The importance and potential of artificial insemination in CANDES (companion animals, non-domestic, endangered species)

Artificial insemination (AI) is the least invasive assisted reproductive technology, and is therefore of great interest to breeders of companion animals, non-domestic, and endangered species (CANDES). This most fundamental artificial breeding technique circumvents physical or behavioral impediments to natural mating and provides the means for genetic exchange between populations without transfer of live animals. In addition, because oocytes grow, mature and are fertilized in vivo and embryos are not subjected to in vitro culture conditions, AI eliminates the epigenetic effects on the female gamete that are inherent in more invasive assisted reproductive technologies. Although the management of CANDES differs significantly from current livestock husbandry practices, the cattle industry is a powerful example of the potential for AI to enhance the genetic health and sustainability of animal populations. Ultimately, successful AI requires sperm of adequate quality and quantity, oocytes that have attained nuclear maturation and cytoplasmic competence, operational gamete transport systems, accurate timing, and proper placement of sperm in the female reproductive tract. Increased understanding of semen collection, evaluation and preservation techniques, estrus synchronization and superovulation, estrus and ovulation detection, and insemination instrumentation is needed for each CANDES before AI success rates will approach those of the livestock industry. Concentrated, collaborative research in these areas must be encouraged among private breeders, universities and zoological institutions to realize the full potential of AI in the management of CANDES.     

Application of sperm sorting and associated reproductive technology for wildlife management and conservation

Efforts toward the conservation and captive breeding of wildlife can be enhanced by sperm sorting and associated reproductive technologies such as sperm cryopreservation and artificial insemination (AI). Sex ratio management is of particular significance to species which naturally exist in female-dominated social groups. A bias of the sex ratio towards females of these species will greatly assist in maintaining socially cohesive groups and minimizing male-male aggression. Another application of this technology potentially exists for endangered species, as the preferential production of females can enable propagation of those species at a faster rate. The particular assisted reproductive technology (ART) used in conjunction with sperm sorting for the production of offspring is largely determined by the quality and quantity of spermatozoa following sorting and preservation processes. Regardless of the ART selected, breeding decisions involving sex-sorted spermatozoa should be made in conjunction with appropriate genetic management. Zoological-based research on reproductive physiology and assisted reproduction, including sperm sorting, is being conducted on numerous terrestrial and marine mammals. The wildlife species for which the technology has undergone the most advance is the bottlenose dolphin. AI using sex-sorted fresh or frozen-thawed spermatozoa has become a valuable tool for the genetic and reproductive management of captive bottlenose dolphins with six pre-sexed calves, all of the predetermined sex born to date.     

Factors affecting commercial application of embryo technologies in New Zealand: a modelling approach

New reproductive technologies include sexed sperm and embryo-based technologies. The technology of sperm sexing, for various reasons, is not available in New Zealand and its use has not been modelled. Embryo technologies are however already in use on a limited scale and various scenarios for their use in both the dairy and beef industries in New Zealand have been modelled. This review briefly discusses the various technologies available and some of their potential strengths and weaknesses. In the dairy industry, modelling has been used to simulate the production of breeding bulls for large breeding companies and the production of replacement heifers in dairy herds. For the beef industry, similar modelling has been carried out to determine the opportunities for more efficient beef production.All the models confirmed that at current levels of performance, embryo-based reproductive technologies are usually not profitable in New Zealand except in niche market situations where the returns from the resulting offspring are significantly greater than can be obtained from natural mating or artificial insemination (AI) reproduction systems. This is confirmed by the low uptake of these technologies in this country to date. Even if performance lifts to levels similar to AI, profitability is likely to occur only if the costs of pregnancies to embryo-based reproductive technologies can occur at prices less than two to four times greater than AI or natural mating. This break-even requirement depends on the returns that can be achieved and the advantages that can be captured by the technology over and above those available from AI or natural mating. Two new uses for reproductive technologies in dairy cattle could be the proliferation of novel or rare genotypes from gene discovery programs and improving the female reproductive rate for optimal marker assisted selection. In both these uses the technology is not at present competing with AI or natural mating. The challenge exists therefore for the biological scientists to satisfy these requirements, coupled with the ethical and human factors involved in the introduction of any new technology.Potential end users of the technologies have been surveyed. They are quite positive about the technologies provided they can use them profitably and are keen to obtain more information about them.     

