鸡胚无壳孵化研究初报

本试验旨在对鸡胚无壳孵化的效果进行研究.选择胚龄相同的40枚红波罗红羽肉鸡种蛋(蛋重在55～60g之间),随机均分为2组,对照组和试验组种蛋经过清洗消毒后同时入孵,试验组种蛋经过55 h预孵化后去壳转移至人工培养容器于孵化器中继续孵化.将人工培养容器中去壳鸡胚的生长发育情况与对照组同胚龄剥壳剖视鸡胚的生长发育情况作对比...     

第X期至“蚊虫珠”期鸡胚的易壳培养

将发育至第X期的鸡胚转和代用蛋壳内，分为4组分别添加蛋清至满或空余一定气室，正立或倒置孵化，观察鸡胚发育至“蚊虫珠”期胚胎的比较。结果发现鸡胚仅在代用蛋壳气室充盈无气泡、并与壳膜充分接触量，85%（34/40）的鸡胚能发育至“蚊虫珠”期。     

鸡胚大头开孔原壳体外培养方法的建立

通过原壳体外培养和抽蛋清的方法,就胚龄、蛋清抽取量对鸡胚发育的影响进行了研究。结果表明:①0日龄鸡胚抽取1mL、3mL和6mL蛋清后,其孵化率分别为38.6%,30.35%和8.6%。抽取蛋清1mL组(P<0.01)和3mL组种蛋(P<0.05)的孵化率明显优于抽取6mL组。②对3日龄鸡胚,抽取9mL蛋清后,鸡胚的发育受到的影响最大,其孵化率仅为33.3%,明显低于3mL和6mL组(P<0.01);而抽取3mL和6mL蛋清对3日龄鸡胚的影响较小,其孵化率仍高达74.3%和76.5%。③抽取6mL蛋清并在大头顶端开直径为1.0cm的圆孔进行鸡胚原壳体外培养获得成功,3日龄鸡胚的孵化率为18.5%。     

鸡胚大头开孔加抽泵清原壳体外培养的研究

试验以3日龄种鸡胚蛋为实验材料,通过在小头抽取不同量的蛋清和在大头顶端开3种直径分别为0.5、1.0、1.5 cm圆孔的方法对3日龄鸡胚原壳体外培养进行研究.结果表明:(1)抽取9 ml蛋清后,鸡胚的发育受到的影响最大,孵化率为33.3%,明显低于3 ml和6 ml组(P＜0.01);抽取3 ml和6 nd蛋清对3日龄鸡胚的影响较小(P＞0.05).孵化率分别为74.3%和76.5%.(2)在小头抽垠6 ml蛋清后再在大头顶端开直径分别为0.5、1.0、1.5 cm的圆孔,以开孔直径为0.5 cm孵化率最高,随着开孔直径增大,鸡胚的孵化率呈现极显著下降的趋势.     

不同时期鸡胚原始生殖细胞的分离及存活时间的研究

采用Ficoll密度梯度离心，提取第14期(孵化53h)血液、第19期(孵化72h)和第28期(孵化132h)性腺中的PGCs．以比较这3个时期的PGCs在相同的体外培养条件下存活时间的差别。培养基为TCM-199，添加10％的小牛血清，不添加其他的细胞因子，培养后采用台盼蓝染色，以比较其在这种培养液中存活时间的差别。结果发现，第14期提取的PGCs存活时间最短，第19期的最长，第28期的介于两者之间。     

鸡胚大头开孔加抽蛋清原壳体外培养的研究

试验以3日龄种鸡胚蛋为实验材料,通过在小头抽取不同量的蛋清和在大头顶端开3种直径分别为0．5、1．0、1．5em圆孔的方法对3日龄鸡胚原壳体外培养进行研究。结果表明：（1）抽取9ml蛋清后,鸡胚的发育受到的影响最大,孵化率为33．3％,明显低于3“和6ml组（P〈0．01）;抽取3ml和6ml蛋清对3日龄鸡胚的影响较小（P〉0．05）,孵化率分别为74．3％和76．5％。（2）在小头抽取6ml蛋清后再在大头顶端开直径分别为0．5、1．0、1．5cm的圆孔,以开孔直径为0．5cm孵化率最高,随着开孔直径增大,鸡胚的孵化率呈现极显著下降的趋势。     

鸡胚血液中PGCs的分离及原代培养

在鸡胚孵化48,50,54,56,60h的不同时期，分离血液中原始生殖细胞（PGCs)，分离前的浓度分别为0.011%,0.007%,0.006%,0.006%,0.004%,0.001%,分离后的浓度分别为0.245,1.21%,1.02%,0.64%,0.27%,孵化48－54h，血液中PGCs浓度差异不显著，但孵化48h的鸡胚液较少，PGCs绝对量较低。孵化52－54h的鸡胚血液量较大，获取PGCs的绝对值较高，故分离PGCs的时间以孵化52－54h为宜，鸡胚血液中PGCs在未中任何外源生长因子而含10％FCS的TCM－199培养基中体外培养，能够存活3－4d。     

