Effects Of Plant Protein Sources Replacing Fishmeal On Growth Performance,Intestinal Microflora And Immunity Of Juvenile Hybrid Grouper(Epinephelus Fuscoguttatus♀×E. Lanceolatu♂)

In this paper,the hybrid grouper（Epinephelus fuscoguttatus♀×E.lanceolatu♂）was studied.The growth performance,enzyme activity,intestinal inflammation-related antiinflammatory factors,pro-inflammatory factors m RNA expression,and the expression of PI₃K/Akt protein in the intestine of the hybrid grouper were studied to investigate the effects of TLRs/My D88-PI₃K/Akt signaling pathway on the growth performance,enzyme activity,related genes and protein expression of the hybrid grouper.The research contents and main research results of this paper are as follows:1.60% of the feed fishmeal content was set as the control group（FM）,and the concentrated cottonseed protein（CPC）was used to replace 12%,24%,36%,48% and 60%（R12,R24,R36,R48 and R60）fishmeal protein of the FM group,respectively.Six isonitrogenous（50% crude protein）and iso-lipidic（10% crude lipid）diets were formulated,and methionine（Met）and lysine（Lys）were added to the replacement groups at the same level as the FM group.By exploring the growth performance,enzyme activity,intestinal TLRs/My D88 and PI₃K/Akt signaling pathways of the hybrid grouper,the optimal replacement level of CPC in the hybrid grouper diet was sought.The hybrid grouper juveniles with an initial weight of（11.00±0.23）g were randomly divided into 6 groups,3 replicates in each group,30 fishes were cultured in each 0.5 m3 fiberglass barrel,and lasted 8 weeks of the experiment.At the end of the experiment,10 fish were randomly selected from each barrel,and Vibrio parahaemolyticus was injected for a one-week challenge test.The results showed that the specific growth rate（SGR）was the most significant（P<0.05）when the replacement level reached 48%,and when the replacement level was 24%,the feed coefficient ratio（FCR）was significantly increased（P<0.05）.When the replacement level reached 36%,the serum malondialdehyde（MDA）content increased significantly（P< 0.05）.The height of the proximal intestine（PI）,mid intestine（MI）and distal intestine（DI）plica height decreased significantly at the replacement level of 48%（P<0.05）.When the replacement level reached 36%,liver fat deposition increased.The composition of intestinal flora was determined by illumina high-throughput sequencing technology.From the level of the family,when the level of substitution increased,the pathogenic bacteria such as Mycoplasmataceae in the PI increased,the abundances of beneficial bacteria,such as Comamonadaceae and Lactobacillaceae were decreased;the abundance of Lactobacillaceae was decreased in the MI;the abundances of Alcaligenaceae and Xanthomonadaceae were reduced in the DI.When the replacement level reached 60%,the abundances of flora in the PI and MI have been dysregulated,there were no dominant bacteria,and when replacement level reached 24%,the dominant bacteria in the DI has been lost and a serious flora imbalance has occurred.The expressions of IL-6,TNF-α and IL-1β m RNA in the PI,MI and DI were significantly up-regulated（P<0.05）,and the expression of hepcidin m RNA was significantly down-regulated（P<0.05）.IFN-γ m RNA expression was significantly upregulated in PI and MI（P<0.05）.The protein expression of P-PI₃K/PI3 K,P-Akt/Akt and midgut P-PI₃K/PI3 K in the foregut were significantly up-regulated with the increase of the replacement level（P<0.05）,P-Akt/Akt in the MI and P-Akt/Akt in the DI were significantly up-regulated when the replacement level reached 60%（P< 0.05）.The above results indicate that CPC replacing fish meal could reduce the growth performance and intestinal immunity of hybrid grouper.The abundance of beneficial bacteria in the PI,MI and DI was reduced,and the abundances of harmful bacteria were increased.At the same time,different inflammatory functional responses in PI,MI and DI are partially mediated by the TLR-2/My D88-PI₃K/Akt signaling pathway.Based on the SGR criteria,the optimal substitution level and maximum replacement level of CPC in the hybrid grouper were 17.18% and 34.36%,respectively.2.60% of the feed fishmeal content was set as the control group（FM）,soybean meal（SBM）,peanut meal（PM）,cottonseed meal（CSM）and cottonseed protein concentrated（CPC）were used to replace 45% of the fish meal protein in the control group.Five isonitrogenous（50% crude protein）and iso-lipidic（10% crude lipid）diets were formulated.By exploring the growth performance,enzyme activity,intestinal TLRs/My D88 and PI₃K/Akt signaling pathways of the hybrid grouper,the differences in the mechanism of action of different plant protein sources were sought.The hybrid grouper juveniles with an initial weight of（11.00±0.23）g were randomly divided into 5 groups,3 replicates in each group,30 fishes were cultured in each 0.5 m3 fiberglass barrel,and lasted 8 weeks of the experiment.The results showed that The SGR of the FM group was significantly higher than that of the SBM,PM,CSM and CPC groups（P<0.05）,while the SGR of the CPC group was significantly higher than that of the SBM,PM and CSM groups（P<0.05）.Serum CAT content in SBM,PM,CSM and CPC groups were significantly higher than that in FM group（P<0.05）.The MI and DI diameter/ plica height ratio（Id/Ph）of the SBM,PM and CSM group were significantly lower than those of the FM and CPC groups（P<0.05）,while the CPC group was significantly lower than the FM group（P<0.05）.Compared with the SBM,PM and CSM groups,the abundances of Rhodobacteraceae and Microbacteriaceae in the CPC group of MI were increased,and the abundance of Lachnospiraceae of the DI was decreased.Compared with the FM group,the expression levels of TNF-α,IFN-γ and IL-12 m RNA in SBM,PM and CSM groups were significantly up-regulated（P<0.05）,while those in the CPC group were significantly lower than those in SBM,PM and CSM groups（P<0.05）.The expressions of IL-6 in MI and MI,hepcidin in MI,TLR-3 and TLR-3 m RNA in DI were significantly lower than those in FM group（P<0.05）.In PI,MI and DI,the expression of PPI₃K/PI3 K protein in the SBM group and PM group was significantly higher than that in FM group（P<0.05）.The expression of P-Akt/Akt protein in PM and CSM group was significantly higher than that in FM group（P<0.05）.The above results indicate that SBM,PM,CSM,CPC could significantly reduce the growth performance and antioxidant capacity of the hybrid grouper,destroy the PI,MI,DI morphological structure,resulting in PI,MI,DI intestinal flora disorder.On the other hand,plant protein sources could cause intestinal inflammation by partially activating the TLR-2/My D88-PI₃K/Akt signaling pathway in PI,MI and DI.