Antioxidative Defence Mechanism Of Glutathione On Litopenaeus Vannamei

Litopenaeus vannamei is one of the excellent aquaculture varieties in the world, and aquaculture of this white shrimp has become an important export industry in developing countries in Asia and America. In China’s around-sea area, Litopenaeus vannamei has been developed to be the dominant cultured products which is one of the most important exported commodities. However, the white shrimp Litopenaeus vannamei are challenged with crowded, nutritional, environmental, and metabolic stress during the intensive aquaculture processes, resulting in significantly reduced disease resistance, thus disease broken epidemicaly and new plague emerged uninterruptedly. It’s too difficult for veterinarian to diagnose and therapy these maladies so that the farmers keen on abusing the antibiotics in feedstuff. Long-time and large-dose antibiotics abusing in shrimp would lead to many side-effects, such as dysbacteriosis in intestinal tract, disruption microenvironment, drug residues ect. Eventually due to humankind’s allergic reaction, immunosuppression, distortion, canceration, mutation, thus the sustainable development of Litopenaeus vannamei aquaculture industry is threaten by its susceptibility to disease outbreak.Now, it is considered that one of the effective ways to solve the problem is to increase or activate the antioxidative capacities of Litopenaeus vannamei by nutritional regulation, such as using highly efficient and safe antioxidative feed additives.In this research, we emphasized on the roles of reduced glutathione (GSH) playing in: (1) improving the growth performance of Litopenaeus vannamei; protecting the shrimp from oxidative stress; increasing non-specific immunologic factors level in vivo; (2) being an accelerator proliferation of primary cultured hepatopancreas cells; mediating the diverse biological effects of primary cultured hepatopancreas cells of Litopenaeus vannamei; (3) improving the expression of mRNA in SOD and CAT in vitro. This research comprises of four parts.1. Distribution of peroxidative enzymes and lipid peroxidation product in the tissues of litopenaeus vannamei before and after ammonia stressIn order to reveal the distributed characteristics of the antioxidant enzymes ----superoxide dismutase (SOD), catalas (CAT), glutathione peroxidase (GSH-Px); total antioxidant capacity (T-AOC), glutathione (GSH) and lipid peroxidation product-malonydialdehyde (MDA) in various tissues of Litopenaeus vannamei, such as gills, muscles, hepatopancreas and serum, an experiment was designed to assess the changes of these indexes before and after ammonia stress.72 shrimps, with initial weight of 6.322±0.221g were randomly selected from the representative South China Litopenaeus vannamei to sample their gills, muscles, hepatopancreas and serum to analyze the activities of SOD, GSH-Px, CAT, T-AOC, the content of GSH and MDA before and after ammonia stress.The results indicated that hepatopancreas and serum were the sensitive tissues while gills and muscles were not as so sensitive as others. Ammonium-N stress experiments showed that CAT, GSH-PX activities, T-AOC, GSH and MDA content were not sensitive in gills and muscles to NH4Cl treatment whereas all these indexes (except T-AOC) were significantly (p<0.05) changed in hepatopancreas and SOD, GSH-Px and MDA were significantly (p<0.05) changed in serum of Litopenaeus vannamei. Furthermore, CAT activities and GSH content in hepatopancreas and GSH-Px activities in serum were significantly (p<0.01) changed after stress. These results suggest that hepatopancreas and serum are the tissues sensitive to ammonium-N stress, therefore, CAT、GSH-PX、T-AOC、GSH and MDA content could be regarded as antioxidative stress indexes of Litopenaeus vannamei and these antioxidant enzymes and lipid peroxidation product in hepatopancreas and serum could be regarded as the effective indexes of Litopenaeus vannamei when the shrimp suffer from a stress.2. Effects of dietary GSH on the growth performance, antioxidant indexes and lipid peroxidation content, nonspecific immune factors in Litopenaeus vannamei6 levels of glutathione (GSH) (0,60,120,180,240 and 300mg/kg), being named G0, G60, G120, G180, G240 and G300 respectively, were supplemented to a basal diet to test the effects of different dietary GSH levels on the growth performance, hepatopancreas antioxidant indexes and lipid peroxidation content, non-specific immune factors and activities of AKP, ACP, AST/GOT and ALT/GPT in litopenaeus vannamei (IBW of 1.12±0.01g).The feeding experiment ran for 8 weeks. After 24hs the shrimp were weighed, taken into account total dead rate, total feed consumption. Then,15 shrimps were sampled per replication. The rest shrimps were sent back to the circulation system. The Litopenaeus vannamei were treated with 20 mg/L of NH4Cl without water circulating for one week. At the end of 9th week, the feeding was stopped for 24 hours, the dead rate was calculated