中国的动物胚胎技术研究进展

胚胎移植技术最早是由英国的Walter Heape率先研究的,随后经过了胚胎学家几十年的努力才在上个世纪来期逐渐开始广泛应用于医学生殖,畜牧业,动物保种等领域。本文集中论述中国的胚胎移植技术发展以及它在畜牧业领域的应用。 在中国商业性的动物胚胎移植正在蓬勃发展,其成功率已经和国外同行相差不大。通过胚胎移植工作者的努力,一些从事商业性胚胎移植的公司已经能够做到鲜胚移植后奶牛的妊娠率55％～70％,冷冻胚胎的妊娠率达到45％～60％。不仅如此,在各个省还建立了自己的专门的冷冻胚胎中心,为本省的畜牧产业提供胚胎或者相关的服务。 在动物保种方面,胚胎技术给人们提供了一种全新的思路：通过胚胎技术将活的胚胎冷冻在液氮罐以实现永久保存。例如中国科学院动物所陈大元先生提出通过胚胎移植技术实现对大熊猫的物种保存,并取得了一定的成果。 随着经济和社会的进一步发展,人民生活水平的提高对于肉和奶的需求会进一步提高。依靠常规的选育技术来改良牲畜是无法满足人民对于畜牧产品急切要求,要实现畜牧业的跨越式发展,就必须依靠科技的力量来实现畜牧业的大发展,满足人民生活需要。胚胎技术等新技术会为我国的畜牧业的产业提高提供了一个新的机遇,可以相信,在不远的将来,动物胚胎技术必将在中国有一个较大发展。     

The current status of equine embryo transfer

The use of embryo transfer in the horse has increased steadily over the past two decades. However, several unique biological features as well as technical problems have limited its widespread use in the horse as compared with that in the cattle industry. Factors that affect embryo recovery include the day of recovery, number of ovulations, age of the donor and the quality of sire's semen. Generally, embryo recoveries are performed 7 or 8 d after ovulation unless the embryos are to be frozen, in which case recovery is performed 6 d after ovulation. Most embryos are recovered from single-ovulating mares. Because there is no commercially available hormonal preparation for inducing multiple ovulation in the horse, equine pituitary extract has been used to increase the number of ovulations in treated mares, but FSH of ovine or porcine origin is relatively ineffective in inducing multiple ovulation in the mare. Factors shown to affect pregnancy rates after embryo transfer include method of transfer, synchrony of the donor and recipient, embryo quality, and management of the recipient. One of the major improvements in equine embryo transfer over the last several years is the ability to store embryos at 5 degrees C and thus ship them to a centralized station for transfer into recipient mares. Embryos are collected by practitioners on the farm, cooled to 5 degrees C in a passive cooling unit and shipped to an embryo transfer station without a major decrease in fertility. However, progress in developing techniques for freezing equine embryos has been slow. Currently, only small, Day-6 equine embryos can be frozen with reasonable success. Additional studies are needed to refine the techniques for freezing embryos collected from mares 7 or 8 d after ovulation. Demand for the development of assisted reproductive techniques in the horse has increased dramatically. Collection of equine oocytes by transvaginal, ultrasound-guided puncture and the transfer of these oocytes into recipients is now being used to produce pregnancies from donors that had previously been unable to provide embryos. In vitro fertilization, however, has been essentially unsuccessful in the horse. One alternative to in vitro fertilization that has shown promise is intracytoplasmic sperm injection. However, culture conditions for in vitro-produced embryos appear to be inadequate. The continued demand for assisted reproductive technology will likely result in the further development of techniques that are suitable for use in the horse.     

State of the art in sheep-goat embryo transfer

Considerable advances have been made in the last 25 yr in sheep and goat embryo production and transfer technology. This presentation covers the procedures used to overcome the variability of ovarian response after treatment with exogeneous gonadotropins, the asynchrony of ovulations, failure of fertilization in females showing a high ovulatory response, and the side-effects of repeated treatments (surgical trauma, gonadotropins and their antibodies). In the ewe, prior antigonadotrophic pretreatment results in a significant gain in ovulation rate due to the elimination of nonresponses and in a two-fold increase in embryo yield. A better comprehension of the relationships between oocyte quality and follicular characteristics after superovulation can be gained using in vitro techniques. This knowledge will subsequently be used for the optimization of embryo production needed for the genetic improvement of livestock and the development of new biotechnologies.     

