Indian J. Anim. Res.

, 52 (8) 2018: 1146-1150 AGRICULTURAL RESEARCH COMMUNICATION CENTRE

Print ISSN:0367-6722 / Online ISSN:0976-0555 www.arccjournals.com/www.ijaronline.in

Effects of negative pressure on boar semen quality during liquid storage at

17°C
Jingchun Li1,2, Qi Li2, Guosheng Wei2, Jiabao Zhang*1 and Yanbing Li*2
College of Animal Sciences,
Jilin University, Changchun, P. R. China.
Received: 19-06-2017 Accepted: 12-10-2017 DOI: 10.18805/ijar.B-791
ABSTRACT
This study aimed to investigate the influence of negative pressure on boar semen quality during liquid preservation at
17°C. Semen samples from ten large white boars were collected and pooled, divided into four equal parts, and diluted with
Modena containing 0.4% (w/v) of bovine serum albumin. The semen samples were placed in a closed container with valve,
and a negative pressure was applied for 2–5 minutes using a vacuum pump with a barometer. The control group had no
treatment, the P200 group was treated at 200 mbar, the P400 group at 400 mbar, and the P800 group at 800 mbar. During
liquid preservation, sperm motility, total antioxidant capacity, and semen H2O2 content were analyzed every 24 h. The
effective survival time of boar semen during preservation was evaluated. The results indicated that a suitable negative
pressure decreased the effects on reactive oxygen species on boar sperm quality during liquid preservation compared with
the control group. Among all the groups, the 400 mbar negative pressure group had the highest sperm motility, total
antioxidant capacity, and the percentage of spermatozoa with high mitochondrial membrane potential. The P400 group
also had semen H2O2 content than the other groups. A suitable negative pressure improves sperm quality by reducing
oxidative stress and the respiratory metabolism of sperm, and the optimum negative pressure is 400 mbar.
Key words: Boar semen, Hydrogen peroxide, Negative pressure, Sperm quality, Total antioxidant capacity.

INTRODUCTION Hu et al., 2009). To extend the survival time in vitro,

In pig farming, the use of artificial insemination
(AI) is widespread and semen that has been preserved at 15
to 20 °C is predominantly used for this (Fantinati et al.,
2009). Of the estimated 19 million AIs of pigs that are
conducted worldwide, >99% use freshly diluted liquid
semen, whereas frozen semen accounts for only 1% of all
pig AI because of low sperm survival rates after thawing
(Gerrits et al., 2005; Frydrychová et al., 2015). Boar sperm
is extremely vulnerable to low temperatures because of its
low cholesterol to phospholipid ratio (Zhang et al.,2015),
and the quality of sperm after cryopreservation is lower than
that after liquid preservation, which limits the use of frozen
semen in pig AI (Yi et al., 2008). Semen in the liquid state is
either used on the same day it is collected, or stored at 15–
20 °C for 1–5 days (Johnson et al., 2000). The quality of
boar semen after liquid storage should be investigated
because it is important in pig AI.
To preserve spermatozoa for long periods, their
metabolic activity needs to be reduced, and this is achieved
by dilution with an appropriate medium and by lowering the
temperature. The diluents balance pH, maintain sperm
membrane integrity, and defend against oxidative damage
from reactive oxygen species (ROS) (Watson et al., 1990;
*Corresponding author’s e-mail: elj863@163.com
1
College of Animal Sciences, Jilin University, Changchun, P. R. China, 2Department of Animal Science, College of Animal Science and Veterinary
Medicine, Heilongjiang Bayi Agricultural University, Daqing, Heilongjiang, P.R. China.
antioxidants and different nutrients can be added to the
diluents. With this in mind, several commercial semen
extenders have been proposed for boar semen (Zhang et al.,
2016). The most widely used extender is the Beltsville-
Thawing Solution developed by Johnson and Pursel for
thawing boar spermatozoa frozen in a pellet, and this
extender has been adapted for liquid storage (Pursel et al.,
1978). Several antioxidants have been used successfully as
additives in semen extenders, catalase and superoxide
dismutase (Roca et al., 2005; Pandey et al., 2001), -
tocopherol (Cerolini et al., 2000) and L-Glutamine
(Funahashi et al., 2005). In addition to extender additives,
Casali et al. (2014) reported that quality of sheep semen
and fertilization rates were improved by negative pressure
treatment.
