| The development and mode of action of antioxidation about vitamin C on juvenile Jian carp are explored in this study. It has been studied that the variations about the index of the enzymatic antioxidation system and the content of the non-enzymatic antioxidation substances in blood, hepatopancreas, intestinal tract and muscle during the different developmental stages of juvenile Jian carp using the means of internal and in-vitro experiment, which showed the change regularity of internal antioxidation at different developmental stages of juvenile Jian carp. On this basis, the antioxidational regularity and its mode of action in blood, hepatopancreas, intestinal tract and muscle of juvenile Jian carp on different level of vitamin C, and the effection on achievement and efficiency of nutrient of vitamin C in juvenile Jian carp are studied and defined its right amount. Otherwise, systematic study has been conducted that the effection on the development of digestive organs and the digestion-absorption function of juvenile Jian carp about vitamin C, which reveals the mode of action that influences the digestion and absorption of the nutrients. Researching into the effection on the proliferation, differentiation and antioxidation of intestinal epithelial cells through cell model, which is to quest the mode of action of antioxidation about vitamin C at the cell level. Main research contents and results are as follows:1. The variation regularity research of antioxidation index in blood, muscle, hepatopancreas and intestinal tract of juvenile Jian carpChoosing 750 healthy juvenile Jian carps whose weight are 18.0±0.2g (mean±standard deviation), which are set up 5 stages,150 tails per stage, 3 repetitions per stage,50 tails each repetition. Feeding on the same experimental diet on every stage. Sampling once per 14 days when begins during each stage, which are used to measure the index of antioxidation in serum, hepatopancreas, intestinal tract and muscle of juvenile Jian carp. The result shows:the weight of juvenile Jian carp shows highly significant effects (P<0.01) or decreased significantly (P<0.05) on their MDA content and protein hydroxyl content in their organism, hepatopancreas, intestinal tract and muscle. When the body weight 60.3g,52.6g,48.6g,50.6g respectively, the MDA levels of the corresponding organs and tissues reduce to minimum and achieve the platform (P>0.05), When the body weight 68.1g, 58.2g,60.4g and 63.6g respectively, the protein carbonyl content of the corresponding organs and reduce to minimum and achieve the platform(P>0.05). The weight of juvenile Jian carp shows highly significant effects (P<0.01) or significant increased (P<0.05) on the activity of antioxidation enzymatic index SOD,CAT,GSH-Px,GST. When the weight 49.6g,43.4g and58.4g respectively, the SOD activity reaches stable state in blood, intestinal tract and muscle (P>0.05); When the weight, reaches 57.4g,61.6g,63.7g and 57.6g respectively, the activity of CAT in blood, hepatopancreas, intestinal tract and muscle reach the platform (P>0.05); When the weight reaches 65.7g,46.8g and 51.8g, the activity of GSH-Px reach stable state in blood (P>0.05); but when the weight reaches 59.2g and 55.1g, the activity ofGST in serum and intestinal track achieve the platform (P>0.05); When the weight reach 40.1g,40. 1g,31.4g and 31.4g respectively, the activity of GR in blood, hepatopancreas, intestinal tract and muscle reach the platform (P>0.05). The weight of juvenile Jian carp shows highly significant effects (P<0.01) significant increasedon the content of non-enzymatic antioxidation GSH. When the weight reaches 58.6g,40.1g,58.6g respectively, the content of GSH in hepatopancreas, intestinal tract and muscle reach the platform (P>0.05). The result of study shows:antioxidant capacity of juvenile Jian carp changes associates with its body weight. The change of hepatopancreas, intestinal tract and muscle are basically the same variation and reach stable state at the lower weight, which is identical to the changes of the regularity that the capacity of scavenging free radicals and the index change regularity of enzymatic antioxidation system and non-enzymatic system.2. The effection of Vitamin C on the growth performance, body protein, fat and ash of juvenile Jian carpChoosing weight of 12.63±0.02 g (mean±standard deviation) to build a healthy juvenile Jian carp 900. Choosing 900 healthy juvenile