Effects Of Chronic Ammonia Stress On Growth,Tissue Structure And Physiological Metabolism Of Macrobrachium Rosenbergii

Macrobrachium rosenbergii belongs to tropical freshwater shrimp,and 25-30℃is the appropriate temperature for its growth.But it is also one of four shrimp raised fresh water in our country.Since 2014,our laboratory has continued to study the slow growth phenomenon of Macrobrachium rosenbergii.It was found that the energy metabolism of Macrobrachium rosenbergii with growth disorder was abnormal,and the growth disorder was not closely related to the species,but closely related to the aquaculture water environment.However,ammonia nitrogen is the main toxic pollutant in the water body of shrimp aquaculture.In the inland workshop aquaculture mode,the relatively high density aquaculture mode,overfeeding and a large amount of shrimp excrement will directly and rapidly lead to the increase of ammonia nitrogen in the water body.Due to the instability of the biological filter,nitrogen source pollutants may accumulate rapidly in the newly put into use in the installed culture system.However,in the factory farming mode,the aquaculture water body needs to be recycled repeatedly,so there is a high probability of long-term low concentration of nitrogen in the farming system.Therefore,in this study,Macrobrachium rosenbergii with a size of about 4g was selected as the initial research object.Based on the measurement of its anti-ammonia stress ability（96h half lethal concentration）,the response of Macrobrachium rosenbergii under chronic stress with low concentration of ammonia nitrogen was explored mainly from the aspects of growth,feeding,survival rate,tissue structure,energy metabolism and detoxification metabolism.In order to solve the problem of slow growth of Macrobrachium rosenbergii i and provide certain data support for the future healthy breeding of Macrobrachium rosenbergii.1.Acute toxicity test of ammonia nitrogen to juvenile Macrobrum rosenbergiiThe acute toxicity of ammonia nitrogen on juvenile Macrobrum rosenbergii（4.61±0.35g）was studied by conventional biological acute toxicity test.The results showed that the behavior of Macrobrum rosenbergii was extremely severe after ammonia nitrogen poisoning,showing obvious dyspnea,body color turned gray,and molting was difficult.The semi-lethal concentrations of total ammonia on juvenile Macrobrum rosenbergii at96h were 45.13 mg/L,and the safe mass concentration（SC）was4.51mg/L.The semi-lethal concentrations of non-ionic ammonia nitrogen at 96h for Macrobrum rosenbergii were3.32mg/L,and the safe mass concentration（SC）was0.332mg/L.Macrobrum rosenbergii with this specification has stronger ammonia nitrogen tolerance than other shrimp with similar specification.2.Effects of chronic ammonia nitrogen stress on growth,survival and tissue structure of Macrobrum rosenbergiiThe experimental ecology method was adopted,and 20%of 96h semi-lethal concentration(96H-LC₅₀)was taken as the highest treatment group,and five treatment groups were set with equal logarithmic spacing.Through the cultivation experiment in the cultivation workshop for 48 days,six different ammonia nitrogen concentrations were0.02mg/L（control group）,0.5mg/L（T0.5）,and 0.02mg/L（control group）.Effects of chronic ammonia stress on feeding,growth,survival and tissue structure of Macrobrum rosenbergii were studied in 1.04mg/L（T1.04）,2.14mg/L（T2.14）,4.42mg/L（T4.42）and9.03mg/L（T9.03）groups.The results showed as follows:the survival rate of T9.03 was significantly lower than that of control group（P<0.05）,and there was no significant difference between other stress groups and control group（P>0.05）.These results indicated that the safe concentration range would not significantly affect the survival rate of Macrobrum rosenbergii.The weight gain rate（WGR）,relative growth rate（AGR）and specific growth rate（SGR）showed a decreasing trend with the increase of ammonium-nitrogen stress concentration.The feeding rate and feed efficiency also showed the same trend,while the coefficient of difference in body weight showed an increasing trend.These results indicated that long-term stress storage inhibited the growth and feeding of juvenile Macrobrum rosenbergii,and the inhibitory effect was positively correlated with ammonia nitrogen concentration,and with the increase of ammonia nitrogen concentration in aquaculture water,the growth integrity of Macrobrum rosenbergii would decline sharply.The results of tissue sections after 48days of ammonia-nitrogen stress showed that the gill epithelium in T9.03 was largely exfoliated,capillaries were swollen,and the subchitin layer was destroyed,leading to spatial destruction.The lumen morphology of hepatic tubules