Viability analysis of oocyte-follicle complexes and gonadal fragments of zebrafish as baseline for toxicity testing

To achieve more information about growth and development of oocytes in teleost fish or concerning toxicity testing, it is necessary to develop adequate in vitro oocyte culture conditions. Herein, initial stages of zebrafish oocytes (I, primary; II, cortical; III, vitellogenic) were analyzed under serum-free medium conditions as gonadal fragments or as separated oocyte-follicle complexes. Two vital dye staining methods (MTT, trypan blue) were applied to assess mitochondrial activity and membrane integrity of the oocytes during 4 days, and compared to morphological alterations studied by transmission electron microscopy. Vital dye staining indicated reduced viability at day 4 for all stages in both in vitro culture methods. Additionally, the viability decreased significantly in gonadal fragments at day 2 for stages III (MTT, TB) and II (TB only). Signs of degradation at the ultrastructural level (vacuoles, disintegration of endoplasmic reticulum and detachment of follicular cell layers) appeared in gonadal fragments at day 4 for stages II and III, and in separated oocyte-follicle complexes both at day 4 for stages I-III, and at day 2 for stage III. In conclusion, zebrafish oocytes at stages I and II seemed viable for 2 days as separated oocyte-follicle complexes considering their mitochondrial activity, membrane integrity and ultrastructural morphology. Cultured as gonadal fragments, the majority of analyses indicated similar results for stages I and II oocytes. In contrast, stage III oocytes seemed viable for not longer than 24 h. Results should be taken into consideration for the experimental design of in vitro assays using teleost fish oocytes.