Isolation, culture and bacterial contamination of oogonial stem cells of Brown trout, Salmo trutta macrostigma (Dumeril, 1858)

Abstract

Increased commercial value of brown trout, Salmo trutta macrostigma has lead to its declined natural stock due to over exploitation, and thereby made it an endangered species. Cryopreservation of spermatogonial and oogonial stem cells may help in protecting this species. In this context, we have earlier isolated and cultured spermatogonial stem cells from male S. t. macrostigma. In this study, we report isolation and culture of oogonial stem cells from brown trout (S. t. macrostigma). In addition, bacterial contamination in oogonial cell culture media were identified and described. Wild females were obtained from KIN Trout Fish Farm (Kahramanmaras, Turkey). In order to identify the appropriate size, age and ovary structure for oogonial stem cell isolation and culture, the ovary structure was morphologically and histologically studied. Fish were anesthetized with 0.04% 2-phenoxethanol. The ovary tissue were digested by 0.25% trypsin-EDTA.HBSS, with 1.0 mu g/mL NaHCO3, antibiotics were used to maintain cells in a viable stage. The concentration of cells was measured by hemocytometer. Antibiogram and Gram staining techniques were applied to the culture media contaminated with bacteria. Appropriate age, size and weight of trout for oogonial stem cell isolation and culture were identified as 7+ month old, 14.6 +/- 1.6 cm, 28.2 +/- 7.7 g, respectively. The highest oogonial stem cells were measured in the perinucleolar stage of the ovary. Enterobacter cloacae and Acinetobacter baumannii were identified in the contaminated cell media. Density of oogonial stem cells were measured as 5.48x10(5)+/- 2.6x10(5) cells/mL. In this study, germ cell isolation and culture technique was developed for S. t. macrostigma.