【作者】 金明昌；

【导师】 汪开毓；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2007， 博士

【摘要】 本研究采用纯合日粮，系统研究了幼鲤硒的缺乏症、需要量；系统研究了幼鲤硒缺乏情况下，自由基、抗氧化酶系统、肝细胞凋亡之间的关系；系统研究了硒缺乏与免疫的关系；从分子水平研究了不同水平硒对幼鲤类胰岛素样生长因子(IGF-Ⅰ)、谷胱甘肽过氧化物酶(GPx)基因表达的影响。研究包括四个试验：试验一：选择体重12.6±0.8克的健康幼鲤360尾，随机分为4组，饲喂分别添加硒(以亚硒酸钠形式，AR)0、0.15、0.30、0.45mg／kg硒的4种饵料，经30周试验，对幼鲤硒缺乏进行了系统的组织病理学、血液学的研究。结果表明：幼鲤硒缺乏会导致发病和死亡，且与饵料中硒含量呈负相关，各组发病率分别为46.7％、33.3％、13.3％和0；死亡率分别为26.7％、16.7％、6.7％和0；病鱼表现特征性的“瘦背病”和脊柱弯曲症状。病理剖解，可见背部两侧肌肉萎缩变薄，呈刀刃状，体侧红肌纤维退色变白；肝脏和脾脏肿大、充血、出血。组织学上，最突出的变化表现为营养性肌病、营养性肝病、胰腺的变性、坏死等；骨骼肌纤维变性、坏死，残存的肌纤维萎缩变细；心肌纤维细胞肿胀、变性，肝细胞变性、坏死，甚至发生溶解消失；胰腺腺泡变性、坏死；脾脏出血，淋巴细胞大量减少，巨噬细胞增多形成黑素巨噬细胞中心；肾小管上皮细胞肿胀、变性；神经元细胞肿胀，变性，偶尔可见噬神经现象的出现。超微结构上，表现为骨骼肌纤维、肝细胞和肾小管上皮细胞线粒体肿胀，嵴断裂，甚至发生溶解，整个线粒体囊泡化；脾脏细胞出现广泛性的充血，水肿：淋巴细胞坏死。血液学上，红细胞数量、血红蛋白含量和IgM抗体水平都降低，白细胞数量和红细胞脆性增加，血清丙二醛(MDA)含量升高；幼鲤血清中谷草转氨酶(AST)、谷丙转氨酶(ALT)和乳酸脱氢酶(LDH)活性升高，而谷胱甘肽过氧化物酶(GPx)、超氧化物酶歧化酶(SOD)、过氧化氢酶(CAT)活性降低。试验二：选择体重15.5±0.7克的健康幼鲤270尾，随机分为3组，饲喂分别添加硒(以亚硒酸钠形式，AR)0、0.25、0.50mg／kg的3种不同饵料，试验期16周。分别于试验第4、8、16周，采血和取肝脏进行NO、NOS测定和肝细胞凋亡检测。结果表明：硒缺乏可导致幼鲤血清和肝脏中NOS活性显著增强，NO含量显著升高(P＜0.05)。硒缺乏可导致幼鲤肝细胞凋亡，其特征表现为肝细胞染色质浓缩，边移，呈半月状，在浓缩的基础上发生裂解，呈大小不等的小团块，并可见凋亡细胞形成具有特征性的、质膜完整的凋亡小体。随着试验时间的延长，肝细胞凋亡率增高。幼鲤硒缺乏导致肝细胞的凋亡由NO介导。试验三：选择体重10.4±0.4克的健康幼鲤1200尾，随机分为10组，每组3个重复，每个重复40尾，分别饲喂添加硒(以亚硒酸钠形式，AR)0、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9 mg／kg的10种不同饵料，试验期90d。结果表明：随着饵料中硒添加水平的增加，幼鲤的增重和成活率提高，饵料系数降低，添加硒0.4mg／kg时，幼鲤生长性能最佳(P＜0.05)：添加0.3～0.5 mg／kg硒，可显著提高幼鲤头肾和脾脏重及其体指数(P＜0.05)，增加血液红细胞数量，降低白细胞数量，提高血清IgM水平和攻毒后成活率、血清溶菌酶活力及血清凝集抗体效价(P＜0.05)，从而增强幼鲤免疫功能和对疾病的抵抗力；同时，显著提高血清和肝脏中GPx活力(P±0.05)；血清和组织的硒含量(P＜0.05)。幼鲤体增重、饵料系数、头肾重量、脾脏重量、血清溶菌酶活力和IgM抗体水平与饵料硒添加量呈显著的二次曲线相关。根据上述指标确定的最适硒需要量为0.434～0.517mg／kg(分别为0.517、0.499、0.434、0.447、0.480、0.464mg／kg)。试验四：选择体重15.3±0.8克的健康幼鲤160尾，随机分为4组，每组2个重复，每个重复20尾，分别饲喂添加硒(以亚硒酸钠形式，AR)0、0.15、0.30和0.45mg／kg的4种不同饵料，试验期8周。采用反转录一聚合酶链式反应(RT-PCR)对肝脏中的IGF-Ⅰ和GPx mRNA的表达进行测定。结果表明：饵料中添加硒对肝脏的IGF-Ⅰ和GPxmRNA的表达有促进作用；随着饵料硒水平的提高，IGF-Ⅰ和GPX mRNA的表达量也呈显著增加趋势(P＜0.05)。更多还原

