W4171: Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock
(Multistate Research Project)
At its inception 35 years ago, the primary goal of the W171 Regional Research Project (renewed as project #s W1171, W2171 and W3171) was to establish a cooperative, multistate research group comprised of basic and applied scientists that would uncover the mysteries behind germ cell function and embryo development so that these processes could be manipulated for the improvement of livestock. Since the initiation of this formal research collaboration in 1984, significant advances in techniques, technologies, and basic scientific knowledge have been made to this end. Assisted reproductive technologies (ART) such as artificial insemination (AI), cryopreservation of gametes or preimplantation embryos, superovulation, embryo transfer (ET), in vitro maturation of oocytes (IVM), in vitro fertilization (IVF), in vitro culture (IVC) of embryos, semen sexing and nuclear transfer (NT) continue to be adopted within the livestock production industries . Members of the W3171 Multistate Project have been influential in the improvement and use of these procedures since our last Project revision. However, the efficiency of many of these procedures remains too low for application to commercial agriculture .
Of equal importance, genomic modification of livestock continues to make progress. To date, forward-thinking investigators within and outside this group have produced at least 46 different genetic modifications to domestic livestock animals to enhance production traits . Novel genome editing technologies, such as the zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat (CRISPR) – CRISPR associated nuclease 9 (Cas9) systems, have greatly improved the efficiency of genetic engineering and their application in domestic animals have begun to show considerable promise. Likewise, some of the regulatory and public perception hurdles associated with using livestock produced by genetic engineering or genome editing (GE) as sources for meat and animal products have been cleared. In 2015, a major breakthrough occurred when the U.S. Food and Drug Administration (FDA) approved AquaAdvantage GE salmon for use as food, although commercialization in the U.S. remains stymied . Despite these advances, a significant knowledge gap persists regarding the ability to efficiently produce GE livestock species. These obstacles must be overcome if we are to benefit from the advantages of GE farm animals for human food and fiber production. Herein, we request to continue pursuit of our research priorities and renew the W3171 Regional Research Project (as W4171) with the overall goal of increasing the efficiency of ART in livestock and producing GE animals to improve the efficiency of livestock production systems.
Need as indicated by stakeholders
The Food and Agriculture Organization of the United Nations (FAO) reported that the global population could reach 9.73 billion by 2050 and 11.2 billion by 2100 . Of paramount importance is providing enough food to support the people of the world. Many outlets suggest that food production must double in order to meet the needs of the global population in 2050 , presenting a challenge to agricultural systems. Moreover, urbanization of low- and middle-income countries is expected to escalate dramatically, resulting in higher incomes and increasing the demand for animal products . To accommodate this demand, animal agriculture is tasked to significantly improve the efficiency of livestock production.
Poor reproductive efficiency is a limiting component in all animal production systems, decreasing the profitability and sustainability of livestock producers as well as increasing the cost of animal products to consumers. For example, the U.S. dairy industry alone loses between $424 million and $2.9 billion annually due to poor reproductive performance . In pork production, farms producing the most piglets/sow/year are the most profitable . Given that the vast majority of commercial pork is produced by larger farms (> 1,000 sows), even small improvements in reproductive efficiency can significantly influence profitability . Reproductive efficiency is also vital to profitability of beef cattle operations since the calf is the primary product . Thus, there is a critical need to improve reproductive performance of livestock animals.
The objectives of this Regional Research Project fall under Strategic Goal 2 (Maximize the ability of American agricultural producers to prosper by feeding and clothing the world) of the 2018-2022 Strategic Plan for the U.S. Department of Agriculture (USDA) , which includes a mandate to support a competitive agricultural system (Objective 2.2). Additionally, the aims of this research effort are directly in line with Strategic Goal 1 (Science) of the National Institute of Food and Agriculture (NIFA) 2014-2018 Strategic Plan which promises to catalyze exemplary and relevant research, education and extension programs . Specifically, the work of our group supports Sub-Goal 1.1 (Advance our Nation’s ability to achieve global food security and fight hunger), Sub-Goal 1.2 (Advance the development and delivery of science for agricultural, forest, and range systems adapted to climate variability and to mitigate climate impacts), Sub-Goal 1.3 (Optimize the production of goods and services from working lands while protecting the Nation’s natural resource base and environment), and Sub-Goal 1.7 (Ensure the development of human capital, communities, and a diverse workforce through research, education, extension and engagement programs in food and agricultural sciences to support a sustainable agriculture system). Furthermore, research to increase the practicality of making genetically enhanced livestock animals is directly in line with the Farm Animal Integrated Research (FAIR) 2012 Focus Area 1 (Food Security) . Key topic 1-4 under this area is to improve livestock animal reproductive performance, potentially through assisted reproductive technologies, semen cryopreservation, etc. Key Topic 1-3 (Connecting “-omics” to animal production) is also relevant to the research proposed under this Regional Research Project. The labors of Project members toward these goals and objectives can be classified under the following NIFA Knowledge Areas (KA): KA 301 - Reproductive performance of animals; KA 303 - Genetic improvement of animals; and KA 305 - Animal physiological processes.
