Immune Regulation Of Fermented Soybean Meal Partly Replacing Fish Meal And Dietary Crystalline Arginine And Methionine On Growth And Anti-stress Ability Of Litopenaeus Vannamei

1.The effect of fermented soybean meal replacing fish meal on the growth performance,immune-related enzymes and gene expression of Litopenaeus vannameiIn recent years,the study of fermented soybean meal as a substitute for fishmeal has received more and more attention,but the mechanism for affecting the health and immunity of aquatic animals is unclear.The expression of immune related genes and enzyme are closely linked to the health and immunity of aquatic animals.This study intends to explore the health and immunological mechanism of fermented soybean meal substitute fish meal for the growth of the Litopenaeus vannamei with the effect of the immune-related enzyme activity and gene transcription levels.Five experiment diets were designed,in which 30% of fish meal diet(FM)as the control group,and 4%(FSM4),8%(FSM8),12%(FSM12)and 16%(FSM16)of the fermented soybean meal was used to substitute 9.7%,19.4%,29.1% and 38.8% of the fish meal respectively.After 60-day feeding test for L.vannamei(initial weight =7.62±0.23g),the growth performance,muscle nutrition com-position,serum and hepatopancreas immune enzymes activity,the expression levels of HSP70 mRNA in hepatopancreas and Toll receptor mRNA,IMDmRNA and LZMmRNA in gill were determined.The results indicated that the substitution of fermented soybean meal for fish meal had no significant effect on the survival rate of the shrimp compared with the control group(P > 0.05),both too low and too high level of fermented soybean meal reduced the specific growth rate of shrimp(P> 0.05).Muscle crude protein content was little lower in the addition group of fermented soybean meal than the FM group with no significant difference except FSM12,crude fat has a tendency to decrease with the increase of fermented soybean meal,and the lowest value was found in the FSM16 group.The serum alanine aminotransferase(ALT)in FSM4 and FSM16 groups was significantly higher than that in the control group(P< 0.05),and the aspartate amino transferase(AST)was shown the highest and lowest value in FSM4 group and FSM8 group respectively;except FSM12 group,serum ALP was no marked difference between other FSM groups and the control group;as for TP in serum and MDA in hepatopancreas,there were also no statistic difference between FSM groups(except FSM16 group)and the control.With the increase of the amount of fermented soybean meal,the level of gill Toll receptor mRNA expression increased,and the level of IMDmRNA expression increased first and then decreased,and excessive or less of the added fermented soybean meal would decrease the expression of LZMmRNA in gill,while the expression of HSP70 mRNA in hepatopancreas increased with the increase of fermented soybean meal.To sum up,in the condition of this experiment,optimum amount of the fermented soybean meal was 8% to 12%,i.e replacing 19.4%~29.1% of fish meal without affecting the growth of the tested shrimp,It can improve the immunological activity,increase the expression of immune related genes,and improve the immune function,although high substitution can cause different levels of stress response and increase the expression of HSP70 genes in hepatopancreas.2.The effect of soybean antigen protein on the growth,immune enzyme activity and immunological gene expression of L.vannameiTwo experiments were designed,and experiment 1 included two practical diets containing 24.9% soybean meal(SBM),8% fermented soybean meal and 16.95% soybean meal(FSBM),experiment 2 included two semi-purified diets with high antigen protein(SPD1)and low antigen protein(SPD2)approximately equal to SBM and FSBM respectively.Diets were fed to Litopenaeus vannamei(initial weight: 7.48±0.24 g)for 60 days.The results showed that in the experiment 1,growth performance was not significantly different in the two groups,the immune indexes(AKP,AST,ALT,SOD and LZM)were significantly higher in the SBM group;there were same trend about mRNA expression levels of TLR,LZM,IMD and HSP70.In experiment 2,weight gain and specific growth rate were significantly higher in the SPD2 group,while higher activities of AKP,ALT and LZM,lower expression levels of TLRmRNA,LZMmRNA and IMDmRNA and higher expression level of HSP70 mRNA were found in the SPD1 group.All results inferred that SBM was more likely to induce non-specific immune responses and oxidative stress in L.vannamei than FSBM.The antigen protein content in soybean meal was one of the main factors to increase stress,reduce the immunity and injure hepatopancreas health of