At its inception almost 30 years ago, the primary goal of the W171 Regional Research Project (revised and renewed since as project #s W1171 and W2171) 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. In the three decades since the initiation of this formal research collaboration, significant advances in techniques, technologies, and basic scientific knowledge have been made to this end. Likewise, many of the regulatory and public perception hurdles associated with using genetically modified animals as food and/or fiber sources have been cleared. In fact, forward-thinking investigators (including many active participants in this multi-state project) have produced, to date, at least 23 different genetic modifications to domestic livestock animals to enhance production traits Despite the advances within and outside of this regional research effort, a significant knowledge gap persists regarding the ability to efficiently produce genetic modifications to domesticated livestock species. The efficiencies of these technologies will have to be substantially improved if we are to benefit from the advantages of genetically enhanced farm animals for human food and fiber production. New genome-editing technology is one that will greatly improve the efficiency of genetic engineering but its application in domestic animals has just barely began. Herein, we request to continue pursuit of our research priorities and renew the W-2171 Regional Research Project with the overall goal of producing genetically enhanced animals to improve the efficiency of livestock production systems.

The proposed scope of this Regional Research Project falls under both Strategic Goals 1 and 3 of the Strategic Plan for the entire US Department of Agriculture [1], which include mandates to establish and support a competitive agricultural system (including food animal production; Objective 1.3) and to continue the development of agricultural products derived from biotechnological advances (Objective 3.2). Additionally, and to a more specific point, the aims of this research effort are also directly in line with the vision set forth by the National Institute of Food and Agriculture (NIFA) Strategic Plan (Objective 2.2 of Strategic Goal 2) which promises to provide research, education, and extension to increase the efficiency of agricultural production and marketing systems. The labors of the Project members toward these goals and objectives have been and will continue to be evaluated according to the following performance criteria: 2.2.7 - Increase and improve the reproductive performance of animals (NIFA Knowledge Area 301); 2.2.9 - Develop and apply information and technology for genetic improvement of animals (NIFA Knowledge Area 303); and 2.2.11 - Improve understanding of fundamental animal physiological processes (NIFA Knowledge Area 305). In addition, research to increase the practicality of making genetically enhanced livestock animals is directly in line with the Food Animal Integrated Research (FAIR) 2012 Focus Area 1 [2]. Key topic 1-4 under this goal is to enhance livestock animal reproductive efficiency, including through assisted reproductive technologies. Key Topic 1-3 Goal 5 (Connecting -omics to animal production) is also relevant to the research proposed under this Project.

Beneficiaries of this multistate research endeavor include: 1) individual 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 genetically engineered 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 gene transfer alone or in combination with somatic cell nuclear transfer 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.

The economic significance of genetically enhanced animals to U.S. animal agriculture in the future cannot be estimated with any confidence. However, the livestock and dairy industries within the U.S. generated over 170 billion dollars of on-farm receipts in 2012 [3], 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, 2012) included 28.9 million head of beef cattle, 43.7 million swine, 2 million sheep, and 3.3 million dairy cows (that produced 76.5 billion pounds of milk in 2012); the total value of livestock, poultry and their products within these states was $68.4 billion [4]! 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 $680 million dollars into these local economies! Notably, these numbers do not include the tangible and intangible monetary considerations associated with the burgeoning market for large transgenic animals in biomedical research. Even small increases in the efficiency of transgenic animal production will repay research costs many times over. Production of genetically enhanced animals for the production of food and fiber holds significant promise for consumers, animal producers, and scientists, their respective communities, and our environment as well.

Somatic cell nuclear transfer (SCNT) or cloning, has dramatically advanced animal biotechnology, and significantly enhanced our ability to produce genetically altered livestock. This technology has three broad applications: 1) applied animal breeding to propagate animals with superior quantitative traits and/or pedigrees (prime grade steers, proven dairy bulls, e.g.); 2) a tool for basic research, (i.e., mechanisms of cellular differentiation and dedifferentiation), and 3) a biotechnological tool (more efficient approaches to produce genetically enhanced farm animals). The combination of SCNT technology with genetic modifications of somatic cells has resulted in dramatic advancements in the production of genetically modified animals. This has opened new avenues to produce 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 [5]. Specific examples of genetically enhanced animals with application to the livestock industry include: 1) swine that produce omega-3 fatty acids in their meat, enhancing its health benefits [6]; 2) disease resistant dairy cows (i.e., mastitis, Mad-cow disease) that require less pharmaceutical intervention to produce high quality, safe milk products [7]; 3) sows that lactate milk containing human lysozyme proteins, improving piglet growth and survival [8]; 4) chickens that are resistant to avian influenza, thus improving on-farm animal health and welfare and providing safer and more consumer-friendly poultry products [9]; and 5) males of livestock species that produce mono-sex sperm and therefore, sex-specific progeny [10]. Increased efficiencies in production of animal foodstuffs can be of economic benefit to 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. Consistent with this, investigators have developed swine that produce phytase, an enzyme that breaks down phosphorous, in their saliva, reducing emissions in manure that may be hazardous to the environment [11]. Finally, construction of genetically enhanced livestock animals for use in human biomedicine has also developed considerably [12, 13]. In addition to genetically enhanced animals that serve as models for human biology and disease, the potential for harvest of pharmaceutical proteins, antibodies and tissues/organs from these animals for specific medical applications is certainly impactful [14]. Examples of genetically enhanced livestock with significance to human medicine include pigs modified to aid in transplantation of their organs into humans [15, 16], goats engineered to produce human blood coagulation factors in their milk [17] and cattle that produce human antibodies [18-20].

