超排技术及其在家养鹿中应用的研究进展

超数排卵(简称超排)可诱导大量卵泡成熟排卵,增加可移植胚胎的数量,在家畜胚胎移植中起着至关重要的作用,同时也是进行转基因动物生产和动物克隆等研究的基础手段之一。作者介绍了几种常用超数排卵方法和特点及影响超排效果的影响因素,就超数排卵技术在反刍动物(牛羊和鹿科动物)中的最新应用进行了概括论述并提出了存在的问题和发展前景。     

Somatic Cell Nuclear Transfer in Horses

The cloning of equids was achieved in 2003, several years after the birth of Dolly the sheep and also after the cloning of numerous other laboratory and farm animal species. The delay was because of the limited development in the horse of more classical-assisted reproductive techniques required for successful cloning, such as oocyte maturation and in vitro embryo production. When these technologies were developed, the application of cloning also became possible and cloned horse offspring were obtained. This review summarizes the main technical procedures that are required for cloning equids and the present status of this technique. The first step is competent oocyte maturation, this is followed by oocyte enucleation and reconstruction, using either zona-enclosed or zona-free oocytes, by efficient activation to allow high cleavage rates and finally by a suitable in vitro embryo culture technique. Cloning of the first equid, a mule, was achieved using an in vivo -matured oocytes and immediate transfer of the reconstructed embryo, i.e. at the one cell stage, to the recipient oviduct. In contrast, the first horse offspring was obtained using a complete in vitro procedure from oocyte maturation to embryo culture to the blastocyst stage, followed by non-surgical transfer. Later studies on equine cloning report high efficiency relative to that for other species. Cloned equid offspring reported to date appear to be normal and those that have reached puberty have been confirmed to be fertile. In summary, horse cloning is now a reproducible technique that offers the opportunity to preserve valuable genetics and notably to generate copies of castrated champions and therefore, offspring from those champions that would be impossible to obtain otherwise.     

New Biotechniques and their Consequences for Farm Animal Welfare

Contents This paper considers (potentially) harmful consequences of new biotechnologies for farm animal welfare. The most important new biotechnologies that are currently used in farm animals breeding and husbandry include: multiple ovulation and embryo transfer (MOET) and in vitro embryo production (IVP). Cloning by nuclear transfer (NT) and transgenesis are still in development and mainly applied for experimental purposes with the prospect of a more widespread practical implemention in the future. Evidence is presented showing that generally accepted technologies such as MOET and IVP, relative to in vivo procedures, can result in a host of deleterious side-effects commonly known as the large offspring syndrome (LOS). Likewise, NT and transgenesis, which also typically include several in vitro reproductive manipulations, have clearly been associated with the occurrence of LOS symptoms. It is argued that transgenesis may constitute one additional set of factors that may negatively affect farm animal welfare: the expression of the transgene and the concomitant synthesis and release of a protein. NT might lead to incompletely reprogramming of the transferred genome. It is suggested that the introduction of new biotechnologies into farm animal husbandry should be accompanied by scientifically valid and systematic studies into the effects on animal welfare, with the help of a comprehensive welfare protocol.     

卵丘细胞对卵母细胞IVM及分子机制的研究进展

卵母细胞的成熟是当前胚胎工程及相关动物生物技术的基础及重要环节,对体外受精、转基因、胚胎克隆以及相关生物技术的发展具有重要的意义,本文主要将卵母细胞体外成熟的分子机制、卵丘细胞对卵母细胞成熟影响因素、目前取得成果以及存在的问题等方面进行归纳总结,为进一步的研究提供理论依据。     

Fertilization and Early Embryonic Development in the Porcine Fallopian Tube

Fertilization and early embryo development relies on a complex interplay between the Fallopian tube and the gametes before and after fertilization. Thereby the oviduct, as a dynamic reproductive organ, enables reception, transport and maturation of male and female gametes, their fusion, and supports early embryo development. This paper reviews current knowledge regarding physiological processes behind the transport of boar spermatozoa, their storage in and release from the functional sperm reservoir (SR), and of the interactions that newly ovulated oocytes play within the tube during their transport to the site of fertilization. Experimental evidence of an ovarian control on sperm release from the SR is highlighted. Furthermore, the impact of oviductal secretion on sperm capacitation, oocyte maturation, fertilization and early embryo development is stressed.     

