Effects Of Exogenous Factors On Immune Function And Hemolymph Biochemical Indicators In Adult Chinese Horseshoe Crab Tachypleus Tridentatus

Tachypleus amebocyte lysate?TAL?plays an extremely important role in the field of medical examination,the demand for TAL produced from the raw materials of hemolymph collection is increasing.With the sharp decline of T.tridentatus population,resulting the TAL industry is limited by raw materials in China.How to meet the demand for resources of horseshoe crab and protection has becomes a key problem.Therefore,this study mainly discussed the effects of different exogenous factors on the immune function and hemolymph biochemical indicators of Chinese horseshoe crabs.The aim of this study is to strengthen the rapid recovery of the hemolymp,so as to achieve the purpose of protecting and sustainable utilization of horseshoe crab resources.1.Therefore,in order to study the effects of copper supplement on the immune function and blood-chemistry in adult Chinese horseshoe crab,A28-day indoor culture experiment was conducted to study the effects of adding different levels of copper amino acid chelate(0,50,or 100 mg kg^-1)into semi-finished feed on the nonspecific immune enzyme activity and hemolymph biochemical parameters of Tachypleus tridentatus.Results revealed that the activities of serum antioxidant enzyme indicators,namely,copper–zinc superoxide dismutase?Cu/Zn-SOD?,catalase?CAT?,glutathione peroxidase?GPX?,total antioxidant capacity?T-AOC?,lysozyme?LZM?,alkaline phosphatase?AKP?,and acid phosphatase?ACP?,of copper-fed adult horseshoe crabs increased;on the other hand,malondialdehyde?MDA?activity decreased.Hemolymph biochemical indicators,triglyceride?TG?and cholesterol?CHO?decreased in the copper-fed group,but hemocyanin concentration?HC?levels increased during the experiment.At the end of experiment,all antioxidant enzyme indicators were significantly increased in the copper-fed,there was no significant difference between SOD,CAT,GPX,LZM and HC in the high-copper group(100 mg kg^-1)and the low-copper group(50mg kg^-1).The activity of T-AOC in the low-copper group reached the highest value,the content of MDA reached the lowest value,the activity of AKP and ACP in the high-copper group reached the maximum value,but the content of CHO and TG showed a decreasing trend.The above results showed that supplement of amino acid chelate copper could improve the immune performance of Chinese horseshoe crab,but the immune effect of different amounts of copper was obviously different.2.In order to compare the immune response of three immunostimulants to Chinese horseshoe crab hemolymph,We injected three different doses of?-glucan?G?,peptidoglycan?P?and squalene?S?in into Chinese horseshoe crab,and compared the total hemolymph counts,reactive oxygen species,and non-specific immune enzyme activity changes in Chinese horseshoe crab after injection 0,6,12,24,48 h.The results showed that compared to the control group,the total number?THC?,reactive oxygen species?ROS?,superoxide dismutase?SOD?,catalase?CAT?,total antioxidant enzyme?T-AOC?,lysozyme?LZM?and alkaline phosphatase?AKP?injected with 5mg/kg and 10mg/kg respectively showed an increasing trend during the experiment,malondialdehyde?MDA?activity has a decreasing trend,hemocyanin concentration?HC?did not change significantly during the whole experiment.At 48h,in the low dose group?5mg/kg?and the high dose group?10mg/kg?,in the measured hemolymph parameters THC,ROS,LZM and AKP,the injected P all had the maximum value,G and S at low dose showed no significant difference compared with the control group,ROS?LZM and AKP of G and S showed no significant difference at high dose,but the AKP between the two doses was significantly higher than the control group.In the measured SOD,CAT and T-AOC,both of them were significantly higher than the control group under the condition of high and low dose,SOD of G in the low dose group had a maximum value,while SOD of G and S in the high dose group was significantly higher than the control group.There was no significant difference between CAT and T-AOC injected with G,P and S under high and low dose,but all were significantly higher than the control group.At48h,there was no significant difference in MDA among the three immunostimulants,but all of them were significantly lower than the control group.The above results showed that the injection of three immunostimulants at the end of the experiment could improve the hemolymph parameters of Chinese horseshoe crab,a variety of enzyme activities reached the maximum value in the injection P,however,there was no significant difference between most hemolymph parameters of injection G and S.It can be inferred that the peptidoglycan can be developed into Chinese horseshoe crab's immunostimulants by comparing the three immunostimulants.3.To study the effects of N-acetylcysteine?NAC?and inactivated of vibrio anguillus?V?on hemolymph parameters of Chinese horseshoe crab,we injected n-acetylcysteine?NAC?and vibrio anguillus into Chinese horseshoe crab,and compared the changes of total lymphocyte count,reactive oxygen species content and non-specific immunoenzyme activity of Chinese horseshoe crab after injection 0,6,12,24,48h.The results showed that compared to the control group,after NAC injection,the total number of lymphocytes?THC?,reactive oxygen species?ROS?,and malondialdehyde?MDA?activities of Chinese horsescrab showed a decreasing trend during the experiment,the activities of superoxide dismutase?SOD?,catalase?CAT?,total antioxidant enzyme?T-AOC?,lysozyme?LZM?and alkaline phosphatase?AKP?all showed an increasing trend.When NAC was stimulated with different concentrations of V,the decrease trend of THC,ROS and MDA contents of Chinese horsesulus horsesulus was slower than that of NAC injection alone,the activity of other enzymes was relatively increased.Hemocyanin concentration?HC?did not change significantly during the whole experiment.At 6-48h,THC and ROS of the NAC group all had minimum values,SOD activity under NAC and V stimulation was significantly higher than the other group.At48h,there was no significant difference between CAT and T-AOC groups under NAC,V and co-stimulation,but all were significantly higher than the control group.The MDA content of NAC+10⁶V was the lowest at 48h,the AKP activity was the highest at 12-48h,and the LZM activity of NAC group was the highest at 48h.The above results showed that NAC injection could reduce THC and ROS of Chinese horseshoe crabs,and the co-stimulation of NAC and V could alleviate the downward trend.THC and ROS of inactivated vibrio anguillus group were increased,but all of them increased the parameters of the rest hemolymph.Therefore,it can be speculated that ROS content plays an important role in the proliferation and regeneration of Chinese horseshoe crab hemolymph,so as to improve its immune function.