【作者】 王永侠；

【导师】 占秀安；

【作者基本信息】 浙江大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 本课题研究了新型有机硒源-硒代蛋氨酸(Se-Met)及其不同构型对肉鸡生长性能、饲料利用和肉品质的影响,并从硒沉积、抗氧化和免疫功能、相关激素的合成与分泌、关键代谢酶活力和硒蛋白基因表达等方面探讨其主要作用机理,旨在阐明Se-Met对肉鸡的生物学效应,为其开发利用提供科学依据。试验一DL-硒代蛋氨酸的有效性评价本试验采用2因子×4水平完全随机设计,同比SS,研究DL-Se-Met作为硒源对肉鸡的有效性及最适添加量。选用7日龄Ross 308商品代公母混合雏840只,按照饲养试验要求分为7组,每组120只(含3个重复,每重复40只)。对照组为不加硒的基础日粮组(硒含量实测值0.04 mg/kg),试验组分别在基础日粮中添加0.05、0.15、0.25 mg Se/kg的亚硒酸钠(SS)和DL-Se-Met。试验为期42天。自由采食和饮水。饲养试验结束后,每重复选体重相近公鸡4只,进行屠宰试验,并取样分析。结果表明：(1) DL-Se-Met是有效的硒源补充剂,最适添加水平为0.15mg Se/kg。(2)同比SS, DL-Se-Met显著提高了饲料转化率(P<0.05),但对日增重和采食量均无显著影响(P>0.05)；提高了肉鸡宰后16h肌肉的红色色度Hunter a值和降低了24 h、48 h肌肉滴水损失率(P<0.05)；提高了血清和组织硒含量(P<0.05)；增高了血清和组织T-AOC和GSH含量及肌肉SOD活力,减低了组织MDA含量及肌肉和胰脏GPx活力(P<0.05)；提高了胸腺指数和血清IgA、IgG、IgM含量(P<0.05)；提高了血清TT3浓度并降低了血清TT4/TT3比值(P<0.05)。试验二硒代蛋氨酸的构效研究本试验比较研究Se-Met同分异构体对肉鸡的生物学活性。选用1日龄Ross308商品代公母混合雏800只,按照饲养试验要求分为4组,每组200只(含5个重复,每重复40只)。4个处理组分别为SS对照组,D-、L-、DL-Se-Met试验组,均在基础日粮(硒含量实测值0.04 mg/kg)中添加0.15 mg Se/kg。试验为期42天。自由采食和饮水。饲养试验结束后,每重复选体重相近公鸡4只,进行屠宰试验,并取样分析。结果表明：(1)同比SS,不同构型Se-Met对肉鸡生长性能和饲料利用均无显著影响(P>0.05),但显著降低宰后胸肌24h、48 h滴水损失率和提高血清和组织硒含量(P<0.05)。(2)同比SS,L-和DL-Se-Met均显著提高了血清和肝脏GSH含量、胰脏和肌肉T-AOC及降低了组织MDA含量(P<0.05),升高了血清IgG含量(P<0.05),提高了血清TT3浓度和降低了血清TT4/TT3比值(P<0.05),提高了胰脏蛋白酶活力(P<0.05),降低了肝肾TrxR1活力(P< 0.05); D-Se-Met则显著提高了血清GSH含量和降低了肌肉MDA含量(P<0.05),降低了肝肾GPx1和TrxR1活力及血清和肝脏SelP含量(P<0.05)。(3)互比Se-Met构型,DL型较L型显著提高了胰脏T-AOC,血清GSH、IgA和IgG含量,血清、肾脏和肌肉硒含量,而降低了肝脏MDA含量(P<0.05)；而D型较L、DL型降低了肝脏GPx1活力及GSH含量、肌肉和胰脏T-AOC、胰脏淀粉酶和蛋白酶活力(P<0.05)。(4)同比SS,不同构型Se-Met均显著提高了肝肾GPx1、TrxR1和SelP及肌肉SelW mRNA丰度,并皆呈DL> D> L型(P<0.05)。(5)首次成功克隆了鸡GPx1基因cDNA部分序列,长度为416 bp。试验三硒代蛋氨酸体外抗氧化能力研究本实验采用鸡胚肾细胞原代培养,同比SS,研究不同构型Se-Met的体外抗氧化功能。结果表明：(1)同比SS,不同构型Se-Met均提高了肾细胞SOD、GSH和T-AOC及降低了MDA (P<0.05),而L和D-Se-Met降低了GPx活力(P<0.05)。(2)互比Se-Met构型,DL型较L型显著提高了肾细胞GSH含量(P<0.05),但D型较L、DL型降低了SOD和GPx活力及T-AOC水平,而增高了MDA含量(P<0.05)。试验四原代培养鸡胚肾细胞氧化应激模型建立实验成功建立了体外鸡胚肾细胞氧化应激模型,通过研究表明H202对原代培养鸡胚肾细胞产生氧化应激的最适浓度和作用时间分别为100μmol/L和12h。试验五硒代蛋氨酸对肾细胞氧化损伤的保护作用研究本试验采用4因子×7水平+1因子完全随机设计,研究SS和L、D、DL-Se-Met4种硒源对H202诱导损伤肾细胞的保护作用。结果表明：氧化应激使细胞抗氧化能力下降,表现为细胞GPx活力、T-AOC和GSH水平降低而MDA含量增高(P<0.05)：而Se-Met和SS均可减弱H202诱导的氧化损伤程度,就此同比正常对照组,DL-和L-Se-Met组抗氧化指标能恢复到与其同等水平,而D-Se-Met组却显著降低了GPx活力和T-AOC水平(P<0.05),SS也显著降低了SOD活力、T-AOC和GSH水平,提高了MDA含量(P<0.05)；与此同时,DL-Se-Met较L-Se-Met还显著提高了细胞GSH含量(P< 0.05), D-Se-Met较SS升高了T-AOC和GSH水平,降低了MDA含量(P<0.05)。上述结果提示：(1)不同构型Se-Met均是肉鸡有效的硒源补充剂,DL-Se-Met最适添加水平为0.15 mg Se/kg;(2)同比SS, DL和L-Se-Met可显著增加机体硒沉积量,提高抗氧化系统和免疫功能,促进甲状腺激素和关键代谢酶分泌,达到显著提高肉品质并有改善生长和饲料转化率的功效,D-Se-Met也具有促进硒沉积、增强抗氧化和提高肉品质的功能；(3)在抗氧化系统功能方面,总体趋势是Se-Met优于SS,且呈DL>L>D型。更多还原

【Abstract】 This research was conducted to investigate the effects of a novel organic selenium (Se) source-selenomethionine (Se-Met) and its different forms on growth performance, feed utilization and meat quality in broilers, and approached to the mechanism of effects by Se deposition, antioxidant ability, immunity, hormone level, key metabolism enzymes activities and selenoprotein gene expression, consequently to provide a theoretical basis for the industrialization and scientific application of Se-Met. Five experiments were conducted in this paper.Experiment 1 was conducted in a 2×4 factorial arrangement in a randomized complete block design to investigate the effects of DL-Se-Met on broilers and its optimum supplemented level, sodium selenite (SS) served as control. A total of 840 7-day-old Straight-run Ross 308 broilers with an average body weight of 162±0.59 g were randomLy allotted to seven treatments, each of which included three replicates of 40 birds. There were a control diet containing 0.04 mg Se/kg and six additional diets that included 0.05,0.15,0.25 mg Se/kg from SS or DL-Se-Met. The experiment lasted 42 days. Feed and water were provided ad libitum throughout the experiment. At 49 days of age, four male broilers per replicate were randomLy selected based on the similar body weight for slaμghter experiment, and serum, liver, kidney, pancreas and breast muscle were sampled for laboratory analysis. The resμlts showed that:(1) DL-Se-Met coμld act as an effective Se source in broilers, and the optimum supplemented level was 0.15 mg Se/kg.