| Zinc (Zn) is an essential trace element for animal that presents multiple physiological functions and antibacterial effect. The inorganic antibacterial agents prepared by loaded zinc ion onto aluminum silicate clay mineral have a good antibacterial effect. However,whether the zinc-bearing inorganic antibacterial agents can serve as a zinc source and alternatives to antibiotic growth promoters is rarely reported. Therefore, we used palygorskite and zinc chloride to prepare Zn-bearing palygorskite (Zn-Pal) by the solid-state ion exchange method. The optimum conditions were determined and the structure of Zn-Pal was characterized. Moreover, the main metal elements of Zn-Pal desorption and antibacterial effects of Zn-Pal were investigated in vitro. The structure of Zn-Pal was characterized before and after digestion. The bioavailability of Zn-Pal as a zinc source for broiler chickens was evaluated. The effects of Zn-Pal on growth performance,immunity and cecal microflorae serving as a zinc source and alternatives to antibiotic growth promoters were evaluated. The study was composed of six trials as below,Trial 1 was conducted to investigate the optimum conditions of the solid-state ion exchange method for preparing Zn-Pal. The structure of palygorskite and Zn-Pal was characterized by DEXRF, XRD, FTIR, BET analysis, Zeta potential, SEM and TEM. The results showed that the mass ratio of raw materials, reaction temperature and reaction time affected significantly the zinc content of Zn-Pal (P<0.05),which was ranked in the order as temperature>time> mass ratio of materials. The optimum conditions of the solid-state ion exchange method for preparing Zn-Pal were palygorskite and zinc chloride mass ratio of 4:1, reaction temperature 300? and reaction time 3 h. The process of preparing Zn-Pal did not affect the crystal structure of palygorskite. The specific surface area of Zn-Pal was lower than that of palygorskite. Zeta potential of Zn-Pal was enhanced as compared with palygorskite.Trial 2 was conducted to investigate the main metal element desorption of Zn-Pal by simulating crop, gizzard and small intestinal pH value of broilers in vitro. The structure of Zn-Pal was characterized before and after treating by different pH value. The results showed that the desorption rate of loaded-zinc was 68.22%?71.79%. The loaded-zinc released quickly in pH 5.0, which decreased in pH 2.5. The desorption rate of loaded-zinc was the lowest in pH 6.0. Mg, Al, Fe of Zn-Pal could release, where the release amount of Mg was the highest. The release amount of Mg, Al, Fe was the highest in pH 2.5.The crystal structure of palygorskite was not changed after treated by different pH value. The specific surface area of Zn-Pal enhanced while Zeta potential of Zn-Pal decreased after treated by different pH value.Trial 3 was conducted to investigate the antibacterial effect and adsorption of lipopolysaccharide (LPS) of Zn-Pal. The results showed that Zn-Pal had antibacterial activity against avian Escherichia coli (E. coli). The activity increased as the concentration of Zn-Pal. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Zn-Pal were 17.5 mg/mL and 40 mg/mL and the respective zinc ion concentration was 0.83 mg/mL and 1.89 mg/mL. The fluorescence intensity of DNA and RNA decreased after E. coli was treated with Zn-Pal for 2 h (P<0.05). The fluorescence intensity decreased as the concentration of Zn-Pal (P<0.05). Zn-Pal reduced the LPS concentration as dosage.Trial 4 was conducted to evaluate the bioavailability of Zn-Pal in broilers. A total of 432 one-day-old Arbor Acres broilers were randomly assigned to nine groups administered different dietary treatments for 42 days. The broilers were fed a basal diet supplemented with 0, 20, 40, 60 or 80 mg/kg of Zn in the form of either feed-grade ZnSO4 or Zn-Pal,respectively. The results showed that the growth performance was not affected by the addition of Zn (P>0.05). The contents of Zn in the pancreas, tibia and breast muscle increases with supplemental Zn levels at 21 d and 42 d (P<0.01). At 21 days of age, the content of Zn in the blood of broilers fed diets supplemented with Zn-Pal was higher than that of broilers fed diets supplemented with ZnSO4 (P<0.01). Supplementation of Zn-Pal in 40?80 mg/kg zinc increased zinc content in the blood of broilers at 21 d (P<0.05). The broilers fed diets supplemented with 60 mg/kg zinc as Zn-Pal and 80 mg/kg zinc as ZnSO4 had the highest tissue zinc content and there was no difference between them (P>0.05 ).With ZnSO4 as a reference, at 21 days of age, the relative biological values (RBV) of Zn-Pal were 109.64%, 100% and 100%, as determined using the contents of Zn in the pancreas, tibia and breast muscle, respectively, as response parameters. At 42 days of age,the RBV of Zn-Pal was 86.32% and 119.55% based on the contents of Zn in the pancreas and tibia.Trial 5 was conducted to investigate the effects of Zn-Pal on growth performance and immunity of broilers as a zinc source and alternatives to antibiotic growth promoters. A total of 336 one-day-old Arbor Acres broilers were randomly assigned to seven groups,each with six replicate cages of eight birds, administered seven dietary treatments as follows, the basal diet (Control group, no zinc supplementation or antibiotic growth promoters), the basal diet+1.28 g/kg heat modified palygorskite (PAL group), the basal diet+ZnSO4 (ZnSO4 group,supplemented with 60 mg/kg zinc),the basal diet+ZnSO4+40 mg/kg chlortetracycline (NOR group, supplemented with 60 mg/kg zinc), the basal diet+0.43 g/kg Zn-Pal (Zn-Pal-20 group, supplemented with 20 mg/kg zinc),the basal diet+0.85 g/kg Zn-Pal (Zn-Pal-40 group, supplemented with 40 mg/kg zinc), the basal diet+1.28 g/kg Zn-Pal (Zn-Pal-60 group, supplemented with 60 mg/kg zinc).The results showed that the broilers in NOR and Zn-Pal groups had higher average body weight (ABW)and average daily gain (ADG) than that in control group (P<0.05). The broilers inZn-Pal-40 had bigger spleen index than that in control group (P<0.05). The supplementation of Zn-Pal increased the bursa index compared with control group (P<0.05).No differences were observed in the bursa index between NOR and Zn-Pal groups (P>0.05).The interleukin 6 (IL-6) content in the serum of broilers in control group was lower than that of broilers in other groups (P<0.05). The complement 3 (C3) content in the serum of broilers in Zn-Pal-40 group, as well as Zn-Pal-60, was higher than that of broiler in control group (P<0.05) while there was no difference compared with NOR group (P>0.05).Compared with control group, the complement 4 (C4) contents in the serum of broilers was increased in ZnSO4 group, NOR group, Zn-Pal-40 group and Zn-Pal-60 group (P<0.05).The lysozyme activity in the serum of broilers in NOR group was higher than that of broilers in control group (P<0.05). The content of immunoglobulin G (IgG) in duodenal mucosa of broilers in Zn-Pal-40 group and Zn-Pal-60 group was higher than that of broilers in control group (P<0.05) while there was no difference compared with NOR group(P>0.05). The content of IgG in jejunal mucosa of broilers in control group was the lowest among all groups (P<0.05). The slgA secreting cells positive area in duodenum of broilers in Zn-Pal groups and ZnSO4 group was larger than that of broilers in control group and PAL group (P<0.05) while there was no difference compared with NOR group (P>0.05).The mRNA expression of related genes in the Toll-NF-?B signaling pathway in the ileum mucosa of broilers in control group was enhanced compared with other groups (P<0.05).Trial 6 was conducted to investigate the effect of Zn-Pal on cecal microorganism florae of broilers by plate count and high-throughput sequencing technology. A total of 336 one-day-old Arbor Acres broilers were randomly assigned to seven groups, each with six replicate cages of eight birds, administered seven dietary treatments as follows, the basal diet (Control group, no zinc supplementation or antibiotic growth promoters), the basal diet+1.28 g/kg heat modified palygorskite (PAL group),the basal diet+ZnSO4 (ZnSO4 group, supplemented with 60 mg/kg zinc), the basal diet+ZnSO4+40 mg/kg chlortetracycline (NOR group, supplemented with 60 mg/kg zinc), the basal diet+0.43 g/kg Zn-Pal (Zn-Pal-20 group, supplemented with 20 mg/kg zinc), the basal diet+0.85 g/kg Zn-Pal (Zn-Pal-40 group, supplemented with 40 mg/kg zinc), the basal diet+1.28 g/kg Zn-Pal (Zn-Pal-60 group, supplemented with 60 mg/kg zinc). The results showed that E.coli, Salmonella and Lactobacillus colonies of cecal content of broiler in NOR group decreased by 6.00%,1.14% and 4.54% compared with control group (P>0.05). Compared with control group, E. coli and Salmonella colonies of cecal content of broiler in Zn-Pal-40 group decreased by 5.75% and 3.27% (P>0.05) while Lactobacillus colonies increased by 4.04%. In phylum and class level, the supplementation of Zn-Pal to supply 20, 40, or 60 mg/kg of Zn had no effects on the abundance and diversity of cecal microorganism florae of broilers.Compared with Zn-Pal-60 group, the relative abundance of Lactobacillus in cecal content of broiler in control group, PAL group, ZnSO4 group, NOR group, Zn-Pal-20 group and Zn-Pal-40 group decreased by 58.49%, 23.13%, 19.71%, 71.73%, 24.48% and 9.43% in genus level (P>0.05).Based on those results, it can be concluded as follows:1) The optimum conditions of the solid-state ion exchange method for preparing Zn-Pal were palygorskite and zinc chloride mass ratio of 4:1, reaction temperature 300?and reaction time 3 h, which was ranked in the order as temperature>time> mass ratio of materials. The process of preparing Zn-Pal did not affect the crystal structure of palygorskite.2) The desorption rate of loaded-zinc was 68.22%?71.79% in vitro. Low pH value promoted the release of magnesium, aluminum and iron in Zn-Pal to release. The crystal structure of palygorskite was not changed after treated by different pH value.3) Zn-Pal had antibacterial activity against avian Escherichia coli (E. coli) by loaded-zinc. Zn-Pal had the adsorption against lipopolysaccharide by changed surface charge of palygorskite. The MIC and MBC were 17.5 mg/mL and 40 mg/mL meaning zinc ion concentration was 0.83 mg/mL and 1.89 mg/mL.4) With ZnSO4 as a reference, the zinc bioavailability of Zn-Pal was 100% and 119.55% at 21 d and 42 d, respectively, as determined using the contents of Zn in the tibia as response parameters. The optimal level of supplemented Zn was 60 mg/kg in the form of Zn-Pal for broilers from 1 to 42 days of age.5) Zn-Pal could enhance growth performance and immunity of broilers as a zinc source and alternatives to antibiotic growth promoters. The optimal dosage of Zn-Pal was 40?60 mg/kg zinc.6) The immunomodulatory mechanism of Zn-Pal might be associated with improved intestinal mucosal immune function by TLRs-NF-?B signaling pathway and balancing cecal microflora. |
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