CSIRO PUBLISHING

Reproduction, Fertility and Development

http://dx.doi.org/10.1071/RD15118

Effects of reduced glutathione on acrosin activity

in frozenthawed boar spermatozoa
lamo A,
Efren Estrada A, Joan E. Rodrguez-Gil A, Maria M. Rivera del A
A
A,B,C
Alejandro Pena and Marc Yeste
A

Unit of Animal Reproduction, Department of Animal Medicine and Surgery,

Faculty of Veterinary Medicine, Autonomous University of Barcelona,
E-08193 Bellaterra (Cerdanyola del Valle`s), Barcelona, Spain.
B
Present address: Nuffield Department of Obstetrics and Gynaecology, University of Oxford,
Level 3, Womens Centre, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK.
C
Corresponding author. Email: marc.yeste@obs-gyn.ox.ac.uk

Abstract. In pigs, acrosin activity in extended semen is correlated with reproductive performance and has recently been
identified as a freezability marker. Reduced glutathione (GSH) is known to decrease sperm cryodamage and increase the
reproductive performance of frozenthawed boar spermatozoa. However, the effects of GSH on the acrosin activity of
good and poor freezability ejaculates (GFE and PFE, respectively) is yet to be examined. The present study investigated
how supplementing cryopreservation media with GSH affected acrosin activity in GFE and PFE, as well as the relationship
between acrosin activity and reproductive performance in frozenthawed boar spermatozoa. In addition, we examined
whether the increase in fertility rates and litter sizes observed after the addition of 2 mM GSH to cryopreservation
extenders was related to acrosin activity. Supplementing freezing media with 2 mM GSH partially counteracted the
cryopreservation-related decrease in acrosin activity in GFE but not PFE. Acrosin activity was found to be significantly
correlated with in vivo reproductive performance of frozenthawed boar semen. In conclusion, the effects of adding GSH
to freezing extenders on the acrosin activity of frozenthawed boar spermatozoa rely on the intrinsic freezability of the
ejaculate. Furthermore, the maintenance of proper acrosin activity could contribute to the increase in reproductive
performance mediated by GSH.
Additional keywords: cryopreservation, reproductive performance.

Received 26 March 2015, accepted 27 June 2015, published online 21 July 2015

Introduction
The use of AI in the swine industry is widespread worldwide,
because it allows for a significant increase in reproductive performance and offers many advantages, including access to highquality genetic material (Holt 2000; Knox 2011). Although AI
can be performed using both extended and frozenthawed boar
spermatozoa, extended semen is used in most cases (RodrguezGil and Estrada 2013). In fact, although sperm cryopreservation
is the best method to store spermatozoa for long periods, it is only
used in 2% of cases (Roca et al. 2011). Among other reasons, this
is because sperm survival is reduced following cryopreservation
as a result of cryodamage to the sperm membrane and nucleus
(Holt 2000; Casas et al. 2009; Benson et al. 2012; Yeste et al.
2013a), which may ultimately result in lower fertility rates and
litter sizes (Almlid and Hofmo 1995; Johnson et al. 2000; Casas
and Flores 2013). In addition, not all ejaculates have the same
ability to withstand freezethawing and individual differences
between ejaculates exist (Thurston et al. 2001, 2002); consequently, ejaculates are classified as good freezability ejaculates
Journal compilation CSIRO 2015

(GFE) and poor freezability ejaculates (PFE; Casas et al. 2009;

Yeste et al. 2013b).
Since the first cryopreservation attempts in the 1970s, efforts
have been made to improve the quality and reproductive
performance of frozenthawed boar spermatozoa (Polge et al.
1970; Pursel and Johnson 1975; Westendorf et al. 1975). Some
have tried to improve sperm quality by adding antioxidants to
the freezethawing media or changing the packing conditions
and cryopreservation protocols, others have tried depositing the
spermatozoa closer to the site of fertilisation and closely timed
the moment of ovulation (Rath 2002; Casas et al. 2010).
With regard to antioxidants, reduced L-glutathione (GSH) has
been shown to increase the quality and fertilising ability of
frozenthawed boar spermatozoa (Gadea et al. 2004, 2005;
Estrada et al. 2014), primarily because it stabilises the
nucleoprotein structure and increases sperm survival at the
post-thawing stage (Yeste et al. 2013a). However, the beneficial
effects of GSH on post-thaw sperm function and survival have
been reported to depend on the intrinsic freezability of the
www.publish.csiro.au/journals/rfd

Reproduction, Fertility and Development

ejaculate, because PFEs need higher GSH concentrations than

GFEs (Yeste et al. 2014a).
Proacrosin is a zymogen present within the acrosome that
is activated to acrosin following interaction of capacitated
spermatozoa with glycoproteins of the zona pellucida. Thus,
acrosin plays a vital role in the penetration of the zona pellucida
(for a review, see Yeste 2013) and its activity is related to
reproductive performance in boar semen stored at 178C (Pinart
et al. 2013). In addition, acrosin activity has been found to
predict ejaculate freezability, with GFE exhibiting higher values
of activity than PFE before starting cryopreservation (Pinart
et al. 2015). However, it remains to be determined whether GSH
supplementation has any effect on acrosin activity of GFE and
PFE following freezethawing, and whether acrosin activity
underlies the increase in the reproductive performance of boar
cryopreserved mediated by GSH.
Against this background, the aim of the present study was to
evaluate whether GSH supplementation had any effect on
acrosin activity of GFE and PFE after freezethawing, and
whether this effect, if any, could ultimately result in increased
reproductive performance.
Materials and methods
Experimental design
Two sets of experiments were performed as part of the present
study. First, 29 different ejaculates were used, each coming from
a different boar. Upon arrival to our Laboratory from the farm,
each ejaculate was split into three fractions. One (extended) was
used to evaluate conventional sperm parameters and acrosin
activity, whereas the other two were used for cryopreservation.
One aliquot was cryopreserved without any additives following
the Westendorf method (Westendorf et al. 1975), whereas
the other aliquot was cryopreserved using conventional cryopreservation extenders (lactose egg yolk (LEY) and LEY
glycerol (LEYGO)) supplemented with 2 mM GSH.
The second experiment evaluated the relationship between
reproductive performance and acrosin activity in frozenthawed
boar spermatozoa after AI of sows with frozenthawed spermatozoa that were either supplemented or not with 2 mM GSH.
Because the second experiment was conducted later, only GFEs
were used in the AI trials. Up to 13 ejaculates, each ejaculate
coming from a different boar, were used.
All experiments adhered to the guidelines set by the Ethics
Committee, Autonomous University of Barcelona (Bellaterra,
Cerdanyola del Valle`s, Spain), the Animal Welfare Directive of
the Regional Government of Catalonia, Spain (D 214/1997,
DOGC 1997; 2450: 91699174) and the Spanish Welfare and
Protection Standards in Swine (RD 1392/2012, BOE 2012; 241:
7138071382).
Animals and semen samples
Sperm samples were obtained from a local farm (Servicios
Geneticos Porcinos, Roda de Ter, Barcelona, Spain) from boars
of proven sperm quality and reproductive performance. Animals
were housed under controlled temperature and humidity and fed
a standard diet with water provided ad libitum. The gloved-hand
technique was used to collect semen from the boars (Hancock

