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.     

Effects of maternal age on oocyte developmental competence

The widespread use of a variety of assisted reproductive technologies has removed many of the constraints that previously restricted mammalian reproduction to the period between onset of puberty and reproductive senescence. In vitro embryo production systems now allow oocytes from very young animals to undergo fertilization and form embryos capable of development to normal offspring, albeit at somewhat reduced efficiencies compared to oocytes from adult females. They also can overcome infertility associated with advanced age of animals and women. This review examines oocyte developmental competence as the limiting factor in applications of assisted reproductive technologies for both juvenile and aged females. Age of oocyte donor is a significant factor influencing developmental competence of the oocyte. Age-related abnormalities of oocytes include a) meiotic incompetence or inability to complete meiotic maturation resulting in oocytes incapable of fertilization; b) errors in meiosis that can be compatible with fertilization but lead to genetic abnormalities that compromise embryo viability; and c) cytoplasmic deficiencies that are expressed at several stages of development before or after fertilization. In general, oocytes from juvenile donors and the embryos derived therefrom appear less robust and may be less tolerant to suboptimal handling and in vitro culture conditions than are adult oocytes. Research to identify specific cytoplasmic deficiencies of juvenile oocytes may enable modifications of culture conditions to correct such deficiencies and thus enhance developmental competence. Use of oocytes from aged donors for assisted reproduction can have a variety of applications such as extending the reproductive life of individual old females whose offspring still have high commercial value, and conservation of genetic resources such as rare breeds of livestock and endangered species. In general, female fertility decreases with advancing age. Studies of women in oocyte donation programs have established reduced oocyte competence as the major cause of declining fertility with age, although inadequate endometrial function can also be a contributing factor. Most research has emphasized the importance of chromosomal abnormalities because of the well established increase in aneuploidy with increasing maternal age but little is known about the underlying cellular and molecular mechanisms. Research aimed at identifying the specific developmental deficiencies of oocytes from juvenile donors and abnormalities of oocytes from aged females will assist in overcoming present bottlenecks that limit the efficiency of assisted reproduction technologies. Such research will also be crucial to the development of new oocyte-based technologies for overcoming infertility and possibly subverting chromosomal abnormalities in women approaching menopause.     

Microfluidic technology for assisted reproduction.

The physical tools used in assisted reproduction have changed little over several decades. Microfluidics is an emerging technology that allows a fresh examination of the way assisted reproduction is performed. Here we review our work to develop microfluidic devices to perform the functions required in assisted reproduction. These functions include loading/unloading, culture, chemical manipulation, and mechanical manipulation of embryos and oocytes. Basic microfluidic theory and microfluidic device design and operation are discussed. Results are presented for mechanical removal of cumulus cells and for embryo culture. Results suggest that microfluidic systems will lead to improved efficiencies in assisted reproduction.     

Sampling techniques for oviductal and uterine luminal fluid in cattle

Analysis of luminal fluid microenvironments in the reproductive tract is pivotal to elucidate embryo-maternal signaling mechanisms responsible for successful reproduction in mammals, including cattle. Besides facilitating production of an optimized medium for in vitro fertilization and embryo culture in assisted reproductive technologies, screening of luminal fluid constituents in the oviduct and uterus could also provide critical information for elucidation of mechanisms underlying developmental programming. A key issue in this type of research is the sampling of luminal fluids. In this review we discuss the sampling techniques available for bovine species, including a recent in situ technique developed with the Ghent device, which allows rapid recovery of measurable amounts of pure uterine luminal fluid with minimal disturbance to the donor animal.     

