Study On The Effects Of Fermented Soybean Meal In Fish Meal Replacement And The Improvement Of Glutamine And Clostridium Butyricum On Its Effectiveness In The Diet Of Largemouth Bass(Micropterus Salmoides)

The purpose of this study was to investigate the application and nutritional improvement strategy of fermented soybean meal in the diet of largemouth bass.Firstly,the effects of fish meal replaced by various levels of soybean meal and fermented soybean meal on the growth,nutrient utilization,serum index and intestinal health of largemouth bass were investigated.Then,the nutritional value of fermented soybean meal was evaluted in the diet of largemouth bass.At last,glutamine and clostridium butyrate were supplemented in a low fish meal diet containing high level of fermented soybean meal,and their effects on growth and feed utilization were investigated.This study can provide some theoretical basis for the application of fermented soybean meal in the diet of carnivorous fish.Exp.1 Effects of fish meal replaced by fermented soybean meal on growth performance,nutrients utilization and serum indexes of largemouth bassA 56-day feeding trial was conducted to investigate the effects of replacing fish meal(FM)with soybean meal(SBM)and fermented soybean meal(FSBM)on growth performance,nutrition utilization and intestinal histology of largemouth bass.The basal diet contained 350g/kg FM(control),and then FM was replaced with SBM or FSBM of 15%,30%,45%and 60%,respectively.The results showed that the specific growth rate of fish fed FSBM-60,SBM-45and SBM-60 diets significantly decreased,and the feed conversion ratio of SBM-30,SBM-45,SBM-60,FSBM-45 and FSBM-60 groups increased when compared to the control group(P<0.05).The apparent digestibility coefficients of dry matter and crude protein of SBM-45,SBM-60 and FSBM-60 groups were significantly lower than those of the control group(P<0.05),and the substitution of FM with SBM(30%,45%and 60%)and FSBM(45%and60%)significantly reduced the protein efficiency ratio(P<0.05).Serum total protein contents of SBM-60 and FSBM-60 groups and serum cholesterol contents of SBM-45,SBM-60 and FSBM-60 groups were significantly lower than those of the control group(P<0.05).The SBM-30 and SBM-60 groups showed significantly higher alanine aminotransferase activity when compared to the control group(P<0.05).In conclusion,FSBM could replace 30%FM in diet of largemouth bass(350 g/kg FM)without negative effects on growth performance,nutrients utilization and serum indexes,while the substitution level of FM with SBM was only 15%.Exp.2 Effects of fish meal replaced by fermented soybean meal on intestinal histology and microbiota of largemouth bassBased on experiment one,the effects of dietry fermented soybean meal on the intestinal structure of largemouth bass were studied by tissue secting technique.The result showed that the villus length of SBM-60 group and the villus width of SBM-45,SBM-60 and FSBM-60groups decreased when compared to the control(P<0.05).Two methodology were used to evaluate the intestinal microbiota of largemouth bass in CON,SBM-30,SBM-60,FSBM-30and FSBM-60 groups.The total bacteria,lactobacillus,Escherichia coli and bacillus in intestinal tract were counted using plate culture method,which indicate that the total bacteria in SBM-30,Escherichia coli in SBM-60 and FSBM-60 groups were significantly decrease;the lactobacillus and total bacteria in FSBM-60 were significantly higher than in SBM-60 group(P<0.05).An Illumina-Mi Seq high-throughput sequencing analysis on hind-gut showed that The Shannon and simpson diversity index was significantly decreased in FSBM-30 group when compared to the CON group(P<0.05).The taxonomic distribution showed that Tenericutes,Proteobacteria,Fusobacteria,Firmicutes and Actinobacteria were the main microbiome of hind-gut of largemouth bass at phylum level,and Mycoplasma,Cetobacterium,plesiomonas and Vibrio were the dominant microbial communities at genus level.The replacement of FM with SBM significantly increased the ratio of Cetobacterium,and FSBM-30 group had higher ratio of Mycoplasma than the CON group(P<0.05),but FSBM-60 group showed no significantly difference with the CON group(P>0.05).In conclusion,FSBM could replace 45%FM in diet of largemouth bass without negative effects on intestinal histology,while the substitution level of FM with SBM was only 15%,the substitution of FM with SBM and FSBM could significantly affect the intestinal microbiota of largemouth bass(P<0.05).Exp.3 Evaluation of nutrient value of fermented soybean meal in the diet of largemouth bassThis experiment was conducted to compare and evaluate nutrient value of soybean meal(SBM)and