However, here is little information available on the
effect of negative pressure on boar sperm quality, and the
effect of negative pressure on sperm motility has not been
evaluated.
This study aimed to investigate the effect of
negative pressure on sperm metabolism and boar sperm
quality during liquid storage at 17 °C by measuring the sperm
mitochondrial membrane potential (MMP), total antioxidant
 Volume 52 Issue 8 (August 2018)
capacity (T-AOC), and the semen H2O2 content during liquid
storage at 17°C.
MATERIALS AND METHODS
Chemical agents: All chemicals were purchased from
Sigma-Aldrich (Shanghai, China) unless otherwise noted.
Semen collection and semen processing: Semen-rich (80-
100 mL) were collected from mature large white boars (n =
10) by the gloved hand method at the pig breeding farm of
Heilongjiang Bayi Agricultural University, Daqing, China.
The samples were transferred to the laboratory within 2 h.
After evaluating the quality of the fresh semen samples, they
were centrifuged at 750g for 3 min at 17°C. The supernatant
was then removed, and the semen pellet was re-suspended at a
concentration of 1.0g 108 spermatozoa/mL in modified
Modena solution (Zhang et al., 2015). Finally, the semen
samples were all stored in an incubator at 17°C. Only ejaculates
with sperm concentrations of 1×10 8 /mL, 80% motile sperm,
ande 80% normal morphology were used.
After evaluating the quality of the each diluted
semen samples, it was divided into four for the following
experimental groups: control group, no treatment, stored at
normal pressure and 17°C; P200, negative pressure of 200
mbar; P400, negative pressure of 400 mbar; and P800,
negative pressure of 800 mbar. For negative pressure
samples, the semen was placed in a closed container with
valve, and the negative pressure was applied for a set time
by a vacuum pump with barometer. Then, all the semen
samples were placed in an incubator at 17°C.
Analysis of sperm motility: After different periods of
preservation, 10 µL of each semen sample was removed and
put on a glass slide, covered with a glass cover slip, and
analyzed at 37°C at 200 x magnification. The motility and
sperm morphology were determined using a computer
assisted sperm analysis system (CASA, Nanning Song Jing
Tianlun Bio-technology Co., Ltd, Guangxi, China) (Li et al.,
2016)
T-AOC assay: T-AOC was determined using a T-AOC assay
kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu,
China) according to the manufacturer’s instruction. The T-
AOC was measured at 520 nm on the spectrophotometer
(Shanghai Spectro photometer Co., Ltd., Shanghai, China).
The total T-AOC of each semen sample was converted into
units per ml of total protein in 107spermatozoa/mL and then
expressed as mol.
H2O2 content assay: The H2O2 content of the semen samples
was measured using a H2O2 test kit with horseradish
peroxidase and phenol red, by monitoring the change in
absorbance at 610 nm with a spectrophotometer (T6, Beijing
PERSEE General Instrument Co., Ltd.) according to the
manufacturer’s instructions (Nanjing Jiancheng Bioengineering
Institute). The semen sample H2O2 contents are expressed
as mol/L.
1147
Assessment of sperm MMP: Changes in the MMP and
spermatozoa viability were evaluated using the JC-1 probe
(Huo et al., 2002). The samples were observed and analyzed
under a fluorescence microscope. Spermatozoa stained with
JC-1 displayed either green fluorescence (low/moderate
MMP) or a red–orange fluorescence (high MMP or active
mitochondria). The percentage of orange stained cells, which
represents the population of sperm with high MMP, was
recorded (Fig 1).
