W4171: Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock
(Multistate Research Project)
W4171: Germ Cell and Embryo Development and Manipulation for the Improvement of Livestock
Duration: 10/01/2019 to 09/30/2024
Statement of Issues and Justification
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.
Related, Current and Previous Work
As outlined in the Statement of Issues and Justification, understanding the underlying biological mechanisms associated with gamete development and preservation are key for improved embryo production systems. In the following subsections, we provide a brief review of specific areas related to our Project objectives.
Successful oocyte maturation is dependent on two events, nuclear and cytoplasmic maturation . Within the oocyte, levels of cAMP [70, 71] and a Gs-coupled receptor [72, 73] regulate meiotic arrest. Characteristics of the meiotic spindle can be used to evaluate oocyte quality [74, 75]. More recently, several oocyte-secreted factors have been identified as important for communication between the oocyte and cumulus cells and thus valuable in IVM protocols [76, 77] However, the process of nuclear maturation occurs spontaneously upon removal of the cumulus-oocyte complex (COC) from the follicle and does not represent a significant problem using established culture conditions. In contrast, the process of cytoplasmic maturation appears to be the critical factor that determines the success of subsequent embryo development. Determining the indicators of cytoplasmic maturation in the oocyte would enable more efficient selection of oocytes for IVF and IVP. Currently, there is no defined method of measuring cytoplasmic maturation other than successful fertilization and embryo development [78, 79]. Although much more research is required, several cellular and molecular predictors of oocyte quality show promise [80-82] including inadequate distribution of mitochondria and organization of endoplasmic reticulum [83-85], mitochondrial DNA deletions [86, 87] and reduced glucose-6-phophate dehydrogenase levels .
Successful fertilization in mammals is dependent on a number of factors including meiotic/morphological changes during spermatogenesis, acquisition of epidydymal/seminal plasma proteins, attainment of motility, capacitation, binding of acrosome-intact sperm to the zona pellucida of the oocyte, acrosome reaction, fusion of the acrosome-reacted sperm with the oocyte plasma membrane, cumulus cells and oocyte activation [89-94]. Although the use of IVF to produce farm animal embryos has become routine, subsequent embryonic development in vitro remains far below that of in vivo embryos, suggesting that more research is required to optimize these factors. Despite some progress in identification of spermatozoa proteins playing a critical role in fertilization, more research is required to determine the underlying functional mechanisms associated with these proteins [95-97]. As the oocyte matures, the protein composition of the plasma membrane changes including an increased ability to bind and/or fuse with sperm. Integrins located on the plasma membrane of bovine oocytes have been implicated in both fertilization and oocyte activation .
Early embryogenesis is regulated by developmentally controlled events including proper changes in transcriptional machinery, activation of embryonic genes to support further development, metabolic determinants and oviductal proteins [99-103]. It is important to improve IVC systems in order to produce embryos with high developmental competence for use in agricultural and biomedical research, as well as animal biotechnology [104-107]. Although the methodology for maintaining embryos from livestock in culture has existed for many years, the ability of the present systems to support normal development is limited. Studies have been performed to remove, replace or delay the addition of serum into the culture systems . However, the results indicated that removal of serum from the culture medium does not always prevent the occurrence of large offspring syndrome , suggesting other causes of this severe side effect of IVP. A number of different types of semi-defined and defined media have been designed utilizing a variety of supplements [110-113]. The primary problem associated with current culture systems is that they do not mimic the changing oviductal/uterine environment. 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 embryos [37-40, 114] and the quality of IVP embryos, by every metric, is much more variable than in vivo produced embryos . Embryo culture media formulations are generally very good at supporting embryo development to the blastocyst stage [41-43]. However, it is abundantly clear that in vitro manipulations during early development can alter gene expression [44-48] and the epigenetic control thereof [49-54, 115, 116] in implantation IVP embryos, and that these alterations can even persist into postnatal life. Microfluidics and small mechanical systems were created a means for dynamic culture on a volumetric scale and more consistent with the needs of the embryo . Even with those improvements, lack of biologic culture conditions necessitates further innovation in tissue culture methodology and further research in this area.
At the time of fertilization, two of the most differentiated cells in the body, sperm and oocyte, meet to form a totipotent embryo. Dynamic changes in transcriptome, methylome, and chromatin configuration allow the fusion and precise reprogramming of maternal and paternal genomes . Toward this end, aberrant reprogramming has been clearly linked to failed embryonic development [119-122]. Moreover, inadequate epigenetic reprogramming during early mammalian development may result in LOS and other developmental abnormalities [123-126]. However, the underlying master regulators behind this reprogramming, and how they interact, are not well understood. Moreover, systematic understanding of genomes/epigenomes variations in gametes and embryos and their associated phenome traits remain poorly characterized . These gaps in the knowledge base are important because they are hindering advances in fundamental science of mammalian developmental biology and technology.
With significant advancements in the area of SCNT , many experimental stations have evaluated the procedures and underlying biology associated with SCNT. Although scientists can successfully reprogram somatic cells prior to NT and produce live animals, many questions remain regarding the inefficiency of the process, the molecular and cellular effects reprogramming has on somatic cells subsequently used as karyoplasts, and the impact of SCNT on the health of resultant clones. In NT, the somatic nucleus has to be reprogrammed in order to restart and continue the developmental process. This requires that tissue-specific genes be inactivated and embryo-specific genes necessary for normal embryo development be re-activated within the donor nucleus [122, 129]. The factors affecting the efficiency of NT are: type of recipient oocyte, type of donor cell, treatment of the donor cells prior to fusion, enucleation of the recipient oocyte, fusion of the transplanted nucleus to the enucleated oocyte cytosol, activation of the oocyte, treatment of the NT embryos following fusion and activation, and "reprogramming" of the transferred nucleus .
