| Largemouth bass is one of the main species of freshwater fish farming in China.With the continuous development of the largemouth bass farming industry,the importance of research and development of special compound feeds for the precise nutritional needs of largemouth bass has become increasingly prominent.The use of cheap rapeseed meal（RM）replace expensive fishmeal and soybean meal is still necessary.However,the anti-nutritional factors in them are easy to have a negative impact on aquatic animals and become an important factor limiting their application.Condensed tannin is one of the main anti-nutritional factors,and as some other anti-nutritional factors,condensed tannin at low doses can promote animal growth,enhance the body’s antioxidant capacity and immunity,regulate intestinal microecological balance,and improve growth performance,and it can also become a natural antibiotic.In order to explore the maximum tolerance of RM in largemouth bass feed and the effects of condensed tannins in the corresponding RM usage gradient on largemouth bass,based on traditional feeding experiments,the effects of different levels of RM on largemouth bass juveniles were investigated.Effect analyses of growth performance,body composition,plasma indicators,nutrient metabolism,antioxidant immunity,liver and intestinal health,etc.,have been made to determine the maximum tolerance of largemouth bass to RM in feed,and finally through the analyses of intestinal microbes and liver metabolomics,the response and mechanism of juvenile largemouth bass to dietary RM and condensed tannins have been explored.It was further explored whether condensed tannins in RM were the main limiting factor for the application of RM in compound feed,and whether there was an interaction with other antinutritional factors in RM.1 Effects of replacing fishmeal with rapeseed meal and dietary condensed tannins on growth performance,digestibility,metabolic parameters and muscle composition of M.salmoides.In order to evaluate the effects of RM in feed on the growth,digestibility,nutrient metabolism and body composition of largemouth bass,and the role of condensed tannins in this process,eight groups of isonitrogenous and isoenergetic feeds were formulated,of which 6 groups were practical feed groups,setting one group as the control group（CG）,and the other 5 groups as the rapeseed meal（RM）usage group,and the usage gradients were 5%（RM5）,10%（RM10）,15%（RM15）,20%（RM20）,25%（RM25）,the other two groups were semi-purified feeds,adding 0.062%condensed tannin（CT5）（≈5%condensed tannin content in RM）,0.31%condensed tannin（CT25）（≈25%condensed tannin content in RM）.After 55 days of feeding with the eight groups of feed,the results of the RM substitution experiment have shown:with the increase of the level of RM substitution,the weight gain rate,survival rate and specific growth rate showed a downward trend.5%RM had no significant effect on growth performance of largemouth bass.When the dietary RM level reached 10%and above,the weight gain rate and specific growth rate decreased significantly,and when the RM level reached 20%and above,the survival rate also decreased significantly.The 25%RM group showed the worst performance in all indicators of growth performance such as protein efficiency and feed coefficient.Compared with the control group,there were no significant changes in condition factor,hepatosomatic index,viscerosomatic index and intestinal index in each group of rapeseed meal（P>0.05）.With the increase of RM substitution,there was no significant change in pepsin activity,and the activity of gastric amylase showed a trend of first increasing and then decreasing,and the RM5was the highest.When the level of rapeseed was 10%or more,gastric lipase activity was inhibited.Except for the RM20,the intestinal trypsin activity of other RM groups was inhibited.Enteric lipase was not affected by RM,and RM had a promoting effect on intestinal amylase activity.When the level of RM was 15%or more,the plasma TG content was significantly increased.The plasma BUN and LDL contents were the highest in the RM25,and there were no significant differences in the remaining groups.When the level of RM was 20%or more,the content of plasma ammonia was significantly increased.With the increase of RM substitution amount,plasma glucose content showed a trend of first decreasing and then increasing,reaching the lowest when the RM amount was 5%,the plasma glucose content of the control group was significantly higher than that of the RM5 group,and the plasma glucose level of feed RM was higher than that of the control group when the level of feed RM was 10%and above.Compared with the control group,the liver GPT activity was reduced in the RM groups.When the level of RM did not exceed 10%,the liver glycogen content had no significant difference between the control group and the RM groups,and when the level of RM exceeded 15%,its