Efficient long-term cultivation of chicken primordial germ cells (cPGCs) is essential for various avian research and biotechnology applications. Our study aimed to address the challenge of inconsistent culture success by investigating strain-specific variations and optimizing culture conditions using two distinct media: Ovotransferrin-enriched medium (OTM) and chicken serum-supplemented medium (CSM). We demonstrated that each chicken strain has unique nutritional requirements, with Hubbard cPGCs thriving in OTM and Bovans cPGCs favoring CSM. This strain-specific variation was effective in derivation and proliferation rates and the expression of stem cell-specific markers such as POU5F3/OCT4 and NANOG. Furthermore, our study confirmed the sustained germ cell identity of long-term cultured cPGCs through the expression of DAZL, DDX4, and EMA1 germ cell markers. We also showed that cultured cPGCs retained their migratory abilities and transfectability, successfully generating G0 germline chimeras and G1 transgenic Bovans chickens. These findings highlight the importance of optimized culture conditions depending on the genotype to enhance the viability and genetic stability of cPGCs, paving the way for more effective genetic modifications and conservation strategies in avian species.

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