Study On The Biological Functions And Related Mechanisms Of Zinc Bearing Palygorskite In Blunt Snout Bream

Zinc(Zn)is an essential trace element required for normal growth,several biological functions and antibacterial effect for fish.Palygorskite(Pal)is a layered silicate clay mineral with a large specific surface area and good adsorption,cation exchange,adhesion,and carrying capacity.Zn-bearing palygorskite(Zn-Pal)is an inorganic antibacterial agent prepared by ion-exchange method based on the characteristics of Pal and has the ability to release zinc ions slowly.This study aimed to investigate that Pal inclusion in the diet effects on the pelleting technological characteristics,growth performance and tissue trace elements content of blunt snout bream,(Megalobrama amblycephala),evaluated dietary Zn-Pal inclusion effects on the tissue trace elements content,antioxidant capability,immunity,and its resistance to transport stress.Then,the effects of Zn-Pal and zinc sulfate on the intestinal microflora,zinc resistance gene abundance,and antibiotic resistance gene abundance were compared.This study consisted of the following 6 trials:Trial 1 Effects of palygorskite inclusion on pelleting technological characteristics,growth performance and tissue trace elements content of blunt snout breamDiets without(control group)or with 2%palygorskite were pelleted and were measured for pelleting technological characteristics.Then the diets were fed to two groups of blunt snout bream(average weight:11.61±0.05 g)in four replicates(30 fish per replicate)for six weeks.Compared with the control group,dietary 2%palygorskite inclusion enhanced pellet production rate,pellet hardness and the degree of starch gelatinization of pellet,and reduced the percentage of fines(P<0.05).There were no difference on final body weight,percent weight gain,feed conversion rate,condition factor,hepatosomatic index,and viscera/body ratio of fish between groups(P>0.05).Palygorskite supplementation significantly enhanced the intestinal amylase activity,Fe contents in the blood and muscle,and Zn content in the muscle,but reduced muscular Cd concentration(P<0.05).Trial 2 Effects of Zn-Pal supplementation on the growth performance,nutrient retention,and meat quality in blunt snout breamA total of 600 fish(average weight:6.58±0.13 g)were randomly assigned to five groups and distributed into 15 tanks(400 L each)at a rate of 40 fish per tank.The fish were fed a basal diet without an exogenous Zn source and the basal diet supplemented with 125 mg/kg Zn as Zn sulfate(ZnSO4)or 35,80,or 125 mg/kg Zn as Zn-Pal.Each diet was tested using three replicates for seven weeks.The results showed that the X-ray diffraction patterns of Zn-Pal shows no obvious change compared with the raw Pal.Dietary Zn-Pal supplementation enhanced the percent weight gain(quadratic,P=0.009),specific growth rate(quadratic,P=0.009),hepatosomatic index(quadratic,P=0.034),increased the number of leukocytes(linear,P=0.001),lymphocyte(linear,P=0.003),neutrophil(linear,P=0.023),and erythrocyte(linear,P=0.034),plasma total protein(quadratic,P=0.005)and globulin contents(quadratic,P=0.002),the nutrients retention of organic matter(quadratic,P=0.001)and crude protein(quadratic,P=0.002),the whole body of organic matter(quadratic,P=0.025)and crude protein(linear,P=0.049;quadratic,P=0.021)contents,decreased the feed conversion rate(linear,P=0.015;quadratic,P=0.003),plasma urea nitrogen level(quadratic,P=0.031)and the moisture content of the whole body(quadratic,P=0.025)in blunt snout bream.Dietary Zn-Pal inclusion decreased the muscular cooking loss(quadratic,P=0.047),increased the total superoxide dismutase(T-SOD)and copper/zinc superoxide dismutase(Cu/Zn-SOD)activities of muscle in blunt snout bream.Dietary 0.17%Zn-Pal(48 mg/kg