Impact Of Diet Supplementation With Three Autochthonous Strains On Growth Performance,Immune Indicators And Intestinal Health In Juvenile Cobia (Rachycentron Canadum)

It has been proven that probiotics can benefit aquaculture,but the success of probiotics as post-treatments has been questioned,which is worrying.Parallel gene transfer via Gram-negative probiont may facilitate the transmission of virulence genes and enhance antibiotic resistance.It is common in third-world countries for probiotic products to not specify the strains and species of probiotics that are present or the exact number of cells in each.This raises serious concerns about the quality of products sold in these countries.The most effective way to improve aquaculture probiotics’efficacy and efficiency is to use a native isolate aimed at quality and certainty.Therefore,the present study aimed to isolate the indigenous bacterial species from healthy cobia fish and then selected 3 strains,including Bacillus sp.RCS1（MW560712）,Pantoea agglomerans RCS2（MW560713）and Bacillus cereus RCS3（MW560714）from the gut of juvenile’s cobia having advantageous assets or positive characteristics.Their analysis indicated the presence of similar biochemical profiles,and all could effectively utilize carbon sources.After ten days of injecting the fish with isolated bacteria,the biosafety assessment did not show any pathological symptoms.The results showed that all the isolated bacteria in the present study had a low auto-aggregation capacity within the first three hours of incubation.The isolated bacteria showed strong tolerance when exposed to a range of pH.Although asymmetrically,a slow rise in the growth of isolated bacteria was observed within the pH range of 1-8 for RC1,1-7 for RC2 and 1-6for RC3.The antagonistic effects of isolated bacterial strains on the development of pathogens,including Vibrio alginolyticus,Vibrio harveyi,Streptococcus iniae,and Streptococcus agalactiae,were investigated using Luria-Bertani（LB）agar plates.All the isolated bacteria exhibited inhibitory effects against the pathogens,including V.alginolyticus,V.harveyi,S.iniae,and S.agalactiae.These isolated bacteria were characterized by a wide range of antagonistic activities,non-hemolytic activities,high survivability after heat treatments,safety confidence,and antibiotic susceptibility.Cobia（Rachycentron canadum）juveniles were utilised as an aquatic model to explore growth performance and immune indices after 70 days of feeding with supplementation of viable isolated microbes’concentrations of 1×10¹⁰ and 1×10¹² CFU mL^-1 indigenous isolates Bacillus sp.RCS1 and Bacillus cereus strain RCS3,respectively.Regarding growth performance,fish provided with the indigenous isolates exhibited a significant（P<0.05）enhancement.The hematological indices such as red blood cells,white blood cells,corpuscular haemoglobin concentration,mean corpuscular volume,haemoglobin,and Mean corpuscular haemoglobin were all increased（P<0.05）in the juvenile cobia fed with indigenous isolates Bacillus sp RCS1 and B.cereus RCS3 as a nutritional supplement.This study also significantly differed in serum analysis（albumin,globulin,lysozyme,total protein）between all the treatments with the Bacillus sp RCS1 1×10¹⁰ proven to be the best.In the present study,the expression of myostatin and insulin-like growth factor 2 showed no significant change（P>0.05）among the treatment and control groups tested nonetheless,their mRNA expression exhibited variations in all trial groups.In the present study,there were significant differences（P<0.05）among groups in the case of insulin-like growth factor1,peroxisome proliferator-activated receptors.It is possible that the improved antioxidant enzyme activities（CAT and SOD）,digestive enzyme activities（amylase,trypsin,pepsin,lipase,and cellulose）are linked to the enhanced resistance of fish against Vibrio harveyi infection.The enhanced activities of digestive enzymes in indigenous isolates treatments might result in improved digestion and enhanced food absorption that ultimately help enhance feed utilisation and growth performance.The results showed that indigenous isolates addition with a commercial diet had the prospective to enhance cobia juveniles’growth and immune response regardless of their dosage.Additionally,this study also explored the possible effects of dietary additives of indigenous Pantoea agglomerans RCS2 on growth,haematological parameters,biochemical blood indices,antioxidant enzyme activities,disease resistance,digestive enzyme activities,and molecular immune response in juvenile’s cobia fish.After 70 days,the growth parameters（i.e.,specific growth rate,weight gain,and final weight）and feed conversion ratio significantly improved（P<0.05）compared to those fed diets without supplementation.Also,haematological parameters such as red blood cells,white blood cells,corpuscular haemoglobin concentration,mean corpuscular volume,haemoglobin,and Mean corpuscular haemoglobin were high significantly（P<0.05）in supplemented groups.The serum blood biochemical indices aspartate aminotransferase,alanine aminotransferase,alkaline phosphatase,triglyceride,cholesterol,and glucose recorded no significant difference（P>0.05）while globulin,total protein,and albumin were high significantly（P<0.05）in supplemented groups as compared to the group without supplement.The current results displayed that the activities of superoxide dismutase,glutathione peroxidase,and catalase in the liver and head kidney were affected by dietary supplements of indigenous isolated microorganisms.An exceptionally low mortality rate was observed in the treatment groups than the group without supplemented diets when challenged with pathogenic bacteria.Similarly,light microscopy revealed an increase in the intestinal villus length in fish fed with the supplemented diet compared to those without supplements.In this study,the higher digestive enzyme activities show that supplemented bacteria isolates have impacted digestive enzyme secretion in juvenile cobia.Regarding the gene expression,Interleukin-1 Beta（IL-1β）,Tumour necrosis factorα（TNFα）,and Interleukin-8（IL-8）were all up-regulated by indigenous isolates.The current results show that the dietary additive of indigenous Pantoea agglomerans RCS2 could constitute a treasured nutritional approach in the path of a sustainable cobia aquaculture regardless of the dosage used in this study.Furthermore,this study also assessed the effects of three autochthonous strains mixture on cobia fish（Rachycentron canadum）.In the trial group,juvenile cobia received diets supplemented with 1×10¹² CFU mL^-1 autochthonous strains for ten weeks and a control diet without supplementation.Juvenile cobia receiving diets supplementation exhibited significantly improved growth than those without supplementation（control）.Haematological indices,such as Red blood cells,white blood cells,corpuscular haemoglobin concentration,mean corpuscular volume,haemoglobin,and mean corpuscular haemoglobin,were higher in the supplemented group.Similarly,digestive enzymes（amylase,lipase,cellulose,pepsin,and trypsin activities）were higher in supplemented diet with an indigenous isolates mixture.Serum biochemical parameters albumin,globulin,and total protein were significantly higher,while aspartate aminotransferase,alanine aminotransferase,alkaline phosphatase,triglyceride,and cholesterol showed no significant difference.On the other hand,glucose was more elevated without supplementation.On gene expression in the midgut,Immunoglobulin,Colony-stimulating factor receptor 1,and Major histocompatibility complex 1 were all up-regulated by indigenous isolates mixture while T cell receptor beta,and Major histocompatibility complex 2 showed no significant difference.The results in the present study showed that autochthonous strains mixture has significantly enhanced the growth,survival,and innate and adaptive immunities of Juvenile cobia.Finally,gut microbiota plays several roles in fish well-being,promoting health and growth,resistance to bacterial invasion,regulation of energy absorption,and lipid metabolism.However,studies on fish gut microbiota face practical challenges due to a large number of fish varieties,fluctuating environmental conditions,and differences in feeding habits.In this study,chromatography,mass spectrometry,and high throughput sequencing were combined to explore gut microbiota’s composition and metabolite profile in juvenile cobia fed with a basal diet or a supplemented diet with autochthonous strains Bacillus sp.RCS1,Pantoea agglomerans RCS2,and Bacillus cereus RCS3 mixture.Gut bacterial composition was altered in fish receiving a supplemented diet with autochthonous strains.Metabolomics also revealed that certain metabolic pathways were considerably enriched in fish fed with supplemented diet;pathway analysis based on Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment revealed that differentially expressed metabolites were involved in galactose metabolism,tryptophan metabolism,carbohydrate digestion and absorption,purine metabolism,and ABC transporters.Functional analysis of the bacterial community showed that differences in enriched metabolic pathways generally comprised carbohydrate and its metabolites,nucleotide and its metabolites,amino acid and its metabolites,heterocyclic compounds,and tryptamines,cholines,pigments.Understanding the relationship between gut microbiota and the metabolic content in fish fed with beneficial microbes provides the basis for applying the use of supplementation with autochthonous strains to influence the immunity and growth of juvenile cobia（Rachycentron canadum）.The present study provides vital information and contributes to the general understanding of the structure and composition of microbiota in vertebrate organisms.The fish immune responses enhancement and the gut morphology are vital in enhancing growth,absorption of nutrients,and disease resistance;hence the results in the present study are essential outcomes for the development and competitive aquaculture sector.Generally,the characteristics displayed by these strains indicated that they could be used as potential probiotics in the aquaculture industry.