【Abstract】 Four experiments were conducted to study Se requirement and deficiency symptom and molecular mechanism in juvenile carps with purified diets.Experiment 1: 360 juvenile common carps（Cyprinus carpio） with initial body weight 12.6±0.8g were randomly divided into 4 groups with triplicate and 30 carps per replicate, and were fed purified diets supplemented with selenium（Se, NaSeO₃, AR） 0, 0.15, 0.30, 0.45 mg/kg respectively for 30 weeks. By establishing experimental model of carps with selenium deficiency, the pathology and hematology were studied. The results indicated that the morbidity and mortality were 46.7%, 33.3%, 13.3%, 0 and 26.7%, 16.7%, 6.7%, 0 respectively.The carps fed Se deficient diet showed thin back disease and lordosis. Anatomical pathology showed that the muscle at the both side of the carps back became thin and looked like knife blade, muscular dystrophy and white muscle. The liver and spleen were swollen with hyperemia and hemorrhage. The examination of histopathology showed nutritional muscular dystrophy, hepatosis, degeneration and necrosis of pancreas. The skeletal muscles showed degeneration, necrosis and muscular atrophy. The cardiac muscle fibers showed swelling and necrosis. The cardiac muscles showed cellular swelling and degeneration. The hepatocyte showed degeneration and necrosis, even disappeared. The pancreas follicle showed degeneration and necrosis. The spleen showed haemorrhage, lymph cells were reduced, phagocyte increased and formed black phagocyte centre. The renal tubular epithelial cells showed swollen and deteriorative. The neuron cells showed swollen and deteriorative nerve was observed occasionally. In ultra-structure, the mitochondria of the skeletal muscle fibers, liver cells and renal tubular epithelial cells showed swelling, broken back, even disappeared, shaped follicles. The spleen cells showed hemorrhage and dropsy, lymph cells were necrosis. In hematology, erythrocyte haemoglobin amount and IgM were reduced, leukocyte amount and erythrocyte fragility increased, MDA, AST, ALT and LDH in serum increased, but the activities of GPx, SOD and CAT were reduced.Experiment 2: 270 juvenile common carps（Cyprinus carpio） with initial body weight 15.5±0.7g were randomly divided into 3 groups with triplicate and 30 carps per replicate, and were fed purified diets supplemented with selenium（Se,NaSeO₃, AR） 0, 0.25, 0.50 mg/kg respectively for 16 weeks. NO content, NOS activities in serum and liver and hepatocyte apoptosis were studied at 4th, 8th, 16th week respectively. The results showed that Se deficiency increased NOS activity and NO content in serum and liver（P<0.05）. Se deficiency induced hepatocyte apoptosis. The characteristics of apoptotic hepatocyte showed that the chromatin of hepatocyte concentrated and moved to one side, and looked like half-moon shape and then cracked, the characteristic apoptosis bodies with the complete cytoplasm membrane were seen. The hepatocyte apoptosis percentage increased with the test time. The hepatocyte apoptosis induced by Se deficiency was mediated by NO free radicle in juvenile carps.Experiment 3: 1200 juvenile common carps（Cyprinus carpio） with initial body weight 10.4±0.4g were randomly divided into 10 groups with triplicate and 40 carps per replicate, and were fed diets supplemented with selenium（Se,NaSeO₃, AR） 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 mg/kg respectively for 90 days.The results showed that as Se supplemented was raised,the weight gain and survival rate of carps increased, feed conversion efficiency were reduced, and the performance was best in the group with 0.4 mg/kg Se（P<0.05）. When the diets were supplemented with 0.3～0.5 mg/kg Se, head kidney weight: body weight and spleen weight: body weight of carps were higher（P<0.05）, blood red cell amount increased and leukocyte amount decreased, survival rate and serum lysozyme activity 15d after vaccination against Aeromonas hydrophila increased significantly（P<0.05）, valence of agglutinating antibody 17d after vaccination against Aeromonas hydrophila increased.Thus the immune function and resistance to disease of juvenile carps were improved. GPx activities in serum and liver increased significately（P<0.05）. Se contents in blood and tissue were also raised significately（P<0.05）. The relationship between Se and the weight gain, feed conversion efficiency, head kidney weight, spleen weight, serum lysozyme activity, IgM showed the remarkable quadratic curve relation. The most suitable Se requirement was 0.517, 0.499, 0.434, 0.447, 0.480, 0.464 mg/kg respectively according to the upper indices.Experiment 4: 160 juvenile common carps（Cyprinus carpio） with initial body weight 15.3±0.8g were randomly divided into 4 groups with 2 replicates and 20 carps per replicate, and were fed purified diets supplemented with selenium（Se,NaSeO₃, AR） 0,0.15, 0.3 and 0.45 mg/kg respectively for 8 weeks. The IGF-ⅠmRNA and GPx mRNA in the liver of carps were determined with RT-PCR. The results showed that the dietary Se increased IGF-ⅠmRNA and GPx mRNA expression significantly（P<0.05）, as Se supplemented was raised, IGF-ⅠmRNA and GPx mRNA expression increased also.更多还原