Solexa测序技术分析鸡与鹌鹑属间杂交雌性和雄性胚胎的差异microRNAs

分别从孵化3d的已鉴定性别的鸡与鹌鹑属间杂交早期雌性和雄性胚胎组织中提取小RNA,并构建各自的cDNA文库,通过Illumina／Solexa二代测序平台对两样本SmallRNA进行高通量深度测序,结合生物信息学的方法对其属间杂交早期的雌性和雄性胚胎中已知的和预测的miRNAs的类型、长度、丰度以及参与的KEGG通路等进行了初步的统计和分析。结果显示,鸡与鹌鹑属间杂交早期雌性胚胎组织的比对序列有16058009条,雄性组织有17943294条;雌性胚胎预测得到132种miRNA,雄性胚胎预测得到460种miRNA;雌性和雄性样本间差异显著的miRNA有117个（P〈0．01）;KEGG通路分析表明,这些差异miRNAs的靶基因主要参与了wnt信号通路、MAPK信号通路、TGF-beta信号通路等与胚胎生长发育相关的途径。结果表明,就已知的miRNAs而言,在鸡与鹌鹑属间杂交雌性和雄性胚胎问存在着一定的差异,这些差异的miRNAs将为后续的功能和调控机理实验研究提供依据。     

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。     

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。     

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。     

鸡与鹌鹑属间杂交种胚胎早期死亡可能机制的分析

动物的远缘杂交会产生杂交不育问题,主要表现在胚胎早期死亡、异种生殖细胞不易受精、杂种不育、种间和属间杂交种出现极端不平衡的性别比例.已有学者从组织形态学、生殖生理学、组织化学和染色体遗传学等不同角度进行过研究,多数学者认为远缘杂交染色体及基因结构的变异是导致杂交不育的主要原因.鸡与鹌鹑的杂交是典型的远缘杂交,其不育主要表现在胚胎早期出现大量死亡,在出壳前雌性全部死亡,出壳的全部为雄性但其睾丸不发育而无法正常繁育.本文从胚胎早期死亡的表现、染色体异型配对、胚胎死亡引起的蛋白表达异常、器官分化及相关基因表达异常对胚胎死亡的影响等方面进行了研究与分析.     

基于高通量测序技术比较雄性和雌性黑貉肠道菌群间的差异

性别不同导致肠道微生物组成和多样性存在差异,肠道微生物影响宿主的营养和免疫功能.貉是一种重要的经济动物,但是不同性别貉的肠道微生物的差异尚未见报道.通过比较冬毛期雌貉与雄貉肠道微生物的差异,探究性别对冬毛期貉肠道微生物的影响.试验于2月份,在同一饲养场采集了 24只(雌雄各半)日 粮及年龄相同、健康的冬毛期黑貉的新鲜粪...     

视黄酸诱导鸡胚胎干细胞向雄性生殖细胞分化的研究

体外胚胎干细胞（embryonic stem cells,ESCs）向生殖细胞分化可用于治疗不育症,同时也为揭示种系世代的分子机制提供最佳模型。试验旨在探讨视黄酸（retinoic acid,RA）诱导鸡胚胎干细胞（chicken embryonic stem cells,cESCs）向雄性生殖细胞（male germ cells,MGCs）分化的作用效果。利用胰蛋白酶消化法从新鲜种蛋X期鸡胚中分离胚胎干细胞,以鸡胚成纤维（chicken embryo fibroblast,CEF）细胞为滋养层,进行体外培养,利用形态法、碱性磷酸酶（alkaline phosphatase,AKP）染色和胚胎阶段特异性表面抗原（embryo specific surface antigen 1,SSEA-1）检测对获得的胚胎干细胞进行鉴定。结果表明,获得典型的呈巢状或岛状的cESCs克隆,细胞AKP染色呈蓝紫色,表明其具有较高的内源性AKP活性;SSEA-1鉴定结果呈阳性,显示cESCs克隆具有多能性。采用10^-5 mol/L RA诱导鸡胚胎干细胞向雄性生殖细胞分化,镜下观察细胞形态变化,分别于诱导第0、2、4、6、8、10天提取细胞总RNA,反转录成cDNA,用于实时荧光定量PCR检测生殖细胞标志基因的表达。结果表明,在此诱导过程中,作为胚胎干细胞标志基因Nanog、Sox2表达量持续显著下降,而生殖细胞特异性基因Dazl、Stra8、c-kit、integrin α6表达量呈持续上升趋势;免疫细胞化学检测可观察到特异基因相关蛋白的阳性克隆。本研究成功分离出cESCs,可体外培养并保持未分化状态及多能性。10^-5 mol/L RA能够促进cESCs向雄性生殖细胞方向分化,可以引起生殖细胞相应基因的表达,为进一步研究雄性生殖细胞的形成和调控机制提供参考。     

