【作者】 何敏；

【导师】 汪开毓；

【作者基本信息】 四川农业大学 ， 预防兽医， 2009， 博士

【摘要】 维生素E在动物体内具有广泛的生理功能,如促生长、改善肉质、抗氧化作用、提高繁殖力、增强免疫力等等。目前维生素E在水生动物机体内的作用及在水生动物饲料中的最适添加量已成为研究的重点之一。斑点叉尾鮰为淡水温水性鱼类,现已经成为我国淡水养殖的主要品种之一,该鱼具有易饲养、肉质鲜嫩、起捕率高、易加工等特点。近年来,国内外对斑点叉尾鮰的研究主要集中在育苗繁殖生物学和病害防治方面,而关于维生素E对斑点叉尾鮰生长性能、免疫功能及肉质等方面的影响至今未见报道。本试验首次系统研究了饲料中不同含量维生素E对斑点叉尾鮰生长性能、消化酶活性、消化道形态结构、肌肉品质、血液生理生化指标、血清抗氧化系统和非特异性免疫功能等方面的影响,并对其机理进行了初步探讨;从分子水平研究了不同添加量维生素E对斑点叉尾鮰血清中IL-2含量的影响,以期为今后开展相关研究和进一步推进斑点叉尾鮰无公害产业化生产积累资料。研究包括七个试验:试验一:试验选取900尾健康的斑点叉尾鮰,平均体重（5.00±0.50）g,随机分成4个处理组(Ⅰ、Ⅱ、Ⅲ和Ⅳ处理依次分别添加维生素E 0、50、100和1000 IU·kg^-1饲料),每个处理3个重复,每个重复75尾。试验期105d。试验结果显示:在一定添加水平内,维生素E能够显著提高斑点叉尾鮰成活率、特定生长率（SGR）、蛋白质效率和摄饵量（P＜0.01）。当维生素E添加量为到100IU·kg^-1时,以上四个指标均达最高,分别为97.78±0.96%,2.99±0.50%,163.71±5.62%和7478.00±62.01 g/尾,均极显著高于维生素E添加量为0IU·kg^-1组（P＜0.01）;维生素E显著影响斑点叉尾鮰胃肠道、肝胰脏蛋白酶和脂肪酶活性（P＜0.01 or P＜0.05）。100 IU·kg^-1添加组胃蛋白酶和脂肪酶活性最高,分别达29.33±1.66 U·min^-1·mg^-1,196.52±17.28 U·min^-1·mg^-1,极显著高于0 IU·kg^-1添加组（P＜0.01）和显著高于1000 IU·kg^-1添加组（P＜0.05）。结果表明,维生素E在一定添加水平内能显著提高斑点叉尾鮰生长性能和消化酶活性,促进鱼体生长。试验二:应用组织学、免疫组织化学和图像分析技术,探讨饲料中不同含量维生素E对斑点叉尾鮰肠皱襞高度、肠粘膜厚度、粘液细胞数量、生长抑素（SS）表达的影响。结果显示:在一定添加水平内,维生素E能显著提高斑点叉尾鮰肠皱襞高度、肠粘膜厚度、粘液细胞数量、SS的表达强度及阳性细胞数量（P＜0.01orP＜0.05）。当维生素E添加量为100IU·kg^-1时,以上指标均达最高,显著高于维生素E添加量为0IU·kg^-1组（P＜0.01or P＜0.05）。但与添加50IU·kg^-1和1000 IU·kg^-1组差异不显著（P＞0.05）。当维生素E添加量达到1000 IU·kg^-1时,各指标似乎均有下降趋势,但与50 IU·kg^-1和100 IU·kg^-1组差异不显著（P＞0.05）。试验三:试验研究了不同水平维生素E对斑点叉尾鮰肌肉品质的影响,并对其作用机理进行了初步探讨。试验结果显示:维生素E能显著影响斑点叉尾鮰肉质的感官评定指标、物理特性和营养成分。维生素E可改善斑点叉尾鮰肉质气味、色泽、质地、风味等感官评定指标,提高斑点叉尾鮰肌肉中粗蛋白、肌苷酸和鲜味氨基酸的含量,并降低丙二醛含量。实验结果同时显示,维生素E能提高斑点叉尾鮰肌肉组织中胶原蛋白的含量。其对胶原蛋白含量的影响规律和对肌原纤维耐折断力的影响相同,而对失水率的影响相反。肌原纤维耐折断力、失水率与胶原蛋白含量分别是正相关和负相关关系,其回归直线分别为y=1.1506+0.5188x（R²=0.9851）和y=27.375-0.4385x（R²=0.9525）。试验四:本试验通过在基础日粮中添加不同水平的维生素E,以探讨其对斑点叉尾鮰血液生理生化指标的影响。结果显示:在一定添加水平内,维生素E对斑点叉尾鮰血液的生理生化指标具有显著或极显著影响（P＜0.01or P＜0.05）。当维生素E添加量为1OOIU·kg^-1时,红细胞（RBC）、血红蛋白（Hb）、白蛋白（ALB）、球蛋白（GLO）、甘油三酯（TG）、胆固醇（TC）、谷草转氨酶（AST）、谷丙转氨酶（ALT）、尿素氮（BUN）、碱性磷酸酶（AKP）、丙二醛（MDA）等各指标均使鱼体达最佳状态,其值与0IU·kg^-1添加组差异显著或极显著（P＜0.01or P＜0.05）。但与50 IU·kg^-1和1000 IU·kg^-1添加组差异不显著（P＞0.05）。试验五:利用单因素实验设计研究了在饲料中添加不同水平维生素E对斑点叉尾鮰血清抗氧化系统中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）、谷胱甘肽转移酶（GST）等4种抗氧化酶活力的影响。结果显示:在一定添加水平内,维生素E对斑点叉尾鮰血清中超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽转移酶（GST）的活力有显著影响（P＜0.05）,但对谷胱甘肽过氧化物酶（GSH-Px）的影响不显著（P＞0.05）。试验六:在斑点叉尾鮰饲料中添加不同水平的维生素E,通过测定实验鱼的免疫器官重量、白细胞吞噬活性、吞噬细胞杀菌指数、血清溶菌酶含量、补体C3含量和攻毒成活率,比较不同添加量的维生素E对斑点叉尾鮰非特异性免疫功能的影响。结果显示:在一定添加水平内,维生素E能极显著影响斑点叉尾鮰头。肾体指数、脾体指数、白细胞吞噬活性、吞噬细胞杀菌指数、血清溶菌酶含量、补体C₃含量、攻毒成活率（P＜0.01）,但维生素E对斑点叉尾鮰中肾体指数等指标无显著影响（P＞0.05）。试验七:利用RT-PCR技术扩增出斑点叉尾鮰血清IL-2的部分基因序列并进行克隆,构建重组质粒作为标准阳性模板,从而建立斑点叉尾鮰IL-2基因的荧光定量PCR检测方法。应用该方法对不同维生素E添加试验组中的斑点叉尾鮰IL-2含量进行测定。结果显示:在一定添加水平内,维生素E能显著提高斑点叉尾鮰IL-2的含量（P＜0.05）,增强斑点叉尾鮰免疫力。综合试验结果表明:维生素E在一定添加量内可通过促进消化器官生长发育,增强消化酶活性,提高肠道吸收能力从而提高斑点叉尾鮰生产性能和饲料利用率;维生素E可起到改善斑点叉尾鮰肉质品质的作用;维生素E可提高斑点叉尾鮰血清抗氧化系统的抗氧化能力及非特异性免疫力。更多还原

【Abstract】 It is discovered that vitamin E is a substance in animal body with general physiological functions,such as growth promoting,improving meat quality,anti-oxidization,promoting the reproductive ability and enhancing immune function,etc.At present,the function and the most suitable dosage of vitamin E for the aquatic animals would be the highlights of our researches.Channel catfish,a kind of fresh water fish living in warm water,has become one of main variety in our fresh water aquaculture in China with the characteristics of easy to raise and process, tender meat.In recent years,the researches of channel catfish at home and abroad were mainly focusing on the reproductive biology and disease control,with few reports about the effect