鸡、鹌鹑及其杂交种早期胚胎bcl-2基因甲基化的分析

采用甲基化特异性PCR(MSP)方法进行鸡、鹌鹑及其属间杂交种早期胚胎发育期60、66、72、84、96、108、120 h 7个不同胚龄胚胎组织bcl-2基因启动子区CpG岛甲基化状态的对比分析,探讨bcl-2基因甲基化对鸡与鹌鹑属间杂交种早期胚胎发育的影响。结果显示,正常发育的鸡胚和鹌鹑胚龄在60、66、72、120 h均呈高甲基化状态,84和96 h呈非甲基化状态,而鸡与鹌鹑属间杂交种胚胎在60、66、72、96、108和120 h则与鸡、鹌鹑不同,呈现出甲基化或非甲基化无规律性并存,甚至检测不到甲基化状态;84 h则只检测到非甲基化状态。鸡与鹌鹑杂交种早期胚胎组织中bcl-2 基因启动子区CpG岛的异常甲基化有可能是引起鸡与鹌鹑属间杂交种胚胎早期死亡的一个重要影响因素。     

鸡、鹌鹑和杂交种胚胎期生肌调节因子MyoG、Pax7表达规律的研究

为了研究MyoG、Pax7在鸡、鹌鹑和杂交种胚胎期的表达规律,试验采用RT-PCR技术对MyoG、Pax7两个基因在鸡、鹌鹑和杂交种胚胎期不同时间点的表达量进行了检测。结果发现:Pax7在鸡、鹌鹑和杂交种胚胎期的表达特点相似;MyoG在杂交种上的表达特点与鸡和鹌鹑不同,这种不同可能与杂交种胚胎期高死亡率有关。     

鸡、鹌鹑及其杂交禽胚胎肌肉发育过程中MSTN和p21基因的表达规律

旨在研究MSTN和p21基因在鸡、鹌鹑和杂交禽胚胎肌肉发育过程中的表达规律,比较这2个基因在杂交禽与亲本鸡和鹌鹑中的差异表达,探讨MSTN和p21基因与肌肉发育的关系。通过人工受精获得鸡(♂)与鹌鹑(♀)杂交禽蛋,与鸡蛋、鹌鹑蛋按照鸡标准孵化条件同批入孵,采集第7～17天活胚胸肌组织,应用实时荧光定量PCR技术检测MSTN和p21基因在3个物种中每一天mRNA的相对表达量。MSTN和p21基因在胚胎肌肉发育的第7～17天均有表达,这2个基因mRNA的表达在鸡胚中第9天到达第1次峰值,在鹌鹑中第7天到达第1次峰值,后都随胚胎的发育表达水平上升,并维持相对稳定的高水平表达。杂交禽的表达规律与鸡一致,也在第9天达到峰值,而p21mRNA表达极显著高于鸡和鹌鹑(P<0.01)。MSTN mRNA表达规律与成肌细胞退出细胞周期的时间规律一致;在胚胎肌肉发育过程中,MSTN基因特异性上调p21的表达。     

鹌鹑早期胚胎bcl-2基因表达的差异及发育性变化

采用RT-PCR方法,用Wpkci和β-actin引物进行多重PCR鉴定66～120 h鹌鹑胚胎样本性别后,选取不同时间点雌、雄胚胎各4枚,以β-actin为内标,测定bcl-2 mRNA的相对丰度;探讨bcl-2对鹌鹑早期胚胎发育的影响。结果表明：①雄性胚胎在66～90 h bcl-2 mRNA表达量一直维持在较低水平,96～102 h bcl-2 mRNA有所增加,但96与102 h间差异不显著（P＞0.05）,在108 h显著降低（P＜0.05）;与108 h相比,114 h显著升高,120 h维持在较高水平。雄性胚胎在66～108 h（72 h除外）bcl-2 mRNA表达均比雌性胚胎的表达量低,在114～120 h bcl-2 mRNA表达水平均比雌性胚胎的高。②雌性胚胎在66～90 h bcl-2 mRNA表达量一直维持在较低水平;与90 h相比,96 h显著升高,96～120 h逐渐降低,其中96～108 h下降,但三者间差异不显著（P＞0.05）;与108 h相比,114～120 h显著下降（P＜0.05）。③相同时间点雌、雄胚胎间的比较结果表明,84 h雌性胚胎bcl-2 mRNA表达显著高于雄性胚胎（P＜0.05）,90、108 h雌性胚胎bcl-2 mRNA表达极显著高于雄性胚胎（P＜0.01）;而在120 h雄性胚胎bcl-2 mRNA表达极显著高于雌性胚胎（P＜0.01）。鹌鹑胚胎发育早期,雌、雄样本bcl-2 mRNA表达存在一定的时序性,说明bcl-2对雌、雄鹌鹑早期分化及早期胚胎发育有重要影响。     

