title: The use of fetal bovine serum for cryopreservation of stage III zebrafish (Danio rerio) ovarian follicles.
abstract: Although several studies on fish ovarian follicles cryopreservation have been carried out, their cryopreservation still remains unsuccessful. In this study, for the first time the effect of Fetal Bovine Serum (FBS) in combination with several concentrations of methanol (1, 2 or 4M) as cryoprotectant on stage III ovarian follicles viability was investigated and cryopreservation using controlled slow cooling was undertaken. Membrane integrity assessed by trypan blue (TB) staining and ATP level of stage III ovarian follicles were evaluated following cryoprotectant treatment and cryopreservation. The results showed that the survival of ovarian follicles after cryopreservation, assessed by TB staining, was significantly decreased using all three concentrations of CPAs solutions when compared to room temperature controls. The results showed a further decrease of viability when the TB test was performed 2h post-thaw. 2M methanol+10 percent FBS resulted in higher survival rate compared with the other methanol concentrations in combination with 10 percent FBS, viability results obtained 10 min and 2 hours post-thaw were 70.2 +/- 4.2 percent and 46.2 +/- 10.4 percent respectively. Whilst results obtained with 1M methanol supplemented with 10 percent FBS 10min and 2 hours post-thaw were 51.8 +/- 2.4 and 3.4 +/- 1.5 and those obtained with 4M supplemented with 10 percent FBS were 36.0 +/- 5.3 and 6.4 +/- P content was observed with all three cryoprotectant solutions indicating a compromised metabolic status. The addition of 10 percent fetal bovine serum to media used for cryopreservation zebrafish ovarian follicles did not allow the achievement of successful cryopreservation.
authors.affiliation: iBEST Institute of Biomedical and Environmental Science and Technology, University of Bedfordshire, Bedfordshire, UK.
authors.forename: T
authors.initials: T
authors.lastname: Zampolla
authors.forename: D M
authors.initials: DM
authors.lastname: Rawson
authors.forename: T
authors.initials: T
authors.lastname: Zhang
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000378:metabolism*; D005260:Female; D000432:Methanol / Q000378:metabolism*; D006080:Ovarian Follicle / Q000166:cytology* / Q000254:growth & development / Q000378:metabolism; D044967:Serum / Q000378:metabolism*; D015027:Zebrafish / Q000254:growth & development / Q000378:metabolism*
chemical_list: D003451:Cryoprotective Agents; D000255:Adenosine Triphosphate; D000432:Methanol