Surprisingly, there is evidence of a significant degree of reprogramming at the gene expression level in cloned preimplantation embryos compared to their IVF counterparts [122, 131-133]. However, evidence seems to suggest that most of the incomplete reprogramming is related to epigenetics  an area of research that has moved to the forefront of science. The general consensus of those in the field is that nuclear transfer efficiency is much too low (< 15%) to be economically viable except in very limited applications. Any increase in efficiency will greatly enhance the value and practical application of this technology, as well as contribute to our understanding of changes that must occur in chromosomes to allow appropriate embryonic gene expression patterns. It is also critical to understand how potentially subtle modifications in the nuclear structure, as well as nuclear-cytoplasmic interactions, impact the ability of a cell to contribute to production of offspring. Despite the challenges, nuclear transfer offers unique opportunities in agriculture, endangered species conservation, biomedicine, and basic research [134-136].
In vivo produced embryo cryopreservation has been quite successful [137-139]. Alterations in IVM and IVC can affect the susceptibility of in vitro-produced embryos to cryo-damage. With increased utilization of embryo manipulation procedures, it is critical that we gain a better understanding of the impact these technologies have on successful cryopreservation and develop strategies to prevent the occurrence of detrimental alterations to embryos exposed to manipulation . Reduction of the cytoplasmic lipid content of bovine embryos with phenazine ethosulfate (PES) improved cyrotolerance . Furthermore, advances in vitrification have allowed for successful cryopreservation of goat , porcine , equine , ovine  and bovine embryos . Successful vitrification of porcine , equine and bovine oocytes  has been achieved.
Several different systems have been developed for the vitrification of oocytes and embryos. These systems consist of three basic parameters 1) the cryoprotectant molecule or combination of molecules utilized 2) the rate of cooling and subsequent warming and 3) the apparatus or tool used to hold the oocytes or embryos during the process. These three parameters are interrelated as the apparatus or tool used to hold the oocytes/embryos effects the cooling and warming rate possible and the cryoprotectant(s) used effect the desired cooling and warming rates. These three parameters have been investigated and numerous discussions have ensured concerning the relative merits of the different systems utilized [147-149]. Virtually all of these investigations have been centered on the survival of oocytes and /or embryos. There is evidence from somatic cells, embryonic cells and oocytes that the different cryoprotectant molecules utilized and the vitrification process itself can affect cellular function in ways far more subtle than just survival. These changes in cellular function include mitochondrial function, metabolic function and epigenetic modifications effecting expression of [119, 150, 151]. It has been observed that damage to the meiotic spindle is repaired more quickly following vitrification than with slow rate freezing and that mitochondria function can be partially repaired with culture following warming after vitrification [152, 153]. Numerous combinations of cryoprotectants and vitrification devices have been utilized but two prevalent systems have been ethylene glycol and DMSO as cryoprotectants  and ethylene glycol and glycerol  utilizing the cryotop vitrification device to achieve a rapid cooling and warming rate. Both systems have resulted in viable oocytes after warming which have been fertilized and resulted in live births. While the combination of ethylene glycol and DMSO is the most commonly used cryoprotectant solution for human and mouse oocytes , it has been reported that the combination of ethylene glycol and glycerol was more effective for vitrification of sheep  and bovine  embryos. This is consistent with the established use of glycerol as the preferred cryoprotectant in cryopreservation of embryos by slow rate freezing in these species.
Stem Cell Biology
Despite the historic success with isolation and subsequent gene targeting of mouse ES cells, isolation of pluripotent stem cells for agricultural species has been extremely challenging . This lack of success has been offset by dramatic improvements in SCNT, potentially bypassing the need for ES cells. However, as described in the Nuclear Transfer subsection, many difficulties associated with SCNT remain to be solved, especially the inefficiency of the process. Another concern with SCNT is the ability to maintain somatic cells in culture long enough for successful gene targeting and subsequent selection . Multiple laboratories have characterized ES-like or EG (derived from primordial germ) cells for livestock animals [23, 161-163], however, germline chimerism has not been established . More recently, investigators have focused on the isolation of stem cells from a multitude of tissue sources such as adipose, bone marrow, and testis [103, 165, 166]. Induced pluripotent stem cells (iPSC) in which somatic cells are reprogrammed using pluripotency-associated transcription factors may provide an attractive alternate approach, as demonstrated in pigs [167, 168]. The identification of very small embryonic-like stem cells (VSEL) as an alternative source of stem cells for reprogramming has led to their use in restoring fertility in human and mouse studies as well as the in vitro production of gametes . In addition, the potential of ES and iPS cells to differentiate into germ cells represents an important germ cell source to further our understanding of the mechanisms of gametogenesis and for production of genetically enhanced animals [167, 170, 171]. Advances in spermatogonial stem cell (SSC) isolation and transplantation  have made the applications of this technology in the production of gene edited animals more feasible . The challenge of isolating ES cells from large animal models has hindered animal genetics, however, with the efficiency of gene editing technologies the persistent expression of reprogramming genes can be overcome  and the combinatorial use of both iPSC and gene editing has promise in the production of transgenic animals [175, 176].
