Beruflich Dokumente
Kultur Dokumente
Theriogenology
journal homepage: www.theriojournal.com
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
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
48
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
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].
50
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.
51
52
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
54
Table 1
Markers that predict the boar sperm resilience to withstand cryopreservation in extended/fresh semen.
Protein
Marker
Function/action
Reference
Acrosin activity
Sperm
Sperm
Seminal plasma
Sperm
Seminal plasma
Sperm
Sperm
[145]
[148]
[103]
[147]
[102]
[149]
[103]
[150]
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
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
Freezing
Cholesterol-loaded
cyclodextrins (CLC)
Freezing and
thawing
L-cysteine
Freezing
Lutein
Freezing
Reduced GSH
Freezing and
thawing
Trolox
Freezing
[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.
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
10.1. Vitrication
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]
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.
Cryobiology 2015;70:1649.
[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
[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.
Anim Reprod Sci 2010;121:11523.
[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.
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.
Cryobiology 2013;66:6975.
[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]
[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.