【作者】 程佳月；

【导师】 彭克美； 李奎；

【作者基本信息】 华中农业大学 ， 基础兽医学， 2011， 博士

【摘要】 非洲鸵鸟(Struthio camelus)是世界上现存体形最大的鸟,现已列为世界濒危动物范畴。非洲鸵鸟原产于非洲草原和阿拉伯沙漠,属于脊索动物门,脊椎动物亚门,鸟纲,鸵鸟目,鸵鸟科,鸵鸟属。成年鸟身高240-280cm,体重130-200kg,腿长而粗,肌肉发达。鸵鸟已经失去飞翔能力,其主要运动是在陆地上行走和奔跑,奔跑时速一般为50 km/h左右,最高可达90～100 km/h。所以推测其腿骨在组织结构和生理功能上应与其它鸟类的有所不同。有关非洲鸵鸟骨骼的解剖学特征,前人已进行了研究,表明鸵鸟骨骼在结构形态上与其它鸟类的骨骼基本相似。但是关于非洲鸵鸟腿骨的是否有特殊的组织学特征,至今未见更为深入的相关报道。在鸵鸟的养殖过程中,6月龄以内的小鸵鸟很容易发生胫骨骨折,影响育成率,降低鸵鸟养殖业的经济效益。导致雏鸵鸟发生腿病的最重要病因就是营养因素,即日粮中钙、磷、维生素D等营养成分不足、或日粮中钙、磷含量的比例不当、或肠道对营养元素的吸收受到阻遏。同时,鸵鸟是世界上最大的鸟类,身体躯干部分的重量也很大；与其它鸟类相比,鸵鸟的生长速度很快,这就对钙、磷等营养元素有着更高的要求；同时又由于骨的生长发育要略慢于肌肉的生长发育。这就使得雏鸵鸟极易发生腿病,即胫骨的骨折或关节的变形等。雏鸵鸟的腿病在国内外每个鸵鸟场均有不同程度的发生,给鸵鸟产业造成了很大程度的威胁和经济损失。虽然国内外对鸵鸟的研究不断深入,但是这一问题仍然没有被有效地解决。硼,原子序数5,原子量10.811。越来越多的证据表明硼可以促进骨的生长发育。硼对骨生长的影响表现在影响软骨和骨结构、维生素D代谢和矿物质代谢等方面,它可以增加骨量和骨的抗骨折能力、可提高缺钙和缺镁大鼠大腿骨中骨灰分含量、能够提高血中钙、磷含量,并且对微量元素铁、铜、锌的代谢也有一定的促进作用。但尚未报道硼对雏鸵鸟的腿病问题是否有改善作用。综合以上三方面的原因,本课题利用大体解剖、骨磨片技术(结晶紫染色)、扫描电镜(SEM)技术、骨脱钙石蜡切片HE染色技术、细胞凋亡检测及图像分析技术、以及骨密度测定、蛋白质双向电泳技术、放射免疫法、原子吸收分光光度、流动注射分析综合手段,对非洲鸵鸟胫骨的形态学、雏鸵鸟胫骨发育的特征、患腿病雏鸵鸟胫骨病理变化及硼对雏鸵鸟胫骨发育的影响进行全面、系统的研究,旨在为非洲鸵鸟(尤其是雏鸟)的疾病防控及养殖业发展提供研究基础和理论依据。主要研究内容和研究结果包括以下4个方面：1.非洲鸵鸟胫骨形态学特征本试验以1岁非洲鸵鸟为试验动物,采用大体解剖、骨密度测量、骨磨片制作、骨脱钙石蜡切片制作、结晶紫染色、HE染色技术,研究非洲鸵鸟胫骨的形态学结构特征。结果显示：在非洲鸵鸟的胫骨外侧附着有腓骨,但腓骨呈现一种退化的迹象,在距远端关节7～10cm左右的地方与胫骨愈合在一起。在非洲鸵鸟的体内,胫骨独自支撑着身体的全部重量。鸵鸟胫骨的远端关节不能像人类的踝关节一样左右旋转,只能前后伸曲活动,这一解剖学特征更有利于承重,并有利于提高奔跑的速度和效率。1岁非洲鸵鸟胫骨的骨密度值很高,平均值为5.656 g/cm2,不同部位的骨密度值差别十分显著。胫骨组织中有大量骨盐沉积,十分坚硬。鸵鸟胫骨具有一个“过渡区”,位于外环骨板与骨单位的交接处,这个区域着色特殊。2.雏鸵鸟胫骨发育的特点本研究以1d、45d和90d健康非洲雏鸵鸟为试验动物,采用大体解剖、骨密度测量、扫描电镜、骨脱钙石蜡切片制作、HE染色技术,研究雏鸵鸟胫骨的发育特征。研究结果显示：1～90日龄雏鸵鸟胫骨的生长十分迅速。随着日龄的增长,雏鸵鸟胫骨的长度、重量、骨密度(BMD)的增长均为十分显著(P<0.01)。从显微水平和超显微水平上观察发现：在1日龄雏鸵鸟胫骨中,过渡型骨小梁构成了初级骨松质,由初级骨松质构成最初形成的骨干,大量成骨细胞贴附在软骨表面形成薄层初级骨松质,过渡型骨小梁十分菲薄。45日龄雏鸵鸟的胫骨中,骨组织的重吸收活动十分活跃,过渡型骨小梁正在被破骨细胞吸收并重建。同时,骨小梁中央的软骨基质钙化过程也十分活跃,软骨基质钙化区中软骨细胞逐渐退化,留下空泡状的宽大的陷窝。90日龄雏鸵鸟的胫骨中已经形成较厚的骨密质的雏形,但还不是真正意义的骨单位。在新形成的类似骨单位结构的最外周,已经形成了类似成型骨单位的粘合线,可以此来识别新形成的类似骨单位的结构。因此,在雏鸵鸟的饲养过程中,应加强对雏鸟饲养环境的维护和日粮的合理配比,保证雏鸟骨骼生长发育的需要。3.90日龄正常鸵鸟与患腿病鸵鸟胫骨差异本研究以90日龄健康正常的和患有腿病的雏鸵鸟为试验动物,利用骨密度测量、扫描电镜、骨脱钙石蜡切片制作、蛋白质二维电泳技术,揭示正常与患病雏鸵鸟的胫骨组织学与蛋白质水平的差异。试验结果显示：患有腿病的雏鸵鸟的骨密度值显著低于正常的雏鸵鸟。与正常雏鸵鸟胫骨的组织结构相比,患腿病雏鸵鸟胫骨骨小梁面积明显减少,骨小梁变细,且出现断裂消失的现象,骨小梁中间的软骨基质钙化活动也不如正常组的那样活跃；经扫描电镜观察,正常90日龄的雏鸵鸟胫骨的内表面有许多球状新生的骨组织,在新形成的类似中央管的管壁上,有许多骨小管的开口。而患腿病雏鸵鸟胫骨中的骨小梁多处出现微骨折现象,在骨组织观察面上、布满了大大小小的吸收坑。根据这些结果分析,患有腿病的雏鸵鸟虽然腿病各有不同,但基本上都会出现骨量变少和骨小梁微断裂现象。根据试验结果分析显示,患腿病的雏鸵鸟其病因可能为骨质疏松症。此外,本实验通过荧光差异凝胶电泳技术,对正常组与患病组的蛋白质样本进行了进行2D DIGE检测,并用DeCyder v.5.02图像分析软件对DIGE图像进行分析,通过对相匹配的蛋白质点丰度进行差异比较,筛选出154个具有显著性意义(P<0.05)的1.5倍以上的差异蛋白质点。之后计划对已筛选到的154个差异表达的蛋白质点进行酶切肽提取,之后进行质谱鉴定,并通过工具软件对蛋白质进行分类和功能分析,利用Western blot验证DIGE的结果。4.硼的添加对雏鸵鸟胫骨发育的影响本试验选取身高体重近似的45日龄雌性非洲鸵鸟饲喂45d之后,采用扫描电镜(SEM)技术、骨脱钙石蜡切片HE染色技术、细胞凋亡检测及图像分析技术、以及骨密度测定、放射免疫法、原子吸收分光光度、流动注射分析等方法观察硼对雏鸵鸟胫骨发育的影响。