Effects Of Phytosterols On Growth,Immunity,Intestinal Health And Muscle Fatty Acids In Procambarus Clarkii

Procambarus clarkii belongs to Arthropoda,Crustacea,Decapoda.It is a carnivorous omnivorous crustacean.It is the largest and highest-yield freshwater crustacean in China.Because of its delicious taste,it wins people’s love and plays an extremely important role in aquaculture.Cholesterol is an important component of the cell membrane in eukaryotic organisms,and it is also the precursor of ecdysone synthesis in crustaceans.If the body lacks cholesterol,it will affect the molting activity of crustaceans and cause death.Crustaceans are unable to synthesize cholesterol from lipids or carbohydrates due to the lack of key enzymes necessary for the sterol synthesis pathway in the body,and can only be supplemented from exogenous sources,which leads to an increase in feed costs during artificial breeding.Phytosterols are naturally occurring sterols,which are distinguished from cholesterol by having a hydroxyl side chain on the C24 atom.Studies have shown that phytophagous insects and some crustaceans can convert phytosterols into cholesterol.At the same time,phytosterols can reduce serum low-density lipoprotein cholesterol without affecting other lipoproteins,and have physiological functions such as anti-cancer,anti-inflammatory,regulating immunity and bacteriostasis.At present,the related research on phytosterols in Procambarus clarkii has not been reported.Whether Procambarus clarkii can use phytosterols to replace cholesterol in feed to achieve good breeding results,what is the appropriate level of phytosterols in Procambarus clarkii feed,whether Procambarus clarkii has a tendency to different types of phytosterols,and the effect of phytosterols on the health of Procambarus clarkii itself and the effect of Procambarus clarkii on human healthy diet after feeding phytosterols are still unknown.In order to answer the above questions of phytosterols and crayfish,two feeding experiments were designed in this study.Different levels of commercial mixed phytosterols were added to the feed,and the same levels of cholesterol,sitosterol,stigmasterol and ergosterol were added to the feed.In this study,Procambarus clarkii was used as the research object to explore the effects of different phytosterol levels and different types of phytosterols in feed on the growth,digestion,immunity,hepatopancreas antioxidant,hepatopancreas morphology,intestinal flora and muscle fatty acid composition of Procambarus clarkii.It provides new basis and ideas for breeding crayfish.1.Study on the suitable phytosterol level of Procambarus clarkiiThis experiment was conducted to investigate the effects of dietary phytosterols on growth,digestion,antioxidant,immune indexes,hepatopancreas and intestinal morphology of Procambarus clarkii.Five diets were prepared by adding 0%,0.10%,0.19%,0.38% and 0.76% phytosterols to the basal diet,which were named as CON,P1,P2,P3 and P4,respectively.The crayfish with an average body weight of(9.37 ± 0.02 g)cultured for 6 weeks.The results showed: weight gain rate and specific growth rate of P1 and P2 were significantly higher than those of CON(P < 0.05),and the growth performance of P2 was the best.Protease activity in gut of P3 were significantly higher than CON,lipase activity in P3 was significantly lower than in CON(P < 0.05).Acid phosphatase and alkaline phosphatase activity of P1 were the highest in hepatopancreas,acid phosphatase and alkaline phosphatase activity of P2 were the highest in hemolymph,but there was no significant difference compared with CON(P > 0.05).With the increase of phytosterol level,total superoxide dismutase and catalase activity in hepatopancreas and hemolymph increased,and the content of malondialdehyde decreased.Compared with CON,intestinal structure of P1 was improved.When the phytosterol level exceeded0.10%,hepatopancreas and intestinal damage increased with the increase of dietary phytosterol level.With the increase of phytosterol level,the expression of nf-kb in hepatopancreas increased.The expression of hsp70 in P1 was significantly higher than in other groups(P < 0.05).With the experimental conditions,0.10% and 0.19% phytosterol was beneficial to the growth of Procambarus clarkii.With the increase of the phytosterol level,the antioxidant and immune ability of Procambarus clarkii increased.2.Effects of different phytosterols on growth,digestion,tissue biochemistry,immunity of Procambarus clarkiiIn order to study the effects of different phytosterols on the growth,digestion,tissue biochemistry,immunity and intestinal and liver health of Procambarus clarkii,eight isoegg diets were formulated and named CON,SIT1,STI1,ERG1,CHO,Sit,STI2 and ERG2,respectively.Procambarus clarkii with an initial body weight of(10.14±0.01)g were fed for 6 weeks.The results showed that:(1)The final body weight(FBW),weight gain rate(WG)and specific growth rate(SGR)of SIT2 group were significantly higher than those of CON,STI2 and ERG2 groups(P < 0.05).The FCR of ERG1 was significantly lower than that of CON,STI2 and ERG2 groups(P < 0.05).