Vieira 2013

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Animal Reproduction Science 136 (2013) 280– 288
Contents lists available at SciVerse ScienceDirect
Animal Reproduction Science
journa l h omepa g e: www.elsev ier .com/ locate /an i reprosc i
quine spermatozoa stored in the epididymis for up to 96 h at 4 ◦C can
e successfully cryopreserved and maintain their fertilization capacity
.A. Vieira, J. Gadea, F.A. García-Vázquez, K. Avilés-López, C. Matás ∗
epartment of Physiology, Faculty of Veterinary, University of Murcia, Murcia 30071, Spain
 r t i c l e i n f o
rticle history:
eceived 25 July 2012
eceived in revised form 4 October 2012
ccepted 5 October 2012
vailable online 1 November 2012
eywords:
tallion
pididymal
permatozoa
torage time
ryopreservation
a b s t r a c t
After injury or death of a valuable male, recovery of epididymal spermatozoa may be the
last chance to ensure preservation of its genetic material. The objective of this research was
to study the effect of sperm storage, at 4 ◦C up to 96 h, in the epididymides obtained from
castrated horses and its effect on different functional sperm parameters. Aims were to study
the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation
of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability
and chromatin condensation; and (3) freezing–thawing on viability, chromatin condensa-
tion, ROS generation, protein tyrosine phosphorylation and heterologous fertilization rate
(ICSI and IVF using bovine oocytes) of sperm recovered from the epididymis up to 96 h
post castration. The average volume (720 ± 159 �L) and the concentration (6.5 ± 0.4 × 109
spermatozoa/mL) of sperm recovered from the epididymis were not affected by storage.
Sperm viability after refrigeration at 4 ◦C for up to72 h was similar (Pto the laboratory in an
insulated container at room temperature within 1 h after
collection. After transport to the laboratory, the testis and
epididymides were washed with physiological saline solu-
tion (0.9% NaCl). Epididymides were wrapped in foil to
prevent desiccation and stored in a 4 ◦C refrigerator. Each
epididymal sample was randomly assigned to an exper-
imental group for storing during 0, 24, 48, 72 or 96 h at
4 ◦C.
After storage, the cauda region of the epididymis was
dissected and the spermatozoa were obtained by retro-
grade air flushing in a 4 ◦C refrigerated chamber. For this
purpose, a 21-G needle attached to a 5 mL syringe filled
with air was introduced into the vas deferens. Spermato-
zoa were then flushed in a retrograde direction from
the vas deferens through the cauda epididymis into a
1.5 mL eppendorf tube. Epididymal fluid volume, sperm
concentration, viability and chromatin condensation were
measured. The rest of the epididymal fluid was used for
cryopreservation.
2.2. Freezing procedure
Lactose-EDTA-egg yolk medium was used as sperm
freezing extender. The composition of this extender was
100 mL deoinized water (Milli-Q, Millipore), 11% (w/v) lac-
tose, 0.1% (w/v) EDTA, 0.089% (w/v) sodium bicarbonate,
3 mL egg yolk and 3.5 mL of glycerol, pH = 7.4 (Tischner,
1979). After preparation the extender was stabilized at 4 ◦C.
This medium was freshly prepared on the day of use.
Spermatozoa were extended in the freezing media at
4 ◦C (final concentration 100 × 106 cells/mL) for 30 min.
After that time an aliquot was taken to assess sperm viabil-
ity and chromatin condensation before cryopreservation.
Sperm samples were loaded in polyvinyl chloride straws
(0.5 mL, IMV, France) at 4 ◦C, frozen over nitrogen vapor
plugged (4 cm) for 15 min, and stored in liquid nitrogen
until analyzed.
2.3. Thawing procedure
Straws were thawed in a water bath at 37 ◦C for 30 s and
the content diluted in 4 mL Ca2+ and Mg2+-free PBS (phos-
phate buffer saline) (Merkies and Buhr, 1998) previously
warmed at 37 ◦C.
2.4. Sperm evaluation procedures
2.4.1. Sperm concentration and viability
The sperm concentration was evaluated by haemocy-
tometer (Neubauer counting chamber; VWR International,
Haasrode, Belgium). Sperm membrane integrity was
assessed by incubating sperm in a solution containing
20 �L of carboxifluorescein diacetate (CFD, 0.46 mg/mL),
to which 20 �L of propidium iodide (PI, 200 mg/mL),
10 �L of formaldehyde saline solution (1%), 100 �L of the
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82 L.A. Vieira et al. / Animal Repr
ample of semen and 900 �L of physiological saline
olution were added (Harrison and Vickers, 1990). At
east 200 cells per sample were evaluated using a
icroscope equipped with epi-fluorescence (100× magni-
cation, Leica® DMLS). Spermatazoa were classified into
wo groups: (1) cells with green fluorescence: intact mem-
rane integrity; and (2) cells with red fluorescence: altered
embrane integrity.
.4.2. Analysis of seminal parameters by flow cytometry
Flow cytometric analyses were performed on a Coul-
er Epics XL cytometer (Beckman Coulter Inc., Miami,
L, USA). A 15 mW argon ion laser operating at 488 nm
xcited the fluorophores. Data from 10,000 events per
ample were collected in list mode, and four meas-
res per sample were recorded. Flow cytometric data
ere analyzed using the program Expo32ADC (Beck-
an Coulter Inc.) using a gate in forward and side
catter to exclude eventual remaining debris and aggre-
ates from the analysis. Flow cytometry was used for
ssessment of chromatin condensation, acrosomal status,
iability, and ROS generation. The frozen–thawed sam-
les were washed by centrifugation (700 × g for 3 min),
he supernatant was removed, and the pellet was re-
uspended in 1 mL of fresh PBS medium before sperm
ssessment.
