Reference Gene Selection for Normalization of PCR Analysis in Chicken Embryo Fibroblast Infected with H5N1 AIV

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV).In this study,the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system.CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID50 of H5N1 AIV and harvested at 3,12,24 and 30 hours post-infection.The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR.Based on expression stability and expression levels,our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection.However,for the study of replication levels of H5N1 AIV in CEFs,the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.

Canine Distemper Virus Utilizes Different Receptors to Infect Chicken Embryo Fibroblasts and Vero cells

Inducing animal viruses to adapt to chicken embryos or chicken embryo fibroblasts(CEF) is a common method to develop attenuated live vaccines with full security.Canine distemper virus(CDV) also does this,but the mechanisms and particular receptors remain unclear.Virus overlay protein blot assays were carried out on CEF membrane proteins,which were extracted respectively with a Mem-PER TM kit,a radioimmunoprecipitation assay buffer or a modified co-immunoprecipitation method,and revealed a common 57 kDa positive band that differed from the 42-kDa positive band in Vero cells and also from those receptors reported in lymphocytes and 293 cells,indicating a receptor diversity of CDV and the possibility of the 57-kDa protein acting as a receptor that is involved in adaptive infection of CDV Kunming strain to CEF.

The m6A methylation regulates gonadal sex differentiation in chicken embryo

Background:As a ubiquitous reversible epigenetic RNA modification,N6-methyladenosine(m6A)plays crucial regulatory roles in multiple biological pathways.However,its functional mechanisms in sex determination and differentiation during gonadal development of chicken embryos are not clear.Therefore,we established a transcriptome-wide m6A map in the female and male chicken left gonads of embryonic day 7(E7)by methylated RNA immunoprecipitation sequencing(MeRIP-seq)to offer insight into the landscape of m6A methylation and investigate the post-transcriptional modification underlying gonadal differentiation.Results:The chicken embryonic gonadal transcriptome was extensively methylated.We found 15,191 and 16,111 m6A peaks in the female and male left gonads,respectively,which were mainly enriched in the coding sequence(CDS)and stop codon.Among these m6A peaks,we identified that 1013 and 751 were hypermethylated in females and males,respectively.These differential peaks covered 281 and 327 genes,such as BMP2,SMAD2,SOX9 and CYP19A1,which were primarily associated with development,morphogenesis and sex differentiation by functional enrichment.Further analysis revealed that the m6A methylation level was positively correlated with gene expression abundance.Furthermore,we found that YTHDC2 could regulate the expression of sex-related genes,especially HEMGN and SOX9,in male mesonephros/gonad mingle cells,which was verified by in vitro experiments,suggesting a regulatory role of m6A methylation in chicken gonad differentiation.Conclusions:This work provided a comprehensive m6A methylation profile of chicken embryonic gonads and revealed YTHDC2 as a key regulator responsible for sex differentiation.Our results contribute to a better understanding of epigenetic factors involved in chicken sex determination and differentiation and to promoting the future development of sex manipulation in poultry industry.

Identification of the Sex of Earlier Embryos from Generic Hybrids of Chicken-Quail by Wpkci

In this study, a protocol was developed to identify the sex of earlier embryos of chicken （♂）-quail （♀） hybrids and successfully tested the sex proportion of each period （66-120 h）. We acquired cross bred eggs by artificial insemination, hatched them in the same batch according to the standard hatching condition of chicken, and collected earlier living embryos at 66, 72, 78, 84, 90, 96, 102, 108, 114, and 120 h randomly. We adopted RT-PCR protocol and multiple PCR, made the known sex quail as the external control, employed fl-actin as the internal control, and used primers that were designed according to conservative area of gene Wpkci of quail to identify the sex of earlier hybrid embryos. The results indicated that the primer of Wpkci can be used to identify the sex of hybrid embryos accurately; there were more male than female in earlier embryos, the sex proportion of earlier embryos compared with academic numerical value was significantly different （P〈0.01）, and there was no difference between different periods （P〉0.05）. In the present study, we concluded that a simple, fast, credible and stable protocol to identify the sex of earlier hybrids embryos had been established by using primer of Wpkci; in earlier embryos, the death rate of female was higher than that of male and there was no fluctuant peak.

Research on the appropriate way to transfer exogenous substances into chicken embryos

In biological research, chicken embryos are a classic experimental model for the exploration of the embryonic development and cell differentiation. Transferring exogenous substances into chicken embryos for producing medical antibodies has been widely used in the production practice. However, there are few studies about the effect of the different injection site and dosage on chicken embryos. The aim of this study was to explore the effects of different injection sites and dosages on chicken embryo hatching rate and development, so as to provide a basis for further studies using the chicken embryo model. Freshly laid eggs （Rugao yellow chicken） were injected with different doses of saline at the tip, equatorial plane and the blunt end of the egg shell, respectively. Egg hatching rate was recorded and compared among injection sites and different doses. A trypan blue stain was also injected at the aforementioned sites and the growth of chicken embryos was observed. The SPSS （statistical package for the social science） software was used to analyze the relationship between the chicken eggs hatching rate and the different injection sites or the different dosages. The experimental results showed that there were significant differences on egg hatching rates among the different injection sites and doses （P〈0.05）. The hatchability of the blunt end injection group was significantly higher than that of the other two sites. The egg hatching rate decreased with increased saline doses. The egg hatching rate of the 100 pL saline injection group was higher than the 200 and 300 μL dosage groups. Ultimately, we suggest that the optimal chicken embryo injection process is during early development, at the blunt end site with a dose less than 100 μL to minimize damage to the egg.

Chicken Embryo Inhibition Test of Recombinant Freeze-drying Chicken Interferon against Newcastle Disease Virus( NDV) F_(48)E_(10)

The paper was to study the inhibitory effect of recombinant freeze-drying chicken interferon against Newcastle disease virus （NDV） F48E10. Nine-day-old chicken embryos were inoculated with recombinant freeze-drying chicken interferon via allaotoic sack, while 10-day-old chicken embryos were inoculated with NDV F48E10, and in vivo protective efficacy of interferon on chichen embryos was studied. The results showed that the recombinant freeze-drying chicken interferon at the dose of 1.28 mg/embryo reached the protection ratio of 90% on chicken embryo infected by F48E10.

Adaptability of Fowlpox Virus in Chicken Embryo Passage Cells

[Objective] To observe whether fowlpox virus （FPV） can proliferate in chicken embryo passage fibroblasts or not and then try to use chicken embryo passage fibroblasts to replace primary chicken embryo cells for FPV culture. [Method] Primary chicken embryo fibroblasts were prepared and subcultured. After FPV were inoculated on the 20th passage fibroblasts, cytopathy was observed. Then, the FPV culture was identified and determined quantificationally. [Result] Specific cytopathy appeared in the FPV-inoculated chicken embryo passage fibroblasts. The titer of the yielded FPV culture reached the standard for production of fowl pox vaccine. Further analysis reveals that the chorioallantoic membrane lesions were caused by FPV. [ Conclusion] FPV can reproduce in chicken embryo passage fibroblasts, and the Uter of FPV cell culture can meet the pro- duction requirements of fowl pox vaccine.

The Quality Characteristics of Eggs: Impact on the Volume of Allantoin-Amniotic Fluid of Chicken Embryos

In this paper, the eggs＇ weight, quality characteristics （ albumen density, shell quality, yolk size）, and egg weight loss during incubation having impact on volume of allantoin-anmiotic fluid are considered. The major parameters that should be taken into account when creating specialized population of chickens for the production of DEC （ developing embryos of chickens） are determined.

