| 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.
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