Effects Of Replacement Of Dietary Fish Meal With Clostridium Autoethanogenum Protein And Yellow Mealworm (Tenebrio Molitor) On Growth And Intestinal Health In Large Yellow Croakers (Larimichthys Crocea)

Due to the shortage of fish meal and soybean meal,China’s feed industry cannot develop healthily and steadily,while feed grains cannot take up more arable land,so new feed ingredients need to be developed urgently.Clostridium autoethanogenum protein（CAP）is a new kind of bacterial protein produced on CO as a unique carbon source;chlorell meal（CM）is a single-cell protein source that grows and reproduces by photosynthetic autotrophy;Hermetia illucens meal（HM）and Tenebrio molitor meal（TM）are both new insect protein sources that can convert waste into high-quality protein;cottonseed protein concentrate（CPC）is a new plant protein source produced from cottonseed meal through extraction,oil extraction,and dephenolisation processes.In this study,we investigate the apparent digestibility coefficients of five new non-grain protein sources of dry matter,crude protein,crude lipid,gross energy,and amino acids in large yellow croakers（Larimichthys crocea）,and the effects of CAP and TM replacing fish meal（FM）on the growth and gut health of Larimichthys crocea,respectively.The main results of the research were as follows:（1）Apparent digestibility of five new non-grain protein ingredients for Larimichthys croceaThis study was conducted to evaluate the apparent digestibility of dry matter,crude protein,crude lipid,gross energy,and amino acid of these five protein sources by large yellow croakers（Larimichthys crocea）to provide a theoretical basis for the design of artificial compound feed formulations for large yellow croakers.Triplicate groups of fish（initial weight=154.0±5.3 g）were fed the test diets to apparent satiation two times daily for eight weeks.The test diets consisted of 70%basal diet and 30%test ingredients,and 0.1%yttrium oxide（Y₂O₃）was used as an indicator.The results showed that dry matter coefficients of five test ingredients ranged from56.77%to 75.53%（CAP>TM>CM>HM>CPC）;The apparent digestibility of the crude protein ranged from 69.93%to 89.59%（CAP>CM>CPC>HM>TM）;The apparent digestibility of crude lipid ranged from 58.58%to 93.77%（CAP>CM>TM>CPC>HM）;The apparent digestibility of the gross energy ranged from 63.39%to 84.33%（CAP>HM>CM>TM>CPC）;The apparent digestibility of the total amino acids ranged from 76.62%to93.24%（CAP>CM>CPC>HM>TM）.In conclusion,CAP was the most suitable protein source among the five ingredients,followed by CM,while large yellow croakers did not digest TM,CPC,and HM well.（2）Effects of replacement of dietary fish meal with Clostridium autoethanogenum protein on growth and intestinal health in Larimichthys croceaFour iso-nitrogenous and iso-lipidic diets were formulated,with 0%（CAP0）,15%（CAP15）,30%（CAP30）,and 45%（CAP45）CAP added to the feed to replace fishmeal at 0%,6%,12%and 18%respectively.Fish were fed with 4 diets for 8 weeks,samples were collected and analysed.The results showed that there was no significant difference between the CAP0 and CAP15 groups in terms of final body weight（FBW）,weight gain rate（WGR）,and feed efficiency（FE）（P>0.05）.However,compared to the CAP0 group,the CAP30 and CAP45groups had significantly lower FBW,WGR,and lipid deposition rate（LDR）（P<0.05）,and the CAP45 group had significantly lower FE and protein deposition rate（PDR）（P<0.05）.The whole-body moisture was significantly increased by CAP replacement of FM while crude lipid content was decreased（P<0.05）.No significant difference in crude protein,ash,and liver crude lipid was observed among all groups（P>0.05）.Additionally,serum C4 levels in the CAP30 and CAP45 groups were significantly lower than in the CAP0 group（P<0.05）.Compared to CAP0,CAP30,and the CAP45 groups significantly decreased C4 levels（P<0.05）,and CAP45 group significantly decreased alkaline phosphatase（AKP）activity（P<0.05）but significantly increased lysozyme（LZM）activity（P<0.05）.C3 levels were significantly increased by the CAP15 group（P<0.05）.Analysis of intestinal indicators,intestinal lipase activity in the CAP45 group was significantly higher than that in the CAP0 group（P<0.05）,while intestinal amylase and trypsin activities in the CAP0 group were not significantly different compared to the CAP0 group（P>0.05）.Intestinal villus thickness increased with increasing CAP levels and was significantly higher in the CAP15,CAP30,and CAP45 groups than in the CAP0 group（P<0.05）,while intestinal muscularis thickness was significantly higher in the