【作者】 温安祥；

【导师】 周定刚；

【作者基本信息】 四川农业大学 ， 特种经济动物饲养， 2008， 博士

【摘要】 中华鳖（Trionyx sinensis）是一种古老的次生性水生爬行动物,具有重要的研究价值和经济价值。80年代以来,不断扩大的市场需求刺激了养鳖业的迅猛发展。各种环境因素、人为的管理因素以及病原微生物的影响,使中华鳖经常处于应激状态,致使其抗病能力减弱,肌肉品质下降。Gln是一种条件性必需氨基酸,具有重要的营养和药理作用。本论文通过腹腔注射E.coli LPS初步建立中华鳖免疫应激模型,并通过注射或在饲料中添加Gln,从机体内分泌、抗氧化能力、免疫功能和生产性能等方面考察Gln对中华鳖的免疫保护作用及其机理。为深入探索免疫应激机理,促进中华鳖的健康养殖提供理论与实践依据。本论文分三章,设计并实施了以下7个试验。第一章,应用LPS诱导中华鳖免疫应激的研究。分设三个试验,即试验一至试验三。试验一,选择体重接近的健康中华鳖42只（339.46±25.54）g,随机分成7组（A组-G组）,每组设2个重复,每个重复3只。上午10:00时,分别给A组-G组鳖从腹腔注射LPS（剂量分别为0,50,100,250,500,1000,5000μg/kg BW）,LPS用0.5mL 0.7%NaCl配制成注射液。下午3:00时,将各组鳖断头取血,用0.5%肝素钠抗凝,2000r/min离心10min制备血浆。用放射免疫法测定血浆COR含量。结果表明:E组（500μg/kg BW）血浆COR水平极显著高于A组（对照组）、B组（50μg/kg BW）和C组（100μg/kg BW）（P＜0.01）,显著高于D组（250μg/kg BW）（P＜0.05）。试验二,选择中华鳖30只（329.66±23.98）g,随机分成5组（A组-E组）,每组设2个重复,每个重复3只。上午10:00时,A组（对照组）注射0.7%NaCl,B组-E组（试验组）分别注射500μg LPS/kg BW,注射剂量均为0.5mL。分别于注射当日下午3:00时A组、B组),注射后1 d（C组）、4 d（D组）和7 d（E组）的下午3:00时断头取血,制各血浆。用放射免疫法测定各血浆样品COR、TNF-α、IL-1β和IL-6的含量;用免疫浊度法测定血浆样品IgM、IgG和IgA的含量。结果表明:给中华鳖成鳖腹腔注射500μg LPS/kg BW 5h时,血浆COR、TNF-α和IL-1β的含量显著或极显著地高于对照组(P_COR＜0.01,P_TNF-α＜0.05,P_IL-1β＜0.01);注射1 d时,血浆IL-6的含量极显著高于对照组（P＜0.01）;注射4 d时,血浆IgM的含量显著高于对照组（P＜0.05）。试验三,选择幼鳖54只（106.46±19.53）g,随机分成3组（A组-C组）,每组设3个重复,每个重复6只。A组（对照组）注射0.7%NaCl,B组注射500μg LPS/kg BW,C组注射1 000μg LPS/kg BW。LPS用0.2mL 0.7%NaCl配制成注射液。试验2周后测试各组鳖的增重率和饲料系数。结果表明:对照组和500μg LPS/kg BW组幼鳖的增重率、饲料系数未出现显著差异,饲养过程中亦未观察到明显的行为变化。但1000μg LPS/kg BW组幼鳖的摄食能力减弱,增重率极显著降低（P＜0.01）,且注射处理后反应强烈,相互咬噬裙边现象严重。综上,通过对不同剂量的LPS处理5 h时中华鳖成鳖血浆COR含量变化规律的观察（试验一）,对注射500μg LPS/kg BW后不同时段成鳖血浆COR、TNF-a、IL-1β、IL-6、IgM、IgG和IgA含量变化的比较（试验二）,以及对比注射不同剂量LPS后幼鳖摄食、生长和行为学上的变化（试验三）,可见腹腔注射500μgLPS/kg BW能诱导中华鳖成鳖产生免疫应激反应;腹腔注射1000μgLPS/kg BW可显著抑制幼鳖生长;血浆COR含量的变化可作为监测中华鳖免疫应激反应的重要指标之一。第二章,Gln缓解中华鳖免疫应激反应机制的研究。分设三个试验,即试验四至试验六。试验四,选择中华鳖60只（317.86±22.57）g,随机分为3组,A组设4个重复,每个重复3只,B、C组设8个重复,每个重复3只。上午10:00时,A组注射0.7%NaCl,B组注射500μg LPS/Kg BW,C组注射500μg LPS+100mgGln/Kg BW。LPS和Gln用0.5mL 0.7%NaCl配制成注射液。分别于注射当日（A组-C组）、注射后1 d（A组-B组）、4 d（A组-B组）和7 d（A组-C组）的下午3:00时从各组随机取2个重复6只鳖断头取血,制备血浆;解剖取相同部位肝脏组织20 g。检测不同时段（5h,1d,4d,7d）血浆COR、TNF-α、IL-1β、IL-6、GSH、GSH-PX、MDA以及肝脏PRO、GSH、GSH-PX、MDA含量的变化。结果表明:注射LPS后,血浆中COR、TNF-α、IL-1β、IL-6、MDA和肝脏组织PRO、MDA含量在相应时段显著或极显著升高（P＜0.01）,而血浆GSH含量显著降低（P＜0.05）;补充Gln不仅缓解了血浆COR、TNF-α、IL-1β和IL-6等的上升趋势,减缓了血浆GSH的下降,而且使肝脏MDA水平极显著降低。提示Gln可通过抑制有关细胞因子（如TNF-α、IL-1β、IL-6）和糖皮质激素（如COR）的分泌,以及增强机体的抗氧化能力而对免疫应激的中华鳖产生保护作用。试验五,选择中华鳖72只（332.35±13.75）g,随机分成4组（A组-D组）,每组设6个重复,每个重复3只。A组（对照组）喂基础饲料,1周后腹腔注射0.7%NaCl;B组喂基础饲料,1周后腹腔注射500 ug LPS/kg BW;C组在基础饲料中添加2%Gln,1周后腹腔注射500 ug LPS/kgBW;D组在基础饲料中添加2%Gln,1周后腹腔注射0.7%NaCl。上午10:00时注射,注射剂量均为0.5mL。分别于注射处理后的第4d、11d和25d下午3:00从各组随机取2个重复共6只鳖称重,全身灭菌,断头取血。取表层淋巴细胞检测CD4⁺/CD8⁺和淋巴细胞转化率;取血浆测定IgM,IgG,lea和溶菌酶含量;解剖取小肠15cm（自幽门始）,用1.5 mL0.7%NaCl反复灌洗,3000r/min离心10min,取上清液检测S-IgA。结果表明:免疫应激极显著提高中华鳖成鳖血浆IgM水平（P＜0.01）,补充Gln可使LPS诱导的血浆IgM水平显著降低（P＜0.01）;免疫应激抑制了中华鳖的细胞免疫功能（P＜0.05）,补充Gln能缓解免疫应激对中华鳖细胞免疫功能的抑制（P＜0.05）;补充Gln能显著促进肠道S-IgA的分泌（P＜0.01）。试验六,选择中华鳖36只（318.53±10.26）g,随机分成6组（A-F组）,每组设2个重复,每个重复3只。A组注射0.7%NaCl;B组注射500μgLPS/kg BW;C组注射100mgGln/kgBW;D组注射100mgGln+8mgQ/kgBW;E组注射500μgLPS+100mgGln/kgBW;F组注射500μgLPS+100mgGln+8mgQ/kgBW。LPS、Gln和Q用0.5mL 0.7%NaCl配制成注射液。上午10:00时注射,下午4:00时分别取脾脏、肝脏和肠道组织,采用免疫组化法检测HSP70的表达。取血浆测定各组COR含量的变化。结果表明:注射500 ug LPS/kg BW显著促进了中华鳖肝脏和脾脏组织HSP70的合成,而注射100 mg Gln/kg BW或8 mg Q/kg BW抑制了中华鳖肠道、肝脏和脾脏组织HSP70的表达;Gln对免疫应激中华鳖的保护作用,与其诱导机体组织HSP70的表达无关。综合试验四、试验五和试验六的结果,表明Gln可通过抑制炎性细胞因子（如TNF-α、IL-1β、IL-6）和糖皮质激素（如COR）的分泌、调节免疫功能以及增强机体的抗氧化能力等途径对免疫应激中华鳖产生保护作用。第三章,Gln/LPS对中华鳖生产性能的影响。设计一个试验,即试验七,选择中华鳖72只（101.92±17.53）g,随机分成4组（A-D组）,每组设6个重复,每个重复3只。A、B组用基础饲料饲喂;C、D组饲料中添加2%Gln。第8d B、C组注射1000ugLPS/kg BW,A、D组注射0.7%NaCl。第51d进行同样处理。第95d检测各组鳖的增重率、相对生长率、饲料系数,脂体比、裙边比、肌肉的水分、粗蛋白、粗脂肪和氨基酸含量,取小肠、肝脏和脾脏切片,、观察其组织结构变化。结果表明:腹腔注射1000 ug LPS/kg BW,使幼鳖肠绒毛发育受损,肝细胞萎缩变性,显著抑制其生长,并降低肌肉品质。添加2%Gln,能促进肠道发育,显著促进生长,阻止免疫应激对中华鳖肝脏的损伤,改善免疫应激导致的中华鳖生产性能的下降。更多还原

【Abstract】 Trionyx sinensis,an old secondary aquatic reptiles,are of research importance. Since 1980s,the increasing market demand has stimulated the rapid development of Trionyx sinensis breeding.Trionyx sinensis is always under the state of stress under the impact of various kinds of environment,human management and pathogeny,so that its ability to resist disease and muscle quality are reduced.Gln is a conditionally necessary amino acid,which has not only nutrition,but also important pharmacological action.The thesis sets up the mode of the immunological stress in Trionyx sinensis by using E.coli LPS,and makes an on-the-spot investigation into the protection and mechanism of Gln on internal secretion of the mechanism,ability to resist oxidation,immunologic function, and production performance of the immunological stressed Trionyx sinensis by injecting and adding Gln in the fodder.In order to profoundly probe into immunological stress mechanism,the application of Gln to strengthen the immunological protection lays a theoretical foundation for the healthy cultivation of Trionyx sinensis.In the thesis,seven tests are set for the research on easing of immunological stress mechanism in Trionyx sinensis.Forty-two healthy Trionyx sinensises（339.46±25.54）g whose weight is close to each other are chosen in test one and divided into seven groups at random,with 2 subgroups and 3 Trionyx sinensis in each subgroup.Each Trionyx sinensis was respectively injected into different doses of LPS from abdominal cavity,and heads were cut to get plasma after 5h. COR content in the blood plasma was measured using radioimmunoassay.It is proved that the plasma COR level content in group E is strikingly higher than that in group A（comparison group）,B（50μg/kg BW） and C（100μg/kg BW）（P＜0.01）,and is much higher than that in group D（250μg/kg BW）（P＜0.05）.Thirty healthy Trionyx sinensises （329.66±23.98）g are chosen in test two and divided into five groups at random,with 2 subgroups and 3 Trionyx sinensis in each subgroup.We injected 0.7%NaCl in the comparison group and 500μgLPS/kgBW in each test group respectively,and cut their heads to get plasma at the same time after 5h、1d、4d and 7d.We measured COR、TNF-α、IL-1βand IL-6 content in each sample blood plasma using radio-immunoassay, and IgM、IgG and IgA content in the sample blood plasma using immunoturbidimetery.It is proved that when injected 500μgLPS/kgBW