DOI: 10.1111/rda.

12838

ORIGINAL ARTICLE

Freeze-­dried spermatozoa: A future tool?

M Olaciregui | L Gil

Obstetric and Reproduction

Area, Universidad de Zaragoza, Zaragoza,
Spain
Correspondence
Maite Olaciregui, Obstetric and
Reproduction Area, Universidad de
Zaragoza, Zaragoza, Spain.
Email: maiteor88@gmail.com
Funding information
Government of Aragon Research Groups
(Fondo Social Europeo, DGA); IA2
Contents
Cryopreservation has been routinely used to preserve sperm of human and different
animal species. However, frozen sperm storage for a long time brings many inconven-
iences because of liquid nitrogen. Many attempts have been made to overcome the
disadvantages of the current cryopreservation method. Freeze-­drying has been pro-
posed as alternative method for sperm preservation to achieve the ability to store
sperm doses indefinitely at ambient temperature or in ordinary refrigerators. At pre-
sent, it has been reported successfully sperm freeze-­drying on many animal species
including canine and feline. It is well known that during freeze-­drying process, sperm
DNA could be damaged, but if suitable protection is provided, the sperm nucleus could
preserve the ability to activate the oocyte and embryos could be generated by intra-
cytoplasmic sperm injection (ICSI). Many factors influence the freeze-­drying efficacy,
so current researches have been conducted to find strategies to control these factors
to maintain the sperm DNA integrity. This review describes the latest method of sperm
freeze-­drying for practical application in preserving and transporting genetic re-
sources. In addition, the approaches to improve the efficiency of the technique were
studied. We demonstrated that the DNA integrity of freeze-­dried dog sperm is af-
fected by the composition of the freeze-­drying solution as well as the temperature
and period of storage. Further studies are necessary to refine freeze-­drying protocol
in order to protect the DNA and maintain the sperm functionality and obtain offspring
from freeze-­dried sperm.

1 |  INTRODUCTION
1.1 | Freeze-­drying
Lyophilization or freeze-­drying (FD) is a preservation method in
which frozen material is dried by sublimation of ice. The main pur-
pose of the FD is to remove water from the system to values that
will no longer support biological growth or chemical reactions. During
the process, the substrate is first frozen, and then, the quantity of
solvent is reduced, first by sublimation (primary drying) and then by
desorption (secondary drying; Jennings, 2002). The first attempts to
preserve cells by dehydration were performed using bacteria, viruses
and fungi (Flosdorf & Kimball, 1939). Since that, FD has been used to
preserving microorganisms, animal tissues, drugs, blood plasma and
antibiotics (Flosdorf & Mudd, 1935). In 1958, lyophilization was ap-
plied to the food industry, especially milk, eggs, yeast, coffee, soup
and juices (Ramirez & Cañizares, 2003). The first time that FD technol-
ogy was applied to sperm cell was six decades ago using fowl semen
(Polge, Smith, & Parkes, 1949) followed by others attempts in human
(Sherman, 1954) and bull spermatozoa (Bialy & Smith, 1957). The first
successful offspring derived from freeze-­dried spermatozoa was in
1998 using mouse sperm (Wakayama & Yanagimachi, 1998) and a
novel mouse ICSI technique (Kimura & Yanagimachi, 1995).
1.2 | Advantages of lyophilization vs cryopreservation
The freeze-­drying of spermatozoa has been established as a new
method for storing genetic resources instead of the cryopreservation.
Until now the most revolutionary and widely used method to preserve
sperm of human and different animal species has been the cryopreser-
vation. However, frozen sperm storage for a long time brings many

Reprod Dom Anim 2016; 51 (Suppl. 3): 1–7 wileyonlinelibrary.com/journal/rda © 2016 Blackwell Verlag GmbH  |  1
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2       Olaciregui and Gil
inconveniences because of liquid nitrogen. FD sperm results in lower
costs and increased safety, as specialized cryoprotectants and a con-
stant supply of liquid nitrogen is not required. Long-­term preservation
of freeze-­dried sperm allows saving storage and space costs as well
as to facilitate transport of preserved samples. A further advantage is
that freeze-­dried sperm can be temporarily stored at room tempera-
ture. Kaneko (2014) reported that it is possible to transport freeze-­
dried mouse sperm overseas at ambient temperature that requires no
special container.
