Abstract Spermatogonial stem cells (SSCs) are expected to participate in male infertility therapy, endangered species preservation, and transgenic animal technology by their unique unipotency to differentiate into spermatozoa. The main challenges, however, remain to be addressed including the appropriate conditions to reach good number of these cells and how to derive, culture, and maintain them in vitro. In the present study, the testicular tissues were isolated from 1-d-old male chickens to establish primary cell cultures. This culture led to development of distinguished colonies which were further characterized by alkaline phosphatase (AP) activity assay and gene expression analysis. They were shown to be positive for AP activity and expressed two main transcription factors of OCT4 and STRA8 as indicated by reverse transcription-polymerase chain reaction. These were indications of carrying characteristics of SSCs by these colonies. The cultures were also exposed to different concentrations of glial cell line-derived neurotrophic factor (GDNF), basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) growth factors to seek optimum colony-forming conditions. Colony-forming activity assay indicated that they were able to propagate in vitro with an increased self-renewal property when cultured in the presence of 15 ng/mL of GDNF, 20 ng/mL of bFGF, and 15 ng/mL of LIF. The present work provides an easy and practical method for isolation, culture, and in vitro maintenance of chicken spermatogonial stem cells and introduces appropriate cell culture conditions to improve and maintain their self-renewal property based on supplying the necessary growth factors.

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