【作者】 王晗；

【导师】 窦忠英；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2005， 硕士

【摘要】 家畜胚胎的性别鉴定是胚胎移植的重要组成部分。通过移植已知性别的胚胎,可使家畜达到理想的性别比例,这在畜牧业生产上具有重要的意义。动物的某些生产性状是受性别限制（泌乳等）或性别影响的（产肉、产毛等）,通过性别控制可以使这些性状在更多的理想性别中获得更大的经济效益,加快优良母畜的繁殖速度,促进高效畜牧业的发展。如在养牛业上,结合胚胎分割、胚胎冷冻保存等动物繁殖技术,可以选育优良母牛,增加牛奶产量,提高肉牛产肉率。本研究以小鼠和牛为研究对象,分别建立了聚合酶链式反应（Polymerase Chain Reaction, PCR）鉴定小鼠和牛性别的方法,并讨论了影响PCR 扩增的几个因素。主要内容有以下几点: （1）　依据小鼠和牛SRY 基因序列,分别设计合成其巢式PCR 引物;同时根据小鼠和牛ZFY-ZFX基因的序列,设计合成其各自的内对照引物。（2）　分别采集雌雄小鼠和公母牛少量组织样品,试剂盒抽提基因组DNA,保存于TE溶液中,作为反应模板,进行PCR 扩增,其性别鉴定结果与已知性别完全一致。（3）　超排处理母鼠,分别收集2-细胞、4-细胞、8-细胞胚胎和早期桑椹胚,视为微量细胞样品,热处理后进行PCR 扩增。比较常规和巢式、复合式PCR 方法,并对Mg²⁺浓度进行梯度选择（1.5、2.5、3.0 mmol/L）,确立了合适的扩增体系及反应条件。实验结果显示,在扩增微量样品时,巢式PCR 胚胎可鉴别率（94.1%,111/118）高于常规PCR（64.3%,36/56）,复合式PCR 则更有利于排除假阴性结果的干扰,而2.5 mmol/L 的Mg²⁺浓度扩增效果较好。（4）　超排处理母鼠,收集致密桑椹胚和囊胚,进行活检。采用简易切割法,将致密桑椹胚切为一大一小两部分,囊胚则切取少量滋养层细胞。胚胎主体进行染色体分析,其鉴定结果作为性别标准。半胚或取样细胞相应编号,热处理后进行PCR鉴定。实验结果为:通过核型分析法,共鉴定21 枚胚胎,12 枚为雄性,9 枚为雌性;?b通过PCR 法鉴定,其中12 枚胚胎判为雄性,8 枚判为雌性,与核型分析结果一致,1 枚为假阳性结果;PCR 鉴定的准确率为95.2%（20/21）。（5）　收集已知性别的公母牛微量细胞样品。在高倍实体显微镜下分别取5-10 枚细胞,热处理后进行PCR 扩增,确立合适的扩增体系及反应条件。（6）　对牛精液、血清、操作液及操作者头屑等可能对鉴定造成影响的几个因素进行了研究和检测,PCR 扩增结果表明,这些因素不会对PCR 鉴定造成污染。更多还原

【Abstract】 Sex identification in livestock embryo is an important part of livestock embryo transfer. It is to achieve desired offspring sex ratio by means of sex control through the transfer of known sex embryo. It is significant in livestock husbandry. As we all know, some of the production characteristic are limited （secretion of milk） or affected （produce of meat and hair） by the sex of livestock. With the sex control, those production characteristics can be expressed in desired sex offspring, which can accelerate the propagation of high quality dam and improve the development of livestock husbandry. Take cattle for example, selection of high quality dam, increase of the milk yield and the meat ratio can be achieved by the combination with embryo dissection, cryopreservation and sexing. In this experiment, the polymerase chain reactions （PCR） were established to identify the sex of the mouse and bovine, and the aspects which could affect the amplify results were discussed. The main contents were as follows: 1. SRY gene and ZFY-ZFX gene nestsd primers of mouse and bovine were designed respectively. 2. The tissue DNA of mouse and bovine were extracted and identified. All the results were identical with the sex of the tissues. 3. In vivo derived embryos （2-cell, 4-cell, 8-cell embryo and early morula） which were collected from superovulated mouse were identified by PCR. The conventional PCR and nested PCR、multiplex PCR were compared and the system of PCR was established. At the same time, the density of Mg²⁺ was selected from 1.5、2.5、3.0 mmol/L. The results indicated that the ratio of the identified embryos by the nested PCR （94.1%,111/118） was higher than by the conventional PCR （64.3%,36/56）. And the multiplex PCR was helpful to avoid the interference of the false negative result. And the results were advantageous when the density of Mg²⁺ was 2.5mmol/L. 4. The morulae and blastocyst were collected from superovulated mouse. The morula stage embryos were cut into two parts, and few trophectoderm cells were removed from blastocyst stage embryos. The embryos were sexed by karyotype analysis and PCR. The results were that 12 of 21 embryos were identified for male, and 8 of 21 for female. The accurate rate of PCR was 95.2% （20/21）. 5. Few cells （5-10 cells） from bovine were collected and identified, and the system of PCR was established. 6. The sperm, the serum, the operating solution and the operator factor were researched. And the results indicated that those factors did not result in contaminate.更多还原