title: Cryopreservation of zebrafish (Danio rerio) oocytes using improved controlled slow cooling protocols.
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.
authors.affiliation: LIRANS Institute of Research in the Applied Natural Sciences, University of Bedfordshire, 250 Butterfield, Great Marlings, Luton, Bedfordshire LU2 8DL, UK.
authors.forename: M
authors.initials: M
authors.lastname: Guan
authors.forename: D M
authors.initials: DM
authors.lastname: Rawson
authors.forename: T
authors.initials: T
authors.lastname: Zhang
mesh_terms: D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D002021:Buffers; D002470:Cell Survival / Q000502:physiology; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000737:chemistry; D005260:Female; D005947:Glucose / Q000737:chemistry; D000432:Methanol / Q000737:chemistry; D009865:Oocytes / Q000502:physiology*; D009930:Organic Chemicals / Q000737:chemistry; D011189:Potassium Chloride / Q000737:chemistry; D013194:Staining and Labeling; D014343:Trypan Blue / Q000737:chemistry; D015027:Zebrafish
chemical_list: D002021:Buffers; D003451:Cryoprotective Agents; C095240:L15 culture medium; D009930:Organic Chemicals; D011189:Potassium Chloride; D000255:Adenosine Triphosphate; D014343:Trypan Blue; D005947:Glucose; D000432:Methanol