Effects Of Partially Replacing Fish Meal With Fermented Cottonseed Meal And Adding Crystal Lysine On The Growth And Health Of Litopenaeus Vannamei

As an animal protein source with high balance of amino acid composition,fish meal is widely used in the aquaculture feed industry.However,in recent years,due to the shortage of fish meal supply and the soaring price,fish meal resources have become unstable.In addition,it limited the healthy and sustainable development of the shrimp farming industry of Litopenaeus vannamei to a certain extent.It is urgent to find a new diet protein source with high protein content,low price and rich sources.Fermented cottonseed meal has all of the above characteristics.Therefore,this paper aimed to study the effects of fermented cottonseed meal replacing fish meal on the growth performance of Litopenaeus vannamei,the nutritional composition of muscle and whole shrimp,the antioxidant capacity of serum and hepatopancreas,the health of the immune system,intestinal digestive system,and the mechanism of hepatopancreas metabolic pathway.The content of lysine in cottonseed meal is low.Although the content of lysine in fermented cottonseed meal has increased,gossypol will combine with lysine in the process of feed processing,which still results in low utilization of lysine by aquatic animals.In theory,crystal lysine needs to be added to optimize the nutritional value of fermented cottonseed meal.Different protein sources cause different demands for lysine in the shrimp diet.To achieve the purpose of precise nutrition,this paper carried out a preliminary exploration of lysine demand after fish meal replacement,hoping to provide a reference for the use of fermented cottonseed meal in the shrimp diet.1.Effects of partially replacing fish meal with fermented cottonseed meal and adding crystal lysine on the growth and antioxidant capacity of Litopenaeus vannameiThe purpose of this experiment was to explore the most appropriate amount of fermented cotton meal replacing fish meal in the diets of Litopenaeus vannamei and initially investigate necessity of supplemental lysine in a practical diet.A total of 8kinds of isonitrogenous and isoenergetic experimental diets were designed,among which 5 groups were gradient levels of fermented cottonseed meal（FCSM 0%,7.5%,15%,22.5%and 30%）partially substituting fish meal,and the other 3 diets were added with 0.25%,0.50%and 0.75%crystal lysine based on the diet containing7.5%FCSM.All 8 diets were fed to shrimp with the initial body weight of（2.55±0.016）g for 60days.The results showed that the survival rate had no significant difference among the FCSM groups（P>0.05）,however,the survival rate exhibited a decreased trend with increasing FSCM,as well as the weight gain rate and specific growth rate.The growth performance was best in the FCSM7.5 group and worst in the FCSM30 group.Supplemental crystal lysine didn’t further improve growth performance.Regarding serum biochemical indexes,there was no significant difference in the activity of alanine aminotransferase among FCSM groups（P>0.05）,but the lowest value appears in the FCSM7.5 group.Among the lysine supplemented groups,the activity of alanine aminotransferase and aspartate aminotransferase in serum of 0.5%group and 0.75%group were significantly lower than that of the FCSM7.5（P<0.05）.In terms of antioxidant capacity,the total antioxidant capacity of hepatopancreas in each FCSM containing group was significantly higher than that in the control group（FCSM0）（P<0.05）,among which the highest value appeared in the FCSM22.5 group and FCSM7.5 group followed by.Catalase,aspartate aminotransferase and glutathione S-transferase activities of hepatopancreas in each FCSM group were not significantly different（P>0.05）.With the increase of lysine content,MDA in serum showed a decreasing trend,and the MDA in the 0.75%group was the lowest.The results of tissue sectioning showed that with the increase of fermented cottonseed meal in the diet,the normal structure of liver corpuscles was gradually lost,and the boundary of basement membrane was not clear.The lumen of hepatic tubules was deformed,and the hepatopancreas tissue was vacuolized.The addition of crystal lysine made the liver corpuscles fuller and tighter.Fermented cottonseed meal partially substituting fish meal induced cell apoptosis in hepatopancreas via CYC,Caspase-3 and AIF gene pathways.The results indicated that excessive use of fermented cottonseed meal will damage the hepatopancreatic cells of Litopenaeus vannamei.The use of 7.5%fermented cottonseed meal in the diet was beneficial to the growth performance of Litopenaeus vannamei,and had no negative impact on the health of hepatopancreas.When the amount of fermented cottonseed meal in the diet was 7.5%,the addition of crystal lysine had no significant effect on the growth performance of Litopenaeus vannamei,but it was beneficial to its health and anti-stress ability.2.Effects of fermented cottonseed meal partially replacing fish meal and adding crystal lysine on nonspecific immunity and digestion of Litopenaeus vannameiIn order to further