【作者】 张挺；

【导师】 彭健；

【作者基本信息】 华中农业大学 ， 动物营养与饲料科学， 2016， 博士

【摘要】 养猪生产中运输应激的发生,给猪肉屠宰、零售及相关产业造成巨大的经济损失。运输应激发生时,动物应激激素指标,各组织器官生理生化指标、抗氧化指标和肠道功能均发生显著改变,肉质的pH值、滴水损失和肉色等指标也发生相应变化。因此,我们推测,在运输应激过程中,上述指标和功能的变化存在必然的联系,并对肉质造成影响。植物提取物止痢草油具有抗氧化、抗菌和改善肠道功能等多种特性,在动物肠道及体内均会发生作用,并影响机体生理代谢功能。此外,止痢草油(Oregano essential oils,OEO)还具有改善肉质,延长肉品的货架期的作用。本研究的目的是通过相应试验研究探索止痢草油在运输应激时改善肉质的作用。本研究由三部分试验构成。首先模拟运输构建大鼠运输应激模型,确认模拟运输能够影响大鼠肌肉氧化状态和形态特征,通过对应激激素、抗氧化酶活和肌肉形态等指标检测,验证大鼠对运输应激具有敏感性及重复性,明确模拟运输模型用于肌肉能量代谢和形态方面研究的可行性。然后,利用此模型研究止痢草油对运输应激大鼠血液应激激素指标,血液、肠道及肌肉抗氧化指标,肠道炎性因子及肌肉能量代谢等指标的影响,揭示止痢草油缓解大鼠肌肉损伤的机制。最后,将止痢草油应用在育肥猪生产中,通过在育肥猪饲料中添加止痢草油,研究止痢草油对育肥猪生长性能的影响,并通过设定屠宰前不同的运输强度,研究止痢草油对宰前育肥猪血清、肌肉及肝脏抗氧化能力和肉质相关指标的影响,确定止痢草油对缓解运输应激及改善肉质的效果。主要结果如下：1、运输应激大鼠模型用于肌肉状态改变研究的可行性选择6周龄健康雄性SD大鼠,模型温度设定35℃,摇床转速60 r/min,持续摇摆2 h。与对照组大鼠相比,模拟运输应激大鼠血清中皮质醇、去甲肾上腺素及促肾上腺皮质激素释放因子(Corticotropin releasing factor,CRF)水平极显著升高(P<0.01),血清中脂质过氧化产物丙二醛(Malondialdehyde,MDA)含量极显著升高(P<0.01),而谷胱甘肽过氧化物酶(Glutathione peroxidase,GSH-Px)的活性极显著降低(P<0.01),过氧化氢酶(Catalase,CAT)的活性显著降低(P<0.05)。同时,模拟运输应激大鼠肌肉中MDA含量极显著升高(P<0.01),GSH-Px酶活性极显著降低(P<0.01),CAT的活性显著降低(P<0.05)。肌肉组织中活性氧(Reactive oxygen species,ROS)含量极显著升高(P<0.01)。从肌肉组织形态学观察到,肌肉纤维有明显的断裂、破碎现象,说明肌肉形态已经发生损伤。这些结果表明：模拟运输应激大鼠已发生明显的应激反应,此模型可以用于研究影响肌肉状态的因素。2、止痢草油对运输应激大鼠肌肉状态的影响及机制的研究利用模拟运输应激大鼠模型,采用双因素试验设计。选择6周龄健康雄性SD大鼠36只,随机分为4组,每组9只。试验分组如下,非应激组、应激组、非应激组+OEO(灌服5mg/t.bw)和应激组+OEO(灌服5mg/t.bw)。试验期为20 d,记录每天的采食量和试验末重。试验结束后,采集血样、肌肉和空肠等样品。检测应激激素指标、生理生化指标、机体抗氧化指标、肌肉形态学指标和肠道免疫等指标,研究了止痢草油对大鼠肌肉状态的影响及机制。结果表明：尽管添加止痢草油不能显著提高大鼠日增重,但可以降低料重比(P<0.05)。与非应激组相比,运输应激极显著提高了大鼠血清中的皮质醇、去甲肾上腺素、CRF、乳酸脱氢酶(Lactate dehydrogenase,LDH)、肌酸激酶(Creatine kinase,CK)、IL-6、TNFα、ROS、MDA和CAT的水平(P<0.01)。与未灌服止痢草油组相比,灌服止痢草油可以极显著降低血清中皮质醇、去甲肾上腺素、CRF、LDH、IL-6、TNFα、ROS、MDA和GSH-Px等的水平(P<0.01)。此外,皮质醇、去甲肾上腺素、CRF、MDA、GSH-Px和SOD存在显著的交互作用(运输×OEO, P<0.05)。与应激组相比,灌服止痢草油可以显著提高宰后0h及48 h肌肉中SOD酶活性(P<0.05)；极显著降低0h及48 h肌肉MDA和乳酸浓度(P<0.05)；极显著升高0h及48 h肌肉糖原浓度(P<0.01)。此外,灌服止痢草油可以提高显著提高肠道SOD与GSH-Px酶活性(P<0.01),显著降低MDA含量(P<0.05)。肌肉切片结果显示,灌服止痢草油后,肌肉形态结构更完整,解剖后内脏组织无充血、胀气现象。这些结果显示,(1)止痢草油通过提高肠道抗氧化能力,降低炎性因子的产生,从而降低糖皮质激素的水平,调节肌肉能量代谢,减少糖无氧酵解及乳酸的产生,防止pH值快速下降；(2)止痢草油通过提高机体抗氧化酶的活性,减少体内自由基的产生；(3)止痢草油由于自身具有抗氧化能力,在体内代谢过程中,能够淬灭自由基,减少氧化产物的产生,从而减少对肌肉的损伤。3、止痢草油对肥育猪生产性能的影响及对肉质的干预作用选择日龄为150±3 d、体重78±5kg、公母各半的杜长大商品猪288头,随机分成3组,每组96头,分别饲养在6个栏里面(16头/栏)进行饲喂和管理。分别饲喂基础日粮或添加200mg/kg维生素E、500mg/kg的止痢草油产品.(含OEO 5%)的日粮30 d。记录初始重、末重和采食量等数据。饲喂结束后,每组挑选出60头体重接近,共计180头猪,根据模拟运输应激条件,进行高密度(252 kg/m2)长时间(5h)或低密度(168 kg/m2)短时间运输(1 h)运送至屠宰场进行屠宰。采集血液和相关组织。结果显示：与对照组相比,止痢草油组结束体重(P=0.06)和总增重(P=0.08)均有提高的趋势,能够显著降低料肉比(P<0.05)。与非应激组相比,运输应激处理血清中皮质醇水平(P=0.09)、去甲肾上腺素水平(P=0.06)和MDA水平(P=0.09)有增加趋势；而OEO和VE处理,极显著抑制了血清中皮质醇和GSH-Px的水平(P<0.01)；且皮质醇和去甲肾上腺素存在极显著的交互作用(P<0.01)。与非应激组相比,运输应激处理肝脏中MDA水平有提高的趋势(P=0.05),极显著的提高了SOD和T-AOC的活性及ROS的水平(P<0.01)：而止痢草油处理,则极显著降低了MDA水平(P<0.01),显著提高了SOD的活性(P=0.01)。在肌肉组织中,运输应激处理极显著提高了MDA、SOD和GSH-Px的水平(P<0.01)；且MDA水平存在显著的交互作用(P=0.04)。运输应激,极显著降低了pH(45min) (P<0.01),显著降低了导电率(72 h)(P=0.04),显著提高肌肉滴水损失(P=0.05)。日粮处理及运输方式对肌肉色值及肌肉导电率的影响差异不显著(P>0.05)；基础日粮、VE和止痢草油处理组肌肉滴水损失差异显著(P=0.05)。添加止痢草油极显著地降低了肌肉中HSP27和HSP70mRNA的丰度(P<0.01)；并且运输方式和日粮处理对HSP27的表达存在极显著的交互作用(P<0.01)；添加止痢草油组空肠绒毛高度、绒毛表面积、绒毛／隐窝比值显著高于对照组(P<0.05)。