Accomplishments of the Previous Project
- The roles of OCT4 and KDM6B during bovine early embryogenesis were determined.
- Generated a list of genes suitable to serve as quantitative references during embryonic development.
- Systematically compared dosage compensation of the X chromosome in ovine fetuses developed under different nutrition statuses.
- Developed a bioinformatics tool for cross-stage mining of genes that are co-regulated and conserved among different species. This tool is available for any scientist to use without charge.
- RNA sequencing data comparing oocytes collected by ovum pick up (OPU) or from slaughterhouse ovaries identified differentially expressed genes in both the germinal vesicle and metaphase II stages.
- Determined that it is possible predict the future development of in vitro produced bovine embryos after only 2 days of culture using 1H-nuclear magnetic resonance (NMR).
- Evaluation of embryo metabolism by gas chromatogrpahy-mass spectrometry (GC-MS) demonstrated that this approach is capable of measuring substrate use and metabolic pathway fluxes in as few as 4 embryos and that substrate balance in the media has a profound influence on pathway activities (e.g., glycolysis).
- CRISPR/Cas 9 gene editing was utilized to knockout pig conceptus IL1B2 expression and the secretion of IL1B2 during the time of conceptus elongation.
- Developed a new electric channel device method to select high motility sperm without harming sperm characteristics.
- Analysis of hormone levels in the blood of GnRH-II receptor knockdown and littermate control boars during pubertal development indicated that testosterone secretion was impaired in GnRHR-II KD males, however, LH levels were similar between swine lines.
- Initial characterization of global epigenetic marks (histone modifications) in oocytes derived from small vs. large (pre-ovulatory) follicles revealed that there may be no difference in H3K4me3, but that H3K9me3, H3K27me3, and DNA methylation levels may be different between oocyte from the different sized follicles.
- Methods for efficient delivery of solutions into livestock oocytes and zygotes as well as robust derivation of bovine embryonic stem cells were developed.
- Established a reprogramming assay to study mechanisms/factors for naïve-state pluripotent iPSC generation.
- Determined that novel collagen-glycosaminoglycan hydrogel (CG) scaffolds support bone and cartilage growth from mesenchymal stem cells.
- Ascertained that zinc has a positive effect on bone formation of adipose stem cells in vitro.
- Targeting and genome editing constructs for KDM1A were designed, tested in vitro, and used to study their function within the placenta in vivo.
- Demonstrated that site directed insertion of a large transgene construct can be efficiently attained by using CRISPR/Cas9.
- Determined the roles of Akt3 in embryonic stem cell survival and proliferation.
- Developed and optimized smaller-sized variants of the Dracula micromanipulation system for use on embryos of other species.
- Modification of the CFTR gene in sheep cells has been remarkably efficient. Very high pregnancy rates resulted from embryo transfers of SCNT embryos derived from CFTR-modified karyoplast donor cells.
- Using the CRISPR/Cas9 gene editing system, we disrupted up to three genes simultaneously at near 100% efficiency in vitro.
- Exposure of immature and mature bovine oocytes to DMSO- and glycerol-based vitrification solutions, as well as vitrification with these solutions, resulted in minor or no change in epigenetic marks such as DNA methylation and histone acetylation.
Related Regional Research Projects
A search of NIMSS projects revealed a number of multistate research projects with the goal of improving reproductive efficiency in livestock. These projects include: NC1201 – Methods to Increase Reproductive Efficiency in Cattle; NCERA_temp57 – Swine Reproductive Physiology; W3112 – Reproductive Performance in Domestic Ruminants; NE1727 – Influence of Ovary, Uterus, and Embryo on Pregnancy Success in Ruminants; S1064 – Genetic Improvement of Adaptation and Reproduction to Enhance Sustainability of Cow-Calf Production in the Southern United States; S1081 – Nutritional Systems for Swine to Increase Reproductive Efficiency; SCC81 – Sustainable Small Ruminant Production in the Southeastern U.S.; and W3112 – Reproductive Performance in Domestic Ruminants. Search results suggested that only the W1727 multistate project had some similarities. Unlike the NE1727 group, however, the W4171 group is primarily focused on gamete biology, early embryonic development and technologies for the production of genetically enhanced (transgenic) and gene-edited livestock animals. Thus, our proposed research studies do not duplicate the efforts of any other multistate research projects.
Understand the biology of gamete development, fertilization and embryogenesis including the underlying cellular and molecular mechanisms.
Refine methods to produce animals by genetic engineering or genome editing for the improvement of livestock production efficiency and development of human biomedical models.
Objective 1: Understand the biology of gamete development, fertilization and embryogenesis including the underlying cellular and molecular mechanisms.
The overall aim of this research area is to gain a better understanding of the biological requirements for successful gamete maturation, fertilization, and subsequent embryonic development. The production of live offspring is dependent upon all of those events occurring in a well-orchestrated fashion. More importantly, perhaps, is the fact that a better understanding of the mechanisms underlying these biological processes (i.e., epigenetics) will lead to the production of more healthy, viable offspring. This, in turn, will improve many of the in vitro systems even further, advancing them yet closer to conditions present in vivo. A number of member experiment stations will be investigating this objective including: AR, CA, CO, CT, IA, IL, LA, MD, MO, MT, NE, SC, UT, VA, WA.
