| In this study ,150 Tianfu ducks were divided into 30 groups as follows: 17 groups in the embryonic period (one group per day from 12 to 28 days embryonic stage), 13 groups after hatching(0,1,3,5,8,11,14,17,20,23,26,29,32 weeks) . Using morphological method, light and electron microscopy, immunohistochemisty, terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and in sita hybridization techniques, the morphological structures, cell proliferation and apoptosis, apoptotic proteins and genes and neuropeptide expression during the embryonic and post embryonic development of the central immune organs of Tianfu duck were studied. All results are shown as follows:1. The morphological and histological structures of the duck bursa of Fabricius and thymus underwent obvious age-related changes during the embryonic and post embryonic development. The post embryonic development of these organs was divided into three stages as follows: the continuous development period (from 0 to 8 weeks), the matured and sustained period (in the bursa: from 8 to 14 weeks; in the thymus: from 8 to 17 weeks) and the regressive period (in the bursa: from 17 to 32 weeks; in the thymus: from 20 to 32 weeks).At 26 days embryonic stage(ES), the bursa and thymus had almost possessed the matured histological structure. The bursa and thymus grew very rapidly from 1 to 3 weeks and 3 to 5 weeks after hatching (AH), respectively, and gained their maximum in size and absolute weight at 8 weeks AH. The growth of bursal follicle associated epithelium (FAE),taking part in the post embryonic growth of the follicle cortex and being related to the bursal regression, showed age-related changes during the post embryonic development. The thymic corpuscle and epithelial cyst, with the latter outnumbering the former and presenting several features, were important functional components of the thymic matrix in the duck thymus. The bursa of Fabricius regressed earlier and more quickly than the thymus.2. In the bursa, the apoptosis ratios of lymphocytes in the follicle cortex followed a series of changes as rising (from 22 days ES to neonatal stage), dropping (from 3 to 5 weeks AH) and maintaining at a certain level (from 0 to 3 weeks and from 5 to 14 weeks AH), and increasing again(from 14 to 29 weeks AH). Increasing from 1 to 3 weeks AH, the changing pattern of the apoptosis ratios of lymphocytes in the follicle medullar in other groups was similar to those in the follicle cortex. The apoptosis ratios of busal lymphocytes in the medullar were significantly higher from 22 days ES to 14 weeks AH or lower from 17 to 29 weeks AH than those in the cortex. The apoptosis ratios of thymic lymphocytes in the cortex and medullar kept unchanged (from 22 to 26 days ES and from 8 to 17 weeks AH) and increased (from 26 days ES to 8 weeks AH, and from 17 to 32 weeks AH).In each group, the apoptosis ratios of thymic lymphocytes in the cortex were significantly higher than those in the medullar. The ultrastructure and process of lymphocytes apoptosis in the bursa were similar to those in the thymus. The nuclei of apoptotic lymphocytes with great changes presented different features. In the bursa, the proliferative indexes of lymphocytes in the follicle cortex and medullar increased during the embryonic development and decreased during the post embryonic development. Moreover, the proliferative indexes of bursal lymphocytes in the medullar were significantly higher than those in the cortex in each group. In the thymus, the proliferative indexes of lymphocytes in the cortex and medullar decreased during both the embryonic and post embryonic development. The proliferative indexes of thymic lymphocytes in the cortex were significantly higher than those in the medullar in each group.3.In the bursa, the Bcl-2 positive ratios and the ratios of Bcl-2 and Bax of lymphocytes in the follicle cortex and medullar paralleled reversely with the apoptosis ratios of lymphocytes (except 26 days ES to 14 weeks AH and from 5 to 14 weeks AH in the follicle cortex, and from 5 to 14 weeks AH in the follicle medullar ). The Caspase-3, Bax, Fas and FasL positive ratios of lymphocytes in the follicle cortex and medullar paralleled positively with the apoptosis ratios of lymphocytes (except Bax positive ratios from 22 days ES to 5 weeks AH). Furthermore, the ratios of Bcl-2 and Bax of