结果显示：添加硼饲喂之后使鸵鸟血清中的钙水平呈现下降趋势,血清中的磷水平先下降再升高,血钙和血磷的比值([Ca]/[P])出现先升高后降低的情况。血清中的瘦素水平逐渐升高,在添加200mg/L硼之后不再升高,而雌二醇的水平波动十分剧烈。添加少量硼时,血清中雌二醇的水平显著升高；但当硼的添加量达到200 mg/L时,其含量陡然下降,只比对照组略高；而当硼的添加量继续升高时,雌二醇水平又随之升高。在饮水中添加不同剂量的硼(A、B、C、D组鸵鸟的饮水中分别添加0 mg/L、100 mg/L、200 mg/L、400mg/L)之后,4组鸵鸟的平均体重A组和C组基本持平,B组略高,D组最低；胫骨长度先出现显著的增长,之后又出现明显的下降；胫骨的重量也逐渐升高,C组的胫骨重量达到最高峰值,之后下降；胫骨周长持续增加：胫骨的灰分也随之持续增加；胫骨的骨密度先升高,而后陡然下降；胫骨骨髓腔的直径显著缩减,同时骨质的厚度也显著增加；此外,胫骨的骨髓腔不再居于胫骨的中央,而是位于靠近边侧的位置,即骨密质的最大值与最小值相差极为悬殊；4组的胫骨脱钙石蜡切片上用Tunel法检测出细胞凋亡情况,并用类流式细胞仪图像分析系统分析,得到的细胞凋亡检测结果显示,A、C两组细胞凋亡情况类似,而B、D两组中的细胞凋亡情况却显著高于B组,但T检验结果显示差异并不显著。由数据分析得出：B组的血清钙磷比最高,C、D组血清瘦素水平最好,C组雌二醇水平最优,B组体重数值最高,B、C组胫骨长度数值最高,C组胫骨重量数值最高,加硼组的胫骨周长均优于对照组,C、D组灰分占优,C组骨密度数值最高,骨密质厚度B、C组占优,A、C组凋亡细胞数量最少。综上所述,推测C组中雏鸵鸟胫骨发育的情况最为良好,即推测在4组不同剂量中,最适硼的添加量为200 mg/L。但真正的最适添加量仍需要更为严格的加硼梯度试验来进一步确定,以便更加有效地应用于鸵鸟饲养的生产实践中。更多还原

【Abstract】 African Ostrich is the existing biggest birds, originated from African grassland and Arabian desert, and is classified within the kingdom Animalia, phylum of Chordata, class of Aves, superorder of Paleognathae, Struthioniformes order of ratites, genus of Struthio, and family of Struthionidae. Adult ostriches are 240-280cm high and 130-200kg weight, have long legs with developed muscle. The ostrich has lost flying ability, but running at speed around 50 km/h, and the top is 90～100 km/h. It is inferred that ostrich shank would have special structure compared with other birds. Some studies have indicated ostrich skeletal characteristics, but seldom further reports.Tibia fracture occurs on the ostrich chicks under 6 months frequently, which reduces the economic benefits of ostrich breeding. The main reason for tibia fracture is nutritious factors, including the diet calcium, phosphorus and vitamin D deficiency, inappropriate diet calcium phosphorus proportion, or nutrition uptake repression. These reasons cause osteoporosis in ostrich chicks, which induces leg diseases (eg. tibia fracture or transformation). Ostrich leg disease occurs frequently, and cause economic losses in ostrich husbandry. There are some researches about ostrich leg diseases resently, but the problem is still not solved.Boron, atomic number is 5, atomic weight is 10.811. More and more evidence shows that boron can promote the growth of bones. Boron may influence cartilage and bone structure, vitamin D metabolism and minerals metabolism, for instance. It can increase the bone mass and the ability of anti-fracture, can improve the ash content in the femur of calcium and magnesium deficiency rats, can improve metabolism of blood calcium and phosphorus, and copper and zinc. But high dosage of boron would also bring adverse effects on animal development and reproduction. There are no reports referred to the effects of boron