Considerations for recipient male selection in a livestock Surrogate Sire breeding strategy

Abstract

Surrogate Sires is a novel breeding technology for increasing the rate of genetic gain in food animal production settings through natural mating. As an overarching concept, a surrogate sire is a male possessing testes devoid of germ cells yet capable of harboring spermatogenesis following introduction of donor germline. The utility of this technology relies on recipient males that are not only germline ablated and capable of harboring donor-derived spermatogenesis but also otherwise phenotypically normal and readily produced. Since the advent of spermatogenic stem cell transplantation in mice, many methods have been used to generate surrogate males by either removing germline with cytotoxic treatments or preventing germ cell survival and/or development through gene editing. At present, the use of gene editing to cause germline ablation shows the most promise as the precise inactivation of genes involved in germ cell development allow for testis-specific effects and have been assessed for the ability to harbor spermatogenesis from introduced donor germline. Of particular interest is the knockout of NANOS2 as the consistent germline ablation phenotype and proven production of donor-derived spermatogenesis combined with the fertility of both knockout females and heterozygous males establishes a clear and simple means for the scaling and use of Surrogate Sires in livestock production.

Introduction

In the age of assisted reproductive technologies, current approaches to accelerating livestock production efficiency through genetic improvement have become increasingly labor intensive and limited to confinement production systems where animals can be meticulously managed [1, 2]. While the use of artificial insemination in swine, cattle, sheep, and goats has allowed for selection and dissemination of high merit genetics in intensive management settings, widespread use of this technology is obstructed by conventional large scale production practices. Range-based operations where the majority of ruminant food animal production occurs are not easily converted into confinement systems, nor would they benefit when considering both animal welfare and economic impacts. It is therefore necessary to develop new genetic improvement strategies that may be applied within the boundaries of current production practices.

A key to improving the dissemination of high value genetics within livestock populations at large is capitalizing on the male contribution to sexual reproduction. While both sexes provide genetic material for the creation of offspring, the female impact on generational improvement is limited by uterine carrying capacity and gestation length. Males, however, produce millions of sperm daily and are regularly used to breed many females in a season, therefore, increasing the weight of their contribution to genetic gain of a production population. As natural mating is most common in traditional range- and pasture-based production systems [1, 2], the challenge of accelerating the improvement of food animal genetics is in making “elite” or “desirable” genetics available for dissemination via a multitude of males globally, a concept termed Surrogate Sires [3]. In the same way that a surrogate mother carries offspring for another female, a surrogate sire is a male that produces sperm containing the genetics of another male. By generating males to be vessels for customizable sperm production, the range of an elite male’s genetics is not limited by geographical area or labor-intensive practices and further opportunities for introducing genetic modifications through germ cells becomes available for global livestock production improvement [3].

At a basic level, a surrogate sire is a male with properly formed testicles, including normal seminiferous tubule structure and somatic support cell populations, that are devoid of endogenous germline [3]. The initiation of spermatogenesis is then dependent on the introduction of cells from a donor male that can give rise to the spermatogenic lineage; for example, spermatogenic stem cell transplantation (SSCT). Integration of a donor’s germline within a surrogate recipient’s seminiferous tubules allows attainment of fertility via donor-derived spermatogenesis [4, 5]. In this way, Surrogate Sires can be a mechanism for producers to access desirable genetics and achieve large scale dissemination in a natural breeding scheme that is conducive to all manner of production settings.

Phenotypic considerations of a surrogate sire

An essential aspect of a Surrogate Sire breeding concept is the reproductive development of the sire. To prevent competition with transplanted spermatogenic stem cells (SSCs) and ensure all offspring produced have the donor’s genetics, the testes of an ideal surrogate sire must be devoid of endogenous germ cells, regardless of stage. In addition, the structure and function of the testicular soma must be intact to harbor engraftment of donor SSCs and subsequent establishment of normal spermatogenesis. Any strategy employed to generate recipient males must, therefore, restrict cell damage to the germline. Furthermore, specificity to the gonads is essential to avoid negative health consequences that may arise from treatments or genetic alterations with systemic effects. Beyond lacking germ cells, the growth, behavior, and health of a surrogate male should have limited deviation from unaltered, wild type counterparts.