1.3 | Freeze-­drying spermatozoa
When mammalian spermatozoa are freeze-­dried, their motility is
lost, and therefore, they are unable to fertilize oocytes both in vivo
and in vitro. However, the fertilizing ability of reconstituted freeze-­
dried spermatozoa has been demonstrated using the technique of
ICSI (Kwon, Park, & Niwa, 2004). Recently, FD has been applied to
several animal species as an alternative method to sperm cryopreser-
vation: rabbit (Liu et al., 2004), rat (Hirabayashi, Kato, Ito, & Hochi,
2005), primate (Sanchez-­Partida, Simerly, & Ramalho-­Santos, 2008),
dog (Olaciregui, Luño, Gonzalez, de Blas, & Gil, 2015; Watanabe,
Asano, Abe, Fukui, & Suzuki, 2009), cat (Ringleb, Waurich, Wibbelt,
Streich, & Jewgenow, 2011), horse (Choi, Varner, Love, Hartman, &
Hinrichs, 2011; Olaciregui, Luño, Marti, Aramayona, & Gil, 2015),
hamster (Muneto & Horiuchi, 2011), mouse and rat (Kaneko &
Serikawa, 2012a,b), boar (Garcia Campos, Gil, Malo, Mart_ınez, & de
Blas, 2014; Men et al., 2013) and bull (Hara, Tagiri, Hwang, Takahashi,
& Hirabayasi, 2014) including wild animals (Kaneko, Ito, Sakamoto,
Onuma, & Inoue-­Murayama, 2014). Yet, production of live offspring
derived from ICSI with freeze-­dried sperm has just been reported in
mouse, rat, hamster, rabbit and horse, while in the other species the
results did not go further from blastocyst. The main problem is the
DNA damage that occurs during the FD by the action of endonu-
cleases and the reactive oxygen species (ROS). It is well known that
if the DNA integrity of the sperm nucleus could be maintained, the
sperm would maintain the ability to activate the oocyte and embryos
could be generated by ICSI (Kusakabe, Szczygiel, Whittingham, &
Yanagimachi, 2001). Therefore, current researches have been con-
ducted to refine FD protocol in order to protect the DNA and maintain
the sperm functionality.
1.4 | Critical points that affect the effectiveness of
sperm freeze-­drying
Protection of sperm DNA is one of the most important points for the
subsequent development of oocytes after fertilization. Many factors in-
fluence the FD process, so further studies are necessary to find the strat-
egies to control these factors so as to maintain the sperm DNA integrity.
1.4.1 | Condition during freeze-­drying
The maintenance of sperm cellular function can be influenced by
the drying conditions during the FD preservation (Hara et al., 2014).
The interaction between temperature, vacuum pressure and drying
period regulates the kinetics and degree of drying, and these factors
have a marked impact on the spermatozoa (Hochi, Abdalla, Hara, &
Hirabayashi, 2011). In addition, Kawase, Hani, Kamada, Jishage, and
Suzuki (2007) suggested that the condition of FD such as vacuum pres-
sure and the process length has an influence on the ability of freeze-­
dried spermatozoa to participate in embryonic development in vitro.
1.4.2 | Freeze-­drying solution
The FD solution acts as a protective medium with an important role
in preserving sample integrity. As the level of sperm DNA fragmenta-
tion depended on the solution used during FD, it should be able to
support the integrity of at least the nucleus of the spermatozoa (Nakai
et al., 2007). Various groups of substances have been tested because
of their protective capability (Kaneko, Whittingham, Overstreet, &
Yanagimachi, 2003; Kaneko, Whittingham, & Yanagimachi, 2003;
Kusakabe et al., 2001; Martins, Bao, Dodea, Correa, & Rumpf, 2007;
McGinnis, Zhu, Lawitts, BhowmickmS, & Biggers, 2005; Men et al.,
2013). It has been suggested that to preserve freeze-­dried spermato-
zoa for the long term, it is indispensable to protect sperm DNA from
physical damage caused by activity of endogenous nucleases during
storage (Kaneko & Serikawa, 2012a). The addition of a chelating agent,
such as EDTA or EGTA, to the FD solution inactivates sperm DNase
and protects against disruption of sperm DNA during FD and sub-
sequent storage (Kaneko & Nakagata, 2006; Kusakabe et al., 2001).
