through the intermediate liquid phase again. This latter phase
transition is known as sublimation and can be accomplished in
theory by either further lowering the pressure (see Fig. 1, path C
to D) or supplying heat to the system (see Fig. 1, path C to E). The
latter path is more practical and commonly used in freeze-drying
protocols to sublimate ice. To achieve efficient sublimation, a concentration gradient of water vapor has to be accomplished between
the drying front/chamber and condenser, wherein the temperature
is considerably lower than that in the drying front/chamber, and
thus water vapor migrates to the condenser and freezes there. The
primary drying ends by removal of the frozen unbound water and
results in around 8–10% moisture content depending on sample
composition. To ensure sample stability, the remaining unfrozen
bound water can be removed by desorption in the secondary drying
step, wherein the samples are further heated under the lowest
attainable vacuum to form water vapor from the bound water.
The resulting samples can potentially be stored at ambient temperature and be reconstituted by adding water, whenever needed.
Early studies on freeze-drying of sperm reported encouraging
results, including up to 50% post-rehydration motility with fowl
sperm [2] and bull sperm [11], birth of 12 litters with rabbit sperm
[12], and pregnancies after artificial insemination with freeze-dried
bull sperm [13, 14]. However, subsequent studies failed to reproduce the initial success [15–19] and led to a loss of interest in
freeze-drying of sperm for a while. A breakthrough came in 1998
when Wakayama and Yanagimachi demonstrated development of
normal mice from oocytes fertilized with freeze-dried sperm
[20]. Interestingly, their freeze-drying approach was not extraordinary and resulted in 0% motile sperm after rehydration. In fact, all
rehydrated sperm were dead based on a red/green dual fluorescence viability assay. The use of intracytoplasmic sperm injection
(ICSI) to introduce freeze-dried immotile sperm into oocytes was
the key to their success. Clearly, their results showed that freezedrying of mouse sperm in a cell culture medium (i.e., DMEM and
CZB) was sufficient to preserve integrity of sperm DNA and
oocyte-activating factor(s) for up to 3 months at 4
C and for a
few weeks at ambient temperature. It should be noted that in an
earlier study, similar findings (i.e., development to term) were
reported by Goto et al. [21] after microinjection of bovine sperm
that were dead as a result of cryoprotectant-free freezing and thawing. Taken together, these studies revealed that to support full-term
development, mammalian sperm does not need to be viable in
the conventional sense as long as its genetic integrity is intact.
The breakthrough study of Wakayama and Yanagimachi revived
the interest in freeze-drying of mammalian sperm, and several
studies reporting successful results with freeze-dried sperm from
different species followed (see Table 1 for details). The species
included rat [22, 23], hamster [24], rabbit [25], bovine [26–28],
Freeze-Drying of Mammalian Sperm
723
transition is known as sublimation and can be accomplished in
theory by either further lowering the pressure (see Fig. 1, path C
to D) or supplying heat to the system (see Fig. 1, path C to E). The
latter path is more practical and commonly used in freeze-drying
protocols to sublimate ice. To achieve efficient sublimation, a concentration gradient of water vapor has to be accomplished between
the drying front/chamber and condenser, wherein the temperature
is considerably lower than that in the drying front/chamber, and
thus water vapor migrates to the condenser and freezes there. The
primary drying ends by removal of the frozen unbound water and
results in around 8–10% moisture content depending on sample
composition. To ensure sample stability, the remaining unfrozen
bound water can be removed by desorption in the secondary drying
step, wherein the samples are further heated under the lowest
attainable vacuum to form water vapor from the bound water.
The resulting samples can potentially be stored at ambient temperature and be reconstituted by adding water, whenever needed.
Early studies on freeze-drying of sperm reported encouraging
results, including up to 50% post-rehydration motility with fowl
sperm [2] and bull sperm [11], birth of 12 litters with rabbit sperm
[12], and pregnancies after artificial insemination with freeze-dried
bull sperm [13, 14]. However, subsequent studies failed to reproduce the initial success [15–19] and led to a loss of interest in
freeze-drying of sperm for a while. A breakthrough came in 1998
when Wakayama and Yanagimachi demonstrated development of
normal mice from oocytes fertilized with freeze-dried sperm
[20]. Interestingly, their freeze-drying approach was not extraordinary and resulted in 0% motile sperm after rehydration. In fact, all
rehydrated sperm were dead based on a red/green dual fluorescence viability assay. The use of intracytoplasmic sperm injection
(ICSI) to introduce freeze-dried immotile sperm into oocytes was
the key to their success. Clearly, their results showed that freezedrying of mouse sperm in a cell culture medium (i.e., DMEM and
CZB) was sufficient to preserve integrity of sperm DNA and
oocyte-activating factor(s) for up to 3 months at 4
C and for a
few weeks at ambient temperature. It should be noted that in an
earlier study, similar findings (i.e., development to term) were
reported by Goto et al. [21] after microinjection of bovine sperm
that were dead as a result of cryoprotectant-free freezing and thawing. Taken together, these studies revealed that to support full-term
development, mammalian sperm does not need to be viable in
the conventional sense as long as its genetic integrity is intact.
The breakthrough study of Wakayama and Yanagimachi revived
the interest in freeze-drying of mammalian sperm, and several
studies reporting successful results with freeze-dried sperm from
different species followed (see Table 1 for details). The species
included rat [22, 23], hamster [24], rabbit [25], bovine [26–28],
Freeze-Drying of Mammalian Sperm
723