| Zinc is a trace element essential for animal growth.Substantial studies have shown that zinc is closely related to the intestinal health of animals,and the deficiency of zinc leads to the destruction of intestinal morphology and barrier function,while zinc supplementation can alleviate the compromised intestinal barrier function induced by pathogen via inhibiting the occurrence of intestinal inflammation.China is the largest country in the world in duck husbandry.In the background of thorough prohibition on the use of antibiotics in the animal industry,how to improve the growth performance and maintain the intestinal health of duck by means of nutrition is of great importance for all the nutritionist.Until now,the study on the growth performance and intestinal health of duck is few,while the report on the influence of zinc on the intestinal barrier function of duck is even rare.Three experiments were carried out to investigate the effect and the mechanism of zinc on the intestinal barrier function of meat duck,combining with the study on the in vitro duck intestinal epithelial cells culture,the mechanism of zinc regulate the intestinal barrier function was studied.The main contents and results are presented as follow:Experiment 1 The effect of zinc on the growth performance,intestinal morphology and intestinal barrier function of meat ducksThe present study was conducted to investigate the effect of dietary zinc level on growth performance,meat quality,intestinal morphology and intestinal barrier function of meat ducks.Seven-hundred and sixty-eight 1 d old Pekin ducks were randomly assigned into six dietary treatments.Each treatment had eight replicates with sixteen ducks per replicates.The ducks were fed either a corn-soybean meal basal diet or basal diets supplemented with 15,30,60,120,240 mg Zn/kg from zinc sulfate.This experiment lasted for 5 weeks,the jejunum sample were harvested at the end of starter phase(14 d)and grower phase(35 d).Results shown that:1)The body weight,daily weight gain of the 120 and 240 mg Zn/kg treatment group were significantly higher than the control group at 14 and 35 d(P?0.05).Ducks treated with more than 60 mg Zn/kg had a significantly higher feed intake than the control group in the starter stage and the whole period(P?0.05).Ducks treated with 120 and 240 mg Zn/kg had a significantly reduced feed to gain ratio in the grower stage and the whole period(P?0.05).At 35 d,120 and 240 mg Zn/kg treatment group had higher slaughter weight,carcass weight,eviscerated weight,breast muscle weight and leg muscle weight than that of the control group and 15 mg Zn/kg treatment group(P?0.05).There was no significant difference in dressing percentage,percentage of eviscerated weight,percentage of breast muscle and percentage of leg muscle among all treatment groups(P>0.05).2)Dietary zinc level had no significant effect on p H value of the breast muscle at 45min posts-laughter(P>0.05),while,the 120 and 240 mg Zn/kg treatment group had a significantly higher p H value of the breast muscle at 24 h post-slaughter than that of control(P?0.05).The post-slaughter lightness value of breast muscle of 120 and 240 mg Zn/kg treatment group was significantly decreased(P?0.05).The dietary zinc level had no significant effect on the redness and yellowness value of breast muscles at 45 min and 24 h post-slaughter(P>0.05).120 and 240 mg Zn/kg treatment group had a lower drip loss,shear stress and a higher intramuscular fat content in breast muscle compared with the control and15 mg/kg group(P?0.05).3)Supplemental zinc significantly improved the antioxidant enzyme activity of SOD,GPX,GR,CAT and the content of GSH in the breast muscle of ducks(P?0.05).Compared with the control and 15 mg/Zn kg treatment group,120 and 240 mg Zn/kg treatment group significant decreased the MDA content in the breast muscle(P?0.05).Ducks treated with more than 120 mg Zn/kg significantly improved the gene expression of SOD,GPX,GR,CAT and Nrf2 in the breast muscle(P?0.05).4)The zinc content in the tissues of duck at 35 d increased accordingly as the increasing dietary zinc level.120 and 240 mg Zn/kg treatment group had higher zinc concentration in the breast muscle and blood than that of control group(P?0.05);The levels of zinc in the liver and tibia of 120 and 240 mg Zn/kg treatment group were significantly higher than those in the control group and the 15 mg Zn/kg treatment group.5)At 14 and 35 d of age,the jejunum villus height(VH)was significantly higher in the120 and 240 mg Zn/kg treated group than the control(P?0.05).The jejunum crypt depth(CD)of the 120 and 240 mg Zn/kg treated group was significantly lower than the control group(P?0.05),and the ratio of villus height to crypt depth ratio(VCR)of jejunum in the120 and 240 mg Zn/kg treated group were significantly higher than that of control group at14 d(P>0.05).6)The transcription of physical barrier related gene CLDN-1,OCLD,ZO-1 and ZO-3were significantly higher in the