Oocyte Maturation and Developmental Competence. Traditionally, this area of investigation has been emphasized and will continue as a strong collaborative subgroup of this multistate research project (CA, CT, IL, LA, NE, SC, UT). The primary goal of this subgroup will be to investigate factors necessary for oocyte development. The efficiency of IVM systems is still sub-optimal. A better understanding of the molecular and cellular mechanisms underlying oocyte maturation will help increase the efficiency of IVM procedures. One focus area of the group will be regulation of nuclear and cytoplasmic maturation. Although questions regarding the molecular mechanisms underlying nuclear maturation remain, nuclear maturation does not present a significant problem with current IVM culture systems. However, there is no defined method of measuring cytoplasmic maturation other than successful fertilization and embryo development . This group will evaluate changes that occur during cytoplasmic maturation using standard IVM protocols for respective livestock species in an attempt to isolate specific predictive indicators of mature oocyte cytoplasm. Similarly, a better understanding of the factors and mechanisms underlying oocyte maturation will lead to the isolation of biomarkers for the ability of the oocyte to fertilize and develop as an embryo, or developmental competence. Researchers will identify markers for developmental competence of oocytes in livestock species by: 1) comparing IVM- vs. in vivo-derived oocytes; 2) evaluating the micro-environment of maturing oocytes (i.e., follicular fluid, granulosa cells); 3) examination of oocytes from females at different stages of reproductive maturity (i.e., pubertal vs. pre-pubertal); 4) comparing oocytes exposed to hormones/growth factors/supplements known to enhance development (i.e., FSH, AMH) either in vivo or in vitro with controls; and 5) utilizing genetic strains of animals (i.e., GnRH-II receptor knockdown vs. littermate controls) that may be divergent for quality and/or rates of oocyte/embryo development. The search for markers of developmental competence will utilize functional genomic (RNA-Seq, ddPCR), proteomic (immunofluorescence, immunocytochemistry, stains/dyes for cellular organelles and viability, nanoparticles, immunoblotting, mass spectrometry, real-time bioimaging), metabolomics, and other molecular approaches. Non-invasive approaches, such as examining the relationship between follicular dynamics and oocyte maturation, will also be employed. Metabolic needs of oocytes will be evaluated with proton nuclear magnetic resonance (1H-NMR) and Gradient Light Interference Microscopy (GLIM) during oocyte maturation and gamete co-incubation.
Sperm Physiology. This workgroup (AR, CA, IA, IL, LA, NE, SC) will evaluate the biology associated with sperm development including meiotic and morphological changes. Methods to increase production of fertile sperm are critical to improving the efficiency of IVF procedures and subsequent embryonic development. Males from multiple livestock species will be evaluated for fertility measures using computer assisted semen analysis (CASA), standard staining protocols, immunocytochemistry, immunoblotting, nanoparticles, hormone assays and advanced microscopic analyses. Similar to the Oocyte Maturation and Developmental Competence group, this subgroup will utilize functional genomic approaches to identify markers of fertility in bulls/boars and implement these into functional genetic tests to predict fertility/subfertility/infertility in males. Genetic markers will be correlated to measures of fertility and subsequent embryo development from the same animals. The IA station plans to separate sperm from the highly viscous seminal plasma in ejaculates from the llama and alpaca using enzymatic digestion of proteins in the seminal plasma and centrifugation in various density gradient solutions. The NE station has localized the GnRH-II receptor to the connecting piece of boar sperm and detected GnRH-II within porcine seminal plasma. They will further explore this relationship by examining the effects of GnRH analogues on sperm characteristics of extended semen. In addition, they will determine GnRH-II concentrations in seminal plasma of boars with high or low fertility and correlate GnRH-II levels in seminal plasma with sperm characteristics. The NE and IL stations will work together to examine differences in sperm physiology of GnRHR-II knockdown and littermate control males using label-free, phase-sensitive imaging to evaluate spermatozoon viability. They will employ spatial light interference microscopy (SLIM) to perform high-accuracy single-cell phase imaging and decouple the average thickness and refractive index information for the sperm population.
Fertilization Mechanisms. The cellular and molecular aspects of fertilization and oocyte activation will be explored by this group (AR, CA, IA, IL, NE, UT, VA). One mechanism identified by this group for further investigation is the regulation of polyspermy. Although its importance may vary between species, polyspermy remains a significant difficulty in successful porcine IVF procedures. Researchers will try to isolate sperm/oocyte factors as well as conditions that may contribute to polyspermy. Similarly, the group is also interested in examining basic mechanisms of fertilization in domestic livestock species. Despite some progress in identification of spermatozoa proteins playing a critical role in fertilization, more research is required to determine the underlying functional mechanisms associated with these proteins . Collaborative efforts of the group will continue to isolate fertilization-specific proteins using proteomic approaches. As the oocyte matures, the plasma membrane increasingly gains the ability to bind and/or fuse with sperm. Integrins located on the plasma membrane of bovine oocytes have been implicated in both fertilization and oocyte activation . In addition, the CA station has identified gamete membrane proteins that may be directly involved in sperm oocyte interaction in the pig. They will use affinity chromatography of partially purified gamete plasma membrane proteins and mass spectrometry to expand and characterize the potential receptors/ligands. The IL station will investigate the three methods of sperm selection for bovine in vitro fertilization, using SLIM to evaluate the morphology and CASA to evaluate motility. The VA station will conduct porcine IVF experiments to increase efficiency of fertilization and culture in vitro.