in the abdominal cavity of mature Trionyx sinensis for five hours,the COR、TNF-αand IL-1βcontent is much higher or strikingly higher than that in the comparison group(P_COR＜0.01,P_TNF-α＜0.05,P_IL-1β＜0.01),if injected for 1d,IL-6 content in the blood plasma is strikingly higher than that in the comparison group（P＜0.01）,if injected for 4d,the IgM content in the blood plasma is much higher than that in the comparison group（P＜0.05）.Fifty-four young Trionyx sinensises（106.46±19.53） g are chosen in test three,and divided into three groups at random,with 3 subgroups and 6 Trionyx sinensis in each subgroup.They were respectively injected into 0.7%NaCl,500μgLPS/kgBW and 1000μgLPS/kgBW.We tested weight gain ratio（WGR） and feed conversion ratio（FCR） of each group after two week’s test.It is proved that there is neither obvious difference in WGR and FCR of young Trionyx sinensis in comparison group and group 500μg LPS/kg BW,nor obvious behavior difference in the process of feeding.However,young Trionyx sinensis in group1000μgLPS/kg BW has weakened food intake ability and remarkably decreased WGR（P＜0.01）.Furthermore, there is strong reaction,frequent aggression of mutual biting skirt lateral after injection.In conclusion,through COR content change in the blood plasma of mature Trionyx sinensis by injecting different doses of LPS after 5h（test one）,change of the COR、TNF-a、IL-1β、IL-6、IgM、IgG and IgA content in th blood plasma at different periods after injection of 500μg LPS/kg BW（test two）,and changes in the food intake,growth and behavioristics of young Trionyx sinensis after injection of different doses of LPS（test three）,the injection of 500μgLPS/kg BW into the abdominal cavity could cause immunological stress reaction in mature Trionyx sinensis,while injection of 1000μg LPS/kg BW into the abdominal cavity could inhibit its growth.in young Trionyx sinensises and COR content in the blood plasma could be regarded as one significant index to supervise the immunological stress reaction of Trionyx sinensis.Sixty Trionyx sinensises（317.86±22.57） g are chosen in test four and divided into three groups at random,with 4 subgroups and 3 Trionyx sinensis in each subgroup in group A,with 8 subgroups and 3 Trionyx sinensis in each subgroup in group B and C.group A,B and C were respectively injected 0.7%NaCl、500μgLPS/Kg BW、500μg LPS+100mgGln /Kg BW into abdonamal cavity to inspect the content variation of COR、TNF-α、IL-1β、IL-6、GSH、GSH-PX、MDA in blood plasma and protein、GSH、GSH-PX、MDA in lever at different times.The result indicates that after injection of LPS,the content of COR、TNF-α、IL-1β、IL-6、MDA in blood plasma and PRO、MDA in the liver organization strikingly rise at the corresponding period（P＜0.01）,while the GSH content in blood plasma remarkably lower（P＜0.05）.The supplement of Gin not only eases the rising trend of COR、TNF-α、IL-1βand IL-6,the decrease of GSH in the blood plasma,but also prominently lowers the MDA level of liver.Gln could protect the immunological stressed Trionyx sinensis by restraining the secretion of concerned cell factors such as