Reproductive biology in the era of genomics biology

Current and emerging technologies in reproductive biology, including assisted reproductive technologies and animal cloning, are discussed in the context of the impact of genomics era biology. The discussion focuses on the endocrinology associated with establishment and maintenance of pregnancy, fetal-placental development, lactation, and neonatal survival. Various aspects of uterine biology, including development during the neonatal period and function in adult females, are discussed with respect to reproductive efficiency. It is clear that combining strategies for use of conventional animal models for studying the reproductive system with new genomics technologies will provide exceptional opportunities in discovery research involving data integration and application of functional genomics to benefit animal agriculture and the biomedical community. New and emerging biotechnologies and comparative genomics approaches will greatly advance our understanding of genes that are critical to development of the reproductive system and to key events at each stage of the reproductive cycle of females and males.     

Live rhesus offspring by artificial insemination using fresh sperm and cryopreserved sperm

Artificial insemination (AI) and the cryopreservation of sperm with full reproductive capabilities are vital in the armamentarium of infertility clinics and reproductive laboratories. Notwithstanding the fantastic successes with AI and sperm cryopreservation in numerous species, including humans and cattle, these assisted reproductive technologies are less well developed in other species of importance for biomedical research, such as genetically modified mice and nonhuman primates. To that end, AI at high efficiency in the rhesus macaque (Macaca mullata) and the successful cryopreservation of rhesus sperm is presented here, as are the complexities of this primate model due to differences in reproductive tract anatomy and gamete physiology. Cryopreservation had no effect on the ability of sperm to fertilize oocytes in vitro or in vivo. Post-thaw progressive motility was not affected by cryopreservation; however, acrosome integrity was lower for cryopreserved (74.1%) than for fresh sperm (92.7%). Fertilization rates did not differ when fresh (58.1%; n = 32/55) or cryopreserved sperm (63.8%; n = 23/36) were used for in vitro fertilization. Similarly, pregnancy rates did not differ significantly after AI with fresh (57.1%; n = 8/14) or cryopreserved sperm (62.5%; n = 5/8). Seven live rhesus macaques were born following AI with fresh sperm, and three live offspring and two ongoing pregnancies were obtained when cryopreserved sperm were used. Cryopreservation of rhesus sperm as presented here would allow for the cost-effective storage of lineages of nonhuman primates with known genotypes. These results suggest that either national or international centers could be established as repositories to fill the global needs of sperm for nonhuman primate research and to provide the experimental foundation on which to explore and perfect the preservation of sperm from endangered nonhuman primates.     

Ultrasonography as an important tool for the development and application of reproductive technologies in non-domestic species

Ultrasound imaging in reproductive sciences offers new opportunities regarding optimization of the induction of the sexual cycle and ovulation, superovulation regimes, contraception programs, semen collection and testicular sperm extraction techniques, ovum pick up and ovarian transplantation procedures, as well as the application of artificial insemination, embryo collection and transfer. In non-domestic species, most of which lack basic data, ultrasonography is an ideal tool to study reproductive biology in both captive and wild populations. The use of this imaging modality led us to develop new, or modify established, reproductive technologies. Ultrasonography has been an integral part of over 200 assisted reproduction procedures in 17 mammalian species performed by our research team between 1992 and 1999. These procedures included the initial characterization of sexual cycles, hormonal cycle induction, semen collection by electroejaculation or manual stimulation, non-surgical artificial insemination (AI), non-surgical embryo transfer and temporary hormonal contraception. For these investigations, a variety of newly developed equipment was applied and species-specific hormonal treatments designed. We used several commercial and customized ultrasound systems with a variety of technical features. Some relevant improvements of these applications will be described and the role of ultrasonography elucidated to.     

Artificial insemination and the assisted reproductive technologies in non-human primates

The experience with artificial insemination (AI) and the more invasive ARTs (assisted reproductive technologies) in the propagation of non-human primates (NHPs), although limited, has included representation from the Great Apes and both Old World and New World Macaques. The application of these technologies in NHPs is impacted by high cost, substantial technical requirements and the limited captive populations of available animals. A major incentive for their use would be to propagate endangered, underrepresented individuals or valuable founder animals. Detailed protocols and a substantial experience base for the ARTs are available for rhesus and cynomolgus macaques and form the basis of this review, including sperm recovery, processing and long-term storage at low temperatures, insemination techniques and timing. Controlled ovarian stimulation and subsequent oocyte recovery required for the invasive ARTs such as intracytoplasmic sperm injection (ICSI), is also described. Three recent AI reports in Old World Macaques are reviewed, along with examples of the use of the ARTs in the propagation of valuable founder animals, in the preservation of endangered macaques, and finally in the creation of neurodegenerative disease models for biomedical research purposes.     

Highly efficient vitrification for cryopreservation of human oocytes and embryos: the Cryotop method

Vitrification is frequently referred to as a novel technology of cryopreservation in embryology, although some young embryologists were born after its first successful application. Unfortunately, in spite of the accumulated evidence regarding its enormous potential value, most domestic animal and human laboratories use exclusively the traditional slow-rate freezing with its compromised efficiency and inconsistency. The purpose of this paper is to clarify terms and conditions, to summarize arguments supporting or disapproving the use of vitrification, and to outline its role among assisted reproductive technologies. To provide evidence for the potential significance of vitrification, achievements with the Cryotop technology, an advanced version of the "minimal volume approaches" is analyzed. This technology alone has resulted in more healthy babies after cryopreservation of blastocysts than any other vitrification technique, and more successful human oocyte vitrification resulting in normal births than any other cryopreservation method. The value of this method is also demonstrated by achievements in the field of domestic animal embryology. A modification of the technique using a hermetically sealed container for storage may help to eliminate potential dangers of disease transmission and open the way for widespread application for cryopreservation at all phases of oocyte and preimplantation embryo development in mammals.     

人工智能时代发酵优化与放大技术的机遇与挑战

人工智能(artificial intelligence, AI)技术正引发一场新的产业革命,其成功应用正从信息产业迅速渗透到各行各业。传统的发酵工程技术受到巨大挑战的同时更多地迎来了发展变革的机遇。首先,合成生物技术飞速发展使高性能菌株的可获得性及获取效率显著提升,对传统低效的发酵优化放大技术提出很大挑战,亟需对发酵优化放大技术进行升级,以满足高通量菌种性能验证及工艺开发能力的需求;其次,发酵装备技术的持续发展为高效发酵优化技术的进步奠定了良好基础,加之人工智能技术特别是数字孪生与知识图谱等技术的应用,将为传统发酵技术的颠覆性发展带来巨大推动力。本文分别从合成生物时代对发酵优化技术的挑战、发酵优化与放大的核心技术、高通量发酵装备技术、数据可视化技术、数字孪生及知识图谱等智能技术在发酵优化放大中的应用等几个方面进行综述,并对未来工业发酵优化技术的场景以及未来发酵技术对人才培养等提出的新要求进行了展望。     

Issues affecting commercialization of sexed sperm

A decision tree for genetics or sperm-sexing entities considering sales of sexed sperm is discussed in terms of: (a) how best to avoid harm; (b) how best to do good; (c) needed synergy with other assisted reproductive technologies; (d) constraints on biotechnology; and (e) costs with current and likely technologies versus potential benefits to producers. The sexed-sperm industry might wish to take a pro-active stance on societal issues potentially affecting use of sexed sperm. For most sales in animal agriculture, cost of added value must be < 50% of benefit. Cost is less important for emotionally-driven uses with horses and human beings. Current procedures for flow-sorting allow most sperm to retain their fertilizing potential. Added cost to produce and package 2 x 10(6) sperm is estimated at US $30 to US $46 with flow sorted sperm. Estimating cost of any alternative technology is premature. For IVF/embryo transfer (ET), cost and numbers of flow-sorted sexed sperm are appropriate for commercial use. For use in low-dose AI, however, added cost to supply one insemination dose must be near US $12. Flow-sorting instruments with higher throughput and lower purchase and operating costs are obligatory for economic application in most AI situations. Developers of antibody-based separations also will face issues of retention of fertilizing potential while minimizing cell loss, separation of living from dead sperm concurrent with sperm sexing, output, and cost. To benefit producers and consumers in a changing world, genetics and sperm-sexing companies will have to collaborate and interface to provide funding for needed research and development and to recover these costs, using mechanisms not yet obvious.     

Possibilities with today's reproductive technologies

Reproductive efficiency is critical to economic viability for cow/calf producers; however, very few producers take advantage of available reproductive technologies that can increase profitability. Today, more opportunities are available for producers who want to capture value from known genetics. Through the use of artificial insemination (AI), the average producer has access to a wide range of high-accuracy sires that can be selected to match production goals. Systems to synchronize estrus and ovulation can now produce pregnancy rates to a single fixed-timed AI that are 10-15% greater than those of the previous generation. Increased age and weight of calves at weaning is sufficient in some situations to pay for the cost of synchronization and AI. As a result of synchronization, more cows calve early the next year and in subsequent years of synchronization. The breeding season can be shortened without reducing end-of-season pregnancy rates, since synchronized cows have one more chance to conceive than unsynchronized cows in a 22-25 day interval. Cow nutrition can be more economically and precisely managed with a shorter breeding period. Producers that establish AI programs now will be prepared to take advantage of newly identified superior genetics or other technologies, e.g. sexed semen, when they become available. Trends towards more value-based marketing and improvements in pregnancy rates from synchronization systems, make this a key time to be aware of the possibilities using reproductive technologies.     

