| This study systematically disscussed the interaction between Vitamin C on the immune performance and the demand for VC, used Cherax quadricarinatus as objective. Adding different VC levels of dietary, to analysis the changes of LSZ, PO and SOD activity in hepatopancreas, gill, muscle and hemacyte; the changes of total hemacyte count and hemolytic activity, agglutination activity, phagocytosis activity and inhibitory rate; effect of VC on the immune enzyme activity and immune gene expression after WSSV infection of Cherax quadricarinatus; Microscopic hemacyte and ultrastructural hemacyte analysis and explore the immune protective of VC. Provide solutions for the optimum amount of VC in the process of breeding, and provide a theoretical basis for the healthy culture and WSSV of Cherax quadricarinatus’s defense.1. Effects of diet VC content on the growth index and tissue VC content of Cherax quadricarinatusVC plays an important role in the growth and immunity performance in decapod crustacean animal. A 8-weeks feeding trial was studied the effect of VC on Cherax quadricarinatus growth and immune performance. The experimental diets included seven levels of vitamin C (100,200,400,800,1600,3200 mg/kg), the result showed, when the adding amount of 0-400 mg/kg, the weight gain rate and specific growth rate increased significantly, when the diet was added to 800-3200 mg/kg, inhibition of Cherax quadricarinatus the weight gain rate and specific growth rate, at this time, the amount of VC was higher and the crayfish weight gain rate was low, but the weight gain rate and specific growth rate in the VC group were higher than 0 mg/kg group. But VC can’t effect of survival rate and hepatosomatic index. The VC content in hepatopancreas was highest, and muscle tissue VC content, hemolymph VC content was relatively low, in a certain range, the content of VC in tissue increased with the increasing amount of VC in diet.When the diet amount of VC was more than 400 mg/kg, the content of VC in hepatopancreas were not increased, VC supplement at 400mg/kg can enhance the weight gain rate and specific growth rate.2. Effects of diet VC content on immune enzyme activityA 8-weeks feeding trial was conducted to explore the effect of immune enzyme ability of Cherax quadricarinatus fed diets with different levels VC. The results showed that VC can significantly enhance the LSZ and PO activity, and LSZ and PO activities significantly increased in hepatopancreas, gill, muscle, hemacyte when the diet amount of VC was 800 mg/kg, and the enzyme ability in 0 mg/kg group was significantly lower than the supplemented group (P< 0.05). After addition of VC, the SOD activity reduced and the hepatopancreas SOD activity decreased significantly. The decrease of SOD activity have different levels in different tissues, SOD activity had no significant difference between the experimental group (P> 0.05).3. Effects of diet VC content on activity of the hemolymphA 8-weeks feeding trial was conducted to explore the effect of the hemolymph activity of Cherax quadricarinatus fed diets with different levels VC. The results show that VC can significantly enhance the hemolytic activity and antibacterial activity, the serum inhibitory rate was 26.89% in 0 mg/kg group, after added VC a maximum inhibitory rate is 59.31%, hemolytic activity and antibacterial activity in VC group were higher than 0 mg/kg group, significant difference between groups (P< 0.05), but the difference was not significant between the added VC groups (P> 0.05). The phagocytic activity of hemacyte agglutination activity increased when the VC content continued to increase in 0-800 mg/kg groups, but when the VC content more than 800 mg/kg, the phagocytic activity of hemacyte agglutination activity increased when the VC content continued to reduce. Phagocytic activity and agglutination activity in VC group were all higher than 0 mg/kg group. Many small granule hemacyte in hemacyte, and the count of small granule hemacyte increased in VC group. The count of total hemacyte increased in VC group of Cherax quadricarinatus.4. Effects of diet VC content on immune enzyme activity and gene expression after WSSV infectionThe Realtime-PCR results showed that C-type lectin gene was widely distributed, with the highest expression level in haemocytes, small amount of expression in hepatopancreas, antennal gland and heart, while expression was almost undetectable in muscle, brain and stomach. The Realtime-PCR results showed that LSZ gene was widely distributed, with the highest expression level in hepatopancreas, small amount of expression in antennal gland, gills and intestines, while expression was almost undetectable in heat, brain and stomach. After WSSV infection, PO, LSZ activity and the expression level of C-type lectin and LSZ gene with virus infection time were decreased.0-24 h, in the early stage of infection, immune enzyme and gene expression were increased in different degree; 24-48 h, in the middle of infection, with the continuous replication of WSSV. The expression of immune enzyme activity and mRNA decreased; 72-96 h expression in the late stage of infection, enzyme activity and gene significantly decreased (P<0.01). Immune enzyme activity and gene expression in VC groups were all higher than 0 mg/kg group, maybe that VC can enhance the strength of Cherax quadricarinatus for WSSV resistance, and have the protective role for Cherax quadricarinatus.5. Effects of diet VC content on haemocytes pathology after infection of WSSVUsed of Wright-Geimsa staining and electron microscope techniques to researched the pathological in haemolymph after infection of WSSV of Cherax quadricarinatus. The results showed that hyaline hemocyte (HC) and small granule hemocyte (SGC) in diseased shrimps was lower and the count of large granule hemocyte (GC) was higher, after infection of WSSV the sizes of three blood cells were all increasing trend, and the nuclear cytoplasmic ratio (NP) of GC and HC significantly decreased (P<0.05). With the extension of the infection time, the total haemocyte count was significantly lower (P<0.05), and the number of haemocyte remained at a high level in 800 mg/kg group. The main micro-pathological and microscopic structure changes of haemocyte showed, haemocyte damage, no obvious nucleus and nuclear membrane structure in 0 mg/kg group; appeared significantly nucleus heterochromatinization in haemocyte in 400 mg/kg group; parts of nuclear membrane were anamorphic but the nuclear membrane is clear and appeared nucleus heterochromatinization in haemocyte in 800 mg/kg group. |