TNF-α、IL-1β、IL-6,glucocorticoid（COR） and improving the oxidatin resistance ability. Seventy-two Trionyx sinensises（332.35±13.75） g are chosen in test five and divided into four groups at random,with 6 subgroups and 3 Trionyx sinensis in each subgroup.Group A and B are fed with basal feed,group C and D are fed with basal feed plus 2%Gln.After being fed for one week,group B and C are injected into 500ugLPS/kgBW,group A and D are injected into 0.7%NaCl.4d,11d and 25d After injection the content of CD₄⁺/CD₈⁺ in peripheral blood,transformation ratio of T lymph,IgM、IgG、IgA in the blood plasma, muramidase and S-IgA content in intestinal canal were examined.The result proves that the immunological stress caused by LPS strikingly improves the IgM level（P＜0.01） in the blood of Trionyx sinensis.Supplement of Gln not only improves the immunologic function of cells and intestinal canal,but also prohibits the rising of IgM level in the blood plasma conducted by LPS.Thirty-six Trionyx sinensises（318.53±10.26）g are chosen in test six and divided into six groups at random,with 2 subgroups and 3 Trionyx sinensis in each subgroup.We used LPS、Gln and Q to treatt them.After 6h,we got spleen,liver and intestinal canal organization and inspected the expression of HSP70 by immunochemistry.At the same time,we inspected the COR content variety of each group. It is proved that injection of 500ugLPS/kgBW remarkably improves the expression of HSP70 in spleen and liver organization of Trionyx sinensis,while injection of 100mgGln/ kgBW or 8mgQ/kgBW inhibits the expression of HSP70 in intestinal canal,spleen and liver organization.To sum up,Gln could protect the immunological stressed Trionyx sinensis by regulating the immunological function,inhibiting the secretion of inflammatory cell factor and improving the oxidation resistance ability,while the protection might have nothing to do with the expression of HSP70.Seventy-two Trionyx sinensises（101.92±17.53） g are chosen in test seven and divided into four groups at random,with 6 subgroups and 3 Trionyx sinensis in each subgroup.Group A and B are fed with basal feed,and group C and D are fed with basal feed plus 2%Gln.On the eighth day,group B and C are injected into 1000ugLPS/kg BW and 0.7%NaCl is injected into group A and D.On 51th day,they are under the same treatment,on 95th day,we inspected the weight gain ratio,comparison growth ratio, feed conversion ratio,the content of body fat ratio,body skirt ratio,muscle moisture, coarse protein,coarse fat and amino acid,and got small intestine,liver and spleen to observe their structure variation.It is proved that injection of 1000ugLPS/kgBW caused the damage of the villus growth of the intestine,the shrinkage and degeneration of liver cells,which strikingly inhibits the growth of the young Trionyx sinensis and lowers the quality.Supplement of 2%Gln could improve the growth of intestine,remarkably enhance the growth,restrain the damage of immunological stress to the liver of Trionyx sinensis,and improve the production performance of Trionyx sinensis reduceded by immunological stress.更多还原