E. Estrada et al.

and Howell 1959). The resting period between collections was

3 days. An insulated, warmed container was used and only the
sperm-rich fraction was collected and filtered through gauze.
For the first experiment, sperm-rich fractions were filtered
through gauze and immediately diluted in a commercial extender
(Androstar Plus; Minitub Iberica, Tarragona, Spain). Extended
semen samples were cooled to 178C and split into seminal doses
intended for post-cervical AI (each 3  109 spermatozoa per
dose). For each boar, four seminal doses were sent to our
laboratory within 4 h of collection. In total, 29 different boars
were involved in this experiment. Upon arrival at the laboratory,
each four doses coming from the same boar were pooled and
acrosin activity, plasma and acrosome membrane integrity,
sperm morphology, motility and concentration were evaluated.
Results from these analyses are shown as being for extended
semen. Apart from providing data for the initial step, this
assessment allowed us to confirm that all samples presented
a quality that fulfilled the minimal requirements for sperm
cryopreservation, namely .80% spermatozoa exhibiting an
intact plasma membrane (SYBR14 positive and propidium
iodide (PI) negative), .85% morphologically normal spermatozoa and .80% total motile spermatozoa (Yeste et al. 2013a).
After evaluating these sperm parameters, semen samples
were stored at 178C for 20 h, because sperm survival following
freezethawing is better when the spermatozoa are held at 178C
for 24 h after collection (Yeste et al. 2014b). At the time of
cryopreservation, the semen volume pooled from the doses was
split into two fractions of equal volume. Both fractions were
frozen; the FT-GSH fraction was frozen using the cryopreservation extenders (LEY and LEYGO) supplemented with 2 mM
GSH, whereas the FT-C fraction served as a control and was
frozen using LEY and LEYGO only. Samples were frozen and
thawed following the protocol described below, and acrosin
activity, sperm motility and plasma and acrosome integrity
were evaluated 30 and 240 min after thawing. The 29 ejaculates
were classified into two groups (GFE and PFE) based on sperm
membrane integrity (SYBR14/PI test) and motility data from
controls (FT-C) evaluated at 30 and 240 min after thawing. This
allowed us to later determine the effects of supplementing the
cryopreservation extenders with 2 mM GSH on acrosin activity
in GFE and PFE.
Following on from the results of the first experiment, the
second experiment was performed to determine whether acrosin
activity was positively correlated with reproductive performance of frozenthawed boar spermatozoa. To this end, 13
GFE from boars of proven fertility were collected and diluted
1 : 2 (v/v) using the same commercial extender (Androstar Plus;
Minitub Iberica). Then, a fraction of these prediluted ejaculates
was diluted again with Androstar Plus and used to inseminate
sows (inseminations with extended semen; 3  109 spermatozoa
per dose, 60 mL doses). The remaining volume was packed with
plastic bags and transported to our laboratory within 4 h of
collection. Again, samples were kept at 178C for an additional
20 h. As in the case of the first experiment, acrosin activity,
sperm membrane integrity, morphology, motility and concentration were evaluated to check that the samples met the
minimal sperm quality requirements for sperm cryopreservation
(i.e. 80% SYBR14/PI spermatozoa, 85% morphologically

Acrosin activity, GSH and cryopreserved boar spermatozoa

normal spermatozoa and 80% total motile spermatozoa). Ejaculates were split into two fractions of the same volume, and
the cryopreservation extenders of one of these fractions were
supplemented with 2 mM GSH. Finally, both fractions were
cryopreserved following the protocol described below. Samples
were stored in liquid nitrogen for 23 months and all straws were
then thawed and used either for determination of sperm parameters 30 and 240 min after thawing or for AI.
Cryopreservation of boar spermatozoa
Boar spermatozoa were cryopreserved using the Westendorf
method (Westendorf et al. 1975) as modified by Yeste et al.
(2013a). As mentioned above, in both experiments, sperm samples were cryopreserved after a holding time of 24 h at 178C.
Then, samples were centrifuged at 600g for 5 min at 178C (JP
Selecta, Barcelona, Spain) and the pellets resuspended in LEY
medium made up of 80% (v/v) 310 mM b-lactose (SigmaAldrich, St Louis, MO, USA), 20% (v/v) egg yolk and 100 mg
mL1 kanamycin sulfate (Gibco, Life Technologies, Carlsbad,
CA, USA). The final concentration of spermatozoa in the LEY
medium was adjusted in a Neubauer chamber (Paul Marienfeld,
Lauda-Konigshofen, Germany) to 1.5  109 spermatozoa mL1.
Spermatozoa were subsequently cooled to 58C over 120 min
using a programmable freezer (Icecube14S-B; Minitub, Tiefenbach, Germany) at a rate of 0.18C min1. Then, samples
were rediluted to 1  109 spermatozoa mL1 in LEY medium
containing 2% glycerol (Sigma-Aldrich) and 0.5% Orvus ES
Paste (OEP; Equex STM; Nova Chemical Sales, Scituate, MA,
USA). For aliquots of FT-GSH, 2 mM GSH (C10H17N3O6S;
Sigma-Aldrich) was added to both LEY and LEYGO media.
Sperm samples were finally packed in 0.5-mL plastic straws
(Minitub) previously labelled with the following information:
treatment (either FT-C or FT-GSH), boar, collection date,
freezing date and ejaculate code. Straws were subsequently
cooled to 58C before sperm packing. Next, sperm straws were
transferred to a programmable freezer (Icecube14S-B; Minitub)
and SY-LABORATORY software (Minitub) was used to run a
cryopreservation program specific for boar spermatozoa. This
program, which lasted for 5 min and 13 s, consisted of the following cooling steps and rates: first, sperm samples were cooled
from 58C to 58C over 100 s at a cooling rate of 68C min1. The
cooling rate was then changed to 39.828C min1 for 113 s and
the temperature decreased from 58C to 808C. This was followed by a holding step (i.e. no temperature changes) at 808C
for 30 s. Finally, the temperature was decreased at a rate of
608C min1 for 70 s from 808C to 1508C. Following this,
straws were immediately plunged into liquid nitrogen (1968C)
and stored for 23 months.
When used to evaluate acrosin activity, sperm motility and
the integrity of plasma and acrosome membranes (i.e. first
experiment), as well as determination of frozenthawed sperm
quality (second experiment), four straws per ejaculate and
treatment (FT-C or FT-GSH) were thawed at 378C for 20 s
(Casas et al. 2012) and immediately diluted with three volumes
of warmed Androstar Plus at 378C to obtain a final dilution of
1 : 4. Sperm parameters were determined 30 and 240 min after
thawing.

Reproduction, Fertility and Development

When used for AI in the second experiment, six straws per

dose, boar and treatment (FT-C or FT-GSH) were thawed at
378C for 20 s and diluted with three volumes of previously
warmed Androstar Plus at 378C (final dilution 1 : 4). Then,
48 mL Androstar Plus was added to the diluted frozenthawed
spermatozoa to reach a final volume of 60 mL (i.e. 3  109
spermatozoa per dose in 60-mL doses, the same concentration
and volume as for extended semen).
Evaluation of sperm concentration and morphology
Sperm concentration was assessed when semen samples were
received in the laboratory and during cryopreservation steps
(dilution in LEY, dilution in LEYGO and thawing). In all
cases, sperm concentration was determined in triplicate using
a haemocytometer (Neubauer chamber; Paul Marienfeld). To
this end, samples were previously diluted with 4% formalinbuffered solution and the sperm count adjusted for the dilution
factor.
Sperm morphology was evaluated in both experiments only
upon arrival of semen samples at the laboratory. The aim of
this assessment was to confirm that the percentage of morphologically normal spermatozoa was .85%, because this was the
threshold set for samples to be considered as having standard
sperm morphology. Briefly, spermatozoa were fixed with 4%
paraformaldehyde (Sigma-Aldrich) and three replicates (100
sperm counted per replicate) per sample were evaluated.
Counts were made after 5 mL of each fixed sperm sample was
placed on a slide and covered with a coverslip. Samples were
evaluated under a phase contrast microscope (BX41; Olympus,
Hamburg, Germany) at 200 magnification (20  0.40 PLAN
objective lens, positive phase contrast field; Olympus). Each
spermatozoon was classified as either morphologically normal,
with cytoplasmic droplets or aberrant (coiled tails, tails folded at
the connecting piece, at the intermediate piece or at Jensens
ring) according to Yeste et al. (2008a). The mean  s.e.m.
number of spermatozoa in each category was calculated for the
three replicates.
Evaluation of plasma and acrosome membrane integrity
Sperm membrane integrity was evaluated before and after
freezethawing using SYBR14/PI and the fluorescein
isothiocyanate-conjugated lectin peanut agglutinin (PNAFITC)/PI tests (Garner and Johnson 1995; Nagy et al. 2003)
in a flow cytometer (Cell Laboratory QuantaSC cytometer;
Beckman Coulter, Fullerton, CA, USA). Prior to any
assessment, the sperm concentration was adjusted to 1  106
spermatozoa mL1 in a final volume of 0.5 mL. The electronic
volume (EV; equivalent to forward scatter) channel was periodically calibrated using 10-mm Flow-Check fluorospheres
(Beckman Coulter). Each assessment for each sample and
parameter was repeated three times in independent tubes before
calculating corresponding mean  s.e.m. values.
Two filters of the flow cytometer were used to detect
SYBR14 and PNAFITC (green) fluorescence (FL-1, dichroic/
splitter; Dichroic Long Pass (DRLP), 550 nm; band pass filter,
525 nm; detection width 505545 nm) and PI (red) fluorescence (FL-3, long pass filter, 670/30 nm). Samples were

