BIOPRESERVATION AND BIOBANKING

Volume 00, Number 00, 2018

ª Mary Ann Liebert, Inc.
DOI: 10.1089/bio.2018.0113

Spermatozoa Cryopreservation:
State of Art and Future in Small Ruminants

Chunrong Lv,1–3 Guoquan Wu,1–3 Qionghua Hong,1–3 and Guobo Quan1–3

The cryotolerance of farm animal spermatozoa varies according to their specific features, such as size, shape,
and lipid composition. Thus, it is impossible to develop a standardized freezing procedure for different kinds of
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livestock species. The establishment of an efficient semen cryopreservation procedure will facilitate long-term
conservation of small ruminant genetic resources and extension of artificial insemination in daily production.
Different from sheep, goat seminal plasma contains a phospholipase, which can affect spermatozoa viability
through interaction with milk or egg yolk. Currently, soybean lecithin is a viable alternative that replaces the
components of animal origin in freezing extenders for goat semen. In addition, vitrification or freeze-drying
may act as another alternative to replace traditional cryopreservation. However, these two methods, especially
freeze-drying, may require the aid of the intracytoplasmic spermatozoa injection technology. Furthermore, the
cryoinjury mechanism of mammalian spermatozoa has remained unclear until now. The emergence of pro-
teomics and transcriptomics may provide some inspiration concerning this problem. In this review, we sum-
marize the state of art relating to small ruminant semen cryopreservation, mainly focusing on the current status
of the freezing procedures. In the meantime, some highlights such as protectants, vitrification, and freeze-drying
are also reviewed. Finally, the future perspectives in the field of small ruminant spermatozoa preservation are
discussed.

Keywords: small ruminant, semen, spermatozoa, cryopreservation, vitrification, freeze-drying

Introduction of domestic animal species.3 However, this method requires

a significant investment of money and labor. To resolve

A s an important part of modern livestock industry,

small ruminants, including sheep and goat, can provide
meat, wool, skin, or milk for our society. However, with the
fast development of modern intensified agriculture, the ge-
netic diversity of farm animal species is rapidly being re-
duced in many regions worldwide.1 According to the
Second Report on the State of the World’s ‘‘Animal Genetic
Resources for Food and Agriculture’’ (2000–2014), *99
farm animal species have disappeared from the planet for-
ever. About 17% of livestock breeds are on the brink of
extinction.2 Currently, it is believed that small ruminant
species are facing more serious risk of extinction, mainly
due to the commercial extension of high productive breeds
derived from high-intensity artificial selection and breeding.
Therefore, to conserve the genetic diversity of small rumi-
nant is becoming a necessity.
At present, the establishment of a living conservation
herd is a routine strategy for protecting the genetic resources
these problems, some researchers have attempted to
preserve male or female genetic information through
cryopreservation of spermatozoa or oocytes. However, a
standardized freezing procedure for mammalian oocytes has
not been established because they are sensitive to cryoin-
juries caused by the freezing and thawing process. By
contrast, semen cryopreservation has been applied in the
daily livestock industry, especially in cows. Currently, se-
men cryopreservation has been applied as an important
strategy for establishing the genetic resource bank. Semen
cryopreservation does not need to raise animals, largely
reducing the cost. Furthermore, this technology can con-
tribute to the extension of artificial insemination (AI), which
is a landmark technology in the modern livestock industry.
Semen cryopreservation results in AI being free of the limits
of operating time or site. Now, this method has been widely
used for conservation of the biological diversity of live-
stock.

1
Yunnan Animal Science and Veterinary Institute, Kunming, China.
2
Yunnan Provincial Engineering Laboratory of Animal Genetic Resource Conservation and Germplasm Enhancement, Kunming, China.
3
Yunnan Provincial Meat Caprine Engineering Research Center, Kunming, China.

1
 2 LV ET AL.
In 1937, the early experiments on freezing of sheep se-
men were performed by Bernstein and Petropavlovsky. In
their studies, 9.2% glycerol was used as the permeable
cryoprotectant to freeze semen at -21C.4 Then in 1949, a
milestone breakthrough in the cryobiological history oc-
curred when Polge et al. confirmed the cryoprotective effect
of glycerol on poultry semen.5 Inspired by this finding, the
effects of glycerol on goat semen were first assessed during
the 1950s.6 Another critical transition in semen cryopres-
ervation during the 1950s is that dry ice (-79C) was re-
placed by liquid nitrogen (-196C). Spermatozoa viability
at -196C can be maintained indefinitely. However, the
metabolic activity of spermatozoa does not completely halt
at -79C, which consequently injures the fertility of sper-
matozoa.7 Since then, numerous studies have attempted to
mitigate cryoinjuries on mammalian spermatozoa via the
selection of cryoprotectants and optimization of the freez-
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ing/thawing processes. However, regardless of these dis-
coveries, up to 50% of spermatozoa lose their viability after
freezing and thawing.8 In sheep, 40%–60% of post-thaw
ram spermatozoa remain motile. However, only 20%–30%
are biologically functional.9 In addition, after cervical
FIG. 1. The general procedures used for
cryopreservation of small ruminant semen.
