【作者】 何健；

【导师】 张克英；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2011， 博士

【摘要】 本研究选择以黄曲霉毒素(AF)为主、同时含有多种其他霉菌毒素的自然霉变玉米作为试验材料,考查了在日粮中不同自然霉变玉米用量对肉鸭生产性能、血液学与血液生化指标、抗氧化功能、免疫学功能的作用与影响,并就自然霉变玉米对肉鸭细胞增殖与细胞凋亡进行了研究,最后还探讨了日粮中补充营养性添加剂(VE和有机硒)对采食自然霉变玉米肉鸭的保护作用。研究共包括五个试验。试验一：不同用量自然霉变玉米对肉鸭生产性能、血液学及血液生化指标的影响试验采用单因子设计,用霉变玉米替代日粮中未霉变的正常玉米,替代比例分别为0%(对照组)、25%、50%、75%和100%,共五个处理组,每个处理8个重复,每个重复16只肉鸭,公母各半,试验期5周,其中1-14d饲喂小鸭日粮,15-35d饲喂生长鸭日粮。试验测定了试验日粮中霉菌毒素的含量、各处理组的重量、采食量；记录了各处理组的死亡只数与重量；在14d和35d分别采血进行血液学和血液生化指标测定。结果表明：(1)试验日粮所含霉菌毒素是以AF为主,其AFB1含量均随日粮中霉变玉米用量增加,而呈梯度递增,达到试验设计要求。(2)随着日粮中霉变玉米用量增加,肉鸭的ADFI呈极显著的线性下降(P<0.01)、ADG呈极显著的线性降低(P<0.01)、F/G呈显著的二次曲线降低(P<0.05)、死亡率呈极显著的增加(P<0.01)。(3)在1-2周龄时肉鸭MGR为2.05%/10ug AFB1/kg,5GRC为33.82ug AFB1/kg；在3-5周龄时,MGR为2.15%/10ug AFB1/kg,5GRC为57.22 ug AFB1/kg。(4)自然霉变玉米使肉鸭血液RBC减少,WBC增加,在小鸭阶段,鸭血液的HGB、HCT和MCH降低；血液中ALT、AST和LDH活性均显著或极显著增加。随肉鸭的日龄增加,血液中RBC、GHB、HCT、MCH、MCHC和RDW均呈极显著升高(P<0.01),而PLT、MCV极显著下降(P<0.01)。结论：自然霉变玉米抑制肉鸭采食,使肉鸭生长减慢、F/G降低,饲料利用率提高,死亡率增加。在本试验条件下,霉变玉米替代正常玉米比例不超过25%,不会显著影响肉鸭生产性能。试验二：自然霉变玉米对肉鸭生产性能、抗氧化功能及免疫功能的影响采用单因子试验设计,用霉变玉米替代日粮中未霉变的正常玉米,替代比例分别为0%、50%和100%,共三个处理组,每个处理8个重复,每个重复16只肉鸭,公母各半,试验期5周,其中1-14d饲喂小鸭日粮,15-35d饲喂生长鸭日粮。试验测定各处理组的增重、采食量,记录了死亡鸭的只数和重量,并在14d和35d采血、剖杀试验鸭,测定血液和肝脏抗氧化指标、血液生化指标、免疫器官指数和特异性抗体滴度。结果表明：(1)在整个试验期内,随着日粮中霉变玉米用量增加,各处理组鸭的ADG、ADFI、F/G均呈极显著的线性降低(P<0.01),死亡率呈极显著线性增加(P<0.01)。与试验—研究结果相似。(2)在整个试验期内,100%霉变玉米组鸭血清中SOD活性均极显著低于对照组(P<0.01)；随日粮中霉变玉米用量增加,血清中GSH-PX和CAT活性而呈下降趋势,MDA和蛋白质羰基含量呈增加的趋势。(3)在14d时,0%霉变玉米组鸭肝脏中CAT活性显著高于50%霉变玉米组和100%霉变玉米组(P<0.05),肝脏中SOD、GHS-PX活性呈下降趋势；MDA和蛋白质羰基呈增加趋势。在35d时,各处理组鸭肝脏中的SOD、GSH-PX、CAT、MDA和蛋白质羰基差异不显著(P≥0.05)。结论：霉变玉米使肉鸭产生氧化应激,导致肝脏损伤,肝脏功能改变,从而影响肉鸭生产性能。试验三：维生素E和酵母硒对采食自然霉变玉米肉鸭生产性能和免疫功能的影响采用2x2析因试验设计,其中玉米2种：未霉变正常玉米、自然霉变玉米；在基础日粮(含而有VE 10IU/kg、无机Se 0.2mg/kg)中额外添加维生素E(VE)+酵母硒(YSe),添加量为0和VE 50IU/kg+YSe0.2mg/kg两个水平,共四个处理组。每个处理7个重复,每个重复16只肉鸭,公母各半,试验期4周,其中1-14d饲喂小鸭日粮,15-28d饲喂生长鸭日粮。试验测定了试验日粮中霉菌毒素含量、各处理组的重量、采食量；记录了各处理组的死亡只数与重量；并在14d和28d采血、剖杀试验鸭,测定免疫器官指数、体液免疫、细胞免疫、细胞因子含量。结果表明：(1)在整个试验期内,霉变玉米极显著降低鸭ADG和ADFI(P<0.01),补充VE+YSe显著提高鸭的ADG和ADFI (P<0.05)。(2)霉变玉米不同程度的增加了鸭的肝脏指数、脾脏指数和法氏囊指数,并极显著的降低了鸭的胸腺指数,补充VE+YSe能显著提高霉变玉米组鸭脾脏指数和法氏囊指数。(3)霉变玉米和补充VE+YSe对血液中IgG、IgA、IgM含量均无显著影响(P≥0.05),但是霉变玉米使特异抗体滴度显著下降,而补充VE+YSe可提高抗体滴度。(4)霉变玉米使外周血T淋巴细胞转化率、ANAE阳性率显著或极显著降低,补充VE+YSe使T淋巴细胞转化率、ANAE阳性率升高。结论：采食霉变玉米的肉鸭补充VE+YSe能缓解对采食和生长的抑制作用,死亡率降低,但对F/G没有作用；霉变玉米抑制肉鸭细胞免疫和体液免疫,但对细胞免疫影响比体液免疫更大；补充VE+YSe能部分缓解对免疫的抑制作用。试验四：自然霉变玉米与维生素E、酵母硒对肉鸭Toll样受体和β-防御素基因表达的影响采用2x2析因试验设计,其中玉米2种：未霉变正常玉米、自然霉变玉米；在基础日粮中额外添加VE+YSe,添加量为0和VE 50IU/kg+YSe0.2mg/kg两个水平,共四个处理组(基础日粮中含而有VE 10IU/kg、无机Se0.2mg/kg)。每个处理7个重复,每个重复16只肉鸭,公母各半,试验期4周,其中1-14d饲喂小鸭日粮,15-28d饲喂生长鸭日粮。采用实时荧光定量PCR测定了14d肉鸭肝脏和脾脏细胞TLR-1mRNA、TLR-4 mRNA和β-防御素mRNA的表达。结果表明：(1)霉变玉米使14d肉鸭肝脏细胞TLR-1mRNA极显著上调(P<0.01),补充VE+YSe有上调肝脏细胞TLR-1mRNA的趋势,但差异不显著(P≥0.05)；霉变玉米也使肝脏细胞TLR-4mRNA上调,饲喂霉变玉米的鸭补充VE+YSe显著或极显著上调TLR-4mRNA表达；霉变玉米使β-防御素mRNA表达极显著上调,饲喂霉变玉米的鸭补充VE+YSe极显著上调肝脏细胞β-防御素mRNA表达。(2)霉变玉米使14d肉鸭脾脏细胞中TLR-1 mRNA表达上调、TLR-4 mRNA下调,但是差异不显著,补充VE+YSe有下调脾脏细胞TLR-1 mRNA和TLR-4 mRNA的趋势；霉变玉米和补充VE+YSe均使脾脏细胞β-防御素mRNA显著或极显著上调。(3)脾脏细胞TLR-1mRNA、β-防御素mRNA均极显著高于肝脏细胞的表达量,而肝脏细胞TLR-4mRNA又极显著高于脾脏细胞。