Animal Reproduction Science 112 (2009) 107–118

The cryoprotective effect of trehalose supplementation

on boar spermatozoa quality
Jian-Hong Hu a , Qing-Wang Li a,b,∗ , Gang Li a ,

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Zhong-Liang Jiang a , Shu-hai Bu c ,
Hai Yang a , Li-Qiang Wang a

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aCollege of Animal Science and Technology, Northwest A & F University, Yangling, ShaanXi 712100, China
b College of Environment and Chemistry Engineering, Yanshan University, Qinhuangdao, HeBei 066004, China
c College of Life Sciences, Northwest A & F University, Yangling, ShaanXi 712100, China

Received 14 September 2007; received in revised form 2 April 2008; accepted 8 April 2008
Available online 24 April 2008
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Abstract
In order to improve boar sperm quality during frozen–thawed process, the influence of the presence of
trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze–thawing toler-
ance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0,
25, 50, 100 and 200 mmol/l), and tried to determine the optimum concentration of trehalose. We chose sperm
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motility, acrosome integrity, membrane integrity and cryocapacitation as parameters to evaluate cryopreser-
vation capacity of boar spermatozoa. We obtained the best results for 100 mmol/l trehalose-supplemented
extenders, with values of 49.89% for motility, 66.52% for acrosome integrity and 44.61% for membrane
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integrity, while freeze–thawing tolerance was diminished significantly for 200 mmol/l of trehalose. Before
and after capacitation, the CTC score for semen diluted by extender containing 100 mmol/l trehalose was
3.68% and 43.82%, respectively. In conclusion, trehalose could confer a greater cryoprotective capacity to
boar spermatozoa. Trehalose-supplementation with 100 mmol/l concentration in basic extender could sig-
nificantly improve sperm motility, membrane integrity and acrosome integrity parameters, and reduce boar
spermatozoa cryocapacitation during the cryopreservation process.
© 2008 Published by Elsevier B.V.
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Keywords: Boar semen; Cryopreservation; Trehalose; Sperm quality

∗ Corresponding author at: College of Animal Science and Technology, Northwest A & F University, Yangling, ShaanXi

712100, China. Tel.: +86 29 87092102; fax: +86 29 87092164.

E-mail address: ysulqw@126.com (Q.-W. Li).

0378-4320/$ – see front matter © 2008 Published by Elsevier B.V.

doi:10.1016/j.anireprosci.2008.04.009
108 J.-H. Hu et al. / Animal Reproduction Science 112 (2009) 107–118

1. Introduction

Artificial insemination (AI) has been widely used for animal genetic improvement and the
cryopreservation of spermatozoa has offered an effective means for long-term storage of impor-
tant genetic material of livestock. Freezing of boar spermatozoa has been studied more than 30
years ago (Polge, 1956). However, current methods on cryopreservation of boar spermatozoa
are unsatisfactory. To date, the boar semen cryopreservation often involves some cumbersome
processing procedures and AI of swine with frozen–thawed spermatozoa results in farrowing
rates and litter sizes 20–30% below those observed following insemination of fresh spermatozoa
(Johnson et al., 2000). Recent improvements in cryopreservation protocols have contributed to
better post-thaw sperm viability, and fertility with frozen–thawed spermatozoa was more than

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70% and 9 piglets born alive per sow (Eriksson et al., 2002). However, fertility of frozen–thawed
boar spermatozoa is still very variable (Bolarin et al., 2006). These factors or variation have
greatly hampered the application of this technology to all boars, ultimately conspiring against a
wider use of AI to the swine industry (Eriksson et al., 2002; Yi et al., 2004; Hernandez et al.,