content increased significantly.Compared with the control group,the liver T-CHO content had no significant difference when the RMl replacement amount did not exceed 10%,and its content increased when the RM replacement amount was 15%or more.When the level of RM exceeded 20%,the liver TG content increased significantly.RM reduced muscle protein content and EE content,but RM below 5%had no significant effect.RM had no significant effect on muscle∑SFA,∑PUFA,TNEAA and TEAA content,but reduced muscle∑MUFA content.The results of condensed tannin concentration experiments showed that the addition of0.062%CT had positive effects on the weight gain rate,specific growth rate,protein efficiency and feed coefficient of largemouth bass,but had no significant effect（P>0.05）.The addition of 0.31%CT significantly reduced the weight gain rate,specific growth rate,survival rate and protein efficiency.The addition of 0.31%CT inhibited the activities of protease,lipase and amylase in the stomach,as well as the activities of enteric trypsin and lipase.In 0.062%of CT,only gastric lipase activity was inhibited,and intestinal amylase activity was increased.There were no significant differences in plasma T-CHO,LDL,HDL,TG and BUN between the control group and the CT groups.0.062%CT significantly reduced plasma glucose concentration,and 0.31%CT significantly increased plasma ammonia and glucose concentration.The addition of CT improved the activity of liver GPT and had no significant effect on liver T-CHO,GOT and GLN.The content of HG and TG in liver increased in 0.31%of the CT group.0.062%of the CT group decreased the content of liver HG,and there was no significant change in liver TG content.CT addition did not significantly change muscle moisture,protein and ash,and muscle fat content was significantly reduced.CT addition had no significant effect on muscle∑SFA,∑PUFA,∑MUFA,TNEAA and TEAA.According to the results of the comparison experiment between condensed tannins alone and RM,the weight gain rate,protein efficiency and feed coefficient of CT5 were better than those in RM5.Compared with RM5,the intestinal trypsin and amylase activities were increased in the CT5,and the plasma BUN,GLU and TG in the CT5 group were lower than those in the RM5 group,but the difference was not significant（P>0.05）.Compared with RM5,the CT5 had increased liver GPT activity,decreased liver HG and TG content,and had no significant change in muscle composition.The CT25 group compared to the RM25 group,there was no significant change in growth performance,and gastric lipase,enteric trypsin and pepsin activities were decreased,amylase and intestinal lipase activities were increased,plasma content of ammonia,TG and liver HG and T-CHO decreased,plasma content of T-CHO and GLU increased,and muscle composition did not change significantly.Therefore,it can tolerate 5%of RM content without affecting the growth,digestion and metabolic function of largemouth bass.The experiment of condensed tannin concentration showed that 0.062%condensed tannin had a regulating effect on the digestion metabolism of largemouth bass and improved feed utilization.According to comparative experiments,it can be proved that there was an interaction between anti-nutritional factors in RM,and other anti-nutritional factors had obvious synergistic effect at low RM levels,and the antagonism was more obvious at high levels of RM.2 Effects of replacing fishmeal with rapeseed meal and dietary condensed tannins on antioxidants and immunity,liver and intestinal health of M.salmoides.To evaluate the effects of RM and condensed tannins in feed on largemouth bass,and to speculate on the possible effects of condensed tannins on immunity and antioxidant when RM is used in feed,further testing was carried out on the experimental fish in Chapter 1.According to the results of RM substitution experiment,compared with the control group,the plasma MDA content and DAO activity of the RM group were significantly increased,and the plasma SOD and GSH-Px activities of the RM group were reduced.The total plasma antioxidant capacity of RM20 and RM25was significantly higher than that of the control group and other RM groups,and there was no significant difference in the total antioxidant capacity of plasma between other RM groups and the control group.The plasma ROS content in the RM25 group was significantly higher than that in the control group and the rest of the RM groups.The activity of hindgut diamine oxidase in the RM5 group and RM10 group was significantly higher than that in the control group,and there was no significant difference in the other groups in the RM replacement group.The content of liver ROS increased and GST activity decreased in RM groups,the liver MDA content of RM above 10%groups increased significantly,the liver CAT activity decreased in RM groups of 15%and above,and the AKP activity of liver decreased in 25%of RM group.The