explore the effect of fermented cottonseed meal on the health of Litopenaeus vannamei,we measured the serum immune indexes,gene expression of Litopenaeus vannamei,the activity of digestive enzymes in hepatopancreas,intestinal tissue sections and intestinal anti-inflammatory genes.The results showed that the content of serum C₃ and C₄ in each fermented cottonseed meal group decreased with the increase of fermented cottonseed meal content.There was no significant difference in C₃ and C₄ between the FCSM7.5 group and the control group（P>0.05）.Among the lysine group,the serum C₃ of the 0.75%group was significantly higher than that of the control group（P<0.05）.The content of C₄ in 0.75%group was the highest（P>0.05）.In terms of immune gene expression,the expression of lectin and Toll-like receptor genes in gills gradually increased with the increase of fermented cottonseed meal content.The expression of IMD gene in FCSM7.5 group was significantly higher than that in the control group（P<0.05）,but there was no significant difference in HSP70 gene between the FCSM7.5 group and the control group（P>0.05）.The expression of lectin gene in0.75%group was significantly lower than that in the control group（P<0.05）,while the expression of IMD gene was significantly higher than that in the control group.There was no significant difference in the Toll-like receptor and HSP70 gene between the0.75%group and the control group（P>0.05）.In terms of digestive enzymes,with the increase of fermented cottonseed meal,the activity of digestive enzymes in hepatopancreas showed a downward trend.The activity of amylase and trypsin in0.75%group was significantly higher than that in the control group（FCSM7.5）（P<0.05）.The activity of lipase was not significantly different from that in the control group（P>0.05）.Amylase and lipase in 0.25%group were significantly lower than those in control group（P<0.05）.In terms of intestinal tissue health,with the increase of fermented cottonseed meal content,the intestinal epithelial cells were damaged more severely.The length of intestinal villi was gradually shortened and shed.Compared with the control group（FCSM0）,the intestinal epithelial cells of FCSM7.5 group were more tightly connected,and the intestinal villi were more developed,dense and higher.The intestinal epithelium of each lysine group was closely connected.The intestinal villi of 0.5%group were more developed.In terms of intestinal anti-inflammatory genes,the TGF-β1 gene expression decreased with the increase of fermented cottonseed meal.There was no significant difference in TGF-β1 gene expression between 0.75%group and FCSM7.5 group（P>0.05）.In the diet of Litopenaeus vannamei,7.5%of fermented cottonseed meal can promote the immune system,improve the absorption area of intestinal villi and enhance the absorption ability of shrimp to nutrients.Adding crystal lysine to the diet can improve the immune function of Litopenaeus vannamei,improve the morphology of intestinal tissue,and promote the secretion of digestive enzymes in the hepatopancreas.3.Effect of partially replacing fish meal with fermented cottonseed meal on hepatopancreas metabolism of Litopenaeus vannameiIn this study,the non-targeted metabonomics of the hepatopancreas of Litopenaeus vannamei was studied by liquid chromatography-mass spectrometry to further analyze the mechanism of the effect of fermented cottonseed meal on the growth performance and hepatopancreas health of Litopenaeus vannamei.According to VIP>1 and T-test P-value<0.05,1466 differential metabolites were screened in FCSM7.5 group（compared with FCSM0）,1190 differential metabolites were screened in FCSM30group（compared with FCSM0）,and 1240 differential metabolites were screened in FCSM30 group（compared with FCSM7.5）.The results showed that there were 7metabolic pathways in FCSM7.5 group and FCSM0 group with significant difference（P<0.05）,among which 3 metabolic pathways were extremely significant（P<0.01）.There were 10 metabolic pathways between FCSM30 group and FCSM0 group,among which 7 metabolic pathways were extremely significant（P<0.01）.There were 11metabolic pathways between FCSM30 group and FCSM7.5 group,among which 7metabolic pathways were extremely significant（P<0.01）.Through analysis,it was found that the use of fermented cottonseed meal in the diet mainly affected the metabolism of nucleotides,glycerol phospholipids and amino acids in the hepatopancreas of Litopenaeus vannamei.After the body ingested fermented cottonseed meal,it might regulate energy metabolism by promoting the metabolism of nucleotides to meet the needs of rapid growth.When the amount of fermented cottonseed meal was too massive,shrimps may adapt to the plant protein source by affecting the linoleic acid metabolism.Excessive fermented cottonseed meal would lead to metabolic disorder and decline of hepatopancreas function.This study preliminarily explored the differential metabolites and metabolic pathway of the hepatopancreas of Litopenaeus vannamei,providing theoretical support for improving the application of fermented cottonseed meal in the diet of Litopenaeus vannamei.