on ostrich bone development at present.For the above 3 reasons above, this article is about the research of morphology of ostrich tibia, tibia development in ostrich chick, pathological change of leg disease ostrich tibias, and the effects of boron on ostrich tibia development by using anatomy, bone grinding chip(crystalline violet stain), scanning electron microscopy (SEM), bone demineralization paraffin section(HE stain), two-dimensional difference gel electrophoresis(2-D DIGE), apoptosis detection and image analysis technology, bone density determine, radiation immune method, atomic absorption spectrophotometric, flow injection analysis comprehensive method, etc., aiming to provide fundamental research and theoretical foundation for disease control and prevention of ostrich (especially nestlings). The main contents and results of this research include the following 4 aspects:1. Morphology characteristics of African Ostrich tibia1-year-old African ostriches are used in the study to investigate the morphological structure of tibia, by the experiments of anatomy, bone grinding chip (crystalline violet stain), scanning electron microscopy(SEM), bone demineralization paraffin section(HE stain), bone density determination. Result shows that:the bone mineral density of the experimental animals is very high (5.656 g/cm2), and the bone mineral density on different parts of tibia are significantly different. Ostrich tibia is very hard with plenty of mineral deposited. There is a transform area stained specially between outer circumferential lamella and osteon area. In addition, ostrich tibial distal joints cannot left-right rotate as human, these anatomic features may be more advantageous for bearing and improving the running speed.2. Developmental characteristics of ostrich chick tibia1,45,90 days old ostrich chicks are used in this study to investigate the morphological structure of tibia of ostrich chicks, by the experiments of anatomy, scanning electron microscopy(SEM), bone demineralization paraffin section(HE stain), bone density determination. Result shows that:ostrich chicks under 90 days grow very fast, and the length, weight, bone mineral density (BMD) of the tibia all increase significantly and continuously. In the tibia of 1-day-old bird, transitional bone trabeculae constitute the primary cancellous bone, which constitute the early formation of backbone. In the tibia of 45-day-old bird, resorption is very active, transitional bone trabeculae are absorbed by osteoclasts and then rebuilded. In thetibia of 90-day-old bird, rudiment of new born compact bone and osteons already exists, but the mature bones are still not formed. Therefore, in the young ostrich husbandry, nestlings should be kept in a good feeding environment and the feed should be given at a reasonable nutrition proportion to meet the needs of the tibias growth of ostrich chicks.3. Differences between tibias from normal and leg disease ostrich chicks90 days old normal and leg disease ostrich chicks were used in the study to investigate the difference of morphological structure and protein content between disease bird tibia and the normal one, by the experiments of anatomy, scanning electron microscopy(SEM), bone demineralization paraffin section(HE stain), bone density determine and protein two-dimensional electrophoresis technology. Result shows:bone mineral density value of leg disease birds is much lower than the normal one. In the disease tibia, compared with normal tibia, there are less bone trabeculae, which are attenuated and began to disappear. In addition, great number of absorb pits are observed on the surface and micro-fracture appears on bone trabeculae. These results indicate that, the disease tibia has less bone density and trabeculae micro-fracture happens in tibia with different disease. Furthermore, the samples of normal group and leg-disease group are detected by two- dimensional difference gel electrophoresis (2-D DIGE). And the 2-D DIGE results are analyzed to screen out 154 differentially expressed protein spots with more than 1.5 times of significance(P<0.5).4. Effects of boron on the development of ostrich chick tibia90 days old ostrich chicks were used in the study to investigate the effects of boron on ostrich chick tibia development, by the experiments of scanning electron microscopy (SEM), bone demineralization paraffin section(HE stain), apoptosis detection and image analysis technology, bone density determination, radiation immune method, atomic absorption spectrophotometric, flow injection analysis comprehensive method, etc.. Result shows that:with different dosage of additional boron, the ostrich serum calcium and boron level are decreased, but serum calcium phosphate ratio increased, serum leptin levels gradually raised, estradiol level in Group C slightly higher than Group A, Bone density value, length, ash, etc all were improved. Group B has highest body weight, B and C group tibial length numerical highest, Group C tibial weight is highest, Group C, D has more ash content, Group C has highest BMD, Group A and C has less apoptosis cells. In conclusion, it was support that ostrich chick tibias in group C grow best, and the most suitable dosage for the development of tibia is 200 mg/L. However, the real optimum dosage of additional boron still needs further gradient experiments to ascertain to effectively apply in animal production.更多还原