jejunum of 120 and 240 mg Zn/kg supplementation group than that of the other groups in 14 and 35 d(P?0.05),while the gene expression of CLDN-2,a leakage protein,was significantly decreased with the increasing dietary zinc levels(P?0.05).120 and 240 mg/kg of zinc supplementation significantly increased the expression of chemical barrier related gene MUC2 and TFF2,as well as the immune barrier related gene Ig A,p Ig R,LYZ,Av BD2 in jejunum of ducks at 14 and 35 d(P?0.05).These results indicated that zinc supplementation could improve the growth performance,carcass traits,meat quality and the tissue zinc concentration in ducks.Dietary zinc supplementation improved the intestinal morphology and increased the expression intestinal physical barrier,chemical barrier,immunological barrier related genes,which implied an enhanced intestinal barrier function.Base on the broke-line regression analysis,the optimal supplemental zinc should be 105.63 and 121.5 mg/kg according to the ADG and F/G from 1-35 d for meat ducks.Experiment 2 The protective effect of zinc on the intestinal barrier function of ducks challenged with LPSThis aim of the present study is to investigate the protective effect of zinc supplementation on the intestinal barrier function of ducks challenged with LPS.480 1-day-old Pekin ducks were selected and randomly assigned to 6 treatments,8 replicates for each treatment and 10 ducks for each replicate.A 3×2 factor design was used with three dietary zinc levels(0,30,120 mg Zn/kg in the form Zn SO4 was added to the corn-soybean basal diet)and challenged with or without LPS.LPS was intraperitoneal injected to ducks at d 15,17,19,21.The non-challenged group were intraperitoneal injected with saline.FITC-D was gavage to ducks to evaluate the intestinal permeability on day 21.The samples were collected 4 h after last injection.The results were as showed:1)At 1-14 d,the body weight was significantly higher in the 120 mg Zn/kg treatment group than other groups(P?0.05).LPS challenge significantly decreased the body weight at 21 d of age,body weight gain and feed intake from d 15-21(P?0.05).120 mg/kg of supplemental zinc significantly increased the body weight and the body weight gain of meat ducks(P?0.05).A significant interaction between dietary zinc level and LPS injection was observed for body weight and body weight gain,zinc supplementation alleviated the loss of body weight and body weight gain caused by LPS challenge(P?0.05).2)LPS challenge significantly decreased the jejunum VH and the value of VCR and increased the CD(P?0.05).120 mg/kg of supplemental zinc significantly improved VH and VCR,while decreased the CD in jejunum significantly(P?0.05).There was significant interaction between dietary zinc level and LPS injection on the intestinal morphology of the duck,high level of zinc alleviated the decreased VH and VCR,and increased CD of jejunum caused by LPS challenge(P?0.05).3)LPS injection significantly reduced the activities of maltase,AKP,and Na+/K+-ATPase(P?0.05),but improved the mucins-2 content in the jejunum(P?0.05).Zinc supplementation significantly improved the activity of alkaline phosphatase(AKP),Na+/K+-ATPase and the content of MUC2 in jejunal mucosa.There was a significant interaction between dietary zinc level and LPS challenge on the activity of AKP and Na+/K+-ATPase,zinc supplementation significantly alleviated the decreased activity of AKP and Na+/K+-ATPase induced by LPS injection(P?0.05).4)LPS injection significantly increased the intestinal permeability indicated by the increasing permeability maker FITC-D and D-Lactate in serum(P?0.05).Zinc supplementation decreased the FITC-D and D-Lactate in serum(P?0.05).Dietary zinc level and LPS injection had significant interaction effect on the concentration of FITC-D and D-Lactate in serum,zinc supplementation decreased the elevated serumal FITC-D and D-Lactate levels induced by the LPS injection(P?0.05).5)LPS challenge significantly decreased the m RNA level of tight junction related gene CLDN-1,ZO-1,ZO-3,EPCAM and JAM2,and improved the gene expression of CLDN-2and MLCK(P?0.05).Zinc supplementation significantly increased the expression of tight junction related genes in the jejunum CLDN-1,OCLD,ZO-1 and ZO-3,and reduced the expression of CLDN-2 and MLCK(P?0.05).There is an interactive effect between the dietary zinc level and LPS challenge on the gene expression of CLDN-1,ZO-1 and MLCK in jejunum,zinc supplementation could resist the trend of lower CLDN-1,ZO-1 expression and higher MLCK expression in the jejunum caused by LPS injection(P?0.05).6)LPS injection significantly increased the expression of proinflammatory cytokines IL-2,IL-6,IL-8,INF-?,TNF-?,TLR4,i NOS and the expression of anti-inflammatory cytokines IL-10,as well as the apoptosis-related genes caspase 3 and caspase 8 in jejunum(P?0.05).Supplemental zinc significantly decreased the gene expression of IL-2,IL-6,IL-8,INF-?,TNF-?,TLR4,i NOS,caspase-3,caspase-8 in jejunum,while