Duration: 10/01/2024 to 09/30/2029

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Introduction

Established 40 years ago, the primary goal of the W171 Regional Research Project (renewed as project #s W1171, W2171, W3171 and W4171) was to inaugurate 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 attained. Assisted reproductive technologies (ART) continue to be adopted within the livestock production industries including:  1) artificial insemination (AI), including fixed-time AI in cattle and post-cervical AI in pigs; 2) cryopreservation of gametes and preimplantation embryos; 3) superovulation; 4) embryo transfer (ET); 5) in vitro production (IVP) of embryos – encompassing in vitro maturation of oocytes (IVM), in vitro fertilization (IVF), and in vitro culture (IVC) of embryos; 6) sexed semen; and 7) somatic cell nuclear transfer (SCNT) [1, 2]. Members of the W4171 Multistate Project have been influential in the improvement and use of these procedures since our last Project revision. Although improvements are evident, many of these procedures remain too inefficient for application to commercial agriculture [3-5].

Furthermore, assisted reproductive technologies are critical to the production of livestock with intentional genetic alterations. Novel genome editing technologies, such as clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated nuclease 9 (Cas9) systems, have greatly improved the efficiency of producing intentional genetic alterations in livestock. More importantly, application of CRISPR/Cas9 systems in domestic animals could revolutionize livestock production such as enhancing production traits, conferring disease resistance, improving animal welfare and controlling livestock pests [6]. Since our last Project renewal, investigators from the associated research stations have been leaders in this field, producing over 24 domestic livestock animals with intentional genetic alterations. Likewise, some of the regulatory and public perception hurdles associated with using livestock produced with intentional genetic alterations as sources for meat and animal products have been cleared. In 2022, the U.S. Food and Drug Administration (FDA) made a low-risk determination for the marketing of products (including food) from genome-edited beef cattle and their offspring after determining that the intentional genomic alteration posed no safety concerns, opening an accelerated pathway for marketing animals containing low-risk intentional genetic alterations [7]. Although these advances have been noteworthy, a significant knowledge gap persists regarding the ability to efficiently produce livestock species with intentional genetic alterations. To benefit from the advantages of farm animals with intentional genetic alterations for human food and fiber production, these obstacles must be overcome. Herein, we request to continue pursuit of our research priorities and renew the W4171 Regional Research Project (as W5171) with the overall goal of increasing the efficiency of ART in livestock species and producing animals with intentional genetic alterations 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 [8, 9]. In addition, people are living longer; by 2050, 20% of the world’s population will be over 65 years of age, with 80% residing in low- and middle-income countries [10]. Of paramount importance is providing enough food to support the people of the world, between 691 and 783 million people faced hunger in 2022 [11]. Many outlets suggest that food production must double to meet the needs of the global population in 2050 [8, 9], presenting a challenge to agricultural systems. Moreover, the urban population is growing more than three times faster than the rural population in low- and middle-income countries [11], resulting in higher incomes and increasing the demand for meat and milk [8, 9]. 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 loses $473 to $484 million annually due to infertility [9]. In typical production sow farms (> 1,000 sows), profit is determined by the number of piglets/sow/year, therefore, even small improvements in reproductive efficiency can significantly impact profitability [12]. Reproductive efficiency is also vital to profitability of beef cattle operations. Utilizing feeder calf prices of $1.42/lb and a selling weight of 500 lbs, a 1% increase in calf production could save a producer $7.10/cow/year [9], which would have saved U.S. beef producers over $214 million in 2022 [9, 13]. Thus, there is a critical need to improve reproductive performance of livestock animals.  

The objectives of this Regional Research Project fall under the 2022-2026 Strategic Plan for the U.S. Department of Agriculture (USDA) [14]. Strategic Goal 1 (Combat climate change to support America’s working lands, natural resources, and communities) includes a mandate to use climate-smart management and sound science to enhance the health and productivity of agricultural lands (Objective 1.1). Strategic Goal 2 (Ensure America’s agricultural system is equitable, resilient, and prosperous) includes a mandate to foster agriculture innovation (Objective 2.3). Additionally, the aims of this research effort are directly in line with Strategic Objective 1 (Bolster scientific research to enhance the nation’s resilience and response to climate change by embracing innovative and novel approaches) and Strategic Objective 2 (Enhance research and investment in communities to ensure equity, reduce barriers to access, and advance opportunities for underserved communities) of the National Institute of Food and Agriculture (NIFA) 2022-2026 Strategic Plan [15].

Importance of the proposed work and consequences if it is not done

In 2022, cash receipts for animals and animal products within the U.S. totaled $258.5 billion [16]. Within the states comprising this regional research project, livestock numbers (as of January 1, 2023) included 51.1 million head of beef cattle, 41 million swine, 3.9 million sheep and goats, 3.5 billion poultry and 4.2 million dairy cows (that produced 102 billion pounds of milk in 2022) [13]. Moreover, on-farm cash receipts for animals and products totaled $127.6 billion for these states [16]. Thus, even a 1% increase in production would inject an additional $1.27 billion dollars into these local economies.

Reproductive efficiency is a major economic driver of livestock production systems. Assisted reproductive technologies provide powerful tools to overcome infertility or subfertility in animals [3]. The adoption of ART use in livestock production continues to grow rapidly. In 2021, more bovine IVP embryos were recorded (31.5%) and transferred (32.8%) worldwide compared to the previous year [17]. In North America, 78% of the bovine embryos recorded were IVP, whereas 22% were in vivo derived (IVD) in 2021, largely due to the enhanced use of ovum pick-up (OPU) to collect oocytes [17]. Although the use of ART in domestic large animals has increased dramatically since our last Project revision, inefficiencies of these methodologies persist, limiting their use in commercial animal production systems. In cattle, less than 50% of IVP embryos develop into blastocysts [18], whereas IVD embryos have developmental rates of 85-95% [19]. Survival of cryopreserved IVP embryos remains considerably lower than that of IVD embryos as well [18]. In the pig, only 40% of presumptive IVP zygotes will develop to the blastocyst stage and those will have fewer cells than IVD embryos [20]. Superovulation and ET have been widely utilized in beef and dairy cattle, yet the number of transferrable embryos has changed very little [21]. In addition to its role in the production of genome-edited animals, SCNT could benefit producers by improving the average performance of their livestock animals in a single generation, progress that is unmatched in traditional breeding programs [22]. However, the reduced viability of cloned embryos results in substantial pregnancy losses [22].