The gray arrows show that the negative ef-
fects of seminal plasma should be removed
during cryopreservation of goat semen. The
dashed arrows represent the procedure of
the one-step method.
AI using post-thaw semen, the occurrence of embryonic
mortality and abnormal spermatozoa transporting patterns
through the female reproductive tract demonstrates com-
promised functions of frozen spermatozoa.10–12
The current cryopreservation procedure for small rumi-
nant semen is modified from that of other farm animals,
such as cattle. Interestingly, the freezing effect on goat
spermatozoa may be more successful than that of ram
spermatozoa. AI with post-thaw goat semen can result in
satisfactory fertility if semen is injected into or through the
cervix. However, the same method is difficult to obtain
similar results in sheep, which may be due to the difference
in the cervix anatomy between goat and sheep.7 During
freezing and thawing, spermatozoa have to face various
types of stresses, such as ice formation, cold shock, che-
mical toxicity, osmotic stress, and oxidative stress. These
stresses primarily influence the plasma membrane, con-
sequently leading to a lower fertility.13,14 Currently, low
pregnancy rates can be achieved when cervical AI with
frozen/thawed semen is performed.15 However, intrauterine
AI can obtain better fertility in comparison with cervical AI,
especially that a lower number of spermatozoa are required
 SMALL RUMINANT SEMEN CRYOPRESERVATION
for intrauterine AI than for cervical AI.8,14 However, this
method requires a specialized instrument and qualified
technicians. In sheep, intrauterine AI is far more effective
than cervical AI. According to the study of Maxwell in
1986, only 20 million motile post-thaw sheep spermatozoa
were required to achieve fertility of more than 50%. By
contrast, cervical AI is believed to need 10 times that dose.16
The cryoinjury mechanism of spermatozoa is complicated
and unclear. In addition, although the semen cryopreserva-
tion procedures have been applied in the livestock industry,
their progress is tardigrade compared with embryos or oo-
cytes. In this review, the current status of small rumi-
nant semen cryopreservation research is summarized. Some
technological highlights, which may give us some inspira-
tion to improve current freezing procedures, are reviewed.
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Current Status of Small Ruminant
Semen Cryopreservation
Cryopreservation greatly prolongs the storage time of
mammalian spermatozoa in vitro, primarily due to complete
inhibition of metabolic activity in a frozen state. According
to the report of Salamon et al., after cryopreservation for 35
years, ram spermatozoa still have a capacity to fertilize
in vivo with intrauterine AI.17 The mainstream procedures
for cryopreservation of small ruminant semen are demon-
strated in Figure 1.
Semen collection
Semen can be collected using artificial vagina or electric
stimulation. However, it should be noted that electric
stimulation may not be effective for the goat because it can
alter the components of seminal plasma, consequently re-
ducing the capability of spermatozoa to tolerate cryoin-
jury.18 To guarantee the quality of collected semen, some
stresses, such as cold shock or urine contamination, should
be considered. Generally, the semen quality is assessed
immediately after collection. The general parameters used
for assessment of semen quality include spermatozoa con-
centration, motility, and morphological normality. In addi-
tion, the other tests, such as the thermal resistance test,
acrosome and membrane integrity, hypo-osmotic swelling
test (HOST), or in vitro fertilization, can be used for as-
sessment of semen quality.19
It is highly valuable to discover a parameter that can
directly reflect the real fertility of spermatozoa. As a simple
and efficient approach with good reliability and repeatabil-
ity, HOST has been used to predict spermatozoa fertility.20
However, Kasimanickam et al. hypothesized that sperma-
tozoa DNA, mitochondrial membrane potential, and motility
may be more reliable than the other parameters for assess-
ment of spermatozoa fertility and the subsequent embryonic
development.21 In addition, some studies demonstrated a
potential correlation between spermatozoa motility and
fertility after AI.22,23 However, despite these attempts, it is
difficult to determine spermatozoa fertility based on a single
parameter at present. Except for the effects of spermatozoa
quality, their fertilizing capability is also influenced by
several factors, such as female fertility, estrus treatment
(natural or hormone-manipulated), season of AI, and in-
semination site (intrauterine or cervical).19 Considering the
3
great value of finding a parameter to determine spermatozoa
fertility, this topic is still attractive.