Effects of Low Oxygen Tension for in vitro Oocyte Maturation and Early Embryo Development through Hypoxia-inducible Factor (HIF)

Oocytes and early embryo in vitro culture (IVC) was a key technology of mammalian embryo engineering and was the basis of biotechnology such as genetically modified (GM) and cloning research.The factors,which affect the efficiency of in vitro culture for oocytes and early embryo,include the composition,ion concentration and osmotic pressure of the culture medium,and the gas composition and temperature of the culture environment. Recent studies have shown that the oxygen tension in the culture systems can significantly affect the development of embryos,and this effect is mainly regulated by the hypoxia-inducible factor (HIF). This paper reviewed the effects of the oxygen tension on oocyte maturation and early embryo development,and the structure and regulation mechanism of HIF were discussed,in order to provide a theoretical basis for further explore the application of hypoxia method in the in vitro culture of oocytes and early embryo.     

低氧分压通过低氧诱导因子(HIF)影响卵母细胞的体外成熟和早期胚胎的发育

卵母细胞和早期胚胎的体外培养(IVC)是哺乳动物胚胎工程的一项关键技术,是生殖生物学和转基因与克隆技术等生物技术研究的基础.影响卵母细胞和早期胚胎体外培养的因素包括培养液组成成分、离子浓度和渗透压,培养环境气相组成和温度等.最近研究表明,培养系统中的氧分压可以显著影响胚胎的发育,而这种影响主要由低氧诱导因子(hypoxia-inducible factor,HIF)进行调控.作者综述了氧分压及HIF对卵母细胞和早期胚胎体外发育的影响,并对HIF的结构及调控机制进行讨论,为研究低氧培养技术在卵母细胞和早期胚胎体外培养中的应用提供依据.     

Role of the oviduct in early embryo development

This review highlights the role of the oviduct in early embryo development, which has to fulfil many aligned and well-tuned tasks during early embryogenesis. The oviductal lining is subjected to dynamic changes to timely accomplish gamete transport, fertilization and embryo development and to deliver a competent and healthy conceptus to the endometrium which can implant and develop to term. Although knowledge about the role of the oviduct is limited, we know that embryos are very sensitive to the environment in which they develop. The success of in vitro embryo production techniques demonstrates that it is possible to bypass the oviduct during early development and, to a certain extent, replicate the conditions in vitro. However, comparative studies show that embryos developed in vivo are superior to their in vitro produced counterparts, underlining our relatively poor knowledge of the biology of the oviduct. Oviduct activity is orchestrated by various factors, depending on cyclic dynamics, which crucially affect the success of tubal transfer and/or (re-)collection of embryos in embryo transfer studies. This paper reviews data which demonstrate that in vivo culture of embryos in the bovine oviduct is a useful tool for the assessment of embryos developed under various conditions (e.g. superovulation vs single ovulation, lactating dairy cows vs non-lactating cows). It is concluded that more work in the field of early embryo development within the oviduct would contribute to improved ART protocols leading to healthy pregnancies and offspring.     

体内、外卵母细胞对猪体细胞克隆胚胎发育潜力的影响

为探讨猪体内、外成熟卵母细胞对核移植重组胚胎发育能力的影响,试验通过激素促排获得体内成熟卵母细胞和收集废弃卵巢获取体外成熟的卵母细胞,分别构建核移植重组胚,比较其卵裂率、囊胚率及胚胎移植受孕情况。结果显示,PGC+PMSG+HCG组的平均排卵数（27.8枚/头）显著高于PGC+HCG （12.5枚/头）、PMSG+HCG （13.7枚/头）及自然发情组（11.5枚/头）（P<0.05）,体内收集到的卵母细胞,可用于构建核移植重组胚的可用卵率均达到90%以上,与其他处理组差异不显著（P>0.05）,说明通过激素处理可获得更多的可用卵母细胞,而且卵母细胞的质量没有显著差异;以体内和体外成熟卵母细胞作为核移植受体构建的克隆胚胎,二者的胚胎融合率（80.31%和79.29%）和卵裂率（90.40%和86.51%）差异均不显著（P>0.05）,但来自体内成熟卵母细胞克隆的胚胎发育至囊胚期的比例显著升高（P<0.05）;将体内、外成熟卵母细胞构建的核移植重组胚分别移植代孕母猪,头平均移植30或60枚时,体内成熟卵母构建的克隆胚胎移植出生仔猪10头,而体外培养卵母细胞构建的克隆胚胎均未着床受孕,表明通过激素促排获得的卵母细胞质量更好,能显著提高克隆胚胎的囊胚率,减少胚胎移植数量,提高代孕母猪的怀孕率。     