The emergence of genome editing procedures (CRISPR/Cas9) has markedly improved the efficiency of producing intentional genetic alterations in livestock animals for use in agriculture or as biomedical models. Estimating the economic significance of livestock with intentional genetic alterations to U.S. animal agriculture is challenging. A few examples of animals with intentional genetic alterations that have application to the livestock industry include: 1) disease resistance in pigs, cattle and poultry [23-25]; 2) synthesis of omega-3 or -6 fatty acids in pork [23]; 3) production of human lysozyme proteins in the milk of sows [26]; 4) production of phytase in the saliva of pigs [23]; 4) double-muscled sheep and cattle [27]; and 5) hornless dairy cattle [28]. It is easy to imagine how these examples could impact 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.

In biomedical research, there are tangible and intangible monetary considerations associated with the growing market for livestock animals with intentional genetic alterations. Examples of intentional genetic alterations in livestock with clinical applications include genetically modified pigs for organ transplantation into humans [29, 30], goats producing human blood coagulation factors in their milk [31] and cattle that produce human antibodies [32, 33]. In addition, the National Swine Research and Resource Center (NSRRC) at the University of Missouri (established in 2003 upon funding from the National Institutes of Health) has produced over 100 different swine strains to be utilized as biomedical models (K. Lee, personal communication). Our member institutions also collaborate with companies focused on genome editing. For example, Recombinetics, Inc., is a leader in the field and is comprised of subsidiaries including Acceligen (precision breeding for livestock production), Sarxion Biologics (regenerative medicine), Therrilume (preclinical research) and Makana Therapeutics (xenotransplantation).

Thus, in considering gaps in our knowledge, as well as 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 livestock with intentional genetic alterations – are: 1) reproductive inefficiencies in all segments of animal agriculture; 2) millions of dollars lost in opportunity costs associated with reproductive inefficiency; 3) an inability to supply the world’s growing population with high quality animal protein in a responsible and sustainable manner; and 4) a compromised ability to appropriately model human health concerns using genetic or other large animal models of human disease.

Technical feasibility of the research

The production of livestock animals with intentional genetic alterations involves the use of ART (IVM, IVF, IVC, micromanipulation, cell culture, SCNT). These technologies are inefficient, so before intentional genetic alterations in animals can contribute significantly to livestock production systems, construction of these animals will have to be optimized. Inefficiencies occur at many levels including IVP of embryos, SCNT, and establishment of pregnancy. The use of IVP embryos is much more practical than recovery of IVD embryos, but IVM, IVF and IVC methodologies remain suboptimal in many species. For example, the best rates of blastocyst development are 50 and 40% for cattle and pigs, respectively [18, 20]. Consistent with this, development of IVP embryos following ET is inferior to IVD embryos [34-37]. Moreover, the quality of IVP embryos is reduced compared to IVD embryos; IVP blastocysts exhibit vacuoles in trophoblastic cells, fewer microvilli, less intercellular connections, differences in gene expression and changes in lipid metabolism [38]. In 2023, Latham [39] comprehensively reviewed the complexities of gene expression changes in oocytes and preimplantation embryos during mammalian development. Despite significant advances in embryo culture media formulations, in vitro manipulations during early development alter gene expression [40, 41] and epigenetic control [42, 43] in pre-, peri- and post-implantation IVP embryos, that can even persist into postnatal life.

A monumental step in improving the efficiency of producing large domestic animals with intentional genetic alterations can be attributed to engineered nucleases, like CRISPR [44]. Although most genome-edited livestock have been produced using CRISPR/Cas9 and SCNT [45], the CRISPR/Cas9 base editing system allows the introduction of intentional genetic alterations into zygotes (via microinjection or electroporation) [44]. Genome editing of zygotes greatly enhances the overall efficiency of creating intentional genetic alterations in livestock species but remains limited by genetic mosaicism and off-targeting effects. The SCNT approach eliminates the risk of genetic mosaicism and allows the detection of accidental off-target mutations prior to the birth of offspring [45].  However, SCNT is hindered by poor rates of development; often, only 1 to 5% of SCNT embryos develop into live animals [44]. In addition to these inefficiencies, the technique is costly, highly time consuming and labor intensive. Therefore, this methodology needs further improvement before it will be widely adopted into mainstream livestock animal production systems.

Members of the W5171 Multistate Research Project are actively pursuing the techniques and the knowledge that will improve the efficiency of producing livestock animals with intentional genetic alterations, including the biological processes critical to successful ART. These research pursuits include (but are not limited to) the following areas of concern:

• Developmental rates of IVP embryos are considerably lower than that of IVD embryos [18, 20]. Overcoming this obstacle will greatly enhance the efficiency of producing animals with intentional genetic alterations.

• To develop useful biomarkers indicative of an embryo’s ability to establish a successful pregnancy, an improved understanding of the cellular and molecular mechanisms underlying normal gametogenesis and embryogenesis is required. Advances in ‘omics’ approaches provide strong methodologies to assess the molecular differences more comprehensively [46].

• Since our last Project proposal, the role of extracellular vesicles (EVs) in biological processes has moved to the forefront of scientific investigation. Regarding the mechanisms fundamental to gamete and early embryonic development, a wealth of knowledge has been disseminated [47-51]. Further exploration will likely reveal potential avenues to enhance IVP embryo characteristics.

• Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) from livestock species possess great promise to markedly advance animal biotechnology [52]. Successful isolation of stable ESCs in cattle [53], pigs [54, 55] and sheep [56] and reprogramming of somatic cells into porcine and bovine iPSCs [55, 57, 58] have been reported. As a result, a blossoming area of investigation has focused on differentiating ESCs and iPSCs into gametes in vitro. The emergence of efficient systems for in vitro gametogenesis could dramatically transform ART in livestock.

• Cryopreservation of IVP embryos remains a detriment to the efficient production of animals with intentional genetic alterations due to their vulnerability to cryo-damage [59]. Similarly, long-term storage of embryos following micromanipulation procedures [60] requires more investigation.

• Placental defects are a key factor in the reduced embryonic, fetal, and neonatal survival rates of IVP and SCNT embryos. Aberrant epigenetic remodeling in gametes and early embryos can have dramatic effects on these outcomes [61, 62] and are impaired by ART [61, 63]. Analysis of genomic and epigenomic variation (in gametes and embryos) with systematic, comprehensive approaches require further exploration, before appropriate steps of intervention can be undertaken to ensure more successful development.

This renewal proposal will evaluate two critically important areas to the future success of animal biotechnology: 1) elucidate the cellular and molecular mechanisms underlying biological processes that are critical to the success of assisted reproductive technologies in livestock; and 2) advance the production of livestock animals with intentional genetic alterations through the development of more efficient methodologies.

