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Cryobiology. 2017 Oct;78:1-7. doi: 10.1016/j.cryobiol.2017.08.003. Epub 2017 Aug 10.

Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi.

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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. Electronic address:
School of Biological Sciences, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia.


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.


Cryopreservation; Freezing; Mollusc; Sperm survival

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