In order to study cryopreservation of zebrafish (Danio rerio) oocytes, large numbers of oocytes need to be isolated from the ovaries for experimental use. Zebrafish oocytes have been previously separated from ovaries mechanically and the method is laborious and time consuming. The aim of the present study was to develop a simple and rapid method for obtaining large number of morphologically and functionally intact zebrafish oocytes at different stages of development. Zebrafish ovaries were treated with three enzymes-trypsin, collagenase or hyaluronidase at different concentrations (0.2-1.6 mg/ml) for different time periods (5-20 min). Membrane integrity and development competence of the oocytes isolated mechanically and enzymatically from zebrafish ovary were assessed. The results showed that when optimal concentration and treatment time combinations were used, high separation and viability rates of oocytes could be obtained with all the three enzymes. The best conditions were confirmed as 1.6 mg/ml hyaluronidase treatment for 10 min or 0.4 mg/ml collagenase treatment for 10 min at 22 degrees C. Oocytes survivals after optimal hyaluronidase enzymatic separation were 96.6+/-0.7%, 92.9+/-1.3% and 94.6+/-0.9% for stages I, II and III oocytes, respectively using trypan blue staining. Successful enzymatic separation of zebrafish oocytes is reported here for the first time. The method developed in this study will undoubtedly assist investigations on cryopreservation of zebrafish oocytes and studies on zebrafish oocytes in general.
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