Explorations of the optimal method for isolating oocytes from zebrafish (Danio rerio) ovary.

Obtaining oocytes from the adult female zebrafish (Danio rerio) ovary has enormous importance in the studies of developmental biology, toxicology, and genetics. It is vital to establish a simple and effective approach to ensure the quantity and quality of oocytes, which will enable the success of follow-up experimental investigation finally. Usually, oocytes are separated with mechanical or enzymatic methods, however, little studies have been done with concerns about the comparative effects. The present study separated zebrafish oocytes of Stage III with five frequently used methods, including stripping, pipetting, hyaluronidase (1.6 mg/ml), collagenase (0.4 mg/ml), and trypsin (0.1%). The cell viability, oxidative stress, mitogen-activated protein kinase (MAPK) protein phosphorylation, and apoptosis levels were selected as main biomarkers to evaluate the oocytes health status. The results showed that both trypsin and hyaluronidase isolation significantly upregulated germinal vesicle breakdown (GVBD) rates and downregulated p38 MAPK activity simultaneously. GVBD rates and survival rates were decreased notably in oocytes separated by the collagenase method. Above results indicate that zebrafish oocytes in vitro are sensitive to enzymatic treatments and the enzymatic isolation is not the suitable mean for collecting zebrafish oocytes although it is time-saving. The mechanical strategy of pipetting remarkably increased the reactive oxygen species and malondialdehyde level in isolated oocytes. Interestingly, oocytes separated with stripping show less physiological and biochemical damages. Therefore, stripping isolation is comparatively recommended as the optimum method for separating and collecting numerous intact and healthy zebrafish oocytes in vitro for the subsequent developmental research.