Abstract
Zebrafish ovarian follicle is developed in five stages. The best method of isolationg follicle from ovary is enzymatic
treatment with collagenase and hyaluronidase. 90% L-15 media (pH 9.0, temperature 28o) supplemented with
hCG (for stage I and II follicle) for 24 hour or 60% L-15 media (pH 9.0, temperature 26o) supplemented with DHP
(for stage III follicle) for 8-24 hour for can be used to mature ovarian follicle in vitro. But follicles cultured this way
cannot be fertilized. Follicle of third stage cultured in 90% L-15 media supplemented with DHP or BSA for 270
minute can be fertilized successfully. The follicle can be cryopreserved by slow-cooling method in cryoprotectant
solution of DMSO and methanol prepared in KCl buffer. The viability of cryopreserved follicle can be tested by TB,
FDA+PI and GVBD assay and by calculating ADP/ATP ratio.