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. For example, oocyte and embryo vitrification procedures appear particularly laboratory dependent; the optimal exposure time for vitrification of mouse oocytes and mouse blastocysts varied significantly among laboratories [64-66]. Sharing of information and approaches across this multi-state project is critical in advancing ART and improving the efficiency of producing farm animals with intentional genetic alterations. Significant genomic and epigenomic variation exists among gametes and embryos, which can be further compounded by ART [61-63]. Evaluation of ‘omics’ datasets from different research stations examining distinct gametes and embryos is extremely valuable scientifically. Improving developmental rates of IVP embryos, examining the influence of extracellular vesicles on gamete and early embryonic development, developing useful biomarkers of embryo quality, and characterizing the function of primordial germ cell-like cells (derived from ESCs and iPSCs) in livestock are other areas that would benefit from this multiple laboratory approach.

Collectively, our committee stands poised to expand our knowledge of the cellular and molecular mechanisms underlying biological processes (gamete development, fertilization and embryogenesis) critical to the success of ART in livestock as well as advance the production of livestock animals with intentional genetic alterations through the development of more efficient methodologies.

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 because 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 livestock with intentional genetic alterations that are resistant to diseases, facilitating reduced antibiotic use in animal feed. The scientific community will also benefit from the efforts of the Project members. The use of genome editing alone (injection or electroporation of zygotes) 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.  Moreover, the scientists from our member research stations are highly productive. From 2020-2023, scientists representing the current Project (W4171) published 244 peer-reviewed journal articles, 30 book chapters, proceedings, instructional media or theses/dissertations, 168 abstracts and 19 miscellaneous publications. 

Related, Current and Previous Work

As outlined in the Statement of Issues and Justification, understanding the cellular and molecular mechanisms underlying biological processes associated with ART are key for improved embryo production systems. Furthermore, these same processes play an important role in enhancing the efficiency of producing livestock with intentional genetic alterations. In the following subsections, we provide a brief review of specific areas related to our Project objectives.

Oocyte Maturation

Oocyte maturation is a highly dynamic process that remains a significant limitation to the success of in vitro embryo production. It is well documented that oocytes undergo extensive cytoplasmic alterations during final maturation within the dominant follicle after exposure to an ovulation-inducing surge of LH, however, IVM oocytes do not undergo these changes [67]. The developmental competence of oocytes has been linked to both mitochondrial function and subsequent ATP production. Oocytes undergo a drastic increase in their mitochondrial content during the growth phase, which prepares them for metabolic isolation after germinal vesicle breakdown and severing of gap junctions that connect them to surrounding cumulus cells [68]. The unique mechanisms for metabolic coupling between oocytes and cumulus cells are still under investigation but cumulus cell glycolytic activity during oocyte growth and final maturation is associated with increased development [69]. When oocytes are removed from the follicular environment, spontaneous nuclear maturation occurs without appropriate progression of cytoplasmic maturation. Efforts to improve IVM systems have focused on development of two-step culture systems aimed at synchronizing nuclear and cytoplasmic maturation for improved metabolic activity, however significant improvements to maturation media are still required to improve the success of these systems [70]. Metabolomic profiling of bovine follicular fluid after the onset of estrus has further isolated enriched pathways required for energy production in oocytes and cumulus cells, which may contribute to further optimization of maturation conditions [71]. Other efforts have focused on culture of intact follicles to support complete acquisition of oocyte developmental competence occurring after the LH surge with successful generation of blastocysts and viable offspring in mice [72].

Fertilization and Sperm Biology

Improved understanding of events surrounding fertilization and sperm biology are essential for addressing global topics of interest related to paternal contributions to embryo development and environmental intersections of male fertility [73-76]. Unassisted fertilization and methods of assisted fertilization require specific considerations for events that impact structural, mechanical, nuclear, and respiratory functions of sperm [1]. Changes elicited by environmental conditions including the uterine milieu alter sperm transport, oviduct storage, capacitation, the acrosome reaction, gamete binding and subsequent fertilization [77-80]. The molecular mechanisms that impact sperm function are not fully understood [81]. Furthermore, species-specific considerations prudent to the advancement of reproductive technologies requires a more thorough understanding for contributions that increase reproductive efficiency and reduce infertility [82]. In addition to gaps in knowledge that directly address the foundation of sperm biology, recent evidence suggests that novel roles of sperm function in embryo development are influenced by genomic and epigenomic factors [82-85]. The roles of RNAs and sperm-borne factors remain gaps in knowledge that continue to require further study for their contributions to fertilization and subsequent embryo development [82, 86, 87].

Embryonic Development

Early embryogenesis is regulated by developmentally controlled events including proper changes in transcriptional, translational and epigenetic machinery, activation of embryonic genes to support further development, metabolic determinants and oviductal proteins [39, 88-91]. It is important to improve IVP systems to produce embryos with developmental competence for use in agricultural and biomedical research, as well as animal biotechnology [92-94]. During the last few years, comprehensive genome-wide characterization of the molecular events accompanying pre-implantation embryo development including the transcriptome [95, 96], translatome [97], proteome [98], epigenome [99, 100], histone modification [101], chromatin remodeling [102, 103] and non-coding RNAs [104, 105] have emerged in livestock species.  These findings have provided some insight into what can potentially go wrong in pregnancies that fail at these early stages. The developmental program is likely most plastic during the preimplantation period, when genome-wide reprogramming processes may be influenced by external and internal factors that result in improper molecular changes within embryos. In this regard, various environmental stresses (nutrition, heat stress, management practices, high altitude, maternal and paternal age, breed, etc.) early in pregnancy influence embryonic development [46, 106]. These alterations can have long-acting effects on development, even extending into postnatal life. Although, the mechanisms responsible for this phenomenon remain largely unexplored. Overall, there is the potential for strategic environmental interventions during critical times in early life to program embryo health.

We suggest that future research should focus on: 1) systematic gene mutation and phenotyping of livestock embryos; 2) evaluation of embryo cytokines and other beneficial molecules; 3) examination of developmental programming during the offspring’s life and for subsequent generations; 4) investigation into the possible mechanisms for developmental programming and associated molecular changes in embryos that result in visible consequences later in life, and 5) assessment of interventions (i.e., dietary supplementation, zygote interventions, etc.) and how we can take advantage of the positive, adaptive aspects of developmental programming.  

Epigenetics

During fertilization, the highly specialized mammalian germ cells (sperm and oocyte) merge and form a totipotent embryo. This process entails the fusion of distinct epigenetic landscapes from each gamete, encompassing DNA methylation, histone modifications, and non-coding RNAs [107, 108]. The embryo subsequently undergoes critical epigenetic changes, including reprogramming of the DNA methylome, chromatin configuration, and initiation of the embryonic transcriptome through zygotic genome activation [39, 109-111]. Abnormal epigenetic signatures in gametes have been linked to infertility, poor embryogenesis, and pregnancy loss [61, 62].  Moreover, aberrations in epigenetic reprogramming are associated with abnormal embryonic development, a phenomenon that is exacerbated by ART [61, 63]. Insufficient epigenetic remodeling during early mammalian growth can lead to conditions like large offspring syndrome (LOS) and other developmental disorders [112-115]. Epigenetic mechanisms in gametes and the embryo have also been related to phenotypic traits in production animals [116, 117]. The principal mechanisms governing this reprogramming and their interactions remain elusive. A comprehensive analysis of genomic and epigenomic variations in gametes and embryos, along with their phenotypic expressions, is yet to be thoroughly explored [118-120]. Addressing these knowledge gaps is crucial for advancing our understanding of mammalian developmental biology and related technological innovations.