综上所述,本研究的结论是：(1)建立了大鼠运输应激模型,其条件为温度为35℃,摇床转速为60 r/min,持续摇摆时间为2 h。(2)止痢草油通过三个方面影响肌肉能量代谢和形态特征：第一,通过降低肠道氧化损伤,减少炎性因子的产生,从而降低糖皮质激素的分泌,减少肌肉以无氧酵解方式供能,减少乳酸的积累,防止pH值快速下降；第二,止痢草油通过提高机体抗氧化酶的活性,使机体处于氧化还原平衡状态,减少氧化应激产生的ROS对肌肉造成的损伤；第三,止痢草油在体内代谢过程中,通过防止肌肉细胞膜发生脂质氧化,减少MDA的产生。(3)在育肥猪日粮中添加500 g/t的止痢草油产品,可以提高宰前30天肥育猪的生长性能。可以缓解高密度(252kg/m2)、长时间(5h)运输对猪应激反应。降低了应激激素水平,提高了机体的抗氧化能力,改善了肠道的健康,有效缓解了运输应激所带来的负效应。在肌肉组织中,减少了肌肉滴水损失,改善肉色等指标。且止痢草油对肉质的改善效果好于维生素E。更多还原

【Abstract】 Transportation stress can result in significant economic losses to producer due to decreased animal productivity and increased mortality in pig production. Not only the levels of stress hormone, physiological and biochemical of tissue and organ, antioxidation and intestinal function were changed, but also pH, drip loss and colour of meat were changed in transportation stress situation. Thus, we speculate that the changes of these indexes and functions existed inevitable connection and affected meat quality. Plant extracts of oregano essential oils has so many functions such as antioxidant, antibacterial and improving the function of intestinal. All of these can affect the metabolism of the body. In addition, oregano essential oils (OEO)also has to improve meat quality and prolong the shelf life of the meat. The purpose of this study was to determine the effect of oregano on improve meat quality in transportation stress.This study mainly includes three parts. Firstly, the current study was to built a model of transportation stress and to evaluate feasibility of this model used in the study of energy metabolism and morphology of muscle in rats; Secondly, using this model, to research the effect and mechanism of oregano essential oils on muscle damage under transportation stress; Thirdly, to assess the potential of oregano essential oils as a dietary supplement for finishing pigs to alleviate transport stress. Also to evaluate the effects of dietary oregano essential oils on growth performance, meat quality and muscle Hsps expression after transport stress in finishing pigs.1 The feasibility of the model of transportation stress used in the study of muscle damage in ratsSix-week-old healthy male SD rats were placed in 35℃ shaker (60r/min,2 h) to induce transportation stress. Compared with the control group, the levels of cortisol, norepinephrine and CRF in serum was extremelysignificantly increased in the stress control group (P< 0.01), the content of malondialdehyde (MDA) in serum was extremely increased (P< 0.01). At the same time, the content of MDA in muscle was significantly increased (P<0.01), the activity of GSH-Px was extremely significantly decreased (P< 0.01), the activity of CAT was significantly reduced (P<0.05). The content of ROS in the muscle was extremely increased (P<0.01). Muscle fiber have been broken on muscle morphology, these phenomenon indicated that muscle has already damaged. These results showed that the model can be used to study the influencing factors of muscle