MLP在肉鸡、蛋鸡及其与鹌鹑属间杂交禽胚胎肌肉发育过程中的差异表达

本研究旨在探讨MLP基因在蛋种鸡(♂)×鹌鹑(♀)和肉种鸡(♂)×鹌鹑(♀)子代杂交禽胚胎肌肉发育过程中的表达规律,以及该基因在2种杂交禽中的差异表达情况。通过人工受精获得蛋鸡、肉鸡(♂)与鹌鹑(♀)杂交禽蛋,将4种受精蛋在相同条件下孵育。通过实时荧光定量PCR对7~17d胚胎胸肌组织中MLP基因的mRNA相对表达量进行测定,4种胚胎样品每天各测1次。结果显示,在胚胎肌肉发育的7~12d,4个物种的MLP mRNA表达水平很低,且在13d出现瞬时增高。其中,MLP mRNA的表达在鹌鹑胚胎发育14d达到高峰,在肉鸡胚胎发育的15d达到高峰。肉杂交禽在13~17d维持相对稳定的高表达水平,并在14,16d达到高峰。而蛋杂交禽在15d达到高峰后出现极显著性下降。上述结果表明,MLP mRNA的表达规律与肌管融合相一致,可能参与到肌管的融合过程,MLP可能在胚胎肌肉生长和发育过程中起调控作用。     

用异源蛋白和蛋壳体外培养鹌鹑胚胎的研究

将50个鹌鹑胚胎在鸡蛋壳中用鸡稀蛋白培养,对种蛋产出体外阶段裸黄状态的鹌鹑胚胎进行培养的方法进行了探讨。1～2.5d、2.5～14d和15～17d胚胎培养温度分别是38.0℃、37.8℃和37.5℃,相对湿度分别是60%、55%和70%,1～14d每小时翻蛋1次,翻蛋角度1～2.5d为90°,2.5～14d为50°,15d后静止落盘。2.5d和15d胚胎存活率以及孵化率分别为92%、78%和28%。结果表明裸黄状态的鹌鹑胚胎不排斥鸡稀蛋白,用鸡稀蛋白培养鹌鹑胚胎可行。     

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。     

Malpositions in chicken-pheasant hybrid embryos

The occurrence of malpositions in hybrid embryos obtained by two‐way crosses between Ring‐necked pheasants (Phasianus colchicus) and Columbian Rock fowls (Gallus domesticus) was examined and the numbers of the various types of malpositions were recorded. Of the 230 unhatched embryos, the 178 embryos which were detected to be more than 18 d old were classified according to their position in the shell. Of those exhibiting malpositions 68 were males and 52 were females. A X² analysis revealed that the sex of the embryo and the distribution of positions (normal vs malposition) were not correlated, although there were significant differences amongst the frequencies with which the different malpositions occurred (P<0.001). Since some of the embryos in malposition 2, in which the head is in the narrower end of the shell, were alive at the time of examination, while those in malposition 3, in which the head is turned to the left, were all dead, it is postulated that the former malposition is not always lethal, whereas the latter may be. Possibly some of the malpositions observed in these embryos represent earlier stages, or minor deviations, of the normal position. Although the proportion of embryos showing malpositions was large (67.4% of all the unhatched embryos examined), malpositions 2 and 3 together amounted to only 23.4% of the total showing malpositions. These observations, and the fact that 32.6% of the unhatched embryos were in normal position, suggest that malposition per se is not the main cause of the poor hatchability of these hybrids.     

