A Study on Freeze-Drying as a Method of Preserving Mouse Sperm

Kawase, Yoshiaki; Suzuki, Hiroshi

doi:10.1262/jrd.10-199e

Cited by 11 publications

(8 citation statements)

References 20 publications

(40 reference statements)

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“…Assisted Reproductive Technologies in mammals has been steadily progressing. Freeze-drying (FD) procedure, or lyophilisation, has been applied as an alternative technology to preserve gene resources [7][8][9][10][11]13,24]. The feasibility of this method is owing to the evidence that freeze-dried spermatozoa allows viable offspring by intracytoplasmic sperm injection (ICSI) in rabbits [15], mice [5,7,13,14,24,25], rats [3,4] and horses [1].…”

Section: Introductionmentioning

confidence: 99%

Long-term preservation of freeze-dried rabbit sperm by adding rosmarinic acid and different chelating agents

et al. 2018

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A B S T R A C TFreeze-drying (FD) technique has been applied as an alternative technology to preserve gene resources to allow simple sperm preservation and shipment at 4°C. Nevertheless, DNA sperm might be damaged by mechanical or oxidative stress throughout FD procedure. Therefore, suitable protection to maintain DNA integrity is required. The aim of this study was to determine the effect of rosmarinic acid (RA) as an antioxidant and two chelating agents (EGTA and EDTA) on the DNA integrity of freeze-dried rabbit sperm after storage of the samples at 4°C and room temperature for 8 months. Rabbit sperm were freeze-dried in basic medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with 50 mM EGTA (1), 50 mM EGTA plus 105 μM RA (2), 50 mM EDTA (3) or 50 mM EDTA plus 105 μM RA (4). Semen samples were kept at 4°C and room temperature during 8 months. After rehydration, DNA integrity was evaluated with Sperm Chromatin Dispersion test observing that DNA fragmentation was higher when semen samples were freeze-dried with EGTA (10.9%) than with EDTA (4.1%) (p < 0.01). Furthermore, RA acted better under adverse conditions and no significant differences were found in temperature storage. Summarizing, FD is a method that can allow simple gene resources preservation among 4°C to 25°C during 8 months and transportation without the need for liquid nitrogen or dry ice. EDTA chelating agent is the most suitable media for freeze-dried rabbit sperm and the addition of RA protects the DNA against the oxidative stress caused during FD procedure. (P. Domingo).Cryobiology xxx (xxxx) xxx-xxx 0011-2240/

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Section: Introductionmentioning

confidence: 99%

Long-term preservation of freeze-dried rabbit sperm by adding rosmarinic acid and different chelating agents

et al. 2018

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“…If spermatozoa can be preserved at RT, the maintenance cost will decrease and the samples will be easier to handle compared with those in a deep freezer or in LN 2 storage. However, at that time, the rate of full-term development of embryos derived from freeze-dried spermatozoa (FD sperm) was very low compared with fresh spermatozoa [3, 7], and the preservation period at RT was only a few months [8,9,10,11]. Consequently, the preservation of FD sperm at RT has not been reliable and is not routinely practiced.…”

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confidence: 99%

Effect of trehalose on the preservation of freeze-dried mice spermatozoa at room temperature

Ito

Wakayama

Kamada

et al. 2019

Journal of Reproduction and Development

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Freeze-drying of spermatozoa is a convenient and safe method to preserve mammalian genetic material without the use of liquid nitrogen or a deep freezer. However, freeze-dried spermatozoa (FD sperm) are not frequently used because of the low success rate of offspring after intracytoplasmic spermatozoa injection (ICSI). In this study, we determined the optimal concentration and a point of action of trehalose as a protectant for the preservation of FD sperm from different mouse strains at room temperature (RT). Although trehalose demonstrated no potential to protect the FD sperm of ICR mice against the freeze-drying procedure itself, the blastocyst rate was significantly improved when FD sperm was preserved for more than 1 month at RT (56–63% vs. 29% without trehalose). The optimal concentration of trehalose was 0.5 M. Importantly, remarkable results were obtained when spermatozoa of inbred mouse strains (C57BL/6N, C3H/He, and 129/Sv) were used, and many offspring were obtained when FD sperm that was preserved for 3 months at RT (26–28% vs. 6–11% of without trehalose) was used. However, when DNA damage in FD sperm was examined by gamma-H2Ax assays, it was found that trehalose failed to protect the FD sperm from DNA damage. These results suggest that trehalose has the potential to protect other sperm factors rather than sperm DNA during preservation at RT for longer periods and trehalose is more effective for inbred mouse strains.