of vitamin E on growth performance,immune function and meat quality of channel catfish.The effect of vitamin E in different dosage in feed to Channel Catfish（Ietalurus punetaus Rafinesque） was studied on such fields as the growth performance,the activity of digestive enzymes,morphostructure of alimentary tracts,meat quality,physiological and biochemical indexes of blood,anti-oxidization system in the serum and nonspecific immunity, etc.,and initially discusses the relative mechanism.The effect of Vitamin E in different dosage to the content of IL-2 in the serum of Channel Catfish was studied in molecular level,in order to enrich the experimental data on relative researches in the future and furthering the marketing of harmless channel catfish production.This research included 7 experiments as following:Experiment 1:The experiment was conducted to determine the effects of vitamin E on the growth performance and activities of digestive enzymes of Channel Catfish,Ictalurus punctatus Rafinesque,and probe into mechanism tentatively.900 healthy Channel Catfish（5.00±0.50g of average weight）were randomly divided into four groups,each group included three replicates of 75 Channel Catfish.GroupⅠwas supplemented with 0 IU·kg^-1 vitamin E,another three treatments were supplemented consecutively with 50 IU·kg^-1 vitamin E for groupⅡ,100 IU·kg^-1 vitamin E for groupⅢ,1000 IU·kg^-1 vitamin E for groupⅣ.The experiment lasted for 105 days.The result showed that vitamin E could improve the survival rate,special weight growth rate（SGR）,protein efficiency ratio and feed quantity of Channel Catfish notably（P＜0.01）.When vitamin E were 100 IU·kg^-1 diet,the fish had the maximal survival rate,special weight growth rate,protein efficiency ratio and feed quantity（97.78±0.96 %,2.99±0.50%,163.71±5.62%and 7478.00±62.01 g/tail）,higher than the vitamin E adding amount for groupⅠextremely notably （P＜0.01）.What’s more,vitamin E had significant effect on protease, lipase in the gastrointestinal tract and hepato-pancreas of Channel Catfish（P＜0.01 or P＜0.05）.Vitamin B were 100 IU·kg^-1 diet,Channel Catfish had the maximal the activities of protease,lipase(29.33±1.66 U·min^-1·mg^-1,196.52±17.28 U·min^-1·mg^-1),higher than the vitamin E adding amount for groupⅠextremely notably（P＜0.01）and higher than groupⅣnotably（P＜0.05）.Therefore,these results indicate that vitamin E could improve the growth,activities of digestive enzymes of Channel Catfish in a certain level.Experiment 2:The effect of vitamin E in different dosage on the height of intestinal fold,the thickness of intestinal mucosa,the number of mucous cells and the expression of SS of Channel Catfish was studied by the methods of histology,immunohistochemistry and graphical analysis. The test results demonstrated that vitamin E could significantly increase the height of intestinal fold,the thickness of intestinal mucosa,the number of mucous cells,the expression of SS and the number of positive cells（P＜0.01or P＜0.05） in a certain level.When the amount of vitamin E reached to I00 IU·kg^-1,these four indexes above came to the highest, which were extremely higher than those in groupⅠ（P＜0.01or P＜0.05）, while not significant with groupⅡandⅣ（P＞0.05）.When the amount of vitamin E reached to 1000 IU·kg^-1,these indexes in groupⅣhave tended to decline while not significant with groupⅡandⅢ（P＞0.05）.Experiment 3:The effect of vitamin E in different dosage on the quality of meat of Channel Catfish was studied,and the relative mechanism was initially discussed.The test results of meat quality