and samples were taken. The Litopenaeus vannamei tissues were collected for assessing viability and immune factor levels.The results showed that:(1) weight gain rate (WGR), survival rate and feed conversion efficiency (FCE) of the shrimp fed dietary GSH were significantly increased than those of control (p<0.05). Survival rate was increased for 8.53%-31.69%(p<0.05);WGR increased with dietary GSH increasing and reached the highest when dietary GSH was 180mg/kg, but tent to decrease when dietary GSH increasing above 180mg/kg (p<0.05); When dietary GSH was above 120mg/kg, FCE was significantly increased (p<0.05). Protein content in serum, hepatopancreas, muscle of the shrimp were increased when dietary GSH was supplemented, being significantly higher in serum and hepatopancreas than that of control when dietary GSH was above 180mg/kg (p<0.05); Protein content in gills had no significant difference among the 6 groups (p>0.05). The numbers of haemolymphs of the shrimp fed dietary GSH were significantly higher than those of the control (p<0.05), showing a dose-dependent relationship (p<0.05). GSH content in hepatopancreas was increased when dietary GSH was above 60mg/kg (p<0.05). In summation, the optimal level of dietary GSH for litopenaeus vannamei was 174.13 mg/kg.(2) Dietary GSH improved the activities of antioxidant enzymes and total antioxidation capacities, significantly decreased the content of MDA (p<0.05). The increasing dietary GSH had a significant effect on the activities of SOD and GSH-Px when the supplementary GSH was above 120 mg/kg (p<0.05), while had a significant effect on GR activities in 60mg/kg and 120mg/kg (p<0.05). Dietary GSH raised the content of GSH and the total antioxidant ability (T-AOC) in hepatopancreas and showed a dose-dependent relationship, the increase rate was 8.93%-52.57% and 3.02%-37.03%, respectively. With increasing dietary GSH, ROS and MDA level tent to decrease, reached the lowest at 240mg/kg and 300mg/kg (p<0.05) respectively. The ROS in group 180mg/kg,240mg/kg and the MDA content in each experiment group was significantly lower than those of the control (p<0.05). Dietary GSH significantly improved the survival rate and the anti-O2-ability in group 120mg/kg and 180mg/kg (p<0.05).(3) In hepatopancreas, with the increasing of dietary GSH level, activities of MPO, LSZ, AKP and ACP of the shrimp increased, reaching the highest at 120 mg/kg,240 mg/kg,240 mg/kg,120 mg/kg respectively, then significantly decreased at a higher level (when more than 240 mg/kg). While in serum, activities of LSZ, AKP and ACP increased, reaching the highest at 180 mg/kg,240 mg/kg and 240 mg/kg respectively, then significantly decreased. AST/GOT and ALT/GPT activities, both in serum and hepatopancreas of shrimp fed dietary GSH, decreased significantly and achieved the bottom at 240 mg/kg (p<0.05), then increased at 300 mg/kg.After a week’s stress of NH4+, the survival of the shrimp fed with high level of dietary GSH was significantly increased than that of the control and the low dose group (p<0.05). Furthermore, the appropriate dietary GSH had a positive effect on nonspecific immunity factors. But for the different immunity factors, there had the different effects and had different sensitive tissues in Litopenaeus vannamei. LSZ, AST/GOT, ALT/GPT and ACP activities both in hepatopancreas and serum could be the sensitive indexes to reflect the state of non-specific immunity factors in Litopenaeus vannamei, nevertheless, the MPO and the AKP in hepatopancrea was lacking regularity.In conclusion, dietary GSH could increase body weight gain and feed conversion efficiency of Litopenaeus vannamei. Optimal supplementation of GSH in shrimp diet could improve antioxidant capacities and decrease lipid peroxidation products, enhance the immune factors of Litopenaeus vannamei, and improve the antioxidant stress caused by ionized ammon and activate the non-specific immune functions of Litopenaeus vannamei.3. Extraction method of hepatopancreas mitochondria, establishment of lipid peroxidation model and the effect of GSH on the oxidative damage in mitochondria of Litopenaeus vannameiIn this study, the extraction method of hepatopancreas mitochondria of Litopenaeus vannamei was studied. By using different oxidants (VC/FeSO4、NADH) at different conditions (concentration, time, and pH), two lipid peroxidation models to induce hepatopancreas mitochondrial oxidation of the Litopenaeus vannamei were established. The optimized conditions and the effective elicitor to induce hepatopancreas mitochondrial oxidation of the Litopenaeus vannamei were elucidated by analyzing the lipid peroxidation products, swelling rate of mitochondria and DNA double chain ratio. These mitochondrial oxidation models could be suitable for studying the hepatopancreas mitochondrial damage caused by lipid peroxidation, and the roles in protecting oxidation damage conferred by GSH in vitro at the subcellular level.This research includes two parts. Firstly, the