| Yeast culture,consisting mainly of yeast metabolites,yeast cells and denatured media,plays a significant role in improving the growth performance of organisms,improving intestinal health homeostasis and enhancing the immunity of the body.As a probiotic,it is widely used in livestock and aquaculture because it is non-polluting,residue-free and does not produce drug resistance.Therefore,this experiment was conducted to investigate the effects of using yeast cultures in feed as a substitute for fishmeal and the addition of yeast cultures to fishmeal-free feed on the growth performance,digestibility and immune performance of Chinese mitten crabs.The effects of yeast cultures on intestinal health were also investigated through their effects on intestinal digestive enzymes,antibacterial capacity and intestinal flora.Finally,the effect of yeast cultures on the mitigation of ammonia and nitrogen stress in E.sinensis was investigated based on the antioxidant capacity of yeast cultures.In this experiment,0%,1%,2%and 3%of fishmeal were replaced by yeast cultures in the basal diet for the Chinese mitten crab,noted as R0,R1,R2 and R3 groups respectively.In addition 0%,1%,2%and 3%yeast cultures were added to the fishmeal-free feed,noted as groups N0,N1,N2 and N3.All crabs were fed twice a day for 90 d to ensure that all crabs completed two moults.At the end of the culture trial,growth performance,digestibility and immune performance were measured.Intestinal digestive enzyme activity,expression of antimicrobial peptide-related genes and intestinal flora were also measured.Finally,the Chinese mitten crab was subjected to ammonia stress at a concentration of 40 mg/L NH4Cl,with group 0under normal conditions as the control group and the test group under ammonia conditions as the attack group,respectively,for a 24 h acute attack test,with no feeding during the attack period.The test results are as follows:（1）Yeast cultures replacing 1%and 2%fishmeal significantly increased FBW,WGR and SGR,reduced FCR（P<0.05）and significantly increased the crude protein content of the organism（P<0.05）in E.sinensis.Substitution of 2%and 3%fishmeal levels reduced the body crude fat content（P<0.05）.Replacement of fishmeal by yeast cultures in the diet significantly increased the apparent digestibility of dry matter,protein and fat in the organism（P<0.05）.After testing insulin-like growth factor 2（IGF2）,ribosomal protein S6（S6）,ribosomal S6protein kinase（S6K1）,eukaryotic translation initiation factor 4E binding protein 1（4EBP1）and eukaryotic translation initiation factor 4E（e IF4E）in muscle,it was found that the expression of IGF-2 was significantly higher at 1%,and at alternative levels of 1%and 2%,the The expression of m TOR,S6K1 and e IF4E was significantly higher（P<0.05）.In a study of antioxidant properties,it was found that yeast cultures replacing fishmeal significantly increased the activities of SOD,GSH,GST,GSH-PX,T-AOC and CAT in the hepatopancreas of Chinese mitten crab and simultaneously reduced the MDA content.However,at a substitution level of3%,some negative effects may occur,such as a significant decrease in GSH-PX activity（P<0.05）.In a study on immune performance,it was found that substitution levels of 1%significantly increased AKP,ACP and LZM activity in haemolymph（P<0.05）,while GOT activity was significantly higher at substitution levels of 3%（P<0.055）.Substitution of fishmeal by yeast cultures in feed activated the expression of pro PO and GST genes in the E.sinensis organism and inhibited the expression of the pro-inflammatory factor LITAF（P<0.05）,but had no significant effect on the expression of LZM（P>0.05）.In summary,replacing fishmeal with yeast cultures at 1%and 2%in the feed improved the antioxidant capacity and immune performance of Chinese mitten crab,while significantly increasing IGF-2 expression and promoting the body’s ability to digest and absorb nutrients through activation of the m TOR/S6K1 pathway and improving growth performance.（2）In studies on intestinal health it was shown that yeast cultures replacing 1%and 2%of fishmeal significantly increased intestinal AMY,LPS and Trypsin activity（P<0.05）.Yeast cultures replacing fishmeal increased the expression of intestinal antimicrobial peptide-related genes（ALF1,ALF3,Relish and crustin）and periplasm（PM1 and PM2）（P<0.05）,but the expression of crustin and PM2 was significantly lower at a substitution level of 3%（P<0.05）.In study on intestinal flora,it was found that the effect of yeast cultures replacing fishmeal on intestinal flora changed the abundance of Tenericutes,Firmicutes,Firmicutes and Actinobacteria mainly at the phylum level.At the genus level,Acinetobacter,a conditionally pathogenic bacterium,was significantly elevated at a substitution level of 3%.Studies have shown that yeast cultures can modulate intestinal resistance to pathogens by increasing intestinal digestive enzyme activity and inducing the expression of antimicrobial peptide-related genes,while improving the composition of the intestinal flora to promote the digestion and absorption of nutrients and the immune performance of the organism.