【作者】 李燕；

【导师】 康相涛； 田亚东；

【作者基本信息】 河南农业大学 ， 动物营养与饲料科学， 2009， 硕士

【摘要】 应用电子显微镜技术、流式细胞术、免疫组织化学技术等方法,研究断喙应激对雏鸡免疫器官发育、细胞增殖、分化、凋亡及相关凋亡蛋白表达的影响,探讨断喙应激的缓解时间及应激对免疫器官发育抑制的机理,旨在为进一步研究采取措施减少断喙应激对雏鸡生长的影响奠定理论基础。试验一断喙应激对雏鸡免疫器官发育及超微结构的影响采用1日龄的健康绿壳蛋鸡240只(公鸡),随机分为2组(对照组和试验组),每组4个重复,每重复30只鸡。对照组不断喙,试验组于10日龄进行断喙。于断喙后12h、1d、3d、5d、7d随机从每组每重复抽取1只称重,心脏采血致死。取胸腺、脾脏、法氏囊,称重后保存于2.5%戊二醛磷酸盐缓冲液中,用于电子显微镜观察免疫器官淋巴细胞的超微结构。结果表明:(1)试验组雏鸡的体重在1周内均比对照组轻,在断喙后3d时差异显著(P<0.05);试验组雏鸡的胸腺重量均比对照组轻,但差异不显著(P>0.05),且在断喙后5d两者重量相差最大;试验组脾脏的重量在断喙后均比对照组轻,在5d时差异显著(P<0.05);试验组法氏囊的重量在断喙后均比对照组轻,在5d、7d时两者数值相差较大,且在5d显著差异(P<0.05)。(2)试验组雏鸡免疫器官指数均比对照组低。胸腺指数差异不显著(P>0.05);脾脏指数在断喙后5d时差异显著(P<0.05) ;试验组法氏囊指数在5d时呈现显著差异(P<0.05)。(3)试验组雏鸡免疫器官胸腺细胞可看到细胞核明显的应激反应,细胞核凝集较致密,部分细胞核即将溶解,并出现一定数量的凋亡和坏死细胞;脾脏淋巴细胞在断喙后呈现细胞体积较大,细胞核染色质凝集成块,细胞质形成的空泡较多,线粒体并有空泡现象出现;法氏囊淋巴细胞在断喙后细胞间隙较大,细胞的核质排空,核仁高度凝集,核膜消失,凋亡数量多,并有坏死细胞出现。试验二断喙应激对雏鸡免疫器官细胞周期及凋亡率的影响采用1日龄的健康绿壳蛋鸡240只(公鸡),随机分为2组(对照组和试验组),每组4个重复,每重复30只鸡。对照组不断喙,试验组于10日龄进行断喙。于断喙后12h、1d、3d、5d、7d随机从每组每重复抽取1只,心脏采血致死。取胸腺、脾脏、法氏囊,称重后保存于-70℃冰箱用于流式细胞仪分析免疫器官细胞周期、增殖指数及凋亡率。结果表明:(1)对照组和试验组的免疫器官淋巴细胞内都是以G1期为主,其余少量为S期和M期,且试验组免疫器官淋巴细胞内G1期细胞数量均比对照组高,S、M期细胞数量比对照组低;试验组胸腺细胞内在断喙后1d、3d出现异倍体;脾脏淋巴细胞内G1期细胞数量在断喙后1d表现出显著性差异(P<0.05);S期细胞数量在3d时低于对照组,且差异显著(P<0.05),在断喙后1d和3d出现异倍体。(2)试验组雏鸡免疫器官淋巴细胞增殖指数均比对照组低。胸腺细胞增殖指数在断喙后3d时差异显著(P<0.05);脾脏淋巴细胞增殖指数在断喙后3d、5d时差异显著(P<0.05);法氏囊淋巴细胞增殖指数在断喙后3d时差异显著(P<0.05)。(3)断喙后雏鸡胸腺、脾脏、法氏囊淋巴细胞的凋亡率均比对照组高,胸腺淋巴细胞凋亡率断喙后5d时差异显著(p<0.05);雏鸡断喙应激后胸腺细胞和法氏囊淋巴细胞凋亡率呈上升-下降-上升-下降的趋势,在7d时凋亡率最低;脾脏淋巴细胞的凋亡率先上升后下降,在断喙后3d时凋亡率最高,7d时未检测到凋亡细胞。试验三断喙应激对雏鸡免疫器官内相关凋亡蛋白表达的影响采用1日龄的健康绿壳蛋鸡240只,随机分为2组(对照组和试验组),每组4个重复,每重复30只鸡。对照组不断喙,试验组于10日龄进行断喙。于断喙后12h、1d、3d、5d、7d随机从每组每重复抽取1只。取胸腺、脾脏、法氏囊,称重后保存于中性福尔马林溶液中用于免疫组织化学测定免疫器官淋巴细胞相关凋亡蛋白(Bcl-2和Bax)表达量的变化。结果表明:断喙应激没有影响Bcl-2和Bax蛋白在免疫器官内的表达部位;降低了Bcl-2在免疫器官淋巴细胞内的表达量,胸腺细胞、脾脏淋巴细胞、法氏囊淋巴细胞内的表达量分别在断喙后1d、3d、1d时表达量最低;提高了Bax在免疫器官淋巴细胞内的表达量,在胸腺细胞、法氏囊淋巴细胞内的表达量均在断喙后5d时最高,脾脏淋巴细胞内的表达量呈上升-下降-上升的趋势。更多还原

【Abstract】 The electron microscope technique, flow cytometry, immunohistochemical technique and other methods were adopted to study the effects of trimming stress on the development of immune organ, cell proliferation, cell differentiation, cell apoptosis and the expression of apoptosis-related protein in chickens. The remission time of trimming stress and the mechanism of stress to immune organ inbition were also discussed. And all of these researches aimed at laying the theoretical foundation of the further study which contribute to take measures to decreasing effects of trimming stress in chickens growth.Experiment 1: Effects of trimming stress on the development and ultra structure of immune organ in chickens240 healthy green-shell chickens in one day old which were adopted in this study were randomly divided into two treatment, and four repeats which have 30 green-shell chickens were included in every treatment. The chicken in control group were not cut beak, and chickens in experimental group were cutted beak in 10-day-old. Two chickens which were chose randomly from each repeat in every group in 12h, 1d, 3d, 5d, 7d after trimming were weighted, and the thymus, spleen and bursa of fabricius of these chickens were extracted and conserved. The results are as follows: (1) The weight of chickens in experimental group were lighter than that in control group within 1 week, and the difference between two groups were significant in fifth day after debeaking(P<0.05). The weight of thymus in experimental group were lighter than that in control group, and the difference between two groups in fifth day after debeaking was largest, but the difference wasn’t significant(P> 0.05). The weight of spleen in experimental group were lighter than that in control group, and the difference between two groups was significant in fifth days (P <0.05). The weight of bursa of fabricius in experimental group were lighter than that in control group after debeaking, the difference between two groups were larger in fifth day and seventh day, and the data between two groups in the fifth day showed significant differences (P <0.05). (2) The thymus index of chicks in experimental group were higher than that in control group in fifth day after debeaking, in the rest of the time, the thymus index in experimental group were lower than that in control group, and the difference between two groups was not significant(P> 0.05). The spleen index of chickens in experimental group were lower than that in control group after debeaking, and the difference in fifth day after debeaking between two groups was maximal, and it was also significant(P <0.05); the numerical value of spleen index between two groups in seventh day were basically close. The bursa of fabricius index of chicks in experimental group were higher than that in control group in first day after debeaking, but the difference was not significant(P> 0.05); and in the rest of time, the bursa of fabricius index of chicks in experimental group were lower than that in control group, and the bursa of fabricius index of chicks in fifth day after debeaking between two groups was significant (P <0.05), the numerical value of bursa of fabricius index between two groups in seventh day were basically close. (3) The obvious stress response of nucleolus was observed from thymocyte in immune organ of chicks in experimental group, the phenomena was as follows: cell nuclear agglutinated more dense than normal, part of the nucleus were about to dissolve, and a certain amount of apoptosis cells and necrotic cells emerged; the phenomenon such as: cell volume became larger, chromatin of cell nuclear agglutinated conglobation, cytoplasm formed more vacuole and mitochondria emerged vacuolization phenomenon were observed in splenic lymphocyte after debeaking; and the phenomenon such as: intercellular space became larger, nucleoplasm emptying, nucleolus high agglutinated, nuclear membrance disappeared, the number of apoptotic cell increased, and necrotic cells emerged were observed in bursa of fabriciu cell after debeaking.Experiment 2: Effects of trimming stress on the cell cycle and the rate of apoptosis of immune organ in chickens240 healthy green-shell chickens in one day old which were adopted in this study were randomly divided into two treatment, and four repeats which have 30 green-shell chickens were included in every treatment. The chicken in control group were not cut beak, and chickens in experimental group were cutted beak in 10-day-old. Two chickens which were chose randomly from each repeat in every group in 12h, 1d, 3d, 5d, 7d after trimming .the thymus, spleen and bursa of fabricius of these chickens were conserved in negative temperature at 70℃were used to analyse the cell cycle, proliferation index and apoptosis rate of immune organ by flow cytometry. The results are as follows: (1) The lymphocyte within immune organ in control group and experimental