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2007).
Sperm-freezing protocols involved to the use of extenders, the choice of sugars, the cooling
and warming processes and its main aim was to maintain normal sperm motility and viability
by preventing the formation of lethal intracellular ice crystal and reduce acrosome and plasma
membrane damage during cryopreservation. The most popular diluents for boar spermatozoa
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freezing were glucose and egg yolk (Polge et al., 1970); Tris, fructose, EDTA, citric acid, glucose
and egg yolk (Visser and Salamon, 1974); Tris, glycine, citric acid, glucose and egg yolk (Obando
et al., 1984). Currently, disaccharide trehalose had been found to be able to resist dehydration
or freezing in a number of plants and animals (Westh and Ramlv, 1991). Trehalose could form
hydrogen bonds with the polar head groups of phospholipids to prevent fusion events of juxtaposed
membranes (Anchordoguy et al., 1987). Thus, trehalose had been widely used as a cryoprotectant
for spermatozoa (Dalimata and Graham, 1997).
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In diluents without glycerol, the ram sperm motility was higher in the presence of trehalose
than that of glucose, indicating a cryoprotective effect of the disaccharides (Molinia et al., 1994).
Aboagla and Terada (2003) found that higher trehalose concentrations significantly protected
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goat spermatozoa against freezing damage. However, De Leeuw et al. (1993) found a lower
survival of bull sperm with trehalose than that with sucrose. Chen et al. (1993) also investigated
the effect of trehalose in freezing medium for bull semen on the viability of the sperm after
freezing–thawing and reported no positive contribution of trehalose. So far, some study reports
indicated that trehalose, as a cryoprotectant, had significant effects on sperm cryopreservation of
mouse (Sztein et al., 2001), bull (Woelders et al., 1997), dog (Yildiz et al., 2000), ram (Aisen
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et al., 2002) and goat (Eiman and Takato, 2004), and these promising but somewhat conflicting
results warranted more detailed investigation to the effects of trehalose on survival of domestic
animals sperm during freezing–thawing, but no authors evaluated the effect on boar spermatozoa
quality after the use of trehalose-supplementation.
The objective of the present study was to obtain more information on the cryoprotective effects
of trehalose as an extender for boar semen cryopreservation, and establish the range of trehalose
concentration in a Tris-based extender for boar semen, in order to obtain an adequate post-thawing
integrity. The experiment assessed the effects of the presence of trehalose-supplemented at final
concentrations of 0, 25, 50, 100 and 200 mmol/l. Sperm quality was evaluated by examining sperm
motility, acrosome and plasma membrane intactness and cryocapacitation to determine optimal
proportion of trehalose in freezing extender.
 J.-H. Hu et al. / Animal Reproduction Science 112 (2009) 107–118 109

2. Materials and methods

2.1. Semen collection

Semen was collected from six mature and healthy Yorkshire boars (aged between 2 and 2.5
years) once weekly by the gloved hand technique and filtered to remove the gel particles. The
semen samples of the sperm-rich fraction were assessed for volume, sperm concentration, sperm
morphology and percentage of motile spermatozoa. The ejaculated sperm-rich fraction with >75%
motility and >80% normal sperm morphology were used for this study. The experiment was
conducted at the domestic animal improving center of Hebei Province (China) over a 3-month
period from October to December (2006). A total of 48 ejaculates (8 ejaculates every boar) were

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used in this study.

2.2. Extender preparation

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The component of basic diluent, TCF (Tris–citric acid–fructose), was consisted of 200 mM
Tris, 77 mM citric acid and 61 mM fructose. The cooling extenders were composed of basic
diluent plus 250 mg gentamicine, 500,000 IU penicillin and 200 ml egg yolk for 1000 ml distilled
nonpyrogenic water (per liter of medium). The freezing extenders were composed of the cooling
extender supplemented with glycerol and trehalose solution. The initial concentration of trehalose
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was 375 mmol/l (378 mOsM). The osmolality of the media was measured in the absence of
glycerol (replacing the glycerol with water to obtain the right end volume) in order to obtain a
measure of the osmotic contribution of the nonpermeant solutes. The osmolality of the cooling
extenders was 316 mOsm/kg of water. Trehalose solution was used as a freezing extender, and
was added at final concentrations in the diluted semen of 0, 25, 50, 100 and 200 mmol/l, and
osmolality of the medium were 316, 344.5, 373, 430 and 543 mOsm/kg of water, respectively. All
media had the same glycerol concentration. The final glycerol concentration in the diluted semen
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was 3% (v/v).