On the other hand, one way to overcome the detrimental effects of
ROS could be the addition of antioxidant compounds to the extender
to block or prevent oxidative stress (Donoghue & Donoghue, 1997).
Several studies have shown the beneficial effect of antioxidant therapy
on oxidative stress in mammalian spermatozoa (Motlagh et al., 2014).
There is some evidence which proves that the addition of antioxidants
to freezing extenders decreases the detrimental effects of ROS (Luño
et al., 2014). There are many varieties of antioxidants that could be
used in this matter. However, at the moment there are no studies that
evaluate the relation between antioxidants and freeze-­dried sperm.
Our research group is studying the antioxidant effect of rosmarinic
acid added to the FD solution on sperm DNA integrity (Olaciregui,
Gil, Luño, Jerez, & Gonzalez, 2014). Furthermore, sugars have been
studied as a FD medium component in different animal’s species. It
has been suggested that the addition of trehalose to the medium at
suitable concentrations improves sperm DNA integrity after FD pro-
cedure (Garcia Campos et al., 2014; Martins et al., 2007; McGinnis
et al., 2005; Men et al., 2013). McGinnis et al. (2005) claimed that the
presence of trehalose inside of the cell should afford protection to
the internal biomolecules such as proteins, organelles and chromatin.
Other sugars, such as lactose and sucrose, have shown beneficial ef-
fect on DNA integrity of freeze-­dried boar sperm (Garcia Campos
et al., 2014). An alkaline pH of the FD solution also contributes to
DNase inactivation (Kaneko, Whittingham, Overstreet et al., 2003).
Currently, a FD solution that contains 10 mmol/L Tris and
1 mmol/L EDTA with the pH adjusted to 8.0 is the most simple and
stable approach to maintain sperm fertility after FD (Kaneko, 2016).
Olaciregui and Gil
1.4.3 | Temperature and period of storage
Both the FD protocol and subsequent storage temperature of the
spermatozoa are important for suitable preservation (Kaneko, 2016).
Long-­term preservation at room temperatures of freeze-­dried sper-
matozoa would be ideal, and it could make sperm storage and trans-
portation easier. However, Kawase, Araya, Kamada, Jishage, and
Suzuki (2005) indicated that successful preservation of freeze-­dried
spermatozoa for 100 years or more requires storage at temperatures
below −80°C. Currently, 4°C is thought to be the optimal tempera-
ture for long-­term preservation of freeze-­dried spermatozoa (Kaneko
& Serikawa, 2012a,b). In addition, Kaneko and Nakagata (2005) sug-
gested that spermatozoa can be stored for at least 3 months at room
temperature. It has been reported normal offspring obtained from
oocytes fertilized with freeze-­dried mouse spermatozoa that were
air-­transported at ambient temperatures between Japan and the USA
(Kaneko, 2014, 2015). The possibility of short-­term preservation of
freeze-­dried spermatozoa at room temperature is expected to pro-
mote easy and safe transportation worldwide.
Freeze-­dried sperm storage times can be very long, ranging from
days to years. Kaneko and Serikawa (2012a) found no significant differ-
ences in the rate of development for the offspring of embryos produced
from freeze-­dried mouse spermatozoa stored for 3 years when com-
pared with fresh or freeze-­dried spermatozoa stored for a short term.