shrimp.3.Immune regulation of dietary crystalline arginine on growth and anti-stress ability of L.vannamei when fermented soybean meal was a partial substitute for fish mealFermented soybean meal as a substitute for fish meal has been widely used in aqua-feed,but there is usually poor amino acid balance when substitution occurs.Arginine as one of the essential amino acids,it plays an important role in metabolism of aquatic organisms.This experiment aimed to investigate the significance of inclusive arginine in the diet of Litopenaeus vannamei when fish meal was partly substituted by fermented soybean meal.Based on the results of our previous study,we chose the formulated diet containing 1.98% arginine as control,in which 22.5% fish meal and 10% fermented soybean meal was as the main protein source,other four diets with gradient levels of additive crystals of arginine(0.2%,0.4%,0.6%,0.8%)were prepared.All these five diets were randomly fed to L.vannamei with initial weight: 0.62±0.1g for 60-day feeding trial.The growth performance,serum and hepatopancreatic index,related genes expression,resistance ability to hypoxia and vibrio were determined.Results showed that there was no significant difference in the survival rate of shrimp among 5 groups(P>0.05),the weight gain rate and the specific growth rate in the 0.2% group and the 0.8% group were significantly higher than that of the other groups(P<0.05).No significant difference on serum MDA was found among 5 groups,but AST,ALT activity was highest in the 0.2% group,the lowest in 0.8% group(P<0.05).The MDA content of hepatopancreas was lowest in the 0.2% group,the resistance to low oxygen and vibrio was highest in the 0.2% group,followed by the 0.8% group.Three diets group(0.0%,0.4%,0.8%)gene expression was detected,it was found that the addition group of 0.4% was the lowest in IMD,LZM and MyD88 gene expression(P<0.05).The expression of ATF4 gene was the highest in 0.0% group(P<0.05).It concluded that 0.4% of additive crystalline arginine in the diet including 22.5% fish meal and 10% fermented soybean meal could effectively modulate immune system and enhance the anti-stress abilities of shrimp,L.vannamei,protect the hepatopancreas healthy,reduces oxidative stress in hepatopancreas and did not affect the growth of shrimp.4.Immune regulation of dietary crystalline methionine on growth and anti-stress ability of L.vannamei when fermented soybean meal was a partial substitute for fish mealFermented soybean meal as a substitute for fish meal has been widely used in aquatic animals,nevertheless we may face troublesome amino acid balance problems when substitution occurs.As first limited amino acid,methionine theoretically plays a more important role compared to other essential amino acids when plant protein sources replacing fish meal.For this reason,five formulated feeds containing 22.5% of fishmeal and 10% of fermented soybean meal as the main protein source with gradient levels of additive crystalline methionine(0.00%,0.12%,0.24%,0.36%,0.48%)were designed,in which the feed with 0.81% methionine but crystalline methionine excluded was regarded as basic diet based on our former study.Litopenaeus vannamei with initial weight(0.62±0.1)g was fed with above five diets for 60-day feeding trial,then growth performance,biochemical index in serum and hepatopancreas,resistance ability to hypoxia and vibrio were determined.The results showed that there were no significant differences on the survival rate,the weight gain rate,the specific growth rate and serum MDA among the five groups(P> 0.05).AST and ALT activities in serum were significantly higher in the 0.00% and 0.48% groups than other groups(P<0.05),and the content of MDA in hepatopancreas was the lowest in 0.24% group.In addition,the highest value of the ability of anti-vibrio was shown in the 0.12% and 0.24% groups,while the ability of hypoxia tolerance was significantly higher in the 0.00% group than the others.Three diets group(0.0%,0.24%,0.48%)correlated gene expression was detected,found that 0.24% group TLR genes relative expression is significantly higher than other groups,IMD gene in 0.0% group was significantly lower than the other two groups,LZM gene expression with the increase of methionine and reduce the transcription,ATF4 gene group is significantly higher than 0.24% group in 0.0%.In summary,when the amount of fish meal was 22.5% and fermented soybean meal was 10% in the diet,the inclusion of 0.24% of crystalline methionine could effectively regulate immune system and improve anti-vibrio ability of L.vannamei,reduce the oxidative degree and injury possibility of hepatopancreas without affecting shrimp's growth performance.