Somatic Cell Nuclear Transfer

Remarkable progress has been made over the last two decades in the field of livestock genetic engineering. Initially, knockouts of multiple genes in fetal fibroblasts required in some cases years and were accomplished by sequential targeting and fibroblast rejuvenation by cloning [121]. Arrival of engineered nucleases gave us an ability to introduce precise insertions or deletions easily and efficiently and multiple knockouts (KO) and knockins (KI) can be now introduced simultaneously [45]. Recent advancements in engineered nucleases reestablished zygote micromanipulation as a highly effective tool for intentional genetic alterations.  However, SCNT continues to be a crucial method to produce gene-edited livestock with about 70% of KI and 50% of KO farm animals generated by cloning [45]. The primary advantage of genetic engineering via SCNT is the ability to conduct comprehensive cell verification in vitro to ensure that the desired genetic modification is present before production of animals with intentional genetic alterations [44, 122-125]. The edited cells can also be screened for potential off-target mutations, the presence of which is one of the major concerns [126]. Furthermore, the SCNT approach eliminates genetic mosaicism and thus, would likely reduce the time requirements and cost of producing animals with intentional genetic alterations.

Further improvements in SCNT efficiency are warranted. Aberrant epigenetic reprogramming often results in developmental defects in SCNT embryos during embryonic genome activation leading to low cloning efficiency [127]. Availability of more precise ‘omics’ technologies (scRNAseq, ATACseq, etc.) provide us with opportunities to better understand gene expression and genome-wide chromatin landscape during early embryonic development [102], specifically during embryonic genome activation in SCNT embryos and learn how it differs from a fertilized embryo. This knowledge will assist with developing new mechanism-based approaches to enhance epigenetic reprogramming and subsequently cloning outcomes.

Cryopreservation

Cryopreservation of IVD preimplantation embryos from mammalian livestock species has generally been quite successful [128-130], with perhaps the exception of South American camelids [131]. On the other hand, cryopreservation of IVP embryos has not been uniformly successful. Additional research is needed to pinpoint the underlying cause of the increased susceptibility of IVP embryos to cryo-damage (i.e., alterations caused during IVM, in vitro sperm capacitation, IVF, and/or IVC) [59]. With increased utilization of embryo manipulation procedures (including genome editing), it is critical not only to gain a better understanding of the impact these technologies have on successful cryopreservation but also to subsequently develop strategies to prevent the occurrence of detrimental alterations to embryos exposed to manipulation [60].

Although embryo cryopreservation protocols developed decades ago can result in the birth of live offspring after storage of embryos in liquid nitrogen for decades [132], refinements in cryopreservation procedures are needed to enhance post-thaw/post-warming viability. Vitrification has been used to successfully cryopreserve embryos from goats [133], pigs [133], horses [134], sheep [135], cattle [136], and alpacas [131]; successful vitrification of porcine [137], equine and bovine oocytes [138] also has been achieved. Recent reviews highlight some of the progress that has been made in recent years in embryo cryopreservation [139-141].

Several different systems have been developed for the cryopreservation of oocytes and embryos [142].  These systems consist of three basic parameters: 1) the chemical compound(s) used as cryoprotective agents or vitricants; 2) the rate of cooling (slow versus ultra-rapid); and 3) the physical device used to hold the oocyte or embryo during the cryopreservation process.  These three parameters are interrelated as the device used to hold the oocyte/embryo affects the cooling and warming rates that can be attained, and the chemical compound(s) used influences the efficiency and timing of dehydration of embryonic cells. Upon investigation of these parameters, numerous discussions have ensued concerning their relative merits [143-145].  Virtually, all these investigations have been centered on the survival of oocytes and/or embryos.

Evidence from somatic cells, embryonic cells and oocytes suggests that different cryoprotectant molecules and the cryopreservation process itself can affect cellular function in ways far more subtle than just survival [146]. These changes in cellular function include mitochondrial function, metabolic function and epigenetic modifications affecting expression of genes [147-149].  Damage to the meiotic spindle is repaired more quickly following vitrification than with slow rate freezing, and mitochondria function can be partially repaired with culture following warming after vitrification [150, 151].  Numerous combinations of cryoprotectants and vitrification devices have been utilized, but the two most prevalent systems have been ethylene glycol and DMSO as cryoprotectants/vitricants [152] and ethylene glycol and glycerol [153] 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. The combination of ethylene glycol and glycerol was more effective for vitrification of sheep [154] and bovine [155] embryos than ethylene glycol combined with DMSO.  This is consistent with the established use of glycerol as the preferred cryoprotectant in cryopreservation of embryos by slow rate freezing in these species. Species-specific considerations, however, must not be overlooked; South American camelid embryos seem more tolerant to galactose than to either glucose or sucrose [131]. 

Stem Cell Biology

The field of stem cell biology in domestic animals has seen a dramatic change since the publication of reports describing the successful derivation of stable ESCs in cattle [53], pigs [54, 55] and sheep [56] and reprogramming of somatic cells into induced pluripotent stem cells (iPSC) [55, 57, 58]. The establishment of pluripotent stem cells allowed for fast advances in the field of organoids, including the recent publications of successful formation of blastoids [156] created by combining bovine ESCs [53] and trophoblast stem cells [157]; these stem cell aggregates self-organize as blastocyst-like structures containing the main cell lineages found in native blastocysts. Blastoids are capable of undergoing elongation after transfer into recipients, making them a suitable model for the study of pre- and peri-implantation embryonic development and enabling potential applications in advanced assisted reproduction in livestock.

Differentiation of ESCs and iPSCs into gametes in vitro has also been a rapidly developing field enabled by the establishment of these pluripotent cells in livestock. In vitro oogenesis and spermatogenesis from ESCs have been repeatedly demonstrated in rodents [158-161]. Recent progress has been made in primordial germ cell-like cell (PGC-LC) differentiation from pluripotent stem cells in livestock species including rabbits, cows, pigs, horses, sheep, and goats [55, 162-164]. However, despite the demonstration of successful induction based on gene and protein expression of specific markers, there are still no studies demonstrating the functionality of these PGC-LCs in any non-rodent species. This will be a critical step in the establishment of efficient systems for in vitro gametogenesis with the potential to revolutionize animal breeding and genetic improvement [165].

