Advancements in Large Animal Embryo Transfer and Related Biotechnologies

Embryo transfer has been an inherent part of cattle breeding for more than 35 years and has also gained remarkable interest from the equine industry after several breeds allowed registration of more than one foal per year. In both large animal species, non‐surgical embryo recovery and transfer are well‐established techniques. However, success rates after superovulation and cryopreservation of embryos in horses are still lagging behind those of cattle, and more research is needed to address these areas. To address the problem of freezing large equine embryos, we offer a preliminary demonstration of a new cryopreservation method which involves reduction of the blastocoelic volume and microinjection of cryopreservative. Successful cryopreservation will improve the ability of practitioners to preserve and implant embryos in recipient mares. Recent advances in the use of equine FSH to induce superovulation in mares brings to the forefront the issue of how to best preserve the large number of embryos that are produced. Finally, the use of sexed semen after superovulation will provide the bovine and equine breeding industry the offspring of the desired sex.     

肉牛遗传育种与繁殖技术应用现状

通过介绍我国肉牛遗传育种与繁殖技术方法,概述了数量遗传学、育种值估计、常规育种、分子育种技术等在肉牛育种、生产领域中的应用现状,并对人工授精技术、胚胎移植技术、胚胎分割技术、性别控制技术、体外授精技术和克隆技术等繁殖技术在肉牛科研与生产中的应用进行了介绍,以期为肉牛遗传育种与繁殖技术研究及生产提供参考.     

Assisted reproductive techniques for cattle breeding in developing countries: a critical appraisal of their value and limitations

Commercialization of animal biotechnologies, including those related to reproduction [also known as assisted reproductive techniques (ARTS)], is an increasing reality in developing countries, following the enormous flow of information around us and the increasing global commercial interests in areas where cattle production has its major assets. The present review discusses the achievements of various biotechnological tools for reproduction in cattle including semen handling for artificial insemination (AI), superovulation and embryo transfer (MOET), in vitro handling of oocytes and production of embryos, reproductive cloning and emerging technologies (sex selection, gene targeting and nuclear transfer for livestock transgenesis, genomics for marker-assisted selection, etc.). The application of these technologies for cattle breeding is critically discussed in relation to their impact in the improvement of the efficiency of dairy and beef production in developed and - particularly - in developing countries, which ultimately rule the possibilities of a competitive and sound production of food for human consumption. Despite the remarkable progress made and the punctual importance of some of the above-mentioned technologies, AI remains the most important assisted reproductive technology (ART) in developing countries. Any attempt to gain widespread of any other ART under the predominant economical conditions in developing countries ought to match the simplicity and the success of AI as a breeding tool.     

Laboratory Production of Equine Embryos

Assisted reproduction technologies (ART) are well developed in humans and cattle and are gaining momentum also in the equine industry because of the fact that the mare does not respond to superovulation but can donate large numbers of oocytes through ovum pick up (OPU). After collection, the oocytes can be fertilized by intracytoplasmic sperm injection (ICSI) using a variety of stallion semen samples, even of poor quality, and the resulting embryos can establish high pregnancy rates after cryopreservation and transfer. The discoveries that equine oocytes can be held at room temperature without loss of viability and that an increase in vitro maturation time can double the number of embryos produced are fueling the uptake of the OPU technique by several clinics that are shipping oocytes of their client’s mares to specialized ICSI laboratories for embryo production and freezing. In this article, we present a retrospective analysis of 10 years of work at Avantea with a special focus on the last 3 years. Based on our data, an average production of 1.7 to 2 embryos per OPU-ICSI procedure can be obtained from warmblood donor mares with a pregnancy rate of 70% and a foaling rate in excess of 50%. OPU-ICSI offers the added value of freezing embryos that allows the development of embryo commercialization worldwide to the benefit of top horse breeders who are endorsing this technology as never before.     

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.     

黄体对牛胚胎移植效果影响的探究

黄体释放的黄体酮对母畜发情周期、启动发情行为和维持妊娠起着至关重要的作用,但目前国内外对黄体是否影响胚胎移植效果有着不同报道。本文以探究黄体对牛胚胎移植效果的影响为主线,查阅大量文献,归纳黄体的检查和级别判断方法,分别分析黄体大小、黄体位置、黄体质量和黄体生产溶解过程激素水平与胚胎移植受胎率的关系,为提高胚胎移植效果提供科学的参考依据。生产上常通过B超扫描结合直肠检查法判断受体牛黄体发育和位置作为能否移植的依据;由各学者研究结果可见,不同直径黄体的胚胎移植受胎率差异不显著,为提高受体牛的利用,在对受体牛黄体大小选择时可适当放宽标准;胚胎移植到黄体同侧的受胎率比移植到黄体对侧的高,另外,可能因为操作者左右手操作习惯不同,发现左侧黄体移植受胎率比右侧的要高;较多学者认为质量好的胚胎选择黄体质量好的受体移植才能得到更高的受胎率;胚胎移植后能否成功受孕,还与黄体生成溶解过程中孕酮含量及孕酮与雌二醇的比例等有关。     

