【作者】 王岩；

【导师】 彭克美；

【作者基本信息】 华中农业大学 ， 动物遗传育种与繁殖， 2008， 博士

【摘要】 非洲鸵鸟主要生活在非洲和酷热的阿拉伯沙漠地带。雌鸵鸟一般在2-2.5y时才达性成熟,是禽类中性成熟最晚的动物,其育雏期也很长,为3mon。究竟是什么原因使得鸵鸟性成熟这么晚?其性腺必然有其独特的性状。服用雌激素可使血清17β-雌二醇、血清铜蓝蛋白和甘油三酯水平明显升高,补硼后,这些升高更为明显;说明硼有雌激素样作用,并能增强雌激素的功能。因此,能否通过添加硼而提高鸵鸟的的养殖效益具有重要的科学意义。鉴于此,本研究以非洲雏鸵鸟为试验动物,分别添加硼酸0mg/L、100mg/L、200mg/L、400mg/L,利用透射电镜技术、放射免疫测定、组织化学法、免疫组化定位表达及原位缺口末端标记,免疫印迹等多种方法,从细胞、亚细胞、血清激素水平、蛋白分析等多个层面系统地研究雌性雏鸵鸟下丘脑-垂体-卵巢轴（HPO）的组织结构特点,以及添加硼酸后对HPO轴的作用机理。主要研究结果如下:1硼对雌性雏鸵鸟下丘脑的影响本试验以45d雌性雏鸵鸟为试验动物添加硼酸,石蜡切片,HE染色、免疫印迹、免疫组化SABC法对MT1在雏鸵鸟下丘脑内分布进行定位,研究硼对雏鸵鸟下丘脑的影响及作用。结果显示,下丘脑室旁核细胞单位面积数目在400mg/L组最高,差异不显著。大、中型细胞率在200mg/L组最高,显著高于其它3组;下丘脑视上核各组细胞单位面积数目在200mg/L组最高,与对照组相比差异非常显著。大、中型细胞率在200mg/L组最高,400mg/L组主要以小型细胞居多;下丘脑弓状核细胞单位面积数目在对照组最高,200mg/L组次之,差异显著;400mg/L组最少,差异极显著,与对照组相比差异极显著。对照组以小型细胞居多。室旁核褪黑素受体免疫阳性细胞（MT1ir）细胞在阳性强度表达方面,200mg/L组阳性表达最强,差异极显著,400mg/L组MT1ir强度表达最弱,差异极显著;视上核MT1ir数量以400mg/L组最多,排列密集,但是平均积分光度最低,细胞形态不完整,差异极显著;弓状核对照组MT1ir细胞数量最多,排列密集。100mg/L组平均积分光度最高,阳性表达最强,差异不显著。400mg/L组平均积分光度最低,差异显著。结果表明室旁核、视上核在200mg/L组具有显著促进作用,但是400mg/L具有显著抑制作用;弓状核在100mg/L组时具有较小的促进作用,400mg/L组具有显著的抑制作用。2硼对雌性雏鸵鸟垂体的影响采用Peacse垂体染色、免疫印迹法、免疫组化SABC法对添加硼后雏鸵鸟腺垂体3种细胞及MT1在下丘脑内分布进行定位。Peacse法结果显示对照组内嗜酸性细胞数量较多,细胞轮廓清晰,呈三角形或圆形。嗜碱性细胞大小不等,胞质内含有嗜碱性颗粒。嫌色细胞数量多,细胞体积小着色淡,细胞界限不清。添加硼100mg/L组嗜碱性细胞数量增多。200mg/L组嗜碱性细胞数量最多。400mg/L组嗜酸性、嗜碱性细胞成团分布,嫌色细胞数量最少;SABC法结果显示对照组腺垂体中的MT1ir细胞呈深浅不等的棕黄色,胞体呈不规则形,成堆或分散分布,阳性免疫反应颗粒主要分布在细胞核上,胞质不着色。100mg/L组腺垂体中的MT1ir细胞数目增多,免疫阳性细胞较强。200mg/L组腺垂体中的MT1ir细胞数目增多,免疫阳性细胞以强阳性为主。400mg/L组腺垂体中的MT1ir阳性细胞数目减少,免疫阳性细胞以弱阳性为主。结果表明:添加硼100mg/L、200mg/L组都能增加嗜碱性细胞的数目;添加100mg/L、200mg/L硼后MT1ir细胞数目呈递增趋势,强度也增强,但是400mg/L组对MT1ir阳性细胞产生了抑制作用。3硼对雏鸵鸟卵巢的影响制作光镜和电镜样本,提取总蛋白,采用HE、免疫组化SABC、免疫印迹法,研究雌性雏鸵鸟卵巢的组织学结构特点以及添加硼酸对雏鸵鸟卵巢的作用。结果表明,添加添加100mg/L硼能够增大卵巢的初级卵母细胞直径,差异非常显著,100mg/L硼能够显著增加原始卵泡的数目,200mg/L能够显著增加原始卵泡、初级卵泡、次级卵泡数目,并且各级卵泡的卵泡细胞以及卵母细胞MT1ir细胞强度比对照组显著增强,400mg/L能够显著减少原始卵泡数目。4添加硼后血清中E₂、P₄的变化采用放射免疫技术,在添加硼15d,30d,45d后检测雏鸵鸟血清中E₂、P₄的变化。结果显示添加硼15d后,100mg/L、200 mg/L组与对照组相比血清中E₂、P₄水平都升高,差异不显著。400 mg/L组的E2水平降低,差异极显著,P4差异不显著;添加硼30d后,100mg/L、200 mg/L以及400 mg/L组与对照组相比血清中E₂、P₄水平差异都不显著。添加硼45d后,100mg/L、200 mg/L组血清中E₂水平与对照组相比升高,200 mg/L组差异极显著,400 mg/L组的水平降低,差异不显著。200 mg/L、400 mg/L组的P₄水平与对照组相比降低,差异显著。结果表明,适量添加硼能够增加血清中E₂水平,但是在添加量达到400 mg/L的时候会降低血清中E₂水平;添加硼15-30d时对P₄的影响较小,但是添加到45d时血清内P₄水平显著降低。5硼对雌性雏鸵鸟下丘脑-垂体-卵巢轴细胞凋亡的影响采用原位缺口末端标记（TUNEL）技术对添加不同剂量硼后雏鸵HPO轴进行检测,系统研究正常雏鸵鸟细胞凋亡规律及硼对此轴内细胞凋亡的影响,并对雏鸵鸟体内凋亡的组织细胞进行定位。结果表明硼对下丘脑视上核、弓状核、垂体以及卵巢都能产生显著作用,在100、200mg/L组能显著降低其凋亡指数,400mg/L硼能显著增大其凋亡指数。硼对下丘脑室旁核在各组间差异不显著。更多还原

【Abstract】 African ostrich originated from African desert.The female ostrich reach sexual maturity until 2-2.5y,which is the latest one in the avian.And it’s brood time lasts for 3 months.What’s the reason for it? As we know the gonad must be different from others. Taking estrogen can cause serum levels of 17β-E2,copper-protein,triglyeride heightened obviously,and after taking boron,these levels heightened much more.Through the additive of boric acid to ostrich chicks,we can explore the estrogen-like mechanism of boron,especially the effect on hypothalamic- pituitary-ovarium axis.In this research, ostrich chicks were treated by additive of various doses boron acid（0mg/L、100mg/L、200mg/L、400mg/L）.Using the methods of