Statistical analysis: All data were analyzed by one-way
analysis of variance using STATVIEW 5.0 software (Abacus
Concepts, Inc., Berkeley, CA) (Li et al., 2016). If the
ANOVA P-value was less than 0.05, a Bonferroni/Dunn’s
honest significant difference test was carried out using the
same program. All data are expressed as mean ± standard
deviation. Findings were considered significantly different
at P < 0.05.
RESULTS AND DISCUSSION
During storage at 15–18°C, boar spermatozoa
undergo several changes, including decreased motility
(Lalrintluanga et al., 2014), an increase in sperm with
acrosome damage (Leahy et al., 2011), and a decrease in
ATP  concentration  and mitochondrial activity (Gogol et al.,
2009). Many studies have demonstrated that preservation
of boar spermatozoa for long periods requires a decrease in
their metabolic activity, which is achieved by dilution in an
appropriate medium and lowering the temperature to 15–
17°C (Johnson et al., 2000). In addition, acidic or hypoxic
environments can reduce sperm metabolism (Zhang et al.,
2015).
In the current study, the effects of different negative
pressures on sperm motility, T-AOC, and the semen H2O2
content and sperm MMP were investigated during liquid
Fig 1: Fluorescence images of different mitochondrial activity of
spermatozoa using JC-1. Picture A shows sperm with high MMP;
Picture B shows sperm with the low MMP.
1148 INDIAN JOURNAL OF ANIMAL RESEARCH

Fig 2: Effects of different negative pressures on sperm motility (%) during liquid storage at 17°C (n = 4).
Bars with different superscripts are significantly different (P<0.05).
storage. Sperm motility is an important index of semen 2005). To reduce the harmful effects of ROS on sperm quality
quality. The results indicated that application of a suitable during preservation, antioxidants can be added to the
negative pressure (400 mbar) could improve sperm motility extender (Sariozkan et al., 2013). In this study, the results
and increase the effective survival time significantly (P< indicated that a suitable negative pressure (400 mbar) could
0.05) (Fig 2). decrease the H2O2 content of storage semen. These findings
The boar sperm motility during liquid storage at also indicated that a suitable negative pressure could improve
17°C was evaluated at different negative pressures (Fig.2). sperm motility by decreasing H2O2 produced by sperm
Sperm motility decreased with increasing storage time. After metabolism from the harmful effects of ROS. In future
preservation for 5 days, the sperm motility in the P400 group studies, it may be possible to achieve better preservation of
was higher than that in any of the other groups. Therefore, a boar semen in diluents with added antioxidants under an
negative pressure of 400 mbar improved boar sperm motility appropriate negative pressure.
during liquid storage. The effects of different negative pressures on T-
No significant differences were observed among AOC of boar spermatozoa were evaluated (Table 2). The T-
all the groups for the H2O2 content on day 1 (P >0.05). From AOCs of the P400 and P800 groups were higher than those
the day 3 to 13, significant differences were observed of the control and P200 groups. The P400 group had the
between the control group and the groups at different negative highest T-AOC among all the groups (P < 0.05). However,
pressures (200, 400, 800 mbar) (P<0.05). However, no the difference between the P400 and P800 groups was not
significant differences were observed between P400 and significant (P > 0.05).
P800 group (P > 0.05) (Table 1). In this study, the sperm T-AOC was measured and
Oxidative stress is considered to be one of the most analyzed in the presence of different negative pressures. T-
important factors that contribute to poor semen quality AOC is an indicator of the antioxidative capacity of
(Bucak et al., 2010). During semen preservation, oxidative spermatozoa. In our study, some groups treated with different
damage to sperm caused by ROS generated by the cellular negative pressures (400 and 800 mbar) showed higher T-
components of semen, namely superoxide anion radicals (O2-), AOC than the control group. Therefore, a suitable negative
hydrogen peroxide (H2O2), and lipid hydroperoxides, are the pressure could maintain the antioxidant capacity of boar
main causes for the decline in sperm quality (Alvarez et al., sperm (Table 2).
Table 1: Effects of different negative pressures on H2O2 content (mol/L) of the semen during liquid storage at 17°C
Treatment\ time Day 0 Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13
Control 4.9±1.0 12.6±5.2 23.7±1.0a 38.4±1.0a 49.6±2.5a 52.0±1.9a 53.4±1.8a 54.6±1.6a
P200 13.9±2.0 22.5±0.8a 33.4±0.8 b 41.4±0.6 b 44.2±1.7 b 44.4±1.1 b 46.2±1.4 b
P400 11.1±1.8 19.7±2.0 b 27.7±4.1c 34.4±3.8c 38.1±2.3c 40.7±1.5c 42.9±1.0c
b c c c c
P800 11.0±3.4 19.8±1.1 28.4±4.1 32.9±2.7 36.0±1.7 38.3±1.4 41.2±1.8c
The boar semen was preserved at 17 °C during 13 days in Modena containing 0.4% (w/v) BSA at different pressures. Data are given as
mean ± SD from four replicated experiments. Values with different superscripts within columns are significantly different (P<0.05).
 Volume 52 Issue 8 (August 2018) 1149
Table 2: Effects of different negative pressures on T-AOC activity of sperm during liquid storage at 17°C.
Treatment\ time Day 0 Day 1 Day 3 Day 5 Day 7 Day 9
Control 5.72±2.13 3.85±0.99 0.65±0.13b 0.55±0.15b 0.11±0.02c 0.03±0.01c
P200 3.23±1.08 1.25±0.12b 0.75±0.23b 0.55±0.42b 0.18±0.11b
a a a
P400 5.61±1.21 4.34±1.13 3.71±1.24 1.34±0.07 0.74±0.01a
P800 5.66±1.42 3.64±1.50a 2.82±1.76a 1.31±0.07a 0.59±0.09a
(After 9 days preservation, T-AOC (Total antioxidant capacity) of spermatozoa was not detected due to less T-AOC activity). Data are
given as mean ± SD from four replicated experiments. Values with different superscripts within columns are significantly different
(P< 0.05).

Table 3: Effects of different negative pressures on percentage of spermatozoa with hMMP (%) during liquid storage at 17°C.
Treatment\ time Day 0 Day 1 Day 3 Day 5 Day 7 Day 9 Day 11 Day 13
Control 96.9±1.8 94.3±2.1 90.6±2.6 b 71.9±1.8c 13.8±1.6c 2.7±1.2c 1.3±1.0 b 0b
b a b bc b
P200 95.2±1.5 92.8±2.4 78.8±2.9 21.9±2.4 4.2±1.5 2.5±1.4 1.0±0.8 b
P400 96.4±1.3 95.6±1.9a 82.5±3.1a 32.5±3.2a 12.6±1.6a 7.1±0.9a 4.3±0.6a
P800 94.9±4.1 92.5±1.6 b 77.9±2.6 b 22.4±2.7 b 5.8±1.3 b 2.7±1.0 b 1.1±0.5 b
The boar semen was preserved at 17 °C during 13 days in Modena containing 0.4% (w/v) BSA at different pressures. Data are given as
mean ± SD from four replicated experiments. Values with different superscripts within columns are significantly different (P<0.05).

On day 0, 96.9% of spermatozoa had active
mitochondria (Table 3). However, the percentage of
spermatozoa with high MMP decreased with decreasing
storage time in vitro. On day 3, the P400 group had the
highest percentage of spermatozoa with high MMP among
all the groups (P < 0.05). However, there were no significant
differences among the other groups (P > 0.05). On day 5,
there were significant differences between the P400 and
control groups, and the P400 and P800 groups (P < 0.05).
After preservation for 7 days, the percentages of sperm with
high MMP in all groups were drastically reduced compared
with those on day 5. On days 9, 11, and 13, the P400 group
had the highest percentage of spermatozoa with high MMP
among all the groups (P < 0.05). The control group had the
lowest percentage of spermatozoa with high MMP among
all the groups (P < 0.05).
