PCR方法用于奶牛早期胚胎的性别鉴定

根据牛Y染色体上的特异DNA序列合成一对引物, 通过PCR反应对奶牛早期胚胎进行性别鉴定。预测性别与移植胚胎产犊的实际性别相符率为80%。 Abstract:We have obtained the specific DNA segment from the bovine Y-chromosome and used it to design a pair of primer.The sex of embryos at the preimplantation stage have determined by using the polymerase chain reaction.10 months after uterine transfer showed that the rate of accuracy is 80%.     

试管牛和试管羊胚胎性别的鉴定

奶牛和山羊的卵母细胞经过体外成熟、体外受精和体外发育至桑椹期,在显微操作下,吸取4～6个胚胎细胞,应用巢式PCR技术对其DNA进行SRY基因的测定以进行胚胎性别鉴定。共有27枚转有外源基因的试管牛胚胎和207枚转基因试管羊胚胎进行了SRY基因检测。选取10枚经过性别鉴定的牛胚胎移植于8头受体母牛和124枚已知性别的羊胚胎移植于44头受体母羊,移植后妊娠率分别为37．5％（牛,3/8）和61．4％（羊,27/44）。最后产生1头分娩牛犊和2头流产胎牛以及14头分娩羊羔和2头流产胎羊,它们的性别与胚胎性别鉴定的结果完全相符。 Abstract: The oocytes of bovines and goats were subjected to in vitro maturation,in vitro fertilization(IVF)and in vitro development upto morula stage．4～6 embryonic cells were aspirated with micro-manipulating and the embryo sexes were identified by detecting SRY gene with nested PCR procedure．A total of 27 transgenic IVF bovine and 207 transgenic IVF goat embryos were performed SRY gene detection．10 bovine and 124 goat sexed embryos were selected to be transferred into 8 bovine and 44 goat recipients,respectively,leading to the pregnant rates of 37．5％( in bovine,3/8)and 61．4％(in goat,27/44)．In the end,1 cattle and 14 goatlets were born at term but 2 fetal bovine and 2 fetal goats were miscarried．Their sexes were fully in accordance with the embryonic sex predetermination．     

Viability of Fresh and Frozen-Thawed Biopsied Bovine Embryos

Bovine embryos were biopsied using a simplified splitting technique and frozen-thawed according to a standard method with glycerol as cryoprotectant. The viability of fresh and frozen-thawed biopsied and intact embryos were evaluated after in vitro culture, by means of fluorescence test or following transfer to recipients. The survival rates after in vitro culture of fresh intact and biopsied embryos and of frozen-thawed intact and zona free embryos were not significantly different (70%, 60%, 68% and 52%, respectively), but significantly reduced for bipsied frozen-thawed embryos (16%) (p≤0.05). The pregnancy results after transfer of biopsied frozen-thawed embryos were also significantly lower (8%) compared to fresh biopsied embryos (39%) (p≤0.05). Both intact and biopsied embryos fluoresced after incubation with diacetylfluorescin but with higher intensity for the intact embryos. It is suggested that the reduced survivability for the frozen-thawed biopsied embryos might be caused by combined effects of the loss of the zona pellucida and the reduction of cells as a result of the simplified biopsy technique. It is concluded that improved biopsy and/or freezing techniques must be used if biopsied embryos have to be frozen.     

Gonadal Growth in Chick Embryos

During ovarian differentiation of bird embryos, ovaries grow faster than testes. The difference for mammals may reflect the sex chromosome constitution.     

Modelling the Growth of Salmonid Embryos

A mechanistic model for the growth of salmonid embryos (prior to feeding) is developed with coupled differential equations describing anabolism and catabolism. The equations model changes in embryo and yolk sac masses in which the flux of nutrients to the embryo is controlled by geometric properties of the embryo and yolk. The rate parameter describing this flux is a well defined function of temperature. Water absorption is also a factor in determining mass. The model, describing the size and time-to-arrival at developmental stages, is fit to chinook salmon growth data from fertilization to complete yolk absorption.     