(2) Compared with SS, DL-Se-Met significantly increased the feed utilization (P < 0.05). The Hunter a value of breast muscle at 16 h was increased and the drip loss of breast muscle at 24 and 48 h was reduced by DL-Se-Met supplementation in comparison with SS supplementation (P< 0.05). The Se concentration in serum and organs was significantly higher in the DL-Se-Met-treated group than that in the SS-treated group (P< 0.05). DL-Se-Met supplementation significantly increased total antioxidation capability (T-AOC) and glutathione (GSH) content in serum and tissues, superoxide dismutase (SOD) activity in breast muscle, while decreased malondialdehyde (MDA) content in tissues and glutathione peroxidase (GPx) activity in breast muscle and pancreas, compared with broilers fed SS diet (P< 0.05). The thymus index and serum IgG, IgA, IgM contents were greatly improved by DL-Se-Met in comparison with SS (P< 0.05). The addition of DL-Se-Met significantly increased TT3 concentration and decreased the ratio of TT4 to TT3 (P< 0.05), when compared with broilers fed SS diet.Experiment 2 was conducted to compare the effects of different forms of Se-Met on broilers, SS served as control. A total of 8001-day-old Straight-run Ross 308 broilers with an average body weight of 44.30±0.49 g were randomLy allotted to four treatments, each of which included five replicates of 40 birds. These four groups received the same basal diet containing 0.04 mg Se/kg, supplemented with 0.15 mg Se/kg from SS, L-Se-Met, D-Se-Met or DL-Se-Met. The experiment lasted 42 days. Feed and water were provided ad libitum throμghout the experiment. At 42 days of age, four male broilers per replicate were randomLy selected based on the similar body weight for slaμghter experiment, and serum, liver, kidney, pancreas and breast muscle were sampled for laboratory analysis. The resμlts showed that:(1) Average daily gain, average daily feed intake and feed conversion ratio were not affected by the dietary treatments in any period of growth or in the overall data (P>0.05).(2) Se-Met groups were more effective in decreasing drip loss of breast muscle at 24 and 48 h after slaμghter and depositing Se in serum and different organs studied than SS group (P< 0.05).(3) The antioxidant status was greatly improved in broilers of DL-and L-Se-Met-treated groups in comparision with the SS-treated group and was illuminated by the increased GSH concent in serum and liver, T-AOC in pancreas and breast muscle and decreased MDA concent in tissues of broilers (P< 0.05). Besides, supplementation with D-Se-Met was more effective in increasing serum GSH concent and decreasing breast muscle MDA concent than SS (P< 0.05). When compared with broilers fed SS diet, supplemental DL-and L-Se-Met significantly increased serum IgG content and trypsinase activity (P< 0.05). The TT3 concentration was increased and the ratio of TT4 to TT3 was reduced by DL-and L-Se-Met compared with SS (P< 0.05). The SS supplemented diet increased more cellμlar glutathione peroxidase (GPxl) and thioredoxin reductase 1 (TrxRl) activities in liver and kidney as well as selenoprotein P (SelP) content in serum and liver than the D-Se-Met supplemented diet (P< 0.05). In addition, liver and kidney TrxRl activities were significantly decreased by DL-and L-Se-Met in comparison with SS (P< 0.05).