In this study, the percentage of sperm with high
MMP was different in the P400 group compared with the
other groups after preservation for 3 days. The largest
difference in the percentage of viable sperm with an intact
plasma membrane was observed between the groups on day
7, and this difference was significant, whereas on days 1, 3,
and 5 it was not. In addition, our results indicated that a
progressive decrease in the percentage of motile and viable
sperm was associated with a decrease in the MMP (Gil
Ananya et al., 2014). In the present study, the percentage of
spermatozoa with high MMP in the P400 group was the
highest among all the groups over the entire storage period.
A suitable negative pressure could decrease ATP
consumption of sperm mitochondria during storage.
CONCLUSION
Different negative pressures have different effects
on the quality of boar sperm. A suitable negative pressure
decreases the effects of ROS on sperm quality during liquid
preservation compared with a control group. The P400 group
has higher sperm motility, longer effective survival time, and
higher T-AOC than the other groups. The P400 group also
has a lower H2O2 content. In conclusion, a suitable negative
pressure improves boar sperm quality by reducing oxidative
stress and the respiratory metabolism of the sperm during
liquid storage at 17 °C. The optimum negative pressure is
400 mbar.
ACKNOWLEDGMENT
This work was supported by National Natural
Science Foundation of China (No. 31402136), Natural
Science Foundation of Heilongjiang Province of China (No.
C2017053), Innovative Talents Project of Heilongjiang Bayi
Agricultural University (No. CXRC2017004) and Guiding
Science and Technology plan project of the city of Daqing
(No. zd-2016-087). Doctoral Starting up Foundation of
Heilongjiang Bayi Agricultural University (No. B2012-06;
XYB-2016-04).

REFERENCES
Alvarez, J.G., and Storey, B.T. (1995). Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipids
of human spermatozoa. Mol. Reprod. Dev., 42: 334-346.
Bucak, M.N., Sarıözkan, S., Tuncer, P.B., Sakin, F., Ateşşahin, A., Kulaksız, R., Çevikf, M. (2010). The effect of antioxidants on
post-thawed Angora goat (capra hircus ancryrensis) sperm parameters, lipid peroxidation and antioxidant activities. Small
Ruminant Res., 89: 24-30.
Casali, R., Silva, L.G., Arcego, C.C., Mozzaquatro, F.D., Mezzalira, A. (2015). Negative pressure in the pre-freezing of ram semen. Acta
Scientiae Veterinariae, 42: 1–6.
Cerolini, S., Maldjian, A., Surai, P., Noble, R. (2000). Viability, susceptibility to peroxidation and fatty acid composition of boar
semen during liquid storage. Anim. Reprod. Sci., 58: 99-111.
1150 INDIAN JOURNAL OF ANIMAL RESEARCH
Fantinati, P., Zannoni, A., Bernardini, C., Forni, M., Tattini, A., Seren, E., Bacci, M.L. (2009). Evaluation of swine fertilisation
medium (sfm) efficiency in preserving spermatozoa quality during long-term storage in comparison to four commercial
swine extenders. Animal, 3: 269-74.
Frydrychová, S., Lustyková, A., Václavková, E., Lipenský J., Rozkot, M. (2015). Effect of different extenders on quality of frozen-
thawed boar semen. Indian J. Anim. Res., 49: 851-854.
Gerrits, R. J., Lunney, J. K., Johnson, L. A., Pursel, V. G., Kraeling, R. R., Rohrer, G. A., Dobrinskya, J.R. (2005). Perspectives for
artificial insemination and genomics to improve global swine populations. Theriogenology, 63: 283-299.
Gil Ananya, M.C., Barón, F.J., Guerrero, J.M., García-Marín, L.J., Gil, J. (2014). Increasing extender viscosity improves the quality of
cooled boar semen. J. Agri. Sci., 6: 12-22.