damage.2 The effect and mechanism of oregano essential oilson muscle energy metabolism and morphology in transportation stressBy using the model of transportation stress, and adopt double factor experiment design.Six-week-old healthy male SD rats were randomly allotted to 4 treatments arrangement and 9 replicates with 1 rat per group.Each group was randomly allocated to 1 of 4treat containing no stress, stress, no stress with OEO (orally 5mg/t.bw) and stress with OEO (orally 5mg/t.bw). The stress hormone physiological and biochemical indicators, antioxidant, muscle morphological and intestinal immunity index were determinated. The results showed that the oregano essential oils didn’t significantly improve daily gain of rats, but can significantly reduce feed conversion ratio (P<0.05). Compared with the negative control group, experimental group can significantly reduce the levels of cortisol and CRF in serum (P< 0.05) after stress; at the same time very significantly reduced the level of LDH in serum of rats (P< 0.01). The content of ROS was significantly reduced in the experimental group (P< 0.05). The activity of SOD and GSH-Px in serum were extremely significantly increased (P< 0.01) in the experimental group. Compared with the negative control group, the activity of SOD were significantly increased on 0 h and 48 h after slaughter in the muscle (P< 0.05), the content of MDA (P< 0.01) and lactic acid (P< 0.05) were significantly reduced after slaughter in the muscle. The content of glycogen was extremely significantly increased (P< 0.01) in the experimental group. The activity of SOD and GSH-Px were significantly increased in the intestinal feed oregano under stress.The content of MDA were significantly reduced in the intestinal (P< 0.05). The level of TNF-a and IL-6 were significantly reduced (P< 0.05). These results showed that OEO can improve antioxidant capacity of intestinal and reduce the production of inflammatory cytokines. These results showed that reduced the levels of glucocorticoid and regulate the metabolism of muscle energy, and then reduce anaerobic glycolysis and the production of lactic acid, prevent of pH falling fast. And OEO can increase the activity of antioxidant enzymes and reduce the generation of free radicals in the body. Furthermore, OEO can reduce the damage of muscle because of its antioxidant ability.3 Effect of dietary oregano essential oils supplementation on growth performance and meat qualityA total of two hundred and eighty-eight pigs (Landrace×Yorkshire) were randomly allotted to 3 treatments arrangement based on initial body weight (initial body weight BW=77.80±5kg) and 6 replicates with 16 pigs per pen in each replication. Each group was randomly allocated to 1 of 3 finishing diets containing commercial basal diets (the control),200 mg/kg VE, or 25 mg/kg OEO of feed. After 28-d feeding trial,60 pigs from each group were subjected either to no stress (NS) (low stocking density and rest for 20 h) or 5h of transportation stress (TS). OEO supplementation