Genome Editing

Traditionally, pigs with intentional genetic alterations were produced by introducing genetic modifications in somatic cells and subsequently performing SCNT (i.e., cloning) [166]. However, inefficiency in genetic engineering technology limited the use of these pigs. Recent advancements in genome editing technology, such as the CRISPR/Cas9 system, have facilitated the production of livestock with intentional genetic alterations at high efficiency [167, 168]. The CRISPR/Cas9 system also permits introduction of targeted genetic modifications during embryogenesis [167, 168, 169], thus by-passing the need of cloning in generating pigs with intentional genetic alterations. The genome editing technology was used to generate pigs resistant to Porcine Reproductive and Respiratory Syndrome (PRRS) virus [167, 171], which is a great threat to the swine industry. The pigs were generated by disrupting the CD163 molecule known to serve as a key receptor for the viral entry [167]. The production of PRRS virus resistant pigs is just an example and more genome-edited livestock models are available to improve productivity, animal welfare, and sustainability. 

Most of the leading genome editing systems are designed to introduce a locus-specific double strand break (DSB) on the genome. The DSBs trigger activation of the DNA repair mechanism which can be utilized to introduce desired genetic modifications to target genes. Because genome editing technologies often rely on inducing a DSB on the genome, unintended DSBs may introduce random mutations during the DNA repair process. The possibility of introducing this ‘off-targeting event’ (i.e., unintended mutations on the genome) has been considered the main concern of utilizing genome editing systems. Previous reports on the frequency of off-targeting in genome-edited pigs illustrates that off-targeting is, in general low, in the pigs [172, 173]. However, another study identified a high frequency of off-targeting events (>70%) introduced in a line of genome-edited pigs produced using the CRISPR/Cas9 system [174] (Carey et al, 2019). Other genome editing tools are being developed to reduce off-targeting events by not relying on the DSB; however, efficiency of these tools is low, and they are not widely used in livestock species. Studies aimed to refine and improve the efficacy of different genome editing systems will offer more technical tools that can be used to establish animal models or to study gene-based physiology in livestock.

Accomplishments of the Previous Project

Objective 1:

• Systematically characterized the epigenetic landscape (DNA methylome and chromatin modifiers) of bovine in vivo developed pre-implantation embryos.


• Using Raman spectroscopy, metabolomic profile analysis of bovine IVF and SCNT embryos identified metabolomic differences between embryos of different developmental potential.


• Identified potential receptors and ligands functioning during porcine fertilization.


• Designed a practical system to deliver defined photostimulation (wavelength and intensity) to cells and embryos in culture.


• The world’s first cria (baby camelid) produced from a cryopreserved embryo was born.


• Porcine theca cells from GnRH-II receptor knockdown females expressed 40% less GnRH-II receptor protein than theca cells in littermate controls or granulosa cells from either line.


• FLI cytokine-supplemented maturation medium improved bovine oocyte maturation, blastocyst development and initial pregnancy rate following somatic cell nuclear transfer.


• RNA sequencing data generated for bovine germinal vesicle and metaphase-II stage oocytes were used to compare transcriptome changes across species in a meta-analysis strategy to identify conserved mechanisms driving oocyte maturation.


• Follicle growth after ovarian cortex cryopreservation indicates that this can be a viable method to preserve follicles that could later be retrieved from the tissue for further culture.


• Developed a modified semen extender compatible for long-term storage of frozen-thawed bovine sperm at ambient temperature.


• Using mass spectrometry, the lipid profiles of individual porcine oocytes was profiled during in vitro maturation.


• Characterized the expression and localization of PPARgamma in bovine sperm.

Objective 2:

• CRISPR/Cas 9 genome editing was successfully used to knockout expression of specific genes associated with the porcine conceptus, including IL1B2, CYP19A1 and PTGS2.


• Developed an efficient protocol for the introduction of human specific mutations into ovine fetal fibroblast cells using CRISPR/Cas9 genome editing.


• Established putative naïve bovine embryonic stem cells that have shown in vitro and in vivo differentiation capabilities.


• Pretreatment of capacitated sperm with progesterone increased fertilization after ICSI in bovine oocytes.


• Electroporation of CRISPR/Cas9 components into pig zygotes was used to circumvent the drawbacks of microinjection.


• High-resolution ribosome fractionation and low-input ribosome profiling of bovine oocytes and preimplantation embryos has enabled us to define the translational landscapes of bovine early embryo development.


• Developed an efficient method to generate bovine blastocyst-like structures (termed blastoids) via the assembly of trophoblast stem cells and expanded potential stem cells.


• Created a new area of research – investigation of thyroid hormone physiology in growth restricted fetuses induced by knockdown of chorionic somatomammotropin (CSH) using placenta specific RNAi.


• Established bovine trophoblast stem cells that exhibit transcriptomic and epigenetic features characteristic of trophectoderm cells from bovine embryos and retain developmental potency to differentiate into mature trophoblast cells.


• A modification in the method used for bovine intracytoplasmic sperm injection, referred to as vigorous injection, improved subsequent embryo development.


• Created a line of type I interferon receptor (IFNAR) knockout sheep and are using them to study pregnancy recognition in ruminants.

Related Regional Research Projects

A search of NIMSS projects revealed several multistate research projects with the goal of improving reproductive efficiency in livestock. These projects include:  NC1201 – Methods to Increase Reproductive Efficiency in Cattle; NCERA57 – Swine Reproductive Physiology; NE2227 – Contribution of Ovarian Function, Uterine Receptivity, and Embryo Quality to Pregnancy Success in Ruminants; S1093 – Management Systems for Beef Cattle Reared in Subtropical and Tropical Environments; S1081 – Nutritional Systems for Swine to Increase Reproductive Efficiency; and W4112 – Reproductive Performance in Domestic Ruminants. However, the W5171 group is uniquely focused on elucidating the cellular and molecular mechanisms underlying biological processes critical to the success of assisted reproductive technologies and advancing the production of livestock animals with intentional genetic alterations through the development of more efficient methodologies. Thus, our proposed research studies do not duplicate the efforts of any other multistate research projects.

Objectives

1. Elucidate the cellular and molecular mechanisms underlying biological processes that are critical to the success of assisted reproductive technologies.
   
2. Advance the production of livestock animals with intentional genetic alterations through the development of more efficient methodologies.
   

Methods

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 multistate project. In the case of the W5171 group, these interactions have led to some very specific collaborative efforts that are a direct result of the multistate research project.  We have divided this section into working groups (subgroups) under each of our objectives.

Objective 1:  Elucidate the cellular and molecular mechanisms underlying biological processes that are critical to the success of assisted reproductive technologies in livestock.