Early Embryonic Development. Another large collaborative workgroup (AR, CA, CO, CT, IL, MD, MO, MT, NE, SC, UT, WA) of scientists will investigate factors underlying the early stages of embryonic development. One major area of collaboration will be to optimize methods for performing RNA-seq analyses on embryos at early stages of development as well as analysis of single embryos from mice, cattle and pigs. Stations will collect embryos from specific experimental designs to isolate factors vital to these developmental pathways and utilize the expertise of the CT and CA stations for RNA-seq analyses. Shortly after fertilization, mammalian embryos undergo genome-wide epigenetic reprogramming by demethylation, followed later by de novo remethylation . Of primary interest, the effects of epigenetics at this extremely important stage of development will be targeted. The LA and CT stations are collaborating to study the epigenetic regulation of gametes and embryos. They have obtained genome-wide dynamics of DNA methylation during bovine early embryonic development that will serve as an important reference base for embryos produced by ART. The WA station will conduct studies on epigenetic modifications regulating early embryonic development, focusing on skeletal muscle and adipose tissue development. The LA station aims to use a combination of approaches including the unique bovine embryo model, throughput sequencing, bioinformatics analysis, mathematical modeling and state-of-the-art gene editing technologies to identify molecular drivers controlling the viability of embryos. The MD station will use metabolomics and fluxomics to investigate the impact of energy sources on early embryonic development. The IL station will investigate non-invasive nuclear magnetic resonance and GLIM analysis of embryo metabolites during in vitro embryo culture. The MT station will integrate embryo manipulation technologies with their previous work on redox-regulation of early embryonic patterning to advance our understanding of critical regulatory mechanisms in early embryonic development.
Additional studies will pursue peri- and post-implantation development, including genetic regulation of conceptus and placental development. The MO station will investigate key factors regulating early pig conceptus development and survival. Identification of factors influencing this critical step in early preimplantation development will assist the multistate project to improve IVC systems, enhancing the competence of embryos. The CO station has ongoing studies focusing on the molecular and hormonal regulation of placental development and differentiation that is absolutely critical for establishment of pregnancy and embryo survival. This is important as impaired placental development can lead to conditions such as intrauterine growth restriction, which in turn have long-term consequences postnatally.
Objective 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.
The primary aim of this research objective is to enhance the success of each step in the process required for the successful production of both livestock animals and large animal biomedical models by genetic engineering or genome editing (GE). Continual advances in gene transfer techniques and somatic cell nuclear transfer (SCNT) methodology have led to a crucial need for more research that will lead to increased incorporation of these methods into livestock production systems. Research under Objective 2 will be conducted by multiple research institutions comprising the Project: AR, CA, CO, CT, IA, IL, LA, MD, MO, MT, NE, SC, UT, VA, WA.
Genome Editing. This workgroup (CA, CO, CT, IL, LA, MD, MO, NE, UT, VA) will utilize novel genome editing tools (ZFN, TALEN, CRISPR-Cas9) that have resulted in a whole new area of genetic manipulation with different potentials and possibilities. Precise genome editing is based on the ability of engineered nucleases to cut a targeted position in the genome, then a double-stranded break stimulates either homologous recombination or non-homologous end-joining mutagenic repair, which could introduce a targeted mutation into a specific genomic location [177, 178]. This approach provides a powerful tool to generate gene “knock-out” and “knock-in” livestock models. Genome-editing combined with SCNT (or cloning) is the most common approach. However, the efficiency of these systems is high enough to induce mutations during embryogenesis, by-passing the need for SCNT in generating GE livestock. Although the CRISPR/Cas9 system is advantageous and inexpensive compared to the traditional gene targeting approach, a major concern is its off-targeting potential, inducing unintended genome alterations. The specificity of guide RNA that is 20 nucleotides long is not 100%, which may lead to a non-specific mutation elsewhere. The VA station has analyzed potential off-targeting activity in GE pigs produced through genome editing. They determined no off-targeting activity when the CRISPR sequence was designed at high specificity. Moreover, the use of CRISPR/Cas9 system is safe in producing GE pigs. Genome editing approaches are or will be utilized by numerous stations to: 1) introduce the Callipyge (CLPG) mutation  into the goat genome, resulting in a 30-40 % increase in protein production; 2) uncover the role of stem cell factor LIN28, fatty acid transporter FATP4, and the histone lysine demethylase KDM1A in the ruminant placenta; 3) correct DNA sequences that confer genetic diseases, such as the NHLRC22 locus in Angus cattle; 4) modify genes on the X- and Y-chromosomes; and 5) understand the roles of IL1B2 and aromatase gene expression during conceptus development and the establishment of pregnancy in the pig. In addition, the CA station is optimizing transfection delivery of CRISPR-Cas9 for generating single step “knock-out” and “knock-in” livestock.