Deep intra-uterine artificial inseminations using cryopreserved spermatozoa in beluga (Delphinapterus leucas)

Artificial insemination (AI) with liquid-stored spermatozoa and sperm cryopreservation using directional freezing (DF) have been successful in the beluga. This study built on this foundation to develop a deep intra-uterine AI technique with frozen-thawed semen in beluga. Forty-two ejaculates from one male were cryopreserved using DF technology and subsequently used for 10 insemination attempts with seven females. Percentage pre- and post-thaw progressive motility and viability were (mean ± SD) 73.0 ± 12.2, 38.4 ± 8.8, 88.0 ± 0.1, and 59.3 ± 15.7%, respectively. A series of GnRH injections (3 x 250 μg, IV, 1.5 to 2 h apart) were used to induce ovulation, once a growing follicle >2.5 cm in diameter was visualized via trans-abdominal ultrasonography. Artificial insemination was performed at 30.1 ± 3.8 h post-initial GnRH injection with semen deposited in the uterine horn, 92.6 ± 16.2 cm beyond the genital opening using a flexible endoscope. The external cervical os (cEOS) was located beyond a series of 5 to 10 vaginal rings, 44.8 ± 9.3 cm from the external genital opening. The internal bifurcation of the uterus was 27 ± 6.8 cm beyond the cEOS. Ovulation occurred at 8.5 ± 7.6 h post-AI. Two of 10 inseminations (20%) resulted in pregnancy. The first pregnancy resulted in twins; both calves were born 442 d after AI, with one surviving. The second pregnancy is ongoing. These findings represent the first successful application of AI using frozen-thawed semen in beluga, and are important examples of how assisted reproductive technologies can provide tools for the global management of threatened species.     

RETHINKING RADICAL POLITICS IN THE CONTEXT OF ASSISTED REPRODUCTIVE TECHNOLOGY

Radical feminists have argued for both the radical potential of assisted reproductive technology (ART) and its oppressive and damaging effects for women. This paper will address the question of what constitutes a radical feminist position on ART; I will argue that the very debate over whether ART liberates or oppresses women is misguided, and that instead the issue should be understood dialectically. Reproductive technologies are neither inherently liberating nor entirely oppressive: we can only understand the potential and effects by considering how they are actually taken up within a culture. The internal contradictions, tensions, and inconsistencies within ART and the way it is addressed within the law points to a dialectic that resists a simple reductivist understanding.     

Epigenetic perspectives of safety in assisted reproductive technologies

To date, a wide range of assisted reproductive technologies is available for patients with impaired fertility. In general, the current methods of reproductive medicine are considered safe and do not significantly increase the frequency of birth of children with diseases or congenital malformations. However, the evidence has been accumulating in literature on higher risk of genomic imprinting diseases (Beckwith-Wiedemann and Angelman syndromes) as a result of using assisted reproductive technologies. In most cases examined, the appearance of these syndromes was explained by defective methylation status of imprinted genes. It has been suggested that manipulations with gametes and embryos during the period of total epigenetic modification of their genomes may act as potential risk factors of assisted reproductive technologies. Moreover, overcoming many natural reproductive barriers may contribute to the development of some pathological phenotypes. The review summarizes current views on epigenetic risk factors associated with assisted reproductive technologies.     

Epigenetic perspectives of safety in assisted reproductive technologies

To date, a wide range of assisted reproductive technologies is available for patients with impaired fertility. In general, the current methods of reproductive medicine are considered safe and do not significantly increase the frequency of birth of children with diseases or congenital malformations. However, the evidence has been accumulating in literature on higher risk of genomic imprinting diseases (Beckwith-Wiedemann and Angelman syndromes) as a result of using assisted reproductive technologies. In most cases examined, the appearance of these syndromes was explained by defective methylation status of imprinted genes. It has been suggested that manipulations with gametes and embryos during the period of total epigenetic modification of their genomes may act as potential risk factors of assisted reproductive technologies. Moreover, overcoming many natural reproductive barriers may contribute to the development of some pathological phenotypes. The review summarizes current views on epigenetic risk factors associated with assisted reproductive technologies.     

