Toward a dynamic model of deposition and utilization of yolk steroids

The discovery by Schwabl that maternal steroid hormones aretransferred to the egg yolk and have effects on the phenotypeof offspring revealed a new pathway for non-genetic maternaleffects. The initial model relied on passive transfer. The thinkingwas that steroids passively entered the lipophillic yolk duringyolk deposition and then were deposited in the yolk until theywere passively delivered to the embryo as the yolk was used.Subsequent studies revealed that the system is much more dynamicthan that. Here, we explore questions about how dynamic thesystem really is and look at questions like: Is transfer ofmaternal steroids to the yolk passive or is it actively regulated?At what stages of the maternal reproductive cycle are steroidstransferred? During reproduction, how dynamic are the levelsof yolk steroids? Especially in the case of potentially deleterioussteroids (e.g., androgens in female offspring; glucocorticoids),once deposited can they come out of the yolk over time? Canthey be metabolized by the yolk or by the embryo? During incubation,how much do steroid levels in the yolk change? Can steroidsdiffuse from the yolk to the embryo prior to yolk utilization?Does the embryo contribute to yolk steroid levels as it develops?We believe that comprehensive answers to questions like thesewill eventually allow us to generate a much more accurate andcomplete model of the transfer and utilization of yolk steroidsand that this model will be much more dynamic and active thanthe one initially proposed.     

The Holstein cow in embryo transfer today as compared to 20 years ago

Embryo transfer practice and results were examined over a 20-year period in Holstein cows and heifers within four commercial embryo transfer programs located in different areas of North America. Mean embryo production per collection decreased (P < 0.05) in one program over time, but not in the other three. Changes in the type of cows entering embryo transfer programs, the number of times they were superstimulated and changes in the brands of gonadotropins used for superstimulation all complicated the analysis of embryo production over time. Data reveal higher pregnancy rates (P < 0.001) following transfer of embryos into Holstein heifers than into lactating dairy cows. It is not clear whether pregnancy rates have decreased over time as a result of the change from surgical to non-surgical embryo transfer. In the two programs in which pregnancy rates were analyzed, there was a decrease (P < 0.001) when non-surgical transfers were adopted in one program, while no change occurred in the other. One of the biggest changes in all programs was that more than 50% of embryos recovered from donors are now frozen after collection, whereas the majority were transferred fresh 20 years ago.     

Micromanipulation of mammalian embryos: Principles, progress and future possibilities

Numerous advances in development of techniques for manipulating mammalian embryos outside the maternal environment have been made over the past decade. Some techniques were developed primarily for use in research; others were developed in response to problems of practical livestock production but have proven useful in research as well. Embryo micromanipulation procedures are used often in conjunction with embryo transfer, and interest in these procedures was stimulated by growth of the embryo transfer industry. Included in this review are discussions of procedures for manipulation of gametes and embryos, including sperm injection into oocytes, pronuclear and nuclear transfer, embryo biopsy and splitting, experimental chimera production and isolation of embryonic stem cells.     

一种非手术性小鼠胚胎移植技术

为了提高非手术性小鼠胚胎移植技术的成功率,利用塑料移植导管模拟非手术胚胎移植过程,通过观察染料在子宫角的分布而评估胚胎移植效果,并在此基础上将自然妊娠3.5 d的小鼠囊胚经子宫颈移植受体小鼠。结果表明:将CD-1小鼠囊胚移植假孕2.5 d小鼠单侧子宫角,平均70.9%的胚胎能够发育至成活新生仔鼠,建立了高效非手术性小鼠胚胎移植技术。该方法简便快捷、不易污染、费用低,无需专业的手术器械,且符合实验动物伦理原则,完全可以取代手术法胚胎移植技术,更重要的是,它为人类和其他大动物的胚胎移植提供了研究模型。     

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.     

The influence of variation in embryo stage and maternal hormone profiles on embryo survival in farm animals

The physiological mechanisms that lead to the requirement for synchronous embryo transfer have been defined in some species, particularly the sheep. In mated animals, variation in hormone profile and embryo stage have been shown to be sufficient to cause some embryo loss, perhaps by generating an asynchronous relationship. During procedures of embryo transfer, there may be additional divergence between hormone profile and embryo stage. It may be beneficial to supply hormones to recipients and to transfer embryos at a particular stage of development at a precise interval after initiation of this treatment.     

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.     

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.     