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“…When spermatozoa are freeze-dried or evaporatively dried, none of the sperm survive; however, mouse spermatozoa can maintain the ability to generate offspring when added to water and microinjected into fresh oocytes (31,32), and this may possible with human spermatozoa in the future (33,34). More importantly, such dried spermatozoa can be preserved at room temperature for up to 2 y (35,36) and in a freezer almost indefinitely (37). In addition, the samples are very light and occupy a small volume.…”

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Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months

Wakayama

Kamada

Yamanaka

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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If humans ever start to live permanently in space, assisted reproductive technology using preserved spermatozoa will be important for producing offspring; however, radiation on the International Space Station (ISS) is more than 100 times stronger than that on Earth, and irradiation causes DNA damage in cells and gametes.Here we examined the effect of space radiation on freeze-dried mouse spermatozoa held on the ISS for 9 mo at -95°C, with launch and recovery at room temperature. DNA damage to the spermatozoa and male pronuclei was slightly increased, but the fertilization and birth rates were similar to those of controls. Nextgeneration sequencing showed only minor genomic differences between offspring derived from space-preserved spermatozoa and controls, and all offspring grew to adulthood and had normal fertility. Thus, we demonstrate that although space radiation can damage sperm DNA, it does not affect the production of viable offspring after at least 9 mo of storage on the ISS.International Space Station | preservation | freeze-dry | spermatozoa | fertilization S ince the "space dog" Laika (Лайка) was first placed into orbit in 1957 (1), many humans and animals have been to space or stayed on the International Space Station (ISS) for more than 6 mo. In the future, humans likely will live on largescale space stations or in other space habitats for several years or even over many generations. At that time, assisted reproductive technology (ART) likely will be used to produce humans in space habitats, given that the use of ART by infertile couples has increased year by year and that ART can be performed with cryopreserved spermatozoa or embryos (2, 3). In a similar way, domestic animals likely will be generated by artificial insemination (AI) in space, because many domestic animals are already produced by AI using long-term cryopreserved spermatozoa (4). In addition, genetic diversity is very important for maintaining a species, especially in small colonies, and this could be achieved by cryopreserving a diverse range of gamete cells. The environment in space is very different from that on Earth, however, including high levels of space radiation and microgravity, and the effects of these factors on mammalian reproduction are largely unknown. Although with current technology, producing offspring in such an environment can be difficult or dangerous (5), the study of reproduction in space is a very important subject for our future.So far, the effects of microgravity on early development have been studied using sea urchins, fish, amphibians, and birds (6-12). These studies have concluded that microgravity does not prevent animal reproduction. However, because of the difficulty in maintaining mammals and performing experiments in space, studies of mammal reproduction in space have not progressed as well as in other animals, and only a few papers have been published (13-18). Those studies and our previous study (19) have suggested that mammalian reproduction in space under conditions of microgravity cannot be easil...

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A Study on Freeze-Drying as a Method of Preserving Mouse Sperm

Cited by 11 publications

References 20 publications

Long-term preservation of freeze-dried rabbit sperm by adding rosmarinic acid and different chelating agents

Long-term preservation of freeze-dried rabbit sperm by adding rosmarinic acid and different chelating agents

Effect of trehalose on the preservation of freeze-dried mice spermatozoa at room temperature

Healthy offspring from freeze-dried mouse spermatozoa held on the International Space Station for 9 months

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