2.3. Frozen semen processing

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The semen was processed and frozen by the straw freezing method (Pursel and Johnson, 1971).
The collected fresh semen samples were randomly divided into 15 ml pre-warmed tubes, and held
for 30 min at 20–22 ◦ C, and subsequently centrifuged at 20–22 ◦ C for 10 min at 500 × g to remove
the seminal plasma. After elimination of seminal plasma, about 12 ml pre-warmed addition of
BTS solution (BTS was consisted of glucose 3.7 g, EDTA 0.125 g, sodium citrate 0.6 g, sodium
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bicarbonate 0.125 g, and potassium chloride 0.075 g per 100 ml distilled water) was added to all
tubes. The tubes were wrapped with 12–15 layers of sterile gauze and the sperm suspension was
slowly cooled to 17 ◦ C. After centrifugation at 17 ◦ C for 10 min at 800 × g, the supernatant was
discarded.
We diluted the semen in the different extenders in a two-step procedure. The concentrated semen
sample was diluted with the cooling extenders to obtain 1.5 × 109 spermatozoa/ml (Eriksson et
al., 2001). The diluted semen was gently mixed, and all tubes were wrapped with 12–15 layers
of sterile gauze again. Then they were slowly cooled to 5 ◦ C and equilibrated for 1.5–3 h.
The semen was further diluted (2:1, two parts semen to one part extender) with different freez-
ing extenders, respectively. The final sperm concentration was 1.0 × 109 spermatozoa/ml. The
sperm suspension was loaded into 0.25 ml straws immediately. The straws were sealed manually
110 J.-H. Hu et al. / Animal Reproduction Science 112 (2009) 107–118

using metallic sealing balls. Then the straws were horizontally placed on an aluminum rack and
maintained at 5 ◦ C for 2–3 h. The freezing programme had the following steps: from +5 ◦ C to
−5 ◦ C with 1 ◦ C/min by programmable freezing device (Mini Digitcool 1400, IMV, France). All
straws were then placed in contact with nitrogen vapor about 2–3 cm (−120 ◦ C) above the nitro-
gen liquid level for 15 min, and then immersed into the liquid nitrogen (−196 ◦ C) for storage.
After 6 weeks of storage, samples were removed from the liquid nitrogen and thawed in a water
bath to evaluate the sperm quality.

2.4. Sperm quality evaluation

2.4.1. Sperm motility

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For the motility analysis of the post-thaw semen samples, six straws per treatment were thawed
by immersion in a water bath at 37 ◦ C for 45 s. Immediately, all thawed samples were then trans-
ferred into a plastic tube containing 9.5 ml of BTS solution and 0.5 ml of the relevant cooling
extender (pre-warmed) to prevent the spermatozoa from sticking to the glassware during motility

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analysis. After 10 min incubation at 37 ◦ C, 10 ␮l aliquots were transferred into glass slides and
cover-slips were applied. Sperm motility was assessed by determining the percentage of sper-
matozoa showing any movement of the flagellum. The percentage of linear motile sperm was
estimated at 37 ◦ C by light microscope at 400×. At least 300 spermatozoa were counted per slide.
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2.4.2. FITC-PNA staining
Acrosome status was assessed using fluorescein isothiocyanate-conjugated peanut agglutinin
(FITC-PNA) in a procedure slightly modified from the one described by Aboagla and Terada
(2003). After thawing, semen samples were transferred into a plastic tube containing 2 ml 3%
PVP and centrifuged at 20–22 ◦ C for 3 min at 800 × g. The sediment (sperm) was diluted with
37 ◦ C PBS solution to obtain 1–2 × 106 spermatozoa/ml. The 30 ␮l sperm solution was used to
prepare smears on microscope slides. After air-drying, sperm smears were fixed with absolute
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methanol for 10 min at 20–22 ◦ C. About 30 ␮l FITC-labeled peanut agglutinin (FITC-PNA) solu-
tion (100 ␮g/ml) in PBS was spread over each slide. The slides were then incubated in a dark and
moist chamber for 30 min at 37 ◦ C. After incubation, the slides were rinsed with PBS, air-dried,
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and mounted with 10 ␮l of antifade solution to preserve fluorescence. The slide smear was covered
by slip and sealed with colorless nail polish.
The acrosome status of the spermatozoa was examined and photographed using an epifluores-
cence microscope (LEIKA DM-IRB linked up to a Nikon digital camera DXM). All treatments
were replicated with ejaculates from the same boar. All samples were coded before evaluation,
and were evaluated by one observer. The whole acrosome was visualized with strong green flu-
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orescence under a fluorescence microscope and was scored as acrosome-intact sperm cells. The
percentage of fluorescent acrosome-intact spermatozoa was counted in at least 300 sperm cells
per slide.