Jian carps whose weight are 12.63±0.02 g (mean±standard deviation), which are set up 6 treatments,150 tails per treatments,,3 repetitions per treatments,,50 tails each repetitions. Each are fed on the contents of vitamin C Omg/kg, 21.4mg/kg,45. lmg/kg,69.5mg/kg,93.6mg/kg, and 142. lmg/kg, respectively for 60 days. The result indicates:Vitamin C can improve highly significantly specific growth rate and food intake of juvenile Jian carp (P<0.01), when the content of vitamin C increases to 40.9 mg/kg and 45.1 mg/kg, the rate of specific growth and food in taking reach the maximum and platform (P>0.05).To compare with the control group, increases 7%at the feed effiencieny (P<0.05); Vitamin C improves the rate of protein, fat and Phosphorus deposition highly significantly (P<0.01) or significantly (P<0.05),when the vitamin C content reaches 39.4mg/kg and 38.6 mg/kg, respectively, the rate of protein and fat deposition reach platform respectively and the maximum platform(P> 0.05), and there is no significant effect on the deposition of ash and calcium (P> 0.05). The result shows that: Vitamin C promotes the growth of You-Jian carp and improves the rates of Protein, fat, ash and phosphorus depositions. Marked with SGR, Weight increment, food intaking, the rate of protein and fat deposition, the requirement of the juvenile Jian carp for vitamin C is 40.9mg/kg,40.6mg/kg,45. lmg/kg,39.4mg/kg and38.6mg/kg, respectively.3. The research of Vitamin C on digestion and absorption capacity of juvenile Jian carpChoosing 900 healthy juvenile Jian carps whose weight are 12.63+0.02g (mean±tandard deviation),which are set up 6 treatments,150 tails per treatments,3 repetitions per treatments,,50 tails each repetitions. Each are fed on the contents of vitamin C Omg/kg,21.4mg/kg,45. lmg/kg,69.5mg/kg, 93.6mg/kg, and 142. lmg/kg respectively for 60 days. The result indicates: Vitamin C can improve highly significantly specific growth rate and food intake of juvenile Jian carp (P<0.01), when the contents of vitamin C for one increases to 46.9mg/kg,69. lmg/kg and 45.lmg/kg, and the other reaches 44.4mg/kg,48.9mg/kg,48.9mg/kg and 44.8mg/kg respectively for 60 days, The result indicates that:Vitamin C can highly significantly increase on juvenile Jian carp, the activity of pancreas trypsin, chymotrypsin, lipase and amylase in gut liver and pancreasof the juvenile Jian carp (P<0.01), were respectively increased by 46.9mg/kg,69. lmg/kg,45.lmg/kg和44.4mg/kg,48.9mg/kg,48.9mg/kg,44.8mg/kg, the activity of the intestinal trypsin, chymotrypsin and lipase, as well as pancreatic trypsin, chymotrypsin, lipase and amylase, respectively reach the platform and the largest platform (P> 0.05). Vitamin can highly significantly (P<0.01) or significantly (P<0.05) increases anterior, middle and posterior high degree of Plica in intestine of the juvenile Jian carp. When content of the vitamin C infodder reaches 44.5mg/kg, the high degree of the foregut plica reaches to the maximum. Vitamin C can highly and significantly increase the activity of the anterior, middle and posterior intestinal alkaline phosphatase, Na+, K+-ATP,γenzyme,γ-GT enzyme and All intestinal CK enzyme gut CK (P<0.01). When the content of vitamin C reaches 48.5mg/kg, 40.8mg/kg,40.7mg/kg respectively in diet, the activity of Na+, K+-ATPase in the anterior, middle and posterior intestine reached the maximum; while the level of vitamin C was up to 51. Omg/kg,51.5mg/kg,48.4mg/kg in diet, their activity ofγ-GT are the strongest; when up to 44.5mg/kg, the activity of entire intestine CK up to the maximum. So Vitamin C can highly significantly increase the weight of hepatopancreas, the index of the liver somatic, the weight of intestine, the. Vitamin C significantly affects the amount of intestinal lactobacilli and bacillus (P<0.01), while no significant influence to the amount of Aeromonas sp (P>0.005). Research results indicate that:Vitamin C increases the ability of digestion and absorbion of juvenile Jian carp. And it promotes the growth of the liver pancreas and intestine with Vitamin C, the capacity of secretion of pancreatic trypsin, chymotrypsin, lipase and amylase, the absorption area and capacity of intestine, and also it promots the growth of beneficial lactic acid bacteria and Bacillus subtilis, and inhibits the growth of harmful E. coli bacteria.4. The impact of Vitamin C to the index of anti-oxidation in different tissues of juvenile