Nuclear Transfer. This subgroup of the Project (CT, IL, LA, MD, UT) will focus on the procedures and underlying biology associated with significant advancements in the area of SCNT. Although scientists can successfully reprogram somatic cells prior to NT and produce live animals, there is a gap in knowledge regarding the inefficiency of the SCNT process (<15%), especially with respect to GE. The IL station will study CRISPR/Cas9 combined with NHEJ attenuation by RNAi enhanced introgression of transgenes into the ROSA26 locus in fibroblast cells for nuclear transfer. Our findings demonstrate that site directed insertion of a large transgene construct can be efficiently attained by using CRISPR/Cas9 along with the suppression of the NHEJ-dedicated Ku70 protein. Aberrant patterns of DNA methylation, one of the major epigenetic modifications of the genome, have been proposed as a contributing factor in the poor development of embryos following SCNT. Another concern with SCNT is the ability to maintain somatic cells in culture long enough for successful gene targeting and subsequent selection . This group will evaluate methods to enhance the number of cloned embryos that develop to term. Finally, many developmental abnormalities are associated with SCNT procedures and systems for optimal survival post-transfer need further study. This group will attempt to identify why gene expression patterns and morphology are abnormal in clones. For instance, the LA station will examine gene expression and epigenetics in NT embryos produced from fibroblasts and iPSCs in order to identify genetic drivers of embryo development. The UT group is working on identifying factors responsible for LOS phenotype after SCNT in cattle and sheep by assessing gene expression and methylation status of imprinted genes.
Gamete and Embryo Cryopreservation. This subgroup of the Project (AR, CA, CO, IA, LA, MT, SC) will develop methodologies for improvements in cryopreservation of sperm, oocytes and embryos, as well as gain a better understanding of the molecular and cellular processes influenced by freezing and thawing procedures. Developing methods for cryopreservation of oocytes has presented many new challenges. Successful vitrification of porcine , equine and bovine oocytes  has been achieved, although the success of these cryopreservation technologies remains low. The group will try to enhance the efficiency of oocyte cryopreservation. Although the metaphase II spindle can be preserved during the slow freezing process, it is gradually disassembled during thawing of oocytes. Efforts will be concentrated on identifying cryoprotectants/proteins and cellular components that prevent disassembly of the metaphase II spindle during thawing. The LA station will study the effect of oocyte vitrification with different vitrification solutions on epigenetic marks and gene expression in embryos resulting from those oocytes. These studies will also include the effect of different oocyte cryopreservation methods on mitochondria number and function.
Further, this group will analyze differences in mammalian sperm cryopreservation. Male to male variation in the ability of sperm to survive the freezing and thawing process is significant and more investigation is required to identify the factors contributing to these differences in fertility. These researchers will cryopreserve sperm from males of different species/breeds. The IA station plans to investigate methods for the cryopreservation of spermatozoa from various livestock species including South American camelids, pigs, sheep, goats, and horses. The efficacy of unloaded and cholesterol-loaded ß-cyclodextrin will be examined, as will be the efficacy of various permeating cryoprotective agents such as ethylene glycol, propylene glycol, and 1,4-butanediol.
Finally, the group will continue to advance their knowledge base in regard to cryopreservation of embryos. Efficient production of genetically enhanced animals depends on successful protocols for short and long-term storage of manipulated embryos . Consistent with this, cryopreservation of manipulated and/or IVF embryos need to be improved. The group will examine the effects of various manipulation methods (IVF, IVC, microinjection, assisted hatching) on the viability of embryos following freezing and thawing compared to standard in vivo-derived embryos. The CA station is investigating whether inhibiting fatty acid transport into the bovine oocyte during IVM improves cryosurvival of resulting IVP embryos. Novel tools for embryo manipulation and assisted cryopreservation are being developed by the group (MT). These are allowing successful cryopreservation of embryo “types” (i.e., either of different species or of different stages) that were previously refractory to this. These technologies will be further advanced here. Culture conditions will be critically examined, alternative cryoprotectants will be analyzed, molecular and cellular mechanisms will be compared and new protocols identified to make manipulated embryos more similar to in vivo-derived controls. The IA station also plans to investigate methods for cryopreservation of oocytes and embryos from South American camelids, pigs, sheep, goats, and horses. The efficacy of pre-cryopreservation blastocoele cavity volume reduction will be investigated in conjunction with conventional slow-cooling equilibrium methods of embryo freezing as well as ultra-rapid non-equilibrium methods (vitrification).
Stem Cells. This group of investigators (CA, CO, CT, IL, LA, MD, NE, UT, WA) will study biological mechanisms and procedures involved in isolation of both ES cells as well as tissue-specific stem cells. Our understanding of cellular differentiation has greatly expanded, largely due to the dramatic advances in SCNT methodologies. However, one major limitation to the livestock industry is the absence of ES cells, largely due to inappropriate culture conditions to maintain these undifferentiated cells appropriately. Isolation of ES cells in livestock species will continue to be of utmost importance to this scientific group. The group will focus on development of new, unique methods that might lead to an improvement in isolation of these cells in livestock animals as well as understanding the unique pathways of pluripotency maintenance that may be operating in livestock species. The WA Station will define the long-term impacts of epigenetic events that occur in ES cells on the properties of their derived adult stem cells. The LA station will optimize culture conditions for bovine ES cells and induce pluripotency in bovine fibroblasts through activation of endogenous pluripotent genes. As an alternative to ES cells, much research expertise has been dedicated to the isolation of tissue-specific stem cells. This group has already isolated neural, amniotic, mesenchymal, and adipose-derived stem cells as well as pursued iPSCs for livestock species. The CT station is performing RNA-seq analysis on the induction process between human and bovine iPSCs to identify key genes and cellular events/signals that are important for the generation of pluripotent bovine iPSCs. In addition, member stations will collaborate on studies with mesenchymal and adipose-derived stem cells, focusing on epigenetic modification and trans-differentiation of these cells. The IL station is beginning studies with porcine and bovine iPSCs for both tissue engineering and genetic engineering applications. The CA station has developed a novel differentiation culture system that is capable of producing large numbers of germ-like cells that undergo advanced stages of development from ES cells. The group envisions major impacts of this system on the successful outcomes of NT as well as providing a source for renewal of oocytes.
