Physiological Effects Of Hypoxia Stress On Juvenile Chinese Mitten Crab (Eriocheir Sinensis) And Nutritional Alleviation Methods

The rapid development of intensive aquaculture scale and the continuous increase in density have resulted in the problem of low dissolved oxygen(hypoxia stress)in aquaculture waters becoming increasingly prominent.Hypoxia stress can affect the growth and physiological functions of aquatic animals,causing the body's resistance to stress to decline or even die.Chinese mitten crab(Eriocheir sinensis)is a unique economic species in China and has important economic value and scientific research value.It is particularly important to explore the negative physiological effects of hypoxia stress on E.sinensis and and seek nutritional methods for relief.In this paper,E.sinensis are used as the research object to explore the changes of glucose and lipid metabolism-related physiological indexes,metabolic enzymes and the expression of hypoxia-inducible factor 1? genes(HIF1?)under short-term hypoxia stress.Carbohydrate source and level and the effect of addition of salidroside and astragalus polysaccharide on the growth performance and hypoxia tolerance of juvenile crabs also been explore.The research results can be used to supplement the basic biological data of E.sinensis and other crustaceans responding to hypoxia stress,and provide references for the adoption of effective nutrition improvement measures in the production of E.sinensis.The main research results and conclusions are as follows:1.Effect of hypoxia stress on glucose and lipid metabolism of juvenile Chinese mitten crab(Eriocheir sinensis)To explore the effect of hypoxia stress on the glucose and lipid metabolism of E.sinensis,the juvenile crab(4 ± 0.21)g was exposed to(2.0 ± 0.2)mg/L hypoxia environment and the control group is dissolve oxygen(7 ± 0.2)mg/L(normoxia).Three replicates were set for each group.Hepatopancreas and hemolymph of juvenile crabs in the normoxia group and the hypoxia group were collected at 0 h,3 h,6 h,12h,24 h,48 h and 96 h after the experiment started to determine the metabolites,metabolic enzyme activity and metabolic related gene expression of glucose and lipid metabolism.The experimental results showed that the lethal concentration of dissolved oxygen in the juvenile E.sinensis at 12 hours was 0.773 mg/L(95% confidence interval: 0.615-0.926 mg/L).Glucose metabolism indicators showed that the hemolymph glucose concentration under hypoxia was significantly higher than normoxia at 6 h(P < 0.05),and the hemolymph glucose concentration under hypoxia was significantly lower than normoxia at 24 h and 48 h(P < 0.05).3 h,6 h,12 h,24 h and 48 h hepatopancreas glycogen content was significantly lower than normoxia(P < 0.05).3 h,6 h and 24 h hypoxia pyruvate content was significantly higher than normoxia(P < 0.05).The expression of pyruvate dehydrogenase gene in 3 h,6 h,12 h,24 h,48 h and 96 h under hypoxia was significantly lower than that of normoxia(P < 0.05).The activity of lactate dehydrogenase(LDH)at 3 h,12 h and 24 h was significantly higher than that of normoxia(P < 0.05).At 3 h,6 h and 24 h,the lactic acid content of hypoxia was significantly higher than that of normoxia(P < 0.05).Lipid metabolism indexes showed that there was no significant difference in hemolymph triglyceride(TG)content in all groups between hypoxia and normoxia(P > 0.05).Hepatopancreas TG content under 3 h,6 h and 24 h hypoxia was significantly higher than that of normoxia(P < 0.05).Triglyceride hydrolase es_IL m RNA level under 6 h,12 h and 24 h hypoxia was significantly lower than that of normoxia(P < 0.05).Triglyceride hydrolase es_TGL2 m RNA level at 6 h and 12 h hypoxia was significantly lower than that of normoxia(P < 0.05).The results of hepatopancreas HIF1? expression showed that the HIF1? expression was significantly higher at 3 h,6 h,12 h and 24 h hypoxia than that of normoxia(P < 0.05).The results suggest that the 12 h hypoxia lethal concentration of the juvenile E.sinensis was 0.773 mg/L.Short-term(24 h)hypoxia stress can increase the transcription of HIF1? in juvenile crab,leading to changes in hepatopancreas glucose and lipid metabolism,enhanced glycolysis and anaerobic glucose metabolism,weakened aerobic metabolism and inhibited lipolysis.As time goes on(more than 24 h),glucose metabolism weakens,and lipid catabolism gradually recovers.2.Effects of different carbohydrate source and