The zootechnical applications of biotechnology in animal reproduction: current methods and perspectives

The development of the four generations of Reproductive Biotechnology, particularly in cattle and since the last world war, represents one of the best examples of the success story of technology transfer. This review will only refer to the first three generations and will not deal with nuclear transfer nor transgenesis. Based on sound so-called "finalised" research, Artificial Insemination first, then in vivo collected embryo transfer and later in vitro fertilised embryo transfer have been implemented worldwide. Each of these Biotechnologies has many advantages and limitations. In addition to the specificity of each of them, one major point is that farmers and breeders may choose either collectively or individually, the best technology to be used in order to achieve the goals they have set for their industry. It is noteworthy that these technologies have been able to match with the economics demands over the last decades and yet are in a very good capacity to respond to the contemporary demand of sustainable development. In this context, there are further advantages such as potentially contributing to maintaining biodiversity or allowing preservation ex situ of genes otherwise threatened to extinction.     

Aspects of in vivo oocyte production,blastocyst development,and embryo transfer in the cat

A brief overview of the progress made during the past approximately 40 years on the development of methods for in vitro production of cat embryos and intra- and interspecies embryo transfer is described. The presentation is focused primarily on research done over the past 30 years at the Cincinnati Zoo (1980–1995) and at the Audubon Nature Institute, New Orleans (1996–present) beginning with original studies on determining optimal doses of porcine FSH for ovarian stimulation and uterine embryo recovery, cryopreservation, and transfer. A key early finding was the ability of cats to respond to multiple gonadotropin (porcine FSH) treatments by repeated stimulation of follicular development. With a ≥6-month interval between FSH treatments, over the past 15 years (1998–2013), we have done 1603 laparoscopic oocyte retrievals on 337 cats and recovered >38,000 mature oocytes (mean = 24.1 per laparoscopic oocyte retrieval). The limited information available on in vivo blastocyst development in the cat during the latter portion of the preimplantation period (approximately Days 8 to 12 after coitum or approximately Days 7 to 11 after ovulation) was assembled for the purpose of comparing and contrasting it with the growth, expansion, and zona functioning of in vitro-derived blastocysts. Also, results of transferring morulae and/or blastocysts into synchronous recipients are described to emphasize evidence that appears to allude to an essential role for an intact zona pellucida in successful implantation and subsequent development in the cat. Until 2003, our in vitro-derived embryos were transferred into the uterine horns of recipients to determine the feasibility of producing offspring from such primary methods as IVF, intracytoplasmic sperm injection, SCNT, and embryo cryopreservation. With the exception of SCNT embryos, pregnancy rates were satisfactory, but embryo survival rates were not. Subsequently, after finding that SCNT embryo survival rate could be improved using laparoscopic transfer of early cleavage stage embryos into the oviduct, we applied the technique to embryos derived using IVF with sex-sorted sperm, oocyte vitrification, and embryo cryopreservation. Overall, a pregnancy rate of 67% (14/21) has resulted. Most recently, with the oviductal embryo transfer technique, two litters of Black-Footed cat kittens have been born from intra- and interspecies transfer of cryopreserved embryos.     

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。     

HUMAN DIGNITY IN INTERNATIONAL POLICY DOCUMENTS: A USEFUL CRITERION FOR PUBLIC POLICY?

Current developments in biomedicine are presenting us with difficult ethical decisions and raising complex policy questions about how to regulate these new developments. Particularly vexing for governments have been issues related to human embryo experimentation. Because some of the most promising biomedical developments, such as stem cell research and nuclear somatic transfer, involve such experimentation, several international bodies have drafted documents aimed to provide guidance to governments when developing biomedical science policy. Here I focus on two such documents: the Council of Europe's Convention for the Protection of Human Rights and Dignity of the Human Being and the Additional Protocol to the Convention for the Protection of Human Rights and Dignity of the Human Being. I argue that by using human dignity as a criterion to determine the permissibility of particular human embryo research practices, these documents cannot aid in identifying research that would be contrary to human dignity. Thus, they fail to guide public policy on embryo experimentation. Their use of human dignity as a criterion makes their task of offering guidance unfeasible because the concept as used in these documents is too vague and is applied in contradictory ways. I discuss the main goals of these documents and their claims in relation to human embryo research. I then discuss how they have influenced public policy in several countries. Finally, I show that although these Council of Europe treaties attempt to serve as public policy guides in the area of embryo research, they fail to do so.     

Attempts to explain and reduce variability of superovulation

The variability in response to superovulation treatment is a major disadvantage for economical use of embryo transfer and clearly limits use of embryo transfer in animal breeding. Despite considerable efforts, it has not yet been possible to reduce this variability drastically. Less than 40% of the causes for variability has been explained to date. The high proportion of unexplained variability leads to the hypothesis that variability is a special biological function that supports natural selection over the long term. Corresponding data from laboratory animal studies support this hypothesis.     