substitution amount of RM of 10%and above can significantly improve the activity of hindgut catalase.The content of hindgut MDA in all RM groups was significantly higher than that in the control group,and the content of hindgut PC and ROS in the 25%RM replacement group were significantly increased,and the activities of hindgut antimicrobial peptide and complement C3 were significantly reduced.The results of condensed tannin concentration experiments showed that there was no significant change in plasma ROS between the groups,but the lowest in the CT5 group and the highest in the CT25 group.Compared with the control group,there were no significant changes in plasma T-AOC,MDA,DAO content and GOT,GPT and GSH-Px activities in the CT5,no significant changes in the contents of T-AOC,PC,MDA and hindgut ROS,PC and DAO in the liver of the CT5,and plasma SOD activity,CAT activity,GSH-Px activity,GST activity in the liver of the CT5,CAT activity in the hindgut of the CT5,AMP activity,and LZM activity in the head and kidney of the CT5were significantly increased.Moreover,the ROS content and DAO activity in the liver of the CT5 were significantly reduced.In the 0.31%CT group,the MDA content,DAO activity,GOT activity,GSH-Px activity in plasma,the PC content,ROS content,DAO activity,and hindgut ROS activity in the liver were significantly increased,and in the CT25 group,the total antioxidant activity and SOD activity in plasma,CAT,GST in the liver and SOD activity,LZM activity,complement C3 activity in the hindgut decreased significantly.The content of MDA in the hindgut of the CT group increased.According to the experimental results of adding condensed tannins alone and condensed tannins in RM,there was no significant difference in plasma MDA between CT5 group and RM5 group,and the plasma MDA in CT25 group was significantly higher than that in RM25 group.There was no significant difference between the plasma oxygen free radical content of CT5 group and RM5 group,and there was no significant difference between CT25 group plasma oxygen free radical content and RM25 group.The plasma diamine oxidase activity of CT5 group was significantly lower than that of RM5 group,and the plasma diamine oxidase activity of CT25 group had no significant difference compared with RM25 group.The hindgut catalase activity of CT5 group was significantly higher than that of RM5 group,and the hindgut catalase activity of CT25 group was significantly lower than that of RM25 group.The activity of diamine oxidase in hindgut of CT25 group was significantly higher than that of RM25 group.There was no significant difference in hindgut MDA activity between CT5 group and RM5 group,and there was no significant difference between CT25group and RM25 group.Hindgut superoxide dismutase activity of CT5 group was significantly higher than that of RM5 group,and there was no significant difference in hindgut superoxide dismutase activity of CT25 group compared with RM25 group.According to the expression of antioxidant and inflammatory genes in the hindgut,RM5 was significantly lower than the control and RM25 in the expression of intestinal IL-6.There was no significant difference between RM25 and the control group,and CT5 was significantly lower than the control and CT25 groups.In intestinal TGF-β2expression,RM5 was significantly higher than RM25,and RM5 had no significant difference with the control.There was no significant difference between the control group,CT5 group and CT25 group.In the expression of intestinal TGF-β1,RM5 was significantly higher than control and RM25,CT5 was significantly higher than PG and CT25,and CT25 was significantly lower than control.In gut Nrf2 expression,RM5was significantly lower than control but higher than RM25,CT5 was significantly higher than control and CT25 was significantly lower than control.Therefore,adding more than 15%RM may cause oxidative damage to fish liver and intestine,and adding25%RM may aggravate intestinal inflammation.Adding 0.062%condensed tannin can improve the body’s antioxidant capacity and intestinal immune function,while adding0.31%condensed tannin can produce the opposite effects.Based on the results of the comparative test,it was speculated that the antioxidant properties of condensed tannins contained in the 5%rapeseed meal group were inhibited by other anti-nutritional factors,while the antagonistic effect of various anti-nutritional factors in the 25%rapeseed meal group inhibited the pro-oxidative effect of condensed tannins.3 Effects of replacing fishmeal with rapeseed meal and dietary condensed tannins on liver metabolomics and intestinal microbiota of M.salmoides.In order to explore the mechanism of action of RM and CT on nutrient metabolism and antioxidant and immune action,further liver metabolism and intestinal microbial community were carried out on the experimental fish samples in Chapter 1.The results of intestinal