Embryonic origins of health: long-term effects of IVF in human and livestock

Assisted reproduction technologies have been introduced 1) to overcome reproductive failures in the human, 2) to increase the number of offspring from selected females and 3) to reduce generation intervals in livestock in farm animals. The successful introduction of these technologies in clinical practice and in livestock breeding programs is the result of enormous scientific efforts. In general, the offspring generated by IVF (human) and IVP (cattle) is normal, but as numbers increase the restraints and drawbacks of new reproductive technologies become visible with respect to the overall efficiency as well as to the occurrence of abnormalities and/or anomalies in the offspring. The objective of the present symposium on "Embryonic Origins of Health" is to present "the-state-of-the-art" and to discuss the restraints and possible long term effects of the application of assisted reproduction technology in both human and livestock. This introduction to the symposium focuses on the relation between early embryonic development and post-natal health. We hypothesize that IVF in the human and IVP in cattle influence the timing of cell-cell interactions during the early stages of embryogenesis which finally result in a incorrect timing of gene expression during the phylotypic stage and subsequent organogenesis. These deviations in embryonic timing might have consequence for the postnatal homeostasis and health.     

Buffalo and cattle hybrid embryo development is decreased by caffeine treatment during in vitro fertilization

Water buffalo are renowned for difficulties in the implementation of assisted reproductive technologies, with both males and females being problematic. In this study, we used cattle oocytes to assess the effect of treatments with heparin and caffeine on buffalo spermatozoa and subsequent fertilization and embryo development in vitro. There was no significant difference between buffalo and bovine spermatozoa in the events associated with fertilization. Fertilization of cattle oocytes with buffalo spermatozoa resulted in 7.8% of oocytes developing into hybrid embryos. A difference in the developmental capability of hybrid embryos compared with the cattle control was observed. This has not been previously reported. The subsequent transfer of a limited number of hybrid embryos did not produce a viable pregnancy. However, control treatments in this experiment also failed to achieve pregnancy, so objective data is not available to provide conclusions about the developmental competence of the buffalo and cattle hybrid embryos. Optimal spermatozoa capacitation treatments achieved 61% fertilization and 21% zygote cleavage into two cell embryos. There was no significant difference in fertilization or development due to heparin or spermatozoa concentrations. However, treatment of buffalo and cattle spermatozoa with caffeine significantly decreased embryo cleavage but also tended to decrease embryo development to the blastocyst stage. These studies suggest that problems with reproduction in buffalo may reside with biological mechanisms associated with the oocyte that are often complicated by poor male reproductive performance. Selection for bull fertility would prevent some of these complications.     

Bovine embryo technologies

Embryo technologies are a combination of assisted reproduction, cellular and molecular biology and genomic techniques. Their classical use in animal breeding has been to increase the number of superior genotypes but with advancement in biotechnology and genomics they have become a tool for transgenesis and genotyping. Multiple ovulation and embryo transfer (MOET) has been well established for many years and still accounts for the majority of the embryos produced worldwide. However, no progress has been made in the last 20 years to increase the number of transferable embryos and to reduce the side effects on the reproductive performance of the donors. In vitro embryo production (IVP) is a newer and more flexible approach, although it is technically more demanding and requires specific laboratory expertise and equipment that are most important for the quality of the embryos produced. Somatic cell cloning is a rapidly developing area and a very valuable technique to copy superior genotypes and to produce or copy transgenic animals. More knowledge in oocyte and embryo biology is expected to shed new light on the early developmental events, including epigenetic changes and their long lasting effect on the newborn.Embryo technologies are here to stay and their use will increase as advances in the understanding of the mechanisms governing basic biological processes are made.     

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.     

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.     

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.     

Advancements in cryopreservation of domestic animal embryos.

The development of embryo freezing technologies revolutionized cattle breeding. Since then, advancements in cryobiology, cell biology, and domestic animal embryology have enabled the development of embryo preservation methodologies for our other domestic animal species, including sheep and goats. Recently, technologies have been developed to cryopreserve pig embryos, notorious for their extreme sensitivity to cooling; horse embryo cryopreservation is in its infancy. While cryopreservation can enhance the utilization of in vitro embryo production technologies, cryosurvival of in vitro-produced (IVP) or micromanipulated embryos is less than that of in vivo-derived embryos. This review outlines recent efforts in livestock embryo cryopreservation. In the near future, use of preserved embryos could be a routine breeding alternative for all livestock producers providing 1) preservation methods for maternal germplasm, 2) global genetic transport, 3) increased selection pressure within herds, 4) breeding line regeneration or proliferation, and 5) methodology for genetic rescue.     