The application of Surrogate Sires in livestock production systems is impacted by ease of commercialization and scaling to produce a multitude of germline ablated recipient males in a simple and economical manner [6]. Over the last several years, a primary mechanism that has been developed for the generation of germline ablated male livestock is gene editing to inactivate (i.e., knockout) genes that are necessary for germ cell development and function [7,8,9,10,11,12]. With the current state of gene editing technology, embryo manipulation via either direct editing or use of somatic cell nuclear transfer (SCNT) must be employed to produce animals that have germline impacted by the modification [13, 14]. This aspect presents several challenges including the possibility of ranging editing events in the case of direct embryo manipulation and low efficiency in the case of SCNT [7, 14, 15]. For these reasons, engineering of a knockout allele for a gene expressed only in male germ cells would provide a major advantage. In this case, males that are heterozygous and females that are homozygous for the knockout allele would likely be fertile, thus allowing simple crossbreeding to be used to fix the allele within any population and generate a plethora of homozygous germline ablated males to be used as surrogate recipients. This mode of operation would make the production of surrogate sires predictable and scalable to meet the needs of any size livestock operation.

Approaches to germline transplant recipient preparation developed with rodent models

The concept of Surrogate Sires as a breeding technology was borne from the functional demonstration that SSCs exist in mammalian testicular tissue and can be transferred between males [4]. Although initially discovered in mice, the existence of SSCs in testes of all mammalian species is generally regarded as fact. Seminal mouse studies used Kit^W/Kit^WV (W/W^v) mutant and busulfan-treated models, both of which were known to be infertile due to a lack of sperm production, as recipients for transplantation of testicular single cell suspensions from donor male mice [4, 5]. The resulting establishment of spermatogenesis within the recipient testes not only provided evidence for the presence of SSCs but also served as proof of concept for the generation of a surrogate sire.

The use of recipient mice prepared through injection of the chemotoxic compound, busulfan, or bred from the W/W^v line, is a classic representation of the primary methods by which surrogate males may be generated. In the case of busulfan treatment, endogenous germline is depleted via apoptosis of proliferating cells, including those in the spermatogenic lineage. Significant challenges exist for use of busulfan treatment, however, including the need for sublethal doses to achieve a high degree of endogenous germline depletion which has collateral negative impact on the health of the animals and does not lead to death of all endogenous SSCs. Indeed, busulfan-treated mice consistently demonstrate the return of endogenous spermatogenesis over time [16, 17]. Alternatively, W/W^v mice require no treatment to prepare for transplantation as they are depleted of germ cells from birth due to genetic deficiency [18]. A nuance of W/W^v mice is that the genetic mutation not only impacts the germ cells but also causes other health defects including deficiencies in immune cell populations and red blood cell function [19,20,21,22]. Due to the limitations of busulfan treatment and W/W^v mutation, other strategies have been attempted with rodents including irradiation, heat shock, and cold ischemia of the testes to reduce the systemic impact of a chemical treatment or collateral health problems from genetic mutations that also impact somatic cells [23,24,25,26,27]. However, none of these approaches have been shown to result in complete ablation of the endogenous germline.

When considering the translation of these approaches to livestock, discovery of a naturally occurring mutation like the W/W^v mouse model that causes germ cell ablation is unlikely due to the production necessity of eliminating infertility genetics from herds and flocks. In the case of chemical treatment, the collateral negative effects on health and biohazardous nature of chemotoxic drugs is unlikely to be an acceptable approach for preparing large animal recipient males due to the quantity of drug required and the environmental risk of treatment. For these reasons, recent strategies for surrogate sire development with livestock species have centered on the use of gene editing for precise inactivation of genes that are essential for germ cell development.

