| Probiotics are living microbial additives that have beneficial effects on the host by improving the relevant microbial community of the host or the environment,improving feed utilization or its nutritional value,enhancing the response of the host against disease or improving the quality of the surrounding environment.In intensive aquaculture,fish are often subjected to a variety of external adverse stress,resulting in growth retardation,fish disease outbreak and even death in severe cases,which seriously affects the aquatic product quality and the sustainable aquaculture development.Therefore,it is very important to improve fish immunity and reduce diseases.In the face of adverse effects caused by the environment to aquatic animals,chemical drugs or antibiotics are usually used,which may easily cause a series of adverse results such as drug residues,drug resistance,decreased immunity,microecological imbalance,teratogenic,carcinogenic and environmental pollution.As feed additives,probiotics are favorable substitutes for antibiotics.Moderate probiotics in aquatic animal feed can regulate the physiological balance,improve digestion and absorption capacity,promote the growth of aquatic animals,improve the specific and non-specific immunity,and improve the anti-bacteria and anti-virus capacity.Therefore,as feed additives,probiotics have been paid more and more attention and could have a wide potential.Carassius auratus gibelio,also named as gibel carp,is one of the most widely cultivated r species in China.It is characterized by fast growth rate,low oxygen tolerance,tender meat and rich nutrition.However,it is susceptible to be affected by external stress and thus induce a stress response,such as hemorrhage,superficial mucus secretion decreases,and poor feed intake.In this study,we used gibel carp to explore the ameliorative mechanism of Bacillus coagulans(B.coagulans),an effective probiotic,on growth,metabolism,immunity,and antioxidant capacity based on approaches of nutriology,high throughput transcriptome and miRNA sequencing,and bioinformatic analysis.With this research,we are expecting to get deep insights into the molecular regulation and epigenetic mechanism of B.coagulans on the protective effect of gibel carp,uncover the immune-protective effects of B.coagulans,and provide theoretical basis for the use of B.coagulans as an immunostimulant in gibel carp feed.This research is mainly divide into the following six parts:1.Effects of B.coagulans on growth and physiological condition of gibel carpFive isonitrogenous and isoenergetic experimental diets were fomulated containing different contents of B.coagulans(0,125,250,500 and 1000 mg/kg).the effects of different levels of B.coagulans on gibel carp were investigated through 8-week rearing experiment and evaluated by growth,digestive enzyme activity,immune indexes,antioxidant enzyme activity and anti-pathogen infection.Results suggested that 500 mg/kgB.coagulans significantly increased the weight gain ratio(WGR),significantly reduced the feed coversion ratio(FCR),significantly increased the activity of intestinal protease and amylase,significantly increased lysozyme,burst activity,anti-tumor necrosis factor TNF-α,superoxide dismutase(SOD)and anti-superoxide anion(ASAFR)activity,while malondialdehyde(MDA)content was significantly decreased.The cumulative mortality after Aeromonas hydrophilis(A.hydrophilis)infection was significantly reduced.The expression of stress protein HSP70 was significantly increased.These results showed that B.coagulans could effectively improve the growth performance,metabolism,immunity and antioxidant capacity,and improve the pathogen resistance of gibel carp.2.Transcriptome analysis of B.coagulans on gibel carpThe transcriptome analysis was carried out on gibel carp liver with treatment of 0 and 500 mg/kgB.coagulans based on the high-throughput sequencing technology to explore potential regulatory mechanism of B.coagulans on gibel carp.We found that a total of 1969 differentially expression genes(DEGs)in BC500 group,among which 1226 genes were up-regulated and 743 were down-regulated.GO-enriched analysis showed that DEGs were mainly enriched in oxidation-reduction process,proteolysis and metabolic process,cell membrane,and MHC protein complex.The KEGG pathway enrichment analysis indicate that DEGs were mainly enriched in the life process of insulin signaling,endocytosis,phagocytosis,cell adhesion molecules,PPARs signaling and fatty acid synthesis signaling pathways.This indicate B.coagulans regulated the protein metabolism,fat metabolism,immune function and cell signal transduction of gibel carp.In-depth analysis showed that the DEGs induced by B.coagulans mainly affected metabolism and immune function,and the Nrf2-keapl signaling pathway was activated,which was involved in the immune and antioxidant regulation of gibel carp.3.Epigentic analysis of B.coagulans on gibel carp by miRNA sequencingThe miRNA analysis was carried out on