Improving post-transfer survival of bovine embryos produced in vitro: Actions of insulin-like growth factor-1, colony stimulating factor-2 and hyaluronan

Technologies for in vitro embryo production have the potential to enhance the efficiency of cattle production systems. However, utilization of in vitro-produced embryos for transfer remains limited throughout much of the world. Despite improvements over the past two decades, problems associated with the production of bovine embryos in vitro still exist which limit the widespread commercial application of this technology. In particular, bovine embryos produced in vitro have a reduced capacity to establish and maintain pregnancy as compared with their in vivo-derived counterparts. Embryo competence for survival following transfer is improved by in vivo culture in the sheep oviduct, thus indicating that standard embryo culture conditions are sub-optimal. Therefore, one strategy to improve post-transfer survival is to modify embryo culture media to more closely mimic the in vivo microenvironment. The maternal environment in which the bovine embryo develops in vivo contains various growth factors, cytokines, hormones, and other regulatory molecules. In addition to affecting bovine embryo development in vitro, recent research indicates that embryo competence for survival following transfer can also be improved when such molecules are added to embryo culture medium. Among the specific molecules that can increase post-transfer embryo survival are insulin-like growth factor-1 (IGF-1), colony stimulating factor-2 (CSF-2) and hyaluronan. This paper will review the effects IGF-1, CSF-2 and hyaluronan on post-culture embryo viability and discuss the potential mechanisms through which each of these molecules improves post-transfer survival.     

Improving post-transfer survival of bovine embryos produced in vitro: actions of insulin-like growth factor-1, colony stimulating factor-2 and hyaluronan

Technologies for in vitro embryo production have the potential to enhance the efficiency of cattle production systems. However, utilization of in vitro-produced embryos for transfer remains limited throughout much of the world. Despite improvements over the past two decades, problems associated with the production of bovine embryos in vitro still exist which limit the widespread commercial application of this technology. In particular, bovine embryos produced in vitro have a reduced capacity to establish and maintain pregnancy as compared with their in vivo-derived counterparts. Embryo competence for survival following transfer is improved by in vivo culture in the sheep oviduct, thus indicating that standard embryo culture conditions are sub-optimal. Therefore, one strategy to improve post-transfer survival is to modify embryo culture media to more closely mimic the in vivo microenvironment. The maternal environment in which the bovine embryo develops in vivo contains various growth factors, cytokines, hormones, and other regulatory molecules. In addition to affecting bovine embryo development in vitro, recent research indicates that embryo competence for survival following transfer can also be improved when such molecules are added to embryo culture medium. Among the specific molecules that can increase post-transfer embryo survival are insulin-like growth factor-1 (IGF-1), colony stimulating factor-2 (CSF-2) and hyaluronan. This paper will review the effects IGF-1, CSF-2 and hyaluronan on post-culture embryo viability and discuss the potential mechanisms through which each of these molecules improves post-transfer survival.     

Improving fertility in beef cow recipients

In the 1970s, bovine embryo transfer (ET) shifted from research in a laboratory environment to commercialization of this technology for beef producers. With the quarantine requirements and expense of importing Continental breeds of cattle from Europe, embryo transfer became the logical means to reproduce greater numbers of these animals at a lower cost. The ET industry grew very rapidly and soon would become what it is today, a common practice utilized by select ranchers and breeders. Research over the years has primarily focused on methods to increase the number of ovulations and fertilized ova from the donor female, but the total number of transferable embryos has not changed markedly in the last 20 years. More recent advances have been in the area of in vitro production of embryos that allow for greater numbers of embryos to be produced and easier accessibility to incorporate technologies such as sexed sperm, sperm injection, or transgenics. This paper will focus on the second part of the equation, the recipient, and decisions that will enable both the customers and practitioners to most efficiently utilize embryos from superovulation, in vitro production, or nuclear transfer, so that the maximum number of pregnancies can be produced.     

Efficient Activation of Reconstructed Rat Embryos by Cyclin-Dependent Kinase Inhibitors

Background

Over the last decade a number of species, from farm animals to rodents, have been cloned using somatic cell nuclear transfer technology (SCNT). This technique has the potential to revolutionize the way that genetically modified animals are made. In its current state, the process of SCNT is very inefficient (<5% success rate), with several technical and biological hurdles hindering development. Yet, SCNT provides investigators with powerful advantages over other approaches, such as allowing for prescreening for the desired level of transgene expression and eliminating the excess production of undesirable wild-type animals. The rat plays a significant role in biomedical research, but SCNT has been problematic for this species. In this study, we address one aspect of the problem by evaluating methods of activation in artificially constructed rat embryos.