Jian carpChoosing 900 healthy juvenile Jian carps whose weight are 12.63±0.02g (mean±standard deviation),which are set up 6 treatments,150 tails per treatments,3 repetitions per treatments,,50 tails each repetitions. Each are fed on the contents of vitamin C Omg/kg,21.4mg/kg,45. lmg/kg,69.5mg/kg, 93.6mg/kg, and 142. lmg/kg respectively for 60 days. The result indicates that:Vitamin C can induce highly significantly the content of malondialdehyde (MDA) and protein carbonyl (P<0.01) in blood, liver and pancreas, gut and muscle tissue, when the contents of vitamin C in diet increases to 42.9mg/kg,46.6mg/kg,50.4mg/kg and 61.4mg/kg, the content of MDA in blood, liver and pancreas, gut and muscle tissue reduces the lowest and reaches the platform, while the content of protein carbonyl in blood, liver and pancreas, gut and muscle tissue showed a significant linear relationship with level of vitamin C in diet, and with the adding vitamin C, it was significantly decreased (P<0.01). Vitamin C highly significantly (P<0.01) or significantly (P<0.05) increases the scavenging ability of anti-superoxide anion (ASA) and anti-hydroxyl radical (AHR) in blood, hepatopancreas, intestinal tract and muscle tissue of juvenile Jian carp, and with the MDA in corresponding tissue and the content of protein carbonyl is extremely significantly (P<0.01) or significantly (P<0.05) negative correlation; Vitamin C can highly significantly (P<0.01) or significantly (P<0.05) increase the activity index of enzymal Anti-oxidation in superoxide dismutase indicators (SOD), peroxide catalase (CAT), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) and Glutathione reductase (GR) in blood, hepatopancreas, intestinal tract and muscle tissue of juvenile Jian carp, Correlated analysis found that these enzymes are showed extremely significantly (P<0.01) or significantly (P<0.05) positive correlation with the MDA in the corresponding tissue or the protein carbonyl. Vitamin C can highly significantly (P<0.01) or significantly (P <0.05) increase the content of non-enzymatic antioxidants reduced glutathione (GSH) in blood, hepatopancreas, intestinal tract and muscle tissue of juvenile Jian carp, and showed extremely significantly (P<0.01) or significantly (P<0.05) positive correlation with the content of MDA in the corresponding tissue and the content of protein carbonyl. The results of research indicate that:With the content of Vitamin C from 0 to 142.1 mg/kg, the level of lipid peroxidation of body gradually reduce, but the degree of lipid oxidation in hepatopancreas, gut and muscle tissues was only reduced in the Vitamin C increased from 0 to 45.lmg/kg, when the activity of vitamin C levels reaches 45. lmg/kg, it is no longer reduced, and impact of regular pattern in liver pancreas, intestine tract and muscle are same; With the vitamin C level from 0 to 142.1 mg kg, the oxidation of protein reduces in body, liver and pancreas, gut and muscle tissue of juvenile Jian carp; it is related to increase the capacity of superoxide anions, hydroxyl radicals, the activity of enzyme antioxidant system such as SOD, CAT, GSH-Px, GST, GR and the material of non-enzymatic system such as GSH of juvenile Jian carp.5. Effect of VC on the proliferation and differentiation of carp intestinal epithelial cellsSingle factorial experiment designs was used in the research, which was divided into six dealings, four repetitions was set up in each dealing. To prepare the intestinal epithelial cells by picking the healthy juvenile Jian carp weighed around 80g. It was inoculated and cultured for 72 hours when the intestinal epithelial cells were separated. And then cultured continually for 96 hours after appending the growth promoting gene with the concentration of vitamin C, respectively,0,4,10,16,22,28mg/L. After that, collected the culture medium and analysis and tested the related indexes. The result indicates that:it was found that the cell number increased obviously that inhibited to the wall with the rising vitamin C concentration. And the large cell colony and laminating monolayer cells were formed, the individual cells largened and showed obvious ovals with clear outline. It was observed that OD value raised with the increasing vitamin C concentration through MTT. OD value achieved the platform when vitamin C concentration reached 10mg/L (P>0.05). Vitamin C has a significant affection on the activity of protein