fermented soybean meal(FSBM)for largemouth bass.The basal diet contained400mg/kg fish meal(CON),and then SBM and FSBM were mixed with the basal diet at the ratios of 10%,20%and 30%,respectively(SBM-10?SBM-20?SBM-30?FSBM-10?FSBM-20?FSBM-30).Largemouth bass(35.7±0.03 g)were fed with 7 diets for two weeks,and then intact feces were collected for the analysis of nutrient apparent digestibility coefficient(ADC)of SBM and FSBM.The results showed that:The ADC of dry matter,crude protein,phosphorus and total amino acids in SBM decreased significantly with the increase of mixing ratio,(P<0.05),but not FSBM(P>0.05).The ADC of dry matter,crude protein,phosphorus and total amino acids FSBM at the ratio of 30%were 74.71%,76.56%,23.24%,87.34%,respectively,which were higher than that in SBM(81.97%,84.00%,46.94%,92.25%).In conclusion,the apparent digestibility of dry matter,crude protein,total amino acid and phosphorus was significantly increased after soybean meal was fermentated.Exp.4 The effects of Alanyl-glutamine on growth performance,nutrients utilization and intestinal health of largemouth bass fed low fish meal dietThis feeding trial was conducted to investigate the effects of Alanyl-glutamine(Ala-Gln)on growth performance,nutrition utilization,intestinal histology and serum index of largemouth bass fed low fish meal diet.The basal diet contained 400mg/kg fish meal(CON),then fermented soybean meal was used to replace 40%of fish meal(FSBM-40).0.3%,0.6%,0.9%and 1.2%,Ala-Gln were suplemented into FSBM-40,respectively(AG-3,AG-6,AG-9and AG-12).Six diets were used to feed largemouth bass(7.0±0.1g)for eight weeks.The results showed that the weight gain rate and special grow rate were significantly lower,and feed conversion rate was significantly higher in FSBM-40 group when compared to control group(P<0.05).The proximate composition and amino acids of muscle showed no significant difference among all groups(P>0.05).The apparent digestibility coefficients(ADC)of dry matter and crude protein of FSBM-40 significantly lower than control group(P<0.05),and the0.6%-1.2%Ala-Gln supplement significantly improved the ADC of dry matter and crude protein(P<0.05).The intestinal histology analysis resulted that the villus width of FSBM-40group was significantly lower than control group(P<0.05),the supplement of Ala-Gln in diet significantly increased the villus width.The villus height and crypt depth showed no significant difference among all groups(p>0.05).The serum albumin in AG group were significantly higher than that in CON and FSBM-40 group,and surum triglyceride in FSBM-40,AG-3,AG-6 and AG-9 were significantly lower than control group(P<0.05).In conclusion,the 0.9%AG supplement in low fish meal diet can improve the intestinal structure and growth performance.Exp.5 The effects of Clostridium butyricum and Alanyl-glutamine on growth performance,nutrients utilization,intestinal histology and microbiota of largemouth bass fed low fish meal dietThis feeding trial was conducted to investigate the effects of Clostridium butyricum and Alanyl-glutamine(Ala-Gln)on growth performance,nutrition utilization,intestinal health of largemouth bass fed low fish meal diet.The basal diet contained 400mg/kg fish meal(CON),then fermented soybean meal was used to replace 40%of fish meal(FSBM-40).Then 0.2%Clostridium butyricum(CB)powder(1.5×10~8cfu/g),0.6%Ala-Gln and their mixture were supplemented into FSBM-40,respectively(CB,AG and CB-AG).Five diets were used to feed largemouth bass(7.0±0.1g)for eight weeks.The results showed that the weight gain rate,apparent digestibility coefficients(ADC)of dry matter and crude protein were significantly lower,and feed conversion rate was significantly higher in FSBM-40 group when compared to control group(P<0.05).The addition of CB,Ala-Gln and their mixture in FSBM-40 did not improve the growth performance(P>0.05).The ADC of dry matter in AG group was significantly higher than that in FSBM-40 group(P<0.05).The intestinal histology analysis showed that the villus width of FSBM-40 and CB group were significantly lower than that of CON group(P<0.05).The serum albumin in the AG+CB and AG groups were significantly higher than that in FSBM-40 groups(P<0.05).The results of intestinal microbial analysis showed that the relative abundance of bacillus and acinetobacter were significantly increased in the intestinal microbiota community of largemouth bass fed Ala-Gln supplemented diet(P<0.05).In summary,the supplement of 0.2%CB and 0.6%Ala-Gln did not improve the growth performance,and supplementation of Ala-Gln in low fish meal diet can improve the intestinal structure and intestinal microbial composition of largemouth bass.