（3）Ammonia and nitrogen stress significantly reduced antioxidant enzyme activity and increased lipid peroxide content in the hepatopancreas（P<0.05）.However,in the experiment,it was found that the hepatic antioxidant enzyme activity was significantly higher in the yeast culture addition group than in the ammonia-nitrogen stress group,but lower than in the normal environment control group.In the study of MAPK pathway and immune factors,the expression of MAPK,P38,ERK,My D88,LITAF,Relish and IL-16 was found to be activated in the hepatopancreas by ammonia and nitrogen stress（P<0.05）,and these genes were significantly down-regulated in the yeast culture substituted fish meal group（P<0.05）,but still significantly higher than in the normal environment control group（P<0.05）.The above results suggest that replacing fishmeal with yeast culture in the diet can alleviate the damage caused by ammonia stress by improving the antioxidant capacity,reducing ROS production and inhibiting MAPK expression in Chinese mitten crabs.（4）The addition of yeast cultures to fishmeal-free diets significantly increased FEW,WGR and SGR levels,reduced FCR and significantly increased crude protein content of the organism at 2%and 3%（P<0.05）.Apparent digestibility of dry matter was significantly increased at1%and 2%addition levels（P<0.05）,and the highest values for protein and fat were reached at 3%addition（P<0.05）.The addition of yeast cultures to fishmeal-free diets significantly activated the expression of IGF2,S6,S6K1,4EBP1 and e IF4E in the muscles of Chinese mitten crabs（P<0.05）.The addition of yeast culture improved the antioxidant capacity of Chinese mitten crabs,and the antioxidant enzyme activity tended to increase and then decrease with the addition of yeast culture.The malondialdehyde level was significantly lower than that of the control group at 1%and 2%,and was still lower than that of the control group at 3%（P<0.05）,although there was an increasing trend.The activity of non-specific immunoenzymes AKP,ACP and LZM was highest in the hemolymph group at 2%（P<0.05）,and the activity of GOT and GPT was not significantly different between the test and control groups（P>0.05）.The expression of pro PO,GST and LZM in the hepatopancreas was significantly higher in the 1%and 2%groups（P<0.05）.the expression of LITAF tended to decrease with the level of yeast culture addition and was significantly lower in all experimental groups than in the control group（P<0.05）.These results suggest that the addition of yeast cultures to fishmeal-free diets can improve the growth performance and digestibility of Chinese mitten crabs by activating the m TOR/S6K1 pathway,as well as improving antioxidant capacity and immune performance.（5）In a study on intestinal health it was found that the addition of 1%and 2%yeast cultures to fishmeal-free diets significantly increased the enzymatic activity of intestinal AMY,LPS and Trypsin（P<0.05）.The addition of yeast cultures activated the expression of intestinal antimicrobial peptide factors（ALF1,ALF3 and crustin）and periplasm（PM1 and PM2）,but the expression of crustin and PM2 was inhibited at 3%addition（P<0.05）.Expression of Relish increased significantly with increasing levels of yeast culture addition（P<0.05）.Analysis of intestinal 16s RNA revealed that yeast addition at 3%significantly reduced the abundance of Tenericutes and Firmicutes,but increased the abundance of the opportunistic pathogen Clostridium.Also,at the genus level,the addition of 3%yeast culture significantly increased the abundance of the opportunistic pathogen Acinetobacter.This suggests that yeast cultures can improve the digestion and absorption of nutrients by increasing digestive enzyme activity,improving intestinal immunity and antimicrobial capacity and altering the abundance of intestinal microbial composition,thus improving the health of the organism.（6）Ammonia and nitrogen stress significantly reduced the activity of antioxidant enzymes in the hepatopancreas of Chinese mitten crab,increased the MDA content,caused the body to produce large amounts of ROS,resulting in oxidative stress in the hepatopancreas（P<0.05）,while activating the MAPK pathway in the hepatopancreas and promoting the expression of inflammatory factors（LITAF,Relish and IL-16）.SOD,CAT,GSH and T-AOC activities tended to increase with increasing levels of yeast culture addition in fishmeal-free diets.Expression of MAPK pathway and inflammatory factors were significantly down-regulated compared to the stress control.The addition of yeast cultures to fishmeal-free diets can effectively alleviate the negative effects of ammonia and nitrogen stress by increasing the antioxidant properties and inhibiting the activation of the MAPK pathway and the expression of pro-inflammatory factors,which can inhibit the excess ROS produced by ammonia and nitrogen stress.In summary,yeast cultures in diets replacing fishmeal and fishmeal-free diets can improve growth performance,digestibility,antioxidant capacity and immune performance by activating the m TOR/S6K1 pathway,improve intestinal health by increasing intestinal digestive enzyme activity,antibacterial capacity and intestinal flora composition abundance,and alleviate oxidative stress due to ammonia and nitrogen stress by inhibiting the MAPK pathway in Chinese mitten crab. |
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