.4.2.1. Production of reactive oxygen species (ROS). Produc-
ion of ROS was measured by incubating the spermatozoa
n the presence of 2′,7′-dichlorodihydrofluorescein diac-
tate (H2DCFDA) (0.5 �M in thawing medium PBS) at 37 ◦C
Gadea et al., 2008). H2DCFDA is a stable cell-permeable
on-fluorescent probe commonly used to detect cellu-
ar ROS production. It is de-esterified intracellularly and
urns to highly fluorescent 2′,7′-dichlorofluorescin upon
xidation. Green fluorescence was collected with an FL1
ensor using a 525 nm band-pass filter. Measurements
ere expressed as the mean green intensity fluorescence
nits (mean channel in the FL1) and this was used as index
f ROS generation. ROS generation was measured after 30
nd 60 min of incubation at 37 ◦C.
.4.2.2. Determination of chromatin condensation. Sperm
hromatin was stained with PI for the determination of
perm chromatin condensation (Gadea et al., 2011). Sam-
les were incubated in a solution of ethanol and phosphate
uffered saline (PBS) (70/30, v/v) for 30 min for sperm
embrane permeabilization and kept at −80 ◦C until use.
fter thawing and process as was indicated above, the sam-
les were incubated with a PI solution (10 mg/mL). Samples
ere maintained in the darks at room temperature for
 h before flow cytometric analysis. PI fluorescence (red)
as collected with an FL3 sensor using a 650 nm band-
ass filter. Measurements were expressed as the mean
ed intensity fluorescence units (mean channel in the FL3)
nd this was used as index of the state of the chromatin
ondensation, as it is directly related to the PI uptake by
NA.
 Science 136 (2013) 280– 288
2.5. Localization of proteins phosphorylated in tyrosine
residues after capacitation
Immunofluorescence was employed to determine
the localization of proteins phosphorylated in tyrosine
residues as previously has been described (Matas et al.,
2011). Frozen–thawed spermatozoa were washed by Per-
coll 45/60% density gradient and then were re-suspended
in PBS or in sperm–TALP (109 mM NaCl, 3.15 mM KCl,
1.99 mM NaHCO3, 0.347 mM NaH2PO4, H2O, 8.468 mM
Hepes, 8.3 mM PVP, 10 mM sodium lactate, 0.199 mM
sodium piruvate, 0.068 mM streptomycin and 0.086 mM
penicillin) (McPartlin et al., 2009). The spermatozoa re-
suspended in sperm–TALP were incubated under 5% CO2
for 15 min at 37 ◦C and the spermatozoa re-suspended in
PBS were incubated for 15 min at 37 ◦C in air. Samples were
centrifuged for 3 min at 700 × g. Spermatozoa were fixed
in 2% formaldehyde solution for 60 min at 4 ◦C. Then the
samples were washed once in PBS, blocked with 4% (w/v)
BSA–PBS and incubated overnight at 4 ◦C. The spermato-
zoa were washed and re-suspended in PBS, smeared onto a
microscope slide, and allowed to air dry. Slides were then
incubated for 1 h with anti-phosphotyrosine monoclonal
antibody at 4 ◦C (1:200, clone 4G10, Millipore, Madrid,
Spain) rinsed with PBS, and incubated for an additional
1 h with fluorescein-conjugated goat anti-mouse antibod-
ies (1:400, Bio-Rad Laboratories, Madrid, Spain). After
rinsing with PBS, samples were mounted on the slides
with 90% glycerol/PBS (v/v). Sperm were observed using a
microscope equipped with epi-fluorescence (100× magni-
fication, Leica® DMLS) for anti-phosphotyrosine antibody
labeling.
Sperm were classified into two groups according to the
localization of the anti-phosphotyrosine monoclonal anti-
body signal (Pommer et al., 2003): (A) fluorescent presents
in the sub-equatorial region and, (B) fluorescence signal in
the flagellum (Fig. 1). A minimum of two slides per sample
was evaluated, counting a minimum of 200 spermatozoa
per sub-sample.
2.6. Heterologous ICSI and IVF with cattle oocytes
The medium used for in vitro maturation (IVM) bovine
oocytes was TCM-199 with Earle’s salts, 10% (v/v) fetal
bovine serum (FBS), 2 mM l-glutamine, 0.2 mM sodium
pyruvate, 50 mg/mL gentamicin, 10 IU/mL eCG (Foligon;
Intervet International BV), and 10 IU/mL hCG (Veterin
Corion Divasa Farmavic). The IVF medium for this species
consisted of IVF-TALP as previously described by Ikawa
et al. (2010).
2.6.1. In vitro maturationof cattle oocytes
IVM of cattle oocytes were performed as previously
described (Coy et al., 2008). Cow cumulus oocyte com-
plexes (COCs) were collected by aspiration of follicles
(2–6 mm in diameter) of ovaries from the slaughterhouse.
COCs were then washed twice in TCM – previously equil-
ibrated for 5 h at 38.5 ◦C and 5% CO2. Groups of 30–40
oocytes were cultured in 500 mL maturation medium for
24 h at 38.5 ◦C and 5% CO2.
L.A. Vieira et al. / Animal Reproduction Science 136 (2013) 280– 288 283
o grou
Fig. 1. Frozen–thawed epididymal stallion spermatozoa classified into tw
antibody signal: (A) sub-equatorial segment and (B) flagellum.
2.6.2. Intracitoplasmatic sperm injection (ICSI) of bovine
oocytes
Spermatozoa were obtained by centrifugation of
frozen–thawed semen on a 45/60 discontinuous Percoll
(Pharmacia) gradient for 20 min at 700 × g. The pellet was
re-suspended in sperm–TALP medium and washed again
for 5 min at 700 × g. The pellet was finally re-suspended in
1 mL sperm–TALP media. In vitro matured cattle oocytes
with zona pellucida were injected using frozen–thawed
epididymal spermatozoa. The sperm injection was per-
formed as previously has been described (García-Vázquez
et al., 2009, 2010). After ICSI, the oocytes were trans-
ferred to IVF-TALP medium and incubated at 38.5 ◦C and 5%
CO2. Eighteen to 20 h post-injection, injected oocytes were
washed in PBS and fixed for evaluation. The oocytes were
fixed for 30 min (0.5% glutaraldehyde in PBS), stained for
15 min by Hoechst 33342 (1% in PBS), washed in PBS con-
taining 1 mg/mL polyvinylpyrrolidone, mounted on glass
slides, and examined under an epifluorescence microscope
for evidence of sperm status and the pronuclear formation
(Fig. 2A).
2.6.3. In vitro fertilization with zona-free cattle oocytes
In vitro fertilization were performed as previously
described (Coy et al., 2008). Briefly, after the oocytes IVM,
30–40 oocytes were transferred to a well containing a solu-
tion of 0.3% (w/v) pronase to solubilize the ZP surrounding
the oocyte. Zona-free oocytes were washed three times
in IVF-TALP medium and groups of 25–30 oocytes were
transferred to a well of four-well Nunc multidishes (Nunc,
Roskilde, Denmark) containing 250 �L of IVF-TALP medium
previously equilibrated at 38.5 ◦C, 5% CO2.
Spermatozoa were obtained by centrifugation of
frozen–thawed semen on a 45/60% discontinuous Percoll
(Pharmacia) gradient for 20 min at 700 × g. The pellet was
re-suspended in sperm–TALP medium and washed again
Fig. 2. Pronuclear formation after 18–22 h post-injection or insemination evalua
epididymal stallion spermatozoa. (B) Zona-free cattle oocytes incubated with fro
ps according to the localization of the anti-phosphotyrosine monoclonal
for 5 min at 700 × g. The pellet was finally re-suspended
in 500 �L IVF-TALP with procaine (5 mM) (McPartlin
et al., 2009). The sperm concentration was adjusted to
2 × 105 cells/mL and maintained for 15 min at 38.5 ◦C in
humidified atmosphere with 5% CO2 before IVF was per-
formed.
Sperm suspensions (250 �L) were added to the fertil-
ization wells to obtain a final concentration of 105 cells/mL.
Eighteen to 20 h post-fertilization, zona-free oocytes were
washed in PBS and fixed for evaluation, as described above,
in order to evaluate the pronucleus formation (Fig. 2B).
2.7. Experimental design
A total of 82 epididymides were used. The epididy-
mides were stored at 4 ◦C for 0, 24, 48, 72 and 96 h.
After a corresponding time of storage in the epididymides,
sperm was collected and (i) volume, concentration, viabil-
ity and sperm chromatin decondensation were analyzed,
(ii) sperm viability and chromatin decondensation was
assessed in sperm that were further incubated for 30 min in
the freezing extender and (iii) viability, chromatin decon-
densation, ROS production (after 30 and 60 min of sperm
incubation in sperm–TALP), protein tyrosine phosphory-
lation pattern and heterologous fertilization rate (IVF and
ICSI) were finally evaluated in post frozen–thawed sperm.
2.8. Statistical analysis
Data are expressed as the mean ± SEM and analyzed by
ANOVA, considering the specific sperm treatment (stor-
age time) as the main variable. When ANOVA revealed a
significant effect, values were compared by the least sig-
nificant difference pairwise multiple comparison post hoc
test (Tukey). Differences were considered statistically sig-
nificant at P 0.05). When spermatozoa were incu-
bated in capacitating medium (sperm–TALP medium), an
increase in the percentage of tyrosine phosphorylation in
relation to the control group was observed only in the
Total 6.4 ± 0.3
. Results
.1. Sperm evaluation
Spermatozoa were successfully recovered from
pididymides using retrograde flushing with air tech-
ique. The epididymal fluid collected had a mean
olume of 720 ± 159 �L, a mean sperm concentration
f 6.5 ± 0.4 × 109 spermatozoa/mL and a total mean
umber of spermatozoa obtained from each epididymis
.7 ± 0.7 × 109 cells. There were no differences between
hese variables and sperm storage time in the epididymis
rior collection (up to 96 h) (Table 1, P > 0.05).
The viability of the samples obtained was greater than
5% when they were recovered from the epididymides on
he same day of castration (0 h) (Table 2), and viability was
ell conserved in stored samples at 4 ◦C during the first
2 h with values greater than 80%. Only samples stored
n the epididymides for 96 h had a significantly lesser via-
ility compared with the other groups (Table 2, P 0.05), with mean values close
o 35% immediately after thawing (Table 3).
The chromatin condensation of the raw samples were
ot affected by the storage of sperm in the epididymides
Fig. 3, P > 0.05). However, the cold shock occurring during
he cooling (dilution in the freezing media and maintained
0 min at 4 ◦C) and freezing process induced an increase
n the chromatin condensation (less PI uptake) which was
able 2
iability of spermatozoa obtained from epididymides stored at 4 ◦C for up
o 96 h. Raw samples obtained from epididymides by retrograde flushing
ith air. Cooled samples were extended in freezing media and maintained
t 4 ◦C for 30 min.
Storage time (h) Raw samples Cooled at 4 ◦C for 30 min
0 86.5 ± 1.3a 84.9 ± 2.0a
24 84.4 ± 1.3ab 79.1 ± 1.7ab
48 84.0 ± 2.0ab 80.0 ± 2.2ab
72 81.1 ± 2.0ab 76.0 ± 2.4bc
96 77.2 ± 2.6b 67.6 ± 2.8c
P value 0.01P 0.05).
3.2. Sperm tyrosine phosphorylation pattern
An objective in this section was to analyze the influ-
ence of the sperm storage in epididymides kept at 4 ◦C
for up to 96 h on patterns of protein sperm tyrosine phos-
phorylation. The results from frozen–thawed spermatozoa
incubated in PBS (non-capacitating medium) had a sim-
ilar pattern of phosphorylation in all the experimental
Fig. 3. Chromatin condensation of spermatozoa obtained from epididy-
mides stored at 4 ◦C for up to 96 h. (a) White bars: raw samples; (b)
diagonal bars: samples extended in freezing media and cooled to 4 ◦C for
30 min and; (c) black bars: frozen–thawed samples. No differences were
found between storage times.
L.A. Vieira et al. / Animal Reproduction
Fig. 4. ROS production by frozen–thawed spermatozoa obtained from
epididymides stored at 4 ◦C for up to 96 h. ROS production measured after
30 min (diagonal bar) and 60 min (black bars) of incubation. No differences
were found between storage times.
Fig. 5. Protein tyrosine phosphorylation observed in frozen–thawed
spermatozoa obtained straight after storage at 4 ◦C for up to 96 h. No
differences were found between storage times.
sub-equatorial segment of the sperm head (Table 4,
Pwere no differences in ROS generation
etween storage times. The oxygen tension and conven-
ional substrates such as glucose are less in the epididymis
nd, therefore, reduces sperm metabolism (Lone et al.,
011). The low temperature used to preserve the epididy-
ides could explain the lesser amounts of ROS generated
y sperm. Further studies need to be developed to analyze
hanges induced by cryopreservation on the antioxidant
209 17 (8.13)
system of the epididymal spermatozoa and their effect on
sperm structure and functionality (membrane lipoperoxi-
dation, DNA fragmentation, etc.).
In the present study, the evaluation of the protein tyro-
sine phosphorylation was used as a tool for monitoring the
sperm functionality after the capacitation process. Protein
tyrosine phosphorylation has an important role in intra-
cellular signaling, transport or cellular cycle progression.
Stallion spermatozoa display an increase in protein tyro-
sine phosphorylation when they are capacitating in vitro.
Pommer et al. (2003) reported an increase of phosphoryla-
tion in the mid- and principal piece of the sperm flagellum
when freshly ejaculated stallion sperm were capacitated
for 3 h in the presence of dbcAMP, caffeine (phosphodie-
sterase inhibitor) or methyl �-cyclodextrin (cholesterol
acceptor). In contrast, the majority of spermatozoa stored
in the stallion cauda region of the epididymis appear to
be incapable (or less able) of undergoing capacitation and
acrosome reaction (Sostaric et al., 2008). This fact, at least in
part, explains the poor pregnancy rates obtained and/or the
large sperm numbers required to obtain pregnancies when
fresh or frozen–thawed stallion epididymal spermatozoa
used for artificial insemination (Morris et al., 2002; Heise
et al., 2010). In addition, the freezing process promotes
sperm capacitation-like processes (Watson, 1995; Bailey
et al., 2000), hence this might reduce the longevity of cryo-
preserved spermatozoa in the female reproductive tract.
However, there was no previous information about the
effect of epididymal sperm storage over time on capacita-
tion. In the present study, it was observed that the detection
of protein tyrosine phosphorylation in the sub-equatorial
segment was very low in all the experimental groups (30%).
Sperm cryopreservation reduces sperm functional
parameters compared to that of freshly ejaculates sperm
(Thomas et al., 1998; Bilodeau et al., 2000). However, a
reduction in sperm quality does not necessarily directly or
proportional translate into a reduction in fertility. Unfor-
tunately, it is generally not possible to conduct large-scale
fertility trials or homologous IVF studies because the ani-
mals or gametes are not available, even in domestic species,
such trials can be expensive and time-consuming (Roth
et al., 1999). Sperm penetration assays can assess a num-
ber of sperm functions simultaneously (e.g. motility, ability
oduction
L.A. Vieira et al. / Animal Repr
to undergo acrosome reaction, oocyte penetration and DNA
decondensation) and may be a better assay to assess sperm
quality (Gadea, 2005). Therefore, heterologous IVF have
been used to predict the fertility of spermatozoa in dif-
ferent species including equine (Choi et al., 2002, 2003;
Garcia-Alvarez et al., 2009; Clulow et al., 2010).
In the current study, ICSI and IVF was performed in a het-
erologous system to evaluate the quality of frozen–thawed
epididymal spermatozoa, recovered from postmortem
epididymides stored at 4 ◦C over time. Spermatozoa from
all the experimental groups (0, 24, 48, 72 and 96 h) were
able to induce egg activation and develop female and male
pronuclei (Table 5). However, penetration rates were very
low compared to other studies developed with stallion or
donkey ejaculated spermatozoa (Choi et al., 2003; Taberner
et al., 2010). One possible cause for these low rates could be
related to a different pattern of capacitation in epididymal
spermatozoa in comparison to ejaculated spermatozoa, as
it was previously reported in boar spermatozoa (Matas
et al., 2010). That could need changes in the capacitat-
ing media and protocols to be modified and adapted for
epididymal spermatozoa. Penetration rates can vary by
more than 40% depending on the medium used (Choi et al.,
2003). The results in ICSI or IVF systems obtained between
spermatozoa recovered from the different storage time
groups seem to be contradictory because more pronuclear
formation is observed after a prolong storage time than in
control group (ICSI at 96 h and IVF at 48 h; Table 5, P > 0.05).
Although the difference is not statistically different, this
discrepancy could be explained by the fact that each stor-
age time refers to a different male and it is known that
there is an intrinsic fertility and cryopreservation variation
between males (Barbas and Mascarenhas, 2009). In addi-
tion, collection of sperm was done throughout the year and
it is well known that sperm quality is affected by seasonal
variation (Hoffmann and Landeck, 1999).
An interesting fact observed was that in all cases, stallion
frozen–thawed epididymal spermatozoa were able to acti-
vate the oocyte (formation of male and female pronucleus).
This demonstrates the integrity of the factors necessary for
this event to occur. A sperm factor, sensitive to heat and
proteases has been determined, to be responsible for the
generation of IP3 and Ca2+ oscillations. This factor is an iso-
form of phospholipase C, named PLCz (Yoneda et al., 2006)
and it is now widely accepted that PLCz activates the oocyte
(Kashir et al., 2010). In frozen–thawed spermatozoa, a pro-
cess that damages the sperm membrane, the amount of
this PLCz in spermatozoa decreases (Kashir et al., 2011).
Nakai et al. (2011) studied the effect on the male pronucleus
formation after ICSI of the treatment of the spermato-
zoa by various methods to break or modify membranes
(sonication, Triton X-100 and three cycles of freezing and
thawing). Oocytes injected with complete spermatozoa
had greater pronuclear formation than oocytes injected
with treated sperm (under the conditions described above)
suggesting that these sperm contain lower amount of PLCz.
In conclusion, the storage of sperm in the epididymes
at 4 ◦C for up to 96 h could be an efficient system for
the salvage of gametes from genetically important stallion
that may die unexpectedly or are castrated. The retro-
grade flushing by air followed by cryopreservation ensure
 Science 136 (2013) 280– 288 287
the preservation of a number of functional sperm which
can be used to successfully fertilize heterologous oocytes
using techniques such as IVF and ICSI. However, further
evaluation using artificial insemination to produce viable
offsprings is necessary to confirm the fertilization compe-
tence of the conserved gametes.
Acknowledgments
The authors appreciate the generous effort and work
from the veterinary surgeons José Aldebarán Rubio, Pedro
Sánchez, Carlos Galves and Natividad González, who
helped us in recovering the testes and epididymides
derived from castrations. We would also like to thank Dr
Linda Lefièvre for reviewing this manuscript. This work was
supported by Fundación Seneca project 08752/PI/08.
References
Alvarez, J.G., Lasso, J.L., Blasco, L., Nunez, R.C., Heyner, S., Caballero, P.P.,
Storey, B.T., 1993. Centrifugation of human spermatozoa induces
sublethal damage; separation of human spermatozoa from semi-
nal plasma by a dextran swim-up procedure without centrifugation
extends their motile lifetime. Hum. Reprod. 8, 1087–1092.
Bailey, J.L., Bilodeau, J.F., Cormier, N., 2000. Semen cryopreservation
in domestic animals: a damaging and capacitating phenomenon. J.
Androl. 21, 1–7.
Barbas, J.P., Mascarenhas, R.D., 2009. Cryopreservation of domestic animal
sperm cells. Cell Tissue Bank 10,49–62.
Barker, C.A., 1962. Long-term survival of frozen equine epididymal
spermatozoa. Can. Vet. J. 3, 221–222.
Barker, C.A., Gandier, J.C., 1957. Pregnancy in a mare resulting from frozen
epididymal spermatozoa. Can. J. Comp. Med. Vet. Sci. 21, 47–51.
Baumber, J., Ball, B.A., Linfor, J.J., Meyers, S.A., 2003. Reactive oxygen
species and cryopreservation promote DNA fragmentation in equine
spermatozoa. J. Androl. 24, 621–628.
Bilodeau, J.F., Chatterjee, S., Sirard, M.A., Gagnon, C., 2000. Levels of antiox-
idant defenses are decreased in bovine spermatozoa after a cycle of
freezing and thawing. Mol. Reprod. Dev. 55, 282–288.
Blash, S., Melican, D., Gavin, W., 2000. Cryopreservation of epididymal
sperm obtained at necropsy from goats. Theriogenology 54, 899–905.
Bruemmer, J., Moore, A., Squires, E., 2003. Freezing epididymal sperm from
transported testicles. In: Squires, E., Wade, J.F. (Eds.), Proceedings of a
Workshop on Transporting Gametes and Embryos. R & W Publications,
Brewster, MA, USA, pp. 61–62.
Bruemmer, J., Reger, H., Zibinski, G., Squires, E., 2002. Effect of storage
at 5 C on the motility and cryopreservation of stallion epididymal
spermatozoa. Theriogenology 58, 405–407.
Bruemmer, J.E., 2006. Collection and freezing of epididymal stallion sperm.
Vet. Clin. North Am. Equine Pract. 22, 677–682.
Cary, J.A., Madill, S., Farnsworth, K., Hayna, J.T., Duoos, L., Fahning, M.L.,
2004. A comparison of electroejaculation and epididymal sperm col-
lection techniques in stallions. Can. Vet. J. 45, 35–41.
Clulow, J.R., Evans, G., Maxwell, W.M., Morris, L.H., 2010. Evaluation of the
function of fresh and frozen–thawed sex-sorted and non-sorted stal-
lion spermatozoa using a heterologous oocyte binding assay. Reprod.
Fertil. Dev. 22, 710–717.
Coy, P., Grullon, L., Canovas, S., Romar, R., Matas, C., Aviles, M., 2008.
Hardening of the zona pellucida of unfertilized eggs can reduce
polyspermic fertilization in the pig and cow. Reproduction 135, 19–27.
Choi, Y., Love, C., Love, L., Varner, D., Brinsko, S., Hinrichs, K., 2002. Devel-
opmental competence in vivo and in vitro of in vitro-matured equine
oocytes fertilized by intracytoplasmic sperm injection with fresh or
frozen–thawed spermatozoa. Reproduction 123, 455.
Choi, Y.H., Landim-Alvarenga, F.C., Seidel Jr., G.E., Squires, E.L., 2003. Effect
of capacitation of stallion sperm with polyvinylalcohol or bovine
serum albumin on penetration of bovine zona-free or partially zona-
removed equine oocytes. J. Anim. Sci. 81, 2080–2087.
Dacheux, J.L., Castella, S., Gatti, J.L., Dacheux, F., 2005. Epididymal cell
secretory activities and the role of proteins in boar sperm maturation.
Theriogenology 63, 319–341.
Dias, A.J., Maia, M.S., Retamal, C.A., Lopez, M.L., 2004. Identification and
partial characterization of alpha-1,4-glucosidase activity in equine
epididymal fluid. Theriogenology 61, 1545–1558.
2 oduction
F
G
G
G
G
G
G
G
G
G
H
H
H
H
H
H
H
I
J
J
J
K
K
88 L.A. Vieira et al. / Animal Repr
ioratti, E.G., Melo, C.M., Villaverde, A.I.S.B., Martin, I., Felício, G.B., Fer-
reira, H.N., Oliveira, J.V., Alvarenga, M.A., Papa, F.O., 2008. Correlation
of testicular volume and weight with sperm recovery from stallion
epididymis. Anim. Reprod. Sci. 107, 322.
adea, J., 2005. Sperm factors related to in vitro and in vivo porcine fer-
tility. Theriogenology 63, 431–444.
adea, J., García-Vázquez, F., Matas, C., Gardon, J.C., Canovas, S., Gumbao,
D., 2005. Cooling and freezing of boar spermatozoa: supplementation
of the freezing media with reduced glutathione preserves sperm func-
tion. J. Androl. 26, 396–404.
adea, J., Gumbao, D., Canovas, S., García-Vázquez, F.A., Grullon, L.A., Gar-
don, J.C., 2008. Supplementation of the dilution medium after thawing
with reduced glutathione improves function and the in vitro fertilizing
ability of frozen–thawed bull spermatozoa. Int. J. Androl. 31, 40–49.
adea, J., Molla, M., Selles, E., Marco, M.A., García-Vázquez, F.A., Gardon,
J.C., 2011. Reduced glutathione content in human sperm is decreased
after cryopreservation: effect of the addition of reduced glutathione
to the freezing and thawing extenders. Cryobiology 62, 40–46.
adea, J., Selles, E., Marco, M.A., Coy, P., Matas, C., Romar, R., Ruiz, S., 2004.
Decrease in glutathione content in boar sperm after cryopreserva-
tion. Effect of the addition of reduced glutathione to the freezing and
thawing extenders. Theriogenology 62, 690–701.
arcia-Alvarez, O., Maroto-Morales, A., Martinez-Pastor, F., Fernandez-
Santos, M.R., Esteso, M.C., Perez-Guzman, M.D., Soler, A.J., 2009.
Heterologous in vitro fertilization is a good procedure to assess the
fertility of thawed ram spermatozoa. Theriogenology 71, 643–650.
arcía-Vázquez, F.A., García-Roselló, E., Gutiérrez-Adán, A., Gadea, J., 2009.
