The Mechanism Of Ascites Syndrome Of Broilers Induced By Low Temperature And The Effect Of VC Supplemention

The study included experiments in vivo and in vitro. The objective of the study was conducted to investigate the effects of VC dietary supplementation on growth performance, heart index, blood parameters, antioxidant capacity, pulmonary vascular remodeling (PVR) and the expression of genes related to hypoxia in broilers under low temperature and secondly explore the effect of hypoxia induced by CoCl2 on hypoxia inducible factor-la (HIF-1α), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2/Flk-1) of pulmonary arteries smooth muscle cells (PASMC).Experiment 1:Effects of low ambient temperatures and dietary vitamin C supplementation on antioxidative capacity, HIF-la mRNA and pulmonary vascular remodeling of 1-21d broilersA total of 400 one-d-old male Cobb broilers were assigned to one of 4 treatments as follows:1) Low ambient temperature (LAT) and a basal diet; 2) LAT and a basal diet supplemented with 1000 mg/kg VC (LAT+VC); 3) Normal ambient temperature (NAT) and a basal diet; 4) NAT and a basal diet supplemented with 1000 mg/kg VC (NAT+VC). Each treatment contained 10 replicates with 10 birds per replicate. All birds were fed to 21 d of age. Broilers in groups 1 and 2 were raised at 24℃ to 26℃ during 1-7 d, and at 9℃ to 11℃ during 8-21 d, whereas groups 3 and 4 were raised at 29℃ to 31℃ during 1-7 d and at 24℃ to 26℃ during 8-21 d.1) LAT increased the feed to gain ratio (F/G) during the whole experimental period (P<0.01), heart index at 21d (P<0.05), and hematocrit (HCT) and hemoglobin (HGB) level at 14d (P< 0.05). Supplementing the diet with VC increased HCT, HGB, and red blood cell (RBC) count at 21d (P<0.05).2) At 21d, LAT conditions decreased total antioxidative capacity (T-AOC) in the serum, liver, and lungs (P <0.05), and it also increased the levels of VC in the serum and liver, the amount of protein carbonylation in liver and lungs, and malondialdehyde (MDA) level in the lungs (P<0.05). The addition of VC tended to increase the T-AOC level in serum (P<0.1).3) LAT increased the value of vessel wall area to total area (WA/TA,%) and mean media thickness in pulmonary arterioles (mMTPA,%) at 21d (P< 0.05). Dietary VC supplementation decreased the value of mMTPA (P< 0.05), but had no effect on the WA/TA.4) LAT increased the relative mRNA expression of hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2/Flk-1) (P< 0.05), while adding VC to the diet can decrease the relative mRNA expression of HIF-la, VEGF and Flk-1 at 21d (P< 0.05).5) A significant positive correlation existed among the mRNA expression of HIF-1α, VEGF and Flk-1 at 21d (P< 0.01). A significant positive correlation existed between the increase of mRNA for VEGF and the values of the corresponding WA/WT or mMTPA at 21d (P< 0.01). In conclusion, LAT resulted in oxidative stress, pulmonary vascular remodeling, and increased HIF-1α, VEGF and Flk-1 mRNA expression for broilers that were fed from 1-21d of age. Dietary VC supplementation had no significant effect on the antioxidant activity, while it can alleviate the PVR and decrease the hypoxic gene expression.Experiment 2:Effect of hypoxia induced by CoCl2 on HIF-1α, VEGF and Flk-1 of pulmonary arteries smooth muscle cellsPulmonary arterial smooth muscle cells (PASMC) of 21-day-old male Cobb broilers were cultured with the adherent method of tissue explants. The pulmonary artery vessels were removed from broilers. The media tunicas separating from the vessels were cut into small pieces and incubated in 5% CO2 at 37℃. Identification of the cells was performed with the morphological feature and immunofluorescence stain. Cells were cultured in hypoxia condition induced by 0,100,200,250,500 and 1000μmol/L CoCl2 for 6,12,24 and 48h in each CoCl2 concentration. The relative mRNA expression of HIF-1α in cells of all treatments and the relative mRNA expression of VEGF and Flk-1 in cells with 0,250 and 1000μmol/L CoCl2 in 48h were determined. The cell proliferation was detected by MTT assay.1) The cells grew from the tissue explants in 5-6d, and then they formed a typical’hill’and’valley’pattern in 7-10d under light microscope.2) Immunofluorescence staining indicated that a-acitn, which was the specific actin of smooth muscle cells, was stained with red fluorescence.3) The main effects of time and CoCl2 concentration on the relative mRNA expression of HIF-la were significant (P<0.05). The relative mRNA expression of HIF-la were increased in the groups conducted with 250,500 and 1000μ mol/L CoCl2 within 12.24 and 48h, and the group conducted with 250μmol/L within 48h was the highest(P<0.01).4) The main effect of CoCl2 conducted time on the proliferation of PASMC was significant (P<0.001). The proliferation of PASMC conducted by each CoCl2 concentration was increased along with time, and it was the highest in 48h (P< 0.01).5) The relative mRNA expression of VEGF in cells conducted with 250 and 1000μ mol/L CoCl2 in 48h were tended to be higher than these conducted with 0μmol/L CoCl2 in 48h (P<0.1), while effect on the mRNA expression of Flk-1 was not significant (P> 0.05). In conclusion, using tissue block adherent method can successfully obtain large numbers of PASMC. The 250μmol/L concentration of CoCl2 and the time 48h can induce cell hypoxia successfully, and increase the gene expression of HIF-1α, VEGF and Flk-1 in some degress, thus indicated that a closely relationship exsited between the hypoxia and the gene expression of HIF-1α, VEGF and Flk-1.