Zn)inclusion had the better specific growth rate and feed conversion rate.Trial 3 Effects of Zn-Pal supplementation on the trace elements contents and the relative mRNA levels of Zn transporter protein in blunt snout breamThe present study shared the same details of the experiment design as that of Trial 2.The results showed that dietary Zn-Pal inclusion decreased the contents of Cu(linear,P<0.001),Mg(linear,P=0.009),Mn(linear,P=0.008),Cr(linear,P=0.047).and Cd(linear,P=0.001)of blood,reduced the concentration of Cu(linear,P<0.013;quadratic,P=0.001),Mn(linear,P=0.040),Pb(linear,P=0.004),Cr(linear,P=0.003),and Cd(linear,P=0.002)of muscle,enhanced the hepatopancreas Zn(linear,P=0.005)content and vertebra Fe(linear,P=0.046)level,reduced the hepatopancreas Cd(linear,P=0.012)content and vertebra Cu(linear,P=0.043)content,increased the concentration of Zn(quadratic,P=0.007),Fe(linear,P=0.013),and Cu(linear,P=0.049)of scale,decreased the Cd(linear,P=0.018)accumulation of scale in blunt snout bream.Zn-Pal inclusion in the diet enhanced the mRNA levels of metallothionein(linear,P=0.015),metal response element-binding transcription factor-1(quadratic,P=0.020),Zn-transporter proteins 5(linear,P=0.002),and Zn-transporter proteins ZIP 14(linear,P=0.010)of intestine in blunt snout bream.Compared with the ZnSO4 group,dietary 35 mg/kg Zn as Zn-Pal inclusion enhanced the mRNA level of metal response element-binding transcription factor-1(P<0.05).The addition of 80 mg/kg Zn as Zn-Pal in the diet enhanced the mRNA level of metallothionein(P<0.05).Trial 4 Dietary Zn-Pal inclusion effects on intestinal development,antioxidant capability,cytokines expressions,and microflora in blunt snout breamThe present study shared the same details of the experiment design as that of Trial 2.The results showed that Zn-Pal enhanced the intestinal length(quadratic,P=0.030),weight(linear,P=0.047),intestinal T-SOD(linear,P=0.014),Cu/Zn-SOD(linear,P=0.027),and catalase(CAT;quadratic,P=0.014)activities,decreased the number of Escherichia coli(linear,P=0.011)and Aeromonas(linear,P=0.003)in intestinal digesta,improved the hindgut morphology of villus height(linear,P=0.034;quadratic,P=0.034),crypt depth(quadratic,P=0.040),and villus height/crypt depth(quadratic,P=0.007)in blunt snout bream.Fish fed Zn-Pal enhanced the mRNA levels of nuclear factor erythroid 2-related factor 2(Nrf2;linear,P=0.005),Cu/Zn-SOD(linear,P=0.046),manganese superoxide dismutase(Mn-SOD;linear,P=0.023),and CAT(linear,P=0.045),decreased the mRNA levels of toll-like receptors 2(linear,P=0.011),toll-like receptors 4(linear,P=0.007),tumor necrosis factor receptor associated factor 6(linear,P=0.003),nuclear factor-kappa B(linear,P<0.001;quadratic,P=0.002),interleukin 6(linear,P=0.024:quadratic,P=0.030),and tumor necrosis factor-α(quadratic,P=0.018)in intestine.Zn-Pal groups exhibited no significant difference with ZnSO4 group.Trial 5 Dietary Zn-Pal inclusion effects on antioxidant capability,immune response,and the resistance to transport stress in blunt snout breamA total of 600 fish with similar size(average weight:27.78±0.30 g)were randomly distributed into 20 tanks(678 L each)with 30 fish per tank.Fish in each tank were randomly assigned one of four experimental diets containing the basal diet,and the basal diet supplemented with 90 mg/kg Zn as ZnSO4 or 48,or 90 mg/kg Zn as Zn-Pal,and the analyzed levels of Zn were 55.03,143.79,105.49,and 147.15 mg/kg,respectively.Each diet was tested in five tanks for 60 days.Before transport stress,dietary Zn-Pal inclusion enhanced the final body weight,feed utilization,respiratory burst activity of blood,and the activities of plasma alkaline phosphatase,T-SOD,Cu/Zn-SOD,decreased the plasma cortisol,lactate and component 4 contents,increased the mRNA levels of Nrf2,CAT,Cu/Zn-SOD