1.4.4 | Rehydration of freeze-­dried sperm
Rehydration is the process used to restore the lyophilized product to
its original formulation. Freeze-­dried sperm are normally rehydrated
by adding pure water, the volume of which should be the same as the
original volume of the sperm suspension before FD (Gil et al., 2014). It
is known that the DNA damage is induced by mechanical or oxidative
stress not only during FD but also during the holding period before ICSI
after rehydration (Kusakabe et al., 2001). Therefore, the incorporation of
DNA protective agents to the rehydration solution could be interesting.
1.4.5 | Intracytoplasmic sperm injection (ICSI)
The introduction of the ICSI technique has contributed greatly to suc-
cessful production of offspring using freeze-­dried spermatozoa (Kaneko,
2016). After the FD process, spermatozoa lose their motility and mem-
brane integrity, but some of the cells presumably maintain their genetic
integrity. Goto, Kinoshita, Takuma, and Ogawa (1990) obtained live off-
spring when injected oocytes with artificially immobilized spermatozoa.
Thus, ICSI allows live offspring to be obtained from freeze-­dried sperm,
as first described by Wakayama and Yanagimachi (1998). Although suc-
cessful ICSI has been reported in various species, its efficiency could be
increased further by understanding the specific factors related to em-
bryo development after ICSI in each species (Kaneko, 2016). Therefore,
ICSI is another point of study to minimize the damage caused in the
oocyte by the technique itself and improve its subsequent activation.
There are few experiments designed in dogs to evaluate the rela-
tionship between sperm FD procedure and DNA fragmentation. The
|
      3
aim of this study was to examine the dog sperm DNA integrity which
was freeze-­dried in two kinds of chelating agents: EGTA and EDTA,
and whether the supplementation of FD media with rosmarinic acid
and trehalose has beneficial effect on sperm DNA integrity. In addi-
tion, we evaluated the influence of time and temperature of storage.
2 | MATERIALS AND METHODS
2.1 | Chemicals and media
The FD medium was a 10 mmol/L Tris-­HCl + 50 mmol/L NaCl buffer
supplemented with 50 mmol/L EGTA or 50 mmol/L EDTA. These
media were supplemented with 105 μmol/L of rosmarinic acid, with
0.2 mol/L trehalose or with the combination of both. The pH of final
solutions was adjusted to 8.2.
2.2 | Animals
The study was performed following approval by the Veterinary Ethical
Committee of University of Zaragoza. The care and use of animals
were performed according to the Spanish Policy for Animal Protection
RD1201/05, which meets the European Union Directive 86/609 on
the protection of animals used for experimental and other scientific
purposes. All semen samples were obtained from four Beagle dogs,
aged 2 years, whose semen quality had been previously tested in our
laboratory. Fresh sperm samples obtained after ejaculation were ana-
lysed as control.
2.3 | Sperm collection and freeze-­drying procedures
FD was performed as reported by Wakayama and Yanagimachi (1998).
Semen suspension was split into eight equal fractions and centrifuged
700 × g for 10 min at 37°C. The supernatant was discarded and each
fraction was resuspended in the eight experimental FD media (EDTA,
EDTAR, EDTAT, EDTART, EGTA, EGTAR, EGTAT, EGTART) and kept at
37°C for 10 min. A total of 150 μl of sperm suspension from each group
was placed into individual cryotubes (Labcon North America, USA) and
plunged into liquid nitrogen for 5 min. Then, samples were transferred
to a programmable freeze-­dryer (LyoBeta 25, Telstar) previously cooled
to −50°C. Samples from the eight treatments were submitted to a FD
process under the same conditions: a primary drying at a pressure of
0.053 mbar and temperature of −68°C, and a secondary drying at a
pressure of 0.018 mbar and a temperature of 20°C. Finally, the vials
were stored at two different temperatures, 4°C and at room tempera-
ture. Semen samples were evaluated at 1, 6 and 12 months of storage.