Identification of key microRNAs affecting melanogenesis of breast muscle in Muchuan black-boned chickens by RNA sequencing

ABSTRACT

1. Melanin content is considered an important indicator of meat quality in black-boned chickens, which have a high market value. To understand the complex physiological processes underlying muscle melanogenesis in this chicken, differentially expressed miRNAs (DEMs) were detected between black muscle (BM) and white muscle (WM) of chickens using high-throughput sequencing technology. Six small RNA libraries were constructed, and more than 16.75 million clean reads were obtained for each library.

2. A total of 582 known miRNAs and 65 novel miRNAs were identified from the six chicken sequence libraries. A total of 19 DEMs were identified between the two groups, of which nine were upregulated and 10 were downregulated. Furthermore, the DEMs were predicted to target 572 genes.

3. Certain DEMs (such as miR-204, miR-133b, and miR-12 229-3p) and their target genes may play an important role in muscle melanogenesis of chickens. These findings provide a foundation for clarifying the miRNA regulatory mechanisms involved in muscle pigmentation in avian species.     

Pharmacokinetics of sulfamethazine in male, female and castrated male swine

The concentration of sulfamethazine in plasma and sulfamethazine and its metabolites in urine were compared in male, female and castrated male swine. A surgical technique for placement of catheters in the urinary bladder was used to facilitate the collection of urine in males and castrated males. The elimination rate of sulfamethazine from plasma and the excretion of parent drug and metabolites into urine did not differ significantly among females, males and castrated male swine.     

Cystoscopic examination of male and female dogs

The use of cystoscopy is advocated as an aid to the early differential diagnosis of disease of the canine bladder. Techniques are described for carrying out urethroscopy and cystoscopy in both male and female dogs using modern medical diagnostic instruments. Males were examined with flexible paediatric bronchofibrescopes, which permitted urethroscopy and cystoscopy, but to obtain extensive biopsies or undertake cauterisation of the bladder surface with rigid endoscopes, a simple perineal urethrostomy was necessary. The bladder of females, on the other hand, was examined by cystoscopy and biopsied using standard rigid cystoscopes and resectoscopes.     

Choice feeding of the male and female broiler

1. Male and female broilers from 21 d of age were fed on either a complete diet or given a choice of whole wheat and one of two higher‐protein foods formulated by omitting some or all of the cereal from the complete diet.

2. For males receiving the choice treatments, on average 44.7 and 73.1 % by weight, respectively of the food consumed from 21 d consisted of whole wheat. The corresponding figures for the females were 49.9 and 77.3% respectively.

3. Growth rate from 21 d was significantly lower for the males which received wheat and the cereal‐free higher‐protein food and for the females on either choice treatment. Differences in the treatment means, however, were small.     

Genetic investigations on male and female fertility in cattle

More than one million inseminations from Red Danish (RDM), Black and White Danish (SDM) and Danish Jersey (DJ) bulls were analyzed to obtain heritability estimates on non-return rate (NR%). The results ranged from 0.010 ± 0.003 to 0.036 ± 0.009. Three NR% and three methods of calculation are used in the analyses.Investigations on 32,100 first calving intervals (CI) within the same breeds yielded heritability estimates of 0.096±0.014, 0.030±0.011 and 0.031±0.018 for RDM, SDM and DJ, respectively.The phenotypic correlations between the bulls' own (NR%) fertility and that of their daughters' (CI) were ?0.17, ?0.16 and 0.10 for RDM, SDM and DJ, the corresponding genetic correlations being ?0.30, ?0.35 and 0.20.It is suggested that in a possible breeding scheme for fertility, attention should concentrate on the female's side (progeny testing).