Effect of sperm treatment on efficiency of EGFP-expressing porcine
embryos produced by ICSI-SMGT. Theriogenology 72, 506–518.
arcía-Vázquez, F.A., Ruiz, S., Matas, C., Izquierdo-Rico, M.J., Grullon, L.A.,
De Ondiz, A., Vieira, L., Aviles-Lopez, K., Gutierrez-Adan, A., Gadea,
J., 2010. Production of transgenic piglets using ICSI-sperm-mediated
gene transfer in combination with recombinase RecA. Reproduction
140, 259–272.
ranemann, L., Weiss, R., Kozicki, L., Muradas, P., Treml, T., 2006. Total
number of spermatozooa from stud-horses collected by means of arti-
ficial vagina and through feedback flow of the epididymis tail. Arch.
Vet. Sci. 11, 73–77.
amamah, S., Royere, D., Nicolle, J.C., Paquignon, M., Lansac, J., 1990.
Effects of freezing–thawing on the spermatozoon nucleus: a com-
parative chromatin cytophotometric study in the porcine and human
species. Reprod. Nutr. Dev. 30, 59–64.
arrison, R.A.P., Vickers, S.E., 1990. Use of fluorescent probes to assess
membrane integrity in mammalian spermatozoa. J. Reprod. Fertil. 88,
343–352.
eise, A., Kahn, W., Volkmann, D.H., Thompson, P.N., Gerber, D., 2010.
Influence of seminal plasma on fertility of fresh and frozen–thawed
stallion epididymal spermatozoa. Anim. Reprod. Sci. 118, 48–53.
ewitt, D.A., Leahy, R., Sheldon, I.M., England, G.C., 2001. Cryopreservation
of epididymal dog sperm. Anim. Reprod. Sci. 67, 101–111.
offmann, B., Landeck, A., 1999. Testicular endocrine function, seasonality
and semen quality of the stallion. Anim. Reprod. Sci. 57, 89–98.
oltz, W., Smidt, D., 1976. The fertilizing capacity of epididymal spermato-
zoa in the pig. J. Reprod. Fertil. 46, 227–229.
oodbhoy, T., Talbot, P., 1994. Mammalian cortical granules: contents,
fate, and function. Mol. Reprod. Dev. 39, 439–448.
kawa, M., Inoue, N., Benham, A.M., Okabe, M., 2010. Fertilization: a
sperm’s journey to and interaction with the oocyte. J. Clin. Invest. 120,
984–994.
ames, A., Green, H., Hoffman, S., Landry, A., Paccamonti, D., Godke, R.,
2002. Preservation of equine sperm stored in the epididymis at 4 ◦C
for 24, 48, 72 and 96 hours. Theriogenology 58, 401–404.
ames, A.N., 2004. Preservation of sperm harvested from the rat, caprine,
equine and bovine epididymis. Doctoral Thesis. The Interdepartmen-
tal Program of Animal and Dairy Sciences, Texas A&M University.
imenez, C., 1987. Effects of Equex STM and equilibration time on the
pre-freeze and postthaw motility of equine epididymal spermatozoa.
Theriogenology 28, 773–782.
ashir, J., Heindryckx, B., Jones, C., De Sutter, P., Parrington, J., Coward, K.,
2010. Oocyte activation, phospholipase C zeta and human infertility.
Hum. Reprod. Update 16, 690–703.
ashir, J., Heynen, A., Jones, C., Durrans, C., Craig, J., Gadea, J., Turner,
K., Parrington, J., Coward, K., 2011. Effects of cryopreservation and
density-gradient washing on phospholipase C zeta concentrations in
human spermatozoa. Reprod. Biomed. Online 23, 263–267.
 Science 136 (2013) 280– 288
Lone, F.A., Islam, R., Khan, M.Z., Sofi, K.A., 2011. Effect of transportation
temperature on the quality of cauda epididymal spermatozoa of ram.
Anim. Reprod. Sci. 123, 54–59.
Marks, S., Dupuis, J., Mickelsen,W., Memon, M., Platz Jr., C., 1994. Concep-
tion by use of postmortem epididymal semen extraction in a dog. J.
Am. Vet. Med. Assoc. 204, 1639–1640.
Matás, C., Decuadro, G., Martínez-Miró, S., Gadea, J., 2007. Evaluation of a
cushioned method for centrifugation and processing for freezing boar
semen. Theriogenology 67, 1087–1091.
Matas, C., Sansegundo, M., Ruiz, S., García-Vázquez, F.A., Gadea, J., Romar,
R., Coy, P., 2010. Sperm treatment affects capacitation parameters and
penetration ability of ejaculated and epididymal boar spermatozoa.
Theriogenology 74, 1327–1340.
Matas, C., Vieira, L., García-Vázquez, F.A., Aviles-Lopez, K., Lopez-Ubeda,
R., Carvajal, J.A., Gadea, J., 2011. Effects of centrifugation through three
different discontinuous Percoll gradients on boar sperm function.
Anim. Reprod. Sci. 127, 62–72.
McPartlin, L.A., Suarez, S.S., Czaya, C.A., Hinrichs, K., Bedford-Guaus, S.J.,
2009. Hyperactivation of stallion sperm is required for successful
in vitro fertilization of equine oocytes. Biol. Reprod. 81, 199–206.
Melo, C., Papa, F., Fioratti, E., Villaverde, A., Avanzi, B., Monteiro, G.,
Dell’Aqua, J., Pasquini, D., Alvarenga, M., 2008. Comparison of three
different extenders for freezing epididymal stallion sperm. Anim.