Gogol, P., Szczeœniak-Fabiañczyk, B., Wierzchoœ-Hilczer, A. (2009). The photon emission, ATP level and motility of boar spermatozoa
during liquid storage. Reprod. Biol., 9: 39-49.
Hu, J.H., Li, Q.W., Zhang, T., Jiang, Z.L. (2009). Effect of gynostemma pentaphyllum polysaccharide on boar spermatozoa quality
following freezing-thawing. Cryobiology, 59: 244-249.
Huo, L.J., Ma, X.H., Yang, Z.M. (2002). Assessment of sperm viability, mitochondrial activity, capacitation and acrosome intactness
in extended boar semen during long-term storage. Theriogenology, 58:1349-1360.
Johnson, L.A., Weitze, K.F., Fiser, P., Maxwell, W.M. (2000). Storage of boar semen. Anim. Reprod. Sci., 62(1-3): 143-172.
Lalrintluanga, K., Deka, B.C., Nath, K.C., Sarmah, B.C., Biswas, R.K. (2014). Effect of heterospermy on the quality of spermatozoa
during preservation of boar semen at 18°C. Indian J. Anim. Res., 48: 201-203.
Leahy, T., and Gadella, B.M. (2011). Capacitation and capacitation-like sperm surface changes induced by handling boar semen. Reprod.
Dom. Anim., 46 Suppl 2(s2): 7-13.
Li, J.C, Zhu, S.B., He, X.J., Sun, R., He, Q.Y., Gan, Y., Liu, S.J., Funahashi, H., Li, Y.B. (2016). Application of a microfluidic sperm
sorter to in vitro production of dairy cattle sex-sorted embryos. Theriogenology, 85:1211-1218.
Pandey, R.P., Singh, B.K. (2001): Effect of dilutors on extracellular enzyme activity of preserved boar semen. Indian J. Anim. Sci., 71:
826-828.
Pursel, V.G., Schulman, L.L., Johnson, L.A. (1978). Effect of orvus es paste on acrosome morphology, motility and fertilizing capacity
of frozen-thawed boar sperm. J. Anim. Sci., 47: 198-202.
Roca, J., Rodríguez, M.J., Gil, M. A., Carvajal, G., Garcia, E.M., Cuello, C., Vazquez, J.M., Martinez, E.A. (2005). Survival and in
vitro fertility of boar spermatozoa frozen in the presence of superoxide dismutase and/or catalase. J. Androl., 26: 15-24.
Sarýözkan, S., Türk, G., Cantürk, F., Yay, A., Eken, A., Akçay, A. (2013). The effect of bovine serum albumin and fetal calf serum on
sperm quality, DNA fragmentation and lipid peroxidation of the liquid stored rabbit semen. Cryobiology, 67: 1-6.
Watson, P.F. (1990). Artificial insemination and the preservation of semen. In: Lamming G (ed.), Marshall’s Physiology of Reproduction.
Churchill Livingstone, Edinburgh, London, UK: 747–869.
Yi, Y.J., Li, Z.H., Kim, E.S., Song, E.S., Kim, H.B., Cong, P.Q., Lee, J.M., Park, C.S. (2008). Comparison of motility, acrosome,
viability and ATP of boar sperm with or without cold shock resistance in liquid semen at 17°C and 4°C, and frozen-thawed
semen. Asian Australasian J. Anim. Sci., 21: 190-197.
Zhang, X.G., Liu, Q., Wang, L.Q., Yang, G. S., Hu, J.H. (2016). Effects of glutathione on sperm quality during liquid storage in boars.
Anim. Sci. J., 87: 1195-1201.
Zhang, X.G., Yan, G.J., Hong, J. Y., Su, Z.Z., Yang, G.S., Li, Q.W., Hu, J.H. (2015). Effects of bovine serum albumin on boar sperm
quality during liquid storage at 17°C. Reprod. Dom. Anim., 50: 263-269.