The general semen cryopreservation procedures
After the assessment of semen quality, sheep semen can
be directly diluted using the freezing extenders based on
Tris, fructose or glucose, citric acid, glycerol, antibiotic, and
egg yolk. In this study, the dilution process should be pru-
dently performed to avoid a ‘‘dilution effect.’’ In previous
studies, the dilution rate may vary from 11- to 26-fold.24
However, the two- to fivefold dilution rates are generally
accepted for freezing sheep semen according to supple-
mented components in freezing extenders.4,24 In terms of the
two-step method, sheep semen is first diluted by the freezing
extenders containing no glycerol. Then, the diluted semen is
further extended by the freezing extenders containing
glycerol. By contrast, when the one-step method is used,
semen is directly diluted using the freezing extenders con-
taining glycerol before equilibration at a low temperature.
The concentration generally used for is *4%–6%.4
After dilution, sheep semen should be slowly cooled to
5C during a minimum time of 1.5–2 hours, depending on
the cooling rate that is applied.19,25,26 Then, chilled semen is
further equilibrated at this temperature for 2–4 hours. During
equilibration, spermatozoa have to adapt to a reduced metab-
olism. Moreover, the equilibration process makes cryopro-
tectants (mainly glycerol) enter cells, consequently reaching
an equilibrium status between intracellular and extracel-
lular concentrations of glycerol or other osmotically active
components.24
After equilibration, diluted semen, in pellet form (0.1–
0.2 mL), can be prefrozen for 3–4 minutes on the surface of
dry ice (-79C). In addition, chilled semen can also be
frozen in straws using an automatic freezing machine with a
freezing rate of -8C/min or a chilled rack. When a chilled
rack is used, straws are generally prefrozen for 7–10 minutes
in liquid nitrogen vapor (between -75C and -125C) (4–
6 cm above the liquid nitrogen surface). Finally, prefrozen
semen is directly plunged into liquid nitrogen.4,19 Straws are
generally thawed for 30–60 seconds in a 35C–40C water
bath. However, semen pellets can be thawed in either pre-
warmed thawing solution (wet thawing) or dry glass tubes
(dry thawing).19,25,27
For goat semen cryopreservation, glycerol can be sup-
plemented in one or two steps, as indicated in Figure 1. The
final concentration of glycerol is generally 6%–7%.19 Ex-
cept for removal of the negative effects caused by seminal
plasma, the other operations are similar to sheep semen
cryopreservation. With a spermatozoa concentration ranging
from 80 to 500 · 106 cells/mL, acceptable fertility can be
achieved for frozen goat spermatozoa.28–30
The negative effects of egg
yolk-coagulating enzyme
Different from sheep, goat seminal plasma contains a
special egg yolk-coagulating enzyme (EYCE), which can
compromise the viability of spermatozoa in the presence
of milk or egg yolk. Now, EYCE has been identified as
phospholipase A secretion from the bulbourethral gland,
which can coagulate egg yolk and hydrolyze lecithin to fatty
acids and spermicidal lysolecithins.31 These hydrolysates
 4 LV ET AL.
also induce acrosomal reaction and chromatin decondensa-
tion.30 Similarly, a 55–60 kDa glycoprotein lipase (SBUIII)
was identified from goat bulbourethral gland, which can
induce acrosomal reaction, subsequently injuring survival of
goat spermatozoa frozen in milk-based extenders.32 In ad-
dition, both triolein and milk triglycerides can be hydro-
lyzed by SBUIII to free fatty acids, which strongly damage
spermatozoa motility and plasma membrane.32 Some re-
searchers think that EYCE and SBUIII may be the same
protein.18,33 Therefore, the negative interaction between
EYCE or SBUIII and egg yolk or milk should be considered
during goat semen cryopreservation.
The traditional operation is to completely remove seminal
plasma through centrifugation before dilution using freezing
extenders.30,33 In general, goat spermatozoa are washed by
centrifuging at 550–950 g for 10–15 minutes.34,35 However,
although removal of seminal plasma enhances the cryo-
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survival of goat spermatozoa, some components naturally
included in seminal plasma are also lost. It is well known
that the biochemical composition of seminal plasma is
complex. Some studies have demonstrated a nutritive/pro-
tective function of seminal plasma for spermatozoa.36 Some
components in seminal plasma are essential for spermatozoa
metabolism, function, survival, and movement in the female
reproductive tract.36 Furthermore, the proteins presented
in seminal plasma are engaged in membrane stability,37
motility,38 capacitation,39,40 and spermatozoa/egg fertiliza-
tion.40,41 In addition, seminal plasma proteins may be in-
volved in the extrinsic and intrinsic apoptotic pathways.42
According to the report of Maxwell et al., the presence of
seminal plasma in freezing extenders can improve mem-
brane integrity and motility of post-thaw spermatozoa. Low-
molecular-weight proteins (15–25 kDa) are likely to be as-
sociated with these improvements. Particularly, adhesin may
play an important role for the functions induced by seminal
plasma.43
Another way to avoid the negative effects of EYCE or
SBUIII is to use skim milk-based freezing extenders.30
Recent studies indicate that reducing the concentration of
egg yolk to 2.5% cannot damage post-thaw viability of goat
spermatozoa. Therefore, freezing extenders containing low
concentrations of egg yolk may be used for goat semen
cryopreservation.44,45 However, the controversial results
still exist. According to the report of Anand et al., 3% egg
yolk presence in the freezing extender cannot improve the
quality of frozen goat spermatozoa in comparison with 20%
egg yolk.46 Therefore, whether this method is valuable or
not still needs further research.
