黄花棘豆生物碱对体外大鼠胚胎的发育毒性

应用大鼠全胚胎培养（ｗｈｏｌｅｅｍｂｒｙｏｃｕｌｔｕｒｅ，ＷＥＣ）方法，观察了黄花棘豆（Ｏｘｙｔｒｏｐｉｓｏｃｈｒｏｃｅｐｈａｌａ）主要有毒成分生物碱对体外胚胎生长发育、形态分化的影响。首先用醇类溶剂提取法从黄花棘豆中提取生物碱，薄板层析鉴定至少有４种生物碱成分。然后用体内同期胚胎对照及丝裂霉素Ｃ（ＭＭＣ）阳性对照确证了所建立的ＷＥＣ方法的可行性。在选定０．１％ＤＭＳＯ为该生物碱溶剂的基础上，分别在含有２５、５０、１００、２００ｍｇ／Ｌ生物碱的大鼠血清中培养９．５ｄ胚胎４８ｈ，结果，在５０ｍｇ／Ｌ以上可影响胚胎生长发育和器官分化，且呈剂量反应关系，主要表现为头长、颅臀长、体节数、蛋白质含量及１７项形态学总记分低于溶剂对照组，而且随剂量增加，胚胎畸形也增多，２００ｍｇ／Ｌ时畸形率为８０％，主要表现为小头、后脑水肿、无听泡、无视泡、心包积液、肢芽缺失、尾异常、翻转异常等。生物碱还可影响脏壁卵黄囊（ＶＹＳ）的血液循环，抑制其生长。结果提示，黄花棘豆生物碱对体外培养大鼠胚胎有直接发育毒性，ＶＹＳ功能障碍也是引起胚胎发育毒性的重要机制。     

In ovo pathogenicity of Mycoplasma gallisepticum strains in the presence and absence of maternal antibody

Mycoplasma gallisepticum (MG) strains showing marked variation in pathogenicity were examined for virulence in ovo. No correlation was found between in ovo pathogenicity and other in vivo or in vitro methods for pathogenicity evaluation. For certain highly pathogenic strains, there was a clear relationship between the titer of MG inoculated and the embryo mortality and time of death; an LD50 for these strains could be calculated by yolk-sac inoculation. However, not every strain that caused lesions in the respiratory tract in vivo caused embryo mortality. Less pathogenic strains that grow well and colonize the respiratory tract usually caused embryo mortality during the later stages of incubation, and there was no strict correlation between titer of inoculum and embryo mortality. It appeared that embryo death in these cases may have resulted from generalized stress due to mass multiplication of the MG. Embryo mortality due to virulent MG was completely blocked in eggs containing maternal antibody to MG, although the mycoplasma could be reisolated from the yolk-sac membrane of the live embryonated egg after 17 days of incubation. Attempts to mimic the effect of maternal antibody by injecting exogenous MG antiserum were not successful.     

Cdc25B mRNA在早期鸡胚中的动态表达

本试验旨在研究Cdc25B mRNA在早期鸡胚中的时空表达模式。通过分子克隆的方法获得Cdc25B基因特异性目的片段,并用体外转录的方法合成了Cdc25B RNA探针,通过全胚胎原位杂交的方法检测了早期不同发育阶段鸡胚中的Cdc25B mRNA水平。结果表明,Cdc25B的杂交信号主要分布于中枢神经系统(CNS)、体节和肢芽的顶端,在尾部神经上皮中缺乏杂交信号。早期阶段,Cdc25B的杂交信号不对称地分布在亨氏节的左侧。随着胚胎的发育,Cdc25B的杂交信号逐渐增强,并且在即将封闭的神经管中,杂交信号呈现出腹背递减的浓度梯度。然而,在早期各个阶段,脊索都是阴性的。这些结果提示,Cdc25B在前、后期的CNS、体节和肢芽发育中可能发挥不同的功能,这种功能的改变可能与Cdc25B的表达浓度有关。此外,鸡胚胎内部器官的左右不对称性发育可能是Cdc25B与其它基因共同调控的结果,这些调控发育的机制可能是Cdc25B蛋白参与了细胞周期进程,调控细胞增殖或分化。     

Effect of in ovo vaccine delivery route on herpesvirus of turkeys/SB-1 efficacy and viremia