(2)The triglyceride(TG)in CHO group was significantly higher than that in other groups(P <0.05).The total cholesterol(T-CHO)in the cholesterol-added group was significantly higher than that in the non-cholesterol-added group except ERG2(P < 0.05).The low density lipoprotein(LDL)in the CON group was significantly lower than that in the other groups except SIT1(P < 0.05).Alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in CHO group were significantly lower than those in other groups except SIT1 group(P < 0.05).(3)Compared with other groups,the volume and number of vacuoles in intestinal connective tissue of CON,STI2 and ERG2 groups increased.The structure of hepatic tubules of SIT1 and CHO was complete,the stellate structure was obvious,the different cells were obviously distinguished,and the hepatic tubules were closely connected.In the CON and ERG2 groups,the stellate structure disappeared,the hepatic tubules in the CON group were damaged,and the adhesion between the hepatic tubules occurred;(4)The relative expression level of nf-κb in CON group was significantly higher than that in other groups except SIT2 group(P < 0.05).The relative expression level of hsp70 in CHO group was significantly higher than that in other groups(P < 0.05).The relative expression level of fabp in CON group was the highest.The relative expression level of fas in SIT2 group was significantly higher than that in other groups(P < 0.05).The results showed that with the experimental conditions,the addition of three phytosterols alone could significantly enhance the growth performance of Procambarus clarkii,and the effect was similar to that of adding the same level of cholesterol.With the increase of the number of phytosterol double bonds,the antioxidant and immune levels of Procambarus clarkii were improved.When phytosterols and cholesterol were added at a ratio of 1:1,the addition of sitosterol was superior to stigmasterol and ergosterol.3.Effects of different phytosterols on intestinal flora and fatty acid composition of Procambarus clarkiiIn order to study the effect of phytosterols on the intestinal flora of Procambarus clarkii,four groups of CON,CHO,SIT1 and SIT2 were selected for intestinal flora detection.The results showed that:(1)The abundance of flora in SIT2 group was the highest,followed by CON,SIT1 and CHO group.The CON group had the highest bacterial diversity,followed by SIT2,SIT1,and finally the CHO group.(2)In this experiment,the dominant bacteria in the intestinal tract of Procambarus clarkii were Firmicutes,Proteobacteria,Cyanobacteria and Chloroflexi.(3)Compared with CON and SIT2,CHO and SIT1 increased the abundance of Firmicutes and decreased the abundance of Proteobacteria,and the abundance of Aeromonas in SIT2 group increased significantly at the genus level.Studies have shown that a single addition of sterols in feed is more beneficial to the intestinal health of Procambarus clarkii.In order to study the effect of phytosterols on the fatty acid composition of muscle tissue of Procambarus clarkii and evaluate the effect of feeding phytosterols on human healthy diet,the same experimental method was used in Experiment 2.The results showed that:(1)Different phytosterols had significant effects on fatty acids of Procambarus clarkii,and MUFA in CHO group was significantly higher than that in other groups(P <0.05).The PUFA of CHO,SIT1 and SIT2 groups was significantly higher than that of other groups(P < 0.05).The contents of n-3PUFA and n-6PUFA in CHO group were significantly higher than those in other groups(P < 0.05),and the contents of n-3PUFA and n-6PUFA in SIT1 and SIT2 were second only to those in CHO group.(2)IA and IT in ERG2 group were the highest,which were significantly higher than those in other groups(P < 0.05).Studies have shown that crayfish fed the same sitosterol and cholesterol have higher muscle unsaturated fatty acids,and sitosterol can reduce the proportion of saturated fatty acids in crayfish,thereby reducing the occurrence of cardiovascular disease.