鹌鹑早期胚胎bcl-2基因表达的差异及发育性变化

采用RT-PCR方法,用Wpkci和β-actin引物进行多重PCR鉴定66～120 h鹌鹑胚胎样本性别后,选取不同时间点雌、雄胚胎各4枚,以β-actin为内标,测定bcl-2 mRNA的相对丰度;探讨bcl-2对鹌鹑早期胚胎发育的影响。结果表明:①雄性胚胎在66～90 h bcl-2 mRNA表达量一直维持在较低水平,96～102 h bcl-2 mRNA有所增加,但96与102 h间差异不显著(P>0.05),在108 h显著降低(P<0.05);与108 h相比,114 h显著升高,120 h维持在较高水平。雄性胚胎在66～108 h(72h除外)bcl-2 mRNA表达均比雌性胚胎的表达量低,在114～120 h bcl-2 mRNA表达水平均比雌性胚胎的高。②雌性胚胎在66～90 h bcl-2 mRNA表达量一直维持在较低水平;与90 h相比,96 h显著升高,96～120 h逐渐降低,其中96～108 h下降,但三者间差异不显著(P>0.05);与108 h相比,114～120 h显著下降(P<0.05)。③相同时间点雌、雄胚胎间的比较结果表明,84 h雌性胚胎bcl-2 mRNA表达显著高于...     

Bcl-2、PCNA基因在鸡、鹌鹑及其杂交种睾丸组织中的表达研究

为探讨鸡（♂）与鹌鹑（♀）属间杂交种睾丸生长发育不良的分子机理,试验采集新罗曼蛋用型公鸡、朝鲜公鹌鹑、鸡与鹌鹑的雄性杂交种不同发育时期的睾丸组织样共计84份,并用实时荧光定量PCR对公鸡、公鹌鹑及其杂交种各阶段睾丸组织中Bcl-2、PCNA基因的mRNA表达进行了检测。结果显示,鸡和鹌鹑不同生长发育期睾丸组织中Bcl-2、PCNA基因mRNA的表达具有相似性,Bcl-2基因整体呈波动性变化,下降到最低值后,迅速回升到较高水平,PCNA基因均有一个极显著的峰值;与鸡和鹌鹑相比,杂交种Bcl-2、PCNA基因mRNA表达均无明显变化,表明杂交种睾丸发育过程中,Bcl-2基因调控睾丸中细胞凋亡的模式不同于鸡和鹌鹑,也不存在明显的细胞增殖过程,从而推测鸡与鹌鹑的杂交种睾丸生长发育不良的分子影响因素与其睾丸组织中Bcl-2、PCNA基因mRNA的异常表达有关。本试验结果为进一步研究鸡与鹌鹑属间杂交种睾丸发育不良的分子机理提供了参考依据。     

利用DGE筛选早期雌性鸡与鹌鹑属间杂交种性分化相关基因

已有研究表明鸡与鹌鹑属间杂交种胚胎早期死亡与性别分化存在关联性,本试验通过高通量测序构建数字基因表达谱(DGE)筛选雌性鸡与鹌鹑杂交种早期(3d)性别分化的相关基因,有助于揭示胚胎性腺分化时期相关基因与其早期死亡间的作用,利用实时荧光定量PCR技术对筛选出与相关表达差异基因进行验证。结果表明,构建雌雄鸡、雌雄杂交种3d胚胎DGE文库,雌鸡与雄鸡之间差异表达基因中有25个上调,41个下调;雌杂交种与雌鸡之间差异表达基因中有55个上调,175个下调;雌杂交禽和雄杂交禽之间差异表达基因中有36个上调,36个下调;这些差异基因大都与胚胎生长发育、细胞分化繁殖相关基因为代表。GO功能富集分析表明,差异表达基因主要定位在参与细胞增殖、分化、胚胎发育等行使催化、促进生物学过程的功能;Pathway分析得出,雌雄鸡、雌鸡与雌杂交种、雌雄杂交种的差异基因分别富集在12、13、15条pathway中。分析推测得出可能由于这些差异基因在体内紊乱表达,导致调控胚胎发育、分化相关信号通路异常是雌杂交种早期死亡原因之一,筛选出的差异基因将为后续的雌性杂交种早期发育、死亡研究提供依据。     