microbiota showed that in terms of the richness and diversity of the intestinal microbial community,the ACE index of the RM5 group was lower than that of the RM25 group,and there was almost no difference with the control group.The ACE index of CT5 group was higher than that of CT25 group and control group,the ACE index of RM5 group was lower than that of CT5 group,Chao1 index and observed species index showed the same trend.At the phylum level,the top 5 phyla in relative abundance are Proteobacteria,Firmicutes,Bacteroidetes,Actinobacteria,Fusobacteria,these have the highest relative abundance,accounting for more than 95%of the total.Proteobacteria and Firmicutes were the dominant phyla in the samples.Among them,the top ten relative abundance genera are Sphingomonas,Plesiomonas,Muribaculacea,Burkholderia-Caballeronia-Paraburkholderia,Bacteroides,Clostridium sensu stricto 1,Prevotella,Escherichia Shigella,Enterococcus.Kruskal Wallis statistics showed that the relative abundance of Proteobacteria and Firmicutes in RM5 group was significantly higher than that in RM25 group,while the relative abundance of Bacteroidetes and Actinomycetes in RM25 group was significantly lower than that in RM25 group.The relative abundance of Proteobacteria in CT5 group was significantly lower than that in CT25 group,while the relative abundance of Firmicutes,Bacteroidetes and Actinobacteria in CT5 group was significantly higher than that in RM25 group.The relative abundance of Plesiomonas shigelloides in CT25 group was significantly higher than that of other groups,and the relative abundance of Aeromonas veronii in RM5 group was significantly higher than that of other groups and control group.The relative abundance of Acinetobacter junii was significantly higher than that of other groups.The results of metabolomic analysis showed that in largemouth bass liver,a total of 887 differential metabolites were identified between the control group and the RM5 group,479 metabolites were up-regulated in the RM5 group compared with the control group,and 408 down.Compared with the control group,a total of 897differential metabolites were identified in the RM25 group,446 metabolites were up-regulated and 451 metabolites were down-regulated.A total of 880 differential metabolites were identified in the CT5 group compared with the control group,of which 445 metabolites were up-regulated and 435 metabolites were down-regulated.A total of 751 differential metabolites were identified in the CT25 group compared with the control group,of which 379 metabolites were up-regulated and 372 metabolites were down-regulated.Compared with RM25 group and RM5 group,a total of 792differential metabolites were identified,of which 337 metabolites were up-regulated and 455 metabolites were down-regulated.A total of 850 differential metabolites were identified in the CT25 group compared with the CT5 group,of which 446 metabolites were up-regulated and 404 metabolites were down-regulated.Compared with the RM5group,a total of 780 differential metabolites were identified,of which 334 metabolites were up-regulated and 446 metabolites were down-regulated.Compared with CT25group and RM25 group,a total of 782 differential metabolites were identified,of which406 metabolites were up-regulated and 376 metabolites were down-regulated.Among the differential metabolites in the RM group and the control group,most of the marker metabolites were related to lipid metabolism,and only a small part was related to glucose metabolism.Enrichment analysis of these changes in nutrient metabolism can be related to some metabolic pathways,RM group:arachidonic acid metabolism,linoleic acid metabolism,glycerophospholipid metabolism,galactose metabolism,aminoacyl-t RNA biosynthesis,cysteine Acid and methionine metabolism,purine metabolism,m TOR signaling pathway and citric acid cycle（TCA cycle）.Tannin group:arachidonic acid metabolism,linoleic acid metabolism,glycerophospholipid metabolism,aminoacyl-t RNA biosynthesis,citric acid cycle（TCA cycle）,m TOR signaling pathway,β-alanine metabolism,galactose metabolism,arginine biosynthesis.Through the influence of marker metabolites and differential strains on fish,it was found that the use of 5%RM can increase the relative abundance of Firmicutes,thereby promoting bile acid metabolism and reducing lipid deposition,and 25%RM will cause abnormal lipid and protein metabolisms,causing the internal environment of the body disordered and pathogenic bacteria invading,and affecting the health of the body.The addition of 0.062%condensed tannins can increase the abundance of beneficial bacteria in the intestine and maintain the intestinal homeostasis,while 0.31%condensed tannins will destroy the intestinal homeostasis and damage the health of the body.Therefore,high doses of condensed tannins are limiting factors in increasing the use of RM. |
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