carbohydrate levels in feed on hypoxia tolerance of juvenile Chinese mitten crab(Eriocheir sinensis)Carbohydrate is the main source of energy that aquatic animals need to cope with environmental stress.To explore the effect of different carbohydrate sources and carbohydrate levels on the hypoxia tolerance of E.sinensis,corn starch and glucose were selected as carbohydrate source in this study.Six groups of feed contained 15%,25%,35% corn starch(CS15%,CS25%,CS35%)and 15%,25%,35% glucose(GLU15%,GLU25%,GLU35%),feeding juvenile E.sinensis(1.18 ± 0.03 g)for 56 days.After 8-week feeding trial,the survival and growth of juvenile crabs were counted and weighed,and then 6 juvenile crabs from each treatment group were randomly selected to conduct survival time experiments in a closed container to compare the hypoxia tolerance of juvenile crabs in each group.The other crabs were used for 24 h hypoxia(2.0 ± 0.2 mg/L)stress experiment to explore its mechanism of resistance to hypoxia.The results showed that after feeding with different carbohydrate source and carbohydrate levels for 8 weeks,the weight gain rate(WG),specific growth rate(SGR)and hepatopancreas index(HSI)of the corn starch group were significantly higher than that of the same addition level of glucose group.Among the corn groups,The WG and SGR of CS25% group were significantly higher than that of the other group(P < 0.05).Among the glucose groups,the WG and SGR of the GLU25% group was significantly higher than that of the other groups(P < 0.05).The results of the survival time(ST)in the closed container showed that the glucose group ST was significantly higher than the corn starch group in at the same level of addition(P < 0.05).Among the corn starch group,the ST of CS25% group was significantly higher than that in the other group(P < 0.05).Among the glucose group,the ST of GLU25% group was significantly higher than that of other groups(P < 0.05).The results of oxygen consumption rate showed that the oxygen consumption rate of CS35%,GLU25% and GLU35% group was significantly lower than that of normoxia(P < 0.05).After hypoxia stress,the oxygen consumption rate of GLU25% group was significantly lower than that in CS25% group and other glucose groups(P < 0.05).Glucose and lipid metabolism indexes of hepatopancreas showed that under normoxia treatment,the glycogen content of the glucose group was significantly higher than that in corn starch group at the same addition level(P < 0.05),the glycogen,pyruvate and lactic acid content of GLU25% group was significantly higher than that of CS25%,GLU15% and GLU35% group(P < 0.05).The results of hepatopancreas gene expression showed that the HIF1? m RNA level of each group after hypoxia treatment was significantly higher than that of normoxia treatment(P < 0.05).In summary,the corn starch group grew better than the glucose group at the same level.25% level were best for the growth of juvenile crabs in the corn starch group and the glucose group.Compared with corn starch,the addition of glucose in the feed can better improve the hypoxia tolerance of juvenile E.sinensis,and the 25% glucose group has the best effect.Glucose can improve the ability of juvenile crabs to withstand hypoxia stress.The main reason is that the body improves the ability to use feed glucose under early feeding conditions,and stores it in the form of glycogen in the hepatopancreas after conversion,providing a material guarantee for dealing with hypoxia stress.At the same time,the appropriate carbohydrate source and level can also reduce the oxygen consumption rate to cope with the adverse effects of hypoxia on survival.3 Effects of dietary salidroside on hypoxia tolerance of juvenile Chinese mitten crab(Eriocheir sinensis)Salidroside are the main components of common anti-hypoxia drugs.In this study,we supplemented the diet with salidroside(300 mg/kg S300;600 mg/kg,S600;900 mg/kg,S900)to evaluate the effects of dietary salidroside on the hypoxia tolerance of juvenile E.sinensis.In addition,the control group(C)was the basic diet and there were four experimental feeds in total.The juvenile crab(0.23 ± 0.01g)was fed for 8 weeks,followed by 24 hours of hypoxia(2.0 ± 0.2 mg/L)stress.The growth performance results showed that the weight gain rate and specific growth rate of crabs in S300 group was significantly higher than that in other groups(P < 0.05),and the feed coefficient was significantly lower than that in