Chapter 39
Preservation of Mammalian Sperm by Freeze-Drying
Levent Keskintepe and Ali Eroglu
Abstract
Long-term preservation of mammalian sperm at suprazero temperatures is desired to save storage and space
costs, as well as to facilitate transport of preserved samples. This can be accomplished by the freeze-drying of
sperm samples. Although freeze-drying results in immotile and membrane-compromised sperm, intracytoplasmic sperm injection (ICSI) can be used to introduce such an immotile sperm into an oocyte and thus
start the fertilization process. So far, it has been shown that improved freeze-drying protocols preserve
chromosomal integrity and oocyte-activating factor(s) in rodent and mammalian species at 4
C for several
years and at ambient temperature for up to 1 year depending on species, which permits shipping freezedried samples at ambient temperature. This chapter concisely reviews freeze-drying of mammalian sperm
first and then presents a simple freeze-drying protocol.
Key words Sperm, Spermatozoa, Freezing, Freeze-drying, Cryopreservation, Long-term storage,
Intracytoplasmic sperm injection, ICSI, Cryoprotectant, Trehalose, EDTA, EGTA, Rosmarinic acid
1 Introduction
Although interest in sperm preservation goes back several centuries
[1], reliable cryopreservation of sperm was achieved for the first
time in 1949 when Polge et al. discovered cryoprotective properties
of glycerol while working with fowl sperm [2]. This discovery
ultimately led to successful methods for low-temperature storage
of bull [3, 4] and human sperm [5]. Initially, dry ice (À78.5
C)
was used to cryopreserve and store sperm, which resulted in
declined sperm motility with increased storage time. In 1963, this
problem was overcome by storing sperm in liquid nitrogen (LN 2 ,
À196
C) [6]. These developments in sperm cryopreservation
greatly facilitated both treatment of human infertility and artificial
insemination and thus genetic improvement of livestock. Other
applications of sperm cryopreservation include (1) fertility preservation for male cancer patients, (2) conservation of endangered
species and maintenance of genetic diversity, and (3) banking of
genetically engineered animal models for human diseases. Meanwhile, cryopreservation of sperm from different species has been
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_39, © Springer Science+Business Media, LLC, part of Springer Nature 2021
721
Preservation of Mammalian Sperm by Freeze-Drying
Levent Keskintepe and Ali Eroglu
Abstract
Long-term preservation of mammalian sperm at suprazero temperatures is desired to save storage and space
costs, as well as to facilitate transport of preserved samples. This can be accomplished by the freeze-drying of
sperm samples. Although freeze-drying results in immotile and membrane-compromised sperm, intracytoplasmic sperm injection (ICSI) can be used to introduce such an immotile sperm into an oocyte and thus
start the fertilization process. So far, it has been shown that improved freeze-drying protocols preserve
chromosomal integrity and oocyte-activating factor(s) in rodent and mammalian species at 4
C for several
years and at ambient temperature for up to 1 year depending on species, which permits shipping freezedried samples at ambient temperature. This chapter concisely reviews freeze-drying of mammalian sperm
first and then presents a simple freeze-drying protocol.
Key words Sperm, Spermatozoa, Freezing, Freeze-drying, Cryopreservation, Long-term storage,
Intracytoplasmic sperm injection, ICSI, Cryoprotectant, Trehalose, EDTA, EGTA, Rosmarinic acid
1 Introduction
Although interest in sperm preservation goes back several centuries
[1], reliable cryopreservation of sperm was achieved for the first
time in 1949 when Polge et al. discovered cryoprotective properties
of glycerol while working with fowl sperm [2]. This discovery
ultimately led to successful methods for low-temperature storage
of bull [3, 4] and human sperm [5]. Initially, dry ice (À78.5
C)
was used to cryopreserve and store sperm, which resulted in
declined sperm motility with increased storage time. In 1963, this
problem was overcome by storing sperm in liquid nitrogen (LN 2 ,
À196
C) [6]. These developments in sperm cryopreservation
greatly facilitated both treatment of human infertility and artificial
insemination and thus genetic improvement of livestock. Other
applications of sperm cryopreservation include (1) fertility preservation for male cancer patients, (2) conservation of endangered
species and maintenance of genetic diversity, and (3) banking of
genetically engineered animal models for human diseases. Meanwhile, cryopreservation of sperm from different species has been
Willem F. Wolkers and Harrie ¨ tte Oldenhof (eds.), Cryopreservation and Freeze-Drying Protocols, Methods in Molecular Biology,
vol. 2180, https://doi.org/10.1007/978-1-0716-0783-1_39, © Springer Science+Business Media, LLC, part of Springer Nature 2021
721