombinant protein has biological activity of hVEGF. Model rats with acute myocardial ischemia were used to further study the expression of VEGFin vivo. The model rats were divided randomly into three groups: control group, pcDNA3.1 (+) group and pcDNA/V group. 50microL naked plasmid DNA or saline was intramyocardially injected at three sites into the border zone of infarction. The hearts of rats were excised and fixed histologically, then the infarction sizes were studied by immunohistochemical staining and electron microscope after four weeks. Immunohistochemical staining for VEGF appeared to be negative in control and pcDNA3.1 (+) groups. In pcDNA/V group, myocardial cells in infarction border zone showed positive staining for VEGF in cytoplasm. Ultrastructural anaylsis showed that there were visible hyperplasia of vascular endothilium in pcDNA/V group. The control and pcDNA3.1 (+) groups showed less capillary hyperplasia. In this study, VEGF165 gene was successfully cloned and its protein expressed in vitro and in vivo was of bioactivity, which provides a basis for the further study of biological functions of human VEGF.
Animals ; Cell Line ; Chickens ; Chorioallantoic Membrane ; blood supply ; Disease Models, Animal ; Genetic Therapy ; Humans ; Male ; Myocardial Infarction ; metabolism ; pathology ; therapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; biosynthesis ; genetics ; therapeutic use ; Transfection ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

Glycerol monolaurate (GML) has been widely used as an effective antibacterial emulsifier in the food industry. A total of 360 44-week-old Hy-Line brown laying hens were randomly distributed into four groups each with six replicates of 15 birds, and fed with corn-soybean-meal-based diets supplemented with 0, 0.15, 0.30, and 0.45 g/kg GML, respectively. Our results showed that 0.15, 0.30, and 0.45 g/kg GML treatments significantly decreased feed conversion ratios (FCRs) by 2.65%, 7.08%, and 3.54%, respectively, and significantly increased the laying rates and average egg weights. For egg quality, GML drastically increased albumen height and Haugh units, and enhanced yolk color. Notably, GML increased the concentrations of polyunsaturated and monounsaturated fatty acids and reduced the concentration of total saturated fatty acids in the yolk. The albumen composition was also significantly modified, with an increase of 1.02% in total protein content, and increased contents of His (4.55%) and Glu (2.02%) under the 0.30 g/kg GML treatment. Additionally, GML treatments had positive effects on the lipid metabolism of laying hens, including lowering the serum triglyceride and total cholesterol levels and reducing fat deposition in abdominal adipose tissue. Intestinal morphology was also improved by GML treatment, with increased villus length and villus height to crypt depth ratio. Our data demonstrated that GML supplementation of laying hens could have beneficial effects on both their productivity and physiological properties, which indicates the potential application of GML as a functional feed additive and gives us a new insight into this traditional food additive.
Albumins/analysis* ; Animals ; Chickens ; Diet ; Dietary Supplements ; Egg Yolk/chemistry* ; Female ; Gonadal Steroid Hormones/blood* ; Intestines/cytology* ; Laurates/administration & dosage* ; Lipid Metabolism ; Monoglycerides/administration & dosage* ; Oviposition/drug effects* ; Ovum ; Oxidative Stress

Dominant inflammatory cytokines might be different depending on the underlying causes of acute lung injury (ALI). The role of kertinocyte-derived chemokine (KC), a potent chemoattractant for neutrophils, has not been clearly established in hemorrhage-induced ALI. In this study, lung injury and cytokine expressison were evaluated in LPS- or hemorrhage-induced ALI models of BALB/c mice. The myeloperoxidase activities at 4 hr after hemorrhage and LPS-injection were 47.4+/-13.0 and 56.5+/-16.4 U/g, respectively. NF-kappa B activity peaked at 4 hr after hemorrhage, which was suppressed to the control level by anti-high mobility group B1 (HMGB1) antibody. Lung expressions of TNF-alpha, MIP-2, and IL-1beta were increased by LPS injection. However, there was only a minimal increase in IL-1beta and no expressions of TNF-alpha or MIP-2 in hemorrhage-induced ALI. In contrast, lung KC increased significantly at 4 hr after hemorrhage compared to control levels (83.1+/-12.3 vs. 14.2+/-1.6 pg/mL/mg by ELISA) (P<0.05). By immunohistochemical staining, lung neutrophils stained positive for KC. Increased KC was also observed in bronchoalveolar lavage fluid and plasma. KC plays an important role in hemorrhage-induced ALI.
Acute Lung Injury/etiology/*metabolism ; Animals ; Antibodies/immunology/metabolism ; Chemokine CXCL2/analysis ; Chemokines/analysis/blood/*physiology ; Chickens ; HMGB1 Protein/metabolism ; Humans ; Interleukin-1beta/analysis ; Lipopolysaccharides/toxicity ; Mice ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Neutrophils/immunology/metabolism ; Peroxidase/analysis ; Shock, Hemorrhagic/*complications ; Time Factors ; Tumor Necrosis Factor-alpha/analysis

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.
Animals ; Antibodies, Viral ; blood ; Bacteriophage T7 ; genetics ; immunology ; metabolism ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Viral ; Immunization ; Influenza A virus ; genetics ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; immunology ; metabolism ; prevention & control ; Peptides ; genetics ; immunology ; metabolism ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Specific Pathogen-Free Organisms ; Viral Matrix Proteins ; genetics ; immunology ; metabolism

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 microL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4degrees C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.
Animals ; Antibodies, Viral/*blood ; Antigens, Viral/*diagnostic use/genetics/metabolism ; Baculoviridae/genetics ; Chickens ; HN Protein/*diagnostic use/genetics/metabolism ; Hemagglutination Inhibition Tests/*methods/veterinary ; Newcastle Disease/*diagnosis/immunology/virology ; Newcastle disease virus/genetics/*immunology/metabolism ; Poultry Diseases/*diagnosis/immunology/virology ; Recombinant Proteins/diagnostic use/genetics/metabolism ; Sf9 Cells ; Spodoptera