Importance of the proposed work
The livestock and dairy industries within the U.S. generated over 176 billion dollars of on-farm receipts in 2017 , and any increases in animal production efficiencies would be extremely impactful within the Western region as well as nationally. Within the states comprising this regional research project , livestock numbers (as of January 1, 2018) included 45.8 million head of beef cattle, 37.8 million swine, 2 million sheep, and 2.9 million dairy cows (that produced 66.3 billion pounds of milk in 2017). Furthermore, the total value of livestock, poultry and their products within these states was $69.1 billion . Therefore, even an incremental 1% increase in the cumulative value of these animals or a corresponding decrease in the production costs would inject an additional $691 million dollars into these local economies.
As described above, reproductive efficiency is a major economic driver of livestock production systems. Assisted reproductive technologies (ART) provide powerful tools to overcome infertility or subfertility in animals [1, 2]. There are many economic advantages associated with adoption of these techniques ; reports suggest an increased return of $25 to $40 per calf produced from AI . In contrast, the swine industry has benefitted from the adoption of AI as well as other types of ART [7, 16]. Farrowing rate improved by 15% and litter size increased by 2 piglets/litter from 2003 to 2012 within the U.S, although sow reproductive efficiency still has room for considerable improvement . The economics of sexed semen use in dairy production has been evaluated  but this technology is limited by reduced viability following cryopreservation and limited access to the technology . Even though superovulation and ET in beef and dairy cows have been extensively utilized for some time, the number of transferable embryos has not changed . The cost effectiveness and dependability of bovine in vitro embryo production (IVP) has resulted in the increased use of IVM, IVF and IVC worldwide, especially within the dairy industry [20, 21]. Each year, in fact, over 500,000 IVP embryos are produced  and the number of IVP embryos utilized for ET is nearing that of in vivo produced embryos . Almost 70% of the IVP embryos are produced in South America, whereas only 20% are produced in North America .
Somatic cell nuclear transfer (SCNT), or cloning, has dramatically advanced animal agriculture, as well as significantly enhanced our ability to produce GE livestock. This technology has three broad applications: 1) applied animal breeding to propagate animals with superior quantitative traits and/or pedigrees; 2) a tool for basic research to study mechanisms of cellular differentiation and epigenetics; and 3) a biotechnological tool to produce GE farm animals more efficiently. Following FDA approval of cloned animals produced by standard NT methods (i.e., no genetic modifications) to be marketed without special labeling (2008), use of this tool in the beef and dairy industries expanded considerably. Even without implementation of GE, producers could significantly improve the average performance of their animals in a single generation, progress that is unmatched in traditional breeding programs . Although the worldwide use of ART has improved substantially since our last Project revision, inefficiencies of these methodologies persist which limits their adoption and use in commercial animal production systems .
During this same timeframe, however, the efficiency of genetically modified animal production has made significant strides. The successful use of genome editing procedures (ZFN, TALEN, CRISPR-Cas9) for the production of genetically modified livestock and large animal biomedical models has exploded. Although commercialization in the U.S. is not finalized, the FDA approved AquaAdvantage GE salmon for use as food in 2015, representing a significant advancement for the field of animal biotechnology . This has spurred others to pursue FDA approval of GE livestock animals. Despite the regulatory and public perception hurdles that still remain, gene editing techniques could immediately impact the livestock industry. The economic significance of GE animals to U.S. animal agriculture in the future is difficult to estimate. What is the value of livestock with improved carcass characteristics, that yield a leaner, more desirable meat, with increased disease resistance, and that are more efficient in growth, reproduction, and wool or milk production? Specific examples of GE animals with application to the livestock industry include: 1) swine that produce omega-3 fatty acids in their meat, enhancing its health benefits ; 2) disease resistant dairy cows (i.e., mastitis) that require less pharmaceutical intervention to produce high quality, safe milk products ; 3) sows that lactate milk containing human lysozyme proteins, improving piglet growth and survival ; 4) chickens that are resistant to avian influenza, thus improving on-farm animal health and providing safer poultry products ; 5) swine that produce phytase in their saliva, reducing emissions in manure that may be hazardous to the environment ; 6) double muscled sheep and cattle, increasing meat yield per carcass ; 7) swine that are resistant to porcine reproductive and respiratory syndrome (PRRS) virus ; and 8) hornless dairy cattle, enhancing animal welfare by eliminating the need for de-horning . It is easy to imagine how these examples could increase efficiencies in the production of animal foodstuffs, economically benefiting both consumers and producers. In addition, more efficient production of food and fiber has obvious advantages to the environment in terms of reduced use of natural resources.
