Tsai S, Rawson D M, Zhang T
LIRANS Institute of Research in the Applied Natural Sciences, University of Bedfordshire, Great Marlings, Luton, UK.
Cryobiology. 2009 Jun;58(3):279-86. doi: 10.1016/j.cryobiol.2009.02.002. Epub 2009 Feb 20.
Cryopreservation of fish gametes is of great importance in aquaculture, conservation and human genomic research. The creation of gamete cryobanks allows the storage of genetic material of targeted species for almost unlimited time periods. Cryopreservation has been successfully applied to fish sperm of many species, but there has been no success with fish embryos and oocytes. One of the obstacles to fish oocyte cryopreservation is their high chilling sensitivity and especially at subzero temperatures. Although studies on late stage oocyte cryopreservation has been carried out, there have been no reported studies on cryopreservation of early stage ovarian follicles. The aim of this study is to investigate the chilling sensitivity of early stage zebrafish ovarian follicles before developing protocols for their cryopreservation. Experiments were conducted with stage I (primary growth), stage II (cortical alveolus) and stage III (vetillogenesis) ovarian follicles, which were chilled in KCl buffer and L-15 medium for up to 144h at -1 degrees C in a low temperature bath. Ovarian follicles were also exposed to 2M methanol or 2M DMSO in L-15 medium for up to 168h at -1 and -5 degrees C, respectively. Control follicles were kept at 28 degrees C. Ovarian follicle viability was assessed using trypan blue staining. The results showed that stage I and II ovarian follicles are less sensitive to chilling than stage III follicles. These results were also confirmed following in vitro maturation of the chilled ovarian follicles. The results also showed that L-15 medium is more beneficial than KCl buffer for ovarian follicles at all stages. The presence of both methanol and DMSO reduced chilling sensitivity of ovarian follicles at all stages with methanol being the most effective. The study indicated that stage I and II follicles are less sensitive to chilling than stage III follicles, and that early stage zebrafish ovarian follicles may be better candidates for cryopreservation.
鱼类配子的冷冻保存在水产养殖、保护和人类基因组研究中具有重要意义。配子冷冻库的建立使得目标物种的遗传物质能够被储存几乎无限长的时间。冷冻保存已成功应用于许多物种的鱼类精子，但鱼类胚胎和卵母细胞的冷冻保存尚未成功。鱼类卵母细胞冷冻保存的障碍之一是它们对低温的高度敏感性，尤其是在零下温度时。尽管已经开展了关于晚期卵母细胞冷冻保存的研究，但尚未有关于早期卵巢卵泡冷冻保存的报道。本研究的目的是在制定早期斑马鱼卵巢卵泡冷冻保存方案之前，研究其对低温的敏感性。实验使用了I期（初级生长）、II期（皮质泡）和III期（卵黄生成）卵巢卵泡，将其在低温浴中于-1℃的KCl缓冲液和L-15培养基中冷冻长达144小时。卵巢卵泡还分别在-1℃和-5℃下于L-15培养基中暴露于2M甲醇或2M DMSO长达168小时。对照卵泡保持在28℃。使用台盼蓝染色评估卵巢卵泡的活力。结果表明，I期和II期卵巢卵泡对低温的敏感性低于III期卵泡。冷冻后的卵巢卵泡体外成熟后也证实了这些结果。结果还表明，在所有阶段，L-15培养基对卵巢卵泡比KCl缓冲液更有益。甲醇和DMSO的存在均降低了所有阶段卵巢卵泡的低温敏感性，其中甲醇最为有效。该研究表明，I期和II期卵泡对低温的敏感性低于III期卵泡，并且早期斑马鱼卵巢卵泡可能是更好的冷冻保存候选对象。