other groups(P < 0.05).The hemocyanin concentration of S300 group was significantly higher than that in other groups under hypoxia(P < 0.05).The oxygen consumption rate of crabs in the salidroside supplementation groups under normoxia was significantly lower than that in the control(P < 0.05),and the oxygen consumption in the S300 group and S900 group under hypoxia was significantly lower than that in the control(P < 0.05).Glucose and lipid metabolism indexes of hepatopancreas showed that the glycogen content of the S300 group was significantly higher than that in other groups under normoxia(P < 0.05).Under hypoxia,the glycogen content of the S300 group was significantly higher than that in other groups(P < 0.05),the content of lactic acid in all salidroside supplementation groups was significantly lower than that in the control(P < 0.05).Under normoxia,there was no significant difference in triglyceride content among all groups(P > 0.05).Under hypoxia,the content of triglyceride in the S300 group was significantly lower than that in the control and S900(P < 0.05).The m RNA level of carnitine palmitoyl transferase gene(CPT1)in S300 was significantly higher than that in other groups(P < 0.05).Antioxidant ability results showed that under normoxia or hypoxia,the salidroside supplementation groups malondialdehyde(MDA)contents was significantly lower and superoxide dismutase(SOD)activity was significantly higher than that in the control(P < 0.05),glutathion peroxidase(GSH-PX)activity of S300 group and S600 group were significantly higher than the control(P < 0.05).In summary,the dietary supplementation of 300mg/kg salidroside can promote the growth and improve the hypoxia tolerance of E.sinensis.Salidroside improves antioxidant capacity and hypoxia tolerance by increasing HIF1? m RNA level and hemocyanin content,inhibiting oxygen consumption rate,improving glucose and lipid metabolism energy supply.4 Effects of dietary astragalus polysaccharide on hypoxia tolerance of juvenile Chinese mitten crab(Eriocheir sinensis)Astragalus polysaccharides are the active ingredient of some anti-hypoxia drugs.In this study,we supplemented the diet with astragalus polysaccharides(150 mg/kg,A150;300 mg/kg,A300;600 mg/kg,A600)to evaluate their effects on the hypoxia tolerance of juvenile E sinensis.In addition,the control group(C)was the basic diet and there were four feed in total.The juvenile crab(0.23 ± 0.01g)was fed for 8 weeks,followed by 24 hours of hypoxia(2.0 ± 0.2 mg/L)stress.The growth performance results showed that the weight gain rate and specific growth rate of the A600 group was significantly higher than that in other groups(P < 0.05),and the feed coefficient was significantly lower than that other group(P < 0.05).The oxygen consumption rate of crabs in the astragalus polysaccharide supplementation groups under normoxia was significantly lower than that in group C(P < 0.05),and the oxygen consumption rate of the A150 group and A600 group under hypoxia was significantly lower than that in C group(P < 0.05).Antioxidant ability results showed that under normoxia and hypoxia,the MDA contents in the astragalus polysaccharide supplementation groups was significantly lower and the SOD activity was significantly higher than that in group C(P < 0.05),and the GSH-PX activity in the A600 group under hypoxia was significantly higher than that in control group.Nonspecific immunization results show that the total blood cell count(THC),alkline phosphatase(AKP)activity and acid phosphatase(ACP)activity in the control under hypoxia was significantly lower than that in normoxia.Under hypoxia,the THC in all of the astragalus polysaccharide supplemented groups was significantly higher than that in the C group(P < 0.05),and the AKP activity and ACP activity in the A600 group were significantly higher than that in normoxia and other hypoxia group(P < 0.05).The results of HIF1? expression showed that under hypoxia,the m RNA level of HIF1? in all astragalus polysaccharide added groups was significantly lower than that of the control group(P < 0.05).In summary,the dietary supplementation of 600 mg/kg astragalus polysaccharides can promote the growth and improve the hypoxia tolerance of E.sinensis.Astragalus polysaccharide mainly suppresses HIF1? expression and oxygen consumption rate and improve the body's non-specific immunity and antioxidant enzyme activity under hypoxia to improve hypoxia tolerance.