It is true that much, or perhaps even most, of the research toward these goals is performed at separate stations with distinct (not shared) endpoints, outcomes, and impacts desired by that individual station. Yet, the sponsored multistate project format allows for substantial, regular interactions with potential collaborators – interactions that would not likely come about without the multi-state project. In the case of the W4171 group, these interactions have led to some very specific collaborative efforts that are a direct result of the multistate research project. Just a few selected collaborations are mentioned in detail below:
- Ken Bondioli (LA), Irina Polaeva (UT) and Kiho Lee (VA) are preparing a review paper focused on genome editing in livestock.
- Zongliang Jiang, Ken Bondioli (LA), Young Tang and Cindy Tian (CT) are collaborating to study the epigenetic regulation of gametes and embryos. They have obtained genome-wide dynamics of DNA methylation during bovine embryonic development which will serve as an important reference base for embryos produced by ART.
- Brett White (NE) and Matt Wheeler (IL) are examining differences in sperm physiology of GnRH-II receptor knockdown and littermate control males using advanced microscopic techniques available at the IL station.
- Carol Keefer (MD) has supplied plasmid DNA expression constructs essential to the production and/or characterization of induced pluripotent stem cells to Cindy Tian (CT).
- Brett White (NE) and Trish Berger (CA) continue their collaboration to decipher the endocrine basis (if any) of the disruption of male fertility in swine that are deficient in GnRH-II receptor.
- Clay Isom (UT) and Ken Bondioli (LA) started a collaborative venture to utilize a technique for single-cell gene expression analysis to test the expression level of genes involved in oocyte quality.
Measurement of Progress and Results
- Peer-reviewed scientific publications reporting novel contributions to the fields of gamete and embryo biology and generation of GE animals.
- Generate new scientific knowledge about the basic molecular, biochemical and cellular mechanisms of gamete and preimplantation embryo biology.
- New approaches for in vitro oocyte maturation, fertilization and culture of preimplantation embryos.
- Improved understanding on the process of nuclear reprogramming during somatic cell nuclear transfer and normal developmental processes.
- New methodologies for genetic modifications of livestock species, with emphasis on gene editing and directed modifications.
- Improved technologies for gamete and embryo cryopreservation.
- Isolation and characterization of pluripotent stem cells from domesticated animal species.
- Graduate and postdoctoral students trained on areas related to gamete and embryo biology and generation of GE animals.
- Further the understanding of epigenetic reprogramming during early development, after somatic cell nuclear transfer, and during induction of pluripotency by defined factors.
- Provide guidance for implementation of assisted reproductive technologies to veterinarians, practitioners and commercial operations.
Outcomes or Projected Impacts
- The ability to isolate and culture bovine oogonial stem cells will allow the study of oocyte development in vitro and potentially increase the yield of oocytes for in vitro embryo production.
- Unraveling the metabolic basis of normal early embryo development will provide significant benefits to human and animal reproductive health.
- Improved embryonic competency following in vitro production and cryopreservation would stimulate the industry by lowering costs, especially those related to embryo transfer recipient management.
- Novel factors may be determined to improve semen extenders in swine, extending the lifespan of sperm and decreasing the cost of semen doses.
- Methods developed on X chromosome dosage compensation in the sheep will reveal a new area of study in this important agricultural species.
- Endocrine and/or genetic markers to predict fertility would be of great economic benefit to livestock producers, allowing for more timely management decisions.
- New knowledge regarding the epigenomics of oocytes will help inform scientists and practitioners about best practices for selecting oocytes for in vitro maturation and embryo production.
- Identification of subfertile males at a younger age would allow producers to focus resources on reproductively superior animals and market subfertile males prior to sexual maturity, significantly increasing their value.
- A better understanding of how the conditions oocytes are exposed to before collection influence cytoplasmic maturation will improve the developmental potential of in vitro matured oocytes.
- Improved identification of fertile vs. subfertile males would contribute more doses of semen sold per ejaculate, reducing the cost of production.
- Optimized methods for preparing sperm cells from sex-sorted semen will allow more efficient production of sex-specific embryos.
- The availability of embryonic stem cells in livestock species will enable the introduction of complex genome modifications.
- Understanding how different components of vitrification affect efficiency will enhance its utility in production of genetically improved livestock.
- Utilization of the CRISPR/Cas 9 gene editing system provides a powerful tool to evaluate the role of genes during early conceptus development.
- Further evidence of the safety and effectiveness of genome-editing technologies will strengthen the case for using such technologies in livestock production.
- Understanding the genetic regulation of placental development may result in methods to improve the efficiency of somatic cell nuclear transfer.
- Understanding how oocyte and embryo cryopreservation methods induce epigenetic changes will enhance its utility in the production of genetically improved livestock.
Milestones(2021):Submission of a collaborative research proposal by W4171 committee members seeking funding from a federal agency or commodity board.
