2015 yeste sperm cryopreservation update

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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, Women s Centre, John Radcliffe Hospital, Oxford,

UK

a r t i c l e i n f o

Article history:

Received 26 August 2015Received in revised form 20 September 2015Accepted 22 September 2015

Keywords:

SpermPigCryopreservationCryprotectantFreezabilitySeminal plasma

a b s t r a c t

Cryopreservation is the most efcient method for long-term preservation of mammaliansperm. However, freeze-thawing procedures may strongly impair the sperm function andsurvival and thus decrease the reproductive performance. In addition, the sperm resilienceto withstand cryopreservation, also known as freezability, presents a high individualvariability. The present work summarizes the principles of cryoinjury and the relevance ofpermeating and nonpermeating cryoprotective agents. Descriptions about sperm cryo-damage are mainly focused on boar sperm, but reference to other mammalian species isalso made when relevant. Main cryoinjuries not only regard to sperm motility andmembrane integrity, but also to the degradation effect exerted by freeze-thawing on otherimportant components for sperm fertilizing ability, such as mRNAs. After delving into themain differences between good and poor freezability boar ejaculates, those proteinmarkers predicting the sperm ability to sustain cryopreservation are also mentioned.

Moreover, factors that may in

uence sperm freezability, such as season, diet, breed, orejaculate fractions are discussed, together with the effects of different additives, likeseminal plasma and antioxidants. After briey referring to the effects of long-term spermpreservation in frozen state and the reproductive performance of frozen-thawed boarsperm, this work speculates with new research horizons on the preservation of boarsperm, such as vitrication and freeze-drying.

2016 Elsevier Inc. All rights reserved.

1. Introduction

Fertility preservation through cryopreservation ofgametes and reproductive tissues may be advised in severalcases in humans, especially in children and adults suffering

from cancer, and other mammals, including endangeredspecies [1,2]. Although, in general, freeze-thawing ofmammalian sperm harms the cell, the extent of thatdamage varies across species and heavily relies upon thesperm resilience to withstand cryopreservation procedures[3,4].

The rst attempts to cryopreserve mammalian spermdate back to 18th and 19th centuries, with the observations

made by Spallanzani in 1776 and Mantegazza in 1866 withhuman sperm. However, this technology was really devel-oped in the 20th century, when glycerol was used as acryoprotective agent (CPA) for mammalian sperm andother 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 fromfrozen-thawed sperm[9].

The 1970s represented a signicant forward step forboar sperm cryopreservation with the establishment of twomethods: the American or Beltsville method designed byPursel and Johnson[10], and the German or Hlsenbergermethod set by Westendorf et al. [11]. Although these twotechniques initially used carbonic ice and liquid nitrogenvapors, cryopreservation success was further increasedthrough the introduction of controlled-rate freezers [12,13].In contrast to the case of boar sperm, the conventional

* Corresponding author. Tel.: 44 (0)1865 782829; fax: 44 (0)1865769141.

E-mail address:[email protected].

Contents lists available atScienceDirect

Theriogenology

j o u r n a l h o m e p a g e : w w w . t h e r i o j o u r na l . c o m

0093-691X/$ see front matter 2016 Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.theriogenology.2015.09.047

Theriogenology 85 (2016) 4764
mailto:[email protected]://www.sciencedirect.com/science/journal/0093691Xhttp://www.theriojournal.com/http://dx.doi.org/10.1016/j.theriogenology.2015.09.047http://dx.doi.org/10.1016/j.theriogenology.2015.09.047http://dx.doi.org/10.1016/j.theriogenology.2015.09.047http://dx.doi.org/10.1016/j.theriogenology.2015.09.047http://dx.doi.org/10.1016/j.theriogenology.2015.09.047http://dx.doi.org/10.1016/j.theriogenology.2015.09.047http://www.theriojournal.com/http://www.sciencedirect.com/science/journal/0093691Xhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.theriogenology.2015.09.047&domain=pdfmailto:[email protected]


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method (i.e., with nitrogen vapors) may be used in othermammalian species with reasonable yields [14,15]. Afterthe establishment of these two methods, research effortshave been directed toward optimization of cryopreserva-tion protocols and articial insemination (AI), spermpreparation, modication of extenders, and identicationof freezability markers (reviewed in[16]).

The present article seeks to summarize fundamentals of

cryoinjury in boar sperm, with reference being made toother mammalian species when relevant. Apart fromdescribing the main common damages inicted byfreeze-thawing procedures, such as those on plasmamembrane integrity, this work also discusses whether suchprocedures affect other important components for spermfunction during fertilization and beyond, such as mRNA orepigenetic regulation elements. The review also summa-rizes the freezability markers identied thus far, brieydescribes those factors that may affect the sperm resilienceto withstand cryopreservation, and delves into the role ofCPAs and the effects of different additives. The article endswith a discussion about other methods that also allowlong-term preservation of mammalian sperm, such asvitrication and freeze-drying.

2. Principles of cryoinjury during freezing and

thawing

It is well known that cell metabolism decreases at lowtemperatures and that this allows long-term preservationof germ cells, embryos, and tissues. The main inconve-nience of freeze-thawing procedures is the cryoinjuryinicted by low temperatures that, among other causes, isassociated with the phase change of intracellular and

extracellular water [17]. However, rather than storage atlow temperatures, the main challenge for cells duringfreezing and thawing is the lethality of an intermediaterange of temperatures, between 15 C and 60 C. Ac-cording to Gao and Critser [17], cells and extracellularmedium remain unfrozen and supercooled at 5 C. Attemperatures between 5 C and 15 C, ice is formed inthe surrounding medium, but the intracellular contents

remain unfrozen and supercooled. Because chemical po-tential of water is higher in supercooled (intracellular) thanin frozen (extracellular) state, water ows out of the celland freezes externally. The cooling rate determines whatoccurs thereafter (Fig. 1).

If cooling rate is very high, intracellular water does notow out completely, cells freeze intracellularly, and theformation of ice crystals in the cytoplasm ultimately resultsin cryoinjury[18,19]. Because the formation of intracellularice not only depends on cooling rate and temperature butalso on CPA concentration, the use of these agents mayalleviate that formation[17].

If cooling rate is very low, most of the waterows out,intracellular solutes are concentrated, and supercooling iseliminated. Rather than freeze intracellularly, cells aredehydrated, experience volume shrinkage of organellesand membranes, and are exposed to high-solute concen-trations before they reach the temperature at which allsolution components are solidied (Fig. 1). This affectslipid-protein complexes, denatures macromolecules, de-creases the size of unfrozen channels, and induces irre-versible membrane fusion [20,21]. The resulting hypertonicstress can change the electrolyte balance, and this may leadthe cells to swell beyond their normal isotonic volume andsubsequently 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 waterows 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 volumeshrinkage.

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Therefore, albeit due to separate mechanisms, both highand low freezing may lead to cell cryoinjury. For thisreason, Mazur et al. [20]proposed the two-factor theory.According to that theory, cryoinjury occurs: (1) due to le-thal formation of intracellular ice at high cooling rates and,(2) due to solute/electrolyte concentration, cell dehydra-tion and reduction of unfrozen fraction in the extracellularspace at low cooling rates. An optimal cooling rate exists for

each specic cell type and is dened as that is low enoughto avoid formation of intracellular ice but high enough tominimize the cryoinjury due to solute/electrolyte concen-tration (Fig. 1).