Over the last two decades, several studies have identified genes in mice with germ cell specific expression that, when knocked out, lead to endogenous germline depletion or complete ablation. Among these candidates are those from the Nanos gene family which encode for RNA-binding proteins that are required for the establishment of functional germ cells. Systematic knockout of these genes has revealed specific roles for Nanos2 in the survival of precursor spermatogonia (i.e., prospermatogonia) and Nanos3 in the survival of migrating primordial germ cells (PGCs) [28]. Outside of the Nanos gene family, Dazl and Etv5 have also been targeted for the generation of surrogate sires due to the known association of their absence or variant expression with male infertility. Expression of Dazl is limited to the germ cell lineage in both males and females and encodes RNA-binding proteins that are essential for meiosis and differentiation of spermatogonia into spermatocytes [29]. In the absence of Dazl, survival and differentiation of germ cells at multiple phases of development is impaired. Because both genetic sexes are sterile due to germline ablation, scaling of Dazl knockout males for a Surrogate Sire breeding system is restricted. In addition, studies with mice indicate that Dazl knockout males still possess undifferentiated spermatogonia which could limit engraftment of donor germline when attempting to generate surrogate sires [29,30,31,32,33]. Expression of Etv5 in both germ cells and Sertoli cells has been identified as essential for establishment of the spermatogenic lineage through regulating maintenance of the undifferentiated spermatogonial population [34, 35]. Although loss of Etv5 function leads to germline ablation, the critical functional role in Sertoli cells and expression in non-reproductive organs including the heart, lungs, and skeletal muscle reduces the suitability of the testes to host donor-derived spermatogenesis. Due to the high level of conservation across gene function between mice and other mammals, these knockout models established a foundation for the preparation of livestock recipient males (Fig. 1).

Fig. 1.

Comparison of strategies for developing germline ablated recipient males as surrogate sires. The suitability of each reported method for yielding the ideal characteristics of a surrogate sire including eliminating the endogenous germline is contrasted. Ideal outcomes are designated as green, those that produce less than ideal or currently undetermined outcomes are designated as yellow, and outcomes that are unsuitable for application are designated as red. Species-specific outcomes are denoted as follows: C = Cattle, G = Goats, M = Mice, P = Pigs, S = Sheep, and R = Rats. Images created in BioRender.

Germline transplant recipient strategies for livestock

Busulfan

Despite the well-known negative effects on overall health, several studies have attempted to use busulfan treatment as a strategy for preparing livestock as germline transplant recipients. Early studies by Hanaramooz et al. treated prepubertal male pigs with varying doses of busulfan to identify a treatment regimen that would deplete the endogenous germline [36]. Unfortunately, systemic toxicity was high for all doses and, therefore, the treatment was not considered a viable strategy for preparation of recipient male pigs. Similar treatment of male lambs with busulfan showed severe negative impacts on health as well and was not sufficient to eliminate endogenous spermatogonia [37]. To overcome the negative effects on overall health, pregnant sows were treated with busulfan to induce germ cell death in developing fetuses with the intent of producing male piglets that were germline ablated. Outcomes demonstrated varying levels of initial germ cell depletion but, by puberty, endogenous spermatogenesis was present, thus presenting a major limitation on utility as a means for preparing recipients for germline transplantation. Indeed, follow-up studies that performed SSCT in recipient boars that had been exposed to busulfan in utero led to a mixture of donor-derived and endogenous sperm production [38]. Another effort to avoid systemic toxicity in postnatal pigs attempted to localize drug effects to the testes through direct injection into the scrotum [39]. Assessment of seminiferous tubules for the presence of germ cells in cross-sections of biopsied testis tissue revealed the major impact from local busulfan treatment was on survival of meiotic and post-meiotic germ cells [39]. Taken together, outcomes of previous studies that treated livestock with busulfan have clearly demonstrated limited utility in preparing recipients for SSCT due to significant negative effects on health and the inability to ablate the endogenous germline.

Irradiation

Alternative cytotoxic exposures to induce germline ablation have also been explored in rodent models as well as large animals. Localized testicular irradiation of rats, sheep, and goats demonstrated incomplete elimination of spermatogonia which varied based on the age of the animal when treatment was applied [36, 40, 41]. In addition, it was noted that irradiation induces calcification of the seminiferous tubules which presents a major barrier for donor-derived spermatogenesis to occur following SSCT [42]. Despite these well documented challenges with localized testicular irradiation, several studies have used the approach in attempts to prepare livestock for use as SSCT recipients. A major limitation of these studies has been that unequivocal demonstration of donor-derived spermatogenesis is not possible because endogenous germline of the recipient persists. Although DNA polymorphisms might exist between the germ cell donor male and recipient male based on breed differences, accurate detection in the ejaculate of the recipient male is difficult. Accuracy of standard PCR-based assessments relies on fidelity of the design and components that are used. Furthermore, ejaculates contain somatic cells in addition to sperm thus simple detection of a DNA polymorphism cannot be linked directly to donor-derived sperm. For these reasons, use of males that are germline ablated rather than depleted is essential for clear determination that germ cell transplantation leads to the intended outcome of donor sperm production.

Genetic engineering

To demonstrate the concept that germline transplant recipient males could be purposefully developed with gene knockout strategies, NANOS2 has been targeted in multiple species. Studies with mice revealed that Nanos2 function is required specifically for male fertility and inactivation leads to germ cell death early in development [8, 28]; however, the capacity for the somatic cell populations in testicles to support spermatogenesis following introduction of SSCs was unknown. Using CRISPR-Cas9 gene editing, multiple precision edited mouse lines harboring inactivation alleles for Nanos2 were generated [8] and knockout males were used as SSCT recipients. Several months after the transfer of donor SSCs the Nanos2 knockout recipients were found to be fertile by natural mating and capable of siring offspring that possessed genetics of the donor male. Importantly, 100% of offspring from all recipients were donor-derived thus demonstrating that no endogenous spermatogenesis occurs in the testes of Nanos2 knockout males. In addition, as Nanos2 expression is specific to the male germline, knockout animals are of normal health, knockout females are fertile, and males that are heterozygous for a knockout allele are also fertile [8, 9]. These findings firmly established Nanos2 as a prime target for translation to generate Surrogate Sire livestock.

To assess translatability of NANOS2 knockout for engineering Surrogate Sires with livestock, CRISPR-Cas9 gene editing was used to inactivate the gene in pigs, goats, and cattle. Like the phenotype of mice, NANOS2 knockout boars, bucks, and bulls were found to be germline ablated but of otherwise normal health [7, 9]. Importantly, SSCT with NANOS2 knockout boars and bucks was shown to produce donor-derived spermatogenesis that persisted throughout a normal reproductive lifespan [9]. Although reports of SSCT in NANOS2 knockout bulls are yet to appear in the peer-reviewed scientific literature, the expectation is a similar outcome to other species. At present, the challenges hindering the readiness of NANOS2 knockout animals for commercial application involve the generation of clean, biallelically edited founder animals and the optimization of SSCT. Due to the nature of direct embryo editing, the risk of mosaicism, the presence of multiple allelic variants, is high and increases the complexity of genotypic assessment. However, since NANOS2 knockout females and heterozygous males maintain fertility, the breeding of founder animals allows for the segregation of allelic variants to propagate the desired genetic knockout thus dampening the impact of this impediment. Beyond these opportunities for technical improvement, with a consistent phenotypic impact on male testicular development across species, cell-specific complete germline ablation, and male specific effect on fertility, the knockout of NANOS2 bears all the hallmarks of the ideal strategy for surrogate sire development as summarized in Fig. 1.

The exploration of alternatives to NANOS2 inactivation for surrogate sire development has yielded a potential use for NANOS3 knockout in the introduction of donor germline through blastocyst complementation. Studies with mice demonstrated that inactivation of the Nanos3 gene leads to apoptosis of PGCs, thus addition of cells from the inner cell mass from a donor blastocyst stage embryo with intact Nanos3 function provides a means to replace the germline in a resulting fetus rather than through transplantation after birth [43]. To validate the conserved role for NANOS3 in livestock species, the gene was knocked out in both pigs and cattle using CRISPR-Cas9 gene editing [10, 12, 44, 45]. In both species, the male phenotype is similar to the NANOS2 knockouts in that the testes are structurally normal yet devoid of germline. In cattle, the presence of typical testicular cell populations in NANOS3 knockout males was further analyzed using single cell RNA-sequencing and confirmed the absence of germ cells [12]. While the intended studies for developing surrogate sires from blastocyst complementation have not yet been performed, the suitability of NANOS3 knockout pigs as recipients has been evaluated via transplantation. Germ cells isolated from wild-type donor males were transplanted into testes of NANOS3 knockout boars and outcomes revealed relatively low levels of sperm in the ejaculates [44]. If the donor-origin of these sperm produced can be proven, NANOS3 knockout males may be transplantation recipients comparable to NANOS2 knockouts. However, utility of targeting NANOS3 for a Surrogate Sires strategy is confounded by the fact that expression is not germline specific thus knockout animals may have unintended negative phenotypic changes [46] and knockout females are also germline ablated thereby limiting scalability for generating an abundance of knockout males [10, 12, 18].