The effect of internal and external factors on bovine embryo transfer results in a tropical environment

The objectives of this work were to determine the effect of external (synchronization methods, month, embryo origin and farm effects) and internal factors (age and size of CL, embryo development and quality score, synchronization methods, age of recipient, quality of transfer and reuse of recipients) on a commercial embryo transfer program in a tropical environment. In the program 1466 Holstein-Friesian purchased embryos were implanted to zebu/European crossbred recipients under field conditions. There were 502 pregnancies detected in this large-scale extension programme. Synchronization methods, month, embryo origin, and farm effects were found to have affected the success rate of embryo transfer. Due to the hot climate and large distances between recipient farms, seasonal effects, reused recipient pregnancy results and the effect of embryo development stage differed from previously reported results. Investigation by ultrasonograph showed that embryo loss occurred before 35 days of pregnancy. Under field conditions, routine fetal sexing resulted in <5% misidentification. In conclusion, under tropical conditions external factors have a major influence on the results of pregnancy from embryo transfer.     

卵胞质移植的研究进展

许多研究表明,线粒体对卵母细胞的受精和胚胎发育有显著影响,卵胞质中线粒体DNA含量和ATP含量的减少,以及线粒体DNA缺失均能降低卵母细胞的受精和胚胎发育,是老龄妇女和老龄动物生育率下降的重要原因之一。卵胞质移植技术能有效改善老龄卵母细胞的受精能力和早期胚胎的发育能力,在人类已有健康后代出生,它已成为人类辅助生殖生物技术和动物克隆研究的新热点。但是,卵胞质转移也可能会导致线粒体DNA异质,即供体和受体的线粒体DNA同时存在于后代体内。目前,人们对于转入的异质卵胞质中对胚胎的发生和发育造成影响的因素并不完全了解。通过卵胞质移植研究概况、卵胞质与受精和胚胎发育、异种线粒体DNA遗传方式和卵胞质转移遗传物质的检测4个方面对卵胞质移植技术进行讨论。     

Non-surgical transfer of deep-frozen bovine embryos

Preservation of cattle embryos by methods of deep-freezing has recently been established (1, 11, 12) and provides a valuable addition to the possibilities of controlled breeding by embryo transfer in cattle.Already long distance transport of frozen embryos has been demonstrated (2, 6) and adopted by some commercial interests. However, in all publications to date, embryos have been transferred to recipients by surgical methods, even though non-surgical methods of embryo recovery and transfer would be preferred for commercial embryo transfer.The purpose of the present experiments was to utilize non-surgical methods of embryo recovery and transfer of deep-frozen cattle embryos to demonstrate the feasibility of the procedure for a farm service to interested breeders. The particular advantage of non-surgical embryo transfer methods is that neither the donor nor recipient need to leave the farm. Embryo preservation by freezing obviates the necessity for synchronization of recipients for immediate transfer from the donor and allows considerable freedom in the choice of the recipients and the timing of embryo transfers.     

PGF2α levels in Day 8 blood plasma are increased by the presence of one or more embryos in the uterus

In cattle, the detection of very early endometrial responses is considered to be hampered by the presence of only a single embryo. Therefore, we have previously developed a model of multiple embryo transfer to circumvent this hindrance. In this work, we analysed embryo–maternal interactions in the bovine uterus on day 8 of development while comparing the presence of multiple v. single embryos using embryo transfer and artificial insemination, respectively. Concentration of proteins (β-actin, NFkB, clusterin and immunoproteosome 20S β5i subunit–i20S), by western blot, and hexoses (glucose and fructose) were measured in paired samples of uterine fluid (UF) from the same animal with and without embryos in the uterus and were compared with UF obtained after artificial insemination. Prostaglandin (PG) F₂α and PGE₂ concentrations were also analysed in blood plasma. The four proteins analysed and hexoses were unaffected by the presence of one or more embryos in the uterus. However, blood PGF₂α showed similar, significant increases with one or more embryos over cyclic animals; such changes were not observed in blood PGE₂. Although multiple embryo transfer may appear to be non-physiological, we showed that the uterus, at the very early embryonic stages, does exhibit physiological reactions. Multiple embryo transfer can, therefore, be used for studies of very early embryo–maternal interactions in vivo in monotocous species.     

Recent developments in pig embryo transfer.

Porcine embryo transfer has been performed for approximately 50 years, and surgical methods have proven to be reliable for collection and transfer of embryos. However, surgical collection and transfer have the disadvantage of being less useful on the farm. Recently, new procedures for both collection and transfer of embryos have been developed to improve usefulness. The surgical procedure has been refined to a minimally invasive procedure, using endoscopy for collection and transfer of embryos. A nonsurgical procedure for embryo collection has also been devised, but is limited to use in sows with surgically shunted (shortened) uterine horns. Nonsurgical embryo transfer procedures have been developed recently and have proven to be successful. The nonsurgical procedures are preferable to surgical procedures from an animal welfare point of view and because these procedures can be performed on farms without the need for special facilities.