Short-term gonadal cultures are sufficient for germline transmission in a songbird.

Primordial germ cells (PGCs) are reproductive stem cells that are valuable for avian biobanking and germline transmission. However, their long-term culture remains a challenge beyond a few poultry species. Here, we compare the transcriptomic impact and reproductive viability between short- and long-term chicken and zebra finch gonadal PGC culture protocols. Using single-cell transcriptomics, we identified rapid germline differentiation in zebra finch gonadal cultures compared to stable PGC identity in chicken, highlighting divergent signaling pathway responses to in vitro germline culture between species. These differentiation profiles were similarly found in low-serum zebra finch blood PGC cultures. Nonetheless, injections of these short-term zebra finch gonadal PGC cultures demonstrated sufficient gonadal reconstitution of host embryos to produce zebra finch germline chimeras and donor-derived offspring. This study demonstrates the minimal viability of short-term songbird gonadal cultures, and offers a roadmap to improve the longevity and self-renewal of non-poultry avian PGC cultures.