transmission electron microscopy,radioactive immunoassay,histochemical method,immunohistochemistry,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling（TUNEL） and western bolt,the structural characteristics of ostrich chicks’ HPO and the role of boron acid and apoptosis in the HPO after additive different dose of boron acid were illustrated.The main research results are as follows:1 Changes of female ostrich chicks hypothalamus after added B45d femal ostrich chicks with adding boric acid to the hydroposia were used in the present study to investigate the effects of boric acid on hypothalamus of ostrich chicks through locating the MT1 cells in hypothalamus of ostrich chicks after making light microscope sample copy by hematoxylin-eosin staining and immunohistochemistry SABC.The results showed that the number of paraventricular hypothalamic nucleus per unit area was highest in the group of 400 mg/L,but the difference was not significant. The rate of large and middle cells was highest in group of 200 mg/L,which was significantly higher than the other three groups;The number of supraoptic hypothalamic nucleus per unit area in group of 200 mg/L was highest among the groups,which was significantly higher than that of the control group.The rate of large and middle cells was highest in group of 200 mg/L and there were mainly small size cells in group of 400 mg/L; The number of nucleus arcuatus hypothalami per unit area was highest in control group and second highest in group of 200 mg/L,the difference between the two groups was significant;it was lowest in group of 400 mg/L and the difference was extremely significant comparing with the control group.The cells in control group were most small size.The positive MTlir cells in paraventricular nucleus were most in group of 200 mg/L and fewest in group of 400 mg/L,the differences were highly significant respectively. The number of MTlir cells in supraoptic nucleus was highest in group of 400 mg/L and the cells arranged closely with the lowest average integrated optical density and morphological incompletion.The difference between group of 400 mg/L and control group was highly significant.MT1 ir cells in nuclei arciformes were most in control group and arranged intensively.The average integrated optical density was highest in group of 100 mg/L with significant difference and lowest in group of 400 mg/L with no significant difference.The results demonstrated paraventricular nucleus and supraoptic nucleus had significant auxoaction in group of 200 mg/L but possessed significant depressant effect in group of 400 mg/L.The arcuate nuclei could promote slightly in group of 100 mg/L but depress significantly in group of 400 mg/L.2 Changes of female ostrich chicks pituitary after added BThree kinds of cells and MT1 in anterior pituitary of female ostrich chicks added B were located by the means of Peacse and SABC.Peacse showed that,there were more acidophilic cells,which were triangle or triangle with clear cell boundary,in control group.Furthermore,the size of basophilic cell were unequal,and there were many chromophobe cells with small size,light stain,and unclear cell