2.4 | Rehydration of freeze-­dried spermatozoa
Freeze-­dried sperm samples were rehydrated by adding 150 μl of
Milli-­Q water. The sperm suspension was centrifuged for 2 min at
1,000 g, and the supernatant was removed. The sperm pellet was
resuspended in 500 μl phosphate-­buffered saline (PBS), and samples
were submitted to DNA fragmentation analysis.
 |
4       Olaciregui and Gil

The results obtained after running SCD test to analyse the

2.5 | Sperm DNA fragmentation analysis
The freeze-­dried sperm from different treatments and control
samples were evaluated by the sperm chromatin dispersion test
(SCD) specifically designed for dog spermatozoa (Canis-­Halomax
kit; Halotech DNA SL, Madrid, Spain) following the manufacturer’s
instructions. This test is based on the different response that in-
tact and fragmented DNA shows after a deproteinization treatment
(Enciso, López-­Fernández, Fernández, García, & Gosálbez, 2006).
Samples were evaluated using fluorescent microscopy (Olympus
BX-­40, Olympus U-­RFL-­T, Tokyo, Japan) at magnification 400×,
and a minimum of 300 spermatozoa were counted per semen sam-
ple. Spermatozoa showing a small and compacted halo around a
compacted nuclear core contained intact DNA, and spermatozoa
that displayed a large and spotty halo around the nuclear core
corresponded to those spermatozoa with fragmented DNA
(Figure 1).
2.6 | Statistical analysis
The statistical analysis was performed using SPSS, version 17.0, for
Windows. Data concerning DNA fragmentation were expressed in
percentages and analysed using the chi-­square test. At least three rep-
lications were carried out in each group. Differences were considered
statistically significant at p < .05.
3 |  RESULTS AND DISCUSSION
The sperm samples evaluated after ejaculation showed a high per-
centage (96.8%) of sperm with small and compacted halo, which in-
dicate intact DNA.
DNA integrity of the lyophilized dog spermatozoa are shown in
Table 1. The DNA damage levels in the EGTA groups were signifi-
cantly lower than those in the EDTA group (p = .001). Kaneko and
Nakagata (2006) demonstrated that a small amount of chelating
agent in the FD solution is necessary to prevent the damage of DNA
mouse sperm during the process. They studied EDTA as an alterna-
tive to EGTA and showed that EDTA had the same effect as EGTA,
but EDTA prevented the fragmentation of sperm chromosomes
more efficiently than EGTA during FD and subsequent preservation.
Conversely, Nakai et al. (2007) reported that EDTA and EGTA are
equally effective at inhibiting endonuclease activity on freeze-­dried
boar sperm. On the other hand, Kusakabe et al. (2001) reported
that EGTA is a suitable chelating agent for adding to FD buffers for
mice spermatozoa, and Kaneko, Whittingham, Yanagimachi (2003)
showed that combination of EGTA and an alkaline pH may synergis-
tically suppress DNase activity when freeze-­dried spermatozoa no
longer have intact plasma membranes. Similarly, Men et al. (2013)
suggested that there is a possibility that EGTA reduces sperm DNA
fragmentation induced by the activity of endonucleases through its
chelating effect. More recently, Garcia Campos et al. (2014) demon-
strated that the combination of EDTA and lactose offered good
properties for the preservation of freeze-­dried boar spermatozoa.
In a recent study performed on freeze-­dried stallion sperm, we ob-
served that the presence of EGTA in the FD solution provided a
higher protective effect on sperm DNA than the presence of EDTA
(Olaciregui, Luño, Marti et al., 2015). Moreover, we demonstrated
that EGTA and EDTA have different chelating action on freeze-­dried
dog sperm. The results showed that the percentage of spermato-
zoa with fragmented DNA was significantly higher when the semen
T A B L E   1   Effect of the different factors studied on the DNA
integrity of freeze-­dried dog sperm evaluated by SCD test

% DNA fragmentation p-­value

Chelating agent
EDTA 14.4 >.01
EGTA 9.7
Rosmarinic acid
No 14.2 >.01
Yes 9.8
Trehalose
No 13.6 >.01
Yes 10.4
Storage temperature
4°C 13.4 >.01
Room temperature 10.6
F I G U R E   1   Freeze-­dried dog sperm processed with Halomax
kit® and stained with SYBR Green fluorescence; sperm nuclei Storage time
with fragmented DNA exhibit a large and spotty halo of chromatin 1 month 3.2 >.01
dispersion (arrow). Sperm nuclei that exhibited small and compact 6 months 9.3
halos of chromatin dispersion corresponded to spermatozoa with
12 months 23.6
unfragmented DNA
Olaciregui and Gil
samples were freeze-­dried in EDTA medium than in EGTA medium
(Olaciregui, Luño, Gonzalez et al., 2015).