AR、ER基因在鸡、鹌鹑及其杂交种睾丸组织中的表达及其对睾丸生长发育的影响

为探讨鸡（♂）与鹌鹑（♀）属间杂交种睾丸生长发育不良的分子机理,采集新罗曼蛋用型公鸡、朝鲜公鹌鹑、鸡与鹌鹑杂交的雄性杂交种不同发育时期的睾丸组织样共计96份并测定其体质量与睾丸质量。采用实时荧光定量PCR对公鸡、公鹌鹑及其杂交种不同生长发育期睾丸组织中AR、ER基因的mRNA表达进行了检测。结果显示：（1）鸡和鹌鹑的睾丸均能随着日龄、体质量的增长而正常生长发育,但鸡与鹌鹑杂交的杂交种的睾丸却生长缓慢而发育不良;（2）鸡和鹌鹑不同生长发育期睾丸组织中AR、ER基因mRNA的表达具有相似性,AR基因均有一个极显著的峰值,ER基因均呈波动性变化;鸡与鹌鹑的杂交种均为极端性表达,AR基因呈直线下降,ER相反则一直升高;与鸡和鹌鹑相比,杂交种AR、ER基因mRNA的极端性表达均应为异常表达。（3）鸡、鹌鹑及其杂交种的共同点是AR表达为最高峰时,ER则趋于最低点,反之,ER表达为最高峰时,AR则趋于最低点,表明ER基因对AR基因具有上调作用或者AR基因对ER基因具有下调作用,从而推测AR与ER即相对立而又协同作用的表达模式是鸡和鹌鹑睾丸组织正常生长发育的分子调控模式,探明了鸡与鹌鹑的杂交种睾丸组织发育不良的分子影响因素是其睾丸组织中的AR、ER基因mRNA异常表达。     

鸡、鹌鹑及其属间杂交种DMRT1b转录本初步分析

DMRT基因家族是一个与性别决定相关的发育基因家族。以鸡、鹌鹑及其属间杂交种胚胎为试验对象,分别采集孵化72 h的鸡、鹌鹑和杂交种胚胎,以DMRT1b为目的基因进行扩增,对DMRT1b基因原序列、扩增序列分析,应用Ex PASy网站对序列编码蛋白进行蛋白质生物信息学分析,结果发现杂交种DMRT1b基因较其父母本发生部分碱基缺失,预测蛋白质功能存在差异,推测由于基因缺失导致蛋白质不同,这种现象可能与杂交种早期死亡、不育存在相关性。研究结果为鸡与鹌鹑属间杂交种雌性致死与雄性不育的研究奠定基础。     

雌激素受体在鸡与鹌鹑属间杂交早期胚胎中的差异表达及其作用

旨在研究雌激素受体（ER）与鸡-鹌鹑属间杂交早期胚胎发育、性别分化的关系。人工授精获得鸡（♂）-鹌鹑（♀）杂交种蛋，按照鸡的孵化标准同批入孵，连续采集早期活胚（2．75、3．00、3．25、3．50、3．75、4．00、4．25、4．50、4．75、5．00d），采用RT—PCR法，用Wpkci和β-actin引物进行多重PCR，鉴定早期胚胎性别；之后选取各时间点雌、雄胚胎各4枚，以β-actin为内标，测定ER mRNA相对表达丰度。雌、雄胚胎ER mRNA的发育性变化趋势基本一致，2．75～3．00d表达水平均较高；3．25d雄性表达水平显著降低（P〈0．05），雌性则极显著降低（P〈0．01）；3．50d雌、雄表达水平又回复至较高水平；3．75d二者均极显著降低（P〈0．01），此后一直维持在较低水平。不同日龄间比较，2．75～3．50d雌性ER表达高于雄性，差异极显著（P〈0．01）。杂交胚胎性分化时间大致开始于胚胎发育的第2．75～3．50d，性分化期ER的异常表达可能与胚胎死亡、杂种不育有关，这一结论有待于进一步研究发育同期鸡、鹌鹑胚胎ER的表达及其功能后证实。     