2.4.3. Hypoosmoticy swelling test (HOST)

The HOS test was used as an assay to evaluate the functional activity of the sperm membrane
(Osinowo et al., 1982). The straws were thawed in a water bath at 37 ◦ C for 45 s. The 50 ␮l
semen was added to 1 ml hypoosmotic solution prepared by mixing 7.35 g sodium citrate 2H2 O
and 13.51 g fructose in 1 l distilled water. After incubation for 30 min at 37 ◦ C (Garcia-Lopez et
al., 1996), sperm swelling was assessed by placing 15 ␮l of well-mixed sample on a warm slide
(37 ◦ C) under light microscopy at 400× magnification. Viable spermatozoa had coiled tails after
 J.-H. Hu et al. / Animal Reproduction Science 112 (2009) 107–118 111

HOST. At least 300 spermatozoa per slide were observed. The spermatozoa were classified as
positive or negative based on the presence or absence of coiled tail.

2.4.4. CTC staining

The chlortetracycline (CTC) staining was used to evaluate the cryocapacitation of sperm, using
the method described by Gill et al. (2000). After thawing, the semen samples were diluted with
BTS solution (37 ◦ C) to obtain 1–2 × 106 spermatozoa/ml. About 400 ␮l semen samples were
added into a plastic tube containing 1 ml 3% PVP, and centrifuged at 500 × g for 6 min. The
sperm were stained before and after capacitation, respectively. Before capacitation, the sediment
sperm was suspended in 200 ␮l PBS, then mixed with 200 ␮l CTC solution containing 750 ␮M
CTC, 5 mM cysteine and 130 mM NaCl in 20 mM Tris–HCl buffer (pH 7.8), and incubated for

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30 s in the dark. The stained spermatozoa were fixed by adding 36 ␮1 of 12.5% paraformaldehyde
(w/v) in 0.5 M Tris–HCl (pH 7.4). The slides were prepared by placing 10 ␮l sample on a slide
with 10 ␮l of antifade solution to preserve fluorescence. The percentage of the capacitated sperm
was estimated at 37 ◦ C by light microscope at 400×. After sperm capacitation, the sediment

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sperm was suspended in 200 ␮l TCM, and then incubated in a dark and moist chamber for 3 h at
37 ◦ C. Then, the spermatozoa was stained and fixed with the same methods. The B (capacitated,
acrosome-intact), F (uncapacitated, acrosome-intact) and AR (acrosome reacted) patterns were
examined with filter 2 (D) under Nikon fluorescent microscope as the described by Fraser et al.
(1995). Both capacitated and viable spermatozoa were defined as capacitated spermatozoa in this
study. The percentage of the capacitated sperm was estimated at 37 ◦ C by fluorescent microscope
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at 400×. At least 300 spermatozoa were counted per slide.

2.5. Statistical analysis

All treatments were repeated at least twelve times with at least six different ejaculates from six
boars, respectively. Results were expressed as mean values ± S.D. in each case. The mean values
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of the percentages of spermatozoa with different staining in individual experiment, the acrosome-
intact and plasma membrane-intact spermatozoa were compared using Duncan’s multiple range
test by ANOVA procedure, when the F-value was significant (P < 0.05). The comparisons of the
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percentages of motile sperm were performed using the t-test. All analyses were performed using
Statistical Product and Service Solutions (SPSS 11.5 for Windows; SPSS, Chicago, IL, U.S.A).