boundary.Basophilic cell population increases in the group added B at the dosage of 100mg/L,and was maximum at the dosage of 200mg/L.In the group of 400mg/L,eosinocytes and Basicytes were agglomerate,and chromophobe cells were the least.SABC showed that,MT1ir cells stained with light/dark-colored brown.Cell with irregular shape was located stacked or scattered,and positive drops were main located in nucleus.In the group of 100mg/L,both MT1ir cell population and positive cells increased.In group of 200mg/L,MT1ir cell population increased more and most cells were strong positive.In group of 400mg/L, MT1ir cell population decreased and most cells were week positive.The results indicated that,when added B at the dosage of 100mg/L、200mg/L,all basicytes,MT1ir cells and positive cells increase.On the other hand,there was an inhibition to MT1ir cells at the dosage of 400mg/L.3 Changes of female ostrich chicks ovary after added BThe histological structure of ostrich chicks ovary and the effect of boron acid on the ovary were studied by the means of histology,SABC,western blot.The result showed that the diameter of primary oocyte was increased significantly in 100mg/L group,the number of primordial follicle,primary follicle,secondary follicle increased significantly,and the MT1ir intensity in the follicular,oocyte of every follicular,but the number of primordial follicle decreased significantly in 400mg/L group.4 Changes of E₂ and P₄ in serum after added BE₂ and P₄ in serum were detected using radio-immunity technology at 15d,30d,45d after added boric acid.The results showed that the levels of E₂ and P₄ in serum were higher in 100mg/L and 200mg/L groups than that in the controls.And the disparity was not significant.The level of E₂ decreased in the 400mg/L group,and the disparity was highly significant.However,the disparity of P₄ was not significant.At the 30d after adding to boric acid,the disparities of E₂ and P₄ were not significant in 100mg/L, 200mg/L and 400mg/L groups compared to the controls.At the 45d after adding to boric acid,the level of E₂ was higher in 100mg/L and 200mg/L groups than that in the controls. And the disparity of 200mg/L group was highly significant.The level of 400mg/L group decreased,and the disparity was not significant.The level of P₄ was lower in 200mg/L and 400mg/L groups than that in the controls.And the disparity was significant.The results revealed that adding to the appropriate amount of boric acid could increase the level of E₂ in serum,but the level would decrease when the amount up to 400mg/L.The effect on the level of P₄ was slight at the 15-30d after adding to boric acid,but at the 45d, the level of P₄ in serum decreased significantly.5 The changes of HPO apoptotic after added BThe regular of Apoptosis and the effect of boron acid on apoptotic in ostrich chicks were studied by the means of Tunel method.The results showed that there are significant effect of boron acid on SON,ARC,hypophysis and ovary.The Apoptotic index was significant decreased in the 100 and 200mg/L group,but it is contra in 200mg/L group. There have no significant disparation on PVN by add boron acid.更多还原