Principal Findings

We demonstrate that treatment with a calcium ionophore (ionomycin) combined with a variety of cyclin-dependent kinase inhibitors is an effective way to activate rat embryos. This is in contrast to methods developed for the mouse embryo, which tolerates much less specific chemical treatments. Methods developed to activate mouse embryos do not translate well to rat embryos.

Conclusions

Activation methods developed for one species will not necessarily translate to another species, even if it is closely related. Further, the parthenogenic response to chemical activators is not always a reliable indicator of how reconstructed embryos will react to the same activation method. A better understanding of rat oocyte physiology, although essential for developing better models of disease, may also provide insights that will be useful for making the SCNT process more efficient.     

Improving successful pregnancies after embryo transfer

Over the past 20 years the rate of blastocyst development in vitro has improved through the development of sequential defined media, refining the oxygen concentrations during culture and providing substrates to ameliorate free radical accumulation. Despite these advances there has been little progress in improving calving rates after the transfer of in vitro produced embryos. This suggests that the culture conditions have been very effective in enabling those fertilised oocytes to reach the blastocyst stage that otherwise would not occur in vivo.We suggest that the next advance by which the embryo transfer technology gains more acceptance in cattle production will be identifying those cows which are intrinsically superior recipients. This must be coupled to the development of non-invasive assessments of the developmental competence of both the oocyte and the blastocyst. Until these two goals are achieved the ET industry will remain static and unable to overcome the economic loss caused by embryo mortality occurring 7-10 days after transfer.     

A history of farm animal embryo transfer and some associated techniques

Events over the last 125 years that have been particularly important to the development of embryo transfer in farm animals are reviewed, arguing that an appreciation of the history of a discipline helps shape its future. Special attention is paid to how the motivations of the scientists involved have changed over time, and how these changes have influenced the practical application of embryo transfer to animal breeding.     

Gene transfer into eukaryotic cells using activated polyamidoamine dendrimers

The development of efficient methods to transfer genes into eukaryotic cells is important for molecular biotechnology. A number of different technologies to mediate gene transfer have been developed over the last 35 years, but most have drawbacks such as cytotoxicity, low efficiency and/or restricted applicability. Activated polyamidoamine (PAMAM)-dendrimers provide a new technology for gene transfer that offers significant advantages over classical methods. Reagents based on this technology provide high gene transfer efficiencies, minimal cytotoxicity, and can be used with a broad range of cell types. This technology could also be useful for in vivo gene transfer in gene therapy applications.     

Cryopreservation of swine embryos: a chilly past with a vitrifying future.

Since the development of embryo freezing technologies for cattle in the 1980s, advances in cryobiology, cell biology and embryology of domestic animals have enabled the development of embryo preservation methodology for the pig, notorious for extreme sensitivity to cooling. This review outlines recent efforts to understand the biology of pig embryos as related to their extreme sensitivity to cooling. Cellular analyses and molecular approaches are discussed that have enabled pig embryos to survive cryopreservation and after transfer develop into live offspring with normal fecundity at maturity. In the near future, use of preserved embryos will be a routine breeding alternative for swine producers, providing: preservation methods for maternal germplasm; global genetic transport; increased selection pressure within herds; breeding line regeneration or proliferation; and methodology for genetic resource rescue. It took almost 50 years after the first successful embryo transfer to develop embryo preservation in the pig. Nonetheless, by applying novel methods described herein, rapid progress has been achieved.     

Animal reproduction biotechnology in Poland

The key research areas of the Department are: in vitro production of embryos, embryo cryopreservation, animal transgenesis, cloning, cytometric semen sexing and evaluation. Research has been focused on the in vitro production of animal embryos, including the development of complex methods for oocyte maturation, fertilization and embryo culture. Moreover, experiments on long-term culturing of late preantral and early antral bovine ovarian follicles have been developed. Studies on the cloning of genetically modified pigs with "humanized" immunological systems have been undertaken. A cloned goat was produced from oocytes reconstructed with adult dermal fibroblast cells. The novel technique of rabbit chimeric cloning for the production of transgenic animals was applied; additionally, the recipient-donor-cell relationship in the preimplantation developmental competences of feline nuclear transfer embryos has been studied. Regarding transgenic animal projects, gene constructs containing growth hormone genes connected to the mMt promoter were used. Modifications of milk composition gene constructs with tissue-specific promoters were performed. Moreover, pigs for xenotransplantation and animal models of human vascular diseases have been produced. Over the last 15 years, our flow cytometry research group has focused its work on new methods for sperm quality assessment and sex regulation. In the 1970s, our team initiated studies on embryo cryopreservation. As a result of vitrification experiments, the world's first rabbits and sheep produced via the transfer of vitrified embryos were born.     