Furthermore, there are tangible and intangible monetary considerations associated with the burgeoning market for large GE animals in biomedical research. Examples of GE livestock with significance to human medicine include pigs modified to aid in transplantation of their organs into humans [32, 33], goats engineered to produce human blood coagulation factors in their milk  and cattle that produce human antibodies [35, 36]. The latter two examples led to a drug that is sold and used clinically (ATryn®; GTC Biotherapeutics) and a platform for human antibody production (DiversitAb™; SAb Biotherapeutics, Inc.). In addition, the National Swine Research and Resource Center (NSRRC) at the University of Missouri recently received funding for another 5 years, representing a 20-year commitment from the National Institutes of Health (NIH). Established in 2003, the NSRRC has produced over 70 different swine strains to be utilized as biomedical models (K.M. Whitworth, personal communication). Finally, the advent of gene editing has led to start-up companies utilizing this platform (Recombinetics, Inc.); subsidiaries of this company are focused on precision breeding for livestock production (Acceligen), development of models for the study of human disease (Surrogen) and production of animal cells, tissues and organs to be transplanted into human patients (Regenevita).
Technical feasibility of the research
Current procedures for the production of GE animals can involve the use of in vitro oocyte maturation (IVM), in vitro fertilization (IVF), in vitro culture (IVC), cell culture and NT (before or after GE). Combined, these technologies are inefficient, so before GE animals can contribute significantly to livestock production systems, construction of these animals will have to be far more efficient. The inefficiencies occur at many levels including in vitro production (IVP) of embryos, nuclear transfer, and establishment of pregnancy. The use of in vitro-derived embryos is much more practical than recovery of in vivo-derived embryos, but IVM, IVF and IVC methodologies remain suboptimal. In the bovine system, blastocyst production by in vitro methods has plateaued at around 40% despite various attempts to improve culture conditions, falling short of the 85 to 95% development rate that occurs in vivo . Similarly, disappointing results can be observed using the swine model, where only 30-40% of IVP embryos cultured in vitro will develop into blastocysts. Carefully controlled studies have shown that development of IVP embryos following transfer into surrogate recipients is substantially poorer compared to in vivo produced embryos [37-40]. Moreover, the quality of IVP embryos, by virtually every embryo quality metric, is much more variable than in vivo produced embryos [41, 42]. Embryo culture media formulations are generally very good at supporting embryo development to the blastocyst stage [43-45]. However, it is also abundantly clear that in vitro manipulations during early development can alter gene expression [46-50] as well as epigenetic control [51-56] in pre-, peri- and post-implantation IVP embryos, that can even persist into postnatal life.
Although we continue to make significant advances in NT technology for livestock and laboratory species [57, 58], much is still to be learned regarding the biology and application of these methods to produce genetically enhanced animals. Cloning by somatic cell nuclear transfer continues to be inefficient, with current success rates averaging 1-10%, depending on species [59-61]. In addition to the inefficiencies associated with the production of genetically enhanced animals, the methodologies are costly, highly time consuming and labor intensive. Up to 10 hours of labor may be required to produce a single cloned bovine embryo for transfer into a recipient female. When this is coupled with a 1-5% pregnancy rate, an estimated 1,000 hours are required to produce a single transgenic offspring. Clearly, this technology remains relatively inefficient at present and needs improvement before it will be widely adopted into mainstream livestock animal production systems.
Members of the W4171 Multistate Research Project are actively pursuing the techniques and the knowledge that will improve the efficiency of producing GE livestock animals. These research pursuits include (but are not limited to) the following areas of concern:
• A basic understanding of the mechanisms of normal gamete and embryo function are necessary before any meaningful diagnosis of faulty embryogenesis is possible.
• Nuances of oocyte and/or donor cell physiology and the responses of these tissues to their respective environments may have profound effects on the success rates of SCNT. In isolated experiments, cloning success rates of 20-40% have been reported . An appreciation for the circumstances surrounding such successes could result in widespread changes to donor cell, oocyte, or embryo culture protocols that might make survival rates of 30% the norm rather than the exception. Understanding epigenetic changes in both the somatic donor cell and the cloned embryos during SCNT is actively being pursued by members of this project and will continue to be a major area of study.
