Hassan Md Mahbubul, Li Xiaoxu, Qin Jian G
School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia; Department of Fisheries Biology and Genetics, Hajee Mohammad Danesh Science and Technology University, Dinajpur 5200, Bangladesh.
Aquatic Sciences, South Australian Research and Development Institute, 2 Hamra Avenue, West Beach, SA 5024, Australia.
Cryobiology. 2017 Oct;78:1-7. doi: 10.1016/j.cryobiol.2017.08.003. Epub 2017 Aug 10.
Low survival of cryopreserved sperm impedes the application of cryopreservation technique in spermcasting oyster species. This study developed a simple method of liquid nitrogen vapor freezing to improve post-thaw sperm survival in the spermcasting oyster Ostrea angasi. The results indicate that the permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were non-toxic to sperm up to 20% concentration and 90 min exposure whereas methanol at 10% or higher was toxic to sperm for any exposure over 30 min. Among the treatments with permeable cryoprotectants, 15% EG produced the highest post-thaw sperm motility. Sperm motility was further improved by the addition of non-permeable cryoprotectants (trehalose and glucose), with 15% EG + 0.2 M trehalose resulting in the highest post-thaw sperm motility among all the combinations evaluated. The durations of 20, 30 and 60 min equilibrations produced a higher post-thaw sperm motility and plasma membrane integrity (PMI) than 10 min. Higher post-thaw motility and PMI were achieved by freezing sperm at the 8 cm height from the liquid nitrogen surface than at the 2, 4, 6, 10 or 12 cm height. Holding sperm for 10 min in liquid nitrogen vapor produced higher post-thaw motility and PMI than for 2, 5 or 20 min. The cryopreservation protocol developed in this study improved both post-thaw motility and PMI of O. angasi sperm at least 15% higher than those cryopreserved using programmable freezing method. Liquid nitrogen vapor freezing might have greater applicability in improving post-thaw sperm quality of spermcasting oyster species.
冷冻保存精子的低存活率阻碍了冷冻保存技术在采精型牡蛎物种中的应用。本研究开发了一种简单的液氮蒸汽冷冻方法，以提高采精型牡蛎安氏巨蛎解冻后精子的存活率。结果表明，渗透性冷冻保护剂二甲基亚砜（DMSO）、乙二醇（EG）和丙二醇（PG）在浓度高达20%且暴露90分钟的情况下对精子无毒，而10%或更高浓度的甲醇在暴露超过30分钟时对精子有毒。在使用渗透性冷冻保护剂的处理中，15%的EG产生了最高的解冻后精子活力。添加非渗透性冷冻保护剂（海藻糖和葡萄糖）可进一步提高精子活力，在所有评估的组合中，15%的EG + 0.2 M海藻糖产生了最高的解冻后精子活力。20、30和60分钟的平衡时间产生的解冻后精子活力和质膜完整性（PMI）高于10分钟。在距离液氮表面8厘米高度冷冻精子比在2、4、6、10或12厘米高度冷冻获得更高的解冻后活力和PMI。在液氮蒸汽中保存精子10分钟比保存2、5或20分钟产生更高的解冻后活力和PMI。本研究开发的冷冻保存方案使安氏巨蛎精子的解冻后活力和PMI均比使用程序冷冻法冷冻的至少提高了15%。液氮蒸汽冷冻在提高采精型牡蛎物种解冻后精子质量方面可能具有更大的适用性。