Regardless of the chelating agent used, samples freeze-­dried with
media supplemented with rosmarinic acid showed significantly lower
percentage of DNA damage (9.8%) than those in media without ros-
marinic supplementation (14.2%). Sperm DNA might become dam-
aged by both freezing and drying stresses induced by mechanical or
oxidative stress during freezing, during freeze-­drying and during the
holding period before ICSI after rehydration (Kusakabe et al., 2001).
One way to overcome the detrimental effects of ROS could be the
addition of antioxidant compounds to the extender to block or prevent
oxidative stress (Donoghue & Donoghue, 1997). To our knowledge,
there have been no studies focusing on determine the antioxidant
effect of rosmarinic acid on freeze-­dried dog sperm. Recently, Luño
et al. (2014) suggested that the addition of antioxidants to freezing ex-
tenders decreases the detrimental effects of ROS. There are many va-
rieties of antioxidants that could be used in this matter. Among them,
herbal antioxidants have been gaining attention from several research-
ers (Motlagh et al., 2014). Rosemary aqueous extract (Rosmarinus of-
ficinalis) is known to contain important scavengers of free radicals
which may subsequently support the intercellular antioxidant system
(Alvarez, Touchstone, Blasco, & Storey, 1987). This extract has been
successfully added into semen freezing extenders in several species,
including porcine (Malo et al., 2010), canine (González et al., 2010) and
ovine (Gil et al., 2010). Rosmarinic acid (RA), from rosemary, has also
been used as an antioxidant in numerous studies. Luño et al. (2014)
suggested that rosmarinic acid is a potential candidate for use as an
antioxidant in boar sperm cryopreservation extenders. Based on this
study, we supplemented the FD media with 105 μmol/L of rosmarinic
acid, and the results clearly show a protective effect of antioxidant
supplementation on sperm DNA. However, there is a contradictory
report regarding the effects of rosemary, where increasing concentra-
tions of this herb proved to be unsuitable for freezing canine semen
(González et al., 2010).
When trehalose was added to FD medium, the results indicated
a protective effect of trehalose against DNA damage. It was obtained
a significantly higher percentage of sperm integrity in sperm samples
freeze-­dried with trehalose (89.6%) than in media without trehalose
(86.4%). There have been several attempts to reduce DNA damage
during FD procedures. The natural process by which trehalose helps
anhydrobiotic organisms to survive dehydration has attracted much
interest with regard to its potential role in protecting biomolecules,
including sperm DNA (Crowe, Hoekstra, & Crowe, 1992). Trehalose
has been used with some success for the desiccation of mammalian
cells (McGinnis et al., 2005). Lapeña, Pardo, and Gacto (1987) sug-
gested that the number of resulting viable cells can be increased by
the addition of exogenous trehalose immediately before the desicca-
tion treatment. It has been demonstrated that trehalose has beneficial
effects to preserve the genetic material and the proteins of partially
desiccated sperm required for oocyte activation and embryonic de-
velopment (McGinnis et al., 2005). However, reports that used treha-
lose for the in vitro desiccation of cells and biomolecules have been
contradictory. Molina et al. (2004) suggested that trehalose could
|
      5
F I G U R E   2   Effects of interactions between the different factors
studied: media, storage temperature and storage period on the
DNA integrity of lyophilized dog spermatozoa analysed by SCD test
(mean ± SD). The Y-­axis represents the percentage of sperm DNA
fragmentation
not prevent degradation of lyophilized lipid/DNA complexes during
long-­term storage and Garcia de Castro, Lapinski, and Tunnacliffe
(2000) have found trehalose alone unable to preserve desiccated cells.
