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Yet, developing optimal conditions for the isolation, culture, and maintenance of SSCs in\u00a0vitro remains challenging, especially for chicken. The objectives of this study were to (1) find the optimal age for SSC isolation in Huaixiang chicken, (2) develop efficient protocols for the isolation, (3) enrichment, and (4) culture of isolated SSCs. In the present study, we first compared the efficiency of SSC isolation using 11 different age groups (8\u201379\u2009days of age) of Huaixiang chicken. We found that the testes of 21\u2010day\u2010old chicken yielded the highest cell viability. Next, we compared two different enzymatic combinations for isolating SSCs and found that 0.125% trypsin and 0.02\u2009g\/L EDTA supported the highest number and viability of SSCs. This was followed by investigating optimal conditions for the enrichment of SSCs, where we observed that differential plating had the highest enrichment efficiency compared to the Percoll gradient and magnetic\u2010activated cell sorting methods. Lastly, to find the optimal culture conditions of SSCs, we compared adding different concentrations of foetal bovine serum (FBS; 2%, 5%, 7%, and 10%) and different concentrations of GDNF, bFGF, or LIF (5, 10, 20, or 30\u2009ng\/mL). We found that a combination of 2% FBS and individual growth factors, including GDNF (20\u2009ng\/mL), bFGF (30\u2009ng\/mL), or LIF (5\u2009ng\/mL), best supported the proliferation and colony formation of SSCs. In conclusion, SSCs can be optimally isolated through enzymatic digestion from testes of 21\u2010day\u2010old chicken, followed by enrichment using differential plating. Furthermore, adding 2% FBS and optimized concentrations of GFNF, bFGF, or LIF in the culture promotes the proliferation of chicken SSCs.<\/jats:p>","DOI":"10.1111\/rda.14661","type":"journal-article","created":{"date-parts":[[2024,7,9]],"date-time":"2024-07-09T12:20:18Z","timestamp":1720527618000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Optimal isolation, culture, and in\u00a0vitro propagation of spermatogonial stem cells in Huaixiang chicken"],"prefix":"10.1111","volume":"59","author":[{"given":"Fahar","family":"Ibtisham","sequence":"first","affiliation":[{"name":"Agricultural College Guangdong Ocean University  Zhanjiang Guangdong China"},{"name":"Department of Veterinary Biomedical Sciences, Western College of Veterinary Medicine University of Saskatchewan  Saskatoon Saskatchewan 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