Effects Of Dietary Bacillus Velezensis MSP05 On Nonspecific Immunity And Growth Performance Of Loach (Misgurnus Anguillicaudatus)

Loach is an important high-quality aquatic product with a large market demand.In recent years,the large-scale intensive factory breeding of loach has encountered fish disease and aquaculture environmental degradation,which restricts the healthy and sustainable development of loach aquaculture to some extent.It is an ecological priority strategy to screen suitable probiotics as feed additives to improve fish health and enhance the resistance to pathogens.This study was conducted to investigate the effect of dietary B.velezensis MSP05 on growth performance,digestion,and non-specific immune function of juvenile loach(M.anguillicaudatus).360 fishes with an average initial body weight of(3.24±0.14)g were randomly assigned into 4groups in triplicates with 30 individuals per replicate.One control group and three test groups were added 0(CS)、1×10~6(BS1)、1×10~7(BS2)、1×10~8(BS3)CFU/g B.velezensis MSP05 to basal diets respectively.After 8 weeks of feeding,the growth performance,intestinal digestive enzyme activities,intestinal morphology,intestinal microflora,serum antioxidant indices,non-specific immune enzyme activities and immune-related gene expression of juvenile loach were studied respectively,and Aeromonas hydrophila challenge test was conducted finally.The results demonstrated that the weight gain rate(WGR)and specific growth rate(SGR)of the groups adding B.subtilis MSP05 were significantly higher than those of the control group(P<0.05),and there was no significant difference among three test groups(P>0.05).The activities of protease and amylase were increased firstly and then decreased with the B.velezensis level increasing,achieving the highest value in BS2 group and significantly increasing by 76.85%and 324.47%,while the activities of lipase rose continuously and significantly increased by 700.14%in BS3 group(P<0.05).The optimal morphology of foregut,midgut and hindgut of loach were observed in BS1,BS1,BS2 group,respectively.Intestinal mucosal plica was intact and larger in height,the thickness of intestinal wall and muscular layer were thicker than the control group,while the hindgut was damaged in BS1 group significantly.The activities of serum catalase(CAT),superoxide dismutase(SOD),lysozyme(LSZ)and alkaline phosphatase(ALP)in the serum were significantly higher than the control group(P<0.05),while MDA levels showed the opposite trend.Challenged by Aeromonas hydrophila for 15 days,the cumulative mortality rate of juvenile loaches decreased significantly fed with the B.velezensis MSP05.Comprehensive analysis showed that the BS2 group supplemented with 1×10~7CFU/g B.velezensis MSP05 had the best effect on growth,digestion and non-specific immunity of juvenile loach relatively,with little damage to intestinal morphology.The analysis of the intestinal microbiota and gene expression was conducted between the control group(CS)and BS2 group with the best probiotic effect(reffered to as BS group).The addition of 1×10~7CFU/g B.velezensis MSP05(BS)to the feed increased the abundance of the intestinal microbiota and concentrated the distribution of dominant species(P<0.05),only the top two species(Aeromonas veronii and Flavobacterium succinicans)accounted for 58.00%.Compared with the CS group,the abundance of Proteobacteria were significantly decreased(P<0.05)and Bacteroidetes were significantly increased(P<0.05)in BS group at the phylum level;at the genus level,the abundance of Flavobacterium was significantly increased(P<0.05),and the abundance of Aeromonas,Acidovorax,Pseudomonas,Shewanella and Paludibacter were decreased(P<0.05).DEGseq was used to analyze gene expression difference.1582 differential genes were obtained,of which1034 genes were significantly up-regulated and 548 genes were significantly down-regulated.Up-regulated expression of growth-related genes(IGF-1,IGF-1r)and immunity-related genes(C9,GSH-Px1a)were observed in the group BS2(P<0.05),but most immunity-related genes were down-regulated to some extent.The down-regulated differentially expressed genes were enriched in pathways of immune response and immune system process by GO functional enrichment analysis.In conclusion,the addition of B.velezensis MSP05 can significantly enhance the growth performance,digestive enzymes activity,intestinal morphology,antioxidant enzyme activity,non-specific immune function and disease resistance against A.hydrophila.B.velezensis MSP05 can be implemented as an effective loach dietary supplement.