Evaluation of zebrafish (Danio rerio) ovarian follicle viability by simultaneous staining with fluorescein diacetate and propidium iodide.
Reliable fish oocyte quality assessment methods are essential in developing protocols for cryopreservation as well as their in vitro maturation and fertilisation. Current ovarian follicle viability assessment methods either lack sensitivity (e.g. Trypan Blue staining-TB) or are stage dependent (e.g. in vitro maturation and observation of germinal vesicle breakdown-GVBD). The aim of the present study was to develop a new viability assessment method for zebrafish ovarian follicles that is reliable, sensitive and not-stage specific. Fluorescein Diacetate (FDA) and Propidium Iodide (PI) were used for the first time to assess viability of zebrafish ovarian follicles. After preliminary studies to evaluate the efficacy of FDA and PI, a combination of these two fluorochromes was subsequently chosen and compared with TB staining and GVBD test in a series of cryoprotectant toxicity studies and following cryopreservation using stage III ovarian follicles. In all cases the FDA-PI test proved to be more sensitive than TB staining but less sensitive than the GVBD test. Ovarian follicle survivals after 4M Methanol treatment for 30 min at 22 degree C were 67.4 +/- 4.4%, 43.9 +/- 3.8% and 19.6 +/- 1.9% using TB, FDA-PI and GVBD test respectively. Survivals after cryopreservation procedure were 38.9 +/- 4.0% and 28.9 +/- 2.5% using TB and FDA-PI respectively when Hank's solution was used as medium and 45.2 +/- 4.3% and 35.2 +/- 3.5% when KCl buffer was used. The results showed the method to be promising, and it may offer a new approach for viability assessment of fish ovarian follicles.
Zampolla
T
T
LIRANS Institute of Research in the Applied Natural Sciences, University of Bedfordshire, Luton, Bedfordshire, UK.
Zhang
T
T
Rawson
D M
DM
D000818:Animals; D002021:Buffers; D002470:Cell Survival; D004396:Coloring Agents; D015925:Cryopreservation; D022162:Cytoplasmic Vesicles; D005260:Female; D005452:Fluoresceins; D006080:Ovarian Follicle / Q000166:cytology*; D011419:Propidium; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods* / Q000592:standards; D015027:Zebrafish
D002021:Buffers; D004396:Coloring Agents; D005452:Fluoresceins; D011419:Propidium; C018506:diacetylfluorescein