Effects Enteroccus Faecium On Growth Performance,Intestinal Health And Immunity Of Big-belly Seahorses During Diet Conversion

Seahorses(Hippocampus spp.)may face negative impacts on their nutritional absorption and disease resistance during the bait conversion period when consuming frozen bait due to insufficient nutrients,poor palatability,and intestinal stress.To address these issues,this study aims to screen a candidate strain with good in vitro salt-tolerant and antipathogenic activities from collected probiotic candidates.By two combinations of probiotic addition concentrations and two addition frequencies for 48 days cultivation experiment was conducted.Then we analyzed the effects of supplementary concentration and frequency of the probiotic strain on seahorse growth,development,intestinal health,and disease resistance.Comprehensive evaluation systems of both growth performance(GAI)and probiotic effects(PAI)were constructed for the first time to determine the best application strategy of E.faecium supplementation.The results mainly showed that:1.One candidate strain with best salt-tolerant and antipathogenic activities was selected from 17 by probiotic strains and identified as Enterococcus faecium.It was named and preserved as E.faecium LD-NXY01 in CGMCC(China General Microbiological Culture Collection Center)(NO.24041).2.Concentration and frequency of E.faecium supplementation,as well as their interaction,were confirmed to have significant effects on growth performance of big-belly seahorses during bait conversion for the first time(P < 0.05).The results of body weight(BW),specific growth rate(SGR),body weight increase(BWI),standard length(SL),and standard length increase(SLI)H4 and L4 groups were better than that of H2 and L2 groups(P < 0.05).And the growth promoting effects of E.faecium supplementation at the frequency of 4 days was better than that at the frequency of 2days.The GAI of the H4 group was significantly higher than that of the other groups(P < 0.05),indicating the best application strategy of E.faecium supplementation.3.Feed intake(FI)increased in all E.faecium supplemented groups increased significantly(P < 0.05),significant decreased feed conversion rate(FCR)was only found in H4 group(P < 0.05);both of the intestinal mucosal height and width(P <0.05)and digestive enzymes(P > 0.05)were foundincreased in H4 group.It suggests that the increased growth performance of big-belly seahorses maybe attributed to the integrated improvements in FI,digestion,and absorption capacity.4.Compared with group C,seahorses of H4 group had relative higher biodiversity and different structure of intestinal microbiota.The abundance of Phycisphaera,Robiginitomaculum,Amylibacter,Sphingorhabdus,Marinicaulis,unclassi-fied＿f＿Rhodobacteraceae,norank＿f＿Rhodobacteraceae,and Roseovarius significantly increased,while the abundance of Vibrio,Colwellia,and unclassified＿f＿Colwelliaceae decreased significantly in H4 group(P < 0.05).Meanwhile,the activities of bacterial chemotaxis,biofilm formation-Vibrio cholera,and phosphotransferase system significantly decreased,whereas amino acid metabolism and lipid metabolism significantly increased in H4 group(P < 0.05).It indicates that E.faecium can increase the abundance and function of healthy bacteria,while inhibit that of pathogenic bacteria to improve the health of the intestinal microbiota of big-belly seahorses.5.Except for Interleukin-2(IL-2)and Interleukin-10(IL-10),the expression other immune-related genes of Toll-like receptor 5(TLR-5),proinflammatory factor interferon-γ(IFN-γ)and interleukin-1β(IL-1β),and antibacterial peptides piscidin and lysozyme(LYZ)significantly increased in H4 group(P < 0.05).In H4 group,seahorse death occurred 3 days later than that in group C.Final survival rate of H4 group was50% higher than that of the group C.The PAI of the E.faecium-treated groups,particularly H4 group,was significantly higher than that of C and other treated groups(P < 0.05),indicating the optimal probiotic effects.Collectively,the present study confirmed the growth promoting effects and mechanisms of E.faecium supplemention on seahorse aquaculture for the first time,established evaluation systems for comprehensively and accurately assessing the GAI and PAI of big-belly seahorses,and provided a new and applicable research and development ways for health and sustainable development of seahorse aquaculture,as well as other fishes.