The overall aim of this research area is to gain a better understanding of the cellular and molecular mechanisms that are essential to successful ART (gamete maturation, fertilization, and subsequent embryonic development). The production of live offspring, including those with intentional genetic alterations, is dependent upon all 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 more healthy, viable offspring. This, in turn, will improve many of the systems associated with IVP of embryos even further, moving closer and closer to conditions present for IVD embryos. Several member experiment stations will be investigating this objective including AR, CA, CO, CT, FL, IA, IL, MD, MO, MS, NE, LA, OK, SC, TX, UT, WA.

1.1.    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, CO, IL, LA, MO, NE, SC, UT). The primary goal of this working group 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. The LA station will investigate methods to improve the synchronization between nuclear and cytoplasmic maturation. The MO and UT stations will collaborate to investigate how the meiotic spindle formation during oocyte maturation impacts subsequent embryo development. The UT station will study the effect of cytokine supplementation during oocyte maturation on nuclear and cytoplasmic maturation, as well as investigate the seasonal effects on oocyte maturation and embryo development in cattle. The SC station will investigate the mitochondrial bioenergetic fluctuations during oocyte maturation and how alterations in ATP production affect subsequent embryonic development.

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. The LA station in collaboration with the FL station will contribute to this working group by characterizing the molecular differences between in vivo and IVM oocytes and how variants in the IVM system alter these characteristics to mimic IVD oocytes more closely.  The station from CT will examine metabolomic and proteomic profiles of bovine follicular fluid during the window of IVM. A collaboration between the LA and CA stations will use Raman spectrometry imaging to characterize cell populations in preantral growing follicles. The NE station will contribute to this working group by evaluating the micro-environment of maturing oocytes, including follicular fluid as well as granulosa and theca cells. The stations from CO and IL will investigate how exosomes released from follicular fluid and granulosa cells help oocytes mature and deal with stress. The CA station will investigate culture methods for successful development of preantral follicles to the antral stage leading to the development of competent MII stage oocytes.

1.2.    Sperm Biology and Mechanisms of Fertilization. The cellular and molecular aspects of sperm biology, fertilization and oocyte activation will be explored by this group (AR, FL, IA, IL, LA, MS, NE, WA). This group is interested in examining basic mechanisms of fertilization in domestic livestock species. Despite some progress in the 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 ‘omics’ approaches. As the oocyte matures, the plasma membrane increasingly gains the ability to bind and/or fuse with sperm. Epigenetic regulation of the paternal germline, including chromatin modifying enzymes that are involved in histone eviction/retention, will be investigated in multiple species to better understand how paternal chromatin regulates fertility and subsequent embryo development.

The FL station will contribute several projects focused on environmental impacts on postejaculatory function using the bovine as a model permissive to fertilization and embryo development. The effects of environmental stressors on sperm function will be addressed to understand impacts on reproductive efficiency of livestock while also serving as a model for human biomedical research. Complementary projects will be included to advance the development of equine IVF and shipment of cooled semen. The station from IA will investigate zinc signatures of sperm and their role(s) in fertilization of porcine and bovine oocytes. The LA station will investigate how environmental exposures affect sperm viability parameters of goat and bovine and how sperm treatments affect fertilization by intracytoplasmic sperm injection (ICSI) of bovine oocytes. The station from MS will investigate how exosomes of diverse origins (e.g. species and biofluids) and other nanoparticles affect the post-collection handling of semen and sperm quality in various livestock species. Additionally, the station will use spectroscopic approaches (e.g. Raman and near-infrared) to characterize spermatozoa for strategic (cryo)preservation purposes to maintain high artificial insemination results. The IL station will investigate how sperm ultrastructure contributes to individual sperm viability and fertilizing ability using holographic microscopy (SLIM and GLIM). Additionally, stations from AR and IL will continue to examine morphological differences in sperm that influence their ability to be used for AI, IVF, fixed-time AI and natural service. In collaboration with IL, the NE station will examine morphological differences between sperm from GnRH-II receptor knockdown boars vs. littermate control males. Investigators from WA will analyze the impacts of cold exposure of bulls on DNA methylation of sperm cells, which could be passed on to the resulting embryos to affect offspring development.

1.3.    Early Embryonic Development. Another large collaborative workgroup (CO, FL, IL, OK, SC, TX, MD, MT, NY, UT, WA) of scientists will investigate factors underlying the early stages of embryonic development. Shortly after fertilization, mammalian embryos undergo genome-wide epigenetic reprogramming by demethylation, followed later by de novo re-methylation. Of primary interest, the effects of epigenetics at this extremely important stage of development will be targeted. Global changes to chromatin dynamics, initiation of zygotic genome activation, and other transcriptional programs that are necessary for proper development will be investigated through low-input genomic techniques. The station from NY will determine whether the maternal factor "males absent on the first" (MOF), a K(lysine) acetyltransferase of H4K16ac, is enriched in bovine early embryos. The effect of the paternal epigenome on embryo development, including the role of paternal histones and subsequent histone post-translational modification signatures will be studied at the TX station. Transposon elements, integral components of the genome with the capacity to move and replicate within the DNA, also play a critical role in gene regulation and genome stability. The NY station will characterize transposon element expression during bovine embryogenesis.

A collaboration between the CO and IL stations will investigate how exosomes released from oviductal and granulosa cells may transfer molecular regulators that will help early embryos overcome stress. A second study is investigating the prevalence of pathogens in follicular fluid to determine the risk associated with the collection of oocytes from ovaries during the in vitro production of bovine embryos. This work will also investigate if the current mechanisms for embryo washing effectively remove the pathogens, allowing for long-term embryo freezing and international transport. The SC station will investigate the effect of altering mitochondrial bioenergetic capacity at different timepoints during early embryonic development. The MT station will perform experiments to better understand basal redox systems and stress responses during embryogenesis. The station from MD will continue to investigate the metabolism of bovine embryos by employing substrates labeled with heavy stable isotopes (e.g., [¹³C] glucose, [¹³C] fructose, [¹³C] pyruvate or [¹³C] glutamine) as metabolic probes.

Additional studies will pursue peri- and post-implantation development, including genetic regulation of conceptus and placental development. The CO station will investigate the role of thyroid hormone in placental and fetal development during pregnancy. Placenta-specific, lentiviral-mediated in vivo RNA interference (RNAi) to limit the function of genes needed for thyroid hormone regulation in the placenta and fetus will help determine how thyroid hormone is transported across the placenta from mother to the fetus as well as determine the action of thyroid hormone on the placenta. The SC station will study the effect of maternal systemic vasoconstriction on fetal and placental circulation and growth. The FL station will conduct research to determine genomic regulation of early embryo development with specific emphasis on early trophectoderm formation. Research will expand from current projects focused on the roles of PPARG to identify pharmacological intervention strategies that mitigate pregnancy loss. The station from WA will study the impacts of maternal obesity on embryonic development using single-cell RNA-sequencing. Finally, the OK station will explore complex regulatory networks controlled by transcription factors and microRNAs that are involved in regulation of conceptus elongation.