in hepatopancreas of blunt snout bream(P<0.05).The plasma crotisol,glucose,lactate,triiodothyronine 3,and malondialdehyde contents,as well as the aspartate aminotransferase activity,alanine aminotransferase activity,the respiratory burst activity of blood,the malondialdehyde content,and the activities of CAT,T-SOD,Cu/Zn-SOD,and the mRNA level of Nrf2 were increased under transport stress(P<0.05).The CAT and acid phosphatase activities of plasma were decreased under transport stress(P<0.05).After transport stress,dietary Zn-Pal inclusion decreased the contents of plasma crotisol,glucose,and triiodothyronine 3 contents,and the alanine aminotransferase activity(P<0.05),reduced the malondialdehyde content of hepatopancreas,enhanced the lysozyme activity,component 4 content,acid phosphatase activity,total antioxidant capacity(T-AOC),CAT activity,T-SOD activity of plasma,the T-AOC and the activities of CAT,T-SOD,Cu/Zn-SOD in hepatopancreas(P<0.05),up-regulated the mRNA levels of CAT and heat shock proteins 90(P<0.05).The addition of 48 mg/kg Zn as Zn-Pal exhibited higher T-AOC than the ZnSO4 group(P<0.05).Trial 6 Dietary Zn-Pal inclusion effects on the diversity,zinc resistance gene,and antibiotic resistance genes of intestinal microorganism in blunt snout breamThe present study shared the same details of the experiment design as that of Trial 5.Compared with the control group,dietary 90 mg/kg Zn as Zn-Pal or ZnSO4 decreased the shannon indice,the abundance of Aeromonas and Cyanobacteria,increased the simpson indice and the abundance of Cetobacterium of intestinal microorganism in blunt snout bream(P<0.05).The addition of ZnSO4 decreased the sobs indice of intestinal microorganism(P<0.05).However,the Zn-Pal groups exhibited no difference on sobs indice of intestinal microorganism compared with the control group(P>0.05).Dietary 48 mg/kg Zn as Zn-Pal had no difference on alpha diversity index(P>0.05),but increased the abundance of Erysipelotrichaceae of intestinal microorganism(P<0.05).The inclusion of ZnSO4 enhanced the abundance of ZnT A,tet W,aac(6’)-Ib-cr,sul 1 of intestinal microorganism(P<0.05).The inclusion of 90 mg/kg Zn as Zn-Pal enhanced the abundance of ZnT A,czc D,tet W,tet X,aac(6’)-Ib-cr,sul 1 of intestinal microorganism(P<0.05).The inclusion of 48 mg/kg Zn as Zn-Pal just enhanced the abundance of aac(6’)-Ib-cr and sull of intestinal microorganism(P<0.05).In addition,the inclusion of ZnSO4 group exhibited higher abundance of ZnT A than Zn-Pal groups(P<0.05).In conclusion:1.Dietary 2%palygorskite supplementation could enhance pellet production efficiency and pellet quality,increase the tissues Fe and Zn contents,and decrease the Cd accumulations in the tissues without impairing growth performance of blunt snout bream.2.Zn was loaded on Pal in an ionic form by ion-exchange process in Zn-Pal without impairing the crystal phase.3.Dietary Zn-Pal inclusion improved the growth performance,feed utilization,meat quality and trace element content,increased the antioxidant capability,enhanced the immunity capability and the resistance to transport stress of blunt snout bream.Zn-Pal was more efficient than ZnSO4 and could decrease the amount of Zn levels used in the diet of blunt snout bream.The suitable amount of Zn-Pal in the diet is 0.17%(48 mg/kg Zn).4.The addition of Zn could decrease the abundance and diversity of intestinal microorganisms,improve the microflora with the increased zinc level to a certain extent in blunt snout bream.However,the abundance of zinc resistance genes and antibiotic resistance genes will be increased with the increased zinc level.The effects of ZnSO4 on the reduction of intestinal microorganism richness,and the enhancement of zinc resistance gene abundance is stronger than Zn-Pal(48 mg/kg Zn)in blunt snout bream.