The indirect immunofluorescence assay using cardiac tissue from chickens, quails and ducks for identification of influenza A virus during an outbreak of highly pathogenic avian influenza virus (H5N1): a rapid and simple screening tool for limited resource settings

Here we describe the diagnostic utility of the indirect immunofluorescence assay (IFA) during a recent outbreak of highly pathogenic avian influenza (HPAI) subtype H5N1 virus in southern Thailand and demonstrate the usefulness of the cardiac tissue from infected chickens, quail, and ducks for diagnosis. The most reliable sample for IFA diagnosis of influenza A virus was cardiac tissue (83.0%; 44/53) which when divided by species (chicken, quail and duck cardiac tissues) gave respective positivity rates of 88% (22/25), 88.9% (16/18) and 60.0% (6/10). Cardiac tissue also gave the highest IFA intensity for the three species. We believe that the IFA method has wide applicability in developing countries or remote settings where clinically similar avian diseases with high morbidity and mortality such as Newcastle disease and fowl cholera are common and could be rapidly excluded thereby conserving valuable reference laboratory capacity for true HPAI outbreaks.     

Influence of intergeneric/interspecies mitochondrial injection; parthenogenetic development of bovine oocytes after injection of mitochondria derived from somatic cells

Interspecies/intergeneric mitochondrial heteroplasmy can occur in interspecies/intergeneric hybrid embryos or following nuclear transfer. In the present study, intergeneric buffalo (Bubalus bubalis) mitochondria (WB-mt) or interspecies murine (Mus spretus) mitochondria (M-mt) were injected into bovine (Bos taurus) oocytes, and the subsequent embryonic development was characterized. Fibroblast mitochondria (WB-mt or M-mt) were microinjected into in vitro matured bovine oocytes followed by oocyte activation by a combination of electrical stimulation and 6-dimethylaminopurine treatment. After seven days of culture, embryo development was evaluated. The copy number of specific mtDNA populations (introduced and native mtDNA) from heteroplasmic oocytes was estimated using real-time PCR. The results illustrated that oocytes injected with either WB-mt or M-mt can develop to the blastocyst stage (20.6% and 19.6%). Cleavage division rates and development to the morula stage in oocytes injected with WB-mt were lower (76.2% and 45.9%, respectively) in comparison with uninjected oocytes (89.2% and 59.1%, respectively) (P<0.05). However, no differences were found in comparing M-mt injected oocytes and controls (P>0.05). An increase in bovine mtDNA copy number was observed at the expanded blastocyst stage of injected embryos (P<0.01), while the number of injected mtDNA was stable throughout development. This study demonstrates that interspecies/intergeneric mitochondrial injected bovine oocytes have the ability to develop to the blastocyst stage after parthenogenetic activation and that injected mtDNA was neither selectively destroyed nor enhanced through development. Moreover, injected intergeneric mitochondria had a demonstrated influence on bovine parthenogenetic development and mtDNA replication.     

Divergent selection for shape of growth curve in Japanese quail. 7. Effect of egg storage at high temperature on embryo development and hatchability

1. Hatching time, hatchability of fertile eggs and embryo mortality in relation to (1) physical quality of fresh eggs and (2) embryonic development during storage and incubation periods were analysed after egg storage for 1, 5 or 10 d at 30°C in the meat-type lines of Japanese quail, HG and LG, divergently selected for high and low relative weight gain between 11 and 28 d of age, respectively, and constant body weight at 49 d of age.

2. In both lines, the increase of egg storage temperature from 12°C (standard level) to 30°C increased the egg weight loss during storage, shortened the incubation period and reduced the hatching success.

3. Similar to standard egg storage temperature, LG quail hatched earlier than HG quail after egg storage at 30°C and early and late mortality of both lines increased with a prolonged period of egg storage. In contrast to the standard egg storage conditions, no line differences in hatchability were observed.

