【作者】 胡凯；

【导师】 周小秋；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2008， 硕士

【摘要】 本试验主要研究了吡哆醇对幼建鲤抗氧化能力的影响以及幼建鲤吡哆醇缺乏的表观临床症状,组织器官细胞病理学变化。试验选择1050尾健康幼建鲤（初重为11.71±0.05 g）平均分成7组,每组设3个重复,每个重复50尾,分别饲喂吡哆醇水平为0.20、1.71、3.23、4.96、6.32、8.58和12.39 mg kg^-1的半纯合饲粮。试验期为80天。结果表明:吡哆醇可以极显著（P＜0.01）或显著（P＜0.05）降低幼建鲤血清、肠道和后肾组织丙二醛（MDA）含量。当吡哆醇含量从1.71mg/kg分别增加到3.23,4.96和3.23 mg/kg时,幼建鲤血清、肠道和后肾组织MDA含量达到最低,并不随吡哆醇的添加而降低;吡哆醇对血清、肠道和肾脏组织MDA含量有极显著的负直接作用(P_10.1=-0.287,d_10.1=0.0824;P_20.1=-0.455,d_20.1=0.208;P_30.1=-0.885,d_30.1=0.784)。吡哆醇对幼建鲤肠道和后肾组织蛋白质羰基含量有极显著的影响（P＜0.01）,当吡哆醇含量分别为5.91和6.27 mg/kg时,蛋白质的氧化损伤最低。吡哆醇对肠道和肾脏组织蛋白质羰基含量分别有极显著的直接和负直接作用(P_10.1=1.702,d_10.1=2.896;P_20.1=-0.259,d_20.1=0.0670)。说明,毗哆醇能够减少水生动物脂质和蛋白质的过氧化,降低水生动物的氧化损伤。吡哆醇可以极显著（P＜0.01）或显著（P＜0.05）提高血清、肠道和后肾组织抗超氧阴离子活力（ASA）。当吡哆醇含量为3.23 mg/kg时,血清、肠道和后肾组织ASA极显著（P＜0.01）或显著（P＜0.05）提高,并不再随吡哆醇含量的增加而提高。血清、肠道和肾脏组织ASA分别与血清、肠道和肾脏组织MDA含量呈极显著或显著的负相关（r₁=-0.924,P＜0.01;r₂=-0.943,P＜0.01;r₃=-0.825,P＜0.05）;肠组织ASA与肠组织蛋白质羰基含量呈极显著的负相关（r=-0.964,P＜0.01）,说明吡哆醇可以增强幼建鲤清除超氧阴离子的作用,提高鱼类的抗氧化能力。吡哆醇可以极显著影响幼建鲤肾脏组织抗羟自由基活力（P＜0.01）,当吡哆醇水平从1.71 mg/kg增加到4.96 mg/kg时,幼建鲤肾脏组织抗羟自由基活力逐渐升高,进一步增加吡哆醇含量,没有显著变化。相关分析显示,后肾组织抗羟自由基活力与蛋白质羰基含量呈极显著的负相关（r=-0.981,P＜0.01）。毗哆醇对血清和肠组织清除羟自由基能力没有影响,但能增强肾脏组织清除羟自由基能力。吡哆醇对血清超氧化物歧化酶（SOD）活力、过氧化氢酶（CAT）活力、谷胱甘肽过氧化物酶（GSH-Px）活力、谷胱甘肽S转移酶（GST）活力、谷胱甘肽还原酶（GR）活力和还原型谷胱甘肽（GSH）含量有极显著（P＜0.01）或显著影响（P＜0.05）。当吡哆醇含量从1.71 mg/kg分别增加到4.96、3.23、3.23、3.23和6.32 mg/kg时,血清SOD、CAT、GSH-Px、GST和GR活力极显著（P＜0.01）或显著（P＜0.05）提高,进一步增加吡哆醇含量没有影响。血清MDA含量和SOD、CAT、GSH-Px、GST和GR活力含量呈极显著（P＜0.01）或显著（P＜0.05）负相关（r₁=-0.924,P＜0.01;r₂=-0.817,P＜0.05;r₃=-0.968,P＜0.01;r₄=-0.926,P＜0.01;r₅=-0.913,P＜0.05;r₆=-0.824,P＜0.05;r₇=-0.907,P＜0.05）。肠道和后肾组织SOD、CAT、GSH-Px、GST和GR活力与血清中抗氧化酶有类似的趋势。肠道和后肾组织SOD活力、CAT活力和ASA分别与肠道和后肾组织蛋白质羰基含量呈极显著的负直接作用(P_10.2=-1.575,d_10.2=2.4819;P_10.3=-0.705,d_10.3=0.4966;P_10.4=-3.402,d_10.4=11.576;P_20.2=-0.159,d_20.2=0.0252;P_20.3=-0.970,d_20.3=0.0252;P_20.4=-0.327,d_20.4=0.107)。说明,吡哆醇能够通过提高抗氧化酶活力,增强水生动物的抗氧化酶系统的防御能力。吡哆醇对血清、肠道和后肾组织还原型谷胱甘肽（GSH）含量有极显著（P＜0.01）影响。血清、肠道和肾脏组织GSH含量分别与血清、肠道和肾脏MDA含量呈显著负相关（r₁=-0.907,P＜0.05;r₂=-0.861,P＜0.05;r₃=-0.865,P＜0.05）;与肠道和肾脏组织蛋白质羰基含量呈极显著负相关（r₁=-0.930,P＜0.01;r₂=-0.943,P＜0.01）。说明吡哆醇可以通过增加类血清和组织中非酶抗氧化物质GSH含量,提高水生动物非酶抗氧化系统的抗氧化能力。研究结果也表明:吡哆醇缺乏引起幼建鲤死亡率增加,鱼体游动异常,呈螺旋状运动或快速绕圈游动,呼吸困难;出现眼球突出、鳍条出血、头部皮肤糜烂、肛门红肿和鳃丝糜烂等外观变化;头肾、后肾、脾脏、肝胰脏和胆囊肿胀或萎缩,色泽异常;肝细胞溶解,形成大小不一的空泡;头肾有大量的含铁血黄素沉积;后肾上皮细胞肿胀,肾小管上皮细胞变性、坏死、脱落;脾脏淋巴样细胞减少;肠道肠绒毛坏死;心肌纤维肿胀、变性,肌纤维间隙增宽等病理损伤。综上研究结果说明:吡哆醇能够提高幼建鲤抗脂质和蛋白质氧化损伤能力,其能力的提高与吡哆醇提高了机体、肠道和后肾组织超氧阴离子和脂质过氧化产物清除能力有关,也与后肾羟自由基产生能力降低有关。在本试验条件下,对于体重为11～75g的幼建鲤,以血清、肠道和后肾组织脂质过氧化产物MDA含量为标识,饲粮中吡哆醇最适含量分别为3.48、4.72和3.44 mg/kg;以肠道和后肾组织蛋白质羰基含量为标识,饲粮中吡哆醇最适含量分别为5.91和6.27 mg/kg。吡哆醇缺乏引起水生动物死亡增加,神经机能紊乱,眼球突出、鳍条出血、头部皮肤糜烂、肛门红肿和鳃丝糜烂等缺乏症。肝胰脏、肌肉、后肾、肠道、鳃、脾脏和心脏等组织出现病理变化。更多还原

【Abstract】 This experiment was conducted to study the effects of dietary pyridoxine（PN） levels on antioxidant status of juvenile Jian carp（Cyprinus carpio var.Jian） and determine the histopathology observation and effect of digestive enzyme and immune function with dietary pyridoxine deficiency.Semi-purified diets with seven levels（0.20,1.71,3.23,4.96, 6.32,8.58 and 12.39 mg PN/kg） of supplemental calcium d-pantothenate were fed for 80 days to triplicate groups of 50 fish（initial weight 11.70±0.05 g,mean±SD）.The effect of pyridoxine on antioxidant status in