Effect of Oocytes in vivo and in vitro on the Developmental Potential of Porcine Somatic Cell Cloned Embryos

To investigate the impact of porcine oocytes in vivo and in vitro maturation (IVM) on the development of porcine somatic cell cloned embryos,the somatic cell cloned embryos cultured in vitro and the sows were treated with hormones to collect mature oocytes in vivo,and the cleavage rate, blastocyst rate and embryo implantation were compared. The results showed that the average number of ovulation in PGC+PMSG+HCG group was significantly higher than that of PGC+HCG,PMSG+HCG and the natural estrus groups (P<0.05). The oocytes collected in vivo could be used for the construction, and the available oocytes rate reached more than 90%,and there was no significant difference among the four groups (P>0.05),which indicated that groups treated by hormone could obtain more available oocytes and the quality of oocytes was not significant different. In vivo and in vitro matured oocytes were used as nuclear transfer embryos of recombinant receptor,the fusion efficiency (80.31% and 79.29%) and cleavage rate (90.40% and 86.51%) were not significant different (P>0.05), but the proportion of in vivo matured oocytes cloned embryos developed into the blastocyst stage was significantly higher (P<0.05). The reconstructed embryos made from in vivo and in vitro matured oocytes were transplanted into surrogate sows (transferred 30 or 60 embryos),10 piglets were born in in vivo maturation of cloned embryo transfer group,while there was no implantation in in vitro maturation of cloned embryo transfer group. The results showed that high quality oocytes obtained by superovulation could significantly increase the blastocyst rate of embryos,reduce the number of embryos transferred and improve the pregnancy rate of surrogate sows.     

Pregnancies from bovine oocytes matured and fertilized in vitro

Production of transgenic animals and embryo cloning are only a few examples of new biotechnological methods applied to animal embryos. All these techniques require large amounts of oocytes and early embryos. In many laboratory animals, embryos matured and fertilized in vivo are easily obtained, but with larger domestic species it requires laborious surgical procedures and the number of embryos obtained remains relatively small (Bracken et al. 1982). The in vitro maturation of follicular oocytes derived from slaughterhouse ovaries and their in vitro fertilization provides large numbers of oocytes and embryos with considerably less effort. The final proof of the success in the in vitro maturation and fertilization procedure is the birth of healthy progeny. Also the normal preimplantation development of the embryos gives useful information about the efficiency of the method employed.     

水牛繁殖生物技术研究进展

水牛的繁殖受诸多因素的影响。为提高其繁殖力 ,多种繁殖生物技术得到大量研究。水牛精子的稀释、冷冻及人工授精已基本程序化 ,并得到了广泛推广。通过超数排卵和胚胎移植的研究 ,获得了水牛胚胎移植的后代。水牛卵母细胞的采集、体外成熟、体外受精和受精胚的培养也得到大量研究 ,并成功产出体外受精水牛。在水牛胚胎冷冻和移植的研究方面也取得成功并产犊。细胞核移植、基因组和基因图谱等新兴生物技术在水牛上也得到初步研究。随着转基因等生物技术的进一步发展 ,水牛的遗传资源将得到更好的开发     

Embryo cloning in domestic animals]

The cloning of livestock is performed by the nuclear transfer of early embryonic stages into prepared oocytes in order to obtain a high number of genetic identical animals. As the most important technical steps there are maturation and enucleation of the oocytes, isolation of single blastomeres or karyoplasts of the donor embryo, transfer of the nucleus-containing membrane vesicle under the zona pellucida of the recipient cell, and fusion of the recipient cell and blastomere or karyoplast. Until now, the largest clone which is known exists of seven bulls. The efficiencies of the particular methodical steps have to be improved. More knowledge of the activation of oocytes, nucleus differentiation and availability of determined cell cycle-stages of mitosis is required. The combination of embryo cloning, cryopreservation of embryos and non-surgical embryo transfer is required for basic research and animal breeding.     

