Studies On Sensitive Immunology Parameters Screening And Evaluation In White Shrimp Litopenaeus Vannamei And Sea Cucumber Apostichopus Japonicus Selenka

The reports of immunostimulants for aquatic animals are many, but evaluation of them is no uniform standards, thus we need to establish a stable immunostimulant evaluation system. This paper use different approaches (feeding immunostimulants, challenging and hungering) to study non-specific immune response of Litopenaeus vannamei(Litopenaeus vannamei) and sea cucumber (Apostichopus japonicus Selenka), the aim is to complete the selection of sensitive immunology parameters of shrimp and sea cucumber. Main contents and results are as follows:1. An experiment was conducted to screen sensitive immunological parameters of shrimp(Litopenaeus vannamei) on the two immunostimulantsβ-glucan and glycyrrhizin. After continuous feeding and sampling immunology parameters, the results showed that dietaryβ-glucan could significantly increase the phenoloxidase activity (Apo), respiratory burst activity (RB), hemolytic activity (Hemolysin activity), acid phosphatase (ACP) activity, alkaline phosphatase (AKP) activity, total superoxide dismutase (SOD) activity, nitric oxide synthase (NOS) activity and serum protein (Plasma protein) content, the time that increased focused between 3 and 9 days after feeding immunostimulant and then maintained at high levels (P<0.05); Glycyrrhizin in diet could significantly increase blood cell density (THC), phenoloxidase activity (Apo), respiratory burst activity (RB), alkaline phosphatase (AKP) activity, total superoxide dismutase (SOD) activity, nitric oxide synthase (NOS) activity and serum protein (Plasma protein) content. The time that increased focused between 7 and 16 days after feeding immunostimulant and then maintained at high levels (P<0.05).2. An Experiment was conducted to screen sensitive immunological parameters of Litopenaeus vannamei on Vibrio alginolyticus infection. After the two immunostimulants fed 18 d then continuous sampled, immunology parameters of shrimp were measured. The results show that:the feed with 0.2%β-glucan and 0.06% glycyrrhizin for 18 d can significantly improve shrimp survival rate of Vibrio alginolyticus challenging (P<0.05). Within 12～72 h of infection, the total blood cell count (THC), hemolytic activity (Hemolysin activity), nitric oxide synthase (NOS) activity, phenoloxidase (PO) activity, respiratory burst (RB) activity, total superoxide dismutase (SOD) activity, acid phosphatase (ACP) activity, alkaline phosphatase (AKP) activity and serum protein (Plasma protein) content of shrimp with Vibrio alginolyticus infections and deaths have certain regular changes (P<0.01). Correlation analysis showed that survival rate with THC, PO, SOD, NOS and AKP were significant (P<0.01). The results show that these indicators can reflect a more direct response of Litopenaeus vannamei against Vibrio alginolyticus level. 3. The weight of 12.13±1.61 g shrimp for the experimental material, were fed in vitro, in vivo injections infected with white spot syndrome virus (WSSV), then continuous sampled to measure the dynamic changes in immunology parameters. The results showed that compared with the control group, the total number of blood cells (THC), hemolytic activity, serum protein (Plasma protein) content, acid phosphatase (ACP), agglutination activity were significantly decreased after infection (P<0.05); And total superoxide dismutase enzyme (SOD) activity, nitric oxide synthase (NOS) activity had a similar trend, slightly increased and then decreased; Respiratory burst (RB) activity, alkaline phosphatase (AKP) activity, phenoloxidase (PO) activity increased significantly in the 24～48 h, the maximum in dying shrimp. Correlation analysis showed that SOD, NOS, RB, AKP and PO, these five indicators can reflect the immune status of shrimp, which and can be used to evaluate resistance ability of shrimp against WSSV.4. Diet containing 0.2%β-glucan and 0.02% glycyrrhizin had different effects on the immunology parameters of sea cucumber. Sampled consecutively during 45 d feeding period, compared with the control group showed that,β-glucan and glycyrrhizin increased coelomocytes TCC, PC and ISAP of sea cucumber in different time, and LSZ, ACP, AKP, SOD and NOS activity in sea cucumber coelomocyte fluid were significantly increased also (P<0.05). On principle of the time of significant increase focused and maintained a certain level, the sensitive immunology parameters of these two kinds of immunostimulants are PC, LSZ, SOD and NOS.5. After feeding diets containing 0.2%β-glucan and 0.02% glycyrrhizin for 45 days to sea cucumber, then artificially infected with Vibrio splendidus, at 0,5,10 and 15 d. we measured TCC, PC, ISAP, LSZ, ACP, AKP, SOD and NOS activity and recorded the cumulative incidence of sea cucumber, respectively. After infection, only the AKP activity didn't change dramatically in the whole course, other immunology parameters of sea cucumber changed over time. The immune index began to decline on the first 5 d after infection, on 10 d began to recover, to 15 d, almost recovered to the level before infection. The conclusion is that:β-glucan or glycyrrhizin in feed by improving the immunization of sea cucumber body coelomocyte fluid index (TCC, PC, ISAP, LSZ, ACP, AKP, SOD and NOS) enhanced resistance to Vibrio splendidus. In our experimental conditions, the sea cucumber immunology parameters in the time regression analysis after infection can imply that LSZ and SOD activity in sea cucumber body fluid PC and TCC in coelomocytes can be used as sensitive immunology parameters to Vibrio splendidus and these two immunostimulants.6. After feeding diets containing 0.2%β-glucan and 0.02% glycyrrhizin for 29 days to sea cucumber, then started starvation experiment, to study the effects of these two immunostimulants on immunity of hungry sea cucumber. Sampled at 0,5,10,15 and 20 d to test TCC, PC, ISAP, LSZ, ACP, AKP, SOD and NOS activity changes of sea cucumber. The results showed that feeding basic diet,β-glucan and glycyrrhizin, the cell number of body coelomocyte of sea cucumber changed in different degrees in hunger. starvated in a short time such as 5 d, the cell number of control group has not decreased, but an upward trend. The coelomocyte cell ofβ-glucan and glycyrrhizin was gradually decreased throughput the period of hunger, but compared with the control group, the coelomocyte cell of sea cucumber declined in the rate of short-term hunger to be small. Under the starvation stress, the body coelomocyte cells and body fluid enzymes changed, while the immunostimulants can reduce the variation in the rate of short-term hunger. In this experimental condition, obtained by the regression analysis, three types of feed fed to the hungry sea cucumber immunology parameters in close to starvation, can use SOD, NOS and ACP activity to evaluate the immune ability in starvation sea cucumber.