hepatopancreas mitochondria of Litopenaeus vannamei were extracted with three steps of ultracentrifugation. The mitochondria were authenticated by neutral red-Jana’s green B, inspected the purity with the index of OD260/OD280 to choose the suitable extraction method of hepatopancreas mitochondria of Litopenaeus vannamei. Secondly, VC/FeSO4 and NADH were used to induce the peroxidation of hepatopancreas mitochondria of Litopenaeus vannamei, the concentration of different catalysts, the optimal time and conditions for the reaction were established to build two models. In these two models, various concentration of GSH (0, 0.1,0.2,0.4,0.8 and 1.2 mmol/L) were added to the reaction system to observe its inhibition effects of GSH. Finally, these two models were compared objectively, the MDA contents in the reaction system, the swelling rate and double chain of DNA in hepatopancreas mitochondria of Litopenaeus vannamei were the indexes to evaluate these two models.The results showed that:(1) After homogenated, the hepatopancreas with a wet weight:buffer rate is 1:15, in a buffer of STE (mmol/L:Sucrose 250, Tris-HCl 10, EDTA 1, pH8.0), then, centrifuged at 4℃1000gx5 min,10000gx20 min and purified with buffer STM (mmol/L:Sucrose 250, Tris-HCl 50, MgCl25, pH7.4). The hepatopancreas mitochondria were in light green color in the electron microscopy at high magnification after being dyed by neutral red-Jana’s green B, and the OD260/OD280 was 1.73, which is proximity to 1.8.(2) In model VC/FeSO4, when the protein content of hepatopancreas mitochondria was lmg/ml, the VC 0.2mmol/L, the FeSO4 41/4mol/L, the reaction temperature 30℃, reacting for 30min in the buffer of pH 7.4 HEPES, the VC/FeSO4 system induced the peroxidation of hepatopancreas mitochondria effectively. While in the model of NADH, the best reaction conditions were:the protein content of hepatopancreas mitochondria was 1.0mg/ml, FeCl3 0.04mmol/L, ADP 4.0mmol/L, in 25mmol/L HEPES/NaOH buffer system with pH7.4, the start factor was NADH 120μmol/L, the reaction condition was 30℃,30min, then ended the reaction with 20%trichloroacetic acid. The exogenous GSH at 0.4mmol/L inhibited the peroxidation in both of these models obviously.(3) Comparing the NADH with the VC/FeSO4 model of hepatopancreas mitochondrial oxidation of the Litopenaeus vannamei revealed that both the NADH and the VC/FeSO4 could significantly activate mitochondrial lipid peroxidation of Litopenaeus vannamei (p<0.05), whereas there were no significant differences between the two models (p>0.05). In the VC/FeSO4 model, the MDA content, swelling rate of mitochondria and DNA double strands percentage were respectively 2.10 times,1.71 times and 60.07% of the control; in the NADH model, the three parameters were 1.43 times,1.38 times and 55.13%, respectively. From MDA and mitochondrial swelling rate, it was suggested that the NADH model functioned better; however, the percentage of double strand DNA indicated that the VC/FeSO4 model confered better inhibitory effect.4. Study of method for primary culture of hepatopancreatic cells of Litopenaeus vannamei, effects of GSH on the proliferation, physiological and biochemical characteristics and functions, the anti-oxidative effects, and expression of mRNA of SOD and CAT in primary culture cells in Litopenaeus vannameiIn order to reveal the effects of GSH on the proliferation, physiological and biochemical characteristics and functions, on the antioxidant affects and on the expression of anti-oxidase in vitro, thus to have the light on the mechanism insight of micronutrient in regulating aquatic organism’s antioxidant stress at molecular level, we designed the experiment as follows.Firstly, the method for improving the protocol of primary cultured hepatopancreatic cells of Litopenaeus vannamei were studied, including:(1) optimizing the methods of separating, digesting and purifying the hepatopancreatic cells of Litopenaeus vannamei; (2) choosing the culture medium for primary cultured hepatopancreatic cells; (3) selecting the most suitable salt rate and the pH of culture and the seeding density of hepatopancreatic cells of Litopenaeus vannamei.Secondly, the primary cultured hepatopancreatic cells were cultured in the medium with the final concentration of GSH by 0,0.1,0.2,0.4,0.8 and 1.2mmol/L, separately. We collected the supernatant of hepatopancrea cells of all the groups at 24h,48h and 72h separately, to measure the activities of cells, the RNA/DNA, the content of albumin, the nitricoxide synthase (NOS), Insulin-like growth factor-Ⅰ(IGF-Ⅰ), ATPase, alanine aminotransferase (ALT/GPT) and aspartate aminotransferase (AST/GOT).Thirdly, the cells were collected and crushed in the culture media by microwave and then centrifuged (4℃,3000 r/min, 10min) and the supernatants were taken to analysis the antioxidant index, include:SOD, GSH-Px, CAT, MDA and H2O2 quantitatively at 24h,48h and 72h, separately.Finally, after the hepatopancreatic cells were primary cultured in medium with different concentration of GSH for 72 hours, the cells were collected, the RNA of hepatopancreatic cells was extracted and the mRNA was detected by RT-PCR to measure the expression levels of SOD mRNA and CAT mRNA.The results showed that:(1) The method for primary culture of hepatopancreatic cells of Litopenaeus vannamei was established. After being soaked in potassium permanganate, washed by cold PBS and sterilized totally in 75% ethanol, the hepatopancreas were removed away from the Litopenaeus vannamei. The hepatopancreatic cells were isolated in 0.05% typeⅡcollagenase at 27℃for 10 min, then purified with a centrifugation method at 1200 r/min,10 min+1000 r/min,5min, and cultured in the box of 5% CO2, saturated degree of humidity,27℃at a seeding density of 5×105 cells/ml. The cells were at an excellent status characterized by smooth cell surface, better refractive, and higher activities of hepatocytes.(2) GSH boosted the proliferation of Litopenaeus vanhnamei’s hepatocyte in vitro, increased RNA/DNA ratio, accelerated the hepatocytes to excrete IGF-1, indicating that GSH could facilitate the up-growth of cells through regulating the relevant hormone. GSH could take on nutrition action and antioxidant function. GSH could improve the content of albumin and the secretion of nitricoxide synthase (NOS) in vitro, thus enhance the bioactivity of hepatopancreatic cells in vitro. Boost the ATPase activity, and protect the penetrating power of cell membrane while keep the well-balanced physiological function. GSH could reduce the damnification hurt by environment factor, and then accelerate the organizational recovery when the hepatopancrea was damnified.(3) After being cultured for 72h, GSH addition increased the SOD activities in all the experiment groups except 1.2 mmol/L group. The the increase rate was 17.28%、9.05%、37.04% and 45.27% respectively and the group 0.8 mmol/L affected the SOD activities significantly compared with the control (p<0.05). In conclusion, the proper GSH addition affected the SOD activities in the hepatopancreatic cells of Litopenaeus vannamei in vitro, showing a trend of increasing first then declining and increasing slowly until reaching to a platform, finally. After being cultured for 72h, GSH addition improved the GSH-Px activities in all experiment groups, but had no difference between all the groups (p>0.05). Whlie, the CAT hadn’t been detected in some groups because of the insensitivity of the methods for testing in the supernatant preparations from the hepatopancreatic cells in vitro.MDA contents had a obvious decline trend in all groups at 72h obviously (p<0.05) and reach the lowest content at 0.2 mmol/L group. After being cultured for 72h, the GSH addition can decrease the H2O2 contents in all the experiment groups significantly (p<0.05). The the decrease rate was 4.42%,17.85%,10.22%,25.58% and 45.27% respectively and had a difference at distinguish level of group 0.8 mmol/L (p<0.05).(4) Adding GSH evidently boosted the SOD genie expression of litopenaeus vannamei’s hepatopancreatic cells in vitro. Except group 0.1mmol/L, the SOD mRNA expression level were all observably higher than the experiment group (p<0.05). Except group 1.2 mmol/L, adding GSH in hepatopancreatic cells elevated CATmRNA expression level (p>0.05). GSH hoisted SOD enzymatic activities in hepatic separate cells, depressed the lipid peroxide content, effected the expression of SOD and CAT mRNA.The results described above suggests that the supplementation of GSH in culture media be capable of reducing lipid peroxide and modulating the activities of SOD and CAT in hepatopancreatic cells of Litopenaeus vannamei in vitro. Through increasing the effect of the expression capacity of antioxidant enzyme CAT and SOD, especially SODase, regulating antioxidant level, that purged the free radicals, efficiently regulated the free radicals metabolism, prevented lipid peroxidation.In Conclusion, GSH can ameliorate the growth performance of Litopenaeus vannamei in vivi, boost the efficiency of feedstuff transforming, enhance the anti-oxidative level and anti-oxidative capability of Litopenaeus vannamei’s, organic tissue, ease up the oxidation allergic response caused by ammonium stress. The optimal level of dietary GSH for litopenaeus vannamei was 174.13 mg/kg. GSH can also restrain the lipid peroxide of Litopenaeus vannamei hepatopancreatic mitochondrion in vitro, when the culture medium GSH concentration is 0.4mmol/L, there is best protection effect for mitochondrion. Exogenous GSH can improve the growth and proliferation of the hepatopancreatic cell in vitro, enhance it biologic activity. It can protect the cell membrane penetration by regulating the correlative metabolic enzyme and anti-oxidative enzyme, depressing the content of MDA, maintaining the well-balanced physiology function. It can boost the hepatopancreatic cell SODmRNA expression level in Litopenaeus vannamei. The effect reach the prominent level when the culture medium GSH is 0.8 mmol/L.