Postnatal health and welfare of offspring conceived in vitro: a case for epidemiological studies

In vitro fertilization (IVF) has established itself as an important technique in human assisted reproduction and in livestock improvement. In both humans and livestock the possible long-term effects on health and welfare of offspring born after IVF and in vitro culture to the blastocyst stage are still largely unknown. Epidemiological studies in humans, using data collected for individuals born after normal (i.e. non-assisted) pregnancies, have provided evidence for associations between prenatal life events and adult-life disease. Due to the relatively short time that elapsed since the first IVF baby was born, comparable studies for IVF offspring are not yet possible. However, animal experiments and epidemiological studies with the available data from the livestock industry (mainly dairy cattle) may contribute to a better understanding of the risks involved.     

Current progress on assisted reproduction in dogs and cats: in vitro embryo production

The objective of the development of assisted reproduction techniques in dogs and cats is their application to non-domestic canine and feline species, most of which are considered threatened or endangered. Among these techniques, an entirely in vitro system for embryo production is effectively an important tool for conservation of wildlife. In the last decade, progress has been made in embryo production in carnivores. It has been shown that canine oocytes can resume meiosis in vitro and that these oocytes can be fertilized and developed in vitro, although at a much lower rate than most other domestic animal oocytes. The reason lies in the dissimilarities of reproductive physiology of the dog compared to other species and the lack of precise information concerning the oviductal environment, in which oocyte maturation, fertilization and early embryonic development take place. Successful in vitro embryo production in the domestic cat has been attained with oocytes matured in vitro, and kittens were born after transfer of IVM/IVF derived embryos. On the basis of these results the in vitro fertilization of oocytes has also been applied in several non-domestic feline species. The effectiveness of such protocols in the preservation of genetic material of rare species can be improved by developing better techniques for long-term storage of gametes. In dogs and cats sperm cells have been successfully frozen and the cryopreservation of oocytes would greatly increase their availability for a range of reproductive technologies. Cryopreserved cat oocytes can be fertilized successfully and their development in vitro after fertilization is enhanced when mature oocytes are frozen. Thus refined techniques of oocyte maturation and fertilization in vitro coupled with oocyte cryopreservation could allow for an easy establishment of genetic combinations when male and female gametes in the desired combination are not simultaneously available, and the propagation of endangered carnivores would be facilitated.     

Engineering Aspects of Carriers for Immobilized Biocatalysts

Global meat and milk consumption is exponentially increasing due to population growth, urbanization and changes in lifestyle in the developing world. This is an excellent opportunity for developing countries to improve the livestock sector by using technological advances. Biotechnology is one of the avenues for improved production in the “Livestock revolution”. Biotechnology developments applied to livestock health, nutrition, breeding and reproduction are improving with a reasonable pace in developing countries. Simple bio-techniques such as artificial insemination have been well implemented in many parts of the developing world. However, advanced technologies including transgenic plant vaccines, marker assisted selection, solid state fermentation for the production of fibrolytic enzymes, transgenic fodders, embryo transfer and animal cloning are confined largely to research organizations. Some developing countries such as Taiwan, China and Brazil have considered the commercialization of biotechnology in the livestock sector. Organized livestock production systems, proper record management, capacity building, objective oriented research to improve farmer’s income, collaborations with the developed world, knowledge of the sociology of an area and research on new methods to educate farmers and policy makers need to be improved for the creation and implementation of biotechnology advances in the livestock sector in the developing world.     

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.     

