The Study Of Ascorbic Acid Requirement In Juvenile Spotted Halibut (Verasper Variegatus)

This paper studied the effect of vitamin C phosphate ester polymer on spotted halibut(Verasper variegatus),such as demonstrated the effect of VC on embryo hatching,larvae growth,antioxidant,nonspecific immunity and the effect of ammonia nitrogen stress resistance of spotted halibut.The trials were conducted at Shandong Kehe ocean high-technologies company and Yantai Tianyuan aquatic products company.Results of this study are presented as follows:1.In order to explore the membrane time,hatching ratio,survival rate,growth characteristics,the variation of digestive enzyme activities and lipases enzyme activities of spotted halibut during early development,the effects of exogenous VC and the most suitable VC concentration for growth,mature eggs from spotted halibut were obtained by artificial fertilization and selected for subsequent trials.The eggs were divided into five groups,and the VC concentration in water for incubation was 0 、20、25、30 and 35mg/L,respectively.Samples were collected at mature eggs,fertilized eggs,mid-gastrula,crystals appear,mixed nutrition and exogenous nutrition stages.Then a series of trials were conducted to study the variation of digestive enzyme activities,the membrane time,hatching ratio,survival rate and growth characteristics,the digestive enzyme activity and two lipases enzyme activity between control group and 25mg/L VC group.The results showed that 25mg/L VC solution can significantly increase the activities of digestive enzyme and elevate the deposition of protein.Appropriate level of VC could improve lipid metabolism during embryo development of spotted halibut.2.The effects of ammonia-N stress on the gill of juvenile spotted halibut were evaluated.The initial average weight of the juvenile fish was(70±5.5)g.The fish were first exposed to ammonia-N for 96 hours to obtain the 96 hours median lethal concentration.Based on this,the juvenile spotted halibut were reared in the control group(0mg/L)and three ammonia-N levels:35mg/L(low group)、50mg/L(middle group)、65 mg/L(high group).Each ammonia-N level group was respectively sampled at 0、6,12,24,48 and 96h.Gill microstructure observation showed that chloride cell increased;pavement cells fell off;epithelial tissue hyperplasia;blood capillary fractured and red blood cell overflowed;gill coarsen dice became short and curly,gill cavity was broken and congestion.Ammonia nitrogen stress caused liver nuclei dissolved;liver cell degeneration,water sampling blood sinus hyperemia and expansion,;blood sinus expansion and severe congestion.Na+/K+-ATPase andglutathione peroxidase(GSH-PX)activities were first decreased then increased,and both enzymatic activities were significantly different from the control group(P<0.05).Superoxide dismutase(SOD)activity decreased after increased,significantly higher than control group(P<0.05).The malondialdehyde(MDA)concentration in 96 h ammonia nitrogen stress group was higher than the control group,and significantly positive correlated with the concentration of ammonia nitrogen(P<0.05).Ammonia nitrogen had significant effects on serum alanine amino transferase(ALP),aspertate amino transferase(AST)and lysozyme(LSZ)activitities(P<0.05),all the three enzymatic activities were first increased then decreased.Liver ALT and AST activities showed a trend of decline after rising first,96 h group was significantly lower than control group(P<0.05).Liver LSZ activity in 96 h group was significantly lower than the control group(P<0.05).In general,ammonia nitrogen stress caused the fish body antioxidant system and nonspecific immune system declined,normal physiological metabolism was hampered,gill tissue damage,respiratory function was impaired,liver tissue hyperemia and dot lesions appeared,the body’s metabolism and detoxification function declined.3.A 56-day feeding trial was conducted to unravel the suitable amount of VC for juvenile spotted halibut.The studies of VC requirement in spotted halibut has not been reported before.Water temperature was maintained at(12.5±0.5)℃.Juvenile spotted halibut(38±0.8g)were reared in 30 cylindrical tank(d53cm×h60cm)and fed by feed containing 50.2 % protein,8.6 % lipid and supplemented with seven levels of coated-ascorbic acid(CAA)at 0、1500、3000、4500、6000、7500 and 9000 mg/kg.The results of 56 days feeding trial showed that VC concentration at 616.2 mg/kg diet yielded the best growth performance in V.variegatus.The fish had the maximal liver and muscle ascorbic acid level when CAA at 769.5 mg/kg.The fish had the maximal SOD,CAT and lysozyme activity when CAA at 402.8 mg/kg(P<0.05).MDA had the lowest levels when fed at 616.2 mg /kg(P<0.05).VC concentration at 616.2 mg/kg can enhance proteinase activity and immunity(P<0.05).The fish had the maximal lipase activity at 402.8 mg/kg(P<0.05).CAA concentation between 249.1 and 616.2 mg/kg can promote the growth of intestinal plica height,number of mucosa and intestinal wall thickness.These results suggested that dietary VC supplementation within the scope of 402.8 and 616.2 mg/kg may improve the growth performance,the function of antioxidant,nonspecific immunity,digestion and absorption of nutrients in V.variegatus.After 56 days culturing,the juveniles were stressed with 20 mg/L ammonia nitrogen for 24 h.The results showed that the fish had the maximal liver and muscle ascorbic acid levels when fed at 6000 mg/kg,and the maximal SOD and CAT activities when fed at 3000mg/kg(P<0.05)before and after ammonia-nitrite stressing.Na+/ K+-ATP activity significantly increased between 1500 and 6000mg/kg.After ammonia nitrogen stressing,the decline extentwas significantly lower than control group(P<0.05).Cortisol,Glucose and Lactic acid had a significant small upward trend(P<0.05)when VC concentration was between 3000 and 4500 mg/kg.TIBC showed no significantly declined(P<0.05)after stressing.All the results indicated that VC supplements in diet could improve immunity and anti-ammonia-nitrite stress ability of spotted halibut.