Zampolla T, Rawson D M, Zhang T
iBEST Institute of Biomedical and Environmental Science and Technology, University of Bedfordshire, Bedfordshire, UK.
Cryo Letters. 2012 Nov-Dec;33(6):435-42.
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.
尽管已经开展了多项关于鱼类卵巢卵泡冷冻保存的研究，但它们的冷冻保存仍然没有成功。在本研究中，首次研究了胎牛血清（FBS）与几种浓度（1M、2M或4M）的甲醇作为冷冻保护剂对III期卵巢卵泡活力的影响，并采用控制慢速冷却进行冷冻保存。在冷冻保护剂处理和冷冻保存后，通过台盼蓝（TB）染色评估膜完整性，并评估III期卵巢卵泡的ATP水平。结果表明，与室温对照组相比，使用所有三种浓度的冷冻保护剂溶液进行冷冻保存后，通过TB染色评估的卵巢卵泡存活率显著降低。结果显示，解冻后2小时进行TB测试时，活力进一步下降。与其他甲醇浓度与10%FBS组合相比，2M甲醇+10%FBS的存活率更高，解冻后10分钟和2小时获得的活力结果分别为70.2±4.2%和46.2±10.4%。而补充10%FBS的1M甲醇在解冻后10分钟和2小时获得的结果分别为51.8±2.4和3.4±1.5，补充10%FBS的4M甲醇获得的结果分别为36.0±5.3和6.4±P含量，所有三种冷冻保护剂溶液均观察到代谢状态受损。在用于冷冻保存斑马鱼卵巢卵泡的培养基中添加10%胎牛血清并不能实现成功的冷冻保存。