Objective 2: Advance the production of livestock animals with intentional genetic alterations through the development of more efficient methodologies.

The primary aim of this research objective is to enhance the success of each step in the process required for the successful production of intentional genetic alterations in both livestock animals and large animal biomedical models. Continual advances in genomic editing (CRISPR/Cas9) techniques and SCNT methodology have led to a crucial need for more research that will allow incorporation of these methods into livestock production systems. Research under Objective 2 will be conducted by multiple research institutions comprising the Project: CO, FL, IL, MO, MT, NE, OK, SC, TX, UT.

2.1.    Genome Editing. This workgroup (CO, IL, MO, MT, UT, TX) will utilize genome editing tools (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. 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 livestock with intentional genetic alterations. 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 accidental 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 MO and UT stations will develop a streamlined method that can assess frequency of off-targeting events in the livestock genome. The pipeline will be shared with the community and collaborate with other stations to identify potential off-targeting events in other genome-edited animals. In addition, these stations (MO, UT) will conduct experiments aimed to increase efficiency of gene targeting events in somatic cells as well as in embryos by customizing the use of different genome editing tools for livestock species. The UT station will enhance methods for introduction of large expression vectors into the livestock genome using a combination of CRISPR/Cas9 and piggyback approaches that will be beneficial for the development of new livestock models of human diseases and for pharmaceutical protein production. The IL station will investigate the issues surrounding CRISPR/Cas9 editing of single bases surrounded by guanine-rich areas of the genome to manipulate milk synthesis for increased production and the production of biopharmaceuticals. The role of the TX station in this working group will expand considerably upon development of a new reproductive biotechnology center with genome editing facilities for ruminants. The station from CO will investigate the impact of CRISPR/Cas9-mediated knockout of the Keap1 gene in modulating NRF2 activity in sheep. The station from MT will develop adenovirus-associated virus serotype-8 vectors that express sgRNAs targeting key redox- or metabolism-associated genes and co-express a fluorescent protein to mark transduced cells.

2.2.   Assisted Reproductive Technologies. This subgroup of the Project (AR, CA, CO, IA, CT, IL, LA, MD, UT) will focus on the procedures and underlying biology associated with significant advancements in ART (IVM, IVF, IVC, micromanipulation, cell culture, SCNT). The production of livestock with intentional genetic alterations is dependent on improving the efficiency of procedures associated with ART. The FL station will use gene editing approaches to determine the regulation of early embryonic development and roles in cell lineage specification. Techniques will be used to advance extended embryo culture experiments to determine regulatory roles in conceptus elongation using both in vitro techniques and embryo transfer. Collaborations with the CO station to utilize different methodologies targeting the trophectoderm for early placenta development will be developed to advance studies in pregnancy loss. The LA station will investigate modified procedures affecting the development rate of bovine embryos fertilized by ICSI and in collaboration with the AR station will transfer ICSI fertilized embryos to recipients for full term development utilizing animals from a cooperating producer. The UT station will investigate the effect of cytoplasmic polyadenylation on reprogramming of bovine SCNT embryos and optimize IVF protocol in sheep. Consistent with this, the MO station will explore novel oocyte activation methods that can enhance development of SCNT embryos. The CA station will investigate methods for differentiation of in vivo- and in vitro-produced oocytes, including the use of gene-edited sheep to elucidate the mechanisms of oogenesis and folliculogenesis. The IL station will investigate the incorporation of advanced imaging into ART programs in cattle and swine. The stations from AR, IL and LA will further explore optimization of superovulation, fixed-time AI and embryo transfer in cattle.

2.3.   Gamete and Embryo Cryopreservation. This subgroup of the Project (IA, IL, FL, LA, MS, SC, UT) will develop methodologies for improvements in the cryopreservation of sperm, oocytes and embryos (especially IVP embryos), as well as gain a better understanding of the molecular and cellular processes influenced by freezing and thawing procedures. Successful vitrification of oocytes from livestock species has been achieved, although the success of these cryopreservation technologies remains low. This working 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 the 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 investigate disruption of calcium homeostasis during oocyte and embryo cryopreservation and approaches to reduce this disruption and minimize the detrimental effects on mitochondrial function and endoplasmic reticulum stress. In collaboration with the LA station, the SC station will study the effect of photobiomodulation on the recovery of vitrified oocytes after warming. 

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 will investigate methods for efficient removal of spermatozoa from the post-ejaculatory coagulum that is common with South American camelids (alpacas, llamas), which is a vitally important prerequisite to sperm cryopreservation. Viability of extracted spermatozoa will be examined in vitro and in vivo to ensure that the extracted spermatozoa are appropriate for cryopreservation. The MS station will study the suitability of various nanoparticles, biofluid-derived exosomes, and natural plant extracts on sperm freezing/thawing across multiple livestock species. The IL station will investigate the use of various tree saps to vitrify and slow-freeze bovine oocytes.

Finally, the group will continue to advance their knowledge base regarding cryopreservation of embryos. Efficient production of livestock animals with intentional genetic alterations depends on successful protocols for short and long-term storage of manipulated embryos. Consistent with this, cryopreservation of manipulated and/or IVP embryos needs to be improved. The group will examine the effects of various manipulation methods (IVP, microinjection, assisted hatching) on the viability of embryos following freezing and thawing compared to IVD embryos. The IA station will focus on cryopreservation of preimplantation embryos of South American camelids. Alpaca and llama embryos enter the uterus as hatched blastocysts, and their large size is compounded by their high lipid content (lipid is hydrophilic). Both conventional slow freezing and ultra-rapid vitrification approaches will be investigated.  The UT station will investigate the effect of antioxidants and cytokines supplementation on the vitrification of bovine embryos produced during hot and cold seasons.