New advances in somatic cell nuclear transfer: application in transgenesis

The ability to produce live offspring by nuclear transfer from cultured somatic cells provides a route for the precise genetic manipulation of large animal species. Such modifications include the addition, or "knock-in", and the removal or inactivation, "knock-out", of genes or their control sequences. This paper will review some of the factors which affect the development of embryos produced by nuclear transfer, the advantages of using cultured cells as donors of genetic material, and methods that have been developed to enrich gene targeting frequency. Commercial applications of this technology in biomedicine and agriculture will also be addressed.     

Embryo transfer in the dog and cat

The practicality and feasibility of embryo transfer technology in dogs and cats is quickly becoming a clinical reality. Although progress has been slow, I anticipate that embryo transfer will be a practical and an economical technique in the near future. Most importantly, it is essential that the practical lessons learned with equine and bovine embryo transfer be integrated into the development of canine and feline programs.     

Non-invasive nuclear magnetic resonance analysis of male and female embryo metabolites during in vitro embryo culture

Introduction

In the past 20+ years, several studies of bovine embryo production showed how the ratio of male to female embryos changes if embryos are made in vivo or in vitro. It is known that in in vitro systems, the sex ratio is in favor of males when there are high levels of glucose, and favors females when the principal energetic substrate is one other than glucose, like citrate.

Objectives

The aim of this study was to evaluate the embryo metabolism during three important periods of in vitro development: the early development (from day 1 until day 3), the middle of culture (day 3 until day 5), and later development (day 5 until day 7).

Methods

To obtain this information we evaluated the spent medium from each time period by ¹H NMR.

Results

Our results confirm that embryo metabolism is different between sexes. The new information obtained by identifies markers that we can use to predict the embryo sex.

Conclusion

These results open a new, non-invasive method to evaluate sex of the embryos before the transfer. In the first period of embryo culture, valine concentration is good indicator (66.7% accurate), while in the last phase of culture, pyruvate depletion is the best marker (64% accurate) to evaluate the sex of the embryo.

     

Future prospectives on animal biotechnology

Abstract

Farm animal reproduction is entering the era of embryo engineering ‐ a part of the new biotechnology revolution that has been sweeping the nation during the early 1980s. This comes at a time when the $70 billion livestock industry is hard‐pressed for survival. Not since the commercial development of artificial insemination (AI) techniques in the 1950s has any new technical research development caused such a stir in the livestock community. The genetic impact of artificial insemination (AI) in the cattle industry these last 40 years cannot be questioned. Nearly three‐fourths of the dairy cattle in the United States are now being artificially inseminated. Also, commercial processing of bull semen has been and still is a major agribusiness success story, grossing millions of dollars annually. With the development of embryo transfer (ET) technology in the mid‐1970s, animal reproduction again entered a new age of technical advancement. It appears that AI and embryo methodology are just the beginning of a new age in animal reproduction technology. Recent developments in molecular biology and genetic engineering now offer a new dimension in research and development for future application to seed stock farm animals. New molecular technologies will most certainly change the traditional approach to animal breeding, thus allowing the livestock producer to select breeding stock on genotype rather than phenotype. In the future, researchers will be able to study whole animal biology to a depth never before dreamed using molecular biology.     

Toward culture of single gametes: the development of microfluidic platforms for assisted reproduction

During the last few decades in vitro production of mammalian embryos and assisted reproductive technologies such as embryo transfer, cryopreservation, and cloning have been used to produce and propagate genetically superior livestock. However, efficiencies of these technologies remain low. For these technologies to become more commercially viable, the efficiencies must improve. Despite this importance of reproduction for the livestock industry, little progress in decreasing embryonic mortality has been made. The livestock industry has succeeded in achieving large increases in average milk production of dairy cattle, growth rate in beef cattle and leanness in swine but reproductive efficiency has actually decreased. For example, research has provided little progress toward developing an objective method to examine viability of a single living embryo. At the same time, the growth of miniaturization technologies beyond integrated circuits and toward small mechanical systems has created opportunities for fresh examination of a wide range of existing problems. While the investigation and application of miniaturization technologies to medicine and biology is progressing rapidly, there has been limited exploration of microfabricated systems in the area of embryo production. Microfluidics is an emerging technology that allows a fresh examination of the way assisted reproduction is performed. Here we review the progress in demonstrating microfluidic systems for in vitro embryo production (IVP) and embryo manipulation. Microfluidic technology could have a dramatic impact on the development of new techniques as well as on our basic understanding of gamete and embryo physiology.     