improved the IL-10m RNA level(P>0.05).There is an interactive effect between the dietary zinc level and LPS challenge on the gene expression of IL-2,IL-6,IL-8,IL-10,INF-?,TNF-?,TLR4,i NOS,caspase-3 and caspase-8,high levels of dietary zinc significantly reduced the expression of proinflammatory cytokines and apoptosis-related genes,and promoted the expression of IL-10 in jejunum induced by LPS injection(P?0.05).7)LPS injection significantly reduced the m RNA levels of absorptive cell marker AKP and villin(P?0.05),it also significantly improved the expression of secretory cell marker MUC2,Sox9 and LYZ,as well as the stem cell markers Lgr5,Bmi1,Dll1 and Tert(P?0.05).120 mg/kg of supplemental zinc significantly increased the gene expression of AKP,villin,MUC2,LYZ,Sox9(P?0.05).Dietary zinc levels and LPS injection had interactive effect on the gene expression of Lgr5,Sox9,MUC2,LYZ,Bmi1,Tert and?-catenin in jejunum,namely,high dietary zinc significantly alleviate the decreased m RNA level of AKP,elevated the increasing MUC2,Sox9 and LYZ expression in jejunum,while decreased the m RNA level of Lgr5,Bmi1 and Tert induced by LPS challenge(P?0.05).These results showed that zinc supplementation improve the growth performance of ducks challenged with LPS.High level of zinc decreased the expression of pro-inflammatory cytokines and apoptosis-related genes induced by LPS challenge,improved the gene expression related to intestinal barrier function.The reduce apoptosis of intestinal epithelial cells in turn reduced the intestinal stem cells mobilization,maintained the intestinal morphology and the integrity of intestinal tight junction.Experiment 3 The protective effect of zinc on the barrier function of duck intestinal epithelial cells challenged with LPS injection and the possible mechanism(1)The isolation and characterization of duck intestinal epithelial cellsThe present study was conducted to isolated the duck primary intestinal epithelial cells(DIECs)by enzyme digestion.The epithelial origin of DIECs was characterized by morphology,ultrastructure and immunofluorescence.The absorption capacity of DIECs was measured by digestive enzyme activities,and the integrity of DIECs monolayers was evaluated by transepithelial electrical resistance(TEER)analysis.The results were as follows:1)The small intestine originated from 21 d duck embryo were cut up and digested with collagen enzyme?and neutral protease?,and a large number of cell cluster were acquired.These cell clusters are main compose of intestinal epithelial cells,which can attach to the flask,and the DIECs are grow out from the cell cluster,and reached confluence within 3 d.The DIECs monolayers reached confluence and almost all the cells show the cobblestone morphology of the typical epithelium-like pattern.The DIECs could been subculture for limited passages.With the increase of cell generations,DIECs increased in apoptosis and decreased in proliferation,and swelled in size and stretched in shape.2)Immunofluorescence staining showed that tight junction protein ZO-1 and CLDN-1were expressed between DIECs boundaries,presenting a typical"honeycomb"structure.Under the electron microscope,a large number of microvilli were observed on the DIECs apical membrane.Transmission electron microscopy showed the adjacent DIECs were connected by tight junction on the apical side of the lateral membranes.DIECs could express various intestinal epithelial cell-specific genes.Flow cytometry analysis results showed that the vast majority of cells(90.3%)were positive for epithelial marker CK-18.3)DIECs preserved the activity of AKP and Na+/K+-ATPase,but declined with the increasing passages(P?0.05).After seeding in the Transwell insert,the transepithelial electoral resistance value of DIECs reaches 4000?/cm2 within two days.The above results indicate that we have successfully isolated the duck primary intestinal epithelial cells with a high purity.DIECs exhibit the characteristics of intestinal epithelial cells which can be utilized in subsequent experiments.(2)The protective effect of zinc on the barrier function of DIECs challenged with LPS injection and the possible mechanismThe purpose of this study was to investigate the protective effect of zinc levels on the physical barrier function of DIECs challenged with LPS and the possible mechanism.DIECs was used as the experimental subject.A 3×2 trial design was used with 3 zinc levels(zinc deficiency(2?mol/L TPEN,zinc ion chelator),adequate zinc(control medium),high zinc(20?mol/L Zn)combined with 20?g/m L LPS challenge or not.The results were as follows:1)Zinc levels had significant influence on the TEER value of DIECs(P?0.05),zinc deficiency group had significant decreased TEER value than that of other groups(P?0.05).LPS challenge significantly decreased the TEER of DIECs after treated for 4 or 8 h(P?0.05).Zinc levels and LPS challenge had an interactive effect on the TEER of DIECs.Specifically,zinc deficiency aggravated the