Cell survival after cryopreservation not only impliescryoinjuries during freezing, but also along thawing.Indeed, whereas low thawing rates result in recrystalliza-tion, osmotic stress ensues from high rates as CPAs areunable to leave the cell fast enough[21,22]. Such osmoticincrease in the cytoplasm leads the water to enter, and thisultimately disrupts the plasma membrane[12,23]. For thisreason, optimal thawing rates should represent a balancebetween these two scenarios. In the case of boar sperm,thawing is usually performed at 37 C for 20 seconds, butToms et al.[24]have recently shown that faster thawingrates, i.e., 8 seconds at 70 C, give better results.

Hence, the adjustments in sperm cryopreservationprotocols are directed toward nding these optimal coolingand thawing rates, thereby preventing cryoinjuries asmuch as possible[25,26]. Reduction of cryoinjury may beperformed through the use of CPAs or other methods suchas vitrication. Because in the case of mammalian sperm,vitrication is, for the time being, less successful than slowcryopreservation, this review will focus on CPAs in thefollowing section.

3. The relevance of cryoprotectants

As previously stated, the composition of freezing andthawing media plays a crucial role in both slow and rapidfreezing.In addition,although slow-freezing is used to avoidthe formation of ice crystals inside the cell, the formation ofthose crystals is not completely prevented. For this reason,CPAs are used to reduce the stress derived from freezing andthawing protocols, but because these substances may betoxic for sperm, nding the most suitable concentration isvery 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 andegg yolk, sugars, and higher molecular weight compounds,such as polyvinylpyrrolidone, hydroxyethyl starch, poly-ethylene glycols, and dextrans, that contribute to preventice formation and stabilize proteins and cell membranes.These compounds do not pass through plasma membraneand develop its role extracellularly [28]. Despite thesesolutes not being able to fully protect the cell whenpermeating CPAs are absent, they increase the effectiveness

of these permeating CPAs and allow decreasing their con-centration[29].

As main nonpermeating cryoprotectants, freezing ex-tenders contain sugars, mainly disaccharides (mainlylactose or trehalose), as these give better results thanmonosaccharides[3033], and proteins from hen egg yolk.When combined with surfactant Orvus ES Paste (Equex),egg yolk proteins confer better protection because thisdetergent facilitates the interaction of egg yolk proteinswith sperm plasma membrane[23,34].

Different works have investigated whether the egg yolkcomponent can be replaced by low-density lipoproteins(LDL) or soy lecithin. With regard to the former, egg yolk isa mixture of different proteins, but its main protective ef-fect is mediated by LDL. In fact, replacing the entire egg yolkfraction by LDL has benecial effects for boar sperm qualityand DNA integrity after freeze-thawing[3537]. In addi-tion, the egg source inuences the properties of LDL frac-tions, the LDL extracted from pigeon egg yolk having highercryoprotective effects than those extracted from hen, os-trich, duck, and quail eggs[38].

With regard to replacement of egg yolk proteins by soylecithin, there are no studies conducted in pigs, and worksin humans, rams, and bulls have provided different results.In humans, conventional sperm quality parameters, DNAintegrity, and sperm ability to bind to hyaluronate do notsignicantly differ between egg yolk and soy lecithin[39].In contrast, research conducted with stallion spermshowed that replacing egg yolk by soy lecithin resulted in adecrease of reproductive performance[40], and studies inrams found a positive effect on total sperm motility, chro-matin, and acrosome integrity but an impairment ofmitochondrial sperm function at post-thawing[41].

3.2. Permeating CPAs

Permeating CPAs are glycerol, DMSO, ethylene glycol,methanol, propylene glycol, and dimethylacetamide. Allthese solutes are able to permeate the cells, and they arerelatively nontoxic at concentrations of up to 1 M and evenhigher[17]. Their mechanism of action is related to theirability to decrease the concentration of electrolytes and theextent of osmotic shrinkage at low temperatures [42].Concretely, these agents affect cytoplasm viscosity, changediffusion rates, and alter cell membrane properties via itsinsertion in the lipid bilayer [23]. However, these sub-stances may damage the cell and induce osmotic volumechanges at temperatures higher than 5 C. This represents

an inconvenience because they need to permeate beforefreezing, and they have to be quickly removed on thawing[43]. Finally, it can be noted that the suitability of a givenCPA depends on the permeability of the cell to this agentand its specic cytotoxic effects, so that the appropriateCPA concentration relies on the cell type[17].

For boar sperm cryopreservation, glycerol is the mainpermeating CPA, as no CPA has found to yield better resultsthus far [16,44], with an optimal concentration rangingfrom 2% to 3% [27,45]. However, because glycerol maydamage the perinuclear theca of boar sperm [46] andconcentrations higher than 4% affect plasma membraneuidity[47], some attempts have tried to replace glycerol

with other cryoprotectants. Replacing 3% glycerol with80 mM L-glutamine plus 2% glycerol improves post-thaw

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sperm motility [48], and substitution with trehalose(100 mM) increases motility, acrosome integrity, mito-chondrial membrane potential, and in vitro penetrationrates after IVF [49]. In addition, replacing lactose andglycerol by powdered coconut water plus dimethylforma-mide yields higher post-thaw sperm quality [50]. In fact,the benecial effects of glutamine and dimethylformamidehave also been reported for bull and stallion sperm,

respectively [51,52]. In contrast, other permeating CPAs,such as dimethylacetamide or DMSO, yield worse resultsthan glycerol[53,54].

4. Dissecting the sperm cryodamage

Even when both permeating and nonpermeating CPAsare used, cryopreservation inevitably reduces the survival,acrosome integrity, motility, and fertilizing ability offrozen-thawed mammalian sperm [44,5557]. For thisreason, the present section intends to summarize the mostcommon cryoinjuries described after freeze-thawing.Despite descriptions being made in separate subsections,there is a global impact on sperm cell, and damages arerelated (Fig. 2).

4.1. Cryopreservation effects on plasma membrane

The composition and biophysical properties of plasmamembrane are related to cell sensitivity to freeze-thawingprocedures[2]. Plasma membrane of boar sperm is rich inunsaturated phospholipids and poor in cholesterol mole-cules, and this underlies the high sensitivity of boar sperm

to cold shock[12]. Such cold shock includes all dramaticeffects ensuing from destabilization of plasma membraneat temperatures less than or equal to 5 C, thereby affectingCa2 homeostasis, acrosome integrity, and membrane lipiddisorder (Fig. 2)[44,5860].

Any plasma membrane contains phospholipids, whichconfer uidity, and a variable amount of sterols, such ascholesterol, that provide rigidity and stability. At low tem-

peratures, lipids undergo alterations in physical phases (i.e.,uid-and gel-phase lipids). Although thepresence of sterolsinhibits these lipid-phase changes, sperm membranes that,such as that of boar, present a low cholesterol:phospholipidratio andan asymmetric distribution of cholesterol aremoresensitive to damages induced by cooling [23,44,59,61].Therefore, restriction of lateral movements of membranephospholipids occur when temperatures are lower than5 C, and this eventually results in a transition from uid togel phase. Indeed, because different membrane lipids pre-sent different transition temperatures, some unsaturatedphospholipids are jellied earlier than others, and phaseseparations occur. After this phenomenon, integral mem-brane proteins become irreversibly clustered by lipid-phaseseparations, membrane lipids are restructured, and somecholesterol molecules are released[56,62]. As a result ofthese structural alterations, there is a disruption of lipid andprotein interactions, and some proteins, such as ion chan-nels, are translocated and/or lose their function [63]. Thisleads the plasma membrane to destabilize and lose its se-lective permeability, thereby increasing the inux of ions,such as Ca2 and bicarbonate, from the extracellular space[44,59,60]. All these changes haveled someauthors tospeak

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 ofnucleoproteins (protamine 1, P1, and histone 1, H1) and disruption of disul de bridges between cysteine radicals of P1.



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about cryocapacitation or capacitation-like changes, as theevents that are triggered resemble, but are not identical, totrue sperm capacitation[12,64].

As previously stated, the composition of the plasmamembrane plays a crucial role in the sperm ability towithstand freeze-thawing procedures. According toWaterhouse 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 themost abundant fatty acids in boar sperm plasmalemma.Notwithstanding, when the amounts of long-chain poly-unsaturated fatty acids, such as docosapentaenoic acid andDHA are increased, sperm have higher resilience to freeze-thawing procedures[65]. In humans, Martnez-Soto et al.[66] also reported that the content of DHA in spermmembrane is correlated with sperm motility and mem-brane integrity at post-thawing.

4.2. Cryopreservation effects on sperm nucleus

Studies evaluating how cryopreservation affects spermnucleus are focused on chromatin integrity. This can beexplained by distinguishing between DNA, nucleoproteins,and structural interaction between DNA and nucleopro-teins. Sperm chromatin is made up of DNA and nucleo-proteins, which are mainly protamines (P1 and P2) andbetween 2% and 15% histone H1[67]. Not all species pre-sent the same protamines. While boars, bulls, and ramsonly present protamine P1, human, stallion, and mousesperm present P1 and P2. In addition, P1:P2 ratios also varybetween species, and this may also affect the resilience ofthat structure to freeze-thawing procedures[68].

With regard to the impact on protamines, there arechanges in the location and distribution of P1 and H1throughout the nucleus of boar sperm after freeze-thawing(Fig. 2)[69,70], although some changes are already presentat the end of the cooling step (i.e., at 5 C). Moreover, theinteraction through disulde bridges between cysteineradicals of protamines, which constitutes the bone of thesperm chromatin, is disrupted after freeze-thawing (Fig. 2)[70]. The extent of this disruption is related to ejaculatefreezability[71], has a clear impact on reproductive per-formance[72], and is different between species that pre-sent 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 NickEnd Labeling (TUNEL), Sperm Chromatin Structure Assay(SCSA), Sperm Chromatin Dispersion test (SCD), Cometneutral and Comet alkaline, but studies conducted in pigsconcur in similar outcomes, which are obtained regardlessof the method[74]. While there is no signicant increase inDNA fragmentation immediatelyat post-thawing [70], DNAdamage is more apparent when frozen-thawed sperm areincubated at 37 Cforatleast2hours(Fig. 2) [71,73,75].Theextent of DNA-induced cryodamage also differs acrossspecies because those species having P1 and P2 presenthigher levels of DNA fragmentation than those presenting

only P1 [68]. Indeed, while sperm cryopreservation cangreatly affect DNA integrity and condensation in humans

[7678]and stallions[57], there is no such an impact indogs[79].

The mechanisms underlying the increase in DNA frag-mentation after cryopreservation are still unknown andappear to be related to the increase of oxidative DNAdamage[80]. In humans, the increase of DNA fragmenta-tion linked to cryopreservation has been correlated withoxidative stress rather than to activation of caspases[81].

Also in humans, sperm cryopreservation has also been re-ported to induce lesions on crucial genes involved infertilization and early embryo development (ADD1, ARNT,BIK, FSHB, PEG1/MEST, PRM1, SNORD116/PWSAS, andUBE3A), specically to two of these eight genes:SNORD116/PWSASand UBE3A [82]. Although no similar works havebeen conducted in pigs, this clearly warrants furtherresearch.

4.3. Cryopreservation effects on perinuclear theca

The perinuclear theca is a region that surrounds spermnucleus and contains cytoskeletal proteins crucial formaintaining the architecture of sperm head[83]. Becausethis region plays a crucial role during fertilization andcontains relevant sperm proteins such as PLCz and PAWP[8486], its integrity is important for proper sperm func-tion. In this regard, it is worth noting that cryopreservationof boar sperm damages the perinuclear theca and thatchanges in the stabilization of F-actin ultimately result inthe disruption between this theca and actin (Fig. 2). Thisnot only affects these structures but also leads the spermnucleus to decondense[87].

4.4. Cryopreservation effects on mitochondrial function and

ROS production

Mitochondrial activity is reduced after cooling andfreeze-thawing in boar sperm (Fig. 2)[88]. Such reductionis also observed in sperm from other species, such asequine [89,90], especially in poor freezability ejaculates(PFE)[57].

Another interesting point related to mitochondrialfunction regards to reactive oxygen species (ROS) produc-tion. Effects of cryopreservation on ROS production in boarsperm are less clear than in other species [91,92]. WhereasFlores et al.[88]found that cryopreservation decreased thecapacity of mitochondrial of producing ROS and this was

already apparent at the cooling step, Gmez-Fernndezet al. [93] and Yeste et al. [71] showed that good freezabilityejaculates (GFE) and PFE did not differ in terms of intra-cellular levels of peroxides and percentages of viable andnonviable sperm with membrane lipid peroxidation. Infact, although peroxides are considered to be the major freeradical in boar sperm, the marginal increase due to cryo-preservation makes it difcult to assert the real impact ofROS on sperm cryodamage because despite separatestudies reporting some increase in ROS levels after freeze-thawing, such increase is marginal[73,91,94,95]. In spite ofthis, it is worth noting that total antioxidant capacity ofseminal 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



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species. Indeed, in buffalos and bulls, ROS species areknown to affect sperm motility, lipid peroxidation, mito-chondrial membrane potential, and DNA integrity in freshsperm, but this relationship is less clear in the case offrozen-thawed sperm[97]. Again, whereas freeze-thawingdoes not increase ROS levels in buffalo sperm, lipid per-oxidation increases after cryopreservation. In stallion, lipidperoxidation is low in fresh spermatozoa and signicantly

increases after freeze-thawing. However, rather than beinga cryopreservation-induced injury, it is thought to be asublethal cryodamage leading to other injuries that mayultimately compromise the sperm survival[89].

4.5. Cryopreservation effects on sperm vacuoles

There are different vacuoles found in mammalian spermthat are observed in the acrosome and cytoplasm dropletsduring spermatogenesis. Although often observed inejaculated human sperm, they are less frequent in pigs,where they can only be detected under transmission elec-tron microscope, and they appear together withmembrane-bound nuclear invaginations, the most typicalultrastructural nuclear anomalies[98]. In spite of this, onlyfew works have studied the relevance of these vacuoles inpigs, even if an early study reported that heat stressincreased the proportions of sperm showing nuclear vac-uoles in the ejaculate[99]. There are no studies about theeffects of cryopreservation on sperm vacuoles in pigs, andstudies in humans have shown inconsistent results. Indeed,whereas Gatimel et al. [100]showed that freeze-thawingprocedures did not alter total and relative vacuole areas,Boitrelle et al.[77]observed that such procedures increasesperm nuclear vacuolization.

4.6. Changes in levels, location, function, and tyrosine-

phosphorylation of sperm proteins

Different studies have evaluated the effects of spermcryopreservation on levels, localization, and function ofsperm proteins. In a comparison of sperm proteome offresh and frozen-thawed boar sperm, Chen et al. [101] usingisobaric tags for relative and absolute quantication foundthat the amounts of up to 41 proteins involved in multipleprocesses such as sperm premature capacitation, adhe-sions, energy supply, and spermoocyte binding andfusion, were altered. The levels of six of these 41 proteins

were seen to decrease after cryopreservation, whereas theother 35 were seen to increase. Further validation withWestern blotting conrmed that the expression of AKAP3,superoxide dismutase 1 (SOD1), TPI1, and ODF2 wasincreased in frozen-thawed sperm, especially in the case ofSOD1 and AKAP3. It is worth noting that some of theseproteins, such as TPI1, HSP90AA1 and PHGPx have beensuggested to predict sperm freezability in extended ejacu-lates[102,103]. In fact, similar ndings have been reportedfor humans because Wang et al. [104]found that up to 27sperm proteins differed between fresh and frozen-thawedsperm. In this case, ACO2, TEKT1, VIM, OXCT1 were seento decrease in response to cryopreservation, whereas ENO1,

TEKT3, and TEKT4 appeared to signicantly increase. Itdoes not seem that sperm are able to synthesize de novo

proteins in response to freeze-thawing procedures, butrather that the overall cryodamage affects the presence anddetection of some proteins, which could also affect thesperm fertilizing ability if this was a result from proteindegradation. Supporting this, sperm cryopreservation de-creases 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 changesin the location of proteins (Fig. 2), such as actin and mito-fusin 2 [88], and glucose transporter GLUT3 [107]. Withregard to protein function, it has already been mentionedthat membrane cryoinjuries may affect the function of ionchannel proteins. Losing this function could also underliethe reduction of fertilizing ability of frozen-thawed sperm.Indeed, caffeine elicits Ca2-transients, and progesteroneleads to a biphasic increase of Ca2 levels in fresh but not infrozen-thawed boar sperm, which indicates that cryo-preservation reduces the responsiveness of spermatozoa todepolarization, modulators of the internal Ca2 stores andprogesterone[108]. Similar ndings have been observed inother species. For example, mean [Ca2]ibasal values of L-type voltage-gated Ca2 channels are higher in frozen-thawed than in fresh stallion sperm, and increases of[Ca2]i mediated by agonists are higher in frozen-thawedthan in fresh sperm. In contrast, the level of response tothe addition antagonists is lower in frozen-thawed than infresh sperm[109]. All these ndings concur that freeze-thawing procedures affect the function of this channels.

Finally, because capacitation-like changes involvechanges in the phosphorylation of sperm proteins, even ifthe patterns differ between capacitation-like state and truecapacitation already at the cooling step (5 C)[64],it is alsoimportant to pay attention to the effects of cryopreserva-tion on factors involved in this phosphorylation. It is widelyknown that tyrosine-phosphorylation of sperm proteins isone of the features of sperm capacitation that takes placewithin the oviduct or fallopian tube [110112], and isrelated to proteins involved in cAMP/PKA and extracellularsignal regulated kinases pathways[113]. In this regard, it isworth mentioning that tyrosine-phosphorylation of p32 ispresent after cooling and freeze-thawing of boar sperm[114]. This cryopreservation-induced change in tyrosinephosphorylation has also been found in other species,including bull, buffalo, stallion and rhesus macaque, asreviewed by Naresh and Atreja[115]. According to these

authors, alterations in the tyrosine-phosphorylation statusof sperm proteins after cryopreservation could alsocontribute to explain the reduced fertilizing ability offrozen-thawed sperm.

4.7. Any impact on mRNAs and microRNAs?

Parental mRNAs are known to play a crucial role whendelivered to oocyte on fertilization (reviewed in [116]). Twostudies conducted in boars have found that abundances oftranscripts encoding for different relevant proteins, such asB2M, 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 isnot restricted to boar sperm. In humans, sperm mRNAs



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involved in events taking place on fertilization and preg-nancy success (ADD1, PEG1/MEST, PRM1 and PRM2) arereduced after cryopreservation[119]. Similar results withPRM1have been obtained in bulls[120].Therefore, degra-dation of specic sperm mRNAs by cryopreservation pro-cedures could also contribute to explain the reduction inreproductive performance of frozen-thawed sperm. This isa reasonable hypothesis because the relationship between

sperm mRNAs (including MYC, CYP19, ADAM2, PRM1, andPRM2) and early development has also been conrmed inpigs[121]. Further research in this realm is thus warranted.

On the other hand, microRNAs (miRNAs) are smallnoncoding regulatory RNA molecules that modulate geneexpression either via inhibiting mRNA translation or viatargeting mRNA for degradation (reviewed in [122]). Arecent study in pigs has observed that the levels of somemiRNAs are more affected than others by cryopreservationprocedures[123]. Although works conducted in pigs havenot yet addressed the relationship between miRNAs andfertilizing ability[124], data available in humans point to-ward this direction [125,126]. Future studies shouldaddress whether sperm miRNAs degraded by cryopreser-vation are relevant for fertilization success.

4.8. Does sperm cryopreservation affect parental epigenetic

regulation?

The term epigenetics refers to those inheritable changesof gene expression due to mechanisms other than modi-cations in the DNA sequence [127]. Epigenetic signalsinclude DNA methylation, sperm-specic nucleoproteinsand sperm-borne RNAs (discussed in previous subsections),and the DNA loop domain organization by the sperm nu-clear matrix (reviewed in[128]). For example, studies inhumans have found that nucleosomes in which sperm DNAis packed and represent between 15% and 30% of spermDNA contribute to paternal zygotic chromatin and embryoepigenome [129]. Therefore, because, on fertilization,oocyte inherits epigenetic signals from sperm chromatinand these signals are known to play a role in early embryodevelopment, attention to iatrogenic damage inicted bycryopreservation procedures has also been paid.

Data are still scarce with regard to genomic imprinting,and barely a few number of studies conducted in humansexist. In a preliminary study, Klver et al.[130]determinedthe degree of methylation of up to nine genes: maternally

imprinted (LIT1, SNRPN, MEST), paternally imprinted (MEG3,H19),repetitiveelements(ALU,LINE1),onespermatogenesis-specic gene (VASA), and a gene related to male infertility(MTHFR), and found that neither cryopreservation nor thelength of storage (2 days compared to 4 weeks) altered themethylation pattern of those genes. These data indicate thatsperm cryopreservation, at least from the data we have thusfar, is safe in terms of gene imprinting.

4.9. Cryopreservation effects on sperm motility

A dramatic reduction in sperm motility is one of the mostapparent consequences of boar sperm cryopreservation

(Fig. 2) and has been widely reported in the literature[71

73,131]. Another interesting aspect resulting from

alterations of motility due to cryopreservation regards tosperm motile subpopulations. Indeed, whilefreeze-thawingdoes not cause major changes in the structure of spermsubpopulations when dened on curvilinear velocity, thereare signicant variations in the specic percentages of eachsubpopulation[132]. Moreover, in spite of observing foursubpopulations in both cases, kinetic parameters in eachsperm subpopulation in PFE are signicantly lower than in

GFE[133]. The ejaculate variability to withstand cryopres-ervation 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 with-stand freeze-thawing procedures (i.e., freezability), butrather there is a signicant variability between- andwithin-ejaculates, and even between fractions within thesame ejaculate [25,65,71,134,135]. For this reason, boarsand their ejaculates are classied into good or badfreezers, and as GFEor PFE[136,137]. The existence ofGFE and PFE is not restricted to pigs but has also been re-ported in other mammalian species, such as goats [138] andstallions[57]. In addition, the individual boar response isnot only related to cryopreservation, but the success ofother semen-processing techniques, such as liquid storageand gender-sorting, has also been reported to be greatlyinuenced by an individual component[139].

GFE and PFE differ in their post-thawing sperm survivaland motility, and the formers have been reported to pre-sent less resistance of their nucleoprotein structure thanthe latters[71,137]. In contrast, observations under a cryo-scanning electron microscope have revealed that the dif-ferences between GFE and PFE are not related to the degreeof dehydration and ice crystal distribution after freezingand before thawing[140].

The mechanisms underlying differences between GFEand PFE still remain unknown. In a landmark study, Thur-ston et al.[141]found that differences between GFE andPFE were related to polymorphisms in amplied restrictionfragments and identied up to 16 potential geneticmarkers. This indicated that genetic differences could bethe basis for these freezability differences. Notwith-standing, again, this nding does not seem to be exclusive

for pigs because a single nucleotide polymorphism in theposition 191 of HSP70, specically C >G, has been corre-lated with higher sperm freezability in goats [142].More-over, and as discussed in the following section, it appearsthat variations in the expression of genes involved in theresistance of cell against thermal stress are also related toejaculate freezability. However, it is yet to be elucidated bywhich mechanism the abundance of theses proteins isregulated and why.

Freezability is also related to the ability to withstandcooling rates, as Medrano et al. [143] found that cooling ratecompromised sperm function and survival at post-thawingdepending on the intrinsic freezability. In addition, it is

worth noting that variations in the cryopreservation pro-tocol, affect PFE more than GFE[144]and that the sperm



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response to additives in the freezing extender also rely onthe intrinsic freezability[131,145].

Finally, it has been reported that the addition of seminalplasma proteins from GFE increases the freezability of PFE.These positive effects not only regard to viability andmotility of frozen-thawed sperm but also to the percentageof penetrated oocytes after IVF. However, these effects areindependent from the antioxidant capacity [146],and the

seminal plasma proteome of GFE and PFE do not differ interms of antioxidant scavengers[147].

5.2. Freezability markers

One of the main problems for boar sperm cryopreser-vation is the identication of GFE and PFE before startingcryopreservation procedures because conventional spermparameters in fresh/extended sperm fail to predict theejaculate ability to withstand freeze-thawing[16,137]. Thisfact has led researchers to focus on the identication offreezability markers, which are dened as those elements(proteins or not) that may allow the identication of GFEand PFE in fresh/extended semen (i.e., before startingcryopreservation protocols). The following section andTable 1summarize the main freezability markers reportedthus far.

5.2.1. Sperm proteins as freezability markers

Sperm proteins identied as freezability markers aremembrane channels and other proteins that either areinvolved in the response to osmotic and thermal stress orplay a specic role that remains to be fully elucidated. Thusfar, 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 freez-ability when evaluated in extended semen (Table 1).Furthermore, Chen et al.[101]have found that amounts ofSOD1; outer dense ber protein 2, ODF2; and a-kinaseanchor protein 3, AKAP3 differ between extended andfrozen-thawed boar sperm, thereby suggesting that thesethree proteins could also predict the sperm resilience tosustain cryopreservation protocols.

The existence of protein freezability markers is notrestricted to boar sperm. In humans, SOD content at post-thawing after density gradient washing is correlated withsperm motility [151] and levels of enolase-1 and glucose-6-

phosphate isomerase have been reported to be higher inGFE than in PFE[152].

5.2.2. Seminal plasma proteins as freezability markers

Usingdifferentialgelelectrophoresis,Vilagranetal. [147]compared the seminal plasma proteome of fresh GFE andPFE and found that bronectin 1 was a freezability markerfor boar ejaculates (Table 1). A recent study comparing theseminalplasmaproteomeoframGFEandPFEhasfoundthatwhereas 26S proteasome complex, acylamino acidreleasing enzyme, alpha mannosidase class 2C, HSP90AA1,

tripeptidyl-peptidase 2, TCP-1 complex, sorbitol dehydro-genase, and transitional endoplasmic reticulum ATPase arepositively correlated with ejaculate freezability, cystatin,zinc-2-alpha glycoprotein, angiogenin-2like protein,cartilage acidic protein-1, cathepsin B, and ribonuclease 4isoform 1 are negatively correlated[153]. Remarkably, 26Sproteasome has been found within the list. Given the rolesuggest forsperm-borne 26 proteasome forthe degradationof sperm receptor in the zona pellucida of the oocyte[154],this clearly warrants further research.

Another seminal plasma protein that appears to conferhigher cryotolerance to boar ejaculates is the N-acetyl-b-hexosaminidase. The activity of this protein in fresh semi-nal plasma is correlated with the activities of SOD andglutathione peroxidase as well as with mitochondrialfunction and total oxidant status. At post-thawing, the ac-tivity of N-acetyl-b-hexosaminidase is negatively corre-lated to sperm viability, motility, and lipid peroxidation, sothat higher levels ofb-HEX activity in boar seminal plasmaare related to low sperm cryotolerance[149](Table 1).

5.2.3. Other sperm freezability markers

Other freezability markers identied thus far includepatterns of sperm motile subpopulations in extendedsemen[133], specic kinetic parameters evaluated at thecooling step[137], and acrosin activity[145,148].

6. Factors affecting the sperm freezability

As previously mentioned, there is an important indi-vidual component related to each ejaculation that criticallyaffects its cryopreservation success. The present sectionsummarizes six different factors that could inuence thesperm freezability: season, diet, breed, ejaculate fractions,sperm selection, and holding time.

6.1. Season

Temperature and photoperiod affects spermatogenesisand 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 [145][148]

Acrosin binding protein (ACRBP) Sperm Higher levels in GFE than in PFE [103]Fibronectin (FN1) Seminal plasma Higher levels in GFE than in PFE [147]Heat shock protein (HSP90AA1) Sperm Higher levels in GFE than in PFE [102]N-acetyl-b-hexosaminidase (b-HEX) Seminal plasma Enzyme activity negatively correlated with sperm cryotolerance [149]Triosephosphate isomerase (TPI) Sperm Lower levels in GFE than in PFE [103]Voltage-dependent anion channel 2 (VDAC2) Sperm Higher levels in GFE than in PFE [150]

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



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have shown that ejaculate freezability relies on the seasonof ejaculate collection, regardless of breed and boar, so thatboar ejaculates collected during winter and spring havehigher freezability than those obtained during autumn andsummer.

The effects of season on sperm freezability have alsobeen 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. Inthe case of horses, sperm collected between March and

June in the Northern Hemisphere are those that present thehighest freezability [159]. In contrast, in Franches-Montagnes stallions, Janett et al. [160] observed thatsperm collected in autumn presented higher spermmotility at post-thawing than sperm collected in winter.These differences between studies should consider that, incontrast to pigs, horses present breeding and nonbreedingseasons, and the location where studies are conducted mayhave strongly inuenced the nal outcome. In any case,most studies clearly point out that season affects spermfreezability in mammals, except in the case of goats whereseason has not been found to affect the sperm resilience tosustain cryopreservation[138].

6.2. Diet

It has been reported that diet has impact on spermquality in boars[156,161,162]. Unfortunately, there are notmany studies about the impact of boars diet on spermcryotolerance. Supplementing boars diet with omega-3polyunsaturated fatty acids such as DHA and eicosapenta-noic acid (for 67 months) has no effect on the ability ofsperm to be cryopreserved[161]. On the other hand, and asaforementioned, the reduction of sperm quality due tofreeze-thawing procedures has been observed after thereduction of temperature from 15C to 5 C, and there is aninterest 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 supple-menting boar diet with egg yolk.

There are other studies conducted in different livestockspecies. In horses, Brinsko et al. [164]found that supple-menting stallions diet with DHA with a pattern of 14 weeksfeeding, a 14-week resting period, and another 14-weekfeeding period not only increased the DHA amounts inthe resulting sperm cells, but also increased the motility

after freeze-thawing. Another study tried to improve thequality of frozen-thawed stallion sperm in winter becausethe freezability of stallion sperm is known to be reducedduring this season (see previous section). However, addinglinseed oil, and a commercial cocktail of antioxidants(Myostem Protec, Audevard) for a total of 12-week periodhad no effect on this parameter[165].

In all cases mentioned herein, the impact of animalsdiet on sperm freezability seems to be marginal. However,studies conducted in different species clearly indicate thatdiet modications, especially when DHA supplements areused, modulate the lipid composition of sperm plasmamembrane. In addition, in the case of horses, the modi-

cation of stallionsdiet slightly improves the sperm resil-ience to freeze-thawing. Although one should be aware

that results from one species (horses) cannot be extrapo-lated to others (pigs), diet modications not only with re-gard to composition but also to feeding regimens andlength of the trial could modulate the sperm freezabilityalso in boars. This hypothesis requires further research.

6.3. Breeds

It is not clear whether boar sperm from some breeds aremore resilient than others to freeze-thawing procedures,but there is a consensus that, regardless of the breed, in-dividual differences (i.e., between boars) exist and thatthese differences are related to the phospholipid compo-sition of the plasma membrane[65].

The lack of differences between breeds and the exis-tence of an individual-boar component was earlier re-ported in sperm preserved in liquid storage at 15 C[166].In spite of this, a recent report indicates that the amount ofgammaoryzanolenriched rice bran oil required forimproving sperm freezability is higher in Large White andLandrace than in Duroc breeds [167]. Moreover, anotherrecent study has shown that the osmotic resistance ofacrosomal membrane after freeze-thawing is higher inLarge White than in Landrace boars [168]. It is entirelyreasonable that differences between studies are due to theheterogeneity of boar populations used because all worksconcur that individual differences do exist. However, itcould be that breed effects were more apparent in someparameters than others. Therefore, further research isrequired to address these inconsistent ndings.

6.4. Ejaculate fractions

Apart from variability between boars and ejaculatesfrom the same boar, it appears that given fractions within agiven ejaculate are more resilient to freeze-thawing pro-cedures than others[135]. On the one hand, cryopreser-vation of sperm fractions is better than that of the entireejaculate, especially that of the sperm-rich fraction, andthis is attributed to the negative effect of the seminalplasma from the post-spermatic fraction[169].

On the other hand, studies comparing different portionsof the sperm-rich fraction have reported that the rst one(rst 10 mL) is more able to sustain cryopreservation thanthe others, probably because it contains the lowest levels ofbicarbonate [170]. This is consistent with the fact that

percentages of sperm showing high intracellular Ca

2

levels, exocytosed acrosomes, and protein-tyrosine phos-phorylation (including p32) are lower in the rst portionthan in the rest of the sperm-rich fraction[114,171].

6.5. Selection of sperm before cryopreservation

Sperm selection through density gradient washingbefore cryopreservation has also been reported to increasethe ejaculate freezability not only in pigs but also in othermammalian species, such as stallions[172,173]. These posi-tive effects are explained by the necessity of removing deadsperm because these are detrimental for viable sperm[174].

In the case of pigs, the use of single-layer centrifugationthrough porcine-specic colloids, such as Androcoll-P



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(Swedish University of Agricultural Sciences, Uppsala,Sweden), allows removal of cholesterol and seminal plasmaproteins from sperm surface without affecting spermmembrane integrity and improves post-thaw sperm qualityandin 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 to17 C between sperm collection and the start of cryopres-ervation procedures. While some authors have reportedthat the length of this period does not affect the spermresilience to withstand freeze-thawing procedures[24,177], others have shown that its benets, with holdingtimes between 10 and 24 hours being better than those of3 hours [26,169]. It appears that holding time increasessperm cryotolerance through maintaining the lipid archi-tecture of plasma membrane [178], via an unidentiedmechanism that involves phosphorylation of some spermproteins, such as HSP70[179].

7. Increasing the cryopreservation success through

additives

A signicant number of studies have been focused onthe improvement of sperm cryopreservation success. Inthis regard, it is worth noting that most of the additiveshave been proven to be successful in species other than pigsand then tested in this species. However, and as discussedin the following sections, the additives that are benecialfor 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 sem-inal plasma proteins on the sperm resilience to withstandfreeze-thawing procedures. In this regard, it is worthmentioning that concerns on the role of seminal plasmaexist, especially because of its potential detrimental effectson the preservation of boar sperm in liquid and frozenstates. The identity of which seminal plasma componentsare detrimental for boar sperm cryopreservation still re-mains unknown, but it has been reported that removinglow-molecular weight proteins (1214 kDa) by dialysisimproves cryopreservation outcomes[180]. Furthermore, a

bacteria-released endotoxin (lipopolysaccharide) has beenidentied as one of the plasma seminal factors that mayimpair boar sperm freezability because destabilizes theplasma membrane when binds to the Toll-like receptor-4present on the sperm surface[181].

Although most of the studies evaluating the effects ofseminal plasma have supplemented the thawing medium,the addition of 5% seminal plasma from GFE to freezingextenders has been reported to improve the motility,membrane integrity, and IVF outcomes of frozen-thawedsperm in PFE[146]. This positive effect could be mediatedthrough inhibition of in vitro capacitation and cooling-induced 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

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-thawedsperm, some studies have indicated that supplementingthawing medium with seminal plasma is benecial forsperm motility and membrane integrity[181,184], whereasothers have reported a decrease in the integrity of acro-some and plasma membrane and increase ROS levels[185].

The differences between these studies could be explainedby the volume of the seminal plasma added, the extenderused, and the temperature of incubation[186].

Because seminal plasma positively effects the endome-trial reaction after AI in pigs and other species and this mayaffect reproductive outcomes [187189], one couldconsider adding a seminal plasma fraction immediatelybefore AI. Unfortunately, experiments conducted thus farare not conclusive. Indeed, although some reports indicatethat the addition of seminal plasma increases the repro-ductive performance of frozen-thawed boar sperm[181,190,191], others do not[192].

7.2. Cyclodextrins

Cyclodextrins are cyclic heptasaccharides that consist ofb (14) glucopyranose units and have a hydrophobic center.When preloaded with cholesterol (cholesterol-loaded cy-clodextrins, CLC), these substances are able to increase theamounts of this sterol in the plasma membrane. This in-creases the stability of plasmalemma and improves survivalof frozen-thawed mammalian sperm[193]. In the speciccase of pigs, the addition of CLC improves post-thaw spermquality and in vitrofertilizing ability without affectingcapacitation status and DNA integrity (Table 2)[199201].The main inconvenience is that adding CLC may requireincreasing of glycerol concentration (from 3% to 5%;[206]),and this may be detrimental for sperm[46,47].

When added to thawing extender, CLC increase theintegrity of plasma membrane and acrosome of frozen-thawed sperm (Table 2). However, because a detrimentaleffect of CLC supplementation on sperm motility exists atconcentrations higher than 12.5 mg CLC per 500 millionsperm, more research is required to address whether theinclusion of CLC in thawing extenders is benecial or not[202].

7.3. Antioxidants

Supplementing cryopreservation media with antioxi-dants has been reported to be positive in different species,not only in pigs. In humans, e.g., supplementing freezingmedia with vitamin E and hypotaurine and with naturalantioxidants such as those from Opuntia cus-indica im-proves sperm motility and decreases DNA fragmentation atpost-thawing [173,207,208].

In pigs, the list of antioxidants with positive effects forsperm quality at post-thawing includes L-cysteine, a-tocopherol, lutein, butylated hydroxytoluene, Trolox andascorbic acid, the results of the latter being better whencombined with Trolox or with reduced glutathione (GSH;

Table 2) [194

198,203,204]. The benecial effects ofascorbic acid supplementation have also been observed on



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motility and acrosome and membrane integrity of bullsperm[209]. In contrast, other antioxidants such as cata-lase andb-mercaptoethanol have no benecial effects[53].

One antioxidant that has given interesting results isreduced GSH (Table 2). The addition of GSH to both freezingand thawing extenders increases sperm quality at post-thawing, stabilizes the nucleoprotein structure, and im-proves fertilizing ability both in vivo and in vitro[72,73,131,194,205]. These positive results are not exclusiveof pigs. In human sperm, there is a reduction of up to 64% inthe GSH content after freeze-thawing [210], which in-dicates that the antioxidant defense system is challengedby sperm cryopreservation. Furthermore, the addition ofGSH to freezing and thawing media decreases ROS levelsand improves motility of human sperm[210].

7.4. Alginate

The addition of alginate to freezing extenders maintainsbetter the acrosome integrity, increases the activities of

SOD and glutathione peroxidase, and decreases malon-dialdehyde levels after freeze-thawing (Table 2)[96].

7.5. Melatonin

Different studies have evaluated the impact of supple-menting extenders for both fresh/extended and frozen-thawed semen with melatonin, given its antioxidantproperties. In bulls, adding melatonin to freezing extendersat nal concentrations ranging between 2 and 3 mM im-proves sperm survival and motility and reduces lipid per-oxidation at post-thawing [211]. Melatonin also haspositive effects on the lipid peroxidation of cryopreserved

stallion sperm, although this impact on motility is lessapparent[212]. In rams, similar results are observed when

1 mM of melatonin is added to freezing media, the bene-cial effects not only being observed in sperm motility andviability, but also in DNA integrity and embryo cleavagerates after IVF[213]. Finally, while melatonin has not beeninvestigated in cryopreserved boar sperm, studies withextended semen during long-term storage did not showmuch improvement, despite reporting a marginal, positiveeffect on sperm viability [214]. Thus, more research isrequired to address whether melatonin could also bebenecial for cryopreserved boar sperm.

7.6. Insulin growth factor-I

Adding insulin growth factor I (IGF-I) together withvitamin E to extended boar semen improves sperm motilityafter 72 hours of liquid storage at 15 C[215]. However, thebenecial effects of supplementing cryopreservation mediawith IGF-I have been reported in cryopreserved mamma-lian sperm, but not yet in pigs. In rams, the addition of IGF-Ito freezing extenders increases the viability and motility of

frozen-thawed ram sperm but does not have any impactupon fertilizing ability[216]. Again, it would be interestingto investigate whether IGF-I has any positive effect oncryopreserved boar sperm.

7.7. Heat shock proteins: New additives for cryopreservation

media?

A new strategy to improve cryopreservation extenderscould consist of adding proteins that counteract thedamaging effects of these procedures. For example, it isknown that heat shock 70 kDa protein 8 (HSPA8) prolongsthe sperm survival in the oviduct and is upregulated in

oviductal monolayers in response to sperm[217,218]. Themechanism through which this protein maintains sperm

Table 2

Benecial effects on frozen-thawed boar sperm after supplementation of freezing and thawing media with different additives (some of them areantioxidants).

Additive Media Effects Reference

Alginate Freezing Maintains the acrosome integrity. Increases the activitiesof SOD and GPX and decreases malondialdehyde levels.

[96]

Ascorbic acid Freezing andthawing

Improves sperm motility and membrane integrity.Enhances glutathione (GSH) effects.

[194][195]

a-tocopherol Freezing Increases progressive motility, plasma membrane, and acrosome integrity. [196][197]

Butylated hydroxytoluene (BHT) Freezing Improves sperm survival and embryo development toblastocyst after IVF with frozen-thawed sperm.

[198]

Cholesterol-loadedcyclodextrins (CLC)

Freezing andthawing

Improve stability of sperm plasma and maintains the acrosome integrity better.Increase IVF outcomes

[199][200][201][202]

L-cysteine Freezing Improves progressive motility, plasma membrane, and acrosome integrity. [203][204]

Lutein Freezing Improves sperm motility, DNA and acrosome integrity,and the resistance to osmotic stress (HOST).

[195]

Reduced GSH Freezing andthawing

Increases overall sperm quality and stabilizes the nucleoprotein structure.Improves the sperm fertilizing ability both in vivoand in vitro.

[72][73][131][194]

[205]Trolox Freezing Maintains better sperm motility and acrosome integrity. [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.



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survival is related to its ability to maintain the uidity andstability of the sperm plasmalemma[219]. Recently, Holtet al.[220]have shown that the addition of a bovine, re-combinant HSPA8 to freezing and thawing media improvesthe bull sperm survival at post-thawing. Such effects relyon breed, especially when such addition is combined withCLC. While no similar study has been conducted in boarsand that the design of such a medium containing a re-

combinant protein is now far from being marketable due tobenetcost analyses, such nding suggests new researchhorizons on the design of proper freezing and thawingmedia for boar sperm.

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

One of the issues that should be addressed in the futureis whether long-term storage in liquid nitrogen aftercryopreservation is limited and, if this is the case, to whichextent. An article by Fraser et al. [168]has recently indi-cated that when sperm are stored for a period of 4 years,there is a reduction in different sperm parameters, such asmembrane integrity, motility, and mitochondrial function.Although these authors also highlighted the individualcomponent and only their study involved 10 individuals,more research is required to elucidate this point. In fact,separate studies conducted in other species have notobserved the similar outcome. Indeed, a study in a humancryobank involving up to 72 donors found that long-termfrozen storage (0.514.4 years) did not impair progressivesperm motility at post-thawing[221].

9. Fertility of frozen-thawed boar sperm

One of the concerns that have been existing historicallyregards to the lower reproductive performance of frozen-thawed sperm. Indeed, with extended semen, swine in-dustry is used to work with conception rates higher than85% and litter sizes bigger than 11 piglets. However, guresreveal that farrowing rates and litter sizes for AI usingfrozen-thawed semen are signicantly lower[222]. In spiteof this, it is worth noting that since the rst attemptsreporting seven alive piglets born per litter after the use offrozen-thawed boar sperm[44], different advances havebeen made and recent data are far more optimistic as 10piglets 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 similarbetween extended (94.4%) and frozen-thawed (90.9%)semen and also observed that no signicant impact onearly embryo development was found when frozen-thawed sperm was used.

The interval of insemination-to-ovulation is also acrucial factor as too is the use of post-cervical or deep in-trauterine insemination [225,226]. In addition, doubleinsemination yields far better results than single one[227].

Finally, it is worth mentioning that other new technol-ogies, such as sex-sorted sperm, may require or benetfrom the use of frozen-thawed sperm. Furthermore, sperm

cryopreservation may be useful for planning of AI centersand preservation of genetic material in breeds and strains

[12,28]. All this indicates that, even restricted to someparticular cases, boar sperm cryopreservation has its ownniche.

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 andnot many studies in pigs have been conducted, they arementioned herein to suggest further research perspectives.

10.1. Vitrication

Thus far, attempts to vitrify sperm have been mainlyconducted in mice and humans. Freezing/cooling ratesdiffer between slow-freezing and vitrication as well as thecomposition of their media. Indeed, although permeatingCPAs (such as glycerol) are fundamental in the compositionof the former, nonpermeating ones, such as sucrose andserum albumin are the main components in the latter, asIsachenko et al.[228]found in humans.

In rams, a recent study has shown that sperm vitri-cation using a commercial extender (Biladyl) with egg yolkand glycerol gives acceptable results in terms of plasmamembrane and acrosome integrity and DNA fragmenta-tion[229]. This study has also found that the use of su-crose and glycerol is less advisable because cryodamage ishigher. These are interesting results that deserve furtherattention.

Another work in humans that compared conventionalfreezing and vitrication found no effect from the presenceof CPAs on the motility, viability, DNA fragmentation, andhyaluronan-binding ability of post-thawed sperm [230].Because, as previously discussed, the presence of CPAs mayexert cytotoxic effects, the fact that its presence does notmuch affect the cryopreserved outcomes warrants furtherresearch, at least in humans. However, given that humanand pig sperm are different in terms of cryopreservationsuccess, these results may not be extrapolated to pigs. Inany case, as nding a proper method for cryopreservingboar sperm at the beginning was more difcult than inother species (e.g., bulls) and today this is quite a standardtechnique, data available for sperm vitrication in mice,rams, and humans do not discourage further research in

pigs.

10.2. Freeze-drying

Although freeze-drying or lyophilization seems to be atechnique far from being conducted in pigs, the presentsection intends to summarize the recent advances. Most ofthe studies published thus far have been conducted in ro-dent species (mice and rats) to maintain specic animalstrains [231]. The resulting freeze-dried sperm, evenimpaired in their viability, motility and DNA integrity [232],can be used for successful oocyte fertilization throughintracytoplasmic sperm injection (ICSI)[233].

Some new insights have also been obtained from freeze-dried sperm of dogs and wild animals, such as chimpanzee,



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giraffe, jaguar, and weasel, when injected into mouse oo-cytes[234,235]. Besides, it is worth noting that, despite thehigh reduction in sperm membrane integrity, the damageinicted on DNA integrity by freeze-drying in humans islower than that of cryopreservation[236,237].

In 2011, Choi et al. [238] using freeze-dried stallionsperm produced the rst live foals after ICSI and embryotransfer. This was the rst study in a non-laboratory animal

species. In bulls, Hara et al. [239]reported the benecialeffects of adding trehalose to and removing NaCl fromEGTA-based, freezing medium and found that freeze-driedsperm preserved in non-collapsed cakes (i.e., drying tem-perature: 30 C) yield better blastocyst development thanin collapsed cakes (drying temperature: 0 or 15 C).However, Abdalla et al. [240] in cattle showed thatfreeze-drying diminishes the sperm ability to elicit Ca2

oscillations and alleviate the metaphase II arrest in bovineoocytes after ICSI and also impairs the embryodevelopment.

In pigs, a landmark article by Kwon et al.[241]reported,for the rst time, successful ICSI fertilization and embryodevelopment up to blastocyst stage using freeze-dried boarsperm. Men et al.[242]showed that supplementing EGTA-based medium with trehalose had a positive effect on DNAintegrity in freeze-dried sperm but did not nd any in-crease in early embryo development. After this, some ad-vances have been gained from modifying the EDTA-basedmedium with lactose because this variation decreases theincrease in DNA fragmentation and also results in oocytefertilization after ICSI[243].

11. Conclusions

Despite being the most effective method for long-termpreservation of mammalian sperm, cryoinjuries maycompromise greatly sperm function and survival andstrongly decrease the reproductive performance.Mounting evidence indicates that not only cryodamage isrestricted to sperm membrane but also affects the integ-rity of sperm chromatin (nucleoprotein structure andDNA) and mitochondrial function, involves degradation ofmRNAs, and impairs the function of some relevant pro-teins. Furthermore, there is an important individualcomponent that explains the ejaculate freezability. Whiledifferent extrinsic factors, such as season and diet, couldmodulate the ejaculate freezability, intrinsic components,

such as genetic differences between boars and speci

cfeatures of spermatogenesis and epididymal maturation,seem to play a major role. In spite of this, the identica-tion of sperm freezability markers and the positive effectsof some additives have represented an important forwardstep for this technology. Although all this has resulted inan increase in their reproductive performance, the specicfeatures of boar sperm cryopreservation protocols and pigbreeding make this technique to be restricted to researchand germplasm banks. This is in contrast to othermammalian species such as bulls, in which AI is mainlyconducted with frozen-thawed sperm. Finally, vitricationand freeze-drying are currently far from being established

techniques in pigs. However, data from humans and otheranimals are encouraging.

Acknowledgments

The author is funded by the European Commission, FP7-People Programme, Marie Curie-IEF (grant Number:626061). Some diagrams used for producingFigure 2wereobtained and modied fromServier Medical Art.

Competing Interests

The author has no conicts of interest to declare.

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