University of Guilan Aquatic Physiology and Biotechnology 2345-3966 10 2 2022 09 23 Identification and isolation of sprmatogonial stem cells from beluga ( Huso huso) testicular tissue Identification and isolation of sprmatogonial stem cells from beluga (<i> Huso huso</i>) testicular tissue 1 24 5902 10.22124/japb.2021.20253.1430 FA Mahsa Borhani Ph.D. Student in Fisheries Science, Department of Fisheries, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, Noor, Iran Mohammad Reza Kalbassi Professor in Department of Fisheries, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, Noor, Iran Martin Psenicka Associate Professor in Research Institute of Fish Culture and Hydrobiology, Faculty of Fisheries and Protections of Waters, South Bohemia University, Ceske Budejovice, Czech. Bahram Falahatkar Professor in Fisheries Department, Faculty of Natural Resources, University of Guilan, Sowmeh Sara, Iran Alexander Crawford Associate Professor in Department of Preclinical Sciences and Pathology, Faculty of Veterinary Medicine, Norwegian University of Life Sciences, Oslo, Norway Journal Article 2021 07 29 Spermatogonial stem cells (SSCs) are unique cells and able to transmit genetic information to the next generation, therefore, they play an important role in chimeric fish production and preservation of rare species. This study was performed for the first time with the aim of examining morphology of spermatogonial cells through histological method and then isolating them through enzymatic digestion method from beluga (<em>Huso huso</em>) testicular tissue. Histological examination of testicular tissue of one- to three-year-old beluga showed there are two general types of spermatogonial cells, undifferentiated spermatogonia (SSCs) and differentiated spermatogonia. SSCs were often identified by their large size, irregular nuclear envelope, distinct nuclear components and one to two nuclei. Moreover, in one-year-old fish the frequency of SSCs was significantly higher than 2 and 3-year-old beluga (P<0.05). Isolation of testicular tissue cells of one-year-old beluga through enzymatic digestion was indicated that using 0.1% trypsin enzyme with phosphate buffer for 2 to 3 hours at 16-20°C has higher efficiency based on frequency and viability of beluga spermatogonial cells (P<0.05). This study has provided basic information for isolation beluga SSCs for transplantation to produce chimeric fish. Spermatogonial stem cells (SSCs) are unique cells and able to transmit genetic information to the next generation, therefore, they play an important role in chimeric fish production and preservation of rare species. This study was performed for the first time with the aim of examining morphology of spermatogonial cells through histological method and then isolating them through enzymatic digestion method from beluga (<em>Huso huso</em>) testicular tissue. Histological examination of testicular tissue of one- to three-year-old beluga showed there are two general types of spermatogonial cells, undifferentiated spermatogonia (SSCs) and differentiated spermatogonia. SSCs were often identified by their large size, irregular nuclear envelope, distinct nuclear components and one to two nuclei. Moreover, in one-year-old fish the frequency of SSCs was significantly higher than 2 and 3-year-old beluga (P<0.05). Isolation of testicular tissue cells of one-year-old beluga through enzymatic digestion was indicated that using 0.1% trypsin enzyme with phosphate buffer for 2 to 3 hours at 16-20°C has higher efficiency based on frequency and viability of beluga spermatogonial cells (P<0.05). This study has provided basic information for isolation beluga SSCs for transplantation to produce chimeric fish. https://japb.guilan.ac.ir/article_5902_b8f4592ab93c302505790ed5c7ffe303.pdf