Embryo responses to stress induced by assisted reproductive technologies

Assisted reproductive technology (ART) has led to the birth of millions of babies. In cattle, thousands of embryos are produced annually. However, since the introduction and widespread use of ART, negative effects on embryos and offspring are starting to emerge. Knowledge so far, mostly provided by animal models, indicates that suboptimal conditions during ART can affect embryo viability and quality, and may induce embryonic stress responses. These stress responses take the form of severe gene expression alterations or modifications in critical epigenetic marks established during early developmental stages that can persist after birth. Unfortunately, while developmental plasticity allows the embryo to survive these stressful conditions, such insult may lead to adult health problems and to long‐term effects on offspring that could be transmitted to subsequent generations. In this review, we describe how in mice, livestock, and humans, besides affecting the development of the embryo itself, ART stressors may also have significant repercussions on offspring health and physiology. Finally, we argue the case that better control of stressors during ART will help improve embryo quality and offspring health.     

Cross talk between the sporophyte and the megagametophyte during ovule development

In seed plant ovules, the diploid maternal sporophytic generation embeds and sustains the haploid generation (the female gametophyte); thus, two independent generations coexist in a single organ. Many independent studies on Arabidopsis ovule mutants suggest that embryo sac development requires highly synchronized morphogenesis of the maternal sporophyte surrounding the gametophyte, since megagametogenesis is severely perturbed in most of the known sporophytic ovule development mutants. Which are the messenger molecules involved in the haploid–diploid dialogue? And furthermore, is this one way communication or is a feedback cross talk? In this review, we discuss genetic and molecular evidences supporting the presence of a cross talk between the two generations, starting from the first studies regarding ovule development and ending to the recently sporophytic identified genes whose expression is strictly controlled by the haploid gametophytic generation. We will mainly focus on Arabidopsis studies since it is the species more widely studied for this aspect. Furthermore, possible candidate molecules involved in the diploid–haploid generations dialogue will be presented and discussed.     

Perspectives for artificial insemination and genomics to improve global swine populations

Civilizations throughout the world continue to depend on pig meat as an important food source. Approximately 40% of the red meat consumed annually worldwide (94 million metric tons) is pig meat. Pig numbers (940 million) and consumption have increased consistent with the increasing world population (FAO 2002). In the past 50 years, research guided genetic selection and nutrition programs have had a major impact on improving carcass composition and efficiency of production in swine. The use of artificial insemination (AI) in Europe has also had a major impact on pig improvement in the past 35 years and more recently in the USA. Several scientific advances in gamete physiology and/or manipulation have been successfully utilized while others are just beginning to be applied at the production level. Semen extenders that permit the use of fresh semen for more than 5 days post-collection are largely responsible for the success of AI in pigs worldwide. Transfer of the best genetics has been enabled by use of AI with fresh semen, and to some extent, by use of AI with frozen semen over the past 25 years. Sexed semen, now a reality, has the potential for increasing the rate of genetic progress in AI programs when used in conjunction with newly developed low sperm number insemination technology. Embryo cryopreservation provides opportunities for international transport of maternal germplasm worldwide; non-surgical transfer of viable embryos in practice is nearing reality. While production of transgenic animals has been successful, the low level of efficiency in producing these animals and lack of information on multigene interactions limit the use of the technology in applied production systems. Technologies based on research in functional genomics, proteomics and cloning have significant potential, but considerable research effort will be required before they can be utilized for AI in pig production. In the past 15 years, there has been a coordinated worldwide scientific effort to develop the genetic linkage map of the pig with the goal of identifying pigs with genetic alleles that result in improved growth rate, carcass quality, and reproductive performance. Molecular genetic tests have been developed to select pigs with improved traits such as removal of the porcine stress (RYR1) syndrome, and selection for specific estrogen receptor (ESR) alleles. Less progress has been made in developing routine tests related to diseases. Major research in genomics is being pursued to improve the efficiency of selection for healthier pigs with disease resistance properties. The sequencing of the genome of the pig to identify new genes and unique regulatory elements holds great promise to provide new information that can be used in pig production. AI, in vitro embryo production and embryo transfer will be the preferred means of implementing these new technologies to enhance efficiency of pig production in the future.