牛冷冻试管胚胎性别鉴定的应用

利用双引物PCR技术对优质良种牛体外受精的冷冻胚胎进行性别鉴定。并对分割后的半胚分别进行PCR扩增和染色体分析,两种方法的判定结果相吻合,在本实验条件下,利用减少PCR体系中细胞的数量来确定最低的模板DNA的检出量,2－细胞不能检测,4－细胞检出率为62．5％,8－细胞为90％,16－细胞以上为100％。对23牧经过性别鉴定后的胚胎进行移植,获得5头纯种试管牛,产犊率为21．7％,与正常的胚胎移植 结果27．3％（3／11）无显著差异,预测性比为100％符合。     

Xist基因抑制对牛SCNT早期胚胎发育的影响

Xist是与X染色体失活相关的非编码基因,它在合子期基因组开始表达,是胚胎发育早期表达的第一个印记基因。探讨了特异性抑制Xist的TALER-REPRESSOR(TALER)载体转染到胎牛成纤维细胞对Xist基因的抑制作用,并以抑制Xist基因表达的细胞作为核供体制作克隆胚胎,研究Xist基因抑制对牛克隆胚早期发育的影响。结果显示,与对照组细胞相比,TALER载体将Xist相对表达量下调了93.85%,说明本试验设计的载体转染系统能够有效抑制Xist基因的表达。选取Xist抑制表达阳性的转染细胞用于体细胞核移植试验,克隆胚胎发育结果显示,试验组和对照组的卵裂率、8细胞发育率、桑葚胚发育率和囊胚发育率分别为78.8%vs 75.1%(P0.05,无显著差异)、54.4%vs 50.6%(P0.05,无显著差异)、12.3%vs 27.8%(P0.01,差异极显著)、0 vs 26.6%(P0.01,差异极显著)。综上所述,试实验设计的特异性抑制Xist表达的TALER载体可有效抑制雌性胎牛成纤维细胞中Xist的表达。供体细胞Xist这种基因下调可使克隆胚胎2-8细胞率略有提升,但囊胚期和桑葚胚率明显降低。因此,其机制尚待于进一步探讨。     

Prediction of Transgene integration by Noninvasive Bioluminescent Screening of Microinjected Bovine Embryos

Transgenesis in domestic species, as a research tool and in biotechnological applications, has been limited by the expense of producing transgenic offspring by standard microinjection techniques. A major factor is the inefficiency of maintaining large numbers of recipient females, when a high percentage of these carry nontransgenic fetuses. There are two approaches to reduce this cost, the fusion of transfected fetal fibroblasts with enucleated oocytes, and the screening of microinjected embryos for transgene integration in blastocysts, prior to transfer. Here, we develop a luminescent screening system to select transgenic bovine embryos. A transgene with scaffold attachment regions flanking the murine HSP70.1 promoter linked to firefly luciferase cDNA, was microinjected into pronuclei of in vitro produced zygotes. At the blastocyst stage, the transgene was induced by heat shock (45 °C, 15 min) and 4–6 h later, luciferase expression was analyzed by photon counting imaging. Screened blastocysts were transferred to recipients and day 50 fetuses or calves were analyzed by PCR and Southern blot for transgene integration. When nonluminescent blastocysts were transferred, transgene integration was never observed. Of 13 fetuses derived from luminescent blastocysts, 3 contained integrated transgenes that were functional in all tissues examined. Image analysis of the signal emitted by positive blastocysts revealed that 9 nontransgenic fetuses were obtained from blastocysts that exhibited a localized luminescent signal. On the other hand, 3 of 4 fetuses derived from blastocysts that emitted light over more than 70% of their surface were transgenic. Thus, by selecting luminescent blastocysts on the basis of both signal intensity and distribution, the number of recipient females required to produce transgenic offspring can be greatly reduced. Using this technique it should also be possible to improve the efficiency of transgenesis by microinjection through studies in which vector design and integration conditions are examined at the blastocyst stage.     

两温度梯度多重PCR鉴别牛早期胚胎性别的技术

稳定、可靠和快速的牛胚胎性别鉴定方法在生产应用中具有重要意义.通过两温度梯度PCR方法对牛基因组、克隆胚胎、胚胎样品进行性别鉴别实验研究,建立了稳定、简便、快速的牛早期胚胎性别鉴别两温度梯度PCR方法,鉴定时间仅为57分钟.采用两温度梯度PCR方法对30枚奶牛胚胎进行了早期性别鉴别,并将鉴别的15枚胚胎(11枚为雌性,4枚为雄性)移植到同期处理的15头受体母牛体内.60天后妊娠检查,有7个受体成功受孕,5头受体怀孕晚期流产,流产犊牛全部为母犊.结果产下1公1母两头犊牛,流产个体与出生个体的性别与PCR鉴别结果完全相符.     

Patterns of Metabolism and Growth in Avian Embryos

Metabolic rates of embryos of precocial birds increase rapidlyuntil about 80% through incubation, then increase slowly remainconstant or even decline. In altricial species, embryo metabolicrates increase continuously and at an accelerating rate throughout incubation. Total energy cost of development is higher inprecocial than in altricial species. Growth patterns of altricialand precocial embryos differ in the same way as does metabolicrate. Embryo growth rates decline late in incubation in precocialspecies, but increase continuously in altricial species. Embryometabolic rate in cal/hr (P) is related to embryo mass in grams(M) and growth rate in grams/day (GR) by the equation P = 12.17GR+ 1.66M + 1.81. The energy cost of growth in avian embryos is292 cal/g. The energy cost of maintenance is 1.66 cal/g hr andappeals to be independent of embryo mass. Differences in growthpatterns account for the observed differences in metabolic ratesand total energy costs of development. High energy costs ofmaintenance account for high total developmental costs in piecocialspecies and in species that have unusually long incubation periods.     

Gonadal Growth in Embryos of Sex reversed Mice

Gonadal volumes were measured in litters of mouse embryos, aged 15 and 16 days, which were segregating for the Sex reversed factor. The testes of XY embryos were much larger than the ovaries of female embryos and the testes of XX Sex reversed males were almost, but not quite, as large as those of XY males. One of the 13 Sex reversed embryos had ovotestes.
It is concluded that the Sex reversed factor causes an increase in the growth rate of XX gonadal rudiments and that this is a necessary prerequisite for testicular differentiation. It is further postulated that the failure of spermatogenesis seen in adult Sex reversed , XX males is a result of the suboptimal growth rate of the gonads, compared to that of normal testes seen in the embryos. The formation of ovotestes may result from a growth rate which is at a lower limit of that necessary for testicular differentiation.     

碳水化合物对牛体外受精胚胎体外发育的影响

目的　在SOF +PVA(合成输卵管液 +聚乙烯醇 )这一化学成分明确培养系统条件下 ,观察了葡萄糖、丙酮酸和乳酸三种碳水化合物对牛体外受精胚胎体外发育的影响 ,以便为今后进一步探讨影响牛早期胚胎体外发育的因素提供实验依据。方法　牛卵母细胞体外成熟和体外受精后 ,在化学成分明确培养系统内进行体外发育培养。结果　实验 1将牛体外受精卵培养于不含有葡萄糖的SOF +PVA培养系统中 ,培养 12 0h后分别移入含有 0、1 5 0、3 30、5 0 0mmol L的SOF +PVA培养系统中 ,对照组胚胎一直在含有 1 5 0mmol L葡萄糖的SOF +PVA中培养 ,结果囊胚的发育率分别为 9 2 % a、12 1%、19 2 % b、18 9%和 11 7% (a 相似文献   

Survival Rate and Ultrastructure of Vitrified Bovine in vitro and in vivo Developed Embryos

The capacity of different vitrification media and methods was tested onto in vivo and in vitro produced bovine morula/blastocysts and their ultrastructure and survival studied post-thawing. Two vitrification solutions were finally selected, named 40 ES (40% ethylene glycol in PBS containing 0.5 M sucrose) and 35 EFS (composed of 35% (v/v) ethylene glycol in PBS containing 0.5 M/l sucrose and 30% (w/v) Ficoll 70). The straws were either precooled or not precooled in nitrogen vapour, plunged and stored in LN2 for 10–25 days, and then thawed in a 20° C waterbath. The content of the straws was rediluted in 1M sucrose solution in PBS and later cocultured with BOEC for 48 h. The overall survival rates for in vitro and in vivo embryos were 36% (12 of 33) and 20% (3 of 15) after 24 h and 21% (7 of 33) and 33% (5 of 15 ) after 48 h. The survival rates for precooled embryos were significantly higher than for not precooled (48% vs 13% after 24 h and 44% vs 4% after 48 h) when tested across vitrification media. The in vitro-produced embryos presented an ultrastructure similar to the pre-freeze state, irrespective of the vitrification media used. The in vivo developed embryos showed a rather modified post-thaw ultrastructure, with clear signs of osmotic changes at both the trophoblastic and embryonic cells. The results indicated that in vitro and in vivo developed bovine embryos can survive vitrification using ethylene glycol as a cryoprotectant.     

