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.