Effects of "Products of Chicken Embryo" on Growth and Sexual Development in Rats

Products of Chicken Embryo (PCE) such as Ji-Pei-Jing is a kind of food for Chinese children prepard from chicken embryo. Female rats on 21 days were administered with aqueous solutions of Ji-Pei-Jing (1. 2 %, 3 %, 12%, and 48%, respectively) by gavage up to their onsets of puberty.The rats in the control group were treated with distilled water. However, Ji-Pei-Jing treatment exerted some effects on sexual mauration in the immature female rats. Essentially, the effects showed a dose-response tendency with an inverted 'U' shape.The age of vopnal opening for gnup treated with Ji-Pei-Jing was significantly earier than that to the control. Its uterus weight/b. w. ratio also significantly increased on day 30 and at the first estrus. There were significantly increases in the adrenal weight/b. w. raio of 30-day-old rats that were treated with 3%, 12%, and 48% Ji-Pei-Jing. The rats treated with 48% Ji-Pei-Jing had significantly lesser ovary weight.b.w. radio on day 30, too. The rats treated with Ji-Pei-Jing could normally ovulate at the first estrus, and no significant differences were observed during estrous cycles.The effects of PCE on serum levels of K, P, LH in 30-day-old nds and FSH in 28-day-old ras were elevated significantly by 3 % Ji-Pei-Jing treatment. It appeare that the effects of PCE result from interaction of contained complex physiologically active substances. Steriods, especially estradio-17β,possibly Play a key role, and polpeptide hormones may also exert important effects.

Hematomas and Limb Skeletal Malformations in Chicken Embryos Following Exposure to 5-Fluoro-2'-Deoxyuridine

Vascular injury or interruption may play a role in vertebrate limb teratogenesis. Since 5fluoro- 2'- deoxyuridine (FdU) can cause vascular injury in the murine limb and skull prior to the appearance of skeletal malformations in these structures, we studied the effects of this chemical on skeletal development in the chick embryo and noted any vascular injury. The yolk sacs of day three ehick embryos (Hamburger and Hamilton states 17-19) were injected with solutions of vary concentrations of FdU in saline. The embryos developed until the 10th day of incubation when they were fixed for study. Uninjected, saline injected, and sham injected control embryo were similarly fixed. Upon gross inspection, frequent diffuse and saccular hernatomas, as well as fluid-filled blisters, were noted in the limbs of embryos treated with FdU. After the embryos were fixed and cleared, and the skeletons stained, significant skeletal malformations were observed in these limbs. Bony elements of both the upper and lower limbs were affected in at least some of the embryos. The combination of FdU-induced hematomas and blisters with associated skeletal malformations in the same regions of some embryos suggests a relationship between these phenomena.

广东规模化鸡场死鸡胚中鸡毒支原体的分离鉴定、致病性及药物敏感性

旨在从广东某规模化鸡场死鸡胚进行鸡毒支原体(Mycoplasma gallisepticum,MG)的分离,并进行遗传进化分析、致病性及药物敏感性研究。本研究从广东某规模化鸡场的带菌死胚卵黄组织中分离禽支原体,通过菌落观察、血清学试验、16S rRNA支原体通用引物序列鉴定等方法,对分离的菌株进行种属鉴定,同时将分离的毒株对鸡胚和SPF鸡进行攻毒试验及抗菌药物敏感性试验。结果显示:显微镜下菌落呈典型的“荷包蛋”状。平板凝集试验显示其与MG阳性血清有凝集反应,与MS阳性血清不反应;16S rRNA序列测序发现各分离株与MG相似性达99.9%,因此确定分离株为MG。将各分离株感染7日龄SPF鸡胚,结果显示感染鸡胚大多于临近出壳时死亡;感染3周龄的SPF鸡,3周后解剖发现鸡气囊发生显著病理变化,说明其具有较强致病力;药物敏感性试验结果显示,5株分离株对盐酸沃尼妙林、多西环素、泰万菌素及泰妙菌素有较高敏感性,对替米考星、泰乐菌素、恩诺沙星、红霉素、吉他霉素、林可霉素呈现出不同程度耐药性。综上,从广东某规模化鸡场死鸡胚中成功分离到5株MG分离株,各分离株均可引起红细胞凝集,导致鸡胚死亡,对SPF鸡可引起典型的气囊炎症状,且对多种药物存在不同程度耐药性。本研究为临床MG的分离鉴定及用药选择提供了可参考的技术方法及指导,为MG攻毒模型建立提供了研究基础。