decreased the TEER value induce by LPS challenge,while high zinc alleviated the compromised TEER value induced by LPS challenge(P?0.05).Accordingly,LPS challenge lead to an increasing FITC-D permeability of DIECs monolayers(P?0.05).The increased zinc level reduced the FITC-D permeability of DIECs(P?0.05).There was a significant interaction between zinc level and LPS on the FITC-D permeability in DIECs.High levels of zinc decreased the increasing FITC-D permeability of DIECs induce by LPS(P?0.05).2)LPS challenge significantly decreased the gene expression of CLDN-1,OCLD,ZO-1,ZO-3,EPCAM and JAM2 in DIECs,while increased the m RNA level of CLDN-2 and MLCK(P?0.05).High zinc treatment significantly increased the expression of tight junction related gene CLDN-1,OCLD,ZO-1,ZO-3,EPCAM and JAM2 in DIECs,while decreased the m RNA level of CLDN-2 and MLCK(P?0.05).There was an interaction between zinc level and LPS challenge on the gene expression of CLDN-1,OCLD,ZO-1 and MLCK(P?0.05).High zinc treatment improved the compromised gene expression of CLDN-1,OCLD and ZO-1,and decreased the m RNA level of MLCK in LPS challenged DIECs(P?0.05).Western blot results also confirmed that LPS decrease the protein level of tight junction protein CLDN-1,OCLD and ZO-1(P?0.05).High levels of zinc increased the protein expression of CLDN-1,OCLD and ZO-1(P?0.05).There was a significant interaction between zinc and LPS on the tight junction protein levels of DIECs,increasing zinc level alleviated the decreased expression of tight junction protein OCLD and ZO-1(P?0.05).3)LPS challenge significantly increased the expression of pro-inflammatory cytokines IL-2,IL-6,IL-8,INF-?,TNF-?and TLR4(P?0.05),and increased the gene expression of anti-inflammatory cytokine IL-10(P?0.05).Increasing zinc level significantly reduced the gene expression of IL-2,IL-6,IL-8,INF-?,TNF-?and TLR4 in DIECs,while increased the expression of IL-10(P?0.05).Zinc level and LPS treatment had interaction effect on the expression of IL-2,IL-6,IL-8,IL-10,INF-?,TNF-?and TLR4 in DIECs.High zinc treatment significantly decreased the increasing gene expression of IL-2,INF-?,TNF-?,TLR4,and increased the expression of IL-10 in the LPS challenged DIECs(P?0.05).4)LPS significantly improved the m RNA level of apoptosis-related gene caspase-3,caspase-8,BAX,i NOS as well as the cytoprotective gene A20,MT in DIECs(P?0.05).Increasing zinc level significantly reduced the gene expression of caspase-3,caspase-8,BAX and i NOS in DIECs,and increased the expression of MT,A20(P?0.05).There was a significant interaction between zinc level and LPS on gene expression of A20,MT,i NOS,caspase-3.High zinc treatment decreased the increasing m RNA levels of caspase-3 and i NOS induced by LPS treatment(P?0.05),and promoted the increasing gene expression of A20 and MT(P?0.05).Western blot results showed that LPS reduced the phosphorylation of TOR protein in DIECs(P?0.05),which is a molecular plays important part in protein synthesis.Increasing zinc level associated with the increasing phosphorylation TOR in DIECs(P?0.05).Zinc level and LPS challenge had an interaction effect on the phosphorylation level of TOR protein.High dose of zinc alleviated the declining phosphorylation of TOR induced by LPS challenge(P?0.05).5)LPS challenge significantly decreased the activities of AKP,Na+-K+-ATPase(P?0.05),these digestive enzymes not only participated in nutrient absorption,but also played an important part in the maintenance of tight junction structure.High zinc treatment significantly increased the activities of AKP,Na+-K+-ATPase(P?0.05).Zinc level and LPS challenge had an interaction effect on the activities of Na+-K+-ATPase,high zinc treatment increased the compromised activity of Na+-K+-ATPase induced by LPS challenge(P?0.05).In conclusion,zinc supplementation can improve growth performance,intestinal morphology and digestive enzyme activity,and enhance the expression of intestinal barrier function related genes.The optimal level of zinc for meat ducks was estimated to be 121.5mg/kg base on the grow performance from 1-35 d.Zinc supplementation in diet can alleviate the decrease of production performance and the damage of intestinal barrier function caused by LPS stress,which may be due to zinc alleviating the damage of LPS induced inflammatory factors on the expression of tight junction related genes in epithelial cells,reducing the apoptosis caused by inflammatory factors,slowing down the mobilization of intestinal stem cells.Studies on primary duck intestinal epithelial cells also demonstrated that zinc supplementation alleviated the compromised of intestinal barrier function caused by LPS,This might because zinc supplementation decreased the expression of LPS induced pro-inflammatory cytokines,promoted the expression of cytoprotective genes,maintained the activation of TOR signal pathway and prompt the synthesis of intestinal tight junction protein. |
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