The phospholipids in egg yolk, such as phosphatidyl-
choline, are important for cryoprotection of spermatozoa
plasma membrane.47 Therefore, some similar components
such as soybean lecithin may replace egg yolk to be used for
goat spermatozoa cryopreservation.48,49 Currently, com-
mercial extenders containing soybean lecithin have been
used for goat semen cryopreservation.48,49 According to the
report of Vidal et al., soybean lecithin, with a concentration
ranging from 0.04% to 0.16%, benefited the cryosurvival of
goat spermatozoa.50 There are few reports related to the
negative effects caused by soybean lecithin at present. Ac-
cording to the report of Valle et al.,51 soybean lecithin may
interfere with mitochondrial function in post-thaw sperma-
tozoa. In addition, although soybean lecithin resolves the
problems caused by egg yolk, such as contamination, stan-
dardization, and agglutination, it can act as the substrate for
lipid peroxidation because soybean lecithin contains higher
proportions of arachidonic and docosahexaenoic acids and
more of the C18 unsaturated fatty acids.52
Some Highlights Inspiring Small Ruminant
Semen Cryopreservation
To optimize the cryopreservation process, some measures
have been used to reduce cryoinjuries of small ruminant
spermatozoa. Among these attempts, selection of cryopro-
tectants is one of the important aspects, including antioxi-
dants,53–59 oligosaccharides,60–67 and natural or synthetic
ice blockers.68–71 In addition, vitrification and freeze-drying
have emerged as potential approaches for long-term storage
of spermatozoa. The relative research may give us some
inspiration on how to improve the current preservation
procedure of small ruminant semen.
The myth of trehalose
The research related to the functional roles of trehalose
(a-d-glucopyranosyl-a-d-glucopyranoside) is a hot topic in
the field of cryobiology and animal reproduction. In nature,
some organisms, such as bacteria, yeasts, tardigrade, and
plants, can survive through cryobiosis and anhydrobiosis for
a long time. Trehalose accumulates in their bodies during
this process.72 Recently, some encouraging results have
confirmed the role of trehalose during mammalian sperma-
tozoa cryopreservation.73 In small ruminant animals, the
effects of trehalose on cryosurvival of sheep61,63,64,66,74–77
or goat spermatozoa65,67,78,79 have been assessed. In the
presence of trehalose, a better post-thaw viability was ob-
served in sheep66,75,77,80 and goat spermatozoa.78 However,
excessively higher concentrations of trehalose may be
harmful to spermatozoa, which may correlate with severe
osmotic shock.75 In addition, some researchers have con-
cluded that the cryoprotective function of trehalose on small
ruminant spermatozoa is superior to that of other oligosac-
charides, such as sucrose.65,66,80 However, conflicting re-
sults still exist. For example, it has been reported that both
sucrose and trehalose have a similar capability of preserving
the motility of frozen ram spermatozoa.81 In goat, the effects
of trehalose on frozen goat spermatozoa are not superior to
those of other disaccharides, including sucrose, maltose, or
lactose.79 It should be noted that the variable results might
be related to their special experimental conditions.
Different from monosaccharide, trehalose cannot perme-
ate plasma membranes. So it functions primarily as an ex-
tracellular cryoprotectant. According to the report of Crowe
et al., to obtain the optimal protective effect, trehalose
should be presented on both sides of the plasma mem-
brane.82 However, how to load trehalose into cells is a great
challenge. Some technologies, such as thermotropic lipid-
phase transition,83 transfection to express trehalose in mam-
malian cells,84 or microinjection of trehalose into cells,85
have been developed to resolve this problem. However,
whether these technologies can be used for introduction of
trehalose into spermatozoa still needs research.
Currently, the cryoprotective mechanism of trehalose re-
mains largely undetermined. At first, the glass transition
temperature of trehalose (-30C) is much higher than that of
traditional cryoprotectants, such as ethylene glycol (-85C)
 SMALL RUMINANT SEMEN CRYOPRESERVATION
and glycerol (-65C).72 Therefore, trehalose may contribute
to extracellular vitrification formation and reduce ice crystal
production. Second, according to the ‘‘water replacement
hypothesis,’’ trehalose may replace the water shell of mac-
romolecules through the hydrogen bond linkage, conse-
quently reducing cryoinjury.82 Third, trehalose may enhance
fluidity of plasma membrane and mitigate cryoinjuries
caused by the freezing process.65 Furthermore, trehalose is
presumed to insert into plasma membrane and limit exces-
sive cell dehydration, decreasing physical damage induced
by acute alteration of cell volume.86 Interestingly, some
studies have demonstrated the potentially antioxidative roles
of trehalose at lower concentrations during spermatozoa
cryopreservation.67,87 In addition, trehalose is reported to
reduce osmotic sensitivity and block acrosome reaction in-
duced by lysophosphatidylcholine in ram spermatozoa.88
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Can synthetic ice blockers replace
antifreeze proteins?
How to prevent ice crystal formation during the freezing
and thawing process is an essential challenge that cryobi-
ologists have to face. Reducing injuries caused by ice for-
mation is critical for cryosurvival of mammalian cells. In
nature, some particular proteins accumulate in some low-
grade organisms and plants in extremely cold environ-
ments. Subsequent studies demonstrated that these proteins
promote the tolerance of these organisms to coldness or
freezing.
In 1969, DeVries and Wohlschlag discovered some in-
teresting proteins in the blood of Antarctic fish. With the aid
of these proteins, Antarctic fish can survive through some
extreme cold environments.89 Generally, these proteins were
called antifreeze proteins (AFPs), which influence the pro-
cess of ice formation during cooling below the bulk melting
point and protect mammalian tissues or cells in various
ways, such as modifying ice crystal formation,90,91 prohib-
iting recrystallization,92,93 and interacting with the plasma
membrane at low temperature.94,95
At present, AFPs have been used for cryopreservation of
spermatozoa collected from sheep,68 bovine,96 mouse,97 and
chimpanzee.98 Moreover, AFPs have shown a capability to
improve the post-thaw motility of chimpanzee spermatozoa98
and acrosome integrity of sheep spermatozoa.68 However, the
disputes still exist. In mouse, AFP cannot decrease cryoin-
juries in spermatozoa caused by the freezing process.97
Currently, only three types of AFPs (AFP I, AFP III, and
antifreeze glycoprotein) are commercially available.96 Ow-
ing to the high cost and difficult availability of natural AFPs,
some investigators have attempted to discover or design new
ice-inhibiting cryoprotectants to substitute AFPs.99–103 Chou
first designed some inhibitors of ice crystal formation.
However, he chose to slightly modify existing natural AFPs
and did not plan to discover or synthesize nonprotein anti-
freeze molecules.99 Wowk et al. reported the enhancement
of vitrification solutions by the addition of modified poly-
vinyl alcohol.101,103 In addition, the molecular modeling
technique has been used to identify molecular conforma-
tions of some chemicals, which may complement the atomic
spacing of hydrogen-bonding sites on the prism face of ice
crystal and change the ice crystal shape by lattice matching
with available sites on the basal plane surface of an ice
crystal.104 According to the report of Taylor et al., 1,3,5-
5
cyclohexanetriol (1,3,5-CHD), 1,3-cyclohexanediol (1,3-
CHD), and 1,4-cyclohexanediol (1,4-CHD) possess the
required bond angles and distances, which may be used to
change ice growth pattern.104 Cyclohexanetriol has been
found to inhibit apoptosis in sheep spermatozoa induced by
the freezing and thawing process.69 Furthermore, the in-
hibitory capability of 1,3-CHD may be superior to that of
1,4-CHD on ice formation.104
The inspiration from vitrification
Although traditional cryopreservation techniques have
been extensively applied for storage of small ruminant
semen, they cannot completely prohibit ice crystal forma-
tion, which leads to extensive cell shrinkage and structural
damage.13,105 To avoid the negative effects induced by ice
crystal formation, vitrification has been recommended as an
alternative method. Different from traditional freezing pro-
cesses, vitrification involves a direct phase transition of
aqueous solutions from the liquid state to the glassy state,
not experiencing the stage of ice crystal formation.
The main features of vitrification include high cryopro-
tectant concentration and fast freezing/warming velocity.
The combination of the two factors quickly increases the
viscosity of vitrification solutions and consequently blocks
ice crystal formation. Vitrification has been successfully
used for mammalian embryo cryopreservation. However,
direct usage of embryo vitrification procedures in sperma-
tozoa cryopreservation is not suitable, mainly due to the high
sensitivity of spermatozoa to toxic and osmotic stress.106 In
addition, embryos have a multicellular structure. Therefore,
even if some cells in embryos are damaged because of
cryoinjuries, the remaining live cells still can proliferate to
substitute for those dead cells.107 However, spermatozoa are
single cells that lack the capability of transcription and
translation, and so, they cannot recover from cryoinjury.
However, considering the functional role of spermatozoa as a
vehicle of male DNA, the effects of membrane and acrosome
integrity may be neglected when the intracytoplasmic sper-
matozoa injection (ICSI) technology is used for fertilization.