indicated that vitamin E could affected the proper index which affects the quality of meat,physical characteristics and nutrient content of Channel Catfish, such as improving the scent,color,texture and flavor of meat,raising the content of crude protein,inosinic acid and delicious substance of meat,and decreasing the content of malondialdehyde.The results also showed that vitamin E could increase the content of collagen in muscular tissue of Channel Catfish.The influence law of vitamin E to the content of collagen was the same as that to the break-resisting force of myofibril, while contrast to the rate of water loss.The break-resisting force of myofibril was positively associated with the content of collagen,while the rate of water loss have a negative correlation with the content of collagen,with the regression lines as y=1.1506+0.5188x（R²=0.9851） and y=27.375-0.4385x（R²=0.9525） respectively.Experiment 4:The effect of vitamin E in different dosage on physiological and biochemical indexes of blood of Channel Catfish was studied.The test results showed that vitamin E could increase or significantly increase the indexes（P＜0.01or P＜0.05） during a given level.When the amount of vitamin E reached to 100 IU·kg^-1,the indexes of RBC,Hb,ALB,GLO,TG,TC,AST,ALT,BUN,AKP,MDA,etc.made sure the fish were at the top of their form,which were significant or extremely significant with those in groupⅠ（P＜0.01or P＜0.05）,while not significant with groupⅡandⅣ（P＞0.05）.Experiment 5:The effect of vitamin E in different dosage on the activity of SOD,CAT,GSH-Px and GST in anti-oxidization system in the serum of Channel Catfish was studied through the single factor experiment. The test results demonstrated that vitamin E could significantly affect the activity of SOD,CAT and GST（P＜0.05） in a certain level,while not significant with GSH-Px（P＞0.05）.Experiment 6:The effects of vitamin E in different dosage on nonspecific immune function of Channel Catfish were compared,by measuring the weight of immune organs,phagocytic activity of white cells, bactericidal index of phagocytic cells,the content of lysozyme in serum and complement C₃ and survival rate after the challenge of toxic substances. The test results of nonspecific immunity indicated that vitamin E could significantly affect the body indexes of head-kidney and spleen, phagocytic activity of white cells,bactericidal index of phagocytic cells,the content of lysozyme in serum and complement C₃ and survival rate after the challenge of toxic substances（P＜0.01） in a certain level, while not significant with the body index of kidney（P＞0.05）.Experiment 7:A part of IL-2 gene of Channel catfish were cloned by RT-PCR and the recombinant plasmid was constructed as the positive pattern plate in order to establish the FQ-PCR method of IL-2 gene of channel catfish.Through this established method,the contents of IL-2 of channel catfish in different test groups were measured.The results of IL-2 tests showed that vitamin E could significantly provoke the secretion of IL-2（P＜0.05）so as to improve the immunity of Channel catfish.Above all,in a certain level,vitamin E could improve the growing development of alimentary organs,boost the activity of digestive enzyme, and raise the absorbency of intestinal tract so that to increase the production performance of channel catfish and efficiency of feed utilization.Vitamin E could not only improve the meat quality of Channel Catfish,but also enhance the nonspecific immunity and the oxidation resistance of anti-oxidization system in serum.更多还原