Effects Of Vitamin A On Growth Performance And Intestinal Health As Well As Underlying Mechanism

Three experiments in vivo were conducted to investigate the effects of dietary vitamin A on growth performance,intestinal development,and intestinal mucosal damage recovery of Pekin ducks as well as underlying mechanism.Another one experiment in vitro was performed to investigate the effects of all-trans retinoic acid(ATRA)on epithelial cell proliferation and migration using IPEC-J2 cell line.Experiment 1 was conducted to investigate the effects of dietary vitamin A on growth performance,carcass traits,tissue retinol,and plasma biochemical index,and to determine the optimal vitamin A requirement.A dose-response experiment with 8 treatments(0,500,1000,1500,2500,3500,7000,and 14000 IU/kg)and each treatment contained 8 replicate pens of 8 ducks.The results showed that dietary vitamin A significantly increased daily weight gain(ADG),average daily feed intake(ADFI),retinol concentrations in plasma and liver,triglyceride concentration in plasma,and breast muscle yield percentage,but significantly decreased high density lipoprotein cholesterol(HDL-C)and total cholesterol in plasma(P<0.05).According to the first intercept value of the broken-line(on the plateau)and the quadratic fitted line,the optimal vitamin A requirement of pekin duck from hatch to 21 days of age for plasma retinol concentration,plasma tryglyceride and ADG were 3341.6~6347.6IU/kg.Experiment 2 aimed to investigate the effect of dietary vitamin A on intestinal mucosal development and regulatory mechanism.In this study,both of Vitamin A deficiency and abundance models of Pekin ducks were established by experiment 1.On day 21,birds were killed to obtain duodenal and jejunal tissue for histological analysis and intestinal mucosal samples for enzyme activity examination and proteomic analysis using iTRAQ-PRM method.The results showed that vitamin A significantly increased the villi height,villi area and number of goblet cells in duodenum,and significantly increased the villi height,V/C,villi area and number of goblet cells in jejunum(P < 0.05),but had no significant effect on the ileum morphological indicators(P > 0.05).Vitamin A significantly increased the activity of alkaline phosphatase and disaccharidase in duodenum and that of sucrase and maltose in jejunum(P < 0.05).A total of 147 differential proteins were screened,among which 4 were involved in vitamin A metabolism,23 were involved in cell migration,10 were involved in cell proliferation,4 were involved in mucosal immune,and 39 were involved in metabolism.Taken together,dietary vitamin A promoted the development of duodenum and proximal jejunum by enhancing epithelial cell migration through regulating ECM,Arp2/3,and RhoA pathway,and inhibiting cell proliferation through extending cell cycle.Additionally,vitamin A regulated intestinal lipid metabolism and promoted lipid transmembrane absorption and transport.Experiment 3 aimed to investigate dose-dependent effects of ATRA on enterocyte migration and proliferation,as well as the molecular changes in enterocytes in response to ATRA,including activities of ERK1/2 and its downstream targets Elk-1,c-Jun,Creb and Chop.A dose-response experiment was conducted in IPEC-J2 cell model,which was supplemented with ATRA at 4 doses of 0,3,6,12 μM.The results showed that supplementation of ATRA stimulated the intestinal epithelial cell migration in a dose-dependent manner(P<0.05).Expression of migration-related genes,including ZO-1,ECAD,ICAM-1,and Occuldin,were down-regulated by ATRA supplementation(P<0.05).Cell proliferation viability and the mRNA levels of PCNA,Cyclin B1 and Cyclin D1 were decreased dose-dependently by ATRA(P<0.05).Phosphorylation of ERK1/2 was enhanced by ATRA,while phosphorylation of Elk-1,c-Jun,Creb and Chop was decreased(P<0.05).These results indicated that ATRA enabled the cells to attenuate the mitogenic signal of ERK1/2 through Elk-1,c-Jun,Creb and Chop by uncoupling the phosphorylation of Elk-1 and c-Jun from ERK1/2 activation,but the uncoupling mechanism remain to be further investigated.Experiment 4 was conducted to investigate the effects of dietary vitamin A on growth performance and intestinal mucosal morphology in pekin ducks undergoing LPS challenge.This experiment was designed as a 2x3 factorial arrangement(n=7 replicates/treatment;6 birds/replicate)with 3 dietary concentrations of Vitamin A(0,7000,and 14000 IU/kg)and 2 immune treatments(Injection of LPS or saline)which were given by intraperitoneal injection at 14,16,and 18 days of age.The results showed that LPS injection significantly decreased final body weight,ADG,and ADFI(P<0.05).With the increase of dietary Vitamin A concentration,the reduction percentage of final body weight,ADG,ADFI,and feed/gain.LPS injection significantly increased AST but decreased plasma albumin content,while dietary vitamin A significantly enhanced alkaline phosphate activity and total bile acid in plasma.LPS challenge significantly reduced the villi height,villi area and sucrase activity in duodenum(P < 0.05),the villi area and maltase activity in jejunum(P < 0.05),and vitamin A supplementation significantly increased the villi height,V/C,villi surface area and maltase activity in duodenum and jejunum(P < 0.05).In vitro experiments found that LPS inhibited migration,but retinoic acid promoted epithelial cell migration and significantly increased the phosphorylation of ERK1/2 signaling pathway closely related to migration.In addition,ATRA inhibited the activity of NF-kB.Taken together,these results indicated that vitamin A significantly improved the production performance of Pekin duck after LPS challenge and effectively alleviated LPS damage to liver and intestinal mucosa.In conclusion,these results above showed that dietary vitamin A supplementation could significantly improve the growth performance of starter Pekin duck.Vitamin A supplementation promoted the development of duodenum and its proximal jejunum mucosa and maintained the mucosal health by regulating the active migration of intestinal epithelial cells,cell proliferation,immunity and metabolism.ATRA could significantly increase the ratio of phosphorylated ERK1/2 to unphosphorylated ERK1/2 in IPEC-J2 cells,which potentially was one of the mechanisms through which ATRA stimulated the migration of intestinal epithelial cells.