Actually, during ICSI, spermatozoa tails are usually cut by
hand to make the performance more convenient. Therefore,
the structural integrity of spermatozoa is not critical for the
success of ICSI.
In comparison with traditional cryopreservation proce-
dures, vitrification may be more advantageous in some as-
pects.108 Vitrification can prevent intracellular ice formation
and mitigate the detrimental effects induced by high solute
concentrations during the freezing and warming processes.
Another advantage is that the vitrification process is fast and
only takes a few seconds. Moreover, vitrification of mam-
malian spermatozoa may not require the addition of egg
yolk. It is well known that the main limitation of egg yolk is
its undefined components, which may be the primary reason
leading to variable results among different research groups.
Egg yolk may also bring potential bacterial contamination
and disease transmission. Removing egg yolk is more
meaningful for goat semen, due to the toxic interaction
between phospholipase A in seminal plasma and egg yolk.30
In addition, glycerol may be unnecessary when vitrification
is used for spermatozoa storage.109–111 Although glycerol
enhances the cryotolerance of spermatozoa, it also produces
potential toxic and osmotic stress on spermatozoa.
 6 LV ET AL.
However, there are a few limits that may influence the
efficiency of spermatozoa vitrification. At first, the glassy
state of vitrified samples is rather fragile and easily lost.
Therefore, when warming, spermatozoa may face a great
risk of devitrification. Recent studies have demonstrated that
damages caused by the warming process may be more se-
vere than the freezing process.112–114 In addition, vitrified
spermatozoa generally lose their motility, due to their high
sensitivity to osmotic and chemically toxic stresses, and so,
conventional AI may be impossible.113
Spermatozoa vitrification is not a novel concept. The
successful vitrification of frog spermatozoa was reported in
1938.115 However, early experiments on spermatozoa vit-
rification only resulted in low or no cryosurvival.116 Actu-
ally, the opinion that vitrification is not suitable for
cryopreservation of mammalian spermatozoa was popular
for a long time. In 2008, Isachenko et al. found that small
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(30 mL) droplets of human spermatozoa survived through
vitrification and warming after dilution using a freezing
medium containing human tubal fluid, 1% human serum,
and 0.25 M sucrose. Sixty-five percent progressive motility
was obtained and mitochondrial function was maintained in
post-thaw spermatozoa.116 A similar phenomenon was also
found in fish semen.117 These studies are meaningful and
encouraging because they imply that semen vitrification
may be possible. With the emergence and extension of the
ICSI technology, spermatozoa vitrification was revived and
came into favor with researchers again.
Currently, vitrification of human spermatozoa has
achieved some inspiring results.109,118–121 Some researchers
have attempted to vitrify pig122,123 or sheep spermato-
zoa.108,111 However, limited information related to vitrifi-
cation of small ruminant semen is available at present.
According to the report of Arando et al., although the
quality of vitrified sheep spermatozoa was significantly less
than that of fresh spermatozoa, sucrose can improve total
motility, viability, and membrane functionality, when sper-
FIG. 2. The present procedure used for
freeze-drying of mammalian spermatozoa.
‘‘ICSI’’ is the abbreviation of the in-
tracytoplasmic spermatozoa injection tech-
nology.
matozoa were first equilibrated at 5C before vitrification.
The authors presumed that sucrose may act as a potential
cryoprotectant to vitrify sheep spermatozoa. In addition,
equilibration at 5C can mitigate the cryoinjuries caused by
vitrification on sheep spermatozoa.111 In another study,
Jiménez-Rabadán et al. found that vitrification using com-
binations of sucrose and glycerol significantly reduced the
quality of frozen sheep spermatozoa in the absence of egg
yolk. On the contrary, spermatozoa vitrified with combina-
tions of egg yolk and glycerol at the lowest concentration
demonstrated acceptable viability, acrosome integrity, and
DNA fragmentation index, although vitrification still se-
verely damaged spermatozoa motility, demonstrating that
egg yolk may benefit the survival of vitrified sheep sper-
matozoa.108 The main limitation in these two studies is that
egg yolk has proved to be helpful for cryosurvival of vitri-
fied sheep spermatozoa. However, it is known that one of
the benefits brought by spermatozoa vitrification is to avoid
the negative effects caused by egg yolk.
In addition, it should be noted that vitrification may not be
suitable for daily production because a small volume is a
necessary condition to obtain a fast freezing/warming rate
and ensure a glassy state. However, as a way to conserve
farm animal genetic resources, vitrification is valuable and
has great application prospects. Even though the viability of
vitrified spermatozoa is poor or completely lost, an oocyte
still can be fertilized with a sperm with the aid of the ICSI
technology. Therefore, the studies associated with vitrifi-
cation of small ruminant spermatozoa still are meaningful.
Can freeze-drying be used for storage of small
ruminant semen?