gibel carp liver between BC0 and BC500 groups based on the high-throughput sequencing technology,aiming to elucidate the epigenetic mechanism of B.coagulans on gibel carp by differently expressed miRNAs.Results indicated that a total of 779 differentially expressed miRNAs(DE miRNAs)were identified between BC0 and BC500 group,including 466 up-regulated and 313 down-regulated.These DE miRNAs were annotated to 6556 target genes.GO-enriched analysis showed that target genes were mainly enriched in the terms of biological process(regulation of transcription,protein phosphorylation,metabolic process,oxidation-reduction and signal transduction),cellular component(nucleus,cytoplasm,membrane,plasma membrane and intracellular,and molecular function(metal ion binding,DNA binding,nucleotide binding,zinc ion binding and nucleic acid binding,suggesting that the regulatory mechanism of B.coagulans mainly affected transcriptional regulation,metabolism and intercellular information transmission.KEGG enrichment analysis revealed that ingestion of B.coagulans mainly affected MAPK signaling,Wnt antioxidant and cytoskeleton regulation.The results showed that B.coagulans mainly regulated the cellular process,environmental response,metabolism,genetic information transmission and organism regulation of gibel carp.4.Effects of different feeding patterns of B.coagulans on growth,immunity and oxidation resistance of gibel carpDifferent feeding patterns of B.coagulans were established as following:P1,feeding basal diets(0 mg/kg)continuously;P2,feeding 500 mg/kg diet continuously;P3,feeding 1000 mg/kg diet continuously;P4,feeding basic diet for 5 days and 500 mg/kg diet for 2 days;P5,feeding basic diet for 5 days and 1000 mg/kg diet for 2 days.The experiment was conducted for 8 weeks.The effects of B.coagulans on growth,physiology and biochemistry,anti-stress and antioxidant capacity were analyzed.Compared with P1 group,P4 and P5 group increased FBW,WGR and SGR,decreased FCR,increased respiratory burst,myeloperoxidase(MPO)and ASAFR activity,decreased MDA content.The antioxidant signal of Nrf2-keap1 was activated,and the expression of stress proteins such as Prx2 and NOX was up-regulated.The results showed that the optimal feeding mode of B.coagulans improved the growth performance,immunity and antioxidant performance of gibel carp.5.Resistance effects of B.coagulans on gibel carp under high-density stock stressThe above results indicate that B.coagulans exhibited ameliorative function on the growth performance,immunity and antioxidant capacity on gibel carp.In this section,different feeding patterns of B.coagulans(P1,P2,P3,P4 and P5)were applied to evaluate whether it could ameliorate the high-density stock stress.Compared with pre-stress,transcriptional expressions of HSP70 and Prx2 in P3 group were significantly decreased after 24 h and 48 h of high-density stress,indicating that B.coagulans can effectively reduce oxidative stress in gibel carp caused by high density stress.Related blood immunity and serum antioxidant indexes were significantly increased,and serum glucose content was increased.Serum lipid peroxidation product MDA decreased significantly.Transcriptional expression of key stress regulators of HSP70 and Prx2 were down-regulated,the expression of antioxidant related Nrf2-keap1 signaling was effectively alleviated.The results showed that the intermittent feeding of 500 mg/kgB.coagulans could alleviate the metabolic,immune and antioxidant inhibition induced by high density stress.6.Resistance effects of B.coagulans on gibel carp under A.hydrophila infectionBased on the above exprements,here we attempt to evaluate whether different feeding patterns of B.coagulans(P1,P2,P3,P4 and P5)could ameliorate the high-density stock stress.Compared with pre-infection,the serum antioxidant indexes and SOD activity in P5 group were decreased,decreased,and the transcriptional expressions of stress proteins Prx2 and NOX were down-regulated after 24 h and 48 h after A.hydrophilia infection.Meanwhile,the expressions of Nrf2 and Bach1 were significantly down-regulated in different feeding patterns compare with pre-infection,while there have no significant difference among different feeding patterns.After A.hydrophila infection,immune capacity was rescued under 500 mg/kgB.coagulans administration,and antioxidant capacity was rescued under 1000 mg/kgB.coagulans stimulation.This indicate the intermittent feeding of 500 or 1000 mg/kgB.coagulans could effectively reduce the metabolic,immune and antioxidant damage of gibel carp infected with A.hydrophila.Together,this research indicates B.coagulans could improve the growth performance and immune capacity in gibel carp,especially under 500 mg/kg intermittent administration,which significantly improves the anti-resistance and anti-bacterial infection capacity.Therefore,we recommend B.coagulans as an effective immunostimulant for carp aquaculture. |
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