| With the restriction of antibiotics in aquaculture,a large number of immunopotentiators and immunomodulators have been rapidly developed and applied.In recent years,the continuous development of nanotechnology has provided effective ideas and technical guidance for the development of new immunopotentiators and immunomodulators.Nano-Selenium is a nanoparticle forrmed by using the amide plane of the protein or the hydroxyl group of the polysaccharide to attract red element selenium.It has high absorption efficiency,low toxicity,strong biological activity,and low environmental pollution,and thus has broad application prospects.In this study.abalone viscera polysaccharide-protei(AVPSP)and vitamin C were used to reduce sodium selenite to produce elemental selenium,which combined with AVPSP template to form nano-level elemental selenium colloid,with the aim to prepare the abalone viscera polysaccharide-protein(AVPSP-SeNps).The preparation conditions were optimized,and the prepared AVPSP-SeNps were characterized.Tilapia(Oreochromis niloticus),a representative aquaculture fish with important economic value was selected to investigate the biological effects of AVPSP-SeNps.AVPSP-SeNps were added to the feed to study its effects on growth performance,immune genes and intestinal microbiota of tilapia,and the possible impact mechanisms were also discussed,with the aim to lay a foundation for the development of aquatic immunopotentiators,and to promot the the application of AVPSP-SeNps in aquaculture.The contents and results of this study are as follows:(1)AVPSP-SeNps were prepared by binding the elemental selenium,derived from sodium selenite reduction,to the AVPSP templates.The optimal preparation conditions of AVPSP-SeNps were:the concentrations of Na2SeO3,Vc and AVPSP were 1 mM,5 mM and 0.08%,respectively,and the reaction temperature was 25℃,the reaction time was 10 h.The prepared AVPSP-SeNps is uniform and stable.After 14 days of dialysis,the particle size is about 60 nm and could stable for at least 6 months.(2)The physical properties of AVPSP-SeNps synthesized under optimal conditions were characterized.XRD results show that the synthesized nano-selenium particles are amorphous selenium.STEM results show that the synthesized AVPSP-SeNps has a spherical shape with a particle size of about 63 nm and good dispersion.There is no functional group transition between AVPSP and AVPSP-SeNps.The-NH,-CONH2,-COO-,C-O and glycosidic bonds C-O-C in AVPSP are related to AVPSP-SeNps stability.The TSI results show that AVPSP plays a key role in maintaining the stability of SeNps.AVPSP-SeNps is stable in neutral alkaline but unstable under acidic conditions.(3)AVPSP-SeNps were added to fish feed at low(group L,0.5 mg/kg,selenium/feed,the same as below),medium(group M,1.5 mg/kg),high(group H,4.5 mg/kg)doses,and were used to feed tilapia juveniles,with the aim to investigate the effects on growth performance and immune gene expressions of tilapia,the results are as follows:A.Effects on growth performance:In the AVPSP-SeNps added groups,all the average weight,body weight,weight gain rate and specific growth rate of tilapia juveniles were higher than the controls,and most of the liver index and fatness were also higher(except for the H group on the 30th day),while most of the feed coefficients were lower than the controls(except for the groups M and H on the 15th day).The results indicated that the addition of AVPSP-SeNps to the feed promoted the growth of tilapia.The effects of different concentration treatments were different,it seemed that the promotion effects of groups L and M were better than that of the groups L.B.Effects on immune-related genes:In the AVPSP-SeNps added group,all the intestine immune-related genes(HSP70,IFN-γ,lyzc,IL-1β,TNF-α)of tilapia were up-regulated to varying degrees.The amplitude of up-regulation of each immune gene was different among treatment times and treatment concentrations:in the treatment groups L and M,the up-regulation of most of the immune genes reached significant levels at 15 d and 45 d(P<0.05,except for the genes HSP70 and IL-1β),on the 30th day,the expression levels of lyzc of group H,IFN-γ of group M,IL-1β of groups L and M,and TNF-α of group L up-regulated significantly(P<0.05),while the expression levels of other genes were not significant different with contros.In addition,the up-regulation of other immune genes did not reach a significant level.On day 45,in the treatment group H,except for the IL-1β gene,the expression levels of other immune genes were not significant different with controls.(4)The effects of AVPSP-SeNps added feed on the intestinal microbiota of tilapia was studied by Illumina sequencing.The results show:The composition of intestinal microbiota of tilapia juvenile fish in different stages was inconsistent.On the 15th day,the most dominant phyuma in the intestines of tilapia juveniles was Actinobacteria(71.49%),and the subdominant were Proteobacteria,Fusobacteria,Firmicutes and Bacteroidetes.The dominant genus is Micrococcaceae_p_unclassified(66.49%),and the subdominant genera were Cetobacterium,Sphingomonas,Microbacterium,Pseudomonas and Lactobacillus;on the 45th day,the dominant phylum changed to proteobacteria(89.78%),and the subdominant were Fusobacteria,Bacteroidetes,Firmicutes and Actinobacteria.The dominant genus was Pseudomonas(87.10%),and the second dominant genus was Cetobacterium(4.18%).Feeding AVPSP-SeNps added feed affected the composition of intestinal microbiota of tilapia significantly,but the effects of different feeding doses and different feeding durations were different.On the 15th day of the experiment,the abundance of the probiotic genus Cetobacterium increased in the low and medium dose of AVPSP-SeNps treatment groups.On the 45th day,the abundance of the probiotic genus Akkermansia increased significantly in the low and high dose treatment groups.Significant changes in bacterial abundance were also detected in Macellibacteroides,Sphingomonas,Lactobacillus,Pseudomonas,Bacteroidetes,and other six unknown genera. |
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