Recently, it has been demonstrated that trehalose supplementation in
frozen-­thawed semen extender significantly improves the quality of
cryopreserved domestic cat spermatozoa (Susereedamrong, Kunkitti,
Sirisathien, & Manee-­in, 2011). Our result also indicated that sup-
plementation of FD media with trehalose maintained the integrity of
sperm DNA better than no supplemented media.
Regarding influence of storage condition, results showed a signifi-
cantly negative effect of the time but a positive effect of the room
temperature. Freeze-­dried semen samples stored for 1 year reached
the highest level of DNA fragmentation (23.6%). In addition, when the
effect of storage temperature on DNA integrity was analysed, a signif-
icant increase in percentage of DNA damaged sperm was observed
in samples stored at 4°C compared with those at room temperature.
It has been reported that the ability of freeze-­dried mouse sperma-
tozoa to participate in embryonic development was not affected by
storage at 4°C or 25°C for up to 3 months (Wakayama & Yanagimachi,
1998). However, recent studies demonstrated that the storage at a
low temperature is important for the long-­term stable preservation
of freeze-­dried spermatozoa (Hochi, Watanabe, Kato, & Hirabayashi,
2008; Kaneko & Serikawa, 2012a; Kwon et al., 2004). In addition,
Kaneko and Nakagata (2006) showed that 4°C is an optimal tempera-
ture for the preservation of freeze-­dried spermatozoa. In the present
study, freeze-­dried semen samples were stored at 4°C and 22°C, and
the analysis of DNA damage was performed after 1, 6 and 12 months
of storage. We observed that the storage temperature had an influ-
ence on DNA integrity of freeze-­dried dog sperm, and unexpectedly
the lowest level of sperm DNA damage was observed when samples
were stored at room temperature.
Finally, we estimated statistically possible interactions among the
factors simultaneously studied and their influence on sperm DNA in-
tegrity (Figure 2). The results suggested that the level of DNA fragmen-
tation differs significantly (p < .05) among different treatments. Semen
samples freeze-­dried in EGTAT media and stored at 4°C for 1 month
had the lowest rate of DNA fragmentation (1.0%), whereas the highest
 |
6       Olaciregui and Gil
DNA damage rate (65.3%) was observed when semen samples were
freeze-­dried in EDTA media and stored at 22°C for 12 months.
4 |  CONCLUSIONS
Freeze-­drying technology provides a substantial advantage for
biobanking and the maintenance of genetic diversity in laboratory, do-
mestic and wild animal species (Kaneko, 2016). This method has been
proposed to overcome the disadvantages of the current cryopreser-
vation and to achieve the ability to store sperm doses indefinitely at
ambient temperature or in ordinary refrigerators. It is known that if
suitable protection is provided and an intact nucleus is maintained,
spermatozoa could be used to fertilize oocytes through ICSI and are
able to produce live offspring (Martins et al., 2007). Many factors in-
fluence the FD process, so further studies are necessary to find the
strategies to control these factors so as to maintain the sperm DNA
integrity. In the present study, we demonstrated that freeze-­dried dog
sperm DNA integrity is affected by the composition of the FD solu-
tion as well as the temperature and period of storage. We found that
dog spermatozoa lyophilized with EGTA solution had lower DNA frag-
mentation rate than those lyophilized with EDTA solution, and like
rosmarinic acid, trehalose showed a protective effect on sperm DNA.
A negative effect of the time and a positive effect of the room tem-
perature were also observed. Further studies are necessary to refine
FD protocol in order to protect the DNA and maintain the sperm func-
tionality, and consequently to obtain live offspring from freeze-­dried
sperm of the main domestic animals species, and it was obtained in
experimental animals.
ACKNOWLE DG E MEN TS
This study was supported by Government of Aragon Research Groups
(Fondo Social Europeo, DGA) and IA2. The authors would like to
thank the Animal Experimentation Service of Veterinary Faculty of
Zaragoza, especially Gonzalo Romeo, chief of freeze-­drying service.
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