娟姗青年母牛超数排卵及冻胚移植效果试验报告

2005年3月至7月,在广西地区选择25头娟姗母牛作为供体,用不同厂家、不同剂量的促排卵激素进行超排和重复超排,观察了超排及重复超排效果.结果25头供体共采卵46头次,排卵数502枚,回收到278枚胚胎,采卵率为55.4%,获得207枚可用胚胎,平均4.5枚/头次,可用胚率为74.5%;与此同时在2005年4月至2006年1月,开展了娟姗牛冷冻胚胎的移植工作,对185头本地黄母牛和杂交母牛进行了同期发情处理,结果共有166头发情,其中113头可移植,同期发情率为89.7%,可移植率为61.1%.113头可移植牛移植2个月后,经妊娠检查共有47头怀孕,移植妊娠率为41.6%.     

Development of genome engineering technologies in cattle: from random to specific

The production of transgenic farm animals(e.g., cattle) via genome engineering for the gain or loss of gene functions is an important undertaking. In the initial stages of genome engineering, DNA micro-injection into one-cell stage embryos(zygotes) followed by embryo transfer into a recipient was performed because of the ease of the procedure.However, as this approach resulted in severe mosaicism and has a low efficiency, it is not typically employed in the cattle as priority, unlike in mice. To overcome the above issue with micro-injection in cattle, somatic cell nuclear transfer(SCNT) was introduced and successfully used to produce cloned livestock. The application of SCNT for the production of transgenic livestock represents a significant advancement, but its development speed is relatively slow because of abnormal reprogramming and low gene targeting efficiency. Recent genome editing technologies(e.g.,ZFN, TALEN, and CRISPR-Cas9) have been rapidly adapted for applications in cattle and great results have been achieved in several fields such as disease models and bioreactors. In the future, genome engineering technologies wil accelerate our understanding of genetic traits in bovine and wil be readily adapted for bio-medical applications in cattle.     

德系乳肉兼用型西门塔尔牛胚胎移植效果研究

[目的]建立德系乳肉兼用型西门塔尔牛群体,探索文山牛快速扩繁和生产性能提升技术。[方法]在马关县从600头文山黄牛、西本杂、安本杂和短本杂牛母牛中选择270头,用CIDR+PG法进行同期发情处理,选择黄体合格的受体,采用非手术法移植德系乳肉兼用型西门塔尔牛胚胎,在移植后60~90 d通过直肠检查法进行妊娠诊断,确定妊娠率,跟踪调查产犊情况,并测定胚胎移植所产犊牛的初生体重和主要体尺指标。[结果]受体牛同期发情处理270头,胚胎移植87头,妊娠28头,移植妊娠率32.18%;妊娠母牛中产犊17头,产犊率60.71%,产犊19头,成活16头,产犊成活率84.21%。公母犊牛平均的初生重33.00 kg,体高69.50 cm,体斜长62.19 cm,胸围72.69 cm,管围13.50 cm。[结论]首次在文山州开展了牛胚胎移植获得成功,杂交牛受体的移植妊娠率极显著或显著高于文山牛受体,秋季移植的妊娠率极显著高于春季和冬季的。提示以杂交牛为受体在秋季进行胚胎移植可有效提高移植成功率。     

肉用种公牛的后裔测定

肉牛育种工作中对种公牛种用价值最终认定是通过后裔测定完成的。同期同龄比较法是在过去和将来采用并将继续使用的较为普遍的方法。随着动物育种技术的进展以及计算机技术、生物技术的不断革新和完善,ＢＬＵＰ、ＭＯＥＴ和ＩＶＦ技术和手段已应用于肉牛育种,因此肉牛的后裔测定方法也将随之改进。本文介绍肉用种公牛四种后裔测定方法。     

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.     

Cryopreservation of equine embryos: current state-of-the-art

During the past 15 years, embryo transfer (ET) has become increasingly widespread within the sport-horse breeding industry. At present, however, the vast majority (>95%) of horse embryos are transferred fresh or after chilled storage for up to 24 h, whereas cryopreservation is rarely employed despite its obvious potential for simplifying recipient mare management and facilitating long-term storage and international transport of embryos. A number of inter-related factors have contributed to the slow development and implementation of equine embryo cryopreservation, and these include the following: (i) the absence of commercially available products for reliably stimulating superovulation; (ii) very poor pregnancy rates following cryopreservation of embryos >300 μm in diameter; (iii) difficulty in recovering embryos at early developmental stages amenable to cryopreservation; and (iv) interembryo variation in susceptibility to cryodamage. However, acceptable success rates (>55% pregnancy) have been reported for both slow-frozen and vitrified small embryos (<300 μm), and there is renewed interest in cryopreservation, not only in the context of standard ET programmes, but also because it would facilitate pre-implantation genetic testing and allow wider access to techniques for producing embryos in vitro, such as intracytoplasmic sperm injection and nuclear transfer. This article will review the current status of equine embryo cryopreservation.     

新疆塔城地区牛胚胎移植现状问题及对策

本文简要地介绍了塔城地区牛胚胎移植的现状,通过分析胚胎移植工作开展中存在的问题,提出了塔城地区牛胚胎移植的发展思路。     

Manipulation of follicle development to ensure optimal oocyte quality and conception rates in cattle

Over the last several decades, a number of therapies have been developed that manipulate ovarian follicle growth to improve oocyte quality and conception rates in cattle. Various strategies have been proposed to improve the responses to reproductive biotechnologies following timed artificial insemination (TAI), superovulation (SOV) or ovum pickup (OPU) programmes. During TAI protocols, final follicular growth and size of the ovulatory follicle are key factors that may significantly influence oocyte quality, ovulation, the uterine environment and consequently pregnancy outcomes. Progesterone concentrations during SOV protocols influence follicular growth, oocyte quality and embryo quality; therefore, several adjustments to SOV protocols have been proposed depending on the animal category and breed. In addition, the success of in vitro embryo production is directly related to the number and quality of cumulus oocyte complexes harvested by OPU. Control of follicle development has a significant impact on the OPU outcome. This article discusses a number of key points related to the manipulation of ovarian follicular growth to maximize oocyte quality and improve conception rates following TAI and embryo transfer of in vivo- and in vitro-derived embryos in cattle.     

Morphologic stages of the equine embryo proper on days 17 to 40 after ovulation

OBJECTIVE: To describe the gross and histologic changes that develop in the equine embryo proper (ie, the portion of the embryo that becomes the fetus) from days 17 to 40 after ovulation and to compare the external features of equine embryos with those of porcine, ovine, and human embryos. SAMPLE POPULATION: 34 embryos collected from mixed-breed pony mares. PROCEDURE: External features for each embryo proper, including length, number of branchial arches, growth of appendages, face and head features, and body features, were examined, using a dissecting microscope, for embryos collected on days 17 to 40. Internal features were histologically examined by serially sectioning embryos collected on days 20 to 35. RESULTS: Number of embryos recovered for each day ranged from 1 to 5. The initial detection of features was not related closely to age; typically, the first attainment of a given body length or characteristic varied over a 3-day period among embryos. Similarly, the period during which individual characteristics for a given Carnegie stage were attained ranged from 3 to 6 days. Age at first appearance of a characteristic was greater for equine embryos than ages reported for ovine and porcine embryos but less than for human embryos. Indicators of age included number of pairs of branchial arches, all limb buds present, retinal pigmentation, and prominence of the pontine flexure. CONCLUSIONS: No embryologic structures or changes were found that could be considered unique to equine embryos on days 17 to 40 after ovulation.     

国内家畜胚胎移植的现状与展望

统计与分析了1991-2001年国内胚胎移植状况,其中牛、羊胚胎移植所占的比例较大。在1991-1995年间,我国年平均超排处理供体牛300-500头次,羊200-300只;1995年以后,胚胎移植规模有所扩大,超排处理供体牛年平均在400-800头次;羊的胚胎移植在1999年以后,出现了波尔山羊、道赛特和特克赛尔绵羊的移植热潮。国内胚胎移植技术已接近或达到发达国家水平,具备商业化的能力。     

和牛母牛超数排卵及胚胎移植试验研究效果初报

[目的]研究和牛母牛超数排卵及胚胎移植效果。[方法]利用纯种和牛母牛作为供体,荷斯坦母牛及西杂母牛作为受体,通过供体牛的超数排卵、人工授精、采胚、胚胎冷冻及受体牛的同期发情和鲜胚移植,快速获得纯种和牛及和牛胚胎。[结果]对4头青年纯种和牛母牛进行超排,重复冲卵13头次,共采卵数122枚,其中75枚可用胚胎,可用胚率为61.5%,平均5.76枚/头次。对75头荷斯坦牛及西杂母牛同期发情,结果获得89.2%的同期发情率。67头受体牛移植了鲜胚,2个月后妊娠检查有35头怀孕,移植妊娠率为52.23%。[结论]可以利用超数排卵及胚胎移植增加和牛母牛利用效率。