content, c, asparate aminotransferase, aspartate aminotransferase, alt, Na+, K+-ATP enzyme and lactate dehydrogenase in intestinal epithelial cells (P<0.05), and it can improve the protein synthesis in intestinal epithelial cells significantly (P<0.05).The total protein content in cell and culture medium achieved maximum and kept a stable state when vitamin C concentration reached, respectively,22mg/L, 10mg/L; increase aspirate aminotransferase activity in intestinal epithelial cells significantly (P<0.05) and aspirate aminotransferase activity will never improve and achieve a stable state when vitamin C concentration reached 16mg/L;increase aspartame aminotransferase and alt activity significantly (P<0.05),GOT and GPT activity reached the strongest and achieved the platform; Na+, K+-ATP enzyme activity will also never increase and achieve the platform when vitamin C concentration reached 16mg/L;have a significant effection on lactate dehydrogenase (P<0.05), its activity induced to the lowest when vitamin C concentration reached 22mg/L. It showed that:vitamin C can improve proliferation and differentiation of intestinal epithelial cells under this test condition, which was associated to which vitamin C preserved the integrity of intestinal epithelial cell membrane, promoted amino acid turnoverin, increased the activity of cell membrane transporter-Na+, K+-ATP enzyme and promoted proteinosis of intestinal epithelial cells.6. Effect of VC on the antioxidation of carp intestinal epithelial cellsSingle factorial experiment designs was used in the research, which was divided into seven dealings, four repetitions was set up in each dealing. To prepare the intestinal epithelial cells by picking the healthy juvenile Jian carp weighed around 80g. It was inoculated and cultured for 72 hours when the intestinal epithelial cells were separated. And then cultured continually for 16 hours after dealing the growth promoting gene according to the concentration such as Omg/L (blank control),0mg/L+100umol H2O2,4mg/L+100umol H2O2,0mg/L+100umol H2O2,16mg/L+100umol H2O2,22mg/L+ 100umolv H202 and 28mg/L+100umol H202. After that, collected the culture medium and analysis and tested the related indexes. The result indicates that:it was observed that the cell number decreased, iiregular, unclear outline and cell number increased refractivity weakened, adherence capability reduced, after being dialed with 100μmol/L H202 for 16hours compared with the blank control, but being dealed with vitamin C concentration such as 4,10,16 and 22mg/L, the cell shape, refractivity, adherence capability.improved obviously and large amount of intact cells were observed.100 umol of H2O2 leaded to oxidative stress happened in the intestinal epithelial cells, compared with the blank control, which increased lactate dehydrogenase content in the culture medium significantly (P<0.05), decreased cell protein content significantly (P<0.05), the cell growth was prevented, the cell result was breached; compared with the blank control, MDA and protein carbonyl content increased significantly (P<0.05),the scavenging activity of superoxide anion resistance (ASA) and hydroxyl radical (AHR) decreased significantly (P<0.05) antioxidase activity and unenzymatic antioxidation substances content decreased singnificantly (P<0.05).MDA and protein carbonyl content in the culture medium can be induced singnificantly by appending different concentration of vitamin C under oxidative stress (P<0.05); the scavening activity of superoxide anion resistance (ASA) and hydroxyl radical (AHR) can be restored significantly (P<0.05), ASA capacity restored at the blank control level, AHR capacity increased significantly (P<0.05);the activity of SOD, CAT, GST and GR and GSH content were improved significantly (P<0.05), which reached the blank level when vitamin C reached 28mg/L. GSH-Px activity enhanced significantly, which achieved maximum when vitamin C reached 28mg/L, but lower than the blank control (P<0.05).It showed that:vitamin C can protect the oxidation of intestinal epithelial cells and increased the ability with concentration increased; The protect ability is associated to enhancing the scavenging activity of superoxide anion resistance (ASA) and hydroxyl radical (AHR) and improving the activity of antioxidant enzyme system—SOD, CAT, GPx and GST and GSH content in enzyme system.
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