Stress Response Of Chinese Mitten Crab(Eriocheir Sinensis) To PH And Ammonia And Alleviative Effect On It Of Dietary Phospholipid And Astaxanthin

Chinese mitten crab(Eriocheir sinensis)is an important aquaculture species in China,which is very popular with consumers.It is necessary to plant aquatic plants for crabs to hide and climb,but due to the photosynthesis and respiration of aquatic plants,the pH of water body is abnormal.The molecular form of ammonia in water body is affected by pH,and the proportion of ammonia which are with high toxicity increased at high pH.High pH and its interaction with ammonia may affect the growth and physiology of Chinese mitten crab,and then the yield and economic performance.Astaxanthin is a kind of lipid soluble carotenoid with strong ability of scavenging free radicals,which plays a decisive role in the growth,health and quality of Chinese mitten crab.Phospholipid is amphiphilic emulsifier,and also has certain antioxidant effect.Adding phospholipid and astaxanthin in diet could alleviate the stress of crab to pH and ammonia.Based on the actual pH value of crab culture pond,the purpose of this study was to reveal the stress response characteristics of crab to pH and ammonia stress,and to evaluate the alleviative effects of dietary phospholipids and astaxanthin on pH and ammonia stress.The specific research contents and main results are as follows:Part one:Effects of short-term abnormal acid and alkali stress on physiology and growth of Eriocheir sinensisThe photosynthesis and respiration of aquatic plants in the cultural ponds of Eriocheir sinensis caused dramatic fluctuation of pH.Based on the monitored extreme pH value in crab pond,this study explored the effects of short-term acid and alkali stress on the physiology and growth performance of Eriocheir sinensis,in order to provide guidance for the optimization of culture management technology.Crabs with an initial body weight of 50.4± 2.5 g were stocked in acid(pH3.0)and alkali(pH10.3)water for 5 days,and then recovered for 15 days in water of pH7.5.Crabs raised in water of pH 7.5 were used as control.The results showed that the activity of lactate dehydrogenase(LDH)was significantly higher at 6 h and 12 h of acid exposure than that of the control group(P<0.05),and significantly lower at 3 days than that of the control group(P<0.05).The change trend of LDH in alkali exposure group was the same as but weaker than that in acid exposure group.The peak blood glucose was 14 times and 2.7 times higher in acid and alkali exposure group respectively than in control group.The total protein in serum was significantly higher in acid and alkali exposure group at 24 h than in control group(P<0.05).The activity of serum T-AOC was significantly higher than that of the control group(P<0.05)at 6 h and 24 h of both acid and alkali exposure.The content of glycogen in liver decreased significantly(P<0.05)at 5 d of both acid and alkali exposure,and the damage of liver and gill tissue was obvious at that time.Crabs with an initial body weight of 0.87±0.21 g were stocked in acid(pH3.0)and alkali(pH 10.3)water for 5 days,and then recovered for 4 weeks in water of pH7.5.The results showed that acid exposure significantly reduced survival rate and length increase rate of molting of Eriocheir sinensis,shortened molting cycle(P<0.05),while alkali exposure showed the similar trend.These results suggested that extreme acid and alkali exposure resulted in oxidative stress and tissue damage of Eriocheir sinensis.Even if the pH returns to normal,the subsequent survival rate and shelling doubling rate will be reduced.It is necessary to strengthen the control of pH change in crab pond management.Part two:Effects of alkali and ammonia stress on growth,antioxidant and metabolism on Eriocheir sinensisThe existing form of ammonia in water was affected by pH,and the high pH promoted the high proportion of toxic molecular ammonia.The aim of this experiment was to investigate the effects of interaction between different pH and ammonia on the growth,antioxidant and metabolism of Eriocheir sinensis.Crabs were subjected to the controls(pH8.0,pH8),pH9.0(pH9)and pH 10.0(pH10)respectively at three different NH3 levels(0.0 mg/L(A0),0.5 mg/L(A0.5)and 1.0 mg/L(A1)).Nine groups were named as pH8.0AO,pH8.0A0.5,pH8.0A1,pH9.0A0,pH9.0A0.5,pH9.0A1,pH10A0,pH10A0.5 and pH10A1 according to different levels of pH and NH3.The crabs(initial body weight:16.82±0.19 g)were fed commercial diet for 8 weeks.The results of two-way AN OVA showed:the growth performance,HSI and aspartate amino-transferase(AST),alanine aminotransferase(ALT),lactate dehydrogenase,urea and glucose in serum,and Na+-K+-ATP and Carbonic anhydrase(CA)in gill decreased with NH3 level increased compared to control group.However total protein and