The transmission of genetic information across generations depends on the integrity of the germline. Primordial germ cells (PGCs) are the embryonic precursors of mature germ cells, spermatozoa and oocytes, and their proper development is essential for reproductive success. PGCs are initially formed in extraembryonic regions and migrate to the developing gonads. Through a series of mitotic divisions, meiosis, and differentiation, PGCs transform into fully mature gametes that carry the genetic material required for the creation of a new individual upon fertilization. The NANOS gene family has been found to play a critical role in germ cell development across various organisms, including mammals, with specific expression in germ cells. Through murine studies, Nanos3 has been identified as a crucial regulator of germ cell development in both sexes, protecting germ cells from apoptosis during migration and colonization of the gonadal ridge. In contrast, Nanos2 is specifically involved in male germ cell differentiation and maintenance of the spermatogonial stem cell population. These genes exert their control through translational repression of target mRNAs, influencing the expression of key factors involved in meiosis and germ cell differentiation. Recent studies in pigs, sheep, and cattle have shown that NANOS2 knockout (KO) animals replicate the male-specific germline ablation observed in mice, while female germline development remains unaffected. Similarly, NANOS3 KO livestock, including male and female pigs and a female bovine fetus, exhibited a complete loss of germ cells but normal gonadal development. Notably, live NANOS3 KO cattle have not been generated to date, and the specific role of NANOS3 in male cattle has yet to be explored. Collectively, these findings suggest that NANOS3 KO livestock could serve as potential hosts for germline complementation, a technique where donor cells from one genetic background replace the germline of a sterile host with a different genetic background. Germline complementation in livestock offers the potential to generate germ cells from highly genetically valuable donor animals in the gonads of sterile host animals, thereby increasing the availability of gametes from desirable dams and sires (i.e., surrogate sires, or more generally, surrogate hosts). Studies have demonstrated the achievement of germline complementation and the production of live, donor-derived offspring in rodents as well as non-mammalian food species, including chickens and fish. To facilitate the application of germline complementation technology in livestock, it is crucial to develop efficient methods for generating germline-ablated hosts that retain the function and architecture of their somatic cell gonadal support tissues. One promising method involves using genetic tools like gene editing (GnEd) to inactivate essential genes for germline production at the embryo stage. This creates a vacant germline niche within the host, providing an opportunity for donor cells to colonize and develop in the germline in the absence of competition from endogenous germ cells. Among the genes targeted in mammals to achieve germline ablation thus far, the RNA-binding protein gene, NANOS3, stands out as a promising target in cattle for two primary reasons. The disruption of NANOS3, being one of the earliest germline-specific genes expressed, is anticipated to result in the early elimination of PGCs. This would create a favorable environment for exogenous donor cells to migrate to and colonize the gonadal ridge, offering the possibility of germline complementation at an early stage of embryogenesis. Furthermore, if NANOS3 is confirmed to play an essential role in both male and female germ cell development, as hypothesized, the generation of NANOS3 KO cattle could serve as hosts for introducing donor-derived germ cells in both sexes, thus expanding the potential for novel breeding schemes. This study aimed to investigate the consequences of eliminating NANOS3 in bovine germline development and evaluate the potential for NANOS3 KO cattle to serve as hosts for germline complementation. To accomplish this, an approach to achieve a CRISPR/Cas9-mediated KO of NANOS3 via co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes at 6 hours post fertilization in in vitro produced bovine zygotes was successfully optimized. This efficient and repeatable NANOS3 KO method allowed the generation of NANOS3 KO cattle and investigation of the impact of NANOS3 elimination on bovine germline development at different stages of development and at reproductive age. Through embryo transfer, eight pregnancies with NANOS3-presumptively-edited embryos were produced, which were collected at four different timepoints for analysis. These timepoints included during sexual differentiation (41 days of fetal age (d)), after sexual differentiation (90d), at the perinatal stage (283d), and post-puberty (15 months of age (mo)). These samples were comprehensively evaluated using DNA, RNA, protein, and physiological assessments. Long-read sequencing analyses showed the achievement of a remarkable 75% KO rate, with all but one of the edited NANOS3 alleles resulting in a predicted KO (i.e., loss-of-function). Immunofluorescence analyses revealed that male NANOS3 KO bovine gonads exhibited the elimination of PGCs as early as 41d. Further analyses using single-cell RNA sequencing confirmed a complete loss of germ cells in the NANOS3 KO fetal and perinatal testes, while maintaining normal testis cord formation and the presence of somatic support cell populations. Ultimately, three live, healthy NANOS3 edited calves were born without assistance, a heifer calf #854, and two bull calves, #838 and #3964. Heifer #854 and bull #838 were determined to be NANOS3 mosaic KOs. On the other hand, bull #3964 showed editing without complete KO, as he carried an allele with only small, in-frame deletions, that did not result in a complete loss-of-function mutation. The live NANOS3 edited animals exhibited normal growth patterns and were ultimately harvested at 15mo to enable collection of meat samples and their reproductive tracts. The edited bulls both exhibited normal pre-pubertal reproductive hormone profiles. At sexual maturity, KO bull #838 was found to have normal libido and an anatomically normal reproductive tract, although no spermatozoa were present in his ejaculate. Additionally, histological analysis of his testes confirmed the absence of germ cells and the presence of gonadal somatic support cells, indicating successful ablation of the germline while preserving the integrity of the somatic gonad. In contrast, the sexually mature NANOS3 edited bull #3964 exhibited fertility as evidenced by the presence of spermatozoa in his ejaculate and cross-sections of his testes. These findings suggest that bovine NANOS3 is a haplosufficient gene, and that the allele with small, in-frame deletions produced a functional protein. The NANOS3 KO heifer #854 also had a complete loss of germ cells, as confirmed by the absence of oogenesis in her ovarian tissue. However, she also exhibited an anatomically abnormal reproductive tract and irregular gonad development. Additionally, her hormone profiles indicated a lack of reproductive cycling and functional granulosa cells, consistent with the absence of germ cells and follicles in this animal. The relationship between germ cells and supporting somatic cells in the gonad is crucial for the successful development and function of gametes, so any disruption to germ cells can have detrimental effects on the coordinated processes and functions of both germ and somatic cells involved in gametogenesis. In this study, the absence of germ cells in NANOS3 KO cattle had a greater impact on the normalcy of ovarian development as compared to testes development. Finally, the meat composition from these NANOS3 KO cattle at 15mo was within the bounds of the normal variation seen in international meat compositional databases, which is not surprising as NANOS3 is a germline specific gene, and the lack of germ cells would not be expected to have an impact of meat composition. Overall, this study provides insights into the role of NANOS3 in bovine germline development and indicates the potential of NANOS3 KO cattle to serve as hosts for donor-derived exogenous germ cell production in both sexes. This could unlock both an opportunity to reduce the genetic lag between elite seedstock animals and the genetic merit of commercial cattle, and additionally provide an efficient means for the generation and dissemination of genetically improved GnEd donor genetics.