Capitalizing on the promising phenotypes observed in mice, both DAZL knockout sheep and pigs have been evaluated as recipients for SSCT [11, 47]. While the evaluation of the edited sheep testes indicated an absence of prospermatogonia, analysis of the testicular cell composition in piglets suggested that although prospermatogonia were present at 1 week of age, the population was lost by 10 weeks. Following transplantation of donor spermatogonia into the boar testicles, spermatogenesis was detected in 89% of biological replicates but only one male produced sperm with normal motility, and all ejaculates collected had lower than average sperm concentrations [11]. Although promising as a potential means for generating surrogate sires, use of DAZL inactivation does pose limitations (Fig. 1). First, in Dazl knockout mice undifferentiated spermatogonia persist into adulthood [30, 33], and whether this also occurs in livestock is yet to be fully investigated. If undifferentiated spermatogonia are present, the degree of donor SSC engraftment following transplantation will be limited [31]. Second, because both male and female DAZL knockout animals are sterile [29], scalable generation of knockout males for use as surrogate sires relies on editing of embryos directly or SCNT, both of which are inefficient. Lastly, in both mice and pigs lack of DAZL increases the propensity for germ cell tumor formation [48, 49], which could be problematic for developing males intended for natural breeding purposes.

Approaches to transfer of donor germline

Following recipient development, the next aspect of surrogate sire generation is the inclusion of donor germline for the initiation of spermatogenesis. Towards this end, there are two main approaches: SSCT and embryo complementation. Transplantation of SSCs is the delivery of stem cells extracted from the testes of one male into the seminiferous tubules of another male via infusion. The initial transplantation experiments with mice involved microinjection of donor cells directly into accessible regions of seminiferous tubules which were successful in producing donor-derived spermatogenesis, but efficiency was low and translatability to other species limited [4, 5]. In subsequent mouse experiments refinements were made to determine that the rete testis, which serves as a central ductile bundle for connecting all seminiferous tubules, could be accessed via insertion of a microneedle through the efferent duct and donor cell suspension could be infused to fill most of the testis tissue mass [50]. The testes of livestock species have proven to be more challenging to manipulate and access for transplantation than mice because the efferent duct is not an easily accessible structure. While in mice the efferent duct is easily exposed through exteriorization of the testis and manipulation of adipose tissue, efferent ducts in livestock species are embedded in connective tissue making identification and isolation a challenge [51, 52]. To overcome this limitation and achieve widespread dispersion of donor SSCs into the seminiferous tubules, transplantation has been performed via ultrasound guided placement of an infusion needle into the rete testes [9]. Although navigation of the echogenic needle to the rete testis is difficult, successful injection of a donor SSC suspension can be visualized with the ultrasound imaging used during the procedure. Another aspect of the procedure that has been incrementally improved is the composition of the injected single cell solution. Initial SSCT studies involved microinjection of a single cell suspension generated from total testicular tissue of which the relatively rare SSCs were able to engraft in the recipient seminiferous tubules [4, 5]. Following studies yielded both antibody-based and differential plating methods to enrich single cell suspensions for SSCs [53, 54]. This combination of precise injection of cell populations with enrichment for the engrafting SSCs has been proven to result in establishment of donor-derived spermatogenesis in seminiferous tubules of NANOS2 knockout surrogate recipient pigs and goats [9]. To apply this to livestock production settings, however, optimization is still required for determining the optimal age at which recipients should be transplanted to achieve the greatest level of donor SSC engraftment and devising methods for the exponential expansion and maintenance of donor SSCs in culture to provide an abundance of donor SSCs for generating a multitude of surrogate sires.