Effects of Calcium Lactate on the Development of Chicken Embryos in a Shell-less Culture System up to Day Seventeen of Incubation

This study examined the effects of calcium lactate on the development of chicken embryos in a shell-less culture system (cSLCS) up to the seventeenth day of incubation. In the presence of calcium lactate, a significant reduction in embryo viability was observed during the first week of incubation in cSLCS. On day 17 of embryo development, no significant difference was observed in the blood plasma calcium concentration or tibia bone density between cSLCS and intact control embryos, whereas the tibia length was significantly shorter in cSLCS embryos than in the intact control. These results suggest that calcium lactate supplementation in cSLCS supports bone formation in developing chicken embryos, but has adverse effects on the viability of embryos, particularly during the first week of embryo development.     

Ex Ovo Culture System for Avian Embryos and its Application

Ex ovo culture of avian embryos can be applied not only to embryology but also to various fields of basic research such as embryo manipulation, toxicology, and regenerative medicine. The windowing method, which facilitates various manipulations and observations by opening a hole in one part of the eggshell, and culture systems using surrogate eggshells, are widely used. Despite this, biology lessons in high schools cover shell-less culture systems, which involve the development of avian embryos in artificial vessels, such as rice bowls, without using surrogate eggshells. However, as embryo development stops at its early stages in this method, it is not possible to continuously observe the development of the embryo. This led to attempts to develop an embryo culture method using a complete artificial culture vessel that does not use surrogate eggshells, and Kamihira et al. (1998) succeeded in hatching quail embryos in an artificial culture vessel using polytetrafluoroethylene membranes. In addition, Tahara succeeded in hatching chick embryos in artificial culture vessels that used cling film made of polymethylpentene and reported their detailed methodology (Tahara and Obara, 2014). These technologies are being applied not only to school education but also to various fields of research.     

鸡胚胎干细胞的分离和培养

实验从鸡第X期胚胎中分离胚盘,以鸡成纤维细胞为饲养层,用添加了10%的胎牛血清、2%的鸡血清、2mmol/LL-谷氨酰胺、1mmol/L丙酮酸钠、5.5×10-5mol/Lβ-巯基乙醇、10μL/mL非必需氨基酸以及含1000IU/mL白血病抑制因子(LIF)、10ng/mL碱性成纤维生长因子(bFGF)和5ng/mL干细胞生长因子(SCF)的高糖DMEM对细胞进行培养和传代,可以获得传至5～6代的鸡胚胎干细胞(ES)。通过对传代培养后的鸡ES细胞进行AKP染色鉴定和SSEA-1的鉴定,证实细胞未发生分化,具有胚胎干细胞的特征。同时通过不同分离胚盘方法和不同消化时间的比较得出药勺法提取胚盘简单易行,原代消化5～8min的ES细胞适合于传代培养。     

四氯联苯对鸡胚性腺原始生殖细胞的毒性影响

采用四氯联苯处理发育至Ⅹ期的白来航鸡种蛋,孵化5.5天后取出胚胎进行全固定,用石蜡切片和PAS染色(高碘酸希夫试剂)研究四氯联苯对鸡胚性腺原始生殖细胞(PGCs)的影响.结果表明,四氯联苯对发育至5.5日胚龄的鸡胚性腺PGCs数量和结构有明显影响,但雌二醇处理的5.5日胚龄鸡胚PGCs数量变化不显著,其结构无明显变化.表明四氯联苯对5.5日胚龄的鸡胚性腺PGCs的影响主要来自于其毒性作用.     

鸡与鹌鹑属间杂交早期胚胎性别鉴定体系的建立与优化

研究参考鹌鹑Wpkci和β-actin基因设计引物,采用RT-PCR技术,旨在建立鉴定鸡(♂)与鹌鹑(♀)杂交后代早期胚胎性别的方法。Wpkci引物扩增已知性别鹌鹑及未知性别杂交种胚胎的cDNA,从母禽胚胎获得402bp和296bp两条特异性条带,而公禽没有得到条带。为进一步验证该鉴定体系的可靠性和稳定性,采用了多重PCR从母禽胚胎获得490bp、402bp、296bp三条特异性条带,而公禽只有490bp的内标条带。在对试验关键因素进行优化的基础上,建立了简单、快速、可靠、稳定的鉴定杂交种早期胚胎性别的方法。     

微量全血PCR技术鉴定雏鸡及鸡胚性别

为了便于常规PCR技术在鸡性别鉴定上的推广和应用,本研究以成年家鸡全血为模板,应用常规PCR反应缓冲液和Tap DNA聚合酶直接进行PCR扩增,对50 μL PCR反应体系中的血样(0.05~4.0 μL)和Tap DNA聚合酶的使用量(0.05~1.5 μL),以及循环次数(30~40)进行了优化,在此基础上对血样的抗凝剂和保存温度进行了探讨,并对62只1日龄雏鸡、80枚12日龄和80枚16日龄鸡胚的血样直接PCR进行了性别鉴定。结果表明,采集血样时可以使用ACD、肝素或EDTA作为抗凝剂,血样可以在4、－20或－80 ℃至少保存3个月;使用0.1 μL全血就能完成雏鸡和鸡胚性别鉴定,全血PCR鉴定性别与性腺性别的符合度为100%。与常规PCR相比,全血PCR节约了检测成本,提高了检测效率,减少了交叉污染可能。     