Reproduction, Fertility and Development

excited with an argon ion laser (488 nm) set at a power of

22 mW. Sheath flow rate was set at 4.17 mL min1. EV and side
scatter (SS) were recorded in linear mode (EV vs SS dot plots)
for a minimum of 10 000 events per replicate. Sperm-specific
events were gated on EVSS distributions and the EV channel
was adjusted to exclude subcellular debris (particle diameter
,7 mm) and cell aggregates (particle diameter .12 mm).
For the SYBR14/PI test, spermatozoa were stained using the
LIVE/DEAD Sperm Viability kit (SYBR14/PI; Molecular Probes,
Eugene, OR, USA). Sperm samples were incubated at 37.58C for
10 min with 100 nM SYBR14. Then, samples were stained
with 12 mM PI for 5 min at 37.58C. Viable spermatozoa
(SYBR14/PI) were distinguished from non-viable spermatozoa
(SYBR14/PI and SYBR14/PI) and non-DNA-containing
particles (debris; SYBR14/PI). Spillover of green fluorescence
(SYBR14) was compensated into the red (FL-3) channel (2.45%).
The integrity of the acrosome membrane was evaluated using
the PNA-FITC/PI test. To this end, spermatozoa were stained with
2.5 mg mL1 PNA-FITC and 12 mM PI before being incubated
at 37.58C for 10 min. PNA-FITC fluorescence was collected
through FL-1 and PI fluorescence was detected through FL-3.
Because spermatozoa were not previously permeabilised, the
four following populations were identified: (1) spermatozoa with
an intact plasma membrane (PNA-FITC/PI); (2) spermatozoa
with a damaged plasma membrane that had an outer acrosome
membrane that was not fully intact (PNA-FITC/PI); (3) spermatozoa with a damaged plasma membrane and lost outer
acrosome membrane (PNA-FITC/PI); and (4) spermatozoa
with a damaged outer acrosome membrane (PNA-FITC/PI).
Unstained and single-stained samples were used to set the
EV gain, FL-1 and FL-3 photo-multiplier tube (PMT) voltages
and for compensation of PNA-FITC spillover into the PI
channel (2.45%).
It is worth noting that in the case of PNA-FITC/PI staining,
data were corrected following Petrunkina et al. (2010), who
proposed a method to determine the percentages of non-DNAcontaining particles (alien particles) in the first quadrant (q1). To
this end, 5 mL each sperm sample was diluted with 895 mL
MilliQ distilled water. These diluted sperm samples were
stained with 12 mM PI and incubated at 37.58C for 3 min.
Following this, the percentage of alien particles ( f ) was used
to correct the percentages of non-stained spermatozoa (q1) in
each sample evaluated through PNA-FITC/PI, using the following formula:
q01

q1  f
 100
100  f

E. Estrada et al.

Olympus) and captured 25 consecutive digitalised photographic

images per field over a period of 1 s. Aliquots (15 mL) of each
sperm sample, previously incubated at 378C for 15 min, were
placed onto a prewarmed Neubauer chamber (Paul Marienfeld)
at 378C. Different sperm motility parameters, including total and
progressive sperm motility, were recorded as described previously (Yeste et al. 2008b). Total sperm motility was defined as
the percentage of spermatozoa showing an average path velocity
(VAP) .10 mm s1 and progressive sperm motility was defined
as the percentage of spermatozoa showing VAP .45 mm s1.
Three replicates were assessed per sample, with at least 1000
spermatozoa evaluated per replicate.
Evaluation of acrosin activity
Acrosin activity in extended and frozenthawed sperm samples
was determined according to the method of Langlois et al.
(2005) and modified by Puigmule et al. (2011). In the case of
frozenthawed spermatozoa evaluated at 30 and 240 min after
thawing, samples were first centrifuged for 5 min at 158C and
600g, resuspended in phosphate-buffered saline (PBS) and the
concentration adjusted to 2  107 spermatozoa mL1. In the
case of extended semen, the sperm concentration was also
adjusted to 2  107 spermatozoa mL1. Then, 250 mL sample,
containing 5  106 spermatozoa, was then layered onto 500 mL
of 11% Ficoll (Sigma-Aldrich) in 0.12 M NaCl and 0.025
HEPES (pH 7.5) and subsequently centrifuged at 1000g for
30 min at 158C. This allowed removal of remnants of the
extender or freezing media, even though a previous centrifugation step in the case of frozenthawed spermatozoa was
included to help to remove egg yolk and other debris particles.
Each sperm pellet was resuspended in 1 mL buffer substrate
solution containing 23 mM N-a-benzoyl-DL-arginine 4-nitroanilide hydrochloride (BAPNA) substrate and 0.01% Triton
X-100 in 5.5 mM NaCl and 5.5 mM HEPES (pH 8.0). A 5-mL
aliquot was used to evaluate sperm concentration, whereas the
remaining volume was incubated at room temperature for 3 h.
Acrosin activity was measured as the ability to hydrolyse the
BAPNA substrate, evaluating levels of 4-nitroanilin at 410 nM.
Total acrosin activity per sample was determined in triplicate, as
mIU acrosin per million spermatozoa, according to the formula
of Langlois et al. (2005):
Enzyme activity mIU per 106 spermatozoa

Abssample  Absblank  106

E  Tinc =Vt  total spermatozoa

where q10 is the percentage of non-stained spermatozoa after

correction.

where Abs is absorbance at 410 nM, Tinc is incubation time,

Vt is total volume and the extinction coefficient (E) was
9.9 mM1 cm1.

Evaluation of sperm motility

Sperm motility was evaluated using a computer-aided sperm
analysis (CASA) system (Integrated Sperm Analysis System
V1.0; Proiser; Valencia, Spain) and a phase contrast microscope
(BX41; Olympus). This system used a negative phase contrast
field at 100 magnification (10  0.30 PLAN objective lens;

In vivo fertility trials

As mentioned earlier, the aim of the second experiment was to
determine whether there was a correlation between acrosin
activity and the fertilising ability of frozenthawed boar spermatozoa. To this end, fractions coming from the 13 ejaculates and
cryopreserved with (FT-GSH) or without (FT-C) 2 mM GSH

Acrosin activity, GSH and cryopreserved boar spermatozoa

were used to inseminate a total of 156 multiparous Landrace sows

(each ejaculate plus treatment was used to inseminate six sows;
78 FT-C and 78 FT-GSH). All fertility trials were conducted
on a breeding farm (Swine Genetic Services, Sant Sadurn
dOsormort, Barcelona, Spain). All sows were housed in climatecontrolled buildings, provided with water ad libitum and fed with
an adjusted diet. Inseminations were repeated every 2 months up
to three times, according to all-in/all-out production system followed by the farm. Sixty sows were inseminated the first time,
whereas 48 sows were inseminated the second and third times.
Detection of oestrus was monitored from 2 days after weaning by inspection of the vulva for reddening and swelling and
response to a male teaser. Oestrus was confirmed 4 and 5 days
after weaning by checking for the presence of the standing
reflex. All AI were performed using the post-cervical technique
(Watson and Behan 2002) and a Magaplus S catheter (Magapor,
Zaragoza, Spain) and each sow was inseminated twice, with
a 12-h interval between inseminations. This allowed us to
cover the insemination-to-ovulation interval recommended for
frozenthawed boar spermatozoa (Waberski et al. 1994; Casas
et al. 2010). Each sow was inseminated using two doses of six
straws each (0.5  109 spermatozoa per straw, 3  109 spermatozoa per dose) from the same ejaculate. These straws were
thawed for 20 s at 378C and diluted with three volumes of
Androstar Plus extender, previously warmed to 378C. To this
final volume of 12 mL (3 mL spermatozoa and 9 mL extender),
48 mL of the same commercial extender was added to a final
volume of 60 mL.
Pregnancy rates (PR) were evaluated 30 days after AI using
ultrasound (Echoscan T-100; Import-vet SA, Barcelona, Spain).
Farrowing rates (FR) and litter sizes (total number of piglets
born (TP), number of live-born piglets (AP) and number of
stillborn piglets (SP)) were recorded at the time of parturition.
Statistical analyses
Statistical analyses were performed using IBM SPSS 21.0 for
Windows (IBM, Chicago, IL, USA). Each combination of
ejaculate and treatment (control or GSH) was considered as an
independent statistical case.
All data were first tested for normality (ShapiroWilk test)
and homogeneity of variances (Levene test) and, when required,
transformed using logarithmic transformation (log x), square
root (Ox) or arcsine square root (arcsin Ox) to make them match
the parametric assumptions.
Experiment 1: effects of 2 mM GSH on acrosin activity
of GFE and PFE
Sperm motility and membrane integrity (SYBR14/PI test)
evaluated in FT-C samples at 30 and 240 min after thawing
were used to classify ejaculates as GFE or PFE. This was
performed through hierarchical cluster analyses for dissimilarities following the procedure described by Yeste et al.
(2014a). The thresholds to separate GFE and PFE groups were
45% for viable spermatozoa and 35% for progressive motile
spermatozoa at 30 min after thawing, and 30% for viable
spermatozoa and 20% for progressive motile spermatozoa at
240 min after thawing.