孵化过程中鸡胚性别分化期的判定

【目的】研究鸡胚孵化过程中,通过不同方法判断其性别分化的时间。【方法】以黄羽肉鸡胚蛋为研究对象,孵化黄羽肉鸡胚蛋(温度37.8℃,相对湿度60%)。分别通过胚线观察法、生殖腺器官形态观察法、组织学形态学、分子生物学来判断鸡的性别分化期,试验重复3次。【结果】通过胚线观察法分析发现,在鸡胚孵化期第3天,可以通过血管分支的粗细和形态来辨别雌雄。通过生殖腺器官的观察,在孵化期第8天,能够明显关察到雄性性腺对称发育,雌性性腺则不对称发育。通过组织学形态鉴定,在孵化期第7~10天,观察到雄性性腺有类似于曲精细管的团索状结构出现,雌性性腺有皮髓结构出现,能够判定鸡胚性别分化期可能为胚胎孵化期第7天。通过分子生物学鉴定,孵化期第3~10天,观察到雄性个体有1条扩增条带,其大小是为550 bp左右,观察到雌性个体有2条扩增条带,其大小为550 bp和450 bp左右,因此判定性别分化期可能为孵化期第3天。【结论】综上判定鸡性别分化期约在胚胎孵化期第3~8天。

抗鸡传染性支气管炎病毒中药的筛选

【目的】对鸡传染性支气管炎病毒(Infectious bronchitis virus,IBV)进行抗病毒中药的筛选,并验证其治疗效果,为临床用药提供一定参考。【方法】采用鸡胚培养法,通过预防和治疗2个角度对24味单一中药、6种市售复方中药进行抗IBV的有效成分或方剂筛选试验,即在SPF鸡胚中先注射中药2 h后再接种病毒和先接种病毒2 h后再注射中药。使用SPSS 20.0软件对鸡胚重量进行方差分析,利用实时荧光定量PCR法辅助判定治疗效果。【结果】对鸡胚净重进行方差分析显示,桔梗组与阳性对照组差异显著(P<0.05),与阴性对照组差异不显著(P>0.05),表明预防和治疗均有效果。实时荧光定量PCR检测鸡胚尿囊液病毒滴度结果显示,桔梗、鱼腥草、宣肺败毒方、绞股蓝、蒲公英、甘草对IBV增殖均有抑制作用,其中桔梗抑制效果最佳。【结论】桔梗对IBV有显著的预防及抑制效果,结果为深入研究桔梗作用机理提供了依据。

水族鸡胚地龙膏加羼杂疗法对膝骨关节炎患者生活质量的影响

目的探究水族鸡胚地龙膏加羼杂疗法对膝骨关节炎患者生活质量的影响。方法采用随机、平行、安慰剂对照设计,纳入72例患者。治疗组采用羼杂疗法加鸡胚地龙膏,对照组采用假羼杂疗法加鸡胚地龙膏模拟剂,治疗3 w后,再随访4 w,观察两组的生活质量评分(SF-12)及自我评价。结果生活质量评分:两组基线及治疗后3 w、5 w、7 w生活质量评分的差异无统计学意义(P>0.05)。治疗1 w时两组的差异有统计学意义(P<0.05),对照组大于治疗组。自我评价:两组治疗后2、5 w患者自我评价的差异有统计学意义(P<0.05),治疗组明显优于对照组。结论鸡胚地龙膏加羼杂疗法对膝骨关节炎患者生活质量的改善未能表现出明显的促进作用,但在治疗期间能积极提高患者自我感觉。