4. The results did not identify a relationship between the decrease in hatchability or embryo viability and line differences in external egg parameters as well as any important role of undesirable changes induced by a high storage temperature on albumen viscosity.

5. The pattern of embryonic death, low developmental rate of embryos and a dichotomy between the development of the extra-embryonic vascular system and the embryo itself during egg storage at high temperature implied that an insufficient nutrient supply in consequence of developmental delay could represent a key factor in increasing early and late embryo mortality.     

Variance and covariance of hatchability and some of its components in the chickens

Samples of 20 hatching eggs from each of 2004 individual pedigreed pullets were incubated and hatchability recorded. Unhatched eggs were examined for evidence of embryo development and classified as infertile, early embryonic death (died 0–10 d) or late embryonic death (died 11–22 d). Variance analysis yielded a mean heritability value for hatch of all eggs of approximately 0.30. Heritability values for early and late embryo mortality were subject to larger errors but there was a tendency among the estimates of h ² ₈ for late embryo mortality to be higher than those for early embryo mortality.

The genetic correlations between hatchability and both early and late embryonic mortality were high and negative as was to be expected from part × whole correlations. However the genetic correlation between early and late embryonic mortality was also consistently positive indicating common genetic determination of these two independent traits.     

Foetal development in quail during the final stages of incubation

Fertile eggs of the bobwhite and Japanese quail were opened and examined during the last 4 days before hatching and the foetal position and stage of development recorded at known times after the beginning of incubation. A comparison is made between these stages in the quail and similar stages in the domestic fowl.     

家鸡(Gallus domesticus)和鹌鹑(Coturnix coturnix)染色体核型及G带比较分析

采用外周血淋巴细胞培养法和胰酶处理法，对家鸡和鹌鹑染色体的核型和G带进行了研究。结果表明，家鸡和鹌鹑染色体数目均为2n=78，但染色体的形态存在明显差异，主要表现在NO．1、NO．4、NO．6、NO．8和Z染色体上。NO．1染色体，家鸡为m型，而鹌鹑为sm型；NO．4、NO．6、NO．8和Z染色体，家鸡分别为sm、sm、m、m型，而鹌鹑全为t型。G带分析结果显示，家鸡和鹌鹑的G带差异主要反映在NO．1、NO．2染色体上，这可能是由于臂间倒位所致。     

Organic matrix composition and ultrastructure of eggshell: a comparative study.

1. The avian eggshell is a biomineralised composite ceramic consisting of calcium carbonate embedded in an organic matrix. Matrix components are supposed to be involved in the control of mineralisation, crystallographic texture and biomechanical properties of eggshell. 2. The structure and eggshell matrix composition of various domesticated bird species were compared to gain insight into the universality of the eggshell mineralisation process. 3. The SDS-PAGE profiles of soluble eggshell matrix were specific within groups of birds (a: laying hen, breeder hen, quail, pheasant and possibly turkey; b: guinea fowl; c: duck and goose) but some of the protein bands were common to all groups. 4. Analogies between species were confirmed by Western blotting using hen protein antibodies. Ovocleidin-17 (OC-17) and ovalbumin were revealed in all species (except quail for OC-17). Lysozyme was present only in hen eggshell. Another egg white protein: ovotransferrin showed a positive signal in hens, turkey and quail. Osteopontin was observed in laying and breeder hens and quail. 5. Different proteoglycans were localised to discrete regions within the eggshell. Dermatan sulphate was observed within the matrix of the calcified shell of all species except quail which contained chondroitin-6-sulfate. Keratan sulphate was observed in mammillary bodies of breeder and laying hen, quail, pheasant and turkey while chondroitin sulphate was also present in guinea fowl and duck. 6. The general structural organisation of the different avian eggshells was similar but specific differences were observed in the ultrastructure of the mammillary layer. Species of the same taxonomic family could be grouped according to their structural analogies: breeder hen, turkey and pheasant resembled that of the domestic fowl. Guinea fowl was unique. Goose and duck were quite similar with large and confluent mammillary bodies. 7. Some matrix components are therefore common to eggshells of various species but more information is needed to relate differences in matrix composition between taxonomic groups with differences in ultrastructure.