The Effect Of Vitamin E On Key Transporter And Catabolic Enzyme Of Vitamin A In Laying Hens

This thesis includes vivo and vitro two parts of experiments. We investigated the effect of vitamin E on key transporter and catabolic enzyme of vitamin A in laying hens, and we used hepatocytes from laying hens to test the effect ofα-tocopherol on the retinol binding protein and the major subtypes of cytochrome P450 enzymes.Experiment 1 The Effect of Vitamin E on Key Transporter and Catabolic Enzyme of Vitamin A in Laying HensFour hundred and fifty JingHong Brown hens (24 weeks old) were randomly divided to five treatments with six replicates .The control group with fifteen hens in each replicate received corn-soybean meal diet without vitamin E, and the other groups received the same basal diet supplemented vitamin E with 20, 80, 320, 1280 IU/kg respectively. The feeding trail lasted eight weeks.Production performance was recorded daily throughout the experimental period. Feed consumption and feed conversion were measured in each week. Eggs in each replicate were collected and the shell hardness, Haugh units, yolk color, shell thickness and albumen height were measured each 2 weeks. Vitamin E and A in feed were determined at the beginning of the experiment. In 4th and 8th week all egg yolks in each replicate were collected and slaughtered 1 bird in each replicate to get liver and plasma to determined vitamin E, vitamin A. Analysis of RBP and CYP450 in liver was carried out.The result showed that in the 4th week and 8th week, adding different levels of vitamin E in dietary, the content of retinol(and retinol palmitate)was significantly increased (P<0.05) in liver. In the 8th week, higher vitamin E (320, 1280 IU/kg) supplementations decreased vitamin A concentrations of egg yolk significantly (P<0.05).The plasma concentration of retinol was independent of the vitamin E supply. RBP in 320 and 1280 IU/kg dietary vitamin E groups was significantly (P<0.05) depressed in contrast to control group. Supplementation with vitamin E in either group had no detectable effect on plasma RBP concentration over the experimental period. No differences were observed in the content of CYP450.Experiment 2 Optimization of the Methods for Isolation and Preparation of Hepatocytes in Laying HensThis study was to compared two methods of isolation and preparation of hepatocytes, in order to optimize the methods of hepatocytes culture in laying hens.The control group was used the method that ligated posterior mesenteric vein in distal end and free tubal vein as well as pancreaticoduodenal vein. The treatment group was used the method that ligated posterior mesenteric vein in distal end, tubal vein and pancreaticoduodenal vein. The success rate of liver perfusion, total hepatocytes yield and cell viability was recorded during the experiments. Meanwhile, the cell morphology was observed and hepatic lactate dehydrogenase (LDH) activity was determined in 4, 24, 48, 144 and 168 h. After optimizing the method, the success rate of liver perfusion was increased to 100%, and the total hepatocytes yield and cell viability were significantly increased to (1.09±0.21)×109 per liver (P <0.05) and (95.3±1.3)% (P <0.05) respectively. However, there was no significant difference between two methods in the cell morphology and LDH activity (P >0.05).These results indicated that in compared with the old method, the method for isolation and preparation of hepatocytes that established in this experiment was more suitable for hepatocytes culture in egg laying poultry, which can obtain high quality of hepatocytes and meet the requirement of the further study.Experiment 3 The Effect ofα-tocopherol on Retinol Binding Protein and CYP26A1 in Hepatocytes from Laying HensThe laying hens were feed vitamin E free dietary as Experiment 1 described. The methods and materials used in hepatocytes isolation and culture procedures were according to those described in Experimat 2. The culture conditions were identical with normal hepatocytes, and the initial medium was replaced withα-tocopherol (0, 10, 50, 100μM)supplemented William's medium E(serum-free) after 5 hr in culture.Cells were harvested by 20 minutes after addition ofα-tocopherol to the medium. The lactate dehydrogenase activity, total protein and RBP were determined after 20 min. Analysis of RBP and CYP26A1 expression at the mRNA level were used the technology of reverse transcriptase-polymerase chain reaction (RT-PCR).α-tocopherol on LDH activity, total protein had no significant effect (P>0.05).α-tocopherol 100μM group significantly reduced the content of RBP and its mRNA expression (P<0.05). There was no significant effect on CYP26A1 mRNA expression.