was completely destroyed,basement membrane was broken,and vacuolation was serious.The results indicated that the hepatopancreas and gill damage of Macrobrum rosenbergii were serious under long-term ammonia nitrogen stress exceeding safe concentration.However,there was no significant difference between T0.5 and the control group in growth,survival,feeding and tissue structure,indicating that the stress concentration of T0.5（The total ammonia concentration is 0.5 mg/L,and the corresponding non-ionic ammonia concentration is0.036 mg/L）could be used as the maximum concentration without influence of ammonia nitrogen on Macrobrum rosenbergii.3.Effects of chronic ammonia nitrogen stress on serum energy-supplying substances and hepatopancreas energy-metabolism-related enzymes in Macrobrum rosenbergiiSimilarly,in 6 different ammonia nitrogen concentrations of 0.02mg/L（control group）,0.5mg/L（T0.5）,1.04mg/L（T1.04）,2.14mg/L（T2.14）,4.42mg/L（T4.42）and 9.03mg/L（T9.03）groups,The effects of chronic ammonia nitrogen stress on serum energy supply substances and hepatopancreas energy metabolism related enzymes in Macrobrum rosenbergii were studied.The results showed that:In terms of serum energy-supplying substances,the serum Glucose content（Glu）of T9.03 group was significantly lower than that of control group during the whole stress period（P<0.05）,and the serum triglyceride（TG）and total cholesterol（T-CHO）contents of T4.42 and T9.03 were significantly lower than those of control group from day 12 to 48（P<0.05）.The serum protein content（TP）had no significant difference between the 24D stress group and the control group before stress（P>0.05）,but on the 48d,the serum total protein content decreased significantly with the increase of ammonia nitrogen stress concentration（P<0.05）.The results showed that under chronic ammonia-nitrogen stress,the energy materials used by Macrobrum rosenbergii were carbohydrate,lipid and protein.In addition,the activities of hexokinase（HK）,pyruvate kinase（PK）and lactate dehydrogenase（LDH）in the stress group were significantly changed compared with those in the control group during the experiment（P<0.05）,which showed a trend of first increasing and then decreasing.The phenomenon of phosphoenolpyruvate carboxylase（PEPCK）in the stress group was significantly higher than that in the control group in the middle and late stage of stress（P<0.05）,indicating that glycolysis was enhanced in the early stage of ammonia stress,but after adapting to ammonia stress,the metabolic pathway was changed and Glucose heterogenesis was activated.During the whole stress period,the activities of succinate dehydrogenase（SDH）at T4.42 and T9.03 were significantly lower than those in the control group（P<0.05）,indicating that ammonia nitrogen stress decreased the activity of tricarboxylic acid cycle（TCA）and weakened aerobic respiration,which further indicated that chronic ammonia nitrogen stress changed the metabolic energy supply pathway of Macrobrum rosenbergii.4.Effects of chronic ammonia nitrogen stress on genes related to energy metabolism and detoxification metabolism in different tissues of Macrobrum rosenbergiiMacrobrum rosenbergii was exposed to low concentration of ammonia（1.04mg/L）and high concentration of ammonia（9.03mg/L）for 48 days by experimental ecology method.The effects of chronic ammonia stress on blood ammonia and blood urea nitrogen contents,the expression of genes related to energy metabolism and detoxification metabolism in different tissues of juvenile Macrobrum rosenbergii were studied.The results showed that the contents of blood ammonia and blood urea nitrogen in Macrobrachium rosenbergii were positively correlated with the stress concentration and stress time of ammonia nitrogen.The contents of blood ammonia and blood urea nitrogen in T9.03 and T1.04 groups were significantly up-regulated（P<0.01）,and the up-regulation of T9.03 was more obvious.Under ammonia nitrogen stress,AK gene expression in T9.03and T1.04 groups was significantly up-regulated in gill tissues（P<0.01）,and was significantly up-regulated in hepatopancreas for a short time and then significantly down-regulated（P<0.01）.The expression level in muscle tissue was significantly up-regulated（P<0.01）.Groups T9.03 and T1.04 Glutamate dehydrogenase（GDH）and Glutamine synthetase（Glutamine synthetase,GDH）The expression of GS gene was up-regulated in different tissues,and the up-regulation of T9.03 was more obvious（P<0.01）.In conclusion,chronic ammonia stress significantly affected the expressions of AK,GDH and GS genes in the tissues of Macrobrachium rosenbergii,indicating that ammonia stress activated the energy metabolism and ammonia detoxification metabolic pathway in the body.