• The inefficiencies associated with production and selection of genetically modified somatic cells for use as karyoplast donors in SCNT also contribute to the lower success rates of cloning . Incremental progress has been made towards improving the efficiencies of this aspect of SCNT with the adaptation of viral delivery systems (i.e., retroviruses, adenoviruses and lentiviruses) for the production of transgenic donor cells and for the direct virus-mediated transformation of embryo cells [62-63]. The advent of genome editing tools, including ZFN, TALEN and CRISPR-Cas9, promises to revolutionize this process even further . It should be noted, however, that these delivery systems only improve the efficiency of gene transfer, but have little impact on the inefficiencies associated with IVM, IVF and IVC and SCNT procedures.
• Placental defects are a key factor in the low embryonic, fetal, and neonatal survival rates after SCNT in all species studied to date. Moreover, alterations in placental physiology due to embryo culture can result in large offspring syndrome and have long-tern consequences to offspring heath. A more thorough understanding of the differentiation and function of trophoblast and other placental cells in normal and abnormal embryonic development is needed before the necessary and appropriate steps of intervention can be undertaken to ensure more successful development.
• Finally, short and long-term storage of GE embryos is necessary for efficient production of live animals but cryopreservation of manipulated embryos needs to be investigated and improved as well .
Thus, in considering these and other knowledge gaps and critical needs within the fields of production agriculture and biomedical modeling, it is evident that consequences of not addressing basic questions of reproductive efficiency – including the production of GE livestock animals – are:
• continued reproductive inefficiencies at all levels and in all segments of animal agriculture;
• the collective losses of millions of dollars in opportunity costs associated with reproductive inefficiency;
• an inability to supply the world’s growing population with high quality animal protein they need and want using ever-less arable land; and
• a compromised ability to appropriately model human health concerns using genetic or other large animal models of human disease.
This renewal proposal will evaluate two critically important areas to the future success of animal biotechnology: 1) understand the biology of gamete development, fertilization, and embryogenesis including the underlying cellular and molecular mechanisms; and 2) refine methods to produce animals by genetic engineering or genome editing for the improvement of livestock production efficiency and development of human biomedical models.
Advantages for doing the work as a multistate effort
Investigation of challenging questions can be achieved very efficiently via a multistate research project of this nature. The combined expertise and resources of member scientists and institutions from both within the Western region as well as stations residing outside of the region can be utilized. Another advantage to the regional research model is that alternative approaches can be examined in multiple laboratories and the effective procedures further tested in the remaining laboratories. Oocyte and embryo procedures appear particularly laboratory dependent; for example, the optimal exposure time for vitrification of mouse oocytes and mouse blastocysts varied significantly among laboratories [66-68]. Examination of epigenetic alterations in NT-derived embryos compared to in vivo-derived embryos, improvements in NT methods and the development of embryonic/somatic cell lines to serve as nuclear donors are other areas that would benefit from this multiple laboratory approach. Isolation of pluripotent stem cells for agricultural species has been challenging and as yet, has not been fully successful. Sharing of information and approaches across this multi-state project is critical in advancing stem cell biology and its application to farm animals.
Collectively, our committee stands poised to expand our knowledge of the biology and underlying mechanisms of gamete development, fertilization and embryogenesis as well as refine methodologies for production of GE animals with the overall goal of improving livestock production efficiency and developing human biomedical research models.
Likely impacts from successfully completing the work
Beneficiaries of this multistate research endeavor include: 1) livestock producers in the Western states, as well as farmers and ranchers across the country; 2) rural communities of the West; 3) consumers of animal products within the Western region, U.S. and the world; and 4) the scientific community worldwide. Livestock producers will benefit from increased profits as a result of reduced input costs linked to efficient production systems, improved performance of animals, and value-added products. The economic stimulus afforded to a rural community that is located near a profitable and sustainable animal industry can be dramatic, providing many opportunities otherwise unavailable to its residents and enhancing the quality of life. Consumers will be impacted by reduced food prices associated with increased efficiency of livestock production, meat, dairy, and/or other food products with enhanced health benefits, an improved environment resulting from livestock systems producing less waste, and the availability of food sources to meet the demands of an ever-increasing population at both the national and international level. Consumers can also benefit from GE livestock that are resistant to diseases, permitting the use of less/no antibiotics in animal feed. Investigators within the scientific community will also benefit from the efforts of the Project members. The use of GE alone or in combination with SCNT is very useful for obtaining a variety of experimental information. Some examples are insight into the cell cycle, nuclear and cytoplasmic programming or reprogramming, genomic imprinting, gene expression, epigenetics and developmental processes. This information can be used in studies to examine basic biological, biomedical, genetic and evolutionary questions, in addition to agriculture applications.