Conservation of deer: contributions from molecular biology, evolutionary ecology, and reproductive physiology

Molecular phylogenetics, interspecific comparisons, and assisted reproductive techniques are recent approaches to understanding and facilitating conservation of endangered species. This paper reviews the contribution of these approaches to a small but well-studied group of mammals, deer, many of which are endangered in the wild. Conservation efforts require a comprehensive understanding of the biology and history of these animals. The value of assisted reproductive technologies for conservation of deer has received increased awareness especially for captive populations. Such breeding programmes are designed to assist propagation of threatened species and to maximize genetic diversity within populations through the movement of genetic material across the globe, but will only be successful if we understand the genetic and reproductive potential of various lineages. Here we discuss the phylogenetic status of deer, the distinctiveness and evolution of their reproductive patterns, and current approaches for improving the success of controlled breeding programmes for the conservation of endangered lineages. Only by combining both theoretical and practical approaches to conservation efforts can we hope to salvage the remaining organismal diversity of our planet.     

Factors Affecting the Outcome of Artificial Insemination Using Cryopreserved Spermatozoa in the Giant Panda (Ailuropoda melanoleuca)

Artificial insemination (AI) is an important component of captive breeding programs for endangered species, such as the giant panda. The panda has been the subject of increasingly successful captive breeding programs involving a compilation of assisted breeding techniques, including AI using cryopreserved spermatozoa. AI implementation is currently hampered by a lack of understanding of the factors that may cause failure. We investigated factors influencing the probability of success of AI for 14 giant panda females housed at the China Center for Research and Conservation of the Giant Panda (CCRCGP) inseminated in a total of 20 instances using cryopreserved spermatozoa from 11 males currently residing in 6 different captive breeding institutions. One of the pandas was the oldest giant panda female to ever successfully conceive from AI (20.5 years old). The success of AI was significantly affected by the timing of AI in relationship to both timing of peak urinary estrogen of the female and percent decline in urinary estrogen between the peak level and the first AI attempt. Our results suggest that the window for successful AI in giant pandas may be narrower than previously suspected, although individual differences in rates of decline in urinary estrogen may reflect some degree of variation in this crucial window across females. Our results are consistent with recent research on pandas and other species that demonstrates the efficacy of cryopreserved spermatozoa for AI and highlights the need for more in‐depth analysis of factors related to female physiology that may influence its success. Zoo Biol 31:561‐573, 2012. © 2011 Wiley Periodicals, Inc.     

Responses of canine oocytes to in vitro maturation and in vitro fertilization outcome

The potential benefits of assisted reproduction techniques, such as in vitro maturation (IVM) and in vitro fertilization (IVF) in canids, are linked to the protection and saving of species threatened by extinction due to worldwide habitat destruction and pollution. In both domestic and wild species, these technologies will form the basis for the next leap in reproductive performance by improving fertility rates in valuable middle-aged females, by improving pregnancy rate in infertile or sub-fertile populations and by rescuing biological material to replenish populations of endangered species. In vitro techniques are supposed to answer the reproductive questions of canids, to introduce new methods for contraception and to compete with artificial insemination (AI) as the major or predominant method of embryo production, oocyte- and embryo cryopreservation and cloning. The causes affecting in vitro meiosis of dog oocytes are likely to be diverse. Incomplete understanding of the events associated with oocyte developmental competence are imputed to species reproductive physiology, medium composition and source of ovarian oocyte population used for in vitro maturation. This review addresses some issues on the current state of in vitro maturation and in vitro fertilization of canine oocytes.     

Assisted reproductive technologies and arterial hypertension

The effects of assisted reproductive technologies on the development of hypertensive phenotype were reviewed. Special attention was paid to the effects of cultivation and transplantation of preimplantation embryos on arterial pressure in individuals developed from these embryos. The analysis of studies performed on the laboratory models (mostly on hypertensive strains of rats) was performed. These data were discussed in the context of application of assisted reproductive technologies in medicine.     

Assisted reproductive technologies in the reproductive management of small ruminants

In modern agriculture, assisted reproductive technologies are being used for out of season oestrus induction, enhancement of reproductive performance and genetic improvement. In addition, they can have substantial contribution in preservation of endangered species or breeds, as well as in eradication programs of various diseases. While their applications are widespread in cattle, in small ruminants it is almost restricted to artificial insemination. The main limitations of a wider application in small ruminants are the naturally occurring anoestrus period, the variability of response to superovulatory treatments, the fertilisation failure and the need of surgery for collection and transfer of gametes and embryos. Nonetheless, during the last 30 years, considerable progress has been made in sheep and goat embryo technologies, especially in the fields of oestrus synchronisation, superovulation and in vitro embryo production. This paper reviews the status of assisted reproductive technologies in sheep, analysing the prospects offered by recent advances in in vivo and in vitro embryo production from mature and juvenile lambs.