CLK2 mRNA在早期鸡胚中的表达及定位分析

为探究早期鸡胚(孵化后约48 h内)中蛋白激酶CLK2 mRNA的表达特点及部位,用qPCR法测定早期鸡胚中CLK2 mRNA的表达,利用分子克隆技术重组CLK2/pMD18-T质粒,制作CLK2 mRNA原位杂交探针并以地高辛标记,进而通过原位杂交技术测定不同时期鸡胚中CLK2 mRNA的表达和分布。结果表明,早期鸡胚中CLK2 mRNA在各时期均有表达但表达量有所不同;原位杂交结果显示,在鸡胚发育的各个阶段,CLK2 mRNA的表达有所差异,且在各个部位其表达的量也各不相同,头部和尾部的表达最为集中。综上,CLK2 mRNA在鸡胚中存在且表达,其表达分布提示CLK2可能与其头部和神经系统的发育密切相关。

空心莲子草乙酸乙酯部位体内外抗H9N2亚型禽流感病毒活性的研究

[目的]探究空心莲子草乙酸乙酯部位(ethyl acetate extract from Alternanthera philoxeroides,AETAC)体内外抗H9N2亚型禽流感病毒(H9N2 subtype Avian influenza virus, AIV-H9N2)活性及其作用机制。[方法]利用CCK-8法检测不同浓度AETAC作用不同时间对鸡胚成纤维细胞(DF-1)的抑制作用,通过预防感染、影响复制、阻止吸附及直接杀灭4种方式作用后检测AETAC对DF-1细胞的毒性,选择体外最佳抗AIV-H9N2作用方式。尿囊腔接种不同浓度AETAC确定其对鸡胚的最大安全浓度;将AETAC以3种不同给药方式作用于AIV-H9N2感染后鸡胚,选择体内最佳给药方式。经尿囊腔同时接种AETAC和AIV-H9N2,统计各组鸡胚存活数,测定血凝效价,并观察胚胎的发育情况和鸡胚法氏囊病理组织变化。[结果]在一定范围内,随着AETAC浓度升高或作用时间延长,AETAC对DF-1细胞的抑制率升高。与空白对照组相比,4种抗AIV-H9N2作用方式下,病毒对照组和药物组细胞存活率均显著降低(P<0.05);与病毒对照组相比,AETAC浓度为5~30μg/mL时,4种抗AIV-H9N2作用方式下,药物组细胞存活率均显著升高(P<0.05)。4种作用方式下,DF-1细胞存活率分别为8.15%~64.96%(预防感染)、8.56%~83.83%(影响复制)、1.82%~5.90%(阻止吸附)和6.90%~40.12%(直接杀灭)。AETAC对鸡胚的最大安全浓度为16.41 mg/mL,对感染鸡胚的最佳给药方式为AETAC和病毒混合后接种。以最佳体内作用方式给药后,AETAC中、低浓度组鸡胚存活率为40%~60%,高浓度组鸡胚全部存活,胚胎喙部发育明显,鸡胚法氏囊淋巴滤泡发育完整,滤泡大小和数量正常,充血量少,组织病理状态改善。[结论]AETAC在体内外对AIV-H9N2均具有显著的抑制作用,且主要与影响病毒复制作用方式相关。

用正交试验法分析四种表面活性剂的复配性能

用正交试验设计法研究月桂醇聚醚硫酸酯钠、十二烷基硫酸钠、椰油酰胺丙基甜菜碱和月桂酰肌氨酸钠复配的最佳使用比例,摸索四种表面活性剂对泡沫、洗后皮肤水分含量及刺激性的影响。结果显示,在给定的因素水平数里,泡沫高度的优方案为AES:13%,K12:4%,CAB:6%,LS30:5%;洗后皮肤水分含量的优方案为AES:14.5%,K12:3%,CAB:6%,LS30:2%;刺激性最小的优方案为AES:11.5%,K12:1%,CAB:6%,LS30:3%。通过试验数据分析可知,AES对泡沫高度和鸡胚试验评分有显著影响;K12对泡沫高度和洗后皮肤水分含量有显著影响;CAB对泡沫高度有影响;而LS30有助于降低鸡胚评分,在3%时效果最好。