group were mainly in G1 phase, and the remaining small amount were in S phase and M phase, the number of the lymphocyte which are in G1 phase within immune organ in experimental group were more than that in control group, and the number of cells in S phase and M phase were less than that in control group; the heteroploid were found in thymocyte in experimental group in the first day, third day and fifth day after debeaking; the number of spleen lymphocyte in G1 phase showed significant difference(P <0.05); the number of cell in S phase were less than that in control group, and the difference between two groups was significant (P <0.05); the heteroploid were also found in spleen lymphocyte in 1d and 3d after debeaking. (2) Trimming stress brought about some decrease in lymphocyte proliferation index to some extent, and after debeaking 3d, the proliferation index began to rise. The difference of thymocyte proliferation index, spleen lymphocyte proliferation index and bursal of fabriciu lymphocyte proliferation index between the experimental group and control group were significant (P <0.05) after debeaking 3d, and the difference of spleen lymphocyte proliferation index was also significant (P <0.05) in fifth day after debeaking. (3) The apoptosis rate of lymphocyte in thymus, spleen and bursal of fabriciu in experimental group were higher than the rate in control group, and the apoptosis rate of lymphocyte in thymus between experimental group and control group showed significant differences (p<0.05) after debeaking 5d, the difference in the rest of the time was not significant (P> 0.05); the rise-fall-rise trend was found in apoptosis rate of thymocytes and bursal of fabriciu lymphocyte, and the apoptosis rate was lowest in the seventh day; the rise-fall trend was observed in apoptosis rate of spleen lymphocyte, and the apoptosis rate was lowest in the third day after debeaking, apoptotic cells were not detected in the seventh day.Experiment 3: Effects of trimming stress on the expressions of the apoptotic genes(Bcl-2 and Bax) of immune organ in chickens240 healthy green-shell chickens in one day old which were adopted in this study were randomly divided into two treatment, and four repeats which have 30 green-shell chickens were included in every treatment. The chicken in control group were not cut beak, and chickens in experimental group were cutted beak in 10-day-old. Two chickens which were chose randomly from each repeat in every group in 12h, 1d, 3d, 5d, 7d after trimming .the thymus, spleen and bursa of fabricius of these chickens were conserved in netural formalin solution were used to determine the change of the expression of apoptosis protein(Bcl-2 and Bax) in lymphocytes of immune organ . The results are as follows: Trimming stress did not affect the expression sites of Bcl-2 and Bax protein in immune organs; but it reduced the expression level of Bcl-2 in immune organ lymphocytes, the expression level of Bcl-2 in thymocytes, spleen lymphocytes and bursal of fabriciu lymphocytes were lowest in 1 day, 3 days and 1 day after debeaking, respectively. Trimming stress increased the expression level of Bax in immune organ lymphocytes, the expression level of Bax in thymocytes and bursal of fabriciu lymphocytes were up to highest in fifth day after debeaking, and the expression level of Bax in spleen lymphocytes showed a rise- fall-rise trend.更多还原