3. Results

3.1. Spermatozoa motility, acrosome and membrane integrity

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The effect of increasing trehalose concentrations added to the cooling diluent on the boar
sperm motility, acrosome and membrane integrity after freezing–thawing were shown in Fig. 1.
Trehalose 100 mmol/l conferred the highest sperm motility values, the best cryopreservation
on the acrosomal and caudal plasma membrane, with an improvement of 19%, 29% and 14%
over the control group (p < 0.01), respectively. No significant difference was observed for sperm
motility between freezing extenders containing trehalose 50 and 100 mmol/l (p > 0.05). For 50 and
200 mmol/l of trehalose in the diluents, sperm acrosome integrity did not significantly differ from
the extender containing 100 mmol/l trehalose (p > 0.05), and HOS test also did not significantly
differ from the diluent containing 25 mmol/l trehalose (p > 0.05), whereas the sperm membrane
integrity was significantly decreased than that of 100 mmol/l of trehalose (p < 0.05). Addition
112 J.-H. Hu et al. / Animal Reproduction Science 112 (2009) 107–118

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Fig. 1. The percentage of sperm motility, acrosome and membrane integrity in boar semen frozen and thawed in extenders
with increasing trehalose concentrations. Bars represent means ± S.D. Different letters indicate significant differences
(p < 0.05).

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of 25 mmol/l of the trehalose to the cooling extender showed an intermediate effect on motility,
acrosome and membrane integrity preservation. When trehalose concentration was higher than
100 mmol/l, the percentage of linear motile sperm and intact-membrane sperm were significantly
decreased after thawing (p < 0.01), respectively. These results suggested that 100 mmol/l trehalose
could afford better protection ability than that of others during the freeze–thaw process.
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3.2. Sperm cryocapacitation

Sperm cryocapacitation was determined using CTC staining for the CTC-treated spermatozoa
and the results were shown in Table 1. The clear microscopical pictures of sperm capacitation
were generated successfully with very high sensitivity by CTC staining (Figs. 2 and 3).
Before capacitation, we observed that the trehalose concentrations from 25 to 200 mmol/l
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corresponded to a slight enhancement of sperm cryocapacitation, but there was no significant

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Fig. 2. Fluorescent patterns of spermatozoa stained with CTC (spermatozoa observed here were frozen–thawed in
100 mmol/l trehalose extender). no capacitated and intact acrosome sperm capacitated and partially
damaged acrosome sperm.
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J.-H. Hu et al. / Animal Reproduction Science 112 (2009) 107–118
Table 1

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Evaluation of capacitated spermatozoa among boar semen cryopreserved in diluents containing increasing concentrations of trehalose
Trehalose concentration (mmol/l)

0 25 50 100 200

Spermatozoa cryocapacitation (%) (before capacitation) 8.97 ± 2.85 a 4.63 ± 4.26 b 4.41 ± 3.16 b 3.68 ± 2.37 b 4.56 ± 3.62 b
After capacitation
Capacitated spermatozoa (%) 17.68 ± 2.69 d 20.66 ± 3.79 d 28.65 ± 3.66 c 43.82 ± 2.56 a 35.23 ± 2.52 b
Acrosome reacted spermatozoa (%) 36.92 ± 3.02 a 34.83 ± 2.84 a 26.09 ± 3.11 b 21.57 ± 3.57 c 28.18 ± 2.96 b
Uncapacitated spermatozoa (%) R 45.40 ± 2.87 a 44.51 ± 3.19 a 45.26 ± 2.76 a 34.61 ± 3.01 b 36.59 ± 3.43 b

Data with different letters in the same row indicates significant difference at p < 0.05.
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114 J.-H. Hu et al. / Animal Reproduction Science 112 (2009) 107–118

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Fig. 3. Fluorescent patterns of spermatozoa stained with CTC (spermatozoa observed here were frozen–thawed in
100 mmol/l trehalose extender). (left) capacitated and intact acrosome sperm (right) capacitated and
damaged acrosome sperm.