To offset the costs associated with these technologies, development of a nucleus herd of genetically enhanced livestock for implementation into production systems would need to offer a large potential benefit [21]. However, it is not difficult to imagine a cryo-bank of cloned embryos with genetic enhancements available for direct purchase by producers. Even without implementation of genetic modifications, producers could significantly improve the average performance of their animals in a single generation, progress that is unmatched in traditional breeding programs [21].

The current market for products from genetically modified livestock animals is dampened by regulatory and consumer-driven concerns, yet incremental progress is being made to overcome these hurdles. The sale and clinical use of a drug produced by transgenic animals was first approved in Europe in 2006 [14], and subsequently in the US in 2009 (Reuters). This product (ATryn®; GTC Biotherapeutics) is utilized for the prophylaxis of venous thromboembolism in surgery of patients with congenital anti-thrombin deficiency. Also in 2006, the US Food and Drug Administration (FDA) released a comprehensive 678-page report that concluded that food products from animals cloned using SCNT were indistinguishable from those harvested from non-cloned animals, and in 2008 approved the release of cloned animals produced by standard NT methods (i.e., no gene targeting) to be marketed without special labeling. Members of this regional project such as those in Connecticut and other stations provided pivotal data to this assessment. One experiment station (IL) within the W-2171 Regional Research Project verified the absence of a transgene in control animals demonstrating the lack of transmission after co-habitation and post-mating with transgenic animals, providing a critical first step toward rigorous scientific data for risk assessment of transgenic livestock [22]. These findings have been corroborated by others in different circumstances [23]. As a next logical step, legal and scientific mechanisms have recently been drafted to market a genetically enhanced livestock animal (M.B. Wheeler, personal communication), representing a significant advancement for the field of animal biotechnology. Therefore, translation of science performed by members of the Project to livestock producers is already in progress, marking a major impact of research performed by the W-2171 group.

Current procedures for the production of experimental animals with genetic enhancements can involve the use of in vitro oocyte maturation (IVM), in vitro fertilization (IVF), in vitro culture (IVC), cell culture and NT either before or after gene transfer. Combined, these technologies are inefficient, so before genetically enhanced 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. And 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 in model organisms have shown that development of in vitro produced (IVP) embryos following transfer into surrogate recipients is substantially poorer compared to in vivo produced (IVV) embryos [24-28]. Moreover, the quality of IVP embryos, by virtually every embryo quality metric, is much more variable than IVV embryos [26, 27]. Embryo culture media formulations are generally very good at supporting embryo development to the blastocyst stage [29-31]. However, it is also abundantly clear that in vitro manipulations during early development can alter gene expression [32-36] and the epigenetic control thereof [37-43] in pre-, peri- and post-implantation IVP embryos, and that these alterations can even persist into postnatal life.

Although we continue to make significant advances in NT technology for livestock and laboratory species [44-46], 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 [47-49]. 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 this multi-state research project are actively pursuing the techniques and the knowledge that will improve the efficiency of producing transgenic 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 [47]! 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 of epigenetic changes in both the somatic donor cell and the cloned embryos during SCNT is very actively perused 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 [50, 51]. 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 [52-54]. The advent of a new generation of genome editing tools, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regulatory interspaced short palindromic repeat (CRISPR) /Cas-based RNA-guided DNA endonucleases promises to revolutionize this process even further [55]. 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 genetically altered embryos is necessary for efficient production of transgenic animals but cryopreservation of manipulated embryos needs to be investigated and improved as well [56].
A recent report from the National Institute of Food and Agriculture affirms the following:

“Reproductive success in livestock is essential for the economic livelihood of producers and ultimately affects the consumer cost of meat and other animal products. In many livestock production systems, poor fertility is a major factor that limits productivity. The ability of animals to reproduce efficiently is an integral component of animal agriculture. However, infertility is a problem to some degree in all animal production systems, including aquaculture species. Reproductive failure is one of the most significant factors that limit the productivity of animal production systems and result in millions of dollars in lost profits annually. A major challenge facing many producers is finding practical, cost-effective ways to improve reproductive performance without compromising the production of safe, high quality meat and milk products [57].”

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 transgenic 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 (according to reference [58], the US dairy industry alone loses between $424 million and $2.88 billion to poor reproductive performance annually!);
• an inability to supply the world’s growing population (world population predicted to swell to almost 10 billion by 2050; see reference [ 59]) 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.
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 [60-62]. 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.

This renewal proposal will evaluate two critically important areas to the future success of animal biotechnology: 1) understand the biology and underlying mechanisms of gamete development, fertilization, and embryogenesis; and 2) refine methods for production of genetically enhanced animals to improve livestock production efficiency.