水牛卵泡颗粒细胞对卵母细胞体外成熟的影响

为了系统研究颗粒细胞对水牛卵母细胞体外成熟的影响,使用颗粒细胞条件液处理或单层颗粒细胞和卵母细胞共培养的方法,探讨颗粒细胞共培养对水牛卵母细胞体外成熟和早期胚胎发育的影响。结果显示,添加颗粒细胞传代接种第2天收集的20%颗粒细胞条件液到水牛卵母细胞成熟液中能显著提高水牛卵母细胞体外成熟率和囊胚发育率(P<0.05);然而单层颗粒细胞却显著抑制水牛卵母细胞体外成熟和早期胚胎发育(P<0.05)。结果表明,与单层颗粒细胞共培养相比,与颗粒细胞条件液共培养更有利于水牛卵母细胞体外成熟和早期胚胎发育。本研究为水牛卵母细胞体外成熟培养体系的完善提供一定理论依据。     

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.     

转GFP基因核移植牛胚胎的研究

试验采用脂质体转染法与电穿孔法,以携带绿色荧光蛋白（GFP）-新霉抗性（neo-）双标记基因的pMSCV质粒转染胎牛耳成纤维细胞为供体与体外成熟的牛卵母细胞为受体构建克隆胚。研究了体外成熟培养液中添加EGF（表皮生长因子）对转基因胚的影响,不同转染方法构建供体细胞对重构胚发育的影响和在不同体外培养系统中的发育效果。结果显示,体外成熟培养液中添加EGF 30 ng/mL组的卵母细胞成熟率最高,但对后期转基因重构胚的囊胚发育率的影响,以添加EGF 20 ng/mL组的最高;以胎牛耳成纤维细胞为供体细胞,不同转染方法转染供体细胞构建重构胚,其囊胚发育率差异不显著（P>〖JP2〗0.05）;mSOFaa+颗粒细胞单层细胞共培养体系中的转基因囊胚发育率最好,该体系更适合体细胞核移植法生产转基因牛胚胎。     

Factors Affecting the Development of Somatic Cell Nuclear Transfer Embryos in Cattle

Nuclear transfer is a complex multistep procedure that includes oocyte maturation, cell cycle synchronization of donor cells, enucleation, cell fusion, oocyte activation and embryo culture. Therefore, many factors are believed to contribute to the success of embryo development following nuclear transfer. Numerous attempts to improve cloning efficiency have been conducted since the birth of the first sheep by somatic cell nuclear transfer. However, the efficiency of somatic cell cloning has remained low, and applications have been limited. In this review, we discuss some of the factors that affect the developmental ability of somatic cell nuclear transfer embryos in cattle.     

Vitrification of in vitro matured oocytes of Mangalica and Large White pigs

The breeding of Mangalica, a native pig breed in Hungary, had been started in 1833, but this pig breed almost became extinct in Hungary in the past decades. In 1991, the number of sows was only 200. Although in these days the existing Mangalica population consists of more than 6000 animals representing different colour variations, the preservation of this traditional pig breed is still very important. Vitrification is a potential tool for the preservation of gametes and embryos of these animals. The aim of this study was to investigate the effects of vitrification on the developmental competence of Mangalica (M) and Large White (LW) oocytes following fertilisation. The oocytes were vitrified by the Open Pulled Straw (OPS) method using different concentrations of ethylene glycol and dimethyl sulphoxide as cryoprotectants. After rehydration the oocytes underwent in vitro fertilisation; the resultant zygotes were then cultured in vitro for four days to assess embryonic development. In the first experiment, in vitro maturation of M and LW oocytes was compared. No significant difference was observed in the nuclear maturation rate of LW and M oocytes. In the second experiment, the sensitivity of oocytes to vitrification was examined by evaluating oocyte morphology after thawing. A higher percentage of LW oocytes showed normal morphology compared to M oocytes, indicating that Mangalica oocytes are more sensitive to cryoprotectants than Large White oocytes. After warming and in vitro fertilisation, more than 50% of the oocytes started embryonic development and by the end of the incubation period morula stage embryos had developed in both groups. The results show that the OPS vitrification technique is well suited to preserve Mangalica oocytes and from these oocytes morula embryos can be produced.     

牛卵母细胞体外成熟和体外受精技术的研究进展

论述牛卵母细胞体外成熟和体外受精的最新研究进展，包括卵丘卵母细胞复合体、卵母细胞的体内、体外成熟，以及体内、体外的异常成熟和卵母细胞的体外成熟方法，无蛋白质、无血清系统的限定性培养液的研究进展，卵母细胞体外成熟状态与标志的某些理论上的突破；体外受精中精子供体的选择、精子活力和正常形态的选择、冷冻解冻精液的体外获能、精子的体外受精力，以及体外的异常受精；还论述了牛卵母细胞体外成熟和体外受精技术在家畜育种和胚胎克隆等方面的应用前景