结论：鸭采食霉变玉米配制的日粮后,能通过影响固有免疫的模式识别受体TLR-1、TLR-4和β-防御素基因的表达,激活鸭的固有免疫系统；日粮中补充维生素E和酵母硒对固有免疫系统有调节作用。试验五：自然霉变玉米与维生素E、酵母硒对肉鸭肝脏和脾脏细胞凋亡及细胞增殖的影响采用2x2析因试验设计,其中玉米2种：未霉变正常玉米、自然霉变玉米；在基础日粮中额外添加VE+YSe,添加量为0和VE 50IU/kg+YSe0.2mg/kg两个水平,共四个处理组(基础日粮中含而有VE 10IU/kg、无机Se 0.2mg/kg)。每个处理7个重复,每个重复16只肉鸭,公母各半,试验期4周,其中1-14d饲喂小鸭日粮,15-28d饲喂生长鸭日粮。采用流式细胞仪测定了14d和28d肉鸭肝脏和脾脏细胞凋亡率和细胞周期。结果表明：(1)霉变玉米使肝脏和脾脏细胞凋亡率显著或极显著增加,而日粮补充VE+YSe使肝脏和脾脏细胞凋亡率降低,霉变玉米和VE+YSe之间互作效应不显著。(2)霉变玉米无论是对14d还是28d肉鸭肝脏和脾脏细胞增殖均有显著或极显著的影响,使G0/G1期细胞百分率减少,S期和G2+M期细胞百分率增加,细胞分裂指数(PI)增加。(3)日粮中补充VE+YSe有使肝脏和脾脏G0/G1期细胞百分率减少、S期和G2+M期细胞百分率增加的趋势,PI增加。结论：鸭采食霉变玉米配制的日粮后,既促使肝脏和脾脏细胞凋亡,又刺激细胞增殖；补充维生素E和酵母硒可减少细胞凋亡,促进细胞增殖。本研究结果表明,采食自然霉变玉米配制的日粮后,鸭采食量受到明显抑制,生长速度减慢,死亡率增加,但是料肉比降低,饲料转化率提高。鸭的肝脏受到损伤,出现明显病理变化,肝脏功能发生改变,血液中多种与肝功相关的酶活性发生显著升高或降低。霉变玉米导致鸭出现氧化应激反应,与抗氧化有关的酶活性降低,氧化产物(MDA、蛋白质羰基)增加。霉变玉米对肉产生明显的免疫毒性作用,使得鸭特异性抗体滴度降低,外周血T淋巴细胞转化率、ANAE阳性率降低,固有免疫系的TLR-1、TLR-4和p-防御素基因表达发生改变,表现为鸭体液免疫和细胞免疫受到抑制,而固有免疫被激活。霉变玉米还使鸭肝脏和脾脏细胞凋亡率增加,同时,又刺激了细胞增殖。在日粮中补充维生素E和酵母硒能部分消除霉变玉米对鸭所引起的免疫毒性作用。更多还原

【Abstract】 A corn naturally contaminated with different kind of mycotoxins but main with aflatoxin is used in this study. This study was conducted to determine the effects of graded levels of the mycotoxin-contaminated corn on the growth performance, hematology, blood biochemical parameters, anti-oxidant function and immune system of ducks. Furthermore, the cell proliferation and apoptosis, the protective effect of dietary vitamin E and organic selenium for the ducks fed the diet with contaminated corn were evaluated. This study consists of 5 experiments as followed.Experiment 1. Effect of Graded Levels of Corn Naturally Contaminated with Mycotoxins on the Performance, Hematology and Blood Biochemical Parameters in DucksSix hundred and forty 1-day-old ducks were allotted by body weight into 5 treatments with 8 replicates of 16 ducks (8 males and 8 females). Treatment 1(control) was fed diet with normal corn, and treatment 2 to 5 were fed the diets with contaminated corn substituted for normal corn by 25%,50%,75% and 100% respectively. The trial lasted for 5 weeks. The concentrations of mycotoxins in experimental diets, the weight of ducks in each replicate, and the consumption of feed were all evaluated, the number and weight of dead ducks were recorded, and the blood sample was collected at 14d and 35d respectively, to determine the hematology and biochemical parameters. As shown in the results, (1) AFB1 was the predominated mycotoxin in the experimental diet, and gradually increased with the increasing addition levels of contaminated corn, which was according to the experimental design; (2) ADFI, ADG and linearly reduced(P<0.001), F/G decreased in quadratic curve (P<0.05), and mortality linearly increased(P<0.01) with the increment of contaminated corn; (3) during 1-2 wk, the MGR of ducks was 2.05%/10 ug AFB1/kg,5GRC was 33.82 ugAFB1/kg. and during 3-5wk, the MGR was 2.15%/10ug AFB1/kg,5GRCwas 57.22 ug AFB1/kg; (4) the contaminated corn led to the decrease of RBC, increase of WBC, and the decrease of HGB, HCT, MCH during the 1-2wk with the increased significantly of activities for ALT, AST and LDH. When the ducks grew up, the concentration of RBC, GHB, HCT, MCH, MCHC and RDW in blood was reduced significantly (P<0.01). Conclusion:the corn contaminated with mycotoxins could depress the feed intake, slow down the growth, decrease the F/G, and enhance the mortality and the feed efficiency of ducks. In this experiment, there was no significant effect of contaminated corn on the growth performance of ducks when the substitution level of contaminated corn for normal corn was not more than 25%.Experiment 2. Effect of Corn Naturally Contaminated with Mycotoxins on the Performance, Anti-Oxidant Function and Immune System in DucksThree hundred and eighty-four 1-day-old ducks were allotted by body weight into 3 treatments with 8 replicates of 16 ducks (8 males and 8 females). The trial lasted for 5 weeks. The weight of ducks in each replicate and the consumption of feed were all evaluated, the number and weight of dead ducks were recorded, and the blood sample was collected when ducks were killed at 14d and 35d respectively to determine the anti-oxidative parameters in blood and liver, blood biochemical parameters, immune organ index, and specific antibody titer. The results showed that (1) ADFI, ADG and F/G was linearly reduced(P<0.01), and mortality linearly increased(P<0.01) with the increase of contaminated corn, which was similar to the results in experiment 1; (2) in the whole experiment, the serum SOD activities in 100% group was lower than that of the control group(P<0.01); with the increasing addition levels of contaminated corn, the activities of GSH-Px and CAT had the trend to decline, while the activities of MDA and the content of protein carbonyl had the trend to increase; (3) at 14 d, the activities of CAT in liver of the control group was higher than that of 50% group and 100% group (P<0.05), the activities of SOD and GHS-PX had the trend to reduce, and the activities of MDA, the content of protein carbonyl had the trend to increase. At 35d, there was no significant difference in the activities of SOD, GSH-PX, CAT, MDA, and the content of protein carbonyl (P≥0.05). Conclusion:contaminated corn resulted the oxidative stress in ducks, which caused the lesion and function changes, and consequently affected the growth performance of ducks.Experiment 3 Effect of Vitamin E and Yeast Selenium on the Performance and Immune Function in Ducks Fed Diet with Corn Naturally Contaminated with MycotoxinsFour hundred and forty-eighty 1-day-old ducks were allotted by body weight into 4 treatments with 7 replicates of 16 ducks (8 males and 8 females) in a 2x2 factoral experimental design. Normal corn and contaminated corn was used in the basal diet (VE 10 IU/kg、inorganic Se 0.2mg/kg) without or with the addition of VE+yeast selenium (YSe) (VE50IU/kg+YSe0.2mg/kg). The trial lasted for 4 weeks. The concentration of mycotoxins in experimental diets, the weight of ducks in each replication and the consumption of feed were all evaluated, while the number and weight of dead ducks were recorded, and the blood sample was collected at 14d and 28 d respectively to determine the immune organs index, humoral immunity, cellular immunity, and the content of cytokine. The results indicated (1) during the whole experiment, contaminated corn reduced the ADG and ADFI of ducks (P<0.01), but with the supplementation of VE and YSe the ADG and ADFI of ducks was increased (P<0.05); (2) contaminated corn caused varying degrees increase of liver, spleen, and Fabricius bursa index of ducks, and the decrease of the thymus index significantly, however, the supplementation group with VE and YSe could enhance the spleen index, and Fabricius bursa index. (3) there was no significant effect of either the contaminated corn or the supplementation of VE+YSe on the content of IgG, IgA and IgM (P≥0.05). However, the contaminated corn could reduce the specific antibody titer significantly, while the supplementation of VE+YSe could enhance the antibody titer. (4) the contaminated corn caused the significant decrease of peripheral blood T-lymphocyte conversion ratio and positive rate of ANAE, but opposite with the supplementation of VE+YSe. Conclusion:the supplementation of VE+YSe to diet with contaminated corn could relieve the negative effect of contaminated corn on the feed intake,growth performance and mortality, however, there was no effect of it on F/G; contaminated corn could depress the cell immunity and humoral immunity of ducks with more serious to the cell immunity., while the supplementation of VE+YSE could partly relieve the depression of immunity by the contaminated corn.Experiment 4 Effect of Corn Naturally Contaminated with Mycotoxins and Vitamin E, Yeast Selenium on the Expression of Toll-Like Receptor and Defensins Genes in Ducks1-day-old ducks were allotted by body weight into 4 treatments with 7 replicates of 16 ducks(8 males and 8 females) in a 2x2 factoral experimental design. Normal corn and contaminated corn was used in the basal diet (VE 10 IU/kg、inorganic Se 0.2mg/kg) without or with the addition of VE+yeast selenium (YSe) (VE50IU/kg+YSeO.2mg/kg). The trial lasted for 4W. The expression of TLR-1 mRNA、TLR-4 mRNA, andβ-defensins mRNA of livers and spleens of ducks at 14d were determined by fluorescence quantitative PCR. The results showed:(1)the contaminated corn increased the expression of TLR-1 mRNA of liver in ducks(P<0.01), and so did the supplementation group, but not significant (P≥0.05); contaminated corn also increased the expression of TLR-4 mRNA of liver in ducks, and so did the supplementation group(P<0.05); contaminated corn significantly increased the expression of (3-defensins, and supplement of VE+YSe could significantly up-regulate the expression of (3-defensins.(2) contaminated corn up-regulated the expression of TLR-1 mRNA, down-regulated the expression of TLR-4 mRNA of liver in ducks at 14d, without significance, and supplement of VE+YSe could down-regulateTLR-1 mRNA and TLR-4 mRNA of spleen; contaminated corn group and supplementation group both up-regulated the expression ofβ-defensins mRNA;(3)the expression of TLR-1mRNA, P-defensins mRNA of spleen was significantly higher than that of liver, the expression of TLR-4mRNA in which was higher than that of spleen. Conclusion:the innate immune system could be activated by the effect of contaminated corn on the expression of pattern recognition receptor TLR-1、TLR-4 andβ-defensins genes; supplementation of VE+SYe had accommodation for the innate immune system. Experiment 5 Effect of Corn Naturally Contaminated with Mycotoxins and Vitamin E, Yeast Selenium on the Apoptosis and Cell Proliferation of Liver and Spleen in Ducks1-day-old ducks were allotted by body weight into 4 treatments with 7 replicates of 16 ducks(8 males and 8 females) in a 2x2 factoral experimental design. Normal corn and contaminated corn was used in the basal diet (VE 10 IU/kg、inorganic Se 0.2mg/kg) without or with the addition of VE+yeast selenium (YSe) (VE50IU/kg+YSeO.2mg/kg). The trial lasted for 4wk. the apoptosis and the mitotic cycle were determined at 14d and 28d. The results showed:(1)the contaminated corn increased the apoptosis of livers and spleens, but the supplementation of VE+YSe reduced it, and there was no interaction among contaminated corn and VE+YSe; (2) contaminated corn had significant effect on cell proliferation at 14d,28d, which reduced the rate of GO/G1 cells, increased the proportion of S and G2+M period cells, and increased PI; (3) supplementation of VE+YSe could reduce GO/G1 cells of livers and spleens, and increase the proportion of S and G2+M period cells, increase PI. Conclusion:the contaminated corn either stimulated apoptosis of livers and spleens or stimulated cell proliferation, but supplementation of VE+YSe could reduce apoptosis, promote cell proliferation.ConclusionThe results indicated that diet with natural moldy corn could increase the ducks mortality, decrease the feed intake and growth performance, but improve the feed efficiency. Tissue lesion happen in livers, and the function of livers was changed with varied enzyme activities changes. Mycotoxin-contaminated corn caused the oxidative stress, and activities of anti-oxidative enzymes decreased, oxidation products (MDA, protein carbonyl) increased. Mycotoxin-contaminated corn brought about immunotoxicity to ducks with the decrease of specific antibody titer, peripheral blood T-lymphocyte conversion ratio, and positive rate of ANAE, while the expression of TLR-1、TLR-4 andβ-defensins genes of innate immune system was changed. Thus, humoral immunity and cellular immunity of ducks were depressed, and innate immunity was activated. Mycotoxin-contaminated corn also increased the apoptosis of liver and spleen, and the meanwhile stimulated the cell proliferation. Supplementation of Ve+YSe could partially eliminate the immunotoxicity of the moldy corn.更多还原