| With high nutritional value and large market demand,sea cucumber Apostichopus japonicus is one of important species of marine aquaculture in China.However,the intensive farming had led to frequent diseases which seriously restricted the development of the farming of sea cucumber and caused huge economic losses.The use of antibiotics also had negative effects on the farming environment and the health of sea cucumbers.Therefore,it is urgent to find environmentally friendly and economically effective control methods and techniques to reduce the economic losses and promote the sustainable development of sea cucumber aquaculture.As an effective means of preventing the occurrence of diseases in aquatic animals,probiotics have received increasing attention in the farming of sea cucumber in recent years.Previous studies have confirmed that lactic acid bacteria could improve the growth performance,metabolism,immunity,and intestinal microbiota of sea cucumber.As we all know all the beneficial effects attributed to live probiotic may include the effects of metabolites and/or the dead form of this probiotic.However,the effects of different addition forms of Lactobacillus including live bacteria,dead bacteria and metabolites of this bacteria on the intestinal microflora and metabolism of sea cucumber A.japonicus are rarely studied.The present study was designed to explore the effects of a potential probiotic Lactobacillus M2-4 fed with the forms of live bacteria,dead bacteria and metabolites of this bacteria on the intestinal microbiota,intestinal metabolism and intestinal immune related genes in sea cucumber.The experiment included four treatments including control,live M2-4 group,dead M2-4group and metabolites of M2-4 group and the addition amount was 10~9CFU/g.There were three replicates in each treatment.The sea cucumbers with average weight of(0.879±0.034)g(means±SE)were selected for the experiment.The study last for 30 days.The results were as follows:(1)The alpha diversities of the fecal bacteria in sea cucumbers were significantly improved by the live M2-4 and its metabolites.However,there was only an increasing tendency with alpha diversities of intestinal microflora with no significant difference.And the M2-4 was detected in the intestines of sea cucumbers fed with live M2-4.(2)Dietary M2-4 in different forms had a significant effect on the intestinal metabolism of sea cucumbers.The differential metabolites were mostly lipids and peptides,which were concentrated in lipid metabolism and amino acid metabolism.Compared with control group,the most significant pathways of differential metabolite enrichment in the live M2-4 group and the dead M2-4 group were taurine and hypo taurine metabolism,and the concentrated differential metabolite taurine was down-regulated in both groups.The differential metabolites in the dead group were also concentrated in immune-related pathways,such as NF-kappa B signaling pathway and natural killer cell-mediated cytotoxicity pathway,and the concentrated differential metabolite DG(8:0/13:0/0:0)was significantly up-regulated.In the metabolite group,the differential metabolites were enriched in glutathione metabolism.Oxidized glutathione was the concentrated differential metabolites and was significantly up-regulated,but glutathione was significantly down-regulated,and oxidized glutathione was much more than glutathione.Compared with the live group,the most significant pathway in the dead group was vitamin B6 metabolism,and the concentrated metabolite 4-pyridyloxy acid was down-regulated.The most significant pathway in the metabolism group was pyrimidine metabolism,and the concentrated metabolites 2’-deoxyuridine,thymidine,and thymine were all up-regulated.Compared with the dead group,the most significant pathway in the metabolism group was glutathione metabolism,and the changes of concentrated metabolites were similar to those in the comparison of metabolism and control groups.(3)Dietary live M2-4 significantly affected the expression of p105 and Rel genes in intestines of sea cucumber.But dietary inactivated M2-4 and its metabolites had no significant effect on the expression of the four immune related genes(Gpx、Rel、p105、Lys)detected in the experiment in the intestines of sea cucumbers.In summary,live M2-4 and its metabolites can significantly affect the structure of fecal flora and have a positive effect on the diversity of intestinal flora of sea cucumber.The supplementation of three forms of M2-4 could change the intestinal metabolism of sea cucumber,with inactivated M2-4 showing stronger immune stimulation and being more effective in immune regulation than live M2-4 and its metabolites.The live M2-4had the most obvious effect on the expression of intestinal immune related genes of sea cucumber. |
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