To begin, transfer the excised ovaries of a zebrafish to a six-well plate containing two milliliters of cell dissociation solution. Incubate the ovaries at 28.5 degrees Celsius for two to three hours. Add two to three milliliters of pre-warmed L15 medium to the buffer to stop digestion.
Next, place a 70 micrometer cell strainer into another well of the six-well plate. Add L15 medium to the well, ensuring the medium level is higher than the strainer. Now, use a pipette to draw the digestive medium through the cell strainer.
Remove the excess medium with a pipette. Add four milliliters of fresh L15 medium into the well, and gently resuspend the oocytes. After a couple of minutes, pipette out the supernatant.
To select the oocytes for quality control, transfer them into a 35 millimeter wide dish containing fresh L15 medium. Observe the oocytes under a light microscope using a magnification of 10X or more. Use a blunt injection tool to remove any cell fragments, stage one oocytes, or any other stage oocytes.
For confirmation of the stage of growth, add Hoechst 33342 to the L15 medium containing the oocytes and incubate. Observe the oocytes with a fluorescence microscope under UV laser excitation. Carefully pick out the oocytes that do not meet the desired criteria with a needle.
The juvenile ovary showed an abundance of transparent stage one oocytes accompanied by a smaller population of stage two oocytes. Adult fish ovaries showed a predominance of opaque late stage two to three oocytes. The reference method resulted in numerous stained granulosa cell nuclei on the surface of the oocytes densely enveloping the oocytes.
In contrast, the modified method allowed for the separation of stage one oocytes devoid of granulosa cell nuclei staining.