2.4.   Stem Cells. This group of investigators (CA, CT, FL, IL, OK) will study biological mechanisms and procedures involved in isolation of both ESCs and iPSCs. Our understanding of cellular differentiation has greatly expanded, largely due to the dramatic advances in SCNT methodologies. Since our last Project revision, a major limitation to the livestock industry has been overcome upon the successful isolation of ESCs and iPSCs. As a result, a novel area of investigation has begun, with particular focus on differentiating ESCs and iPSCs into gametes in vitro. The emergence of efficient systems for in vitro gametogenesis could dramatically transform ART in livestock. Similarly, significant progress has been made in PGC-LC differentiation from pluripotent stem cells, although there are still no studies demonstrating the functionality of these PGC-LCs in any livestock species. This group will also focus on developing new technologies for the use of stem cells to study gametogenesis and embryogenesis and applying these technologies to enable advanced ART. In collaboration with the FL, IL and CA stations, the OK station will establish and enhance protocols for culture of ESCs derived from both cattle and bison embryos. Recently, the CT station reported the derivation of the putative bovine ESCs from in vitro-fertilized blastocysts. Stations form CA, CT, FL and IL will continue to modify the culture medium of stem cells by modifying levels of growth regulatory factors, nutrients of the base medium and explore the use of nanomaterials. Continued improvement in the culture conditions of ESCs will be critical for the success of downstream biotechnological and biomedical applications.

Measurement of Progress and Results

Outputs

• Peer-reviewed scientific publications reporting novel contributions to the fields of gamete and embryo biology and generation of livestock animals with intentional genetic alterations.
• Generate new scientific knowledge about the cellular and molecular mechanisms underlying gamete and preimplantation embryo biology.
• Improved technologies for gamete and embryo cryopreservation.
• New approaches for IVM, IVF and IVC of preimplantation embryos.
• Improved understanding of how epigenetic reprogramming in IVP and SCNT embryos differs from normal developmental processes.
• New methodologies to ehance the efficiency of producing intentional genetic alterations in livestock species via genome editing.
• Functional characterization of primordial germ cell-like cells differentiated from pluripotent stem cells of domesticated animal species.
• Graduate and postdoctoral students trained in areas related to gamete and embryo biology and generation of animals with intentional genetic alterations.
• Comprehensive genome-wide characterization of the molecular events accompanying gametogenesis and preimplantation embryo development.
• Provide guidance for implementation of ART to veterinarians, practitioners and commercial operations.

Outcomes or Projected Impacts

• Endocrine and/or genetic markers to predict fertility would be of great economic benefit to livestock producers, allowing for more timely management decisions.
• 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.
• Further evidence of the safety and effectiveness of genome-editing technologies will strengthen the case for using such technologies in livestock production.
• Unraveling the metabolic basis of normal early embryo development will provide significant benefits to human and animal reproductive health.
• Time-lapse monitoring and artificial-intelligence-based automated image analysis have the potential for a more accurate evaluation of embryo quality.
• Bovine blastoid technology could lead to the development of new artificial reproductive technologies for cattle breeding, which may enable a paradigm shift in livestock reproduction.
• Research on extracellular vesicle-mediated molecular signaling in ovarian follicles and oocytes will facilitate the development of diagnostic markers associated with maternal physiology and embryo developmental competence, as well as potential therapeutic applications in ART.
• Identification and flux quantification of novel sulfur amino acid-based redox homeostasis mechanisms revealed previously unknown mechanisms cells can use to support redox homeostasis during embryogenesis.
• Understanding the normal mechanisms regulating embryonic and placental development is necessary to acquire basic knowledge that can serve as a foundation to diagnose abnormal embryogenesis.
• Elucidating the differences in prolactin signaling because of a genetic mutation in the prolactin receptor gene (SLICK1) will improve our understanding of the mechanisms involved in thermotolerance.
• As a result of these studies, we will acquire a change in fundamental knowledge regarding how the GnRH-II/GnRHR-II system regulates 17ß-estradiol levels and follicular dynamics in porcine females, representing a potential avenue for future reproductive therapies.
• Overall, our results will provide a unique opportunity to develop a targeted embryo biomarker assessment system for improving animal fertility and reproduction efficiency.
• Improvement in the quality of sheep oocytes and embryos in commercial serum-free medium could have a significant impact on improvement of IVP embryos and reduce large offspring syndrome in lambs.
• Improved outcomes from bovine intracytoplasmic sperm injection (ICSI) will provide an additional method for incorporation of genome editing techniques and fertilization with spermatozoa for which there are limited numbers (i.e., sex sorted sperm).
• Understanding the role of the paternal histone epigenome in embryogenesis is critical as we further appreciate how the health, diet, and environment of the sire can program the embryo for implantation and long-term health and development.
• Our work will provide foundational information to discover essential biological pathways underpinning bovine pre- and peri-implantation development and the molecular causes of early pregnancy failure during this critical period.

Milestones

(2026):Submission of a collaborative research proposal by W5171 committee members seeking funding from a federal agency or commodity board.

(2027):To facilitate dissemination of experimental results to the research community and ART practitioners, the W5171 committee will provide either a pre- or post-conference symposium at the Annual Meeting of the International Embryo Technology Society (IETS). W5171 committee members will deliver scientific presentations related to advances in gamete/embryo technologies. W5171 members (also members of IETS) will initiate discussion with the IETS Planning Committee to organize this outreach activity. Alternatively, the symposium could be held in conjunction with the Annual Meeting of the W5171 Multistate Regional Research Project when it meets at a domestic location in years that the IETS meeting convenes internationally.

(2028):Publication of review articles related to each of the two objectives of the W5171 Regional Research Project.

(2029):Investigators from stations of the W5171 committee will have produced multiple examples of animals with intentional genetic alterations that could improve livestock production efficiency or serve as models for biomedical applications.

Projected Participation

View Appendix E: Participation

Outreach Plan

Project members of W5171 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 W5171 have led the education committee for the International Embryo Technology Society (IETS) for more than two decades. Over the years, many of the graduate student award winners at IETS presented projects that were part of W171, W1171, W2171, W3171 and W4171, the earlier versions of W5171. Furthermore, the CO station is active in training graduate students in ART techniques and currently runs a 1-year Master’s degree program specializing in ART techniques. In complement, the FL station offers a Reproductive Biology Concentration program for graduate students. The IL station is active in on-line training for ART in cattle and swine. Training is targeted at undergraduate and graduate students, as well as continuing education for veterinarians.

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. This report is geared toward livestock producers, extension specialists, and members of the general public. The IA 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 IA station also gives presentations pertaining to research conducted under this multi-state research project to livestock producer groups and community members.

Technology transfer is an important service that we perform related to our individual productivity, but it is also a means of collaboration by inviting W5171 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 ESCs to the high school science curriculum. The Nebraska station has performed over 80 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 to produce livestock with intentional genetic alterations, 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 ART in livestock animals, including SCNT and genome editing. 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.

Restricted budgets from federal and state sources require that we communicate our message and impact to politicians and constituents. Current and former members of W5171 have testified before the U.S. Congress on animal biotechnologies and hosted legislative delegations in our laboratories.

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: IETs, 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 W5171 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.

Overall, W5171 members will use every opportunity to concisely and accurately explain how biological research and technologies can and will increase the efficiency of livestock production.

Organization/Governance

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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