Assessing the tolerance to room temperature and viability of freeze-dried mice spermatozoa over long-term storage at room temperature under vacuum

Abstract

Freeze-drying has been frequently used to preserve food and microorganisms at room temperature (RT) for extended periods of time; however, its application to mammalian species is difficult. Here, we developed a method to prolong the stability of freeze-dried (FD) mice spermatozoa at RT for more than one year without using any cryoprotectant agents. Our data showed that maintaining a vacuum in ampoules is critical to ensuring the viability of FD spermatozoa, as the stability of spermatozoa DNA increased when imperfectly vacuumed ampoules were detected using a non-destructive test and eliminated. Finally a large number of healthy offspring were obtained from mice oocytes fertilized with FD spermatozoa stored at RT for more than one year. Although the birth rate from three-month stored spermatozoa was lower than that from one-day stored spermatozoa, no further reduction was observed even in one-year stored spermatozoa. Therefore, FD spermatozoa preserved in this study were highly tolerant to warm temperatures. This method of storage shows a great potential for the preservation of genetic resources of mammalian species, such as genetically-modified mouse strains, without the use of electric power.

Tolerance of the freeze-dried mouse sperm nucleus to temperatures ranging from −196 °C to 150 °C

“…However, recent advances in reproductive biotechnology allow us to resurrect mammalian species from ‘dead’ cell nuclei. For example, healthy offspring were obtained not only from frozen cadavers 7–9 , but also from FD mouse spermatozoa that had been preserved at RT for more than 1 year 14,15 or even exposed to space radiation by preservation at the International Space Station for 9 months 18 .…”

“…However, previously, we and others have demonstrated that the dead bodies or frozen cadavers of mammalian species could be ‘revived’ when their spermatozoa or somatic cell nuclei were transplanted into oocytes to produce fertilized or cloned offspring 7–10 . In addition, recently, we and others have demonstrated that the complete drying of mammalian spermatozoa—similar to the anhydrobiotic state of Tardigrades—allowed their preservation even at room temperature without losing their potential for supporting development after fertilization 11–15 . These findings suggest that, although the entire mammalian body is susceptible to extreme environments, its cell nuclei can retain vital potential and that healthy offspring can be generated from them using recent reproductive biotechnology.…”

“…These findings suggest that, although the entire mammalian body is susceptible to extreme environments, its cell nuclei can retain vital potential and that healthy offspring can be generated from them using recent reproductive biotechnology. If such nuclei exhibit a strong tolerance against extreme environments for long periods, it might be possible to resurrect extinct species from frozen cadavers 7,8 , rescue endangered species from waste products 9,16,17 , preserve genetically modified mouse strains at room temperature 15 and conserve genetic resources against disasters on Earth (such as the loss of electric power) using storage on the Earth’s moon 18 . Recently, several reports have suggested that the environmental tolerance of DNA from freeze-dried (FD) spermatozoa is increased compared with that of fresh cells; however, it has not been confirmed whether animals could be generated from such spermatozoa 19,20 and the limitations of this technology are not clear.…”

Exploring dry storage as an alternative biobanking strategy inspired by Nature

“…In most freeze-drying studies, the spermatozoa were snap-frozen without any attempt to maintain their membrane integrity (so called 'viability') [e.g. 80,98,99] while in other studies an attempt to obtain viable cells was made by using proper cryopreservation techniques [e.g. 90,100].…”

Effects of temperature and storage time on the motility, viability, DNA integrity and apoptosis of processed human spermatozoa

“…These procedures include evaporating drying, vacuum of TEST‐yolk buffer, electrolyte‐free medium and sodium alginate extender (Bhowmick et al, ; Jaskey & Cohen, ; Kikuchi et al, ; Merino et al, ; Riel et al, ; Yániz et al, ). Recently, freeze‐dried mice spermatozoa under vacuum could fertilise a large number of oocytes and produce healthy offspring (Kamada et al, ).…”