Reproduction, Fertility and Development

After classifying the ejaculates as GFE and PFE, the effects

of supplementing the freezing media with 2 mM GSH on acrosin
activity and other sperm parameters in GFE and PFE throughout
the different cryopreservation steps (extended semen and in
samples 30 and 240 min after thawing (FT30 and FT240, respectively)) were evaluated through a linear mixed-model followed
by Sidaks post hoc test. In this model, the cryopreservation step
(extended, FT30 and FT240) was the intrasubject factor, the
ejaculate was the random effects factor and the treatment
(control or GSH) and the freezability group (GFE or PFE) were
the fixed effects factors. All sperm parameters were considered
as dependent variables.
Experiment 2: GSH, acrosin activity in frozenthawed
spermatozoa and reproductive performance
PR and FR were first transformed using logistic (logit) transformation and the resulting log-odds were used in subsequent
analyses. PR, FR and litter sizes were compared between the
extended, FT-C and FT-GSH groups using a general linear
model with treatment as fixed effects and ejaculate as random
effects factors. This was followed by Sidaks post hoc test for
multiple comparisons. Correlations between reproductive performance parameters using frozenthawed spermatozoa, with or
without 2 mM GSH supplementation (logit PR, logit FR, TP, AP
and SP) and acrosin activity were calculated using Pearson
correlation, as reported by Estrada et al. (2014).
The percentage of variance explained by sperm parameters
(SYBR14/PI and PNA-FITC/PI tests, sperm motility and acrosin
activity) was determined through two factorial analyses that were
run using the values obtained for these parameters after 30 and
240 min incubation at 378C. In each analysis, these parameters
were sorted into some components extracted by principal component analysis (PCA) and the data matrix obtained was rotated
using the Varimax procedure with Kaiser normalisation. Only
those variables with a positive loading factor (aij) .0.6 with its
respective component and ,0.3 with respect to the other components in the rotated matrix were selected from the linear combination of j variables (x) in each component yi (yi ai1x1 ai2x2
y aijxj), for analyses of both 30 and 240 min incubation.
Following this, linear regression analyses were used to
determine the ability of the extracted principal components
from the PCA to predict PR and FR, TP, AP and SP. The
procedure used was the forward stepwise model, as described in
Yeste et al. (2010), and consisted of optimising the regression
equation (y a b1x1 b2x2; k # 2) to increase the determination coefficient (R2). The significance level for introducing each
parameter in the multiple regression model was 0.10 and the
significance level (a) for the model was 0.05.
Data are presented as the mean  s.e.m. and the minimal
level of significance in all cases was set at two-tailed P , 0.05.
Results
Experiment 1: effects of GSH supplementation
on acrosin activity, sperm membrane integrity
and motility of GFE and PFE
Fig. 1 shows acrosin activity before and after freezethawing
of GFE and PFE with or without GSH supplementation.

Acrosin activity (IU acrosin per 106 spz)

Reproduction, Fertility and Development

E. Estrada et al.

120
110

1, a

100
90
80
70

1, b

60
50
2, a

40
30
20
10
0

2, b
GFE
GFE GSH
PFE
PFE GSH
Extended

2, c
2, c
FT30

2, a
2, b
2, c
2, c
FT240

Cryopreservation step
Fig. 1. Acrosin activity in good and poor freezability ejaculates (GFE and
PFE, respectively), with or without 2 mM reduced glutathione (GSH)
supplementation, before cryopreservation and 30 and 240 min after
freezethawing (FT30 and FT240, respectively). Data are the mean  s.e.
m. Different numbers (1, 2) indicate significant differences (P , 0.05)
between cryopreservation steps within a given freezability group and
treatment (i.e. GFE, GFEGSH, PFE and PFEGSH), whereas different
letters (a, b, c) indicate significant differences (P , 0.05) between freezability groups and treatments within a given cryopreservation step.

Table 1. Percentage of SYBR14-positive and propidium iodide

(PI)-negative spermatozoa, fluorescein isothiocyanate-conjugated lectin
peanut agglutinin (PNA-FITC)-negative and PI-negative spermatozoa,
total motile spermatozoa and progressive motile spermatozoa in good
and poor freezability ejaculates (GFE and PFE, respectively), with or
without 2 mM reduced glutathione (GSH) supplementation, before
(extended) and 30 and 240 min after freezethawing (FT30 and FT240,
respectively)
Data are the mean  s.e.m. Within columns (cryopreservation steps
within a given freezability group and treatment), values with different
superscript uppercase letters differ significantly (P , 0.05). Within rows
(freezability groups and treatments within a given cryopreservation
step), values with different superscript lowercase letters differ
significantly (P , 0.05)
Freezability
group
GFE
PFE

GFE
PFE

Before starting cryopreservation protocols and after freeze

thawing, acrosin activity was significantly (P , 0.05) higher for
GFE than PFE. Addition, acrosin activity decreased significantly in both the GFE and PFE groups after freezethawing,
with no significant (P . 0.05) differences observed between the
two post-thawing incubation times (i.e. 30 and 240 min). With
regard to treatments, although supplementing freezing extenders with GSH had a positive effect on acrosin activity of GFE at
30 and 240 min after thawing, it had no effect on acrosin activity
of PFE.
The percentages of SYBR14/PI spermatozoa for all treatment and freezability groups and cryopreservation steps are
given in Table 1. The percentage of SYBR14/PI spermatozoa
did not differ significantly between GFE and PFE (P . 0.05)
before starting cryopreservation procedures. A significant
reduction in the percentage of SYBR14/PI spermatozoa
was observed in all groups and treatments after freezethawing.
However, the percentage of SYBR14/PI spermatozoa was
significantly (P , 0.05) higher in GFE than PFE, and in the FTGSH than FT-C group. At 240 min after thawing, the percentage
of viable spermatozoa in the GFE and PFE with GSH supplementation was 48.1%  1.9% and 31.3%  1.1%, respectively.
The percentages of acrosome-intact spermatozoa (PNAFITC/PI) before and after freezethawing in GFE and PFE,
with or without 2 mM GSH supplementation are given in Table 1.
There was a significant (P , 0.05) decrease in the percentage
of PNA-FITC/PI spermatozoa after freezethawing, with
GFE having a higher percentage of PNA-FITC/PI spermatozoa than PFE. As for the SYBR14/PI test, the percentage of
PNA-FITC/PI spermatozoa at 30 and 240 min after thawing
was significantly higher for both GFE and PFE after addition of
2 mM GSH to the cryopreservation extenders.

GFE
PFE

GFE
PFE

Treatment

Extended

FT30

% SYBR14/PI spermatozoa
51.9  2.1Ba
Control
90.1  3.4Aa
GSH
90.1  3.4Aa
60.4  2.5Bb
Aa
Control
89.4  3.5
30.5  1.2Bc
GSH
89.4  3.5Aa
40.1  1.5Bd


% PNA-FITC /PI spermatozoa
Control
86.3  3.5Aa
48.5  2.2Ba
Aa
GSH
86.3  3.5
56.8  2.6Bb
Control
85.1  3.6Aa
27.6  1.4Bc
Aa
GSH
85.1  3.6
37.0  1.7Bd
% Total motile spermatozoa
Control
89.5  4.5Aa
48.9  2.7Ba
GSH
89.5  4.5Aa
60.6  3.1Bb
Aa
Control
88.3  4.6
28.5  1.7Bc
GSH
88.3  4.6Aa
39.4  2.0Bd
% Progessive motile spermatozoa
39.2  2.1Ba
Control
61.7  3.2Aa
Aa
GSH
61.7  3.2
49.8  2.5Bb
Control
59.8  3.3Aa
23.9  1.3Bc
Aa
GSH
59.8  3.3
32.8  1.8Bd

FT240

37.0  1.4Ca
48.1  1.9Cb
20.6  0.8Cc
31.3  1.1Cd
34.1  1.7Ca
45.0  2.1Cb
17.5  1.0Cc
28.2  1.4Cd
32.7  1.8Ca
45.6  2.4Cb
18.9  1.1Cc
28.7  1.6Ca
25.9  1.4Ca
36.4  2.0Cb
13.7  0.9Cc
22.5  1.2Ca

The percentages of total and progressive motile spermatozoa

before and after freezethawing are given in Table 1. In both
cases, and similar to results of the SYBR14/PI and PNA-FITC/PI
tests, there was a significant (P , 0.05) decrease in the percentages of total and progressive motile spermatozoa after freeze
thawing, with this decrease being more pronounced at 240 min
after thawing. Again, the reduction in total and progressive
sperm motility was more apparent in PFE than GFE. Supplementation of freezing extenders with GSH partially counteracted the cryopreservation-induced decrease in both total and
progressive sperm motility.
Experiment 2: acrosin activity in frozenthawed
spermatozoa and reproductive performance
PR, FR, TP and LP were significantly (P , 0.05) higher in
extended and FT-GSH groups than in the FT-C group (Table 2).
Table 3 gives correlation coefficients between acrosin activity of frozenthawed spermatozoa at 30 and 240 min after
thawing and reproductive performance parameters after AI.
Although no significant correlation was found between PR

Acrosin activity, GSH and cryopreserved boar spermatozoa

Reproduction, Fertility and Development

Table 2. Fertility performance of sows inseminated with extended,

frozenthawed control (FT-C) or frozenthawed semen supplemented
with 2 mM reduced glutathione (FT-GSH)
Data are the mean  s.e.m. *P , 0.05 compared with extended semen

Pregnancy rate at 30 days (%)

Farrowing rate
Total piglets born (n)
Live born piglets (n)
Stillborn piglets (n)

Extended

FT-C

FT-GSH

92.0  3.9
90.1  3.9
13.1  0.9
12.4  0.8
0.7  0.2

73.6  3.0*
73.6  3.0*
8.0  1.3*
7.2  1.0*
0.8  0.2

90.9  3.8
89.3  3.6
12.6  1.0
11.9  1.1
0.7  0.3

Table 3. Correlation coefficients between acrosin activity evaluated at

30 and 240 min after thawing (FT30 and FT240, respectively) and
reproductive performance
*P , 0.05. PR30d, pregnancy rates 30 days after AI, FR, farrowing rates
Acrosin activity (mIU acrosin activity per 106
spermatozoa)

Logit (PR30d)
Logit (FR)
Total piglets born (n)
Alive born piglets (n)
Stillborn piglets (n)

FT30

FT240

0.29
0.27
0.37
0.34
0.12

0.41
0.40
0.53*
0.50*
0.16

Table 4. Principal component analyses run using acrosin activity data

and other sperm parameters evaluated 30 and 240 min after thawing
PI, propidium iodide; PNA-FITC, fluorescein isothiocyanate-conjugated
lectin peanut agglutinin; a2ij, loading factor square
Variance
explained
(%)
30 min after thawing
Component 1

Component 2
Total
240 min after thawing
Component 1

Dependent variable

a2ij

Table 5. Correlations between reproductive performance parameters

and principal component analyses components at 30 and 240 min after
thawing (FT30 and FT240, respectively)
*P , 0.05, **P , 0.01. PR30d, pregnancy rates 30 days after AI; FR,
farrowing rates
FT240

FT30

1st
2nd
1st
2nd
component component component component
Logit (PR30d)
Logit (FR)
Total piglets born (n)
Alive born piglets (n)
Stillborn piglets (n)

0.48*
0.45*
0.45*
0.47*
0.23

0.32
0.36
0.10
0.09
0.02

0.52*
0.55*
0.65**
0.63**
0.21

0.35
0.39
0.32
0.35
0.09

240 min after thawing, two components were found to explain

more than 80% of the variance. It is worth noting that acrosin
activity was found in the first component, together with the
integrity of the plasma (SYBR14/PI) and acrosome (PNAFITC/PI) membranes.
Table 5 gives correlation coefficients between reproductive
performance parameters and the two components obtained in the
PCA at 30 and 240 min after thawing. Although no correlation
between PCA components and reproductive performance parameters was found at 30 min after thawing, PR, FR, TP and LP
were significantly and positively correlated with the first component of PCA analysis (in which acrosin activity was included)
at 240 min after thawing.
Finally, Table 6 provides regression equations worked out to
determine whether PCA components from 30 and 240 min after
thawing could predict PR, FR and litter sizes. In both cases, the
PCA components evaluated at 30 and 240 min after thawing were
able to predict the PR, FR, TP and LP. PCA components obtained
either at 30 or 240 min after thawing failed to predict SP.
Discussion

62.66

20.23

% SYBR14 /PI
% PNA-FITC/PI
Acrosin activity
% Total sperm motility
% Progressive sperm motility

0.81
0.85
0.72
0.94
0.97

% SYBR14/PI
% PNA-FITC/PI
Acrosin activity
% Total sperm motility
% Progressive sperm motility

0.90
0.89
0.85
0.92
0.98

82.89
73.43

Component 2

12.04

Total

85.47

and acrosin activity, TP and LP were significantly correlated

(P , 0.05) with acrosin activity at 240 min after thawing.
Moreover, there was no significant correlation between SP
and acrosin activity at either 30 or 240 min after thawing.
Table 4 lists results of PCA using the sperm parameters
evaluated at 30 and 240 min after thawing. At both 30 and

The aim of the present study was to provide new insights into
acrosin activity in frozenthawed boar spermatozoa. In the first
experiment, supplementing freezing extenders (LEY and
LEYGO) with 2 mM GSH increased acrosin activity in GFE but
not PFE, whereas in the second experiment acrosin activity was
found to be positively correlated with TP and LP.
Despite its advantages, boar sperm cryopreservation is still
regarded as suboptimal because the reproductive performance
of frozenthawed boar spermatozoa is lower than that of
extended semen (Holt 2000; Rodrguez-Gil and Estrada 2013).
In this context, supplementing freezing media with antioxidants
has been shown to be a reliable approach, with GSH partially
counteracting sperm cryodamage (Yeste et al. 2013a). Indeed,
the addition of GSH to freezing and/or thawed media increases
sperm motility and viability, as well as fertilisation outcomes
following IVF and AI (Gadea et al. 2004; Estrada et al. 2014).
This appears to be related to the role of GSH, which stabilises the
whole nucleoprotein structure of the boar sperm head during
cryopreservation procedures (Yeste et al. 2013a; Estrada et al.
2014). However, the GSH-induced improvement in acrosin

Reproduction, Fertility and Development

E. Estrada et al.

Table 6. Regression equations between reproductive performance parameters and principal component analyses components at 30 and 240 min
after thawing
PR30d, pregnancy rates 30 days after AI; FR, farrowing rates; TP, total piglets born; LP, piglets born alive
Regression equation
30 min after thawing
Logit(PR30d) 1.53(1st Component) 1.29(2nd Component) 1.91
Logit(FR) 1.49(1st Component) 1.22(2nd Component) 2.08
TP 0.60(1st Component) 2.02(2nd Component) 10.24
LP 0.47(1st Component) 2.03(2nd Component) 9.76
240 min after thawing
Logit(PR30d) 2.09(1st Component) 1.80(2nd Component) 1.78
Logit(FR) 2.18(1st Component) 1.75(2nd Component) 1.69
TP 4.47(1st Component) 1.86(2nd Component) 9.87
LP 3.97(1st Component) 1.91(2nd Component) 9.34

activity could also be an important factor explaining the beneficial effects of supplementing freezing media with GSH.
Recently, Pinart et al. (2015) found that acrosin activity is a
good freezability maker for boar spermatozoa because GFE
have significantly higher acrosin activity than PFE before
starting cryopreservation procedures. However, although previous studies showed that acrosin activity in boar extended semen
is correlated with reproductive performance (PR and litter sizes;
Pinart et al. 2013), no study has determined whether this is also
true for frozenthawed boar spermatozoa. For this reason, two
separate experiments were conducted in the present study with
the aim of addressing these two issues.
First, we tested whether supplementing freezing media (LEY
and LEYGO) with 2 mM GSH affected acrosin activity of GFE
and PFE after thawing. Following freezethawing, there was a
marked reduction in acrosin activity in both GFE and PFE,
which was in agreement with the results of Pinart et al. (2015). In
fact, sperm cryopreservation is also known to significantly
reduce acrosin activity in other mammals, such as humans
(Cross and Hanks 1991), bulls (Paudel et al. 2010) and bucks
(Sirat et al. 1996). However, the decrease in acrosin activity
following freezethawing has been interpreted in a different
manner by authors conducting their work in different species.
Indeed, whereas in humans, bucks, dogs and boars, the loss of
acrosome integrity has been suggested to underlie the decrease
in acrosin activity (Froman et al. 1984; Mack and Zaneveld
1987; Cross and Hanks 1991; Sirat et al. 1996; Pinart et al.
2015), studies conducted in bucks indicate that higher acrosome
integrity is related to lower acrosin activity (Chelucci et al.
2015). Although whether higher acrosin activity is related to
either a high or low percentage of spermatozoa exhibiting an
exocytosed acrosome needs to be determined, the results of the
present study match those of studies conducted in humans and
boars, because sperm cryopreservation was seen to reduce both
acrosin activity and acrosome membrane integrity.
The present study demonstrated, for the first time, that the
presence of 2 mM GSH in the freezing media has a significant
effect on acrosin activity in GFE but not PFE. Previous studies
have shown that the effects of adding GSH to freezing extenders
rely on the intrinsic freezability of the ejaculate (Yeste et al.
2014a). This could explain why the positive effect was only

R2

P-value

0.20
0.17
0.13
0.25

.0.05
.0.05
.0.05
.0.05

0.45
0.48
0.66
0.60

,0.05
,0.05
,0.001
,0.001

observed in GFE. Moreover, it is worth noting that GSH was

found to be beneficial not only for acrosin activity, but also for
overall sperm function and survival at after thawing. This
finding, apart from agreeing with previous studies from our
group (Yeste et al. 2013a; Estrada et al. 2014; Giaretta et al.
2015), suggests that rather than a specific effect of 2 mM GSH
on acrosin activity, the presence of this antioxidant benefits
general sperm function and integrity during cryopreservation
via an as yet unidentified mechanism. Therefore, the addition of
2 mM GSH to freezing extenders appears to counteract the
decrease in acrosin activity after freezethawing because it
reduces the damage to the acrosome and plasma membranes.
Because membrane damage, especially in the case of the
acrosome, is thought to be the main cause of the decrease in
acrosin activity (Cross and Hanks 1991), this could be the more
reasonable explanation for effects of GSH. Indeed, proacrosin is
activated into acrosin after spermoocyte interaction and integrity of the acrosome membrane is required for the activity of this
enzyme to be maintained (Topfer-Petersen and Cechova 1990;
Lo Leggio et al. 1994; Rosatti et al. 2004; Puigmule et al. 2011).
Although previous studies showed that the decrease in acrosin
activity observed at the cooling step is primarily related to
enzyme deterioration rather than acrosome exocytosis (Pinart
et al. 2015), the present study suggests that GSH increases sperm
survival and membrane integrity following freezethawing rather
than protecting acrosin from deterioration. In fact, the increase in
acrosin activity mediated by GSH is lower than that observed for
plasma and acrosome membrane integrity. Conversely, given that
the main decrease in acrosin activity occurs during cooling from
158C to 48C with no major changes from 48C to FT30 (Pinart et al.
2015) and that our GSH treatment consisted of supplementing
both LEY (used to cool sperm samples from 158C to 48C) and
LEYGO, it is quite unlikely that the addition of GSH at the
cooling step has a major effect on acrosin activity.
The main conclusion of the second experiment is that
acrosin activity is positively correlated with reproductive
performance. Because GSH had no effect on acrosin activity
in PFE after sperm evaluation at post thawing, the second
experiment only involved GFE. Acrosin activity in boar
extended semen has been found to be positively correlated
with FR and litter sizes (Pinart et al. 2013). In the present study,

Acrosin activity, GSH and cryopreserved boar spermatozoa

acrosin activity was significantly correlated with TP and

LP. Nonetheless, higher correlation coefficients were seen
when the evaluation of acrosin activity was performed at
240 min after thawing, thereby indicating that those ejaculates
that were more resistant to cryodamage were also those that
exhibited higher reproductive performance. In contrast with
our data, other studies have found that acrosin activity in boar
frozenthawed spermatozoa is not correlated with penetration
rates following incubation with hamster oocytes (Hammitt and
Martin 1989). Related to this, it is worth mentioning that
although Berger et al. (1996) could not demonstrate any
association between in vivo fertility and spermzona pellucida
binding capacity, they did find that the ability of spermatozoa
to fuse with the oolemma of zona-free hamster oocytes was
correlated with in vivo fertility. This indicates that the results of
heterologous penetration tests should be interpreted cautiously
in the case of boar spermatozoa.
Previous studies from our group showed that supplementing
the freezing media with 2 mM GSH improved the in vivo
fertilising ability of boar spermatozoa subjected to freeze
thawing (Estrada et al. 2014). In the present study, we observed
that GSH improved acrosin activity following freezethawing
and that this is concomitant with higher sperm survival. Indeed,
acrosin activity was found in the first component from PCA
together with the percentage of spermatozoa with intact plasma
and acrosome membranes. This first component at 240 min after
thawing was found to be correlated with PR, FR, TP and LP.
Although this component also allowed prediction of PR, FR and
litter sizes when used in regression equations, it is less clear that
acrosin activity had a strong effect on reproductive performance, because the effect of GSH supplementation on acrosin
activity, even thought it was significant, was marginal and no
apparent correlation between acrosin activity and PR and FR
was observed. Therefore, it seems that the increase in fertility
mediated by the addition of GSH to frozenthawed boar
spermatozoa relies not only on acrosin activity, but also on
other sperm parameters.
The present study is the first to evaluate acrosin activity in
frozenthawed boar spermatozoa after the addition of GSH to
freezing extenders. Although no previous study has addressed
this issue in cryopreserved spermatozoa from other mammalian
species, different studies have evaluated the effects of other
antioxidants. For example, Paudel et al. (2010) showed that
adding ascorbic acid, catalase and chlorpromazine to cryopreservation media not only reduced sperm cryodamage in bulls, but
also better maintained their acrosin activity. In humans, Rao
et al. (2015) recently found that testicular hyperthermia increases
oxidative stress and decreases acrosin activity. Although that
study does not demonstrate an inverse relationship between
acrosin activity and oxidative stress, it shows that defective
spermatogenesis may negatively affect acrosin activity. In the
case of boars, previous studies have shown that freezethawing
procedures do not significantly affect reactive oxygen species
(ROS) levels (Guthrie and Welch 2006), which did not differ
between GFE and PFE (Yeste et al. 2013b). However, despite
GSH having a marginal effect on ROS following sperm cryopreservation (Yeste et al. 2013a), the effects reported herein
seem to be related to another mechanism because the reduction

Reproduction, Fertility and Development

of this activity following freezethawing depends on the intrinsic freezability of the ejaculate and this is independent of ROS
levels (Yeste et al. 2014a). In this context, it is necessary to
discuss the effects of GSH on ROS levels, acrosin activity and
the integrity of the nucleoprotein structure and how this may
affect reproductive performance. As Estrada et al. (2014) found,
supplementing frozenthawed spermatozoa with GSH leads to
increased reproductive performance, with FR and litter sizes
close to those found using extended semen. However, as
observed herein, the effect of GSH supplementation on acrosin
activity and sperm viability is lower than that observed on the
nucleoprotein structure (Yeste et al. 2013a, 2014a). Related to
this, Rosatti et al. (2004) working with frozenthawed bull
spermatozoa suggested that only a minor proportion of the total
of proacrosinacrosin system is required for acrosome reaction
after capacitation. Therefore, under the present experimental
conditions, either the increase in acrosin activity mediated by
GSH was enough to improve the reproductive performance of
frozenthawed boar semen or the relevance of acrosin activity to
reproductive performance of frozenthawed spermatozoa is
lower than for extended semen.
In conclusion, the present study has demonstrated, for the
first time, that supplementing freezing media with 2 mM GSH
results in better maintenance of acrosin activity in GFE after
cryopreservation. In addition, acrosin activity in frozenthawed
boar spermatozoa is positively correlated with reproductive
performance, but the beneficial effects of GSH seem to be
related not only to acrosin activity, but also to overall sperm
function and survival.
Acknowledgements
This work was supported by grants from the Ministerio de Economa y
Competitividad, Spain (AGL-2008-01792/GAN and JCI-2010-08620,
awarded to JER-G and MY, respectively).

References
Almlid, T., and Hofmo, P. O. (1995). A brief review of frozen semen
application under Norwegian AI service conditions. Reprod. Domest.
Anim. 31, 169173. doi:10.1111/J.1439-0531.1995.TB00021.X
Benson, J. D., Woods, E. J., Walters, E. M., and Critser, J. K. (2012).
The cryobiology of spermatozoa. Theriogenology 78, 16821699.
doi:10.1016/J.THERIOGENOLOGY.2012.06.007
Berger, T., Anderson, D. L., and Penedo, M. C. T. (1996). Porcine sperm
fertilizing potential in relationship to sperm functional capacities. Anim.
Reprod. Sci. 44, 231239. doi:10.1016/0378-4320(96)01565-5
Casas, I., and Flores, E. (2013). Gene banking, the freezing strategy. In
Boar Reproduction: Fundamentals and New Biotechnological Trends.
(Eds S. Bonet, I. Casas, W. V. Holt and M. Yeste.) pp. 551588.
(Springer: Berlin.)
Casas, I., Sancho, S., Briz, M., Pinart, E., Bussalleu, E., Yeste, M., and
Bonet, S. (2009). Freezability prediction of boar ejaculates assessed by
functional sperm parameters and sperm proteins. Theriogenology 72,
930948. doi:10.1016/J.THERIOGENOLOGY.2009.07.001
Casas, I., Sancho, S., Briz, M., Pinart, E., Bussalleu, E., Yeste, M., and Bonet, S.
(2010). Fertility after post-cervical artificial insemination with cryopreserved sperm from boar ejaculates of good and poor freezability. Anim.
Reprod. Sci. 118, 6976. doi:10.1016/J.ANIREPROSCI.2009.06.003
Casas, I., Torner, E., Yeste, M., and Bonet, S. (2012). Boar sperm thawing
practices: the number of straws does matter. Theriogenology 77,
14871494. doi:10.1016/J.THERIOGENOLOGY.2011.10.041

Reproduction, Fertility and Development

Chelucci, S., Pasciu, V., Succu, S., Addis, D., Leoni, G. G., Manca, M. E.,
Naitana, S., and Berlinguer, F. (2015). Soybean lecithin-based extender
preserves spermatozoa membrane integrity and fertilizing potential
during goat semen cryopreservation. Theriogenology 83, 10641074.
doi:10.1016/J.THERIOGENOLOGY.2014.12.012
Cross, N. L., and Hanks, S. E. (1991). Effects of cryopreservation on human
sperm acrosomes. Hum. Reprod. 6, 12791283.
Estrada, E., Rodrguez-Gil, J. E., Rocha, L. G., Balasch, S., Bonet, S., and
Yeste, M. (2014). Supplementing cryopreservation media with reduced
glutathione increases fertility and prolificacy of sows inseminated with
frozenthawed boar semen. Andrology 2, 8899. doi:10.1111/J.20472927.2013.00144.X
Froman, D. P., Amann, R. P., Riek, P. M., and Olar, T. T. (1984). Acrosin
activity of canine spermatozoa as an index of cellular damage. J. Reprod.
Fertil. 70, 301308. doi:10.1530/JRF.0.0700301
Gadea, J., Selles, E., Marco, M. A., Coy, P., Matas, C., Romar, R., and Ruiz, S.
(2004). Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and
thawing extenders. Theriogenology 62, 690701. doi:10.1016/J.THERIO
GENOLOGY.2003.11.013
Gadea, J., Garca-Vazquez, F. A., Matas, C., Gardon, J. C., Canovas, S., and
Gumbao, D. (2005). Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves
sperm function. J. Androl. 26, 396404. doi:10.2164/JANDROL.04155
Garner, D. L., and Johnson, L. A. (1995). Viability assessment of mammalian
sperm using SYBR-14 and propidium iodide. Biol. Reprod. 53, 276284.
doi:10.1095/BIOLREPROD53.2.276
Giaretta, E., Estrada, E., Bucci, D., Spinaci, M., Rodrguez-Gil, J. E., and
Yeste, M. (2015). Combining reduced glutathione and ascorbic acid
has supplementary beneficial effects on boar sperm cryotolerance.
Theriogenology 83, 399407. doi:10.1016/J.THERIOGENOLOGY.
2014.10.002
Guthrie, H. D., and Welch, G. R. (2006). Determination of intracellular
reactive oxygen species and high mitochondrial membrane potential in
Percoll-treated viable boar sperm using fluorescence-activated flow
cytometry. J. Anim. Sci. 84, 20892100. doi:10.2527/JAS.2005-766
Hammitt, D. G., and Martin, P. A. (1989). Correlations among assays of
porcine semen quality following cryopreservation. Theriogenology 32,
369384. doi:10.1016/0093-691X(89)90004-6
Hancock, J. L., and Howell, G. J. L. (1959). The collection of boar semen.
Vet. Rec. 71, 664665.
Holt, W. V. (2000). Basics aspects of frozen storage of semen. Anim. Reprod.
Sci. 62, 322. doi:10.1016/S0378-4320(00)00152-4
Johnson, L. A., Weitze, K. F., Fiser, P., and Maxwell, W. M. C. (2000).
Storage of boar semen. Anim. Reprod. Sci. 62, 143172. doi:10.1016/
S0378-4320(00)00157-3
Knox, R. V. (2011). The current value of frozenthawed boar semen for
commercial companies. Reprod. Domest. Anim. 46(Suppl. 2), 46.
doi:10.1111/J.1439-0531.2011.01822.X
Langlois, M. R., Oorlynck, L., Vanderkerckhove, F., Criel, A., Bernard, D.,
and Blaton, V. (2005). Discrepancy between sperm acrosin activity and
sperm morphology: significance for fertilization in vitro. Clin. Chim.
Acta 351, 121129. doi:10.1016/J.CCCN.2004.08.001
Lo Leggio, L., Williams, R. M., and Jones, R. (1994). Some effects of zona
pellucida glycoproteins and sulfated polymers on the autoactivation of
boar sperm proacrosin and activity of beta-acrosin. J. Reprod. Fertil.
100, 177185. doi:10.1530/JRF.0.1000177
Mack, S. R., and Zaneveld, L. J. (1987). Acrosomal enzymes and ultrastructure of unfrozen and cryotreated human spermatozoa. Gamete Res. 18,
375383. doi:10.1002/MRD.1120180411
Nagy, S., Jansen, J., Topper, E. K., and Gadella, B. M. (2003). A triple-stain
flow cytometric method to assess plasma- and acrosome-membrane
integrity of cryopreserved bovine sperm immediately after thawing

E. Estrada et al.

in presence of egg-yolk particles. Biol. Reprod. 68, 18281835.

doi:10.1095/BIOLREPROD.102.011445
Paudel, K. P., Kumar, S., Meur, S. K., and Kumaresan, A. (2010). Ascorbic
acid, catalase and chlorpromazine reduce cryopreservation-induced
damages to crossbred bull spermatozoa. Reprod. Domest. Anim. 45,
256262. doi:10.1111/J.1439-0531.2008.01278.X
Petrunkina, A. M., Waberski, D., Bollwein, H., and Sieme, H. (2010).
Identifying non-sperm particles during flow cytometric physiological
assessment: a simple approach. Theriogenology 73, 9951000.
doi:10.1016/J.THERIOGENOLOGY.2009.12.006
Pinart, E., Yeste, M., Puigmule, M., Barrera, X., and Bonet, S. (2013).
Acrosin activity is a suitable indicator of boar sperm preservation
at 178C when increasing environmental temperature and radiation.
Theriogenology 80, 234247. doi:10.1016/J.THERIOGENOLOGY.
2013.04.001
Pinart, E., Yeste, M., and Bonet, S. (2015). Acrosin activity is a good
predictor of boar sperm freezability. Theriogenology . doi:10.1016/
J.THERIOGENOLOGY.2015.02.005
Polge, C., Salamon, S., and Wilmut, I. (1970). Fertilizing capacity of frozen
boar semen following surgical insemination. Vet. Rec. 87, 424429.
doi:10.1136/VR.87.15.424
Puigmule, M., Fa`brega, A., Yeste, M., Bonet, S., and Pinart, E. (2011). Study
of the proacrosinacrosin activity system in epididymal, ejaculated and
in vitro capacitated boar spermatozoa. Reprod. Fertil. Dev. 23, 837845.
doi:10.1071/RD10345
Pursel, V. G., and Johnson, L. A. (1975). Freezing of boar spermatozoa:
fertilizing capacity with concentrated semen and a new thawing procedure. J. Anim. Sci. 40, 99102.
Rao, M., Zhao, X. L., Yang, J., Hu, S. F., Lei, H., Xia, W., and Zhu, C. H.
(2015). Effect of transient scrotal hyperthermia on sperm parameters,
seminal plasma biochemical markers, and oxidative stress in men. Asian
J. Androl. 17, 668675. doi:10.4103/1008-682X.146967
Rath, D. (2002). Low dose insemination in the sow: a review. Reprod.
Domest. Anim. 37, 201205. doi:10.1046/J.1439-0531.2002.00377.X
Roca, J., Parrilla, I., Rodriguez-Martinez, H., Gil, M. A., Cuello, C.,
Vazquez, J. M., and Martnez, E. A. (2011). Approaches towards efficient
use of boar semen in the pig industry. Reprod. Domest. Anim. 46, 7983.
doi:10.1111/J.1439-0531.2011.01828.X
Rodrguez-Gil, J. E., and Estrada, E. (2013). Artificial insemination in
boar reproduction. In Boar Reproduction: Fundamentals and New
Biotechnological Trends. (Eds S. Bonet, I. Casas, W. V. Holt and M.
Yeste.) pp. 589608. (Springer: Berlin.)
Rosatti, M. I., Beconi, M. T., and Cordoba, M. (2004). Proacrosin-acrosin
activity in capacitated and acrosome reacted sperm from cryopreserved
bovine semen. Biocell 28, 311316.
Sirat, M. P., Sinha, A. K., Singh, B. K., and Prasad, R. L. (1996). Effect of
cryoprotectants on release of various enzymes from buck spermatozoa
during freezing. Theriogenology 45, 405416. doi:10.1016/0093-691X
(95)00377-K
Thurston, L. M., Watson, P. F., Mileham, A. J., and Holt, W. V. (2001).
Morphologically distinct sperm subpopulations defined by Fourier shape
descriptors in fresh ejaculates correlate with variation in boar semen
quality following cryopreservation. J. Androl. 22, 382394.
Thurston, L. M., Siggins, K., Mileham, A. J., Watson, P. F., and Holt, W. V.
(2002). Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following
cryopreservation. Biol. Reprod. 66, 545554. doi:10.1095/BIOLRE
PROD66.3.545
Topfer-Petersen, E., and Cechova, D. (1990). Zona pellucida induces
conversion of proacrosin to acrosin. Int. J. Androl. 13, 190196.
doi:10.1111/J.1365-2605.1990.TB00976.X
Waberski, D., Weitze, K. F., Gleumes, T., Schwarz, M., Willmen, T., and
Petzoldt, R. (1994). Effect of time of insemination relative to ovulation

Acrosin activity, GSH and cryopreserved boar spermatozoa

on fertility with liquid and frozen boar semen. Theriogenology 42,

831840. doi:10.1016/0093-691X(94)90451-N
Watson, P. F., and Behan, J. R. (2002). Intrauterine insemination of sows
with reduced sperm numbers: results of a commercially based field trial.
Theriogenology 57, 16831693. doi:10.1016/S0093-691X(02)00648-9
Westendorf, P., Richter, L., and Treu, H. (1975). Zur Tiefgefrierung von
Ebersperma Labor und Besamungsergebnisse mit dem Hulsenberger
Pailetten-Verfahren [Deep freezing of boar sperm. Laboratory and
insemination results using the Hulsenberger paillete method]. Dtsch.
Tierarztl. Wochenschr. 82, 261267.
Yeste, M. (2013). Boar spermatozoa within the oviductal environment (III):
Fertilisation. In Boar Reproduction: Fundamentals and New Biotechnological Trends. (Eds S. Bonet, I. Casas, W. V. Holt and M. Yeste.)
pp. 343406. (Springer: Berlin.)
Yeste, M., Briz, M., Pinart, E., Sancho, S., Garcia-Gil, N., Badia, E., Bassols, J.,
Pruneda, A., Bussalleu, E., Casas, I., and Bonet, S. (2008a). Boar
spermatozoa and prostaglandin F2alfa. Quality of boar sperm after addition
of prostaglandin F2alfa to the short-term extender over cooling time. Anim.
Reprod. Sci. 108, 180195. doi:10.1016/J.ANIREPROSCI.2007.08.008
Yeste, M., Briz, M., Pinart, E., Sancho, S., Garcia-Gil, N., Badia, E., Bassols, J.,
Pruneda, A., Bussalleu, E., Casas, I., and Bonet, S. (2008b). Hyaluronic acid
delays boar sperm capacitation after 3 days of storage at 15 degrees C. Anim.
Reprod. Sci. 109, 236250. doi:10.1016/J.ANIREPROSCI.2007.11.003

Reproduction, Fertility and Development

Yeste, M., Briz, M., Pinart, E., Sancho, S., Bussalleu, E., and Bonet, S.
(2010). The osmotic tolerance of boar spermatozoa and its usefulness as
sperm quality parameter. Anim. Reprod. Sci. 119, 265274. doi:10.1016/
J.ANIREPROSCI.2010.02.011
Yeste, M., Flores, E., Estrada, E., Bonet, S., Rigau, T., and Rodrguez-Gil,
J. E. (2013a). Reduced glutathione and procaine hydrochloride protect
the nucleoprotein structure of boar spermatozoa during freeze-thawing
by stabilising disulphide bonds. Reprod. Fertil. Dev. 25, 10361050.
doi:10.1071/RD12230
Yeste, M., Estrada, E., Casas, I., Bonet, S., and Rodrguez-Gil, J. E. (2013b).
Good and bad freezability boar ejaculates differ in the integrity of
nucleoprotein structure after freezethawing but not in ROS levels.
Theriogenology 79, 929939. doi:10.1016/J.THERIOGENOLOGY.
2013.01.008
Yeste, M., Estrada, E., Pinart, E., Bonet, S., Miro, J., and Rodrguez-Gil, J. E.
(2014a). The improving effect of reduced glutathione on boar
sperm cryotolerance is related with the intrinsic ejaculate freezability.
Cryobiology 68, 251261. doi:10.1016/J.CRYOBIOL.2014.02.004
Yeste, M., Estrada, E., Rivera, M. M., Bonet, S., Rigau, T., and RodrguezGil, J. E. (2014b). The increase in phosphorylation levels of serine
residues of protein HSP70 during holding time at 178C is concomitant
with a higher cryotolerance of boar spermatozoa. PLoS One 9, e90887.
doi:10.1371/JOURNAL.PONE.0090887

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