(4) Compared with L-Se-Met, DL-Se-Met significantly increased T-AOC in pancreas, GSH, IgA and IgG contents in serum, Se concentration in serum, kidney and breast muscle, while decreased MDA content in liver (P< 0.05). GPx1 activity and GSH content in liver, T-AOC in pancreas and breast muscle as well as amylase and trypsinase activites in pancreas were significantly decreased by D-Se-Met supplementation in comparison with L-and DL-Se-Met supplementation (P< 0.05).(5) In the aspects of GPx1> TrxR1、Se1P and selenoprotein W (SelW) relative mRNA level, DL-Se-Met was superior to D-Se-Met (P< 0.05); D-Se-Met was superior to L-Se-Met (P< 0.05); L-Se-Met was superior to SS (P< 0.05).(6) Partial cDNA sequence of chicken GPx1 gene was first successfμlly cloned by using RT-PCR method, the sequence was 416 bp.Experiment 3 was conducted to study the antioxidant ability of different forms of Se-Met in vitro by primary cultured kidney cell of chicken embryo, SS served as control. The resμlts showed that:(1) The T-AOC, GSH content and SOD activity were greatly improved and MDA content was notably decreased in broilers fed Se-Met compared with those in broilers fed SS (P< 0.05); while GPx activity was significantly decreased by DL-and L-Se-Met in comparison with SS (P< 0.05).(2) Compared with D-Se-Met, L-and DL-Se-Met significantly increased SOD and GPx activities, T-AOC and decreased MDA content (P< 0.05); GSH content in DL-Se-Met group was significantly higher than that in L-Se-Met group (P< 0.05).Experiment 4 was conducted to investigate the effects of different hydrogen peroxide (H2O2) contentrations and exposed time on relative cell viability and lactate dehydrogenase activity in medium of cultured chicken embryo kidney cell. The resμlts showed that oxidative stress of kidney cells of chicken embryo coμld be induced, when they were treated with 100μmol/L H2O2 for 12 h.Experiment 5 was conducted in a 4×7+1 factorial design to investigate the anti-oxidative stress effects of SS and different Se-Met forms on kidney cells damaged by H2O2. The resμIts showed that:Oxidative stress decreased the antioxidant status of kidney cells, GPx activity, T-AOC and GSH content decreased, while MDA content increased (P< 0.05); Se-Met and SS supplementation coμld attenuate H2O2-induced stress injury in kidney cells, but when compared with the normal control group, DL-and L-Se-Met coμId restore the activities of antioxidants to the normal level, while D-Se-Met significantly decreased GPx activity and T-AOC (P< 0.05), SS significantly decreased SOD activity, T-AOC and GSH content as well as increased MDA content (P< 0.05). In addition, DL-Se-Met increased more GSH content than L-Se-Met (P< 0.05); D-Se-Met supplementation significantly increased T-AOC, GSH content and increased MDA content compared with SS (P< 0.05).The resμlts of this study indicated:(1) Se-Met coμld act as effective organic sources of Se supplementation of feed mixtures for broilers, and the optimum supplemented level was 0.15 mg Se/kg for DL-Se-Met; (2) DL-and L-Se-Met had higher nutrient-biological effects than SS in the aspects of meat quality, Se deposition, antioxidant ability, immunity, hormone level and key metabolism enzymes activities; while D-Se-Met possessed higher nutrient-biological effects than SS in the aspects of meat quality, Se deposition and antioxidant ability; (3) With respect to antioxidant ability, the tendency was that:Se-Met was superior to SS, while DL-Se-Met> L-Se-Met> D-Se-Met.更多还原