increased the average daily gain (ADG), and significantly decreased the feed conversion ratio (FCR) (P< 0.05). Serum cortisol concentrations were greatly reduced (P=0.01) in pigs fed VE and OEO diet (transportation stress×diet interaction, P<0.01). ROS levels were significantly increased in the serum (P<0.01) and liver (P=0.01) of TS pigs. Similarly, MDA levels were observably elevated in the serum (P=0.05) and liver (P=0.05) of TS pigs. The SOD activity was slightly increased (P=0.08) and GSH-Px was markedly higher (P=0.01) in pigs fed VE and OEO diet than the control diet in the serum. The SOD activity was dramatically raised no matter transportation (P< 0.01) or dietary treatment (P=0.01), and T-AOC activity was significantly higher (P< 0.01) after transportation than no stress in liver. Muscle MDA, SOD, and GSH-Px levels were significantly increased after 5-h transportation in TS pigs (P< 0.05).However, dietary supplementation with OEO significantly decreased the muscle MDA level (P=0.04), particularly in TS pigs fed OEO than in TS pigs fed the control diet (transport×diet; P=0.04).TS pigs had lowerlongissimus thoracis45 min pH (pHi) and higher longissimus lumborum (LL) drip loss than NS pigs. Notably, dietary OEO supplementationsignificantly decreased LL drip loss under transport stress.The levels of HSP27 and HSP90 mRNA were significantly increased (P<0.01) after transportation compared with the control pigs.Histological analysis revealed intestinal epithelial damage in TS pigs. VE and OEO supplementation increased VH (P<0.01), VCR (P=0.01) and VSA(P=0.03).To sum up, the conclusions of this study was to:(1) The model of transportation stress rats was established. Related parameters for temperature 35℃,the speed of shaking table for 60 r/min, Swing duration for 2 h.(2) Oregano essential oils regulated muscle damage through three aspects:Oregano essential oils lead to reduce the levels of glucocorticoid and regulate the metabolism of muscle energy, and then reduce anaerobic glycolysis and the production of lactic acid, to prevent of pH falling fast; oregano essential oils can increase the activity of antioxidant enzymes and reduce damage of free radicals in the muscle; oregano essential oils can prevent muscle cell membrane lipid oxidation,by reducing the generation of MDA.(3) OEO may exert growth-promoting effects on finishing pigs during heat stress condition. OEO supplementation might be able to alleviate transportation stress andreduce lipid oxidation. Importantly, it is found that dietary supplementation with OEO decreased meat drip loss under transport stress, which may be due to its role in protecting pigs from oxidation stress and thus reducing lipid oxidation during road transportation. These results indicated that supplementation with 25 mg/kg OEO of feed 4 weeks before being transported to the market are beneficial in alleviating TS and in improving antioxidative activity and meat quality. Moreover,25 mg/kg OEO had similar effects as 200 mg/kg VE or even better.更多还原