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difference for the percentage of capacitated spermatozoa among different extenders containing
trehalose (p > 0.05). Furthermore, the percentage of capacitated spermatozoa was significantly
lower in these extenders than that of control (p < 0.05). Trehalose could decrease distinctly the
percentage of spermatozoa capacitated caused by freezing and thawing.
After capacitation, our data revealed that the percentage of capacitated spermatozoa was signif-
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icantly higher in the extender with 100 mmol/l trehalose-supplementation than those in control and
other groups (p < 0.05). Furthermore, increasing trehalose concentrations from 100 to 200 mmol/l
decreased sharply sperm capacitation. Trehalose could significantly increase the proportion of
capacitation B pattern sperm cells, with concomitant decrease in incapacitation F and acrosome
reaction AR pattern sperm cells. Adding 100 mmol/l trehalose might significantly improve sperm
capacitation potential.
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4. Discussion
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In our study, the score of sperm motility, FITC-PNA and HOST for semen diluted by extender
containing 100 mmol/l trehalose was 49.89%, 66.52% and 44.61%, respectively. Before and after
capacitation, the CTC score for semen diluted by extender containing 100 mmol/l trehalose was
3.68% and 43.82%, respectively. Our results showed clearly that adding trehalose in extender
might obtain better results in quality parameter of boar frozen spermatozoa. Furthermore, the
optimum concentration of trehalose-supplementation had been determined to be 100 mmol/l. The
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sperm motility was slight lower than that described by Aboagla and Terada (2003) and Woelders
et al. (1997) in goat and bovine, respectively. The other items were similar to the results described
by Bayarad et al. (1998), Aisen et al. (2002) and Aboagla and Terada (2003) in mouse, ram and
goat, respectively.
Comparing with the results of goat and bovine, boar sperm motility was inhibited in the media
containing trehalose. Boar spermatozoa might have lower ability of resistance osmotic pressure
in hypertonic diluent than that of goat and bovine spermatozoa and sharp dehydrate and die in
hyperosmotic conditions. Because of the loss of part of free intracellular water, the friction in the
sperm tail was increased, which caused an inhibition of sliding of the microtubule filaments or
other structural elements in the flagellum. Irrespective of the medium, the sperm cells must endure
severe dehydration in the hypertonic media and it was more damaging than that of dehydration
 J.-H. Hu et al. / Animal Reproduction Science 112 (2009) 107–118 115

during freezing, which could be due to the fact that the sperm cells must endure dehydration in
the hypertonic media at a superzero temperature (2–5 ◦ C) for quite a long time.
Sperm swelling in response to hypoosmotic conditions resulted in the appearance of a coiled
tail. The HOST procedure might be a useful tool for detecting populations of less viable sperma-
tozoa when it was used in conjunction with another type of membrane integrity test. In present
study, the HOST score was lower than the percentage of motile sperm cells, which was reported by
Vazquez et al. (1997). The difference might be due to the fact that some spermatozoa with mem-
brane damage remained motile. Functional heterogeneity was demonstrated with flow cytometry
of the sperm population which included spermatozoa with a reduced survival capacity despite of
their motility (Morrell, 1991).
Spermatozoa were unable to fertilize the egg immediately after ejaculation in mammalian,

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and they must acquire the capacity to fertilize “capacitation” in the female reproductive tract
(Yanagimachi, 1994). Binding to the zona pellucida stimulated the spermatozoa to undergo acro-
some reaction (AR) in which the outer acrosomal membranes fused with the overlying plasma
membrane, this physiological exocytotic event resulted in the release of hydrolytic enzymes which

