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 22ºC 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.
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Keywords:
FLUORESCEIN DIACETATE;
OVARIAN FOLLICLES;
PROPIDIUM IODIDE;
ZEBRAFISH
Document Type: Research Article
Publication date:
01 November 2008
More about this publication?
CryoLetters is a bimonthly international journal for low temperature sciences, including cryobiology, cryopreservation or vitrification of cells and tissues, chemical and physical aspects of freezing and drying, and studies involving ecology of cold environments, and cold adaptation
The journal publishes original research reports, authoritative reviews, technical developments and commissioned book reviews of studies of the effects produced by low temperatures on a wide variety of scientific and technical processes, or those involving low temperature techniques in the investigation of physical, chemical, biological and ecological problems.