An alternative method for germline integration is embryo complementation. While SSCT is a well-established technique with minimal optimization needed prior to application, embryo complementation for germline inclusion remains largely experimental in regards to both implementation in production and future route to regulatory approval. First used to investigate the function of genes related to lymphocyte development, this technique involves the creation of a chimera from the injection of pluripotent stem cells derived from the inner cell mass of a donor embryo into a blastocyst stage recipient embryo [55]. When pluripotent cells are injected into embryos with a genetic knockout, it is possible for the cells to fill the void and rescue the developmental aspects that require the missing gene, making this a valuable approach to study gene function. In the case of germline integration, the goal is to generate a blastocyst that is devoid of PGCs to allow the injected pluripotent cells from a donor to be the sole contributor of the future germline. Through early chimera research with mice, scientists noted that, though capable of generating modified animals directly from embryo complementation, germline transmission that would allow the modification to be passed to offspring was not guaranteed. While improvements in germline transmission efficiency were made to the process by altering the source of the donor cells, major advancement in this endeavor came with the use of germline-ablated blastocysts [56, 57]. By using embryos homozygous for knockout alleles of genes essential for germline maintenance such as Tsc22d3, Nanos3, and Prdm14, as recipients, complementation with pluripotent cells from a donor embryo was demonstrated to efficiently produce offspring whose germline was fully derived from the donor cells [57,58,59,60].

Despite the feats accomplished using mouse models, translation of embryo complementation for germline replacement in livestock remains largely untested. In terms of the production of functional gametes following complementation, the only evidence of its efficacy has been in cattle oocytes. However, while the complementation of NANOS3 knockout, PGC-ablated embryos with wild type blastomeres produced oocytes with intact NANOS3, suggesting donor-derived gametogenesis, the physiological functionality and true origin of those gametes was not confirmed, thus the ability of the gametes produced to perform the expected role in offspring generation remains unknown [45]. On the male side, the use of embryo complementation for germline transplantation remains theoretical for livestock. With the introduction of donor pluripotent cells, integration into mesoderm, endoderm, and ectoderm lineages of the recipient embryo is uncontrolled and, therefore, could result in varying levels of chimerism [56]. When applied to commercial settings, this variable chimera composition will likely become a barrier to regulatory approval in addition to the unknown effects the random integration may have on development and production of the animal itself. This nuance becomes even more problematic if the strategy for achieving germline ablation of the recipient embryo involves a gene that does not have germ cell specific expression. In such a situation, the resulting offspring could have developmental abnormalities and/or health deficiencies thus potentially negating the purpose of donor germline integration. For these reasons, a strategy that includes inactivation of a gene that has germ cell specific expression such as NANOS2 to ablate the recipient embryo germline is of utmost importance.

Conclusion

The development of Surrogate Sires technology provides an opportunity to overcome geographic and logistical obstacles to genetic improvement for wide-spread dissemination of trait driving genetics including those developed through selective breeding and via gene editing. Generation of males that are devoid of endogenous germline yet otherwise phenotypically normal which can serve as recipients for the germline of donor males that have elite and/or engineered genetics has the potential to be an invaluable tool for achieving unparalleled trait improvements in livestock populations via natural mating. On the back of foundational rodent studies, the development of the ideal germline ablated surrogate sire has been attempted from many angles, from cytotoxic exposure to genetic alteration. At present, evidence suggests that generation of surrogate sires through gene editing, in particular inactivation of NANOS2, is the most promising strategy. With a consistent and durable male specific germline ablated phenotype that translates across mammalian species, the proven ability to harbor donor-derived spermatogenesis, and the ability to use natural breeding for scalable herd expansion, NANOS2 knockout livestock possess the characteristics that are ideal for a Surrogate Sires breeding strategy. As surrogate sire production can be maintained through natural mating, the barrier to regulatory approval for application is limited only by the verification of the founder animals’ minimal risk to human health and safety, a relatively low hurdle given the testis specific impact of the modification. Furthermore, as surrogate sires are only the vessel for the production of high genetic merit cattle for consumption, it is unlikely the offspring will be regulated, unless the donor genetics themselves are modified. Refinement of the approach over the next few years is poised to provide a novel reproductive biotechnology for commercial livestock breeding that will have significant positive impact on the future of food security on a global scale.

Conflict of interests

The authors declare no competing interests.

Funding Statement

The authors’ work in developing Surrogate Sires technology has been supported financially by grants and contracts with the USDA-NIFA and USDA-ARS systems as well as Genus-ABS Global. The publication fee of this article was covered by JSPS KAKENHI Grant Number 22HP2009.