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were essential for the fertilization process. Spermatozoa which acrosome had been induced loss
were unable to fertilize eggs. Acrosomal integrity was a necessary further indicator of potential
sperm function to fertilize the oocytes at AI. The feasibility of using FITC-PNA for studying the
acrosome reaction in boar spermatozoa had been demonstrated previously (Vazquez et al., 1993).
At the level of fluorescence microscopy, the signal representing PNA binding was mainly limited
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to the acrosomal cap of boar spermatozoa. Thus, FITC-PNA could be used as a reliable probe for
detecting acrosome reactions in boar spermatozoa (Fazeli et al., 1997).
The concept of “cryocapacitation” had been introduced to describe the capacitation-like
changes induced by the freezing–thawing procedure by Watson (1995). It was well known that a
number of insults of cold shock, osmotic stress and intracellular ice crystal formation had been
induced to the sperm cells during the freezing–thawing process, which might kill or damage
spermatozoa. Bailey et al. (2000) reported that a subpopulation of surviving spermatozoa had evi-
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dence of cryocapacitation resulting in a shortened life span, both in vitro and in vivo. Additionally,
cryocapacitation and acrosome reaction were closely related. Now the concept which cryocapac-
itation was one of the major factors explaining reduced fertility of frozen–thawed boar sperm had
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been accepted generally (Bailey et al., 2000). In our study, we used the CTC binding test to evalu-
ate spermatozoa cryocapacitation. Before capacitation, the percentage of spermatozoa capacitated
in the extender containing 100 mmol/l trehalose was only 3.68%. After capacitation, the percent-
age of spermatozoa capacitated in the extender containing 100 mmol/l trehalose was 43.82% and
capacitation potential was improved significantly. It was demonstrated that trehalose could signifi-
cantly increase the percentage of capacitation B pattern spermatozoa, and decrease the percentage
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of incapacitation F and acrosome reaction AR pattern spermatozoa in boars. Our results indicated
that the trehalose-supplementation with 100 mmol/l concentration in basic extender could reduce
boar spermatozoa cryocapacitation during the cryopreservation process.
In this study, we showed the concentration-dependent cryoprotective action and the most
protective effect of trehalose were identified to be at a concentration of 100 mmol/l. Non-permeable
trehalose rendered hypertonic media, causing and increasing cellular dehydration before freezing
(Aisen et al., 1990; Fiser et al., 1987). Trehalose had a protective action related to the osmotic
effect and to specific interactions with membrane phospholipids. This osmotic effect decreased
the intracellular freezable water and thus minimized the degree of cell injury by cold shock and
ice crystallization (Storey et al., 1998). The action of trehalose appeared to be connected with
its ability to replace water at the membrane–solution interface (Aisen et al., 2002). In addition,
116 J.-H. Hu et al. / Animal Reproduction Science 112 (2009) 107–118

the protection was believed to result from the formation of hydrogen bonds between the sugar
hydroxyl groups and the phospholipids polar head groups. Trehalose showed a direct interaction
with phospholipid polar head groups of membrane phospholipids, stabilizing the membrane during
the freezing–thawing process. For this reason, trehalose was found to be more efficient in that
respect than other sugars (Aisen et al., 2002; Crowe et al., 1984).
In the present study, high concentrations of trehalose (200 mmol/l) had a detrimental effect
on motility, membrane integrity and spermatozoa cryocapacitation of frozen–thawed boar sper-
matozoa. These could be attributed to water effluxes, and not compatible with an adequate
cellular integrity (Curry and Watson, 1994). The use of hypertonic concentration of disaccha-
ride responded to different physicochemical action, as it did not permeate the sperm plasma
membrane. In addition to its dehydrating action, trehalose conferred a specific cryoprotection

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exerted on the lipidic bilayer. Acrosome integrity was, in turn, not affected significantly by
the different concentrations of trehalose and showed minor decreases after cryopreservation.
The concentration of 200 mmol/l resulted in a high osmolality of the extender, which in itself
was deleterious to the sperm cell. Other researchers obtained similar results with increasing

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osmolarity in the extenders used in horse (Caiza de la Cueva et al., 1997a), boar (Caiza de
la Cueva et al., 1997b) and ram semen (Veksler Hess et al., 1997). Therefore, the protective
action of trehalose on spermatozoa during the freezing–thawing process was due to the different
concentration of trehalose and it decided the value of osmolality of extender. The deleterious
effect of the highest trehalose concentration could be related with the high osmolality of the
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extender.

5. Conclusion

Our results showed that disaccharide trehalose-supplementation could confer a greater cryopro-
tective capacity and remarkable cryoprotective properties to the freeze–thaw boar spermatozoa,
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and it greatly improved the effect for boar sperm motility, membrane integrity and acrosome
integrity parameters. Trehalose could reduce boar spermatozoa cryocapacitation before capacita-
tion and improve boar spermatozoa cryocapacitation after capacitation during the cryopreservation
process. Furthermore, the optimum trehalose concentration had been determined to be 100 mmol/l
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in boar semen extender.

References

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Aisen, E.G., Cisale, H., Fernandez, H., 1990. Criopreservacion do semen ovino. Nueva tccnica. Vet. Argent. 63, 176–182
(in Spanish).
Aisen, E.G., Medina, V.H., Venturino, A., 2002. Cryopreservation and post-thawed fertility of ram semen frozen in
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