Effects Of Fish Meal Replaced By Clostridium Autoethanogenum Protein On Growth,intestinal Health And Muscle Qualityperformance Of Procambarus Clarkii

An 8-week feeding experiment was conducted to determine the optimal requirement of Clostridium autoethanogenum protein(CAP)in the feed of Procambarus clarkia [initial body weight(6.00±0.01)g].Six isonitrogenous and isolipidic(31% crude protein,6% crude lipid)experimental diets containing 0(H0),0.3%(H0.3),0.6%(H0.6),0.9%(H0.9),1.2%(H1.2)and 1.5%(H1.5)Haematococcus pluvialis were designed.The results are shown as follows:(1)Effects of CAP on growth performance and body composition of Procambarus clarkiiWith the increase of the level of CAP replacing fish meal,the weight gain rate and specific growth rate increased first and then decreased.The weight gain rate of Y25 group was significantly higher than that of the control group(P<0.05).The growth broken line model was constructed for the weight gain rate,and the suitable replacement amount of cap in Procambarus clarkii feed was 22.08%.There was no significant difference in the composition activity rate and feed coefficient of each test(P>0.05).There was no significant difference in liver body index,visceral body index and body composition among the groups(P>0.05).CAP instead of fish meal had no significant effect on the whole shrimp moisture,crude protein,crude lipid and crude ash(P>0.05).(2)Effects of CAP on hemolymph biochemical indexes of Procambarus clarkiiWith the increase of the level of CAP replacing fish meal,the activities of aspartate aminotransferase(ALT)and alanine aminotransferase(AST)in hemolymph increased,and reaching the maximum in Y100 group,which were significantly higher than those in the control group(P<0.05).The contents of complement 3(C3),lysozyme(LZM)and the activity of phenol oxidase(PO)activity increased first and then decreased.Among them,the content of complement C3 in Y25 group reached the maximum and was significantly higher than that in other groups(P<0.05);The content of LZM in Y50 group reached the maximum,but there was no significant difference between each experimental group and the control group(P>0.05);The PO activity in Y25 group was the highest and significantly higher than that in the control group(P<0.05).(3)Effects of CAP instead of fish meal on histomorphology and antioxidant enzyme activity of hepatopancreas of Procambarus clarkiiWith the increase of the proportion of CAP replacing fish meal,the transport volume and number of secretory cell B cells increased,the number of storage cell R cells decreased,and the number of vacuoles in cells increased gradually.Among them,in Y100 group,the connection of hepatic tubules was loose,and the basement membrane of some cells in hepatopancreas was broken,resulting in nuclear leakage,indicating that hepatopancreas had lesions.With the increase of the proportion of CAP replacing fish meal in the feed,the SOD activity and GSH content showed a downward trend.Among them,the SOD activity in Y75 and Y100 groups was significantly lower than that in the control group(P<0.05);The content of GSH in Y100 group was significantly lower than that in the control group(P<0.05).There was no significant difference in CAT activity and MDA content among the test groups(P>0.05).(4)Effects of CAP replacing fish meal on intestinal health of Procambarus clarkiiThe intestinal villi of each experimental group were intact.With the increase of the proportion of CAP replacing fish meal in the feed,the height and width of intestinal villi increased first and then decreased.Among them,the height of intestinal villi in Y25 group reached the maximum.With the increase of the proportion of CAP replacing fish meal in feed,the activities of trypsin and Na+K+-ATPase increased first and then decreased.Compared with the control group,the activities of trypsin and Na+K+-ATPase in Y25 group increased significantly(P<0.05),and there was no significant difference in the activities of amylase and lipase among the experimental groups(P>0.05).With the increase of the proportion of CAP replacing fish meal,the m RNA levels of po and alf in the intestinal tract of each experimental group increased first and then decreased.Among them,the expression levels of po in Y25,Y50 and Y100 groups were significantly higher than those in the control group(P<0.05);The m RNA expression level of alf in Y50 group reached the maximum and was significantly higher than that in Y100 group(P<0.05).There was no significant difference in the m RNA expression levels of NF-κB,rel,lzm and tlr(P>0.05).Sequencing analysis of intestinal flora showed that at the phylum level,compared with the control group,the richness of Firmicutes increased and Proteobacteria decreased in each experimental group.At the genus level,compared with the control group,the richness of Candida bacilloplasma and rsahf231 increased,and the richness of Citrobacter decreased.The α Diversity(Chao1,Shannon)and the number of OTUs in each experimental group increased first and then decreased.The β Diversity diversity index principal component analysis(PCo A)found that the composition structure of intestinal microbiota was similar among the groups.(5)Effect of CAP replacing fish meal on muscle quality of Procambarus clarkiiThere was no significant difference in the content of total essential amino acids in muscle among the test groups(P>0.05),but the content of arginine decreased with the increase of CAP substitution ratio,and the content of arginine in Y100 group was significantly lower than that in the control group(P<0.05).CAP had no significant effect on muscle fiber area,muscle fiber diameter and muscle fiber density(P>0.05).With the increase of the proportion of CAP replacing fish meal,the activity of muscle superoxide dismutase(SOD)increased first and then decreased,and Y25 group increased significantly compared with the control group(P<0.05).CAP had no significant effect on catalase(CAT)activity,malondialdehyde(MDA)and reduced glutathione(GSH)content in muscle(P>0.05).With the increase of the proportion of CAP replacing fish meal,the expression levels of nrf2 and mmsod in muscle of each experimental group increased first and then decreased,and the expression levels of nrf2 in each experimental group were significantly higher than those in the control group(P<0.05).The expression level of Keap1 decreased first and then increased,and the expression level of Keap1 in Y25 group was significantly lower than that in the control group(P<0.05).There was no significant difference in the expression levels of gpx,gr and cat in muscles of each group(P>0.05).The expression level of muscle mef2 a increased first and then decreased.The expression level of muscle mef2 a in Y25,Y50 and y75 groups was significantly higher than that in the control group(P<0.05).The expression level of muscle mstn in Y25 group decreased first and then increased,and the expression level of muscle mstn in Y25 group was significantly lower than that in the control group(P<0.05).CAP had no significant difference in the expression of mef2 b,mih in muscle(P>0.05).Conclusion:(1)the complete substitution of CAP for fish meal in basic diet(including10% fish meal)has no significant negative effect on the growth performance,and the substitution of 25% fish meal can significantly improve the growth performance.(2)The appropriate amount of CAP instead of fish meal can improve the digestion and absorption capacity intestinal group,as well as the richness and diversity of flora,and maintain the steady state of intestinal flora.In addition,CAP up regulates the gene expression of intestinal po and alf anti-inflammatory cytokines and improved the function of intestinal mucosal barrier.(3)CAP can improve muscle antioxidant capacity by regulating the expression of antioxidant enzymes and cytokines related to Nrf2/Keap1 signaling pathway,and improve muscle development level and improve muscle quality by up regulating the expression level of muscle development promoting gene mef2 a and down regulating the expression level of myostatin(mstn).(4)The complete substitution of CAP for fish meal in basic diet will reduce the antioxidant level hepatopancreas,increase its oxidative damage,and cause hepatopancreatic tissue lesions,thus damaging liver health.In conclusion,Under the conditions of this test,the complete replacement of fish meal with cap in the feed does not affect the growth performance,but may reduce the antioxidant level of liver and damage the health of liver.The appropriate proportion of CAP to replace fish meal in feed is 25-75%(the content of CAP is 19.85g/kg-79.4g/kg).Based on the weight gain rate,the optimal proportion of CAP to replace fish meal is 22.08%.