CAP15 and CAP45 groups than in the CAP0 group（P<0.05）.Serum diamine oxidase（DAO）activity was significantly higher in the CAP0 group than in all other groups（P<0.05）,and D-lactate（D-La）levels were significantly lower in the CAP15 and CAP30 groups than in the CAP0 group（P<0.05）.The analysis of intestine flora showed that the Sob,Shannon,Simpson,Chao1,and ACE indexes were not significantly different between the groups（P>0.05）.The relative abundance of Proteobacteria increased in the CAP15 group and the relative abundance of Firmicutes and Tenericutes increased in the CAP45 group.The relative abundance of Ralstonia decreased in the CAP15,CAP30,and CAP45 groups compared to the CAP0 group,and the intestinal flora in the CAP15group upregulated levels of amino acid metabolism,lipid metabolism,metabolism of cofactors and vitamins,and membrane transport.Overall,at 40%FM in the basal diet,the optimum level of CAP replacement FM in feed is 15%,which helps to improve humoral immunity and intestinal health with no adverse effects on growth;however,substitution levels of 30%and above inhibited growth and hurt immunity.Additionally,CAP promoted intestinal health by reducing intestinal permeability and improving intestinal flora in large yellow croakers.（3）Effects of replacement of dietary fish meal with Tenebrio molitor meal on growth and intestinal health in Larimichthys croceaFour iso-nitrogenous and iso-lipidic diets were formulated,with 0%（TM0）,15%（TM15）,30%（TM30）,and 45%（TM45）TM added to the feed to replace fishmeal at 0%,6%,12%and18%respectively.Fish were fed with 4 diets for 8 weeks,samples were collected and analysed.The results showed that there was no significant difference in FBW and WGR among TM0,TM15,and TM30 groups（P>0.05）,while the TM45 group significantly decreased the FBW,WGR,and protein efficiency ratio（PER）（P<0.05）.The FE was significantly lower in the TM30and TM45 groups than in the TM0 and TM15 groups（P<0.05）.The PDR and LDR were significantly lower in the TM45 group than in all other groups（P<0.05）.Compared to the TM0group,the whole-body moisture was significantly increased in the TM45 group（P<0.05）,while crude lipid content was significantly decreased in the TM15 group（P<0.05）.No significant difference in crude protein and liver crude lipid was observed among all groups（P>0.05）.Additionally,compared with the TM0 group,AKP activity in serum was significantly decreased in the TM45 group（P<0.05）,while the TM15 group significantly increased LZM activity（P<0.05）.The TM30 group showed significantly higher serum C3 levels compared to the TM0group（P<0.05）,while serum C4 levels were significantly decreased in the TM15,TM30,and TM45 groups（P<0.05）.Analysis of intestinal indicators,intestinal lipase activity was significantly higher in both the TM30 and TM45 groups than in the TM0 and TM15 groups（P<0.05）,while intestinal amylase activity was significantly lower in the TM15 group than in all other groups（P<0.05）,while intestinal trypsin activity was not significantly different between the groups（P>0.05）.The intestinal villus height was significantly higher in the TM15 and TM45 groups compared to the TM0 group（P<0.05）.The intestinal muscularis thickness was significantly higher in the TM15and TM30 groups compared to the TM0 group（P<0.05）,while the villus thickness was significantly lower in the TM0 group compared to all other groups（P<0.05）.Serum DAO activity and D-la levels were significantly higher in the TM0 group than in all other groups （P<0.05）.The analysis of intestine flora showed that the Sob,Shannon,Simpson,Chao1,and ACE indexes were not significantly different between the groups（P>0.05）.The relative abundance of Bacteroidetes decreased in the TM15,TM30,and TM45 groups compared to the TM0 group,while the relative abundance of Firmicutes and Actinobacteria increased.The relative abundance of Ralstonia decreased in the TM15 group compared to the CAP0 group,and the intestinal flora in the TM45 group upregulated levels of carbohydrate metabolism,lipid metabolism,metabolism of terpenoids and polyketides,glycan biosynthesis and metabolism,and nucleotide metabolism.Overall,at 40%FM in the basal diet,the optimum level of TM replacement FM in feed is 15%,which improved humoral immunity and intestinal health with no adverse effects on growth,but the replacement of FM with 30%and 45%TM adversely affects growth and humoral immunity.Furthermore,TM promoted intestinal health by reducing intestinal permeability,improving intestinal villus morphology,and modulating intestinal flora in large yellow croakers.