A study was designed to ascertain the influence of in ovo site of inoculation and embryonic fluid type on the development of Marek's disease (MD) vaccine viremia and efficacy against MD challenge. The experiments were divided into in vitro and in vivo phases. In the in vitro phase, herpesvirus of turkeys/SB-1 vaccine was combined with basal medium eagle (BME) medium (control), amniotic fluid, or allantoic fluid and subsequently titrated on secondary chick embryo fibroblast cultures. There were no significant differences in titer between the virus inoculum carried in BME and the virus inoculum combined with either the allantoic fluid or the amniotic fluid. In the in vivo phase, five routes of inoculation, amniotic, intraembryonic, allantoic, air cell, and subcutaneous at hatch, were compared for generation of protection against virulent MD challenge. Comparisons were made in both specific-pathogen-free and commercial broiler embryos/chicks and, for the amniotic and allantoic routes, injection at either day 17 or day 18 of embryonation. Reisolation of the vaccine virus at day 3 of age was also done for all routes with the exception of the air cell route. Vaccine virus was recovered from all birds tested that were injected in ovo via the amniotic and intraembryonic routes and the subcutaneously at hatch route but was isolated only sporadically from birds inoculated via the allantoic route. Vaccination protective efficacy against virulent MD for all birds vaccinated in ovo via the amniotic or intraembryonic routes and birds vaccinated subcutaneously at hatch was over 90% regardless of day of in ovo injection or bird type. Protective efficacy for vaccines delivered in ovo by either the allantoic or the air cell routes was less than 50% regardless of day of injection or bird type. Therefore, in ovo MD vaccines must be injected either via the amniotic route or the intraembryonic route for optimal performance.     

Antiviral activity of crude extracts from Commiphora swynnertonii against Newcastle disease virus in ovo

Studies were carried out to investigate the effect of crude extracts from resin, leaves, stem barks and root barks of Commiphora swynnertonii against Newcastle disease virus (NDV) using an in ovo assay. Nine-day-old embryonated chicken eggs were divided into seven groups (n?=?6) and received various treatments. Six groups were inoculated with velogenic NDV strain; five groups out of these were treated with different concentrations of the four extracts or a diluent, dimethylsulphoxide. The uninoculated and inoculated groups were left as negative and positive controls, respectively. Embryo survival was observed daily and embryo weights were measured day?5 post-inoculation; a few eggs from selected groups were left to hatch. Allantoic fluid from treated eggs and serum from hatched chicks were collected for hemagglutination and hemagglutination inhibition (HI) tests to detect NDV in the eggs and antibodies against NDV in the hatched chicks respectively. Results showed that embryo survival and mean embryo weight were significantly higher (p?<?0.001) in those groups which were treated with the crude extracts from C. swynnertonii than the positive control group. Also the extracts significantly (p?<?0.001) reduced virus titres, whereas no viruses were detected in the allantoic fluids of the resin-treated group at the highest concentration of 500???g/mL. Furthermore, the HI test results showed very low levels of antibodies against NDV in chicks hatched from resin and root bark extract-treated eggs suggesting that these plant materials were capable of destroying the NDV before stimulating the developing chick??s immunity. The current findings have clearly demonstrated that crude extracts especially that of resin from C. swynnertonii have strong antiviral activity against NDV in ovo. In vivo trials are needed to validate the use of resin from the tree in controlling Newcastle disease in chickens.     

Optimization of in vitro embryo production and zygote vitrification for the indigenous Vietnamese Ban pig: The effects of different in vitro oocyte maturation systems

The Vietnamese Ban pig is a precious genetic resource that needs to be preserved. In vitro embryo production from in vitro matured (IVM) oocytes is an important tool for the utilization of cryopreserved porcine sperm. The aim of this study was to compare two media for the IVM of Ban pig oocytes. Immature oocytes were subjected to IVM either in a non‐defined (TCM‐199 + pig follicular fluid) or in a defined base medium (POM + epidermal growth factor). At the end of IVM, the oocytes were in vitro fertilized (IVF) with frozen Ban sperm. Ten hours after IVF, the oocytes were either subjected to orcein staining to check fertilization and maturation status or cultured in vitro for 7 days. There was no difference between the two IVM media in terms of percentages of oocyte maturation and blastocyst production. However, the percentage of male pronuclear formation after IVF and the total cell numbers in blastocysts were higher with the defined system. Zygotes obtained by the two IVM systems survived vitrification at similar rates. In conclusion, the two IVM systems were both effective for the production of Ban pig embryos; however, better embryo quality was achieved with the defined one.     