牛脑成纤维细胞生长因子的分离纯化与鉴定

新鲜牛脑组织匀浆液经两步硫酸铵沉淀、CM-Sephadex C50 离子交换层析以及肝素-Sepharose 亲和层析,可得到纯化的酸性和碱性成纤维细胞生长因子(aFGF 和bFGF),分子量分别为13.2kD 和15.2—15.8kD.两种因子均可有效促进3T3细胞的 DNA 合成,ED₅₀分别为15.8ng/ml 和 0.32ng/ml.进一步对 aFGF 的等电点及氨基酸组成做了分析.     

The p66Shc Adaptor Protein Controls Oxidative Stress Response in Early Bovine Embryos

The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2–4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2–4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.     

Comparison of Two Semen Extenders in Terms of In Vitro Development of Bovine Embryos Following IVF

In order to conform with current EC standards with regard to antibiotic cover, the Norwegian Cattle Association is currently investigating the use of Biladyl® as an alternative to the milk-based extender which has been traditionally used in Norway. A study was carried out to investigate the effect of using semen frozen with either milk extender or Biladyl® on the outcome of in vitro fertilization and embryo culture. Semen from 6 Norwegian Red bulls was used. There was a significant difference p<0.05 in terms of cleavage rate between the 2 extenders for 1 bull, 78.2% vs 94.9% for milk and Biladyl® extenders, respectively, and for the overall total of 71.3% vs 76.1% for milk and Biladyl® extenders, respectively. There were no significant differences in terms of blastocyst yield amongst any of the bulls. In conclusion, the results suggest that Biladyl® can be used as a replacement for the traditional milk-based extender without any adverse effects on blastocyst yields following in vitro fertilization.     

共培养体系在牛核移植胚体外发育培养中的应用

采用电融合法构建牛体细胞核移植重构胚,分析共培养细胞类型、传代次数、细胞冻-融以及蛋白质添加物(BFF和FBS)对牛体细胞核移植胚体外发育的影响,探讨胚胎体外共培养的条件,以建立优化的共培养体系。结果表明与非共培养组相比,共培养组重构胚的囊胚发育率以及胚胎细胞数显著增加(P<0.05),而输卵管上皮细胞共培养组同颗粒细胞共培养组相比胚胎细胞数显著增加(P<0.05),更适合做共培养细胞;随着共培养细胞传代次数的增加重构胚囊胚发育率及胚胎细胞数显著下降(P<0.05),共培养细胞在冷冻处理后重构胚的囊胚率和胚胎细胞数都显著下降(P<0.05);BFF较FBS更能促进牛核移植胚的囊胚发育率(P<0.05)。表明应用新鲜原代输卵管上皮细胞进行牛核移植胚胎的共培养,并在SOFaa添加10?F能够有效促进核移植胚胎的体外发育。     

牛体细胞克隆胚胎类ES细胞集落的筛选及其核移植

对第7d的牛体细胞克隆囊胚进行体外增殖培养,分离筛选类ES细胞,并对其进行了传代培养,接种在饲养层上的体细胞克隆囊胚细胞,在传代的24h内增殖形成小集落,2～3d有雀巢状的集落出现,筛选形态相同的细胞集落进行传代培养,4～5代后,皿底出现多个大小不等的多细胞单层集落,将传4～5代的细胞集落接种到无饲养层的4孔培养皿中培养,24h出现多细胞单层集落,4～7d长满皿底,并形成上皮样细胞,呈网状,将其作为核供体细胞进行核移植实验。结果有80%（40/50）核-质融合的移核重构胚发生卵裂,5%（2/40）发育至桑椹胚期,2.5%（1/40）发育至囊胚期,92.5%（37/40）停止在2～4细胞期,结果表明：采用牛体细胞克隆胚胎的类ES细胞进行核移植,具发育形成早期胚胎的潜能。