Lymberopoulos A. G. et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
Impact of Storage and Purification on Mitochondrial
Membrane Potential of Boar Spermatozoa
Aristotelis.G.Lymberopoulos1*, Tarek A. Khalifa2, Anastasios Spyridonou1
1
ATEI of Thessaloniki, Faculty of Agricultural Technology, Department of Animal Production, POBox 141, Sindos,
GR-57400 Thessaloniki, Greece
2
EquiBiotech Inc-Research Services in Farm Animal Breeding, Thessaloniki, Greece
Abstract
This study aimed to evaluate the effect of semen purification and storage on sperm mitochondrial membrane
potential (ΔΨm). Gel-free whole ejaculates were collected from five proven fertile Large White boars aged two to
three years. Aliquots of fresh semen were split, diluted in one step with commercial extenders and incubated at 37oC
for 5-10 minutes. Semen was cooled to 18oC and packaged in 15-ml sterile propylene tubes. After 4-10 hours postsemen collection, stored semen was purified by colloidal centrifugation. After 48 hours post-semen collection, stored
semen was incubated at 37oC and evaluated after 45 minutes for motility, velocity and sperm ΔΨm. Samples were
stained with 2.99 μM JC-1 and 2.32 μM EH-1 and assessed by Fluorescence microscopy. After centrifugation a
significant improvement of motility (P<0.035), and velocity (P<0.012) was noticed. The percentage of spermatozoa
with intact plasma membrane and high/low mitochondrial membrane potential was statistical higher after
centrifugation and storage at 18°C for 48 hours. In conclusion, colloidal purification of boar semen can improve
sperm quality and mitochondrial membrane potential.
Keywords: boar, mitochondrial membrane potential, purification, sperm storage
consequences of lipid peroxidation are membrane
damage, inhibition of respiration and leakage of
intracellular enzymes [4]. When the lipid
peroxidation cascade is stimulated, 60% of fatty
acid is lost from the membrane [5], resulting in
loss of membrane integrity. Oxidative damage to
mitochondrial membrane architecture may be a
factor of major important to explain the impaired
fertility and motility of preserved spermatozoa [6].
Mitochondria,
the
sites
of
oxidative
phosphorylation (OXPHOS), are located in the
sperm mid-piece at the extreme anterior end of the
flagellum. As in other cells, sperm mitochondria
produce ATP through aerobic respiration that is
typically required for a cell to survive. Tests for
mitochondrial function are usually highly
correlated with viability assays [7; 8].
Mitochondrial respiration would be the
predominant source of ATP required for flagellar
movements which is the greatest energy
consumption for the sperm cell. Therefore, sperm
1. Introduction

Fertility of boar spermatozoa decreases with
increase in storage time during liquid preservation.
During storage, the boar spermatozoa undergo
several changes including diminished motility,
viability
and
alterations
in
membrane
permeability. Boar sperm seems to be sensitive to
peroxidative damage due to the relative high
content of polyunsaturated fatty acids in the
phospholipids of the membrane [1] and the
relative low antioxidant capacity of boar seminal
plasma [2]. Excessive ROS formation by
spermatozoa during preservation has been
associated with a decrease in the function of
spermatozoa during preservation [3]. The

* Corresponding author: A.G.Lymberopoulos, Tel:
+302310013753, Fax: +302310013753, Email:
[email protected]
58
Lymberopoulos A. G. et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
collected by gloved hand technique with the help
of dummy sow the sperm-rich fraction was
collected in a pre-warmed thermos, while the gel
fraction was held on a gauze tissue covering the
thermos opening. After collection, semen
characteristics (volume, sperm concentration and
subjective sperm motility) were microscopically
evaluated using standard laboratory techniques.
Only gel-free whole ejaculates of ≥ 0.2x109
sperm/ml and 75% sperm progressive motility
were used. The semen then was extended with
commercial extenders and transported to the
laboratory. Once in the laboratory, the semen
samples were incubated at 37oC until analyzed.
motility would indirectly reflect the ability of
mitochondria to propel the sperm and represent a
way to detect the ATP production.
The introduction of fluorescent probes such as JC1, that stain specific sperm compartments, has
enhanced the ability of researchers and clinicians
to evaluate the activity of mitochondria. Since any
changes in mitochondrial function may be
reflected in sperm motility [9] and the proportion
of
5,5΄,
6,6΄-tetrachloro-1,1΄,
3,3΄tetraethylbenzimi-dazolyl-carbocyanine
iodide
(JC-1)-stained spermatozoa are significantly
correlated with motility [10] mitochondrial
activity of boar spermatozoa can be determined
with JC-1, a mitochondria specific stain with a
high sensitivity. JC-1 is a lipophilic cationic
fluorescent carbocyanine dye that is internalized
by all functioning mitochondria, where it
fluoresces
green.
In
highly
functional
mitochondria, the concentration of JC-1 inside the
mitochondria increases and the stain forms
aggregates that fluoresce orange. When human
spermatozoa were divided into high, moderate and
low mitochondrial potential groups, based on JC-1
fluorescence, the in vitro fertilization rates were
higher in the high potential group than in the low
potential group [7].
However, changes in the mitochondrial membrane
potential of boar spermatozoa during liquid
preservation have not been studied in detail. The
aim of the present study was to evaluate the effect
of semen purification and storage on sperm
mitochondrial membrane potential (ΔΨm).
Semen analysis
Sperm concentration of fresh semen was measured
by a precalibrated photometer (SpermaCue,
Minitub-Germany). In preserved samples, 1 μl of
semen was diluