【作者】 郑萍；

【导师】 陈代文；

【作者基本信息】 四川农业大学 ， 动物营养与饲料科学， 2010， 博士

【摘要】 氧化应激明显影响仔猪健康,降低生产性能,但对养分代谢和需要特点的影响还缺乏系统研究。本研究以断奶仔猪为试验动物,以diquat诱导的断奶仔猪氧化应激为模型,研究氧化应激对断奶仔猪精氨酸代谢和需要特点及其可能机制的影响,探明精氨酸的应激代谢和需求特点及其抗应激的效果,为丰富仔猪精氨酸营养原理积累资料,为缓解氧化应激的危害和合理使用精氨酸添加剂提供参考。主要研究内容及结果如下：试验一Diquat诱导的氧化应激对断奶仔猪精氨酸代谢的影响研究diquat诱导的氧化应激对仔猪生产性能和精氨酸代谢的影响。采用单因子试验设计,选用24头28±1日龄的断奶仔猪,分别饲养在24个猪栏中,经过预饲,排除断奶等应激因素后,按体重相近、性别对半的原则随机分成3个处理,每个处理8个重复,每个重复1头猪。氧化应激组：仔猪腹腔注射10mg/kg体重的Diquat,自由采食;正常对照组：仔猪腹腔注射无菌生理盐水,自由采食；采食配对组：仔猪腹腔注射无菌生理盐水,控制采食量与氧化应激组仔猪相同。所有仔猪自由饮水,试验期7天。结果显示：(1)与对照组和采食配对组相比,氧化应激显著降低仔猪的ADG和G/F,降低仔猪血清抗氧化酶的活性,显著增加血清丙二醛的含量,有效诱导了断奶仔猪氧化应激。(2)氧化应激提高仔猪空肠阳离子氨基酸转运载体CAT-1的mRNA水平,增加了精氨酸内源合成关键酶OAT的活性。(3)氧化应激增加了仔猪空肠精氨酸和瓜氨酸的浓度,但是降低血清精氨酸和瓜氨酸的浓度,降低了肝脏和肺的INOS和ENOS的mRNA的表达。本试验表明,氧化应激降低仔猪生产性能,降低仔猪的抗氧化能力,增加仔猪精氨酸转运、内源合成和分解代谢,降低循环中的精氨酸的有效性。试验二氧化应激对仔猪精氨酸需求特点的影响研究精氨酸在仔猪氧化应激中的作用。选择36头21日龄健康PIC断奶仔猪,分别饲养在36个猪笼中。经过一周的预饲,排除断奶等应激因素后,按体重相近、性别对半的原则随机分为3个处理,每个处理12个重复,每个重复1头猪,开始正式试验,分别饲喂3个不同精氨酸水平的日粮。在试验第8天,每个处理一分为二,分别注射diquat和生理盐水。正式试验期共11天。测定仔猪生产性能,应激前0h和应激后6h,24h,48h,96h的血浆抗氧化酶活力,TAOC和MDA的含量,血浆游离氨基酸水平和组织氨基酸水平。试验结果表明：(1)氧化应激降低仔猪ADG (P<0.001),ADFI(P<0.001),增加F/G(P<0.05)。在氧化应激状态下,补充精氨酸(Arg=2.79%)增加仔猪的ADFI(P<0.05)。(2)补充精氨酸(Arg=2.79%)显著增加6h、48h、96h仔猪血浆GPx和SOD的活性,显著增加6h、24h、48h血浆TAOC的含量,显著降低6h、48h、96h血浆MDA的浓度。(3)氧化应激降低血浆精氨酸、谷氨酸、赖氨酸、蛋氨酸、苏氨酸和酪氨酸的浓度,显著增加血浆异亮氨酸和缬氨酸的浓度。在氧化应激条件下,补充精氨酸显著的提高血浆赖氨酸的水平。(4)氧化应激显著增加空肠精氨酸和瓜氨酸水平,增加肾脏中精氨酸的含量,但是降低了肾脏瓜氨酸的水平,对肌肉中精氨酸和瓜氨酸水平无显著影响。补充精氨酸对组织中精氨酸和瓜氨酸无显著影响。本试验表明,补充精氨酸通过缓解仔猪氧化应激诱导的采食量下降,增加仔猪氧化应激下机体的抗氧化能力,改善血液循环中的氨基酸水平。在氧化应激条件下,仔猪对精氨酸的需求量增加。试验三精氨酸抗氧化应激作用的机理初探试验设计同试验二。探讨精氨酸抵抗仔猪氧化应激作用的机理。试验结果表明：(1)氧化应激显著降低仔猪空肠绒毛宽度、固有膜的厚度和隐窝深度,有降低空肠绒毛长度趋势。补充精氨酸,能缓解氧化应激诱导的空肠绒毛宽度的降低,显著抑制氧化应激引起的空肠固有膜厚度的降低。精氨酸与氧化应激对仔猪空肠隐窝深度有显著的交互效应。(2)氧化应激显著增加仔猪空肠CAT-1、CAT-2和CAT-3的相对基因表达量。补充精氨酸则显著抑制氧化应激诱导的CAT-1和CAT-2的基因表达的增加,而对于氧化应激诱导的CAT-3的表达无影响。(3)氧化应激对仔猪空肠和肾脏的ENOS的表达影响不显著,显著降低肝脏ENOS的表达,显著降低空肠和肝脏INOS的表达,增加肾脏INOS的表达。补充精氨酸则抑制氧化应激诱导的肾脏INOS表达量的增加,显著增加空肠和肾脏的ENOS的相对表达量。(4)氧化应激降低肝脏TNOS和INOS的酶活性和NO的产量,而显著增加应激后24h,48h的血浆INOS的酶活性,显著增加血浆48h和96h的NO的产量。补充精氨酸显著增加氧化应激仔猪肝脏TNOS和INOS的酶活性。(5)氧化应激显著增加仔猪应激后48h和96h的血浆皮质醇的浓度,显著降低仔猪血浆胰岛素和IGF-1的浓度。补充精氨酸显著增加氧化应激仔猪的血浆胰岛素水平和IGF-1的水平,降低血浆皮质醇的浓度。(6)氧化应激显著降低肝脏IGF-1、IGF-1R和IGFBP3的基因表达；而对肌肉中这三个生长相关基因的表达无显著影响。补充精氨酸显著增加肝脏IGF-1和IGFBP3的基因表达,对肝脏IGF-1R的基因表达无显著影响；显著增加肌肉IGF-1、IGF-1R和IGFBP3的基因表达。(7)氧化应激显著增加空肠IL-6和TNF-α的表达,而抑制肝脏IL-6和TNF-α的基因表达。精氨酸与氧化应激互作增加空肠IL-6的表达,抑制氧化应激活化的空肠TNF-α的表达,而对PPAR-γ的表达无显著影响。本试验表明,在应激条件下提高精氨酸添加量可以通过维持空肠组织结构、调控精氨酸的代谢、维持精氨酸内源稳定性,增加精氨酸的有效性,调控内分泌、促进蛋白质合成、降低炎症因子的表达等综合途径缓解仔猪氧化应激。通过本试验的研究表明,氧化应激可降低仔猪生产性能,增加仔猪精氨酸内源合成量和转运能力、降低循环中的精氨酸的有效性,改变精氨酸的分解代谢；在应激条件下提高精氨酸添加量可以缓解仔猪氧化应激诱导的采食量下降,增加仔猪氧化应激下机体的抗氧化能力,改善血液循环中的氨基酸水平,从而缓解氧化应激的危害；精氨酸抗氧化应激的作用机制与维持空肠组织结构、调控精氨酸的代谢、维持精氨酸内源稳定性、增加精氨酸的有效性,调控内分泌、促进蛋白质合成、降低炎症因子的表达等途径有关。更多还原

【Abstract】 The health state was significantly influenced and performance was decreased by oxidative stress in piglets. However, there is little research about the effect of metabolism and the characteristic of requirement on nutrients in pigs. This research was conducted to study the effects of oxidative stress by diquat on arginine metabolism and requirement of piglets. We will try to find out the metabolism and requirement of arginine under oxidative stress and the effect of anti-oxidative stress by arginine in piglets. It will accumulate the data of arginine nutrition of piglets and provide the information for relieving the harm by oxidative stress and using arginine rationally.The main results were listed as following:Exp.l Effects of oxidative stress induced by diquat on arginine metabolism of pigletsA total of 24 crossbred postweaning pigs with 10.63±0.21kg BW were individually penned and assigned to three treatments to investigate the influences of diquat-induced oxidative stress on performance and arginine metabolism. Pigs of oxidative stress group were intraperitoneally injected with 10mg/kg BW of diquat and fed ad libitum, those of control group were injected with isotonic saline and fed ad libitum, and those of pair-fed group were injected with isotonic saline and fed the same amount of feed as stress group. The experiment lasted for 7 days. Results indicated that compared to control treatment, oxidative stress induced by diquat significantly decreased average daily gain, feed intake and efficiency of feed utilization, decreased activities of antioxidant enzymes, increased concentration of malondialdehyde in serum, increased cationic amino acid transporter-1 mRNA level and activities of ornithine aminotransferase and concentrations of arginine and citrulline in jejunum, decreased the concentrations of arginine in serum and kidney, decreased induced nitric oxide synthase mRNA level. It is concluded that oxidative stress induced by diquat can reduce performance, influence absorption and metabolism of arginine and consequently modify the requirement of arginine in postweaning pigs.Exp.2 Effects of oxidative stress induced by diquat on arginine requirement of pigletsThis study evaluated whether arginine (Arg) supplementation could attenuate oxidative stress induced by diquat in piglets. A total of 36 PIC postweaning pigs were individually penned and assigned to three treatments including:(1) Arg=1.19%(2) Arg=1.99%(3) Arg=2.79%. On day 8, pigs in each treatment were divided into two to intraperitoneally injected with diquat or sterile saline. At 96 h post-injection, pigs were killed for evaluation of performance, the activities of antioxidant enzymes and TAOC and malondialdehyde in plasma and liver, the concentrations of amid acid in plasma and tissue. The results indicated:(1) Oxidative stress induced by diquat significantly decreased average daily gain(P< 0.001), feed intake (P<0.001)and efficiency of feed utilization(P<0.05). Supplementation of arginine(Arg=2.79%) significantly increased average daily gain(P<0.05).(2) Supplementation of arginine (Arg=2.79%) significantly increased activities of antioxidant enzymes and TAOC, decreased concentration of malondialdehyde in liver.(3) Oxidative stress induced by diquat significantly decreased the concentrations of arginine, glutamic acid, lysine, methionine, threonine and tyrosine in plasma, increased the concentrations of isoleucine and valine in plasma. Supplementation of arginine significantly increased the concentration of lysine under oxidative stress.(4) Oxidative stress induced by diquat significantly decreased the concentrations of arginine and citrulline in jejunum, decreased the concentrations of citrulline in kidney, increased the concentrations of arginine in kidney.Supplementation of arginine have no significantly effect on the concentrations of arginine and citrulline in tissue.It is concluded that arginine can alleviate the growth depression and stress response induced by oxidative stress to some degree through depress the decreasing of food intake, increasing the ability of anti-oxidative stress, improving amino acid level in circulation in piglets. It illustrated that the requirement of arginine was increased under oxidative stress in piglets.Exp.3 The possible mechanism of arginine for anti-oxidative stress of pigletsThis study evaluated the mechanism of arginine (Arg) supplementation attenuate oxidative stress induced by diquat in piglets. A total of 36 PIC postweaning pigs were individually penned and assigned to three treatments including:(1) Arg=1.19%(2) Arg=1.99%(3) Arg=2.79%. On day 8, pigs in each treatment were divided into two to intraperitoneally injected with diquat or sterile saline. At 96 h post-injection, pigs were killed for evaluation of intestinal morphology, arginine metabolism, endocrine secretion, the gene relatively expression of growth factors and inflammatory cytokines. The results indicated:(1)Within 96 h of oxidative stress, oxidative stress induced by diquat significantly decreased villus width, membrane propria thickness and crypt depth in jejunum. Supplementation of arginine significantly mitigated jejunum morphology impairment (e.g. lower villus width and membrana propria thickness).(2) Oxidative stress induced by diquat significantly increased cationic amino acid transporter-1,2,3 mRNA level. Supplementation of arginine significantly suppressed the mRNA level of CAT-1 and CAT-2 induced by oxidative stress.(3) Oxidative stress induced by diquat significantly decreased mRNA level of ENOS and INOS in liver and INOS in jejunum, increased mRNA level of INOS in kidney. Supplementation of arginine significantly suppress the elevation of the mRNA level of INOS in kidney induced by oxidative stress, significantly increased the mRNA level of ENOS in jejunum and kidney suppressed by oxidative stress.(4) Oxidative stress induced by diquat significantly decreased activities of ENOS and INOS and the production of NO in liver, significantly increased activities of INOS in plasma at 24h and 48h. Supplementation of arginine significantly increased activities of TNOS in liver.(5) Oxidative stress induced by diquat significantly increased concentrations of cortisol in plasma at 48h,96h. Oxidative stress significantly decreased concentrations of insulin and IGF-1 in plasma. Supplementation of arginine significantly prevented the elevation of plasma cortisol induced by oxidative stress. Arginine significantly increased plasma insulin and IGF-1 suppressed by oxidative stress.(6) Supplementation of arginine significantly increased the mRNA level of IGF-1 and IGFBP3 in liver. Supplementation of arginine significantly increased the mRNA level of IGF-1, IGF-1R and IGFBP3 in muscle suppressed by oxidative stress.(7) Supplementation of arginine significantly suppressed the mRNA level of TNF-a in jejunum.These results indicate that Arg supplementation has beneficial effects in piglets induced by oxidative stress through keeping morphous of intestine, regulating endocrine section, increasing the availability of arginine, maintaining endogenous stability of arginine, promoting protein synthesis in muscle, depressing inflammatory cytokines.ConclusionsThese results indicate that oxidative stress induced by diquat can reduce performance, influence absorption and metabolism of arginine and consequently modify the requirement of arginine for postweaning pigs. Arginine can alleviate the growth depression and stress response induced by oxidative stress to some degree through depress the decreasing of food intake, increasing the ability of anti-oxidative stress, improving amino acid level in circulation in piglets. Arg supplementation has beneficial effects in piglets induced by oxidative stress through keeping morphous of intestine, regulating endocrine section, increasing the availability of arginine, maintaining endogenous stability of arginine, promoting protein synthesis in muscle, depressing inflammatory cytokines.更多还原