Skeleton pattern and joint formation in chorioallantoic grafts containing the distal parts of the chick wing bud

Skeleton pattern formation was examined in chick wing bud grafts using the chorioallantoic grafting method. The distal parts of the wing bud were excised from the donor wing and transplanted onto the chorioallantoic membrane (the experimental groups). Transplants with intact limb bud material served as the control group. The skeleton pattern formation in the grafts depended on the amount of transplanted material and donor's limb bud stage. The younger the donor's stage and the bigger the piece of the transplanted material the more proximal parts grafts had, more retarded growth and abnormal skeleton in the zeugopod and autopod was. The percentage of the signs of insufficient blood supply in the experimental groups was less than that in the control group. As the amount of the transplanted limb bud material decreased and donor's limb bud aged, post-axial polydactyly changed to the pre-axial one.     

同源物种不同饲养层对鸡精原干细胞体外培养的影响

分别以鸡胚来源的不同细胞为饲养层培养精原干细胞,比较3种不同的饲养层对精原干细胞体外培养的影响。结果显示:精原干细胞在鸡胚成纤维细胞饲养层和鸡睾丸支持细胞饲养层上存活时间较长、生长增殖状况良好。在鸡胚成纤维饲养层上传至四代,每代的AKP阳性克隆率分别为45%、40%、36%和21%。在以睾丸支持细胞为饲养层进行培养时传至三代,每代的AKP阳性克隆率分别是40%、32%、和22%。而以鸡肝细胞作为饲养层,精原干细胞未见有克隆形成,不能进行传代培养,表明鸡胚肝细胞饲养层不适合培养精原干细胞。     

Cryotolerance of Bovine Blastocysts is Affected by Oocyte Maturation in Media Containing Palmitic or Stearic Acid

In this study, non-esterified fatty acids (NEFAs) were added during in vitro maturation at concentrations measured previously in follicular fluid (FF) of high-producing dairy cows in a negative energy status to evaluate their subsequent effect on the embryos cryotolerance. Oocytes were matured for 24 h in serum-free media with or without (negative control) the addition of NEFAs dissolved in ethanol or ethanol alone (positive control). Matured oocytes were fertilized and cultured for 7 days in synthetic oviduct fluid medium supplemented with 5% FCS. Embryos that had at least reached the blastocyst stage were vitrified by open pulled straw (OPS) vitrification. Addition of palmitic (C16 : 0) or stearic acid (C18 : 0) during oocyte maturation had significant negative effects on embryo cryotolerance, whereas ethanol or oleic acid (C18 : 1) had no effect. These in vitro results suggest that high NEFA concentrations in FF during a period of negative energy balance in high-yielding dairy cows can have carry-over effects on embryo quality.     

Development and Status of Cattle Embryo Cloning in Germany

Contents: A review about experiments in bovine embryo cloning performed by different working groups in Germany is given. The procedure is shortly described and the achieved results are specified. Average enucleation rates in the experiments were 58–74%, electrofusion rates were 31 to 85%. Between 3 and 17% of the in vitro cultured embryos cleaved to transferable embryos. The first calf emerging from nuclear transfer in Germany was born in August, 1992. A clone of three identical calves was given birth in April, 1993. Four months later a calf was born, which exclusively emerged from in vitro techniques (in vitro maturation of recipient oocytes, in vitro production of blastomere donor embryo, in vitro culture of cloned embryos). Finally some future aspects of bovine embryo cloning in Germany are illustrated.     

Artificial insemination and fertility in turkeys

Large‐type White turkey hens from a flock with a record of low “fertility” (live embryos at 7 to 10 days’ incubation) were distributed randomly into four groups of 50 hens each and were given treatments involving antibiotic and inseminations on a weekly or fortnightly schedule. During the first 5 weeks of the experiment, semen was introduced, via a plastic tube, at least 5 cm. into the oviduct of all birds in each group. Irrespective of the type of treatment, there was a significant rise in “fertility” in all groups. This was sustained for a 4‐week period, with the highest “fertility” occurring in the group inseminated weekly. When shallow insemination was used with two groups, “fertility” over a second 4‐week period was lowest in these two groups. Since percentage infertile eggs could account for the major share of the decline in percentage live embryos, it is postulated that the low live embryo percentage existing previously in the flock resulted from insufficient numbers of spermatozoa being inseminated rather than from a reaction to an unidentified pathogenic agent, as frequently suspected.     

甜樱桃不同品种需冷量评估初探

研究甜樱桃品种的需冷量,筛选适合南方地区栽植的中、低需冷量品种。通过离体湿沙培养法,将低温积累到一定时间的甜樱桃枝条置于适宜条件下培养,3周后统计萌芽率,确定各品种需冷量区间范围,分析甜樱桃萌芽率变化和品种差异,并对不同地区的相同甜樱桃品种的需冷量进行比较。结果表明,需冷量在400~800h的品种最多,其中<600h的品种28个,发现采集样品时各品种所处的休眠阶段不同,甜樱桃需冷量具有遗传特性,样品间区域差异较小。     

Lipid Content of Non-cultured and Cultured Pig Embryo

The objectives of the study were: (i) to work out a precise and efficient method for quantitative analysis of lipid content and (ii) to quantitatively determine the lipid content in non-cultured and cultured pig embryos . The experiment was carried out on pig embryos from zygote to late blastocyst stages produced in vivo and embryos collected at the zygote stage and then cultured in vitro up to blastocyst stage . Embryos were fixed, dehydrated, embedded in epoxy resin and cut into semi-thin sections to analyse the quantity of lipids in fat droplets. Stained sections were then analysed with Cavalieri and point counting methods to evaluate the following stereological parameters of the embryo: total embryo volume – V(e), volume density of cytoplasm per unit volume of embryo – Vv(c,e), volume density of lipid droplets per unit volume of embryo cytoplasm – Vv(fat,c) and total volume of lipid droplets per whole embryo – V(fat). Values of Vv(fat,c) and V(fat) remained unchanged up to the morula stage, but decreased significantly at blastocyst and late blastocyst stages both in cultured and non-cultured embryos. Volume density of lipid droplets per unit volume of embryo cytoplasm and total volume of lipid droplets for cultured embryos showed statistically significant differences between late blastocyst and almost all other stages. Comparisons of Vv(fat,c) in embryos at the same stages of development but differing in origin of embryos (non-cultured or cultured) show that statistically significant differences exist for all analysed stages. In conclusion, differences in lipid content observed in pig embryos were dependent on the developmental stage of the embryo as well as the culture conditions (i.e. cultured and non-cultured embryos at the same stage of development).     

2,4-D和BAP对蒙古冰草幼胚愈伤组织诱导及生长的影响

以蒙古冰草为材料。在离体培养条件下对其幼胚的发育进行了研究。结果表明：蒙古冰草的幼胚在不含任何激素的培养基上能直接萌发，幼胚发芽率因培养基而异。N6＋7％蔗糖培养基萌发率最高，达96％；其次为MS＋5％蔗糖培养基。萌发率为90％。当幼胚被培养在减半的N6培养基或减半的MS培养基上时，发芽率均显著降低。以上四种培养基均未发生脱分化现象。相反，在含有2，4-二氯苯氧乙酸（2，4-D）的培养基上，蒙古冰草幼胚不同程度地发生了脱分化，并出现了少量再生芽。在继代培养基中，降低2，4D浓度，附加低浓度6-苄氨基嘌呤（BAP）可以改善蒙古冰草幼胚愈伤组织状态，增加胚性愈伤组织诱导率。从而提高分化率。     

Amplification of Bovine Viral Diarrhoea Virus Introduced into an In Vitro Embryo Production System Via Oocytes from Persistently Infected Cattle

The purpose of this study was to determine whether or not embryos derived from in vitro fertilization of oocytes from persistently infected (PI) cattle would contain infectious virus. Three in vitro embryo production treatment groups were assessed: 1) oocytes and uterine tubal cells (UTC) free of bovine viral diarrhoea virus (BVDV) (negative control), 2) oocytes free of BVDV fertilized and cultured in media containing UTC obtained from PI heifers, and 3) oocytes from PI heifers fertilized and cultured in media containing UTC free of BVDV. The developmental media, UTC and embryos (individual or groups of five) were assayed for virus. Virus was not isolated from any samples in treatment group 1. As shown in previous studies, a proportion of embryo samples were positive for BVDV in treatment group 2. In treatment group 3, the virus associated with the oocytes contaminated the developmental media and infected susceptible co-culture cells used during fertilization and culture. In addition, 65% (11/17) of the degenerated ova from treatment group 3 had infectious virus associated with them. While none of the ova developed into transferable embryos, the study did confirm that use of oocytes from PI cows could lead to amplification of BVDV and cross contamination during in vitro embryo production.     

Response of in ovo administration of zinc on egg hatchability and immune response of commercial broiler chicken

An experiment was conducted to determine the effect of in ovo administration of different forms of zinc with respect to hatchability and performance of commercial broiler chicken. In trial 1, the fertile eggs on day 18 were divided into six treatment groups: Group I as control without any supplementation of zinc, group II to IV were supplemented with 0.5 mg zinc per egg as zinc sulphate, zinc methionine or nano zinc, respectively, and Group V with nano zinc at 0.25 mg zinc per egg. Sixth group received 0.5 ml citric acid per egg as sham control. The results of the first trial indicated that in ovo administration of nano zinc at both levels and zinc methionine resulted in complete failure of hatchability. A second trial to validate the result of trial 1 consisted of Group I control (no administration). Group II and Group III were supplemented with zinc sulphate and zinc methionine, respectively, at 0.5 mg zinc per egg. Group IV and Group V were supplemented with nano zinc at 0.04 and 0.08 mg per egg. In the second trial, again there was a similar pattern for zinc sulphate and zinc methionine. Administration of Zn by nano form had around 80% hatchability on fertile eggs in comparison with the unadministered control eggs (92%). There was no difference (p > .05) in body weight gain, feed intake and FCR. No difference (p > .05) was observed between treatments for cell‐mediated immune response and humoral immune response. Nano Zn‐administered group showed a non‐significant downregulation of MUC2 gene. It could be concluded that in ovo administration of higher levels of zinc has to be with caution for the developing embryo of commercial broiler chicken.     

不同方法制备鸡胚成纤维细胞的比较试验

采用传统法、半胚法和全胚法制备鸡胚成纤维细胞(CEF)单层。结果表明:在同等条件下,均以成纤维细胞为主,并伴有少量的上皮细胞,仅有全胚法含有极少量的肝细胞;培养时细胞贴壁和生长状况无明显差异,其产量稍有差异(全胚法>半胚法>传统法)。     

Biological diversity among serotype 2 Marek's disease viruses

Selected biological characteristics were determined for 14 low-passage serotype 2 Marek's disease virus (MDV) isolates. Four of these isolates were also tested after extensive serial passage in chicken embryo fibroblast cultures. Observations were made on replication in vitro and in vivo, pathogenicity by in ovo inoculation, antigenicity, and protection against virulent MDV challenge. Among the low-passage isolates, there were some differences in pathogenicity after in ovo inoculation but relatively little difference in other characteristics, with the exception of the HN-1 strain, which replicated more rapidly in cell culture but produced generally lower in vivo responses than other isolates. After extended in vitro passage, isolates replicated much more readily in cell culture and produced lower pathologic responses in vivo than low-passage isolates, as has been reported for serotype 1 isolates. No antigenic differences among isolates were detected, but high-passage isolates induced lower levels of precipitating antibodies than low-passage isolates, indicating a possible reduction in A antigen production. The observed diversity associated with strain and passage level may be of value in the selection of optimum vaccine strains.     

Differentiation of the crystalline lens in explants of the chicken embryo in the absence of the hypoblast and optical vesicle

In vitro studies of lens formation in chick embryo have suggested the action of two factors leading the lens induction in the cephalic ectoderm in the absence of optic vesicle: preliminary instructive specific stimulus (homotypic endo-mesoderm) and permissive unspecific stimulus (heterotypic mesenchymes). In order to detect the true capacities of tissues that exert this influence in the cultural condition, series of in vitro experiments were planned. Exclusion experiments: explants including presumptive lens ectoderm were cultured previous to a progressive exclusion of adjacent tissues to trigger lens formation (endoderm, mesoderm and neural tissue), from stage 1 to 7 of HAMBURGER/HAMILTON. Recombinant experiments: Recombinations of caudal epiblast with cephalic hypoblast from blastoderms stages 3, 4 and 5; and recombinations of cardiac mesoderm stage 7 with trunk ectoderm stage 11, were cultured in close association. Lens and lentoids were formed in the presumptive lens ectoderm, even when endoderm, neural tissue and optic vesicle were excluded, but always in presence of subjacent mesoderm. Observation of cephalic epiblast after to be separated mechanically from the underlying tissues showed that the presumptive cardiac mesoderm remains in contact with the epiblast. Beside the cardiac area was capable of forming lens bodies in contact with the trunk ectoderm. It was concluded that the cardiac mesoderm is able to exert a instructive specific stimulus and a permissive unspecific stimulus during in vitro lens formation.