lymphocytes in the follicle cortex were significantly higher from 22 days ES to 14 weeks AH, and lower from 17 to 29 weeks than those in the follicle medullar, which paralleled reversely with the apoptosis indexes of lymphocytes in the follicle cortex and medullar. The positive ratios of Caspase-3, Fas and FasL of bursal lymphocytes in the follicle cortex were significantly lower from 22 days ES to 14 weeks AH, and higher from 17 to 29 weeks AH than those in the medullar, which paralleled positively with the apoptosis ratios of lymphocytes in the follicle cortex and medullar. In the thymus, the ratios of Bcl-2 and Bax of lymphocytes in the cortex and medullar paralleled reversely with the apoptosis ratios of lymphocytes from 26 days ES to 8 weeks AH and from 20 to 32 weeks AH. The Bcl-2 positive ratios of lymphocytes in the thymic cortex and medullar paralleled reversely with the apoptosis ratios of lymphocytes from 20 to 32 weeks AH. The positive ratios of Caspase-3 ,Bax and Fas of lymphocytes in the thymic cortex and medullar paralleled positively with the apoptosis indexes of lymphocytes(except Fas positive ratios from 3 to 8 weeks AH).The ratios of Bcl-2 and Bax, the positive ratios of Caspase-3 and Fas of thymic lymphocytes in the cortex were significantly lower or higher than those in the medullar in each group which paralleled reversely or positively with the apoptosis ratios of lymphocytes in the cortex and medullar, respectively. The P53 positive ratios of lymphocytes in the bursa and thymus showed no age-related changes during the embryonic and post embryonic development, so did the FasL positive ratios of lymphocytes in the thymus.4. Only during embryonic development of the bursa did the change patterns of the positive ratios of bcl-2 mRNA of lymphocytes in the follicle cortex and medullar parallel positively with those of the positive ratios of Bcl-2 proteins. During the embryonic and post embryonic development of the bursa, the change patterns of the positive ratios of bax mRNA of lymphocytes in the follicle cortex and medullar were similar to those of the positive ratios of Bax proteins (except 20 to 29 weeks AH in the medullar). bcl-2 and bax mRNA in the bursa were different from those of their proteins in the tissue distribution, whose expression presented the regulative mechanism after transcription. During the embryonic and post embryonic development of the thymus, the bcl-2 and bax mRNA of lymphocytes in the thymic cortex and medullar were similar to those of the positive ratios of Bcl-2 and Bax proteins both in the changing pattern of their positive ratios and in their tissue distribution, whose expression appeared to be modulated transcriptionally.5. The first expression being different, CGRP, VIP, SP, NPY, SS positive cells whose number underwent certain age-related changes were widely located in the bursa and thymus. CGRP, VIP, SP, NPY positive nerves with predominant distribution in the medullar region were occurred in the thymus during the post embryonic development. Only the VIP positive nerves were observed in the bursa. CGRP, VIP, SP, NPY, SS expressions were detected in the bursal FAE and the thymic corpuscles and epithelial cysts.Based on the results mentioned above, it shows that the morphological and histological structures, cell proliferation and apoptosis, apoptotic proteins and genes and neuropeptide expression in the central immune organs of Tianfu duck undergo obvious age-related changes during the embryonic and post embryonic development. The cell proliferation, apoptosis and neuropeptide expression in these organs supervise and control the proliferation and differentiation of T, B lymphocytes cooperatively, which plays significant and regulatory roles in the development and regression of the duck central immune organs. Caspase-3, Bcl-2, Bax, Fas, FasL and P53 apoptotic proteins and bcl-2 and bax mRNA genes modulate the apoptosis of lymphocytes in the bursa and thymus. The changing patterns and regulatory mechanism of the whole development and regression of the central immune organs of Tianfu duck are studied systematically for the first time, which might provide the theoretical evidences for the modulation of their development and stabilization and formulation of the reasonable immune procedures. |
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