PUBLICATION
Cryopreservation of zebrafish (Danio rerio) oocytes using improved controlled slow cooling protocols
- Authors
- Guan, M., Rawson, D.M., and Zhang, T.
- ID
- ZDB-PUB-080429-8
- Date
- 2008
- Source
- Cryobiology 56(3): 204-208 (Journal)
- Registered Authors
- Rawson, David M., Zhang, Tiantian
- Keywords
- Zebrafish oocyte, Controlled slow cooling, ATP content, Trypan blue staining
- MeSH Terms
-
- Adenosine Triphosphate/physiology
- Animals
- Buffers
- Cell Survival/physiology
- Cryopreservation/methods*
- Cryoprotective Agents/chemistry
- Female
- Glucose/chemistry
- Methanol/chemistry
- Oocytes/physiology*
- Organic Chemicals/chemistry
- Potassium Chloride/chemistry
- Staining and Labeling
- Trypan Blue/chemistry
- Zebrafish*
- PubMed
- 18433743 Full text @ Cryobiology
Citation
Guan, M., Rawson, D.M., and Zhang, T. (2008) Cryopreservation of zebrafish (Danio rerio) oocytes using improved controlled slow cooling protocols. Cryobiology. 56(3):204-208.
Abstract
Cryopreservation of gametes provides a promising method to preserve fish genetic material. Previously we reported some preliminary results on cryopreservation of zebrafish (Danio rerio) oocytes using controlled slow cooling and determined the optimum cryoprotective medium and cooling rate for stage III zebrafish oocytes. In the present study, the effects of two different cryopreservation media, cryoprotectant removal method, final sample freezing temperature before LN(2) plunge, warming rate, and the post-thaw incubation time on oocyte viability were investigated. Commonly used cryoprotectant methanol and glucose were used in this study. Stage III zebrafish oocytes were frozen in standard culture medium 50% L-15 or in a sodium-free KCl buffer medium. Oocyte viability was assessed using trypan blue staining and ATP assay. The viability of oocytes frozen in KCl buffer was significantly higher than oocytes frozen in L-15 medium. The results also showed that fast thawing and stepwise removal of cryoprotectant improved oocyte survival significantly, with highest viability of 88.0+/-1.7% being obtained immediately after rapid thawing when assessed by trypan blue staining. However, after 2h incubation at 22 degrees C the viability of freeze-thawed oocytes decreased to 29.5+/-5.1%. Results also showed that the ATP level in oocytes decreased significantly immediately after thawing. All oocytes became translucent after freezing which complicated the use of GVBD test (in vitro maturation of oocytes followed by observation of germinal vesicle breakdown which results in oocytes becoming translucent). New oocyte viability assessment methods are urgently needed.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping