2015 Yeste Sperm Cryopreservation Update

Theriogenology 85 (2016) 4764
Contents lists available at ScienceDirect
Theriogenology
journal homepage: www.theriojournal.com
Sperm cryopreservation update: Cryodamage, markers,

and factors affecting the sperm freezability in pigs
Marc Yeste*
Nufeld Department of Obstetrics and Gynaecology, University of Oxford, Level 3, Womens Centre, John Radcliffe Hospital, Oxford,
UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 August 2015
Received in revised form 20 September 2015
Accepted 22 September 2015
Cryopreservation is the most efcient method for long-term preservation of mammalian

sperm. However, freeze-thawing procedures may strongly impair the sperm function and
survival and thus decrease the reproductive performance. In addition, the sperm resilience
to withstand cryopreservation, also known as freezability, presents a high individual
variability. The present work summarizes the principles of cryoinjury and the relevance of
permeating and nonpermeating cryoprotective agents. Descriptions about sperm cryodamage are mainly focused on boar sperm, but reference to other mammalian species is
also made when relevant. Main cryoinjuries not only regard to sperm motility and
membrane integrity, but also to the degradation effect exerted by freeze-thawing on other
important components for sperm fertilizing ability, such as mRNAs. After delving into the
main differences between good and poor freezability boar ejaculates, those protein
markers predicting the sperm ability to sustain cryopreservation are also mentioned.
Moreover, factors that may inuence sperm freezability, such as season, diet, breed, or
ejaculate fractions are discussed, together with the effects of different additives, like
seminal plasma and antioxidants. After briey referring to the effects of long-term sperm
preservation in frozen state and the reproductive performance of frozen-thawed boar
sperm, this work speculates with new research horizons on the preservation of boar
sperm, such as vitrication and freeze-drying.
2016 Elsevier Inc. All rights reserved.
Keywords:
Sperm
Pig
Cryopreservation
Cryprotectant
Freezability
Seminal plasma
1. Introduction
Fertility preservation through cryopreservation of
gametes and reproductive tissues may be advised in several
cases in humans, especially in children and adults suffering
from cancer, and other mammals, including endangered
species [1,2]. Although, in general, freeze-thawing of
mammalian sperm harms the cell, the extent of that
damage varies across species and heavily relies upon the
sperm resilience to withstand cryopreservation procedures
[3,4].
The rst attempts to cryopreserve mammalian sperm
date back to 18th and 19th centuries, with the observations
* Corresponding author. Tel.: 44 (0)1865 782829; fax: 44 (0)1865
769141.
E-mail address: marc.yeste@obs-gyn.ox.ac.uk.
0093-691X/$ see front matter 2016 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.theriogenology.2015.09.047
made by Spallanzani in 1776 and Mantegazza in 1866 with

human sperm. However, this technology was really developed in the 20th century, when glycerol was used as a
cryoprotective agent (CPA) for mammalian sperm and
other somatic cells and tissues [5,6]. During the 1950s, boar,
horse and bull sperm were successfully cryopreserved [7,8]
and, for the rst time in 1957, piglets were born from
frozen-thawed sperm [9].
The 1970s represented a signicant forward step for
boar sperm cryopreservation with the establishment of two
methods: the American or Beltsville method designed by
Pursel and Johnson [10], and the German or Hlsenberger
method set by Westendorf et al. [11]. Although these two
techniques initially used carbonic ice and liquid nitrogen
vapors, cryopreservation success was further increased
through the introduction of controlled-rate freezers [12,13].
In contrast to the case of boar sperm, the conventional
48
M. Yeste / Theriogenology 85 (2016) 4764
method (i.e., with nitrogen vapors) may be used in other

mammalian species with reasonable yields [14,15]. After
the establishment of these two methods, research efforts
have been directed toward optimization of cryopreservation protocols and articial insemination (AI), sperm
preparation, modication of extenders, and identication
of freezability markers (reviewed in [16]).
The present article seeks to summarize fundamentals of
cryoinjury in boar sperm, with reference being made to
other mammalian species when relevant. Apart from
describing the main common damages inicted by
freeze-thawing procedures, such as those on plasma
membrane integrity, this work also discusses whether such
procedures affect other important components for sperm
function during fertilization and beyond, such as mRNA or
epigenetic regulation elements. The review also summarizes the freezability markers identied thus far, briey
describes those factors that may affect the sperm resilience
to withstand cryopreservation, and delves into the role of
CPAs and the effects of different additives. The article ends
with a discussion about other methods that also allow
long-term preservation of mammalian sperm, such as
vitrication and freeze-drying.
2. Principles of cryoinjury during freezing and
thawing
It is well known that cell metabolism decreases at low
temperatures and that this allows long-term preservation
of germ cells, embryos, and tissues. The main inconvenience of freeze-thawing procedures is the cryoinjury
inicted by low temperatures that, among other causes, is
associated with the phase change of intracellular and
extracellular water [17]. However, rather than storage at

low temperatures, the main challenge for cells during
freezing and thawing is the lethality of an intermediate
range of temperatures, between 15 C and 60 C. According to Gao and Critser [17], cells and extracellular
medium remain unfrozen and supercooled at 5 C. At
temperatures between 5 C and 15 C, ice is formed in
the surrounding medium, but the intracellular contents
remain unfrozen and supercooled. Because chemical potential of water is higher in supercooled (intracellular) than
in frozen (extracellular) state, water ows out of the cell
and freezes externally. The cooling rate determines what
occurs thereafter (Fig. 1).
If cooling rate is very high, intracellular water does not
ow out completely, cells freeze intracellularly, and the
formation of ice crystals in the cytoplasm ultimately results
in cryoinjury [18,19]. Because the formation of intracellular
ice not only depends on cooling rate and temperature but
also on CPA concentration, the use of these agents may
alleviate that formation [17].
If cooling rate is very low, most of the water ows out,
intracellular solutes are concentrated, and supercooling is
eliminated. Rather than freeze intracellularly, cells are
dehydrated, experience volume shrinkage of organelles
and membranes, and are exposed to high-solute concentrations before they reach the temperature at which all
solution components are solidied (Fig. 1). This affects
lipid-protein complexes, denatures macromolecules, decreases the size of unfrozen channels, and induces irreversible membrane fusion [20,21]. The resulting hypertonic
stress can change the electrolyte balance, and this may lead
the cells to swell beyond their normal isotonic volume and
subsequently lyse upon thawing [17].
Fig. 1. Cooling rates and physical events during freezing (Adapted from [17]). When temperature is decreased up to 5 C, ice is formed in the surrounding
medium, and water ows out of the cell. If subsequent cooling rates are high, intracellular water does not ow out completely, and ice crystals are formed inside
the cell. If cooling rates are low, most of the water ows out, cells dehydrate and experience volume shrinkage of organelles and membranes. Optimal cooling
rates are those that are low enough to avoid formation of intracellular ice but high enough to minimize the cryoinjury due to solute concentration and volume
shrinkage.
49
Therefore, albeit due to separate mechanisms, both high

and low freezing may lead to cell cryoinjury. For this
reason, Mazur et al. [20] proposed the two-factor theory.
According to that theory, cryoinjury occurs: (1) due to lethal formation of intracellular ice at high cooling rates and,
(2) due to solute/electrolyte concentration, cell dehydration and reduction of unfrozen fraction in the extracellular
space at low cooling rates. An optimal cooling rate exists for
each specic cell type and is dened as that is low enough
to avoid formation of intracellular ice but high enough to
minimize the cryoinjury due to solute/electrolyte concentration (Fig. 1).
Cell survival after cryopreservation not only implies
cryoinjuries during freezing, but also along thawing.
Indeed, whereas low thawing rates result in recrystallization, osmotic stress ensues from high rates as CPAs are
unable to leave the cell fast enough [21,22]. Such osmotic
increase in the cytoplasm leads the water to enter, and this
ultimately disrupts the plasma membrane [12,23]. For this
reason, optimal thawing rates should represent a balance
between these two scenarios. In the case of boar sperm,
thawing is usually performed at 37 C for 20 seconds, but
Toms et al. [24] have recently shown that faster thawing
rates, i.e., 8 seconds at 70 C, give better results.
Hence, the adjustments in sperm cryopreservation
protocols are directed toward nding these optimal cooling
and thawing rates, thereby preventing cryoinjuries as
much as possible [25,26]. Reduction of cryoinjury may be
performed through the use of CPAs or other methods such
as vitrication. Because in the case of mammalian sperm,
vitrication is, for the time being, less successful than slow
cryopreservation, this review will focus on CPAs in the
following section.
As main nonpermeating cryoprotectants, freezing extenders contain sugars, mainly disaccharides (mainly
lactose or trehalose), as these give better results than
monosaccharides [3033], and proteins from hen egg yolk.
When combined with surfactant Orvus ES Paste (Equex),
egg yolk proteins confer better protection because this
detergent facilitates the interaction of egg yolk proteins
with sperm plasma membrane [23,34].
Different works have investigated whether the egg yolk
component can be replaced by low-density lipoproteins
(LDL) or soy lecithin. With regard to the former, egg yolk is
a mixture of different proteins, but its main protective effect is mediated by LDL. In fact, replacing the entire egg yolk
fraction by LDL has benecial effects for boar sperm quality
and DNA integrity after freeze-thawing [3537]. In addition, the egg source inuences the properties of LDL fractions, the LDL extracted from pigeon egg yolk having higher
cryoprotective effects than those extracted from hen, ostrich, duck, and quail eggs [38].
With regard to replacement of egg yolk proteins by soy
lecithin, there are no studies conducted in pigs, and works
in humans, rams, and bulls have provided different results.
In humans, conventional sperm quality parameters, DNA
integrity, and sperm ability to bind to hyaluronate do not
signicantly differ between egg yolk and soy lecithin [39].
In contrast, research conducted with stallion sperm
showed that replacing egg yolk by soy lecithin resulted in a
decrease of reproductive performance [40], and studies in
rams found a positive effect on total sperm motility, chromatin, and acrosome integrity but an impairment of
mitochondrial sperm function at post-thawing [41].
3. The relevance of cryoprotectants
Permeating CPAs are glycerol, DMSO, ethylene glycol,

methanol, propylene glycol, and dimethylacetamide. All
these solutes are able to permeate the cells, and they are
relatively nontoxic at concentrations of up to 1 M and even
higher [17]. Their mechanism of action is related to their
ability to decrease the concentration of electrolytes and the
extent of osmotic shrinkage at low temperatures [42].
Concretely, these agents affect cytoplasm viscosity, change
diffusion rates, and alter cell membrane properties via its
insertion in the lipid bilayer [23]. However, these substances may damage the cell and induce osmotic volume
changes at temperatures higher than 5 C. This represents
an inconvenience because they need to permeate before
freezing, and they have to be quickly removed on thawing
[43]. Finally, it can be noted that the suitability of a given
CPA depends on the permeability of the cell to this agent
and its specic cytotoxic effects, so that the appropriate
CPA concentration relies on the cell type [17].
For boar sperm cryopreservation, glycerol is the main
permeating CPA, as no CPA has found to yield better results
thus far [16,44], with an optimal concentration ranging
from 2% to 3% [27,45]. However, because glycerol may
damage the perinuclear theca of boar sperm [46] and
concentrations higher than 4% affect plasma membrane
uidity [47], some attempts have tried to replace glycerol
with other cryoprotectants. Replacing 3% glycerol with
80 mM L-glutamine plus 2% glycerol improves post-thaw
As previously stated, the composition of freezing and

thawing media plays a crucial role in both slow and rapid
freezing. In addition, although slow-freezing is used to avoid
the formation of ice crystals inside the cell, the formation of
those crystals is not completely prevented. For this reason,
CPAs are used to reduce the stress derived from freezing and
thawing protocols, but because these substances may be
toxic for sperm, nding the most suitable concentration is
very important [27]. As detailed in the following section,
CPAs can be classied as nonpermeating and permeating,
depending on their ability to permeate the cell.
3.1. Nonpermeating CPAs
Nonpermeating CPAs are proteins found in the milk and
egg yolk, sugars, and higher molecular weight compounds,
such as polyvinylpyrrolidone, hydroxyethyl starch, polyethylene glycols, and dextrans, that contribute to prevent
ice formation and stabilize proteins and cell membranes.
These compounds do not pass through plasma membrane
and develop its role extracellularly [28]. Despite these
solutes not being able to fully protect the cell when
permeating CPAs are absent, they increase the effectiveness
of these permeating CPAs and allow decreasing their concentration [29].
3.2. Permeating CPAs
50
sperm motility [48], and substitution with trehalose

(100 mM) increases motility, acrosome integrity, mitochondrial membrane potential, and in vitro penetration
rates after IVF [49]. In addition, replacing lactose and
glycerol by powdered coconut water plus dimethylformamide yields higher post-thaw sperm quality [50]. In fact,
the benecial effects of glutamine and dimethylformamide
have also been reported for bull and stallion sperm,
respectively [51,52]. In contrast, other permeating CPAs,
such as dimethylacetamide or DMSO, yield worse results
than glycerol [53,54].
4. Dissecting the sperm cryodamage
Even when both permeating and nonpermeating CPAs
are used, cryopreservation inevitably reduces the survival,
acrosome integrity, motility, and fertilizing ability of
frozen-thawed mammalian sperm [44,5557]. For this
reason, the present section intends to summarize the most
common cryoinjuries described after freeze-thawing.
Despite descriptions being made in separate subsections,
there is a global impact on sperm cell, and damages are
related (Fig. 2).
4.1. Cryopreservation effects on plasma membrane
The composition and biophysical properties of plasma
membrane are related to cell sensitivity to freeze-thawing
procedures [2]. Plasma membrane of boar sperm is rich in
unsaturated phospholipids and poor in cholesterol molecules, and this underlies the high sensitivity of boar sperm
to cold shock [12]. Such cold shock includes all dramatic

effects ensuing from destabilization of plasma membrane
at temperatures less than or equal to 5 C, thereby affecting
Ca2 homeostasis, acrosome integrity, and membrane lipid
disorder (Fig. 2) [44,5860].
Any plasma membrane contains phospholipids, which
confer uidity, and a variable amount of sterols, such as
cholesterol, that provide rigidity and stability. At low temperatures, lipids undergo alterations in physical phases (i.e.,
uid- and gel-phase lipids). Although the presence of sterols
inhibits these lipid-phase changes, sperm membranes that,
such as that of boar, present a low cholesterol:phospholipid
ratio and an asymmetric distribution of cholesterol are more
sensitive to damages induced by cooling [23,44,59,61].
Therefore, restriction of lateral movements of membrane
phospholipids occur when temperatures are lower than
5 C, and this eventually results in a transition from uid to
gel phase. Indeed, because different membrane lipids present different transition temperatures, some unsaturated
phospholipids are jellied earlier than others, and phase
separations occur. After this phenomenon, integral membrane proteins become irreversibly clustered by lipid-phase
separations, membrane lipids are restructured, and some
cholesterol molecules are released [56,62]. As a result of
these structural alterations, there is a disruption of lipid and
protein interactions, and some proteins, such as ion channels, are translocated and/or lose their function [63]. This
leads the plasma membrane to destabilize and lose its selective permeability, thereby increasing the inux of ions,
such as Ca2 and bicarbonate, from the extracellular space
[44,59,60]. All these changes have led some authors to speak
Fig. 2. Main damage inicted by freeze-thawing procedures on boar sperm. Cryopreservation affects acrosome integrity and the uidity and permeability of
plasma membrane, and leads to degradation of mRNAs and proteins. The detrimental effects on sperm nucleus include DNA fragmentation, translocation of
nucleoproteins (protamine 1, P1, and histone 1, H1) and disruption of disulde bridges between cysteine radicals of P1.
about cryocapacitation or capacitation-like changes, as the

events that are triggered resemble, but are not identical, to
true sperm capacitation [12,64].
As previously stated, the composition of the plasma
membrane plays a crucial role in the sperm ability to
withstand freeze-thawing procedures. According to
Waterhouse et al. [65], palmitic acid (16:0), stearic acid
(18:0), oleic acid (18:1, n-9), docosapentaenoic acid (22:5,
n-6), and docosahexaenoic acid (DHA, 22:6, n-3) are the
most abundant fatty acids in boar sperm plasmalemma.
Notwithstanding, when the amounts of long-chain polyunsaturated fatty acids, such as docosapentaenoic acid and
DHA are increased, sperm have higher resilience to freezethawing procedures [65]. In humans, Martnez-Soto et al.
[66] also reported that the content of DHA in sperm
membrane is correlated with sperm motility and membrane integrity at post-thawing.
51
[7678] and stallions [57], there is no such an impact in

dogs [79].
The mechanisms underlying the increase in DNA fragmentation after cryopreservation are still unknown and
appear to be related to the increase of oxidative DNA
damage [80]. In humans, the increase of DNA fragmentation linked to cryopreservation has been correlated with
oxidative stress rather than to activation of caspases [81].
Also in humans, sperm cryopreservation has also been reported to induce lesions on crucial genes involved in
fertilization and early embryo development (ADD1, ARNT,
BIK, FSHB, PEG1/MEST, PRM1, SNORD116/PWSAS, and
UBE3A), specically to two of these eight genes: SNORD116/
PWSAS and UBE3A [82]. Although no similar works have
been conducted in pigs, this clearly warrants further
research.
4.3. Cryopreservation effects on perinuclear theca
4.2. Cryopreservation effects on sperm nucleus

Studies evaluating how cryopreservation affects sperm
nucleus are focused on chromatin integrity. This can be
explained by distinguishing between DNA, nucleoproteins,
and structural interaction between DNA and nucleoproteins. Sperm chromatin is made up of DNA and nucleoproteins, which are mainly protamines (P1 and P2) and
between 2% and 15% histone H1 [67]. Not all species present the same protamines. While boars, bulls, and rams
only present protamine P1, human, stallion, and mouse
sperm present P1 and P2. In addition, P1:P2 ratios also vary
between species, and this may also affect the resilience of
that structure to freeze-thawing procedures [68].
With regard to the impact on protamines, there are
changes in the location and distribution of P1 and H1
throughout the nucleus of boar sperm after freeze-thawing
(Fig. 2) [69,70], although some changes are already present
at the end of the cooling step (i.e., at 5 C). Moreover, the
interaction through disulde bridges between cysteine
radicals of protamines, which constitutes the bone of the
sperm chromatin, is disrupted after freeze-thawing (Fig. 2)
[70]. The extent of this disruption is related to ejaculate
freezability [71], has a clear impact on reproductive performance [72], and is different between species that present P1 and those that present P1 and P2 [57,73].
As far as DNA fragmentation is concerned, there are
different methods to evaluate the sperm DNA integrity,
such as Terminal deoxynucleotidyl transferase dUTP Nick
End Labeling (TUNEL), Sperm Chromatin Structure Assay
(SCSA), Sperm Chromatin Dispersion test (SCD), Comet
neutral and Comet alkaline, but studies conducted in pigs
concur in similar outcomes, which are obtained regardless
of the method [74]. While there is no signicant increase in
DNA fragmentation immediately at post-thawing [70], DNA
damage is more apparent when frozen-thawed sperm are
incubated at 37 C for at least 2 hours (Fig. 2) [71,73,75]. The
extent of DNA-induced cryodamage also differs across
species because those species having P1 and P2 present
higher levels of DNA fragmentation than those presenting
only P1 [68]. Indeed, while sperm cryopreservation can
greatly affect DNA integrity and condensation in humans
The perinuclear theca is a region that surrounds sperm

nucleus and contains cytoskeletal proteins crucial for
maintaining the architecture of sperm head [83]. Because
this region plays a crucial role during fertilization and
contains relevant sperm proteins such as PLCz and PAWP
[8486], its integrity is important for proper sperm function. In this regard, it is worth noting that cryopreservation
of boar sperm damages the perinuclear theca and that
changes in the stabilization of F-actin ultimately result in
the disruption between this theca and actin (Fig. 2). This
not only affects these structures but also leads the sperm
nucleus to decondense [87].
4.4. Cryopreservation effects on mitochondrial function and
ROS production
Mitochondrial activity is reduced after cooling and
freeze-thawing in boar sperm (Fig. 2) [88]. Such reduction
is also observed in sperm from other species, such as
equine [89,90], especially in poor freezability ejaculates
(PFE) [57].
Another interesting point related to mitochondrial
function regards to reactive oxygen species (ROS) production. Effects of cryopreservation on ROS production in boar
sperm are less clear than in other species [91,92]. Whereas
Flores et al. [88] found that cryopreservation decreased the
capacity of mitochondrial of producing ROS and this was
already apparent at the cooling step, Gmez-Fernndez
et al. [93] and Yeste et al. [71] showed that good freezability
ejaculates (GFE) and PFE did not differ in terms of intracellular levels of peroxides and percentages of viable and
nonviable sperm with membrane lipid peroxidation. In
fact, although peroxides are considered to be the major free
radical in boar sperm, the marginal increase due to cryopreservation makes it difcult to assert the real impact of
ROS on sperm cryodamage because despite separate
studies reporting some increase in ROS levels after freezethawing, such increase is marginal [73,91,94,95]. In spite of
this, it is worth noting that total antioxidant capacity of
seminal plasma and lipid peroxidation of sperm membrane
are known to be affected during freeze-thawing procedures
[96]. This apparent contradiction is also found in other
52
species. Indeed, in buffalos and bulls, ROS species are

known to affect sperm motility, lipid peroxidation, mitochondrial membrane potential, and DNA integrity in fresh
sperm, but this relationship is less clear in the case of
frozen-thawed sperm [97]. Again, whereas freeze-thawing
does not increase ROS levels in buffalo sperm, lipid peroxidation increases after cryopreservation. In stallion, lipid
peroxidation is low in fresh spermatozoa and signicantly
increases after freeze-thawing. However, rather than being
a cryopreservation-induced injury, it is thought to be a
sublethal cryodamage leading to other injuries that may
ultimately compromise the sperm survival [89].
4.5. Cryopreservation effects on sperm vacuoles
There are different vacuoles found in mammalian sperm
that are observed in the acrosome and cytoplasm droplets
during spermatogenesis. Although often observed in
ejaculated human sperm, they are less frequent in pigs,
where they can only be detected under transmission electron microscope, and they appear together with
membrane-bound nuclear invaginations, the most typical
ultrastructural nuclear anomalies [98]. In spite of this, only
few works have studied the relevance of these vacuoles in
pigs, even if an early study reported that heat stress
increased the proportions of sperm showing nuclear vacuoles in the ejaculate [99]. There are no studies about the
effects of cryopreservation on sperm vacuoles in pigs, and
studies in humans have shown inconsistent results. Indeed,
whereas Gatimel et al. [100] showed that freeze-thawing
procedures did not alter total and relative vacuole areas,
Boitrelle et al. [77] observed that such procedures increase
sperm nuclear vacuolization.
4.6. Changes in levels, location, function, and tyrosinephosphorylation of sperm proteins
Different studies have evaluated the effects of sperm
cryopreservation on levels, localization, and function of
sperm proteins. In a comparison of sperm proteome of
fresh and frozen-thawed boar sperm, Chen et al. [101] using
isobaric tags for relative and absolute quantication found
that the amounts of up to 41 proteins involved in multiple
processes such as sperm premature capacitation, adhesions, energy supply, and spermoocyte binding and
fusion, were altered. The levels of six of these 41 proteins
were seen to decrease after cryopreservation, whereas the
other 35 were seen to increase. Further validation with
Western blotting conrmed that the expression of AKAP3,
superoxide dismutase 1 (SOD1), TPI1, and ODF2 was
increased in frozen-thawed sperm, especially in the case of
SOD1 and AKAP3. It is worth noting that some of these
proteins, such as TPI1, HSP90AA1 and PHGPx have been
suggested to predict sperm freezability in extended ejaculates [102,103]. In fact, similar ndings have been reported
for humans because Wang et al. [104] found that up to 27
sperm proteins differed between fresh and frozen-thawed
sperm. In this case, ACO2, TEKT1, VIM, OXCT1 were seen
to decrease in response to cryopreservation, whereas ENO1,
TEKT3, and TEKT4 appeared to signicantly increase. It
does not seem that sperm are able to synthesize de novo
proteins in response to freeze-thawing procedures, but

rather that the overall cryodamage affects the presence and
detection of some proteins, which could also affect the
sperm fertilizing ability if this was a result from protein
degradation. Supporting this, sperm cryopreservation decreases the detectable levels of PLCz in fertile men [105]
and those of HSP90 in bulls [106].
Apart from changes in the content of different proteins,
cryopreservation has also been reported to induce changes
in the location of proteins (Fig. 2), such as actin and mitofusin 2 [88], and glucose transporter GLUT3 [107]. With
regard to protein function, it has already been mentioned
that membrane cryoinjuries may affect the function of ion
channel proteins. Losing this function could also underlie
the reduction of fertilizing ability of frozen-thawed sperm.
Indeed, caffeine elicits Ca2-transients, and progesterone
leads to a biphasic increase of Ca2 levels in fresh but not in
frozen-thawed boar sperm, which indicates that cryopreservation reduces the responsiveness of spermatozoa to
depolarization, modulators of the internal Ca2 stores and
progesterone [108]. Similar ndings have been observed in
other species. For example, mean [Ca2]i basal values of Ltype voltage-gated Ca2 channels are higher in frozenthawed than in fresh stallion sperm, and increases of
[Ca2]i mediated by agonists are higher in frozen-thawed
than in fresh sperm. In contrast, the level of response to
the addition antagonists is lower in frozen-thawed than in
fresh sperm [109]. All these ndings concur that freezethawing procedures affect the function of this channels.
Finally, because capacitation-like changes involve
changes in the phosphorylation of sperm proteins, even if
the patterns differ between capacitation-like state and true
capacitation already at the cooling step (5 C) [64], it is also
important to pay attention to the effects of cryopreservation on factors involved in this phosphorylation. It is widely
known that tyrosine-phosphorylation of sperm proteins is
one of the features of sperm capacitation that takes place
within the oviduct or fallopian tube [110112], and is
related to proteins involved in cAMP/PKA and extracellular
signal regulated kinases pathways [113]. In this regard, it is
worth mentioning that tyrosine-phosphorylation of p32 is
present after cooling and freeze-thawing of boar sperm
[114]. This cryopreservation-induced change in tyrosine
phosphorylation has also been found in other species,
including bull, buffalo, stallion and rhesus macaque, as
reviewed by Naresh and Atreja [115]. According to these
authors, alterations in the tyrosine-phosphorylation status
of sperm proteins after cryopreservation could also
contribute to explain the reduced fertilizing ability of
frozen-thawed sperm.
4.7. Any impact on mRNAs and microRNAs?
Parental mRNAs are known to play a crucial role when
delivered to oocyte on fertilization (reviewed in [116]). Two
studies conducted in boars have found that abundances of
transcripts encoding for different relevant proteins, such as
B2M, BLM, HPRT1, PGK1, S18, SDHA, YWHAZ, PPIA, RPL4,
DNMT3A, DNMT3B, JHDM2A, KAT8, and PRM1, are affected
by cryopreservation protocols (Fig. 2) [117,118]. This effect is
not restricted to boar sperm. In humans, sperm mRNAs
involved in events taking place on fertilization and pregnancy success (ADD1, PEG1/MEST, PRM1 and PRM2) are
reduced after cryopreservation [119]. Similar results with
PRM1 have been obtained in bulls [120]. Therefore, degradation of specic sperm mRNAs by cryopreservation procedures could also contribute to explain the reduction in
reproductive performance of frozen-thawed sperm. This is
a reasonable hypothesis because the relationship between
sperm mRNAs (including MYC, CYP19, ADAM2, PRM1, and
PRM2) and early development has also been conrmed in
pigs [121]. Further research in this realm is thus warranted.
On the other hand, microRNAs (miRNAs) are small
noncoding regulatory RNA molecules that modulate gene
expression either via inhibiting mRNA translation or via
targeting mRNA for degradation (reviewed in [122]). A
recent study in pigs has observed that the levels of some
miRNAs are more affected than others by cryopreservation
procedures [123]. Although works conducted in pigs have
not yet addressed the relationship between miRNAs and
fertilizing ability [124], data available in humans point toward this direction [125,126]. Future studies should
address whether sperm miRNAs degraded by cryopreservation are relevant for fertilization success.
4.8. Does sperm cryopreservation affect parental epigenetic
regulation?
The term epigenetics refers to those inheritable changes
of gene expression due to mechanisms other than modications in the DNA sequence [127]. Epigenetic signals
include DNA methylation, sperm-specic nucleoproteins
and sperm-borne RNAs (discussed in previous subsections),
and the DNA loop domain organization by the sperm nuclear matrix (reviewed in [128]). For example, studies in
humans have found that nucleosomes in which sperm DNA
is packed and represent between 15% and 30% of sperm
DNA contribute to paternal zygotic chromatin and embryo
epigenome [129]. Therefore, because, on fertilization,
oocyte inherits epigenetic signals from sperm chromatin
and these signals are known to play a role in early embryo
development, attention to iatrogenic damage inicted by
cryopreservation procedures has also been paid.
Data are still scarce with regard to genomic imprinting,
and barely a few number of studies conducted in humans
exist. In a preliminary study, Klver et al. [130] determined
the degree of methylation of up to nine genes: maternally
imprinted (LIT1, SNRPN, MEST), paternally imprinted (MEG3,
H19), repetitive elements (ALU, LINE1), one spermatogenesisspecic gene (VASA), and a gene related to male infertility
(MTHFR), and found that neither cryopreservation nor the
length of storage (2 days compared to 4 weeks) altered the
methylation pattern of those genes. These data indicate that
sperm cryopreservation, at least from the data we have thus
far, is safe in terms of gene imprinting.
4.9. Cryopreservation effects on sperm motility
A dramatic reduction in sperm motility is one of the most
apparent consequences of boar sperm cryopreservation
(Fig. 2) and has been widely reported in the literature [71
73,131]. Another interesting aspect resulting from
53
alterations of motility due to cryopreservation regards to

sperm motile subpopulations. Indeed, while freeze-thawing
does not cause major changes in the structure of sperm
subpopulations when dened on curvilinear velocity, there
are signicant variations in the specic percentages of each
subpopulation [132]. Moreover, in spite of observing four
subpopulations in both cases, kinetic parameters in each
sperm subpopulation in PFE are signicantly lower than in
GFE [133]. The ejaculate variability to withstand cryopreservation is discussed in the following section.
5. Individual variability to sustain cryopreservation
and freezability markers
5.1. Good and poor freezability ejaculates
Not all ejaculates present the same resilience to withstand freeze-thawing procedures (i.e., freezability), but
rather there is a signicant variability between- and
within-ejaculates, and even between fractions within the
same ejaculate [25,65,71,134,135]. For this reason, boars
and their ejaculates are classied into good or bad
freezers, and as GFE or PFE [136,137]. The existence of
GFE and PFE is not restricted to pigs but has also been reported in other mammalian species, such as goats [138] and
stallions [57]. In addition, the individual boar response is
not only related to cryopreservation, but the success of
other semen-processing techniques, such as liquid storage
and gender-sorting, has also been reported to be greatly
inuenced by an individual component [139].
GFE and PFE differ in their post-thawing sperm survival
and motility, and the formers have been reported to present less resistance of their nucleoprotein structure than
the latters [71,137]. In contrast, observations under a cryoscanning electron microscope have revealed that the differences between GFE and PFE are not related to the degree
of dehydration and ice crystal distribution after freezing
and before thawing [140].
The mechanisms underlying differences between GFE
and PFE still remain unknown. In a landmark study, Thurston et al. [141] found that differences between GFE and
PFE were related to polymorphisms in amplied restriction
fragments and identied up to 16 potential genetic
markers. This indicated that genetic differences could be
the basis for these freezability differences. Notwithstanding, again, this nding does not seem to be exclusive
for pigs because a single nucleotide polymorphism in the
position 191 of HSP70, specically C > G, has been correlated with higher sperm freezability in goats [142]. Moreover, and as discussed in the following section, it appears
that variations in the expression of genes involved in the
resistance of cell against thermal stress are also related to
ejaculate freezability. However, it is yet to be elucidated by
which mechanism the abundance of theses proteins is
regulated and why.
Freezability is also related to the ability to withstand
cooling rates, as Medrano et al. [143] found that cooling rate
compromised sperm function and survival at post-thawing
depending on the intrinsic freezability. In addition, it is
worth noting that variations in the cryopreservation protocol, affect PFE more than GFE [144] and that the sperm
54
response to additives in the freezing extender also rely on

the intrinsic freezability [131,145].
Finally, it has been reported that the addition of seminal
plasma proteins from GFE increases the freezability of PFE.
These positive effects not only regard to viability and
motility of frozen-thawed sperm but also to the percentage
of penetrated oocytes after IVF. However, these effects are
independent from the antioxidant capacity [146], and the
seminal plasma proteome of GFE and PFE do not differ in
terms of antioxidant scavengers [147].
5.2. Freezability markers
One of the main problems for boar sperm cryopreservation is the identication of GFE and PFE before starting
cryopreservation procedures because conventional sperm
parameters in fresh/extended sperm fail to predict the
ejaculate ability to withstand freeze-thawing [16,137]. This
fact has led researchers to focus on the identication of
freezability markers, which are dened as those elements
(proteins or not) that may allow the identication of GFE
and PFE in fresh/extended semen (i.e., before starting
cryopreservation protocols). The following section and
Table 1 summarize the main freezability markers reported
thus far.
5.2.1. Sperm proteins as freezability markers
Sperm proteins identied as freezability markers are
membrane channels and other proteins that either are
involved in the response to osmotic and thermal stress or
play a specic role that remains to be fully elucidated. Thus
far, four sperm proteins: heat shock protein 90, HSP90AA1
[102]; acrosin binding protein, ACRBP; triosephosphate
isomerase, TPI [103]; voltage-dependent anion channel 2,
VDAC2 [150] have been found to predict ejaculate freezability when evaluated in extended semen (Table 1).
Furthermore, Chen et al. [101] have found that amounts of
SOD1; outer dense ber protein 2, ODF2; and a-kinase
anchor protein 3, AKAP3 differ between extended and
frozen-thawed boar sperm, thereby suggesting that these
three proteins could also predict the sperm resilience to
sustain cryopreservation protocols.
The existence of protein freezability markers is not
restricted to boar sperm. In humans, SOD content at postthawing after density gradient washing is correlated with
sperm motility [151] and levels of enolase-1 and glucose-6phosphate isomerase have been reported to be higher in
GFE than in PFE [152].
5.2.2. Seminal plasma proteins as freezability markers

Using differential gel electrophoresis, Vilagran et al. [147]
compared the seminal plasma proteome of fresh GFE and
PFE and found that bronectin 1 was a freezability marker
for boar ejaculates (Table 1). A recent study comparing the
seminal plasma proteome of ram GFE and PFE has found that
whereas 26S proteasome complex, acylamino acid
releasing enzyme, alpha mannosidase class 2C, HSP90AA1,
tripeptidyl-peptidase 2, TCP-1 complex, sorbitol dehydrogenase, and transitional endoplasmic reticulum ATPase are
positively correlated with ejaculate freezability, cystatin,
zinc-2-alpha glycoprotein, angiogenin-2like protein,
cartilage acidic protein-1, cathepsin B, and ribonuclease 4
isoform 1 are negatively correlated [153]. Remarkably, 26S
proteasome has been found within the list. Given the role
suggest for sperm-borne 26 proteasome for the degradation
of sperm receptor in the zona pellucida of the oocyte [154],
this clearly warrants further research.
Another seminal plasma protein that appears to confer
higher cryotolerance to boar ejaculates is the N-acetyl-bhexosaminidase. The activity of this protein in fresh seminal plasma is correlated with the activities of SOD and
glutathione peroxidase as well as with mitochondrial
function and total oxidant status. At post-thawing, the activity of N-acetyl-b-hexosaminidase is negatively correlated to sperm viability, motility, and lipid peroxidation, so
that higher levels of b-HEX activity in boar seminal plasma
are related to low sperm cryotolerance [149] (Table 1).
5.2.3. Other sperm freezability markers
Other freezability markers identied thus far include
patterns of sperm motile subpopulations in extended
semen [133], specic kinetic parameters evaluated at the
cooling step [137], and acrosin activity [145,148].
6. Factors affecting the sperm freezability
As previously mentioned, there is an important individual component related to each ejaculation that critically
affects its cryopreservation success. The present section
summarizes six different factors that could inuence the
sperm freezability: season, diet, breed, ejaculate fractions,
sperm selection, and holding time.
6.1. Season
Temperature and photoperiod affects spermatogenesis
and epididymal maturation [155,156]. Barranco et al. [157]
Table 1
Markers that predict the boar sperm resilience to withstand cryopreservation in extended/fresh semen.
Protein
Marker
Function/action
Reference
Acrosin activity
Sperm
Enzyme activity positively correlated with sperm cryotolerance
Acrosin binding protein (ACRBP)

Fibronectin (FN1)
Heat shock protein (HSP90AA1)
N-acetyl-b-hexosaminidase (b-HEX)
Triosephosphate isomerase (TPI)
Voltage-dependent anion channel 2 (VDAC2)
Sperm
Seminal plasma
Sperm
Seminal plasma
Sperm
Sperm
Higher levels in GFE than in PFE

Enzyme activity negatively correlated with sperm cryotolerance
Lower levels in GFE than in PFE
[145]
[148]
[103]
[147]
[102]
[149]
[103]
[150]
Abbreviations: GFE, good freezability ejaculates; PFE, poor freezability ejaculates.
have shown that ejaculate freezability relies on the season

of ejaculate collection, regardless of breed and boar, so that
boar ejaculates collected during winter and spring have
higher freezability than those obtained during autumn and
summer.
The effects of season on sperm freezability have also
been observed in other mammalian species. In humans,
Yogev et al. [158] found that sperm freezability was higher
from October to February than from March to September. In
the case of horses, sperm collected between March and
June in the Northern Hemisphere are those that present the
highest freezability [159]. In contrast, in FranchesMontagnes stallions, Janett et al. [160] observed that
sperm collected in autumn presented higher sperm
motility at post-thawing than sperm collected in winter.
These differences between studies should consider that, in
contrast to pigs, horses present breeding and nonbreeding
seasons, and the location where studies are conducted may
have strongly inuenced the nal outcome. In any case,
most studies clearly point out that season affects sperm
freezability in mammals, except in the case of goats where
season has not been found to affect the sperm resilience to
sustain cryopreservation [138].
6.2. Diet
It has been reported that diet has impact on sperm
quality in boars [156,161,162]. Unfortunately, there are not
many studies about the impact of boars diet on sperm
cryotolerance. Supplementing boars diet with omega-3
polyunsaturated fatty acids such as DHA and eicosapentanoic acid (for 67 months) has no effect on the ability of
sperm to be cryopreserved [161]. On the other hand, and as
aforementioned, the reduction of sperm quality due to
freeze-thawing procedures has been observed after the
reduction of temperature from 15 C to 5 C, and there is an
interest to design extenders for liquid-stored sperm at 5 C.
However, in a study that investigated the sperm at 5 C,
Casas et al. [163] found no benecial effects from supplementing boar diet with egg yolk.
There are other studies conducted in different livestock
species. In horses, Brinsko et al. [164] found that supplementing stallions diet with DHA with a pattern of 14 weeks
feeding, a 14-week resting period, and another 14-week
feeding period not only increased the DHA amounts in
the resulting sperm cells, but also increased the motility
after freeze-thawing. Another study tried to improve the
quality of frozen-thawed stallion sperm in winter because
the freezability of stallion sperm is known to be reduced
during this season (see previous section). However, adding
linseed oil, and a commercial cocktail of antioxidants
(Myostem Protec, Audevard) for a total of 12-week period
had no effect on this parameter [165].
In all cases mentioned herein, the impact of animals
diet on sperm freezability seems to be marginal. However,
studies conducted in different species clearly indicate that
diet modications, especially when DHA supplements are
used, modulate the lipid composition of sperm plasma
membrane. In addition, in the case of horses, the modication of stallions diet slightly improves the sperm resilience to freeze-thawing. Although one should be aware
55
that results from one species (horses) cannot be extrapolated to others (pigs), diet modications not only with regard to composition but also to feeding regimens and
length of the trial could modulate the sperm freezability
also in boars. This hypothesis requires further research.
6.3. Breeds
It is not clear whether boar sperm from some breeds are
more resilient than others to freeze-thawing procedures,
but there is a consensus that, regardless of the breed, individual differences (i.e., between boars) exist and that
these differences are related to the phospholipid composition of the plasma membrane [65].
The lack of differences between breeds and the existence of an individual-boar component was earlier reported in sperm preserved in liquid storage at 15 C [166].
In spite of this, a recent report indicates that the amount of
gammaoryzanolenriched rice bran oil required for
improving sperm freezability is higher in Large White and
Landrace than in Duroc breeds [167]. Moreover, another
recent study has shown that the osmotic resistance of
acrosomal membrane after freeze-thawing is higher in
Large White than in Landrace boars [168]. It is entirely
reasonable that differences between studies are due to the
heterogeneity of boar populations used because all works
concur that individual differences do exist. However, it
could be that breed effects were more apparent in some
parameters than others. Therefore, further research is
required to address these inconsistent ndings.
6.4. Ejaculate fractions
Apart from variability between boars and ejaculates
from the same boar, it appears that given fractions within a
given ejaculate are more resilient to freeze-thawing procedures than others [135]. On the one hand, cryopreservation of sperm fractions is better than that of the entire
ejaculate, especially that of the sperm-rich fraction, and
this is attributed to the negative effect of the seminal
plasma from the post-spermatic fraction [169].
On the other hand, studies comparing different portions
of the sperm-rich fraction have reported that the rst one
(rst 10 mL) is more able to sustain cryopreservation than
the others, probably because it contains the lowest levels of
bicarbonate [170]. This is consistent with the fact that
percentages of sperm showing high intracellular Ca2
levels, exocytosed acrosomes, and protein-tyrosine phosphorylation (including p32) are lower in the rst portion
than in the rest of the sperm-rich fraction [114,171].
6.5. Selection of sperm before cryopreservation
Sperm selection through density gradient washing
before cryopreservation has also been reported to increase
the ejaculate freezability not only in pigs but also in other
mammalian species, such as stallions [172,173]. These positive effects are explained by the necessity of removing dead
sperm because these are detrimental for viable sperm [174].
In the case of pigs, the use of single-layer centrifugation
through porcine-specic colloids, such as Androcoll-P
56
(Swedish University of Agricultural Sciences, Uppsala,

Sweden), allows removal of cholesterol and seminal plasma
proteins from sperm surface without affecting sperm
membrane integrity and improves post-thaw sperm quality
and in vitrofertilizing ability [175,176].
6.6. Does the holding time before cryopreservation affect the
sperm freezability?
Holding time is dened as the storage period at 15 C to
17 C between sperm collection and the start of cryopreservation procedures. While some authors have reported
that the length of this period does not affect the sperm
resilience to withstand freeze-thawing procedures
[24,177], others have shown that its benets, with holding
times between 10 and 24 hours being better than those of
3 hours [26,169]. It appears that holding time increases
sperm cryotolerance through maintaining the lipid architecture of plasma membrane [178], via an unidentied
mechanism that involves phosphorylation of some sperm
proteins, such as HSP70 [179].
for sperm freezability has not only been observed in pigs

[147] but also in other species, such as rams [183].
Regarding the effects of seminal plasma on post-thawed
sperm, some studies have indicated that supplementing
thawing medium with seminal plasma is benecial for
sperm motility and membrane integrity [181,184], whereas
others have reported a decrease in the integrity of acrosome and plasma membrane and increase ROS levels [185].
The differences between these studies could be explained
by the volume of the seminal plasma added, the extender
used, and the temperature of incubation [186].
Because seminal plasma positively effects the endometrial reaction after AI in pigs and other species and this may
affect reproductive outcomes [187189], one could
consider adding a seminal plasma fraction immediately
before AI. Unfortunately, experiments conducted thus far
are not conclusive. Indeed, although some reports indicate
that the addition of seminal plasma increases the reproductive performance of frozen-thawed boar sperm
[181,190,191], others do not [192].
7.2. Cyclodextrins
7. Increasing the cryopreservation success through

additives
Cyclodextrins are cyclic heptasaccharides that consist of
b (14) glucopyranose units and have a hydrophobic center.

A signicant number of studies have been focused on
the improvement of sperm cryopreservation success. In
this regard, it is worth noting that most of the additives
have been proven to be successful in species other than pigs
and then tested in this species. However, and as discussed
in the following sections, the additives that are benecial
for some species may not be useful for others.
7.1. Addition of seminal plasma proteins: Good or bad?
Much research has been devoted to the effects of seminal plasma proteins on the sperm resilience to withstand
freeze-thawing procedures. In this regard, it is worth
mentioning that concerns on the role of seminal plasma
exist, especially because of its potential detrimental effects
on the preservation of boar sperm in liquid and frozen
states. The identity of which seminal plasma components
are detrimental for boar sperm cryopreservation still remains unknown, but it has been reported that removing
low-molecular weight proteins (1214 kDa) by dialysis
improves cryopreservation outcomes [180]. Furthermore, a
bacteria-released endotoxin (lipopolysaccharide) has been
identied as one of the plasma seminal factors that may
impair boar sperm freezability because destabilizes the
plasma membrane when binds to the Toll-like receptor-4
present on the sperm surface [181].
Although most of the studies evaluating the effects of
seminal plasma have supplemented the thawing medium,
the addition of 5% seminal plasma from GFE to freezing
extenders has been reported to improve the motility,
membrane integrity, and IVF outcomes of frozen-thawed
sperm in PFE [146]. This positive effect could be mediated
through inhibition of in vitro capacitation and coolinginduced capacitation-like changes via heparin-binding
seminal plasma proteins (DQH, AQN-1, AQN-3, and AWN)
[182]. In fact, the relevance of seminal plasma composition
When preloaded with cholesterol (cholesterol-loaded cyclodextrins, CLC), these substances are able to increase the
amounts of this sterol in the plasma membrane. This increases the stability of plasmalemma and improves survival
of frozen-thawed mammalian sperm [193]. In the specic
case of pigs, the addition of CLC improves post-thaw sperm
quality and in vitrofertilizing ability without affecting
capacitation status and DNA integrity (Table 2) [199201].
The main inconvenience is that adding CLC may require
increasing of glycerol concentration (from 3% to 5%; [206]),
and this may be detrimental for sperm [46,47].
When added to thawing extender, CLC increase the
integrity of plasma membrane and acrosome of frozenthawed sperm (Table 2). However, because a detrimental
effect of CLC supplementation on sperm motility exists at
concentrations higher than 12.5 mg CLC per 500 million
sperm, more research is required to address whether the
inclusion of CLC in thawing extenders is benecial or not
[202].
7.3. Antioxidants
Supplementing cryopreservation media with antioxidants has been reported to be positive in different species,
not only in pigs. In humans, e.g., supplementing freezing
media with vitamin E and hypotaurine and with natural
antioxidants such as those from Opuntia cus-indica improves sperm motility and decreases DNA fragmentation at
post-thawing [173,207,208].
In pigs, the list of antioxidants with positive effects for
sperm quality at post-thawing includes L-cysteine, atocopherol, lutein, butylated hydroxytoluene, Trolox and
ascorbic acid, the results of the latter being better when
combined with Trolox or with reduced glutathione (GSH;
Table 2) [194198,203,204]. The benecial effects of
ascorbic acid supplementation have also been observed on
57
Table 2
Benecial effects on frozen-thawed boar sperm after supplementation of freezing and thawing media with different additives (some of them are
antioxidants).
Additive
Media
Effects
Reference
Alginate
Freezing
[96]
Ascorbic acid
a-tocopherol
Freezing and
thawing
Freezing
Maintains the acrosome integrity. Increases the activities

of SOD and GPX and decreases malondialdehyde levels.
Improves sperm motility and membrane integrity.
Enhances glutathione (GSH) effects.
Increases progressive motility, plasma membrane, and acrosome integrity.
Butylated hydroxytoluene (BHT)
Freezing
Cholesterol-loaded
cyclodextrins (CLC)
Freezing and
thawing
Improves sperm survival and embryo development to

blastocyst after IVF with frozen-thawed sperm.
Improve stability of sperm plasma and maintains the acrosome integrity better.
Increase IVF outcomes
L-cysteine
Freezing
Improves progressive motility, plasma membrane, and acrosome integrity.
Lutein
Freezing
Reduced GSH
Freezing and
thawing
Improves sperm motility, DNA and acrosome integrity,

and the resistance to osmotic stress (HOST).
Increases overall sperm quality and stabilizes the nucleoprotein structure.
Improves the sperm fertilizing ability both in vivo and in vitro.
Trolox
Freezing
Maintains better sperm motility and acrosome integrity.
[194]
[195]
[196]
[197]
[198]
[199]
[200]
[201]
[202]
[203]
[204]
[195]
[72]
[73]
[131]
[194]
[205]
[195]
The effects are described with regard to the results on un-supplemented controls.
Abbreviations: GPX, glutathione peroxidase; HOST, hypoosmotic swelling test; SOD: superoxide dismutase.
motility and acrosome and membrane integrity of bull

sperm [209]. In contrast, other antioxidants such as catalase and b-mercaptoethanol have no benecial effects [53].
One antioxidant that has given interesting results is
reduced GSH (Table 2). The addition of GSH to both freezing
and thawing extenders increases sperm quality at postthawing, stabilizes the nucleoprotein structure, and improves fertilizing ability both in vivo and in vitro
[72,73,131,194,205]. These positive results are not exclusive
of pigs. In human sperm, there is a reduction of up to 64% in
the GSH content after freeze-thawing [210], which indicates that the antioxidant defense system is challenged
by sperm cryopreservation. Furthermore, the addition of
GSH to freezing and thawing media decreases ROS levels
and improves motility of human sperm [210].
7.4. Alginate
The addition of alginate to freezing extenders maintains
better the acrosome integrity, increases the activities of
SOD and glutathione peroxidase, and decreases malondialdehyde levels after freeze-thawing (Table 2) [96].
7.5. Melatonin
Different studies have evaluated the impact of supplementing extenders for both fresh/extended and frozenthawed semen with melatonin, given its antioxidant
properties. In bulls, adding melatonin to freezing extenders
at nal concentrations ranging between 2 and 3 mM improves sperm survival and motility and reduces lipid peroxidation at post-thawing [211]. Melatonin also has
positive effects on the lipid peroxidation of cryopreserved
stallion sperm, although this impact on motility is less
apparent [212]. In rams, similar results are observed when
1 mM of melatonin is added to freezing media, the benecial effects not only being observed in sperm motility and
viability, but also in DNA integrity and embryo cleavage
rates after IVF [213]. Finally, while melatonin has not been
investigated in cryopreserved boar sperm, studies with
extended semen during long-term storage did not show
much improvement, despite reporting a marginal, positive
effect on sperm viability [214]. Thus, more research is
required to address whether melatonin could also be
benecial for cryopreserved boar sperm.
7.6. Insulin growth factor-I
Adding insulin growth factor I (IGF-I) together with
vitamin E to extended boar semen improves sperm motility
after 72 hours of liquid storage at 15 C [215]. However, the
benecial effects of supplementing cryopreservation media
with IGF-I have been reported in cryopreserved mammalian sperm, but not yet in pigs. In rams, the addition of IGF-I
to freezing extenders increases the viability and motility of
frozen-thawed ram sperm but does not have any impact
upon fertilizing ability [216]. Again, it would be interesting
to investigate whether IGF-I has any positive effect on
cryopreserved boar sperm.
7.7. Heat shock proteins: New additives for cryopreservation
media?
A new strategy to improve cryopreservation extenders
could consist of adding proteins that counteract the
damaging effects of these procedures. For example, it is
known that heat shock 70 kDa protein 8 (HSPA8) prolongs
the sperm survival in the oviduct and is upregulated in
oviductal monolayers in response to sperm [217,218]. The
mechanism through which this protein maintains sperm
58
survival is related to its ability to maintain the uidity and

stability of the sperm plasmalemma [219]. Recently, Holt
et al. [220] have shown that the addition of a bovine, recombinant HSPA8 to freezing and thawing media improves
the bull sperm survival at post-thawing. Such effects rely
on breed, especially when such addition is combined with
CLC. While no similar study has been conducted in boars
and that the design of such a medium containing a recombinant protein is now far from being marketable due to
benetcost analyses, such nding suggests new research
horizons on the design of proper freezing and thawing
media for boar sperm.
[12,28]. All this indicates that, even restricted to some

particular cases, boar sperm cryopreservation has its own
niche.
8. Frozen storage for long-term: Any effect on sperm?
10.1. Vitrication
One of the issues that should be addressed in the future

is whether long-term storage in liquid nitrogen after
cryopreservation is limited and, if this is the case, to which
extent. An article by Fraser et al. [168] has recently indicated that when sperm are stored for a period of 4 years,
there is a reduction in different sperm parameters, such as
membrane integrity, motility, and mitochondrial function.
Although these authors also highlighted the individual
component and only their study involved 10 individuals,
more research is required to elucidate this point. In fact,
separate studies conducted in other species have not
observed the similar outcome. Indeed, a study in a human
cryobank involving up to 72 donors found that long-term
frozen storage (0.514.4 years) did not impair progressive
sperm motility at post-thawing [221].
Thus far, attempts to vitrify sperm have been mainly

conducted in mice and humans. Freezing/cooling rates
differ between slow-freezing and vitrication as well as the
composition of their media. Indeed, although permeating
CPAs (such as glycerol) are fundamental in the composition
of the former, nonpermeating ones, such as sucrose and
serum albumin are the main components in the latter, as
Isachenko et al. [228] found in humans.
In rams, a recent study has shown that sperm vitrication using a commercial extender (Biladyl) with egg yolk
and glycerol gives acceptable results in terms of plasma
membrane and acrosome integrity and DNA fragmentation [229]. This study has also found that the use of sucrose and glycerol is less advisable because cryodamage is
higher. These are interesting results that deserve further
attention.
Another work in humans that compared conventional
freezing and vitrication found no effect from the presence
of CPAs on the motility, viability, DNA fragmentation, and
hyaluronan-binding ability of post-thawed sperm [230].
Because, as previously discussed, the presence of CPAs may
exert cytotoxic effects, the fact that its presence does not
much affect the cryopreserved outcomes warrants further
research, at least in humans. However, given that human
and pig sperm are different in terms of cryopreservation
success, these results may not be extrapolated to pigs. In
any case, as nding a proper method for cryopreserving
boar sperm at the beginning was more difcult than in
other species (e.g., bulls) and today this is quite a standard
technique, data available for sperm vitrication in mice,
rams, and humans do not discourage further research in
pigs.
9. Fertility of frozen-thawed boar sperm

One of the concerns that have been existing historically
regards to the lower reproductive performance of frozenthawed sperm. Indeed, with extended semen, swine industry is used to work with conception rates higher than
85% and litter sizes bigger than 11 piglets. However, gures
reveal that farrowing rates and litter sizes for AI using
frozen-thawed semen are signicantly lower [222]. In spite
of this, it is worth noting that since the rst attempts
reporting seven alive piglets born per litter after the use of
frozen-thawed boar sperm [44], different advances have
been made and recent data are far more optimistic as 10
piglets per litter, or even more, may be achieved [222,223].
These outcomes may be improved when additives, such as
GSH, are used [72,222]. Moreover, in a laboratory study,
Almiana et al. [224] reported fertilization rates similar
between extended (94.4%) and frozen-thawed (90.9%)
semen and also observed that no signicant impact on
early embryo development was found when frozenthawed sperm was used.
The interval of insemination-to-ovulation is also a
crucial factor as too is the use of post-cervical or deep intrauterine insemination [225,226]. In addition, double
insemination yields far better results than single one [227].
Finally, it is worth mentioning that other new technologies, such as sex-sorted sperm, may require or benet
from the use of frozen-thawed sperm. Furthermore, sperm
cryopreservation may be useful for planning of AI centers
and preservation of genetic material in breeds and strains
10. New research horizons in long-term preservation

of boar sperm
Alternative methods for long-term preservation of
mammalian sperm include vitrication and freeze-drying.
While all these technologies are still in their infancy and
not many studies in pigs have been conducted, they are
mentioned herein to suggest further research perspectives.
10.2. Freeze-drying
Although freeze-drying or lyophilization seems to be a
technique far from being conducted in pigs, the present
section intends to summarize the recent advances. Most of
the studies published thus far have been conducted in rodent species (mice and rats) to maintain specic animal
strains [231]. The resulting freeze-dried sperm, even
impaired in their viability, motility and DNA integrity [232],
can be used for successful oocyte fertilization through
intracytoplasmic sperm injection (ICSI) [233].
Some new insights have also been obtained from freezedried sperm of dogs and wild animals, such as chimpanzee,
giraffe, jaguar, and weasel, when injected into mouse oocytes [234,235]. Besides, it is worth noting that, despite the
high reduction in sperm membrane integrity, the damage
inicted on DNA integrity by freeze-drying in humans is
lower than that of cryopreservation [236,237].
In 2011, Choi et al. [238] using freeze-dried stallion
sperm produced the rst live foals after ICSI and embryo
transfer. This was the rst study in a non-laboratory animal
species. In bulls, Hara et al. [239] reported the benecial
effects of adding trehalose to and removing NaCl from
EGTA-based, freezing medium and found that freeze-dried
sperm preserved in non-collapsed cakes (i.e., drying temperature: 30 C) yield better blastocyst development than
in collapsed cakes (drying temperature: 0 or 15 C).
However, Abdalla et al. [240] in cattle showed that
freeze-drying diminishes the sperm ability to elicit Ca2
oscillations and alleviate the metaphase II arrest in bovine
oocytes after ICSI and also impairs the embryo
development.
In pigs, a landmark article by Kwon et al. [241] reported,
for the rst time, successful ICSI fertilization and embryo
development up to blastocyst stage using freeze-dried boar
sperm. Men et al. [242] showed that supplementing EGTAbased medium with trehalose had a positive effect on DNA
integrity in freeze-dried sperm but did not nd any increase in early embryo development. After this, some advances have been gained from modifying the EDTA-based
medium with lactose because this variation decreases the
increase in DNA fragmentation and also results in oocyte
fertilization after ICSI [243].
11. Conclusions
Despite being the most effective method for long-term
preservation of mammalian sperm, cryoinjuries may
compromise greatly sperm function and survival and
strongly decrease the reproductive performance.
Mounting evidence indicates that not only cryodamage is
restricted to sperm membrane but also affects the integrity of sperm chromatin (nucleoprotein structure and
DNA) and mitochondrial function, involves degradation of
mRNAs, and impairs the function of some relevant proteins. Furthermore, there is an important individual
component that explains the ejaculate freezability. While
different extrinsic factors, such as season and diet, could
modulate the ejaculate freezability, intrinsic components,
such as genetic differences between boars and specic
features of spermatogenesis and epididymal maturation,
seem to play a major role. In spite of this, the identication of sperm freezability markers and the positive effects
of some additives have represented an important forward
step for this technology. Although all this has resulted in
an increase in their reproductive performance, the specic
features of boar sperm cryopreservation protocols and pig
breeding make this technique to be restricted to research
and germplasm banks. This is in contrast to other
mammalian species such as bulls, in which AI is mainly
conducted with frozen-thawed sperm. Finally, vitrication
and freeze-drying are currently far from being established
techniques in pigs. However, data from humans and other
animals are encouraging.
59
Acknowledgments
The author is funded by the European Commission, FP7People Programme, Marie Curie-IEF (grant Number:
626061). Some diagrams used for producing Figure 2 were
obtained and modied from Servier Medical Art.
Competing Interests
The author has no conicts of interest to declare.
References
[1] Loren AW, Mangu PB, Beck LN, Brennan L, Magdalinski AJ,
Partridge AH, et al. American Society of Clinical Oncology. Fertility
preservation for patients with cancer: American Society of Clinical
Oncology clinical practice guideline update. J Clin Oncol 2013;31:
250010.
[2] Comizzoli P, Wildt DE. Mammalian fertility preservation through
cryobiology: value of classical comparative studies and the need
for new preservation options. Reprod Fertil Dev 2013;26:918.
[3] Mazur P, Leibo SP, Seidel Jr GE. Cryopreservation of the germplasm
of animals used in biological and medical research: importance,
impact, status, and future directions. Biol Reprod 2008;78:212.
[4] Kopeika J, Thornhill A, Khalaf Y. The effect of cryopreservation on
the genome of gametes and embryos: principles of cryobiology
and critical appraisal of the evidence. Hum Reprod Update 2015;
21:20927.
[5] Polge C, Smith AU, Parkes AS. Revival of spermatozoa after vitrication and dehydration at low temperatures. Nature 1949;164:
666.
[6] Grossfeld R, Sieg B, Struckmann C, Frenzel A, Maxwell WM, Rath D.
New aspects of boar semen freezing strategies. Theriogenology
2008;70:122533.
[7] Polge C. Low-temperature storage of mammalian spermatozoa.
Proc R Soc Lond B Biol Sci 1957;147:498508.
[8] Curry MR. Cryopreservation of semen from domestic livestock. Rev
Reprod 2000;5:4652.
[9] Hess EA, Teague HS, Ludwick TM, Martig RC. Swine can be bred
with frozen semen. Ohio Fm Home Res 1957;42:100.
[10] Pursel VG, Johnson LA. Freezing of boar spermatozoa: fertilizing
capacity with concentrated semen and a new thawing procedure. J
Anim Sci 1975;40:99102.
[11] Westendorf P, Richter L, Treu H. Zur Tiefgefrierung von Ebersperma. Labor-und Besamungsergebnisse mit dem Hlsenberger
Pailetten-Verfahren. Dtsch Tierrztl Wschr 1975;82:2617.
[12] Casas I, Flores E. Gene banking: the freezing strategy. In: Bonet S,
Casas I, Holt WV, Yeste M, editors. Boar reproduction. Berlin:
Springer; 2013. p. 55188.
[13] Baishya SK, Biswas RK, Kadirvel G, Deka BC, Kumar S, Sinha S, et al.
Effect of conventional and controlled freezing method on the post
thaw characteristics of boar spermatozoa. Anim Reprod Sci 2014;
149:2317.
[14] Vutyavanich T, Piromlertamorn W, Nunta S. Rapid freezing versus
slow programmable freezing of human spermatozoa. Fertil Steril
2010;93:19218.
[15] Sieme H, Oldenhof H. Cryopreservation of semen from domestic
livestock. Methods Mol Biol 2015;1257:27787.
[16] Yeste M. Recent advances in boar sperm cryopreservation: state of
the art and current perspectives. Reprod Domest Anim 2015;
50(Suppl 2):719.
[17] Gao D, Critser JK. Mechanisms of cryoinjury in living cells. ILAR J
2000;41:18796.
[18] Mazur P. Equilibrium, quasi-equilibrium, and nonequilibrium
freezing of mammalian embryos. Cell Biophys 1990;17:5392.
[19] Muldrew K, McGann LE. The osmotic rupture hypothesis of
intracellular freezing injury. Biophys J 1994;66:53241.
[20] Mazur P, Leibo SP, Chu EH. A two-factor hypothesis of freezing
injury. Evidence from Chinese hamster tissue-culture cells. Exp
Cell Res 1972;71:34555.
[21] Wiest SC, Steponkus PL. The osmometric behavior of human
erythrocytes. Cryobiology 1979;16:1014.
[22] Mazur P, Cole KW. Inuence of cell concentration on the contribution of unfrozen fraction and salt concentration to the survival
of slowly frozen human erythrocytes. Cryobiology 1985;22:509
36.
60
[23] Holt WV. Basic aspects of frozen storage of semen. Anim Reprod
Sci 2000;62:322.
[24] Toms C, Gmez-Fernndez J, Gmez-Izquierdo E, de Mercado E.
Effect of the holding time at 15 C prior to cryopreservation, the
thawing rate and the post-thaw incubation temperature on the
boar sperm quality after cryopreservation. Anim Reprod Sci 2014;
144:11521.
[25] Holt WV, Medrano A, Thurston LM, Watson PF. The signicance of
cooling rates and animal variability for boar sperm cryopreservation: insights from the cryomicroscope. Theriogenology 2005;63:
37082.
[26] Juarez JD, Parrilla I, Vazquez JM, Martinez EA, Roca J. Boar semen
can tolerate rapid cooling rates prior to freezing. Reprod Fertil Dev
2011;23:68190.
[27] Okazaki T, Abe S, Shimada M. Improved conception rates in sows
inseminated with cryopreserved boar spermatozoa prepared with
a more optimal combination of osmolality and glycerol in the
freezing extender. Anim Sci J 2009;80:1219.
[28] Benson JD, Woods EJ, Walters EM, Critser JK. The cryobiology of
spermatozoa. Theriogenology 2012;78:168299.
[29] Fahy GM. The relevance of cryoprotectant toxicity to cryobiology. Cryobiology 1986;23:113.
[30] Gutirrez-Prez O, Jurez-Mosqueda Mde L, Carvajal SU,
Ortega ME. Boar spermatozoa cryopreservation in low glycerol/
trehalose enriched freezing media improves cellular integrity.
Cryobiology 2009;58:28792.
[31] Hu JH, Li QW, Jiang ZL, Yang H, Zhang SS, Zhao HW. The cryoprotective effect of trehalose supplementation on boar spermatozoa quality. Reprod Domest Anim 2009;44:5715.
[32] Malo C, Gil L, Gonzalez N, Cano R, de Blas I, Espinosa E. Comparing
sugar type supplementation for cryopreservation of boar semen in
egg yolk based extender. Cryobiology 2010;61:1721.
[33] Gmez-Fernndez J, Gmez-Izquierdo E, Toms C, Moc E, de
Mercado E. Effect of different monosaccharides and disaccharides
on boar sperm quality after cryopreservation. Anim Reprod Sci
2012;133:10916.
[34] Rodriguez-Martinez H, Wallgren M. Advances in boar semen
cryopreservation. Vet Med Int 2010. http://dx.doi.org/10.4061/
2011/396181.
_
[35] Fraser L, Strzezek R, Strzezek
J. Fertilizing capacity of boar semen
frozen in an extender supplemented with ostrich egg yolk lipoprotein fractionsda pilot study. Pol J Vet Sci 2007;10:1315.
_ J. Effect of different procedures of ejaculate collec[36] Fraser L, Strzezek
tion, extenders and packages on DNA integrity of boar spermatozoa
following freezing-thawing. Anim Reprod Sci 2007;99:31729.
[37] Hu JH, Li QW, Jiang ZL, Li WY. Effects of different extenders on DNA
integrity of boar spermatozoa following freezing-thawing. Cryobiology 2008;57:25762.
[38] Wang P, Wang YF, Wang CW, Bu SH, Hu JH, Li QW, et al. Effects of
low-density lipoproteins extracted from different avian yolks on
boar spermatozoa quality following freezing-thawing. Zygote
2014;22:17581.
[39] Reed ML, Ezeh PC, Hamic A, Thompson DJ, Caperton CL. Soy lecithin replaces egg yolk for cryopreservation of human sperm
without adversely affecting postthaw motility, morphology, sperm
DNA integrity, or sperm binding to hyaluronate. Fertil Steril 2009;
92:178790.
[40] Papa FO, Felcio GB, Melo-Oa CM, Alvarenga MA, De Vita B,
Trinque C, et al. Replacing egg yolk with soybean lecithin in the
cryopreservation of stallion semen. Anim Reprod Sci 2011;129:737.
[41] Mata-Campuzano M, lvarez-Rodrguez M, lvarez M, TamayoCanul J, Anel L, de Paz P, et al. Post-thawing quality and incubation
resilience of cryopreserved ram spermatozoa are affected by
antioxidant supplementation and choice of extender. Theriogenology 2015;83:5208.
[42] Mazur P. Freezing of living cells: mechanisms and implications.
Am J Physiol 1984;247:C12542.
[43] Gao DY, Liu J, Liu C, McGann LE, Watson PF, Kleinhans FW, et al.
Prevention of osmotic injury to human spermatozoa during
addition and removal of glycerol. Hum Reprod 1995;10:110922.
[44] Johnson LA, Weitze KF, Fiser P, Maxwell WM. Storage of boar
semen. Anim Reprod Sci 2000;62:14372.
[45] Zeng C, Tang K, He L, Peng W, Ding L, Fang D, et al. Effects of
glycerol on apoptotic signaling pathways during boar spermatozoa
cryopreservation. Cryobiology 2014;68:395404.
[46] Arenas-Nez MA, Jurez-Mosqueda Mde L, Gutirrez-Prez O,
Anzalda Arce SR, Izquierdo AC, Rodrguez RM, et al. Glycerol
decreases the integrity of the perinuclear theca in boar sperm.
Zygote 2013;21:1727.
[47] Buhr MM, Fiser P, Bailey JL, Curtis EF. Cryopreservation in different
concentrations of glycerol alters boar sperm and their membranes.
J Androl 2001;22:9619.
[48] De Mercado E, Hernandez M, Sanz E, Rodriguez A, Gomez E,
Vazquez JM, et al. Evaluation of L-glutamine for cryopreservation
of boar spermatozoa. Anim Reprod Sci 2009;115:14957.
[49] Athurupana R, Takahashi D, Ioki S, Funahashi H. Trehalose in
glycerol-free freezing extender enhances post-thaw survival of
boar spermatozoa. J Reprod Dev 2015;61:20510.
[50] Silva CG, Cunha ER, Blume GR, Malaquias JV, Bo SN, Martins CF.
Cryopreservation of boar sperm comparing different cryoprotectants associated in media based on powdered coconut water,
lactose and trehalose. Cryobiology 2015;70:904.
[51] Amirat-Briand L, Bencharif D, Vera-Munoz O, Bel Hadj Ali H,
Destrumelle S, Desherces S, et al. Effect of glutamine on post-thaw
motility of bull spermatozoa after association with LDL (low density lipoproteins) extender: preliminary results. Theriogenology
2009;71:120914.
[52] Gibb Z, Morris LH, Maxwell WM, Grupen CG. Dimethyl formamide
improves the postthaw characteristics of sex-sorted and nonsorted
stallion sperm. Theriogenology 2013;79:102733.
[53] Buranaamnuay K, Grossfeld R, Struckmann C, Rath D. Inuence of
cryoprotectants glycerol and amides, combined with antioxidants
on quality of frozen-thawed boar sperm. Anim Reprod Sci 2011;
127:5661.
[54] Malo C, Gil L, Cano R, Martnez F, Garca A, Jerez RA. Dimethylformamide is not better than glycerol for cryopreservation of
boar semen. Andrologia 2012;44(Suppl 1):60510.
[55] Ozkavukcu S, Erdemli E, Isik A, Oztuna D, Karahuseyinoglu S. Effects of cryopreservation on sperm parameters and ultrastructural
morphology of human spermatozoa. J Assist Reprod Genet 2008;
25:40311.
[56] Vadnais ML, Althouse GC. Characterization of capacitation, cryoinjury, and the role of seminal plasma in porcine sperm. Theriogenology 2011;76:150816.
[57] Yeste M, Estrada E, Rocha LG, Marn H, Rodrguez-Gil JE, Mir J.
Cryotolerance of stallion spermatozoa is related to ROS production
and mitochondrial membrane potential rather than to the integrity of sperm nucleus. Andrology 2015;3:395407.
[58] Cerolini S, Maldjian A, Pizzi F, Gliozzi TM. Changes in sperm quality
and lipid composition during cryopreservation of boar semen.
Reproduction 2001;121:395401.
[59] Watson PF. The causes of reduced fertility with cryopreserved
semen. Anim Reprod Sci 2000;60-61:48192.
[60] Bailey JL, Lessard C, Jacques J, Brque C, Dobrinski I, Zeng W, et al.
Cryopreservation of boar semen and its future importance to the
industry. Theriogenology 2008;70:12519.
[61] Leahy T, Gadella BM. Capacitation and capacitation-like sperm
surface changes induced by handling boar semen. Reprod Domest
Anim 2011;46(Suppl 2):713.
[62] Harrison RA, Miller NG. cAMP-dependent protein kinase control of
plasma membrane lipid architecture in boar sperm. Mol Reprod
Dev 2000;55:2208.
[63] De Leeuw F, Colenbrander B, Verkleij A. The role membrane
damage plays in cold shock and freezing injury. Reprod Domest
Anim 1990;1:95104.
[64] Green CE, Watson PF. Comparison of the capacitation-like state of
cooled boar spermatozoa with true capacitation. Reproduction
2001;122:88998.
[65] Waterhouse KE, Hofmo PO, Tverdal A, Miller Jr RR. Within and
between breed differences in freezing tolerance and plasma
membrane fatty acid composition of boar sperm. Reproduction
2006;131:88794.
[66] Martnez-Soto JC, Landeras J, Gadea J. Spermatozoa and seminal
plasma fatty acids as predictors of cryopreservation success. Andrology 2013;1:36575.
[67] Ward WS. Function of sperm chromatin structural elements in
fertilization and development. Mol Hum Reprod 2010;16:30
6.
[68] Goslvez J, Lpez-Fernndez C, Fernndez JL, Gouraud A, Holt WV.
Relationships between the dynamics of iatrogenic DNA damage
and genomic design in mammalian spermatozoa from eleven
species. Mol Reprod Dev 2011;78:95161.
[69] Flores E, Cifuentes D, Fernndez-Novell JM, Medrano A, Bonet S,
Briz MD, et al. Freeze-thawing induces alterations in the
protamine-1/DNA overall structure in boar sperm. Theriogenology
2008;69:108394.
[70] Flores E, Rami-Lluch L, Bucci D, Fernndez-Novell JM, Pea A,
Rodrguez-Gil JE. Freezing-thawing induces alterations in histone
[71]
[72]
[73]
[74]
[75]
[76]
[77]
[78]
[79]
[80]
[81]
[82]
[83]
[84]
[85]
[86]
[87]
[88]
[89]
H1-DNA binding and the breaking of protein-DNA disulde bonds

in boar sperm. Theriogenology 2011;76:145064.
Yeste M, Estrada E, Casas I, Bonet S, Rodrguez-Gil JE. Good and bad
freezability boar ejaculates differ in the integrity of nucleoprotein
structure after freeze-thawing but not in ROS levels. Theriogenology 2013;79:92939.
Estrada E, Rodrguez-Gil JE, Rocha LG, Balasch S, Bonet S, Yeste M.
Supplementing cryopreservation media with reduced glutathione
increases fertility and prolicacy of sows inseminated with frozenthawed boar semen. Andrology 2014;2:8899.
Yeste M, Flores E, Estrada E, Bonet S, Rigau T, Rodrguez-Gil JE.
Reduced glutathione and procaine hydrochloride protect the
nucleoprotein structure of boar spermatozoa during freezethawing by stabilising disulde bonds. Reprod Fertil Dev 2013;
25:103650.
_ J. Comparison of post-thaw
Fraser L, Parda A, Filipowicz K, Strzezek
DNA integrity of boar spermatozoa assessed with the neutral
comet assay and Sperm-Sus Halomax test kit. Reprod Domest
Anim 2010;45:15560.
Alkmin DV, Martinez-Alborcia MJ, Parrilla I, Vazquez JM,
Martinez EA, Roca J. The nuclear DNA longevity in cryopreserved
boar spermatozoa assessed using the Sperm-Sus-Halomax. Theriogenology 2013;79:1294300.
Zribi N, Feki Chakroun N, El Euch H, Gargouri J, Bahloul A, Ammar
Keskes L. Effects of cryopreservation on human sperm deoxyribonucleic acid integrity. Fertil Steril 2010;93:15966.
Boitrelle F, Albert M, Theillac C, Ferfouri F, Bergere M, Vialard F,
et al. Cryopreservation of human spermatozoa decreases the
number of motile normal spermatozoa, induces nuclear vacuolization and chromatin decondensation. J Androl 2012;33:13718.
Ribas-Maynou J, Fernndez-Encinas A, Garca-Peir A, Prada E,
Abad C, Amengual MJ, et al. Human semen cryopreservation: a
sperm DNA fragmentation study with alkaline and neutral comet
assay. Andrology 2014;2:837.
Urbano M, Dorado J, Ortiz I, Morrell JM, Demyda-Peyrs S,
Glvez MJ, et al. Effect of cryopreservation and single layer centrifugation on canine sperm DNA fragmentation assessed by the sperm
chromatin dispersion test. Anim Reprod Sci 2013;143:11825.
Paoli D, Lombardo F, Lenzi A, Gandini L. Sperm cryopreservation:
effects on chromatin structure. Adv Exp Med Biol 2014;791:137
50.
Thomson LK, Fleming SD, Aitken RJ, De Iuliis GN, Zieschang JA,
Clark AM. Cryopreservation-induced human sperm DNA damage is
predominantly mediated by oxidative stress rather than apoptosis.
Hum Reprod 2009;24:206170.
Valcarce DG, Cartn-Garca F, Riesco MF, Herrez MP, Robles V.
Analysis of DNA damage after human sperm cryopreservation in
genes crucial for fertilization and early embryo development.
Andrology 2013;1:72330.
Sutovsky P, Manandhar G, Wu A, Oko R. Interactions of sperm
perinuclear theca with the oocyte: implications for oocyte activation, anti-polyspermy defense, and assisted reproduction.
Microsc Res Tech 2003;61:36278.
Escofer J, Yassine S, Lee HC, Martinez G, Delaroche J, Coutton C,
et al. Subcellular localization of phospholipase Cz in human sperm
and its absence in DPY19L2-decient sperm are consistent with its
role in oocyte activation. Mol Hum Reprod 2015;21:15768.
Wu AT, Sutovsky P, Manandhar G, Xu W, Katayama M, Day BN,
et al. PAWP, a sperm-specic WW domain-binding protein, promotes meiotic resumption and pronuclear development during
fertilization. J Biol Chem 2007;282:1216475.
Yeste M, Jones C, Amdani SN, Patel S, Coward K. Oocyte activation
deciency: a role for an oocyte contribution? Hum Reprod Update
2015. http://dx.doi.org/10.1093/humupd/dmv040.
Gutirrez-Prez O, Jurez-Mosqueda ML, Mota D, Trujillo ME. The
disruption in actin-perinuclear theca interactions are related with
changes induced by cryopreservation observed on sperm chromatin nuclear decondensation of boar semen. Cryobiology 2011;
62:329.
Flores E, Fernndez-Novell JM, Pea A, Rigau T, Rodrguez-Gil JE.
Cryopreservation-induced alterations in boar spermatozoa mitochondrial function are related to changes in the expression and
location of midpiece mitofusin-2 and actin network. Theriogenology 2010;74:35463.
Ortega Ferrusola C, Gonzlez Fernndez L, Morrell JM, Salazar
Sandoval C, Macas Garca B, Rodrguez-Martinez H, et al. Lipid
peroxidation, assessed with BODIPY-C11, increases after cryopreservation of stallion spermatozoa, is stallion-dependent and is
related to apoptotic-like changes. Reproduction 2009;138:5563.
61
[90] Pea FJ, Plaza Davila M, Ball BA, Squires EL, Martin Muoz P,
Ortega Ferrusola C, et al. The impact of reproductive technologies
on stallion mitochondrial function. Reprod Domest Anim 2015;50:
52937.
[91] Guthrie HD, Welch GR. Determination of intracellular reactive
oxygen species and high mitochondrial membrane potential in
Percoll-treated viable boar sperm using uorescence-activated
ow cytometry. J Anim Sci 2006;84:2089100.
[92] Guthrie HD, Welch GR. Effects of reactive oxygen species on sperm
function. Theriogenology 2012;78:17008.
[93] Gmez-Fernndez J, Gmez-Izquierdo E, Toms C, Moc E, de
Mercado E. Is sperm freezability related to the post-thaw lipid
peroxidation and the formation of reactive oxygen species in
boars? Reprod Domest Anim 2013;48:17782.
[94] Awda BJ, Mackenzie-Bell M, Buhr MM. Reactive oxygen species
and boar sperm function. Biol Reprod 2009;81:55361.
[95] Kim S, Lee YJ, Kim YJ. Changes in sperm membrane and ROS
following cryopreservation of liquid boar semen stored at 15 C.
Anim Reprod Sci 2011;124:11824.
[96] Hu J, Geng G, Li Q, Sun X, Cao H, Liu Y. Effects of alginate on frozenthawed boar spermatozoa quality, lipid peroxidation and antioxidant enzymes activities. Anim Reprod Sci 2014;147:1128.
[97] Kadirvel G, Kumar S, Kumaresan A. Lipid peroxidation, mitochondrial membrane potential and DNA integrity of spermatozoa
in relation to intracellular reactive oxygen species in liquid and
frozen-thawed buffalo semen. Anim Reprod Sci 2009;114:12534.
[98] Briz MD, Fbrega A. The boar spermatozoon. In: Bonet S, Casas I,
Holt WV, Yeste M, editors. Boar reproduction. Berlin: Springer;
2013. p. 347.
[99] Malmgren L, Larsson K. Semen quality and fertility after heat stress
in boars. Acta Vet Scand 1984;25:42535.
[100] Gatimel N, Leandri R, Parinaud J. Sperm vacuoles are not modied
by freezingthawing procedures. Reprod Biomed Online 2013;26:
2406.
[101] Chen X, Zhu H, Hu C, Hao H, Zhang J, Li K, et al. Identication of
differentially expressed proteins in fresh and frozen-thawed boar
spermatozoa by iTRAQ-coupled 2D LC-MS/MS. Reproduction 2014;
147:32130.
[102] Casas I, Sancho S, Ballester J, Briz M, Pinart E, Bussalleu E, et al. The
HSP90AA1 sperm content and the prediction of the boar ejaculate
freezability. Theriogenology 2010;74:94050.
[103] Vilagran I, Castillo J, Bonet S, Sancho S, Yeste M, Estanyol JM, et al.
Acrosin-binding protein (ACRBP) and triosephosphate isomerase
(TPI) are good markers to predict boar sperm freezing capacity.
Theriogenology 2013;80:44350.
[104] Wang S, Wang W, Xu Y, Tang M, Fang J, Sun H, et al. Proteomic
characteristics of human sperm cryopreservation. Proteomics
2014;14:298310.
[105] Kashir J, Heynen A, Jones C, Durrans C, Craig J, Gadea J, et al. Effects
of cryopreservation and density-gradient washing on phospholipase C zeta concentrations in human spermatozoa. Reprod Biomed
Online 2011;23:2637.
[106] Zhang XG, Hu S, Han C, Zhu QC, Yan GJ, Hu JH. Association of heat
shock protein 90 with motility of post-thawed sperm in bulls.
[107] Sancho S, Casas I, Ekwall H, Saravia F, Rodriguez-Martinez H,
Rodriguez-Gil JE, et al. Effects of cryopreservation on semen
quality and the expression of sperm membrane hexose transporters in the spermatozoa of Iberian pigs. Reproduction 2007;
134:11121.
[108] Kim JC, Li Y, Lee S, Yi YJ, Park CS, Woo SH. Effects of cryopreservation on Ca2 signals induced by membrane depolarization,
caffeine, thapsigargin and progesterone in boar spermatozoa. Mol
Cells 2008;26:55865.
[109] Albrizio M, Moramarco AM, Nicassio M, Micera E, Zarrilli A,
Lacalandra GM. Localization and functional modication of L-type
voltage-gated calcium channels in equine spermatozoa from fresh
and frozen semen. Theriogenology 2015;83:4219.
[110] Seshagiri PB, Mariappa D, Aladakatti RH. Tyrosine phosphorylated
proteins in mammalian spermatozoa: molecular and functional
aspects. Soc Reprod Fertil Suppl 2007;63:31325.
[111] Yeste M. Boar spermatozoa within the oviductal environment (II):
sperm capacitation. In: Bonet S, Casas I, Holt WV, Yeste M, editors.
Boar reproduction. Berlin: Springer; 2013. p. 281342.
[112] Yeste M, Fernndez-Novell JM, Rami-Lluch L, Estrada E, Rocha LG,
Cebrin-Prez JA, et al. Intracellular calcium movements of boar
spermatozoa during in vitro capacitation and subsequent acrosome exocytosis follow a multiple-storage place, extracellular
calcium-dependent model. Andrology 2015;3:72947.
62
[113] Morgan DJ, Weisenhaus M, Shum S, Su T, Zheng R, Zhang C, et al.

Tissue-specic PKA inhibition using a chemical genetic approach
and its application to studies on sperm capacitation. Proc Natl Acad
Sci U S A 2008;105:207405.
[114] Kumaresan A, Siqueira AP, Hossain MS, Bergqvist AS. Cryopreservation-induced alterations in protein tyrosine phosphorylation of
spermatozoa from different portions of the boar ejaculate. Cryobiology 2011;63:13744.
[115] Naresh S, Atreja SK. The protein tyrosine phosphorylation during
in vitro capacitation and cryopreservation of mammalian spermatozoa. Cryobiology 2015;70:2116.
[116] Jodar M, Selvaraju S, Sendler E, Diamond MP, Krawetz SA. Reproductive medicine network. The presence, role and clinical use of
spermatozoal RNAs. Hum Reprod Update 2013;19:60424.
[117] Zeng C, He L, Peng W, Ding L, Tang K, Fang D, et al. Selection of
optimal reference genes for quantitative RT-PCR studies of boar
spermatozoa cryopreservation. Cryobiology 2014;68:11321.
[118] Zeng C, Peng W, Ding L, He L, Zhang Y, Fang D, et al. A preliminary
study on epigenetic changes during boar spermatozoa cryopreservation. Cryobiology 2014;69:11927.
[119] Valcarce DG, Cartn-Garca F, Herrez MP, Robles V. Effect of
cryopreservation on human sperm messenger RNAs crucial for
fertilization and early embryo development. Cryobiology 2013;67:
8490.
[120] Card CJ, Anderson EJ, Zamberlan S, Krieger KE, Kaproth M,
Sartini BL. Cryopreserved bovine spermatozoal transcript prole as
revealed by high-throughput ribonucleic acid sequencing. Biol
Reprod 2013;88:49.
[121] Hwang JY, Mulligan BP, Kim HM, Yang BC, Lee CK. Quantitative
analysis of sperm mRNA in the pig: relationship with early embryo
development and capacitation. Reprod Fertil Dev 2013;25:80717.
[122] Yao C, Liu Y, Sun M, Niu M, Yuan Q, Hai Y, et al. MicroRNAs and
DNA methylation as epigenetic regulators of mitosis, meiosis and
spermiogenesis. Reproduction 2015;150:R2534.
[123] Zhang Y, Zeng CJ, He L, Ding L, Tang KY, Peng WP. Selection of
endogenous reference microRNA genes for quantitative reverse
transcription polymerase chain reaction studies of boar spermatozoa cryopreservation. Theriogenology 2015;83:63441.
[124] Curry E, Safranski TJ, Pratt SL. Differential expression of porcine
sperm microRNAs and their association with sperm morphology
and motility. Theriogenology 2011;76:15329.
[125] Abu-Halima M, Hammadeh M, Schmitt J, Leidinger P, Keller A,
Meese E, et al. Altered microRNA expression proles of human
spermatozoa in patients with different spermatogenic impairments. Fertil Steril 2013;99:124955.
[126] Kotaja N. MicroRNAs and spermatogenesis. Fertil Steril 2014;101:
155262.
[127] Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature 2004;429:457
63.
[128] Yamauchi Y, Shaman JA, Ward WS. Non-genetic contributions of
the sperm nucleus to embryonic development. Asian J Androl
2011;13:315.
[129] van der Heijden GW, Ramos L, Baart EB, van den Berg IM,
Derijck AA, van der Vlag J, et al. Sperm-derived histones contribute
to zygotic chromatin in humans. BMC Dev Biol 2008;8:34.
[130] Klver R, Bleiziffer A, Redmann K, Mallidis C, Kliesch S, Gromoll J.
Routine cryopreservation of spermatozoa is safedevidence from
the DNA methylation pattern of nine spermatozoa genes. J Assist
Reprod Genet 2012;29:94350.
[131] Yeste M, Estrada E, Pinart E, Bonet S, Mir J, Rodrguez-Gil JE. The
improving effect of reduced glutathione on boar sperm cryotolerance is related with the intrinsic ejaculate freezability. Cryobiology 2014;68:25161.
[132] Flores E, Taberner E, Rivera MM, Pea A, Rigau T, Mir J, et al.
Effects of freezing/thawing on motile sperm subpopulations of
boar and donkey ejaculates. Theriogenology 2008;70:93645.
[133] Flores E, Fernndez-Novell JM, Pea A, Rodrguez-Gil JE. The degree of resistance to freezing-thawing is related to specic changes
in the structures of motile sperm subpopulations and mitochondrial activity in boar spermatozoa. Theriogenology 2009;72:784
97.
[134] Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 2000;
53:4758.
[135] Pea FJ, Saravia F, Nez-Martnez I, Johannisson A, Wallgren M,
Rodrguez Martnez H. Do different portions of the boar ejaculate
vary in their ability to sustain cryopreservation? Anim Reprod Sci
2006;93:10113.
[136] Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing
function. Reprod Fertil Dev 1995;7:87191.
[137] Casas I, Sancho S, Briz M, Pinart E, Bussalleu E, Yeste M, et al.
Freezability prediction of boar ejaculates assessed by functional
sperm parameters and sperm proteins. Theriogenology 2009;72:
93048.
[138] Dorado J, Muoz-Serrano A, Hidalgo M. The effect of cryopreservation on goat semen characteristics related to sperm freezability.
[139] Parrilla I, del Olmo D, Sijses L, Martinez-Alborcia MJ, Cuello C,
Vazquez JM, et al. Differences in the ability of spermatozoa from
individual boar ejaculates to withstand different semenprocessing techniques. Anim Reprod Sci 2012;132:6673.
[140] Hernndez M, Ekwall H, Roca J, Vazquez JM, Martinez E,
Rodrguez-Martnez H. Cryo-scanning electron microscopy
(Cryo-SEM) of semen frozen in medium-straws from good and
sub-standard freezer AI-boars. Cryobiology 2007;54:6370.
[141] Thurston LM, Siggins K, Mileham AJ, Watson PF, Holt WV. Identication of amplied restriction fragment length polymorphism
markers linked to genes controlling boar sperm viability following
cryopreservation. Biol Reprod 2002;66:54554.
[142] Nikbin S, Panandam JM, Yaakub H, Murugaiyah M, Sazili AQ. Novel
SNPs in heat shock protein 70 gene and their association with
sperm quality traits of Boer goats and Boer crosses. Anim Reprod
Sci 2014;146:17681.
[143] Medrano A, Holt WV, Watson PF. Controlled freezing studies on
boar sperm cryopreservation. Andrologia 2009;41:24650.
[144] Hernndez M, Roca J, Gil MA, Vzquez JM, Martnez EA. Adjustments on the cryopreservation conditions reduce the incidence of
boar ejaculates with poor sperm freezability. Theriogenology
2007;67:143645.
[145] Estrada E, Rodrguez-Gil JE, Rivera Del lamo MM, Pea A, Yeste M.
Effects of reduced glutathione on acrosin activity in frozen-thawed
boar spermatozoa. Reprod Fertil Dev 2015. http://dx.doi.org/10.
1071/RD15118.
[146] Hernndez M, Roca J, Calvete JJ, Sanz L, Muio-Blanco T, CebrinPrez JA, et al. Cryosurvival and in vitro fertilizing capacity postthaw is improved when boar spermatozoa are frozen in the
presence of seminal plasma from good freezer boars. J Androl
2007;28:68997.
[147] Vilagran I, Yeste M, Sancho S, Castillo J, Oliva R, Bonet S.
Comparative analysis of boar seminal plasma proteome from
different freezability ejaculates and identication of Fibronectin 1
as sperm freezability marker. Andrology 2015;3:34556.
[148] Pinart E, Yeste M, Bonet S. Acrosin is a good predictor of boar
sperm freezability. Theriogenology 2015;83:152533.
_
[149] Wysocki P, Orzo1ek A, Strzezek
J, Koziorowska-Gilun M,
Zasiadczyk q, Kordan W. The activity of N-acetyl-b-hexosaminidase in boar seminal plasma is linked with semen quality and its
suitability for cryopreservation. Theriogenology 2015;83:1194
202.
[150] Vilagran I, Yeste M, Sancho S, Casas I, Rivera del lamo MM,
Bonet S. Relationship of sperm small heat-shock protein 10 and
voltage-dependent anion channel 2 with semen freezability in
boars. Theriogenology 2014;82:41826.
[151] Buffone MG, Calamera JC, Brugo-Olmedo S, De Vincentiis S,
Calamera MM, Storey BT, et al. Superoxide dismutase content in
sperm correlates with motility recovery after thawing of cryopreserved human spermatozoa. Fertil Steril 2012;97:2938.
[152] Jiang XP, Wang SQ, Wang W, Xu Y, Xu Z, Tang JY, et al. Enolase1
(ENO1) and glucose-6-phosphate isomerase (GPI) are good
markers to predict human sperm freezability. Cryobiology 2015;
71:1415.
[153] Rickard JP, Leahy T, Soleilhavoup C, Tsikis G, Labas V, Harichaux G,
et al. The identication of proteomic markers of sperm freezing
resilience in ram seminal plasma. J Proteomics 2015;126:30311.
[154] Zimmerman SW, Manandhar G, Yi YJ, Gupta SK, Sutovsky M,
Odhiambo JF, et al. Sperm proteasomes degrade sperm receptor on
the egg zona pellucida during mammalian fertilization. PLoS One
2011;6:e17256.
[155] Sancho S, Pinart E, Briz M, Garcia-Gil N, Badia E, Bassols J, et al.
Semen quality of postpubertal boars during increasing and
decreasing natural photoperiods. Theriogenology 2004;62:1271
82.
[156] Yeste M, Sancho S, Briz M, Pinart E, Bussalleu E, Bonet S. A diet
supplemented with L-carnitine improves the sperm quality of
Pitrain but not of Duroc and Large White boars when photoperiod
and temperature increase. Theriogenology 2010;73:57786.

[157] Barranco I, Ortega MD, Martinez-Alborcia MJ, Vazquez JM,
Martinez EA, Roca J. Season of ejaculate collection inuences the
freezability of boar spermatozoa. Cryobiology 2013;67:299304.
[158] Yogev L, Kleiman S, Shabtai E, Botchan A, Gamzu R, Paz G, et al.
Seasonal variations in pre- and post-thaw donor sperm quality.
Hum Reprod 2004;19:8805.
[159] Wrench N, Pinto CR, Klinefelter GR, Dix DJ, Flowers WL, Farin CE.
Effect of season on fresh and cryopreserved stallion semen. Anim
Reprod Sci 2010;119:21927.
[160] Janett F, Thun R, Bettschen S, Burger D, Hassig M. Seasonal changes
of semen quality and freezability in Franches-Montagnes stallions.
[161] Castellano CA, Audet I, Bailey JL, Laforest JP, Matte JJ. Dietary
omega-3 fatty acids (sh oils) have limited effects on boar semen
stored at 17 C or cryopreserved. Theriogenology 2010;74:1482
90.
[162] Yeste M, Barrera X, Coll D, Bonet S. The effects on boar sperm
quality of dietary supplementation with omega-3 polyunsaturated
fatty acids differ among porcine breeds. Theriogenology 2011;76:
18496.
[163] Casas I, Miller-Lux Y, Osborne B, Bonet S, Althouse GC. Testing an
egg yolk supplemented diet on boars to aid in sperm adaptation at
5 C. Syst Biol Reprod Med 2015;61:25362.
[164] Brinsko SP, Varner DD, Love CC, Blanchard TL, Day BC, Wilson ME.
Effect of feeding a DHA-enriched nutriceutical on the quality of
fresh, cooled and frozen stallion semen. Theriogenology 2005;63:
151927.
[165] Schmid-Lausigk Y, Aurich C. Inuences of a diet supplemented
with linseed oil and antioxidants on quality of equine semen after
cooling and cryopreservation during winter. Theriogenology 2014;
81:96673.
[166] Kommisrud E, Paulenz H, Sehested E, Grevle IS. Inuence of boar
and semen parameters on motility and acrosome integrity in
liquid boar semen stored for ve days. Acta Vet Scand 2002;43:49
55.
[167] Chanapiwat P, Kaeoket K. Breed of boar inuences the optimal
concentration of gamma-oryzanol needed for semen cryopreservation. Reprod Domest Anim 2015;50:2216.
_
[168] Fraser L, Strzezek
J, Kordan W. Post-thaw sperm characteristics
following long-term storage of boar semen in liquid nitrogen.
[169] Alkmin DV, Perez-Patio C, Barranco I, Parrilla I, Vazquez JM,
Martinez EA, et al. Boar sperm cryosurvival is better after exposure
to seminal plasma from selected fractions than to those from
entire ejaculate. Cryobiology 2014;69:20310.
[170] Saravia F, Wallgren M, Rodrguez-Martnez H. Freezing of boar
semen can be simplied by handling a specic portion of the
ejaculate with a shorter procedure and MiniFlatPack packaging.
[171] Hossain MS, Johannisson A, Siqueira AP, Wallgren M, RodriguezMartinez H. Spermatozoa in the sperm-peak-fraction of the boar
ejaculate show a lower ow of Ca(2) under capacitation conditions post-thaw which might account for their higher membrane stability after cryopreservation. Anim Reprod Sci 2011;128:
3744.
[172] Macas Garca B, Morrell JM, Ortega-Ferrusola C, GonzlezFernndez L, Tapia JA, Rodriguez-Martnez H, et al. Centrifugation
on a single layer of colloid selects improved quality spermatozoa
from frozen-thawed stallion semen. Anim Reprod Sci 2009;114:
193202.
[173] Brugnon F, Ouchchane L, Pons-Rejraji H, Artonne C, Farigoule M,
Janny L. Density gradient centrifugation prior to cryopreservation
and hypotaurine supplementation improve post-thaw quality of
sperm from infertile men with oligoasthenoteratozoospermia.
Hum Reprod 2013;28:204557.
[174] Roca J, Martinez-Alborcia MJ, Gil MA, Parrilla I, Martinez EA. Dead
spermatozoa in raw semen samples impair in vitro fertilization
outcomes of frozen-thawed spermatozoa. Fertil Steril 2013;100:
87581.
[175] Kruse R, Dutta PC, Morrell JM. Colloid centrifugation removes
seminal plasma and cholesterol from boar spermatozoa. Reprod
Fertil Dev 2011;23:85865.
[176] Martnez-Alborcia MJ, Morrell JM, Parrilla I, Barranco I,
Vzquez JM, Martinez EA, et al. Improvement of boar sperm cryosurvival by using single-layer colloid centrifugation prior
freezing. Theriogenology 2012;78:111725.
[177] Gale I, Gil L, Malo C, Gonzlez N, Martnez F. Effect of Camellia
sinensis supplementation and increasing holding time on quality
of cryopreserved boar semen. Andrologia 2014;47:50512.
63
[178] Casas I, Althouse GC. The protective effect of a 17 C holding time
on boar sperm plasma membrane uidity after exposure to 5 C.
[179] Yeste M, Estrada E, Rivera Del lamo MM, Bonet S, Rigau T,
Rodrguez-Gil JE. The increase in phosphorylation levels of serine
residues of protein HSP70 during holding time at 17 C is
concomitant with a higher cryotolerance of boar spermatozoa.
PLoS One 2014;9:e90887. http://dx.doi.org/10.1371/journal.pone.
0090887.
ska A, Strzezek R, Strzezek J. Dialysis of boar
[180] Fraser L, Dziekon
semen prior to freezing-thawing: its effects on post-thaw sperm
characteristics. Theriogenology 2007;67:9941003.
[181] Okazaki T, Shimada M. New strategies of boar sperm cryopreservation: development of novel freezing and thawing methods
with a focus on the roles of seminal plasma. Anim Sci J 2012;83:
6239.
[182] Vadnais ML, Roberts KP. Seminal plasma proteins inhibit in vitroand cooling-induced capacitation in boar spermatozoa. Reprod
Fertil Dev 2010;22:893900.
[183] Rickard JP, Schmidt RE, Maddison JW, Bathgate R, Lynch GW,
Druart X, et al. Variation in seminal plasma alters the ability of ram
spermatozoa to survive cryopreservation. Reprod Fertil Dev 2014.
http://dx.doi.org/10.1071/RD14123.
[184] Gmez-Fernndez J, Gmez-Izquierdo E, Toms C, GonzlezBulnes A, Snchez-Snchez R, de Mercado E. Inclusion of seminal
plasma in sperm cryopreservation of Iberian pig. Anim Reprod Sci
2012;130:8290.
[185] Fernndez-Gago R, Domnguez JC, Martnez-Pastor F. Seminal
plasma applied post-thawing affects boar sperm physiology: a
ow cytometry study. Theriogenology 2013;80:40010.
[186] Vadnais ML, Kirkwood RN, Specher DJ, Chou K. Effects of extender,
incubation temperature, and added seminal plasma on capacitation of cryopreserved, thawed boar sperm as determined by
chlortetracycline staining. Anim Reprod Sci 2005;90:34754.
[187] Taylor U, Zerbe H, Seyfert HM, Rath D, Baulain U, Langner KF, et al.
Porcine spermatozoa inhibit post-breeding cytokine induction in
uterine epithelial cells in vivo. Anim Reprod Sci 2009;115:27989.
[188] Heise A, Khn W, Volkmann DH, Thompson PN, Gerber D. Inuence of seminal plasma on fertility of fresh and frozen-thawed
stallion epididymal spermatozoa. Anim Reprod Sci 2010;118:48
53.
[189] Mir J, Vils K, Garca W, Jordana J, Yeste M. Effect of donkey seminal
plasma on sperm movement and sperm-polymorphonuclear neutrophils attachment in vitro. Anim Reprod Sci 2013;140:16472.
[190] Garca JC, Domnguez JC, Pea FJ, Alegre B, Gonzalez R, Castro MJ,
et al. Thawing boar semen in the presence of seminal plasma:
effects on sperm quality and fertility. Anim Reprod Sci 2010;119:
1605.
[191] Okazaki T, Abe S, Yoshida S, Shimada M. Seminal plasma damages
sperm during cryopreservation, but its presence during thawing
improves semen quality and conception rates in boars with poor
post-thaw semen quality. Theriogenology 2009;71:4918.
[192] Abad M, Garcia JC, Sprecher DJ, Cassar G, Friendship RM, Buhr MM,
et al. Effect of insemination-ovulation interval and addition of
seminal plasma on sow fertility to insemination of cryopreserved
sperm. Reprod Domest Anim 2007;42:41822.
[193] Purdy PH, Graham JK. Effect of cholesterol-loaded cyclodextrin on
the cryosurvival of bull sperm. Cryobiology 2004;48:3645.
[194] Giaretta E, Estrada E, Bucci D, Spinaci M, Rodrguez-Gil JE, Yeste M.
Combining reduced glutathione and ascorbic acid has supplementary benecial effects on boar sperm cryotolerance. Theriogenology 2015;83:399407.
[195] Varo-Ghiuru F, Miclea I, Hettig A, Ladosi I, Miclea V, Egerszegi I,
et al. Lutein, Trolox, ascorbic acid and combination of Trolox with
ascorbic acid can improve boar semen quality during cryopreservation. Cryo Letters 2015;36:17.
[196] Jeong YJ, Kim MK, Song HJ, Kang EJ, Ock SA, Kumar BM, et al. Effect
of alpha-tocopherol supplementation during boar semen cryopreservation on sperm characteristics and expression of apoptosis
related genes. Cryobiology 2009;58:1819.
[197] Satorre MM, Breininger E, Beconi MT. Cryopreservation with atocopherol and Sephadex ltration improved the quality of boar
sperm. Theriogenology 2012;78:154856.
[198] Roca J, Gil MA, Hernandez M, Parrilla I, Vazquez JM, Martinez EA.
Survival and fertility of boar spermatozoa after freeze-thawing in
extender supplemented with butylated hydroxytoluene. J Androl
2004;25:397405.
[199] Toms C, Blanch E, Hernndez M, Gil MA, Roca J, Vzquez JM, et al.
Treating boar sperm with cholesterol-loaded cyclodextrins widens
64
[200]
[201]
[202]
[203]
[204]
[205]
[206]
[207]
[208]
[209]
[210]
[211]
[212]
[213]
[214]
[215]
[216]
[217]
[218]
[219]

the sperm osmotic tolerance limits and enhances the in vitro
sperm fertilising ability. Anim Reprod Sci 2011;129:20920.
Toms C, Blanch E, Fazeli A, Moc E. Effect of a pre-freezing
treatment with cholesterol-loaded cyclodextrins on boar sperm
longevity, capacitation dynamics, ability to adhere to porcine
oviductal epithelial cells in vitro and DNA fragmentation dynamics. Reprod Fertil Dev 2013;25:93546.
Blanch E, Toms C, Graham JK, Moc E. Response of boar sperm to
the treatment with cholesterol-loaded cyclodextrins added prior
to cryopreservation. Reprod Domest Anim 2012;47:95964.
Toms C, Gmez-Fernndez J, Gmez-Izquierdo E, Moc E, de
Mercado E. Addition of cholesterol-loaded cyclodextrins to the
thawing extender: effects on boar sperm quality. Reprod Domest
Anim 2014;49:42732.
Chanapiwat P, Kaeoket K, Tummaruk P. Effects of DHA-enriched
hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen. Asian J Androl 2009;11:6008.
Kaeoket K, Chanapiwat P, Tummaruk P, Techakumphu M. Supplemental effect of varying L-cysteine concentrations on the
quality of cryopreserved boar semen. Asian J Androl 2010;12:760
5.
Gadea J, Garca-Vzquez FA, Mats C, Gardn JC, Cnovas S,
Gumbao D. Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function. J Androl 2005;26:396404.
Blanch E, Toms C, Hernndez M, Roca J, Martnez EA, Vzquez JM,
et al. Egg yolk and glycerol requirements for freezing boar spermatozoa treated with methyl b-cyclodextrin or cholesterol-loaded
cyclodextrin. J Reprod Dev 2014;60:1439.
Taylor K, Roberts P, Sanders K, Burton P. Effect of antioxidant
supplementation of cryopreservation medium on post-thaw
integrity of human spermatozoa. Reprod Biomed Online 2009;
18:1849.
Meamar M, Zribi N, Cambi M, Tamburrino L, Marchiani S,
Filimberti E, et al. Sperm DNA fragmentation induced by cryopreservation: new insights and effect of a natural extract from
Opuntia cus-indica. Fertil Steril 2012;98:32633.
Hu JH, Tian WQ, Zhao XL, Zan LS, Wang H, Li QW, et al. The
cryoprotective effects of ascorbic acid supplementation on bovine
semen quality. Anim Reprod Sci 2010;121:727.
Gadea J, Molla M, Selles E, Marco MA, Garcia-Vazquez FA,
Gardon JC. Reduced glutathione content in human sperm is
decreased after cryopreservation: effect of the addition of reduced
glutathione to the freezing and thawing extenders. Cryobiology
2011;62:406.
Ashra I, Kohram H, Ardabili FF. Antioxidative effects of melatonin on kinetics, microscopic and oxidative parameters of
cryopreserved bull spermatozoa. Anim Reprod Sci 2013;139:25
30.
da Silva CM, Macas-Garca B, Mir-Morn A, GonzlezFernndez L, Morillo-Rodriguez A, Ortega-Ferrusola C, et al.
Melatonin reduces lipid peroxidation and apoptotic-like changes
in stallion spermatozoa. J Pineal Res 2011;51:1729.
Succu S, Berlinguer F, Pasciu V, Satta V, Leoni GG, Naitana S.
Melatonin protects ram spermatozoa from cryopreservation injuries in a dose-dependent manner. J Pineal Res 2011;50:3108.
Martn-Hidalgo D, Barn FJ, Bragado MJ, Carmona P, Robina A,
Garca-Marn LJ, et al. The effect of melatonin on the quality of
extended boar semen after long-term storage at 17 C. Theriogenology 2011;75:155060.
Mendez MF, Zangeronimo MG, Rocha LG, Faria BG, Pereira BA,
Fernandes CD, et al. Effect of the addition of IGF-I and vitamin E to
stored boar semen. Animal 2013;7:7938.
Padilha RT, Magalhes-Padilha DM, Cavalcante MM, Almeida AP,
Haag KT, Gastal MO, et al. Effect of insulin-like growth factor-I on
some quality traits and fertility of cryopreserved ovine semen.
Theriogenology 2012;78:90713.
Elliott RM, Lloyd RE, Fazeli A, Sostaric E, Georgiou AS, Satake N,
et al. Effects of HSPA8, an evolutionarily conserved oviductal
protein, on boar and bull spermatozoa. Reproduction 2009;137:
191203.
Yeste M, Holt WV, Bonet S, Rodrguez-Gil JE, Lloyd RE. Viable and
morphologically normal boar spermatozoa alter the expression of
heat-shock protein genes in oviductal epithelial cells during coculture in vitro. Mol Reprod Dev 2014;81:80519.
ana C, Maside C, Gil MA,
Moein-Vaziri N, Phillips I, Smith S, Almin
et al. Heat-shock protein A8 restores sperm membrane integrity by
increasing plasma membrane uidity. Reproduction 2014;147:
71932.
[220] Holt WV, Del Valle I, Fazeli A. Heat shock protein A8 stabilizes the
bull sperm plasma membrane during cryopreservation: effects of
breed, protein concentration, and mode of use. Theriogenology
2015;84:693701.
[221] Yogev L, Kleiman SE, Shabtai E, Botchan A, Paz G, Hauser R, et al.
Long-term cryostorage of sperm in a human sperm bank does not
damage progressive motility concentration. Hum Reprod 2010;25:
1097103.
[222] Knox RV. The fertility of frozen boar sperm when used for articial
insemination. Reprod Domest Anim 2015;50(Suppl 2):907.
[223] Didion BA, Braun GD, Duggan MV. Field fertility of frozen boar
semen: a retrospective report comprising over 2600 AI services
spanning a four year period. Anim Reprod Sci 2013;137:18996.
[224] Almiana C, Gil MA, Cuello C, Parrilla I, Caballero I, SanchezOsorio J, et al. Capability of frozen-thawed boar spermatozoa to
sustain pre-implantational embryo development. Anim Reprod Sci
2010;121:14551.
[225] Martinez EA, Vazquez JM, Roca J, Lucas X, Gil MA, Vazquez JL. Deep
intrauterine insemination and embryo transfer in pigs. Reprod
Suppl 2001;58:30111.
[226] Casas I, Sancho S, Briz M, Pinart E, Bussalleu E, Yeste M, et al.
Fertility after post-cervical articial insemination with cryopreserved sperm from boar ejaculates of good and poor freezability. Anim Reprod Sci 2010;118:6976.
[227] Spencer KW, Purdy PH, Blackburn HD, Spiller SF, Stewart TS,
Knox RV. Effect of number of motile, frozen-thawed boar sperm
and number of xed-time inseminations on fertility in estroussynchronized gilts. Anim Reprod Sci 2010;121:25966.
[228] Isachenko E, Isachenko V, Weiss JM, Kreienberg R, Katkov II,
Schulz M, et al. Acrosomal status and mitochondrial activity of
human spermatozoa vitried with sucrose. Reproduction 2008;
136:16773.
[229] Jimnez-Rabadn P, Garca-lvarez O, Vidal A, Maroto-Morales A,
Iniesta-Cuerda M, Ramn M, et al. Effects of vitrication on ram
spermatozoa using free-egg yolk extenders. Cryobiology 2015;71:
8590.
[230] Agha-Rahimi A, Khalili MA, Nabi A, Ashourzadeh S. Vitrication is
not superior to rapid freezing of normozoospermic spermatozoa:
effects on sperm parameters, DNA fragmentation and hyaluronan
binding. Reprod Biomed Online 2014;28:3528.
[231] Kaneko T. Simple sperm preservation by freeze-drying for
conserving animal strains. Methods Mol Biol 2015;1239:31729.
[232] Gil L, Olaciregui M, Luo V, Malo C, Gonzlez N, Martnez F. Current
status of freeze-drying technology to preserve domestic animals
sperm. Reprod Domest Anim 2014;49(Suppl 4):7281.
[233] Keskintepe L, Eroglu A. Freeze-drying of mammalian sperm.
Methods Mol Biol 2015;1257:48997.
[234] Watanabe H, Asano T, Abe Y, Fukui Y, Suzuki H. Pronuclear formation of freeze-dried canine spermatozoa microinjected into
mouse oocytes. J Assist Reprod Genet 2009;26:5316.
[235] Kaneko T, Ito H, Sakamoto H, Onuma M, Inoue-Murayama M.
Sperm preservation by freeze-drying for the conservation of wild
animals. PLoS One 2014;9:e113381.
[236] Kusakabe H, Yanagimachi R, Kamiguchi Y. Mouse and human
spermatozoa can be freeze-dried without damaging their chromosomes. Hum Reprod 2008;23:2339.
[237] Gianaroli L, Magli MC, Stanghellini I, Crippa A, Crivello AM,
Pescatori ES, et al. DNA integrity is maintained after freeze-drying
of human spermatozoa. Fertil Steril 2012;97:106773.
[238] Choi YH, Varner DD, Love CC, Hartman DL, Hinrichs K. Production
of live foals via intracytoplasmic injection of lyophilized sperm and
sperm extract in the horse. Reproduction 2011;142:52938.
[239] Hara H, Tagiri M, Hwang IS, Takahashi M, Hirabayashi M, Hochi S.
Adverse effect of cake collapse on the functional integrity of
freeze-dried bull spermatozoa. Cryobiology 2014;68:35460.
[240] Abdalla H, Hirabayashi M, Hochi S. The ability of freeze-dried bull
spermatozoa to induce calcium oscillations and resumption of
meiosis. Theriogenology 2009;71:54352.
[241] Kwon IK, Park KE, Niwa K. Activation, pronuclear formation, and
development in vitro of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa. Biol Reprod 2004;71:14306.
[242] Men NT, Kikuchi K, Nakai M, Fukuda A, Tanihara F, Noguchi J, et al.
Effect of trehalose on DNA integrity of freeze-dried boar sperm,
fertilization, and embryo development after intracytoplasmic
sperm injection. Theriogenology 2013;80:103344.
[243] Garcia A, Gil L, Malo C, Martinez F, Kershaw-Young C, de Blas I.
Effect of different disaccharides on the integrity and fertilising
ability of freeze-dried boar spermatozoa: a preliminary study. Cryo
Lett 2014;35:27785.

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Theriogenology 85 (2016) 4764

Contents lists available at ScienceDirect

Sperm cryopreservation update: Cryodamage, markers,

Cryopreservation is the most efcient method for long-term preservation of mammalian

made by Spallanzani in 1776 and Mantegazza in 1866 with

M. Yeste / Theriogenology 85 (2016) 4764

method (i.e., with nitrogen vapors) may be used in other

extracellular water [17]. However, rather than storage at

M. Yeste / Theriogenology 85 (2016) 4764

Therefore, albeit due to separate mechanisms, both high

3. The relevance of cryoprotectants

Permeating CPAs are glycerol, DMSO, ethylene glycol,

As previously stated, the composition of freezing and

3.2. Permeating CPAs

M. Yeste / Theriogenology 85 (2016) 4764

sperm motility [48], and substitution with trehalose

to cold shock [12]. Such cold shock includes all dramatic

M. Yeste / Theriogenology 85 (2016) 4764

about cryocapacitation or capacitation-like changes, as the

[7678] and stallions [57], there is no such an impact in

4.2. Cryopreservation effects on sperm nucleus

The perinuclear theca is a region that surrounds sperm

M. Yeste / Theriogenology 85 (2016) 4764

species. Indeed, in buffalos and bulls, ROS species are

proteins in response to freeze-thawing procedures, but

M. Yeste / Theriogenology 85 (2016) 4764

alterations of motility due to cryopreservation regards to

M. Yeste / Theriogenology 85 (2016) 4764

response to additives in the freezing extender also rely on

5.2.2. Seminal plasma proteins as freezability markers

Enzyme activity positively correlated with sperm cryotolerance

Acrosin binding protein (ACRBP)

Higher levels in GFE than in PFE

Abbreviations: GFE, good freezability ejaculates; PFE, poor freezability ejaculates.

M. Yeste / Theriogenology 85 (2016) 4764

have shown that ejaculate freezability relies on the season

M. Yeste / Theriogenology 85 (2016) 4764

(Swedish University of Agricultural Sciences, Uppsala,

for sperm freezability has not only been observed in pigs

7. Increasing the cryopreservation success through

Cyclodextrins are cyclic heptasaccharides that consist of

b (14) glucopyranose units and have a hydrophobic center.

M. Yeste / Theriogenology 85 (2016) 4764

Maintains the acrosome integrity. Increases the activities

Butylated hydroxytoluene (BHT)

Improves sperm survival and embryo development to

Improves progressive motility, plasma membrane, and acrosome integrity.

Improves sperm motility, DNA and acrosome integrity,

Maintains better sperm motility and acrosome integrity.

motility and acrosome and membrane integrity of bull

M. Yeste / Theriogenology 85 (2016) 4764

survival is related to its ability to maintain the uidity and

[12,28]. All this indicates that, even restricted to some

8. Frozen storage for long-term: Any effect on sperm?

One of the issues that should be addressed in the future

Thus far, attempts to vitrify sperm have been mainly

9. Fertility of frozen-thawed boar sperm

10. New research horizons in long-term preservation

M. Yeste / Theriogenology 85 (2016) 4764

M. Yeste / Theriogenology 85 (2016) 4764

M. Yeste / Theriogenology 85 (2016) 4764

H1-DNA binding and the breaking of protein-DNA disulde bonds

M. Yeste / Theriogenology 85 (2016) 4764

[113] Morgan DJ, Weisenhaus M, Shum S, Su T, Zheng R, Zhang C, et al.