The idea of preserving mammalian spermatozoa at room
temperature or in a refrigerator is always attractive. Liquid
nitrogen is not required for storage of freeze-dried sperma-
tozoa, which can bring some advantages, including reduction
 SMALL RUMINANT SEMEN CRYOPRESERVATION
of the storage or shipping costs and avoiding viral con-
tamination.124,125 In comparison with cryopreservation, the
freeze-drying process is more complicated, due to the addi-
tional drying or dehydration process. As indicated in Figure 2,
the freeze-drying process generally includes primary and
secondary drying and two phase transitions.126 During the
primary drying process, samples are first transformed from
the liquid phase into ice crystals by freezing to below their
eutectic temperature. Then, frozen water is evaporated as
water vapor in a vacuum environment without experiencing
the intermediate liquid phase. After primary drying, there is
an *8%–10% moisture remaining in samples based on their
composition. To ensure sample stability at a relatively higher
temperature, such as room temperature, the remaining un-
frozen bound water has to be further removed by desorption
through secondary drying. At this stage, samples are heated in
a lowest vacuum environment to make bound water form
Downloaded by Gothenburg University Library from www.liebertpub.com at 12/01/18. For personal use only.
water vapor. Following the primary and secondary drying
processes, dried samples may be stored at ambient tempera-
ture or in a refrigerator.
Although early studies on freeze-drying of spermatozoa
showed some encouraging results,127–129 the failure to re-
peat these experiments made the research on freeze-drying
of spermatozoa quiet for a long time. A breakthrough
emerged in 1998 when Wakayama and Yanagimachi ob-
tained healthy mice through fertilization of oocytes with
freeze-dried spermatozoa.130 The key point of their success
is that the ICSI technology was used to load freeze-dried
spermatozoa into an oocyte, due to the fact that all rehy-
drated spermatozoa were dead and lost their motility. Fur-
thermore, the authors proved that DNA integrity and oocyte-
activating factors of freeze-drying spermatozoa can be
preserved for 3 months at 4C and for several weeks at room
temperature.130 Their study demonstrated that the posses-
sion of intact genetic information is a necessary condition
ensuring the success of the ICSI technology and critical for
maintaining the full-term developmental capacity of freeze-
dried mammalian spermatozoa.
Encouraged by the study of Wakayama and Yanagimachi,
some researchers attempted to freeze-dry spermatozoa of
various farm animal species, such as bovine,131 pig,132,133
equine,134 rabbit,135 and sheep.2,136 In small ruminants, cur-
rently there are sporadic reports related to freeze-drying of
spermatozoa. Olaciregui et al. assessed the effects of ros-
marinic acid and storage temperature on DNA integrity of
freeze-dried sheep spermatozoa. Meanwhile, the develop-
mental capability of oocytes microinjected with freeze-dried
spermatozoa was also tested.136 These results demonstrated
that freeze-dried sheep spermatozoa can be preserved at 4C
and room temperature for 12 months. Furthermore, ros-
marinic acid can mitigate DNA damage of spermatozoa
induced by the freeze-drying process. No differences were
found between freeze-dried spermatozoa and frozen sper-
matozoa with respect to blastocyst formation.136 Recently,
Anzalone et al. further confirmed that freeze-dried sheep
spermatozoa supported blastocyst development after ICSI,
implying that freeze-drying is an alternative, low-cost stor-
age strategy for biodiversity conservation. In their study,
after ICSI with freeze-dried sperm, oocytes were chemically
activated by ionomycin to start embryonic development.2
The sperm-oocyte-activating-factor (SOAF), locating at
plasma membrane, can activate oocytes by stimulating a
Ca2+ release from ooplasmic stores.137 Therefore, sperma-
7
tozoa with damaged membrane may lose their capacity to
activate oocytes after ICSI, due to lack of SOAF.138
The resurrection of freeze-drying of spermatozoa is
coupled with the development of the ISCI technology. Be-
cause freeze-dried spermatozoa completely lose their mov-
ing ability, they need the ISCI technology to realize the
fertilization with oocytes. Currently, the quality of freeze-
dried spermatozoa has great room for improvement. Without
a doubt, the damage induced by the freeze-drying process on
spermatozoa is more severe than that caused by cryopres-
ervation, due to additional drying treatments.