fish showed that:pyridoxine significantly decreased malondialdehyde（MDA） in serum,intestine and kidney tissue.The MDA content was reduced with increasing dietary pyridoxine levels up to 3.23,4.96 and 3.23mg kg^-1 diet（P＜0.01 or P＜0.05）,respectively,in serum and intestine tissue,and no differences were found with further increase of pyridoxine levels（P＞0.05）.In serum, intestine and kidney tissue path-analysis results indicated levels of pyridoxine had significantly negative direct effects on MDA content(P_10.1=-0.287,d_10.1=0.0824;P_20.1= -0.455,d_20.1=0.208;P_30.1=-0.885,d_30.1=0.784).Pyridoxine significantly decreased protein carbonyl in serum,intestine and kidney tissue（P＜0.01）.Regression analysis results indicated that protein carbonyl content was lowest when pyridoxine concentration≥5.91 and 6.27 mg kg^-1,respectively,in intestine and kidney tissues.Path-analysis results indicated that levels of pyridoxine had negative direct effects on protein carbonyl content, in intestine and kidney tissue(P_10.1=1.702,d_10.1=2.896;P_20.1=-0.259,d_20.1=0.0670). This result suggested that pyridoxine may decrease lipid peroxidation and oxidative damage to proteins in fish.Pyridoxine significantly increased anti-superoxide anion activity（ASA） in serum,intestine and kidney tissue.The ASA was increased with increasing dietary pyridoxine levels all up to 3.23mg kg^-1 diet（P＜0.01 or P＜0.05）, respectively,in serum and intestine tissue,and no differences were found with further increase of pyridoxine levels（P＞0.05）.The correlation-analysis results indicated that there were significant negative correlation between MDA with ASA in serum,intestine and kidney tissue（r₁=-0.924,P＜0.01;r₂=-0.943,P＜0.01;r₃ =-0.825,P＜0.05）,and protein carbonyl was negative related to ASA in intestine tissue（r=-0.964,P＜0.01）. Path-analysis results indicated that ASA had negative direct effect on MDA content,in serum,in intestine and kidney tissue(P_10.4=-0.327,d_10.4=0.107;P_20.4=-0.389,d_20.4= 0.151;P_30.4=-1.127,d_30.4=1.271).These results indicated that pyridoxine may enhance ASA and increased antioxidative defense in fish.Pyridoxine significantly increased anti-hydroxyl radical activity（AHR） in kidney tissue.The AHR was increased with increasing dietary pyridoxine levels all up to 4.96 mg kg^-1 diet（P＜0.01）,in kidney tissue, and no differences were found with further increase of pyridoxine levels（P＞0.05）.The correlation-analysis results indicated that protein carbonyl was negative related to AHR in kidney tissue（r=-0.981,P＜0.01）.Pyridoxine had no effect on AHR in serum and intestine.Pyridoxine significantly increased the activities of superoxide dismutase（SOD）, catalase（CAT）,glutathione peroxidase（GSH-Px）,glutathione S-transferase（GST） and glutathione reductase（GR） in serum.The activities of SOD,CAT,GSH-Px,GST and GR in serum,were increased with increasing dietary pyridoxine levels all up to 4.96、3.23、3.23、3.23 and 6.32 mg kg^-1 diet（P＜0.01 or P＜0.05）,respectively,and no differences were found with further increase of pyridoxine levels（P＞0.05）.The correlation-analysis results indicated that there were significantly negative correlation between MDA with the activities of SOD,CAT,GSH-Px,GST and GR in