Reprod. Sci. 107, 331-331.
Merkies, K., Buhr, M.M., 1998. Epididymal maturation affects calcium
regulation in equine spermatozoa exposed to heparin and glucose.
Theriogenology 49, 683–695.
Monteiro, G.A., Papa, F.O., Zahn, F.S., Dellaqua Jr., J.A., Melo, C.M., Maziero,
R.R., Avanzi, B.R., Alvarenga, M.A., Guasti, P.N., 2011. Cryopreserva-
tion and fertility of ejaculated and epididymal stallion sperm. Anim.
Reprod. Sci. 127, 197–201.
Morris, L., Tiplady, C., Allen, W., 2002. The in vivo fertility of cauda epi-
didymal spermatozoa in the horse. Theriogenology 58, 643–646.
Nakai, M., Ito, J., Sato, K., Noguchi, J., Kaneko, H., Kashiwazaki, N., Kikuchi,
K., 2011. Pre-treatment of sperm reduces success of ICSI in the pig.
Reproduction 142, 285–293.
Papa, F.O., Melo, C.M., Fioratti, E.G., Dell’aqua Jr., J.A., Zahn, F.S., Alvarenga,
M.A., 2008. Freezing of stallion epididymal sperm. Anim. Reprod. Sci.
107, 293–301.
Pommer, A.C., Rutllant, J., Meyers, S.A., 2003. Phosphorylation of protein
tyrosine residues in fresh and cryopreserved stallion spermatozoa
under capacitating conditions. Biol. Reprod. 68, 1208–1214.
Roth, T.L., Bush, L.M., Wildt, D.E., Weiss, R.B., 1999. Scimitar-horned oryx
(Oryx dammah) spermatozoa are functionally competent in a heterol-
ogous bovine in vitro fertilization system after cryopreservation on
dry ice, in a dry shipper, or over liquid nitrogen vapor. Biol. Reprod.
60, 493–498.
Sharma, R.K., Padron, O.F., Thomas, A.J., Agarwal, A., 1997. Factors asso-
ciated with the quality before freezing and after thawing of sperm
obtained by microsurgical epididymal aspiration. Fertil. Steril. 68,
626–631.
Songsasen, N., Tong, J., Leibo, S.P., 1998. Birth of live mice derived by
in vitro fertilization with spermatozoa retrieved up to twenty-four
hours after death. J. Exp. Zool. 280, 189–196.
Sostaric, E., Aalberts, M., Gadella, B.M., Stout, T.A., 2008. The roles of the
epididymis and prostasomes in the attainment of fertilizing capacity
by stallion sperm. Anim. Reprod. Sci. 107, 237–248.
Taberner, E., Morato, R., Mogas, T., Miro, J., 2010. Ability of Catalonian don-
key sperm to penetrate zona pellucida-free bovine oocytes matured
in vitro. Anim. Reprod. Sci. 118, 354–361.
Thomas, C.A., Garner, D.L., DeJarnette, J.M., Marshall, C.E., 1998. Effect of
cryopreservation of bovine sperm organelle function and viability as
determined by flow cytometry. Biol. Reprod. 58, 786–793.
Tischner, M., 1979. Evaluation of deep-frozen semen in stallions. J. Reprod.
Fertil. Suppl. 27, 53–59.
Watson, P.F., 1995. Recent developments and concepts in the cryopres-
ervation of spermatozoa and the assessment of their post-thawing
function. Reprod. Fertil. Dev. 7, 871–891.
Yoneda, A., Kashima, M., Yoshida, S., Terada, K., Nakagawa, S., Sakamoto,
A., Hayakawa, K., Suzuki, K., Ueda, J., Watanabe, T., 2006. Molecular
cloning, testicular postnatal expression, and oocyte-activating poten-
tial of porcine phospholipase Czeta. Reproduction 132, 393–401.
Zomborszky, Z., Zubor, T., Toth, J., Horn, P., 1999. Sperm collection from
shot red deer stags (Cervus elaphus) and the utilization of sperm frozen
subsequently thawed. Acta Vet. Hung. 47, 263–270.
	Equine spermatozoa stored in the epididymis for up to 96h at 4°C can be successfully cryopreserved and maintain their fert...
	1 Introduction
	2 Material and methods
	2.1 Animals and epididymal sperm collection
	2.2 Freezing procedure
	2.3 Thawing procedure
	2.4 Sperm evaluation procedures
	2.4.1 Sperm concentration and viability
	2.4.2 Analysis of seminal parameters by flow cytometry
	2.4.2.1 Production of reactive oxygen species (ROS)
	2.4.2.2 Determination of chromatin condensation
	2.5 Localization of proteins phosphorylated in tyrosine residues after capacitation
	2.6 Heterologous ICSI and IVF with cattle oocytes
	2.6.1 In vitro maturation of cattle oocytes
	2.6.2 Intracitoplasmatic sperm injection (ICSI) of bovine oocytes
	2.6.3 In vitro fertilization with zona-free cattle oocytes
	2.7 Experimental design
	2.8 Statistical analysis
	3 Results
	3.1 Sperm evaluation
	3.2 Sperm tyrosine phosphorylation pattern
	3.3 Heterologous ICSI and IVF
	4 Discussion
	Acknowledgments
	References