Effect of chemically defined culture medium supplemented with beta-mercaptoethanol and amino acids on implantation and development of different stage in vivo- or in vitro-derived mouse embryos

In vitro culture (IVC) systems are required for many biotechnological and assisted reproductive technologies and the researchers have been modifying in vitro embryo culture conditions to reach the comparable efficiencies provided in vivo. In the present study, the effects of beta-mercaptoethanol (Beta-ME) and amino acids (AA) on the development of mouse embryos obtained in vivo or in vitro at different stages were investigated. Chemically defined potassium simplex optimized medium (KSOM) was used as basic culture medium and six experimental groups were established and by supplementation of Beta-ME and AA into KSOM media. The quality of blastocysts was evaluated by counting the cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. In addition, embryo transfer (ET) was performed to investigate the rate of implantation and live fetuses. The results obtained in the present study demonstrated that the combined treatment of Beta-ME and AA to 1-cell stage embryos not only enhanced in vitro development to the blastocyst stage but also improved both the number of blastocysts cells and live fetuses.     

The current status and future of commercial embryo transfer in cattle

A commercially viable cattle embryo transfer (ET) industry was established in North America during the early 1970s, approximately 80 years after the first successful embryo transfer was reported in a mammal. Initially, techniques for recovering and transferring cattle embryos were exclusively surgical. However, by the late 1970s, most embryos were recovered and transferred nonsurgically. Successful cryopreservation of embryos was widespread by the early 1980s, followed by the introduction of embryo splitting, in vitro procedures, direct transfer of frozen embryos and sexing of embryos. The wide spread adoption of ethylene glycol as a cryoprotectant has simplified the thaw-transfer procedures for frozen embryos. The number of embryos recovered annually has not grown appreciably over the last 10 years in North America and Europe; however, there has been significant growth of commercial ET in South America. Within North America, ET activity has been relatively constant in Holstein cattle, whereas there has been a large ET increase in the Angus breed and a concomitant ET decrease in some other beef breeds. Although a number of new technologies have been adopted within the ET industry in the last decade, the basic procedure of superovulation of donor cattle has undergone little improvement over the last 20 years. The export-import of frozen cattle embryos has become a well-established industry, governed by specific health regulations. The international movement of embryos is subject to sudden and dramatic disturbances, as exemplified by the 2001 outbreak of foot and mouth disease in Great Britain. It is probable that there will be an increased influence of animal rights issues on the ET industry in the future. Several companies in North America are currently commercially producing cloned cattle. The sexing of bovine semen with the use of flow cytometry is extremely accurate and moderate pregnancy rates in heifers have been achieved in field trials, but sexed semen currently is available in only a few countries and on an extremely limited basis. As of yet, all programs involving the production of transgenic cattle are experimental in nature.     

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.     

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.     

Improving the safety of embryo technologies: possible role of genomic imprinting

Although developments in mammalian in vitro embryo technologies have allowed many new clinical and agricultural achievements, their application has been hindered by limitations in the developmental potential of resulting embryos. Low efficiencies of development to the pre-implantation blastocyst stage have been consistently observed in most species, including humans, rabbits, pigs and ruminants. Furthermore, in cattle and sheep a wide range of congenital abnormalities currently termed "Large Offspring syndrome" (LOS) are commonly observed as a result of several embryo culture and manipulation procedures. This paper reviews the hypothesis that at least some of the problems associated with embryo technologies may result from disruptions in imprinted genes. Several imprinted genes (i.e. genes which express only the maternal or paternal allele) are known to have significant effects on fetal size and survival in other species and are possible candidates for involvement in livestock LOS. Major changes in putative imprinting mechanisms such as DNA methylation of imprinted genes occur in the mouse embryo during pre-implantation development. Alterations in DNA methylation are stabley transmitted through repeated cell cycles such that changes in the embryo may still act at the fetal stages. Thus any disruption in establishment and/or maintenance of imprinting during the vulnerable periods of embryo culture or manipulation is a plausible candidate mechanism for inducing fetal loss and Large Offspring Syndrome. Identification of these disruptions may provide crucial means to improve the success of current procedures.