serum（r₁=-0.924,P＜0.01;r₂=-0.817, P＜0.05;r₃=-0.968,P＜0.01;r₄=-0.926,P＜0.01;r₅=-0.913,P＜0.05;r₆=-0.824,P＜0.05;r₇=-0.907,P＜0.05）,and there were significantly negative correlation between protein carbonyl and antioxidant enzymes in intestine and kidney tissue(P_10.2=-1.575, d_10.2=2.4819;P_10.3=-0.705,d_10.3=0.4966;P_10.4=-3.402,d_10.4=11.576;P_20.2=-0.159, d_20.2=0.0252;P_20.3=-0.970,d_20.3=0.0252;P_20.4=-0.327,d_20.4=0.107).The effect of pyridoxine on the activites of antioxidative enzymes in intestine and kidney have similar pattern with serum.These results indicated that pyridoxine may enhance the activites of SOD,CAT,GSH-Px,GST and GR,thus increased antioxidative defense systemes in fish. Pyridoxine significantly increased reduced glutathione（GSH） in serum,intestine and kidney tissue（P＜0.01）.Regression analysis results indicated that pyridoxine concentration≥4.78,4.77 and 5.41 mg kg^-1,respectively,in serum,intestine and kidney tissues could achieve the requirement of GSH.Correlation-analysis results indicated that there was significant negative correlation between MDA with the GSH content in serum in serum（r₁=-0.907,P＜0.05;）.In intestine and kidney tissue,correlation-analysis results indicated that levels of GSH were negative correlated with MDA and protein carbonyl contents in intestine and kidney tissue(r₁₁=-0.861,P＜0.05;r₁₂=-0.865,P＜0.05;r₂₁= -0.930,P＜0.01;r₂₂=-0.943,P＜0.01).These results suggested that pyridoxine may increase GSH content,and promoted non-enzymes antioxidative ability in fish.Results indicated that the diet which pyridoxine deficient results in the increased mortality,tetany,convulsions,epiletiform fits and other nervous disorders;exophthalmus, hemorrhage of fins,dissipated in the skin of head and hyperplasia of gill filaments,etc.. Clinic anatomises showed that tumescence or atrophy in the head kidney,definite kidney, spleen,hepatopancreas and gallbladder of fish fed diet lack of pyridoxine.Reduced vacuolization,cytolysis in hepatocytes;deposited siderous heme of head kidney; degeneration of proximal tubules in kidney;decreased lymphoid cells of spleen;intestinal villus were ruptured and degeneration of myocardial fiber in this experiment.In considering these results,pyridoxine can decrease lipid peroxidation and oxidative damage to proteins in Jian carp.Pyridoxine promoted fish antioxidant capacity by removing superoxide anion and lipid peroxidative product in organism,intestine and kidney.Pyridoxine also decreased the ability of kidney to produce hydroxy radical.The optimum dietary pyridoxine requirement for the levels of malondialdehyde（MDA） in serum,intestine and kidney tissue of juvenile Jian carp（11～75 g） was determined to be 3.48,4.72 and 3.44 mg/kg,respectively,and the optimum dietary pyridoxine requirement for the protein carbonyls in intestine and kidney tissue of juvenile Jian carp（11～75 g） was determined to be 5.91 and 6.27 mg/kg,respectively.Fish fed pyridoxine-deficient diets performed increased mortality,tetany,convulsions,epiletiform fits and other nervous disorders;exophthalmus,hemorrhage of fins,dissipated in the skin of head and hyperplasia of gill filaments,etc..The pathologic changes of tissue and cell trauma such as hepatopancreas,head kidney,kidney,spleen,intestinal and fiber were observed.更多还原