Stress Response And Mechanism Of Waterless Preservation After Low-temperature Induced Dormancy On Pearl Gentian Grouper(?Epinephelus Fuscoguttatus×?Epinephelus Lanceolatus)

Pearl gentian grouper(?E.fuscogutus×?E.lanceolatus)is a kind of tropical marine economic fish with high protein,low fat,high nutrition value and cultured yield,which is mainly sold in the form of live products.The problems of low keep-alive density,low survival rate,poor quality and safety existed in live-circulation with water technology of fish are the key scientific and technical problems that restricts the development of the grouper industry.Waterless live-circulation after low temperature induced dormancy,without anesthetic addition,is a safe and efficient way of fish living circulation.Nevertheless,the effects of stress response and physiological regulation on keep-alive transport in low temperature,water-free and transportation affect directly the result of fish live-transport,and differences in fish species.Therefore,pearl gentian grouper was used to study the stress response in the process of low-temperature induced dormancy and waterless preservation.The material metabolism and oxidative stress response of grouper under acclimation without diet,dormancy induced by low-temperature and waterless preservation were studied in this paper.And the molecular regulation mechanism of pearl gentian grouper under the stress of low temperature dormancy and waterless preservation was researched by the TMT differential proteomics analysis.It enriches the theoretical study on stress response and regulation mechanism of warm water fish,and provides a theoretical basis for the anhydrous and living transportation of pearl gentian grouper.The main results were listed as follows:(1)The survival time of pearl gentian grouper was prolonged by 42 % after acclimation at25 ? without diet for 48 h.The stress response caused by the operation is obviously reduced.The activity of antioxidant enzymes(SOD?GSH-Px?CAT)were activated by acclimation stress were decreased the level of lipid peroxide(MDA content)and reached a new redox stable state.The energy metabolism of fish reached a new balance.Liver glycogen was decomposed to maintain the balance of blood glucose.It is no significant effect on muscle glycogen and free amino acid content.(2)The limit temperature of the ecological ice temperature region of pearl gentian grouper is 11 ?.The grouper was induced to the dormancy state at the 12-13.5 ? bygradient low-temperature acclimation.Under the dormancy state,energy metabolism and stress response were decreased.The activity of serum transaminases(ALT and AST)decreased,and the accumulation of metabolites,the content of glucose and lactic acid in the liver increased by 5.02 and 1.79 times,respectively.Alkaline phosphatase(AKP),antioxidant enzyme(SOD,GPX,CAT)activity were activated to resist oxidative damage induced by low temperature stress.Tyr,Phe,and Pro can be used the main source of energy in the low-temperature induced dormancy of the grouper.The redox level in the body reached a new equilibrium state.Furthermore,Serum Glu,ALT,AKP and brain MDA levels can be used as indicators of dormancy induced by low-temperature.(3)The survival time of pearl gentian grouper after dormancy induced by low-temperature can reach 10 h under 15-18 ? waterless preservation.The activity of serum metabolic enzyme(ALT,AST,ACP,AKP)and the level of cortisol were not significantly changed after 5 h of waterless preservation at 15 ?.And serum glucose was increased by 14.32 times by the glycogen decomposition,and the accumulation of LD was caused by anoxic respiration of fish.The activity of CAT enzyme in liver and brain increased significantly to inhibite the increase of MDA.The level of CAT activity was negatively correlated with the temperature and the level of MDA in tissues.The proper low temperature(15-18 ?)can reduce the stress response in the waterless preservation,and improve the survival rate through the upper adjustment of the metabolic adaptation and the anti-oxidation system.(4)Low temperature,waterless and temperature fluctuation are the main stress factors in the process of low temperature dormancy and waterless preservation of pearl gentian grouper.Lipid metabolism,gluco-metabolism,oxidative stress and liver detoxification,immune response,protein and amino acid metabolism and signal transduction were responded to the stress.(5)Under low temperature dormancy stress,the PEPCK,LDH,FAS,ACC,AMPK enzyme activity and m RNA expression level in the liver of Pearl gentian grouper increased significantly,and the glycogen content in liver and brain tissue decreased by 32.28-44.24% after water resuscitation.Therefore,under low temperature and waterless stress,the liver glycogen and fat were mainly used as energy substances,and protein and amino acids were also involved in energy metabolism in the later stage of stress and water recovery.Low temperature stress stimulated the increasing of anoxic respiratory metabolism,and blood glucose was maintained stability in grouper by the gluconeogenesis of the non-sugar substances such as amino acids.Hypothermic waterless stress stimulated the glycolysis of liver glycogen.Respiratory metabolism in the brain was inhibited by coldand waterless tress.The glucose metabolism of the grouper can not be recovered after resuscitation with water at 25 ?.(6)Under low temperature stress,the liver of pearl gentian grouper can down regulate the expression of CPT-1 and ACAC and inhibit the oxidative decomposition of FA by the dephosphorylation of AMPK.Linoleic acid and Linolenic acid were synthetic by up-regulating FAS,ACC enzyme,and transported to the cell membrane for the synthesis of phospholipids by FABP,which resisted the changes of cell membrane mobility induced by low temperature stress.Under the reaction of PB1 after low temperature waterless stress for 8 h,phospholipids in membrane were decomposed to n-3PUFA(EPA and DHA),which were oxidized to ATP used for resisting stress response.After water recovery at 25 ?,FA were decomposed as energy under the action of ACAC enzyme.(7)There was a negative correlation between serum cortisol,ROS levels and the CAT enzyme activity and m RNA expression level under the stress of low-temperature and waterless in pearl gentian grouper.When the CAT enzyme activity and m RNA expression level was increased,the serum cortisol and ROS production levels were decreased.AMPK phosphorylation regulated the up-regulation of the expression of rps6 kb,the effect target protein in the downstream of m TORC1 under low temperature and waterless stress.The CAT and GSH were up-regulated to scavenge ROS,which induced the body injury by lipid peroxidation.After water recovery at 25 ? stress,the antioxidant enzymes can not completely resist the tissue damage caused by oxidative stress.