cholesterol in serum of pH level was in agreement with NH3,AST,ALT,lactate dehydrogenase,urea,glucose in serum and Na+-K+-ATP and CA in gill increased with pH level increased.The activity of total antioxidant capacity(T-AOC)decreased firstly and then increased with pH increased,and was significantly lower in the other groups than the control,which was opposite to NH3(P<0.05).Compared to control group,the activity of superoxide dismutase(SOD)in other group(except pH8A1 group)induced a decrease,and a significant decrease in pH10A0 group(P<0.05).The results of body composition of two-way ANOVA showed:pH significantly affected total essential amino acids(TEFAA)?total amino acids(TFAA)and His?Thr?Val?Met?Phe?Ile?Lys?Asp?Glu?Ser?Ala?Tyr in hydrolyzed amino acids of whole crab dry matter in Chinese mitten crab(P<0.05).KEGG enrichment analysis of pH10A1 group and pH8A0(control)showed that a number of 33 differential genes in the metabolic signaling pathway,in which significant up-regulation of all gene expression related to steroid biosynthesis and lipoic acid metabolism pathway were detected(P<0.05).In conclusion,pH and ammonia stress both reduced the growth performance of Chinese mitten crab,pH promoted the increase of ammonia toxicity and increased the oxidative damage in vivo,and then leaded to the increase of catabolism.Part three:Effects of dietary phospholipids and astaxanthin on the astaxanthin deposition,antioxidation and lipid metabolism of Eriocheir sinensis Crab rearing patterns that depended on water grass unavoidably lead to high pH stress,however astaxanthin had antioxidant properties,which was of great significance to the growth,health and quality of river crab.Phospholipid had certain antioxidant properties,and polar and nonpolar properties of phospholipid may contribute to the absorption and utilization of astaxanthin and increase the antioxidant capacity.The aim of this study was to investigate the effects of phospholipid on astaxanthin utilization in Chinese mitten crab and the synergistic effect of antioxidation.The phospholipid levels were set at 0 g/kg and 40 g/kg,respectively,and the astaxanthin levels were set at 0 g/kg and 4 g/kg,respectively.According to the supplemental level of phospholipid and astaxanthin,four groups were designed:the control group(CON group),PL,AX and AXPL group.Chinese mitten crabs(initial weight:1.61±0.01 g,n=3)were fed with the above diets for 8 weeks,and the results showed:all groups were no significant difference on growth performance of Chinese mitten crabs(P>0.05).The apparent digestibility of astaxanthin in AXPL group was significantly higher than AX group(P<0.05).Feed plus phospholipid increased astaxanthin content in whole crabs and serum of crabs(P<0.05).Meanwhile,dietary astaxanthin supplementation(AX and AXPL groups)significantly promoted the redness(a*)of cooked carapace deposition(P<0.05).Antioxidant indexes showed that phospholipid and astaxanthin addition increased total antioxidant capacity(T-AOC)in serum and significantly reduced malondialdehyde(MDA)of serum(P<0.05).The superoxide dismutase(SOD)and glutathione peroxidase(GSH-PX)activities of hepatopancreas in AXPL group was significantly higher than that in other groups(P<0.05).After acute pH and NH3(pH 10.0 and 2.0 mg/LNH3)stress for 24 h,serum astaxanthin increased rapidly,moreover serum astaxanthin in AX and AXPL groups were significantly higher than control group(P<0.05).After the stress for 24 h,the activity of T-AOC in all groups decreased sharply and test groups were significantly higher than control group(P<0.05).The results of two-way ANOVA in gene expression showed the significant down-regulation of SOD,CAT and peroxiredoxin(PRX)in hepatopancreas with increase of astaxanthin were detected.The Person-correlation results showed that the serum astaxanthin and antioxidant genes expression(SOD,CAT and PRX)of hepatopancreas were negatively correlated and significantly correlated with SOD gene(P<0.05).The results showed that phospholipid and astaxanthin addition down-regulated DGL,up-regulated TGL mRNA expression and significantly increased the deposition of lipid granules in hepatopancreas,however,phospholipid addition significantly down-regulated IL mRNA expression(P<0.05).Meanwhile,Oil red staining of hepatopancreas presented that phospholipid and astaxanthin addition significantly increased lipid granules deposition in hepatopancreas.The above results suggested that phospholipid may promote digestion and absorption of astaxanthin in dietary and increase serum astaxanthin content.Under the condition of stress,the river crab could transfer astaxanthin from the whole body to increase the capacity of antioxidant in serum and reduce oxidative damage.Meanwhile,the phospholipid addition also increased lipid deposition in the hepatopancreas.