(2022):In order to to facilitate dissemination of experimental results to the research community and ART practitioners, the W4171 committee will provide a pre- or post-conference symposium at the annual meeting of the International Embryo Technology Society (IETS). W4171 committee members will deliver scientific presentations related to advances in gamete/embryo technologies. W4171 members (also members of IETS) will initiate discussion with IETS planning committee to organize this outreach activity. In years that the IETS meeting convenes internationally, the annual meeting of the W4171 Multistate Regional Research Project meets at a domestic location. As an alternative, the symposium could be included as part of this meeting.
(2023):Publication of review articles related to each of the two objectives of the W4171 Regional Research Project.
(2024):Stations of the W4171 committee will have produced multiple examples of GE animals to improve livestock production efficiency or serve as models for biomedical applications.
Projected ParticipationView Appendix E: Participation
Project members of W4171 represent a broad geographical region with a varied and diversified agricultural base for each station. Regardless of environmental and production settings, understanding gamete and developmental biology of livestock will be a key factor in improving livestock efficiency. To that end, members are dedicated to communicating our discoveries and research findings to a host of constituency and peer groups.
Students. Mentoring future scientists is one of our primary outreach methods. Undergraduate, graduate, and post-doctoral students conduct research in our laboratories and farms. During their training they practice scientific method, and hone their higher learning skills by analyzing, interpreting, writing, and presenting research findings. As an example of our commitment to student education, members of W4171 have led the education committee for International Embryo Transfer Society for more than two decades. In 2013, our members led IETS to develop an undergraduate student research competition at their 2014 international meeting. Over the years, many of the graduate student award winners at IETS presented projects that were part of W171, W1171, W2171 and W3171, the earlier versions of W4171.
Public and Livestock Producers. Members of our project frequently present research findings and application to extension/teacher in-service education and training venues, and at livestock producer field days and symposia. Many of those events require either or both verbal and written interpretations of our research findings. For example, one of the primary outreach efforts conducted by the Iowa station involves publication of articles in the annual Iowa State University Animal Industry Report (available online at http://www.ans.iastate.edu/report/air/). This report is geared toward livestock producers, extension specialists, and members of the general public. The Iowa station has also published articles in various encyclopedias such as Encyclopedia of Biotechnology in Agriculture and Food and Encyclopedia of Animal Science that are geared toward a similar audience. The Iowa station also gives presentations pertaining to research conducted under this multi-state research project to livestock producer groups and community members. In similar fashion, the Illinois station sponsors Illini PorkNET (http://www.livestocktrail.illinois.edu/porknet/), the online resource for the Pork Industry, for many aspects of pork production, including reproduction and reproductive technologies.
Technology transfer is an important service that we perform related to our individual productivity, but it is also a means of collaboration by inviting W4171 colleagues to speak at events not located at their home station.
Extending our outreach to non-traditional groups includes presenting hands-on learning activities to students in junior high and high schools. The Maryland station has cooperated with a local school to bring laboratory exercises involving the characterization of embryonic stem cells to the high school science curriculum. The Nebraska station has performed over 40 workshops focused on Animal Biotechnology for junior and senior high school students, 4-H and FFA groups, and junior/community college students. The workshops describe methodologies for the production of transgenic livestock, emphasizing the power of such technologies to improve livestock animal production. These workshops are very popular and effective, assisting in the recruitment of many students to the Animal Science major at the University of Nebraska-Lincoln. Also, members of the Utah station pooled resources and effort to put on a university-sponsored hands-on community “science night” focused on assisted reproductive technologies in livestock animals, including cloning by somatic cell nuclear transfer and transgenesis. Utah station members are also major players in the Utah State University (USU) Center for Integrated BioSystems’ Annual Biotechnology Summer Academy for high school students, which is a structured program where current and recently-graduated high school students come to USU and participate in research and training in the life sciences. This year (2019) marks the 18th consecutive year of the summer academy, and will also be the first year for hosting a second academy session specifically targeted for students of Native American heritage from USU’s Blanding campus in the “Four-Corners” area of the American Southwest.
Restricted budgets from federal and state sources require that we communicate our message and impact to politicians and constituents. Current and former members of W3171 have testified before the U.S. Congress on animal biotechnologies and hosted legislative delegations in our laboratories.
The aforementioned examples of outreach will continue as part of our future plan.
Scientific Community. Presenting at scientific meetings (regional, national, and international) and publishing research findings in peer-reviewed journals is a major component of our outreach plan. Members of W4171 hold membership and leadership roles in many professional organizations. Those organizations include: International Embryo Transfer Society, Society for the Study of Reproduction, American Society of Animal Science, American Dairy Science Association, and American Registry of Professional Animal Scientists; therefore, the impact of W4171 research is spread over many organizations. As stated in the milestones section, we will work with meeting organizers to develop symposia at professional meetings, primarily IETS, but others will be considered.
Overall, W4171 members will use every opportunity to concisely and accurately explain how biological research and technologies can and will increase the efficiency of livestock production.
Governance of this Technical Committee will include the election of a Chair and a Secretary. Election of Secretary will occur annually. Secretary will rotate to Chair. The agenda for the annual meeting of the Technical Committee will be set by the Chair and he/she will preside over the meeting. The Secretary will prepare minutes of the annual meeting as well as the annual report. Administrative guidance will be provided by an assigned Administrative Advisor and a NIFA Representative.
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