鸡W染色体基因CTIF在鸡胚组织中的表达研究

该文对CTIF在胚胎发育过程中的表达进行初步研究。通过设计特异性扩增W拷贝、Z拷贝以及同时扩增W和Z拷贝的引物(CTIF-W、CTIF-Z及CTIF-ZW),对其在胚胎的不同日龄的组织的mRNA表达量进行分析,并通过FAD(fadrozole)诱导性反转探索其对性别表型的响应。结果显示,CTIF-W在孵化6 d的胚胎性腺中的表达高于尾部,且随胚胎发育表达量逐渐升高,而CTIF-Z和CTIF-ZW的表达趋势相似;而在6 d鸡胚的其他组织中,CTIF-ZW在肾中的表达显著高于其他组织,说明CTIF在发挥某些基础功能的同时可能还与肾脏的发育或功能相关。CTIF-Z在多数组织中表达量均呈现出雄性高于雌性,而CTIF-ZW在雌雄之间的表达量差异则相对较小,表明W拷贝可能在一定程度上调控了雌性中Z拷贝的表达。CTIF-W和CTIF-Z在12 d的胚胎组织中均表达,且均高度表达于脾脏组织中;CTIF-Z在大多数组织中雄性表达高于雌性。此外,CTIF-W表达受雌性向雄性性反转的影响而显著降低,而CTIF-Z则没有响应。综上,CTIF在鸡胚的肾脏和脾脏中高度表达,且其Z拷贝在雌雄组织之间存在剂量效应,而W拷贝可能在雌性中作为Z拷贝的补偿以保证其生物学功能的发挥。

Lipid transport to avian oocytes and to the developing embryo

Studies of receptor-mediated lipoprotein metabolic pathways in avian species have revealed that physiological intricacies of specific cell types are highly analogous to those in mammals. A prime example for the power of com- parative studies across different animal kingdoms, elucidated in the chicken, is that the expression of different lipo- protein receptors in somatic cells and oocytes are the key to oocyte growth. In avian species, yolk precursor transport from the hen＇s liver to rapidly growing oocytes and the subsequent transfer of yolk nutrients via the yolk sac to the developing embryo are highly efficient processes. Oocytes grow from a diameter of 5 mm to 2.5-3 cm in only 7 days, and the yolk sac transfers nutrients from the yolk stored in the mature oocyte to the embryo within just 2 weeks. The underlying key transport mechanism is receptor-mediated endocytosis of macromolecules, i.e., of hepatically synthesized yolk precursors for oocyte growth, and of mature yolk components for embryo nutrition, respectively. Recently, the receptors involved, as well as the role of lipoprotein synthesis in the yolk sac have been identified. As outlined here, lipoprotein degradation/resynthesis cycles and the expression of lipoprotein receptors are not only coordinated with the establishment of the tbllicular architecture embedding the oocyte, but also with the generation of the yolk sac vasculature essential for nutrient transfer to the embryo.

鸡胚给养调控鸡体健康研究进展

随着无抗养殖和豆粕减量替代行动的不断推进,对肉鸡和蛋鸡的无抗和高效健康养殖方面提出了新的要求,尤其需要关注养殖过程中的健康问题。鸡胚给养技术可以通过人为的营养干预和调控在孵化期提高鸡的早期营养和免疫能力,并对鸡体的生长和健康产生长期和深远的影响。文章对近五年发表的关于鸡胚给养技术对鸡的器官发育、肠道菌群、抗逆性和抗病性等方面的作用进行了系统综述,旨在为鸡胚给养在禽类的健康养殖中的应用和研究提供理论依据。