Spermatozoa DNA may be degraded by DNAases or
oxidative stress during the freeze-drying process. The en-
dogenous nucleases released from freeze-dried spermatozoa
may be the main reason causing chromosome structural ab-
normality.139 Some measures have been used to reduce the
negative effects of freeze-drying on spermatozoa DNA.139–141
Kusakabe et al. found that the chromosome integrity of mouse
spermatozoa could be maintained when they were freeze-
dried in a Tris-HCl-buffered solution containing 50 mM
EGTA and 50 mM NaCl.139 In addition, the stability of
freeze-dried mouse spermatozoa can be improved by using a
simplified Tris-buffered solution supplemented with calcium
chelators, such as EGTA or EDTA.139,140 Therefore, the
chelation of calcium seems to play an important role for in-
hibition of endonuclease activity.139,141 Furthermore, the ef-
fect of a slightly alkaline solution (pH = 8) has been proven to
be superior to that of neutral or acidic (pH = 7.4–6.0) solu-
tions for maintaining chromosomal integrity and subsequent
embryonic development of freeze-dried spermatozoa.142
Disaccharides, especially trehalose, may be helpful for
chromosome stability and DNA integrity of freeze-dried
spermatozoa.143–145 Some studies have demonstrated that
spermatozoa freeze-dried in the presence of trehalose can
fertilize with oocytes.143,145 In addition, oxidative stress can
lead to spermatozoa DNA fragmentation during freeze-
drying and rehydration.139,146 Exposure of DNA to reactive
oxygen species leads to various chemical alterations, such as
crosslinking, base modification, and DNA strand break-
age.147 Since DNA integrity can be influenced by oxidative
stress, whether antioxidants can reduce oxidative stress
on freeze-dried spermatozoa may be an attractive topic for
research.148
The primary purpose of spermatozoa preservation is to
conserve complete male genetic information. Considering
this aspect, freeze-drying may act as a potentially alternative
scheme of traditional cryopreservation processes. Cryopre-
servation requires liquid nitrogen to block spermatozoa
metabolism. The continuous addition of liquid nitrogen and
mechanical maintenance of freezing equipment are critical
for long-term cryopreservation. Once liquid nitrogen pro-
vision is interrupted, frozen spermatozoa will lose their vi-
ability, especially during some unexpected disasters, such as
earthquakes or typhoons.149 Different from cryopreserva-
tion, the storage requirements of freeze-drying are simple
and easily controlled. However, the storage stability of
freeze-dried spermatozoa may be shorter than that of cryo-
preservation, mainly due to the higher storage temperature.
Perspectives
Although the research on cryopreservation of small rumi-
nant semen has lasted for about 60 years, using current
 8 LV ET AL.
methods 50% of post-thaw spermatozoa still lose their via-
bility. A standardized cryopreservation process has not been
established although some measures, including specific
cryoprotectants, have been applied to improve the survival
and fertility of frozen small ruminant spermatozoa.
Factors leading to spermatozoa cryoinjury are complicated
and have not been determined so far. The extensive and severe
cryoinjuries on the structure and physiological function of
small ruminant spermatozoa have been confirmed. According
to the results obtained by transmission electron microscopy,
the plasma membrane is extremely sensitive to cryodamage,
followed by acrosome. Currently, the cryoinjury mechanism
of mammalian spermatozoa is a hot topic in the field of
cryobiology and reproductive science. The technology of
either cytology or thermophysics has been used to explore
this problem. However, these methods still cannot explain the
nature of spermatozoa cryoinjury. The functional role of
Downloaded by Gothenburg University Library from www.liebertpub.com at 12/01/18. For personal use only.
spermatozoa is regulated by proteins and nucleic acid.
Therefore, a molecular explanation may be necessary. At
present, the fast development of the high-throughput se-
quencing technology has provided an opportunity for us to
answer this problem based on RNA or proteins. In addition,
some potential biomarkers obtained by omics analysis can be
used for assessment of spermatozoa quality.
As alternative strategies of traditional cryopreservation,
vitrification and freeze-drying have been used for storage of
small ruminant spermatozoa. These two methods have some
advantages compared with conventional cryopreservation,
such as no ice crystal formation and storage at room temper-
ature. However, their shortages are also evident. For example,
vitrified or freeze-dried spermatozoa completely lose their
mobility, so they need the ICSI technology to help them
complete fertilization with oocytes. In this case, how to protect
the structural and functional integrity of male DNA is essential.
Research related to cryopreservation of small ruminant
semen has progressed greatly in recent years. However,
significant room for improvement still exists. The current
technological advances, such as vitrification, freeze-drying,
and high-throughput sequencing technologies, can provide a
new visual angle to improve the conservation efficiency of
small ruminant spermatozoa.
Acknowledgments
This study was funded by the National Natural Science
Foundation of China (Grant No. 31560635), Applied Basic
Research Project of Yunnan Province (Grant No.
2016FB042), the National Wool Caprine Industrial Tech-
nology System (Grant No. CARS-39), and Middle and
Young Academic Leaders of Yunnan Province (Grant No.
2014HB051). We sincerely thank Dr. Wei Shen from
Qingdao Agricultural University and Dr. Allai Larbi from
Universitad Autonoma de Barcelona for their careful revi-
sion of this article and precious suggestions.
Author Disclosure Statement
No conflicting financial interests exist.
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Address correspondence to:
Guobo Quan, PhD
Yunnan Animal Science and Veterinary Institute
Jindian, Panlong County
Kunming 650224
China
E-mail: waltq20020109@163.com