鸡胚胎干细胞的分离及其嵌合体制备条件探索

分离培养鸡胚胎干细胞(ESCs),体外培养传代后,进行碱性磷酸酶活性检测和SSEA-1染色鉴定;并用线性化的质粒pEGFP-N1转染鸡ESCs。受体种蛋经赤道面开窗后,将经转染的ESCs注射到受体鸡胚胚下腔,以便制作嵌合体鸡;对获得的嵌合体鸡分别提取血液和组织DNA后进行PCR检测。结果表明:5只存活的的嵌合体鸡血液中没有发生EGFP基因嵌合,但有4只嵌合体鸡的部分器官表达了EGFP基因,其中有2只鸡的性腺发生嵌合,表明利用赤道面开窗后对受体鸡进行胚下腔显微注射可以生产嵌合体鸡。     

用数学模型拟合鸡胚胸腺生长发育的研究

观察成都白羽鸡不同胚龄的鸡胚胸腺,结果,鸡胚胸腺在受精卵孵化到第9天时才开始形成.14胚龄时,胸腺长度为20胚龄的50%.18胚龄时,胸腺重量为20胚龄的50%.鸡胚胸腺长度与胚龄关系呈逻辑斯蒂S曲线生长,胸腺的重量呈指数增长模型.两者与胚体的关系分别呈对数函数和幂函数增长.建议采用逻辑斯蒂S曲线生长和指数增长模型分别拟合胸腺的长度、重量与胚龄的关系.     

Experimental Infection of Mycoplasma ovipneumoniae in SPF Chicken Embryos

In order to evaluate the SPF chicken embryos as a candidate of the experimental model of Mycoplasma ovipneumoniae (Mo) infection,the 7-day-old SPF chicken embryos were infected with different concentrations of Mo(10⁸,10⁹ and 10¹⁰ ccu/mL) by injection of vitelline fluid and allantoic cavity.The mortality and Mo detection rate (PCR detection and isolation) of infected chicken embryos were employed to evaluate the optimal inoculation route,dose and sampling site.Three different isolates of Mo were submitted to injection of chicken embryos for pathogenicity comparison.The results showed that the optimal inoculation route and dose were vitelline fluid injection with 0.2 mL (10⁹ ccu/mL) Mo per embryo and the sampling site for detection was also vitelline fluid.Three Mo isolates revealed good infectivity and lethality to the chicken embryo.The chicken embryos mortality caused by FL3 strain (45%) and MoGH3-3 (40%) were both higher than that by A3 strain (25%),but with no significance (P>0.05).The detection rate of Mo from the chicken embryos in FL3 group (100%) was higher than that of MoGH3-3 strain(85%) and that of A3 strain (90%),but with no significance(P>0.05).This experiment preliminarily proved that the SPF chicken embryos could be infected and partially killed by Mo,and the virulence of different strains of Mo to the chicken embryos was different.The results provided the data for further establishment of the chicken embryo infection model of Mo,which would benefit the research on Mo pathogenicity and vaccine development.     

绵羊肺炎支原体人工感染SPF鸡胚的研究

为探索SPF鸡胚作为绵羊肺炎支原体实验室感染模型的可行性,本试验用不同浓度绵羊肺炎支原体（10⁸、10⁹、10¹⁰ ccu/mL）经由卵黄囊和尿囊腔两个部位接种7日龄SPF鸡胚,通过统计鸡胚死亡情况和不同鸡胚组织样品中绵羊肺炎支原体检测阳性率（PCR检测和支原体分离鉴定）,确定绵羊肺炎支原体鸡胚感染方式、感染剂量和最佳分离部位,再用3株不同来源的绵羊肺炎支原体分离株感染鸡胚,观察其对鸡胚的致病力。结果表明,绵羊肺炎支原体感染鸡胚最佳接种途径为卵黄囊接种,感染剂量为10⁹ ccu/mL、0.2 mL/只,最佳分离部位为卵黄液。3株支原体均能感染和致死鸡胚,并均能从卵黄液中分离到绵羊肺炎支原体,但对鸡胚的致病性存在一定差异：FL3株致鸡胚死亡率为45%,略高于MoGH3-3株（40%）,二者均高于A3株（25%）,但差异均不显著（P>0.05）;FL3株鸡胚检测阳率为100%,高于MoGH3-3株（85%）及A3株（90%）,但差异也不显著（P>0.05）。本试验初步确定了绵羊肺炎支原体可感染和致死SPF鸡胚,不同分离株对SPF鸡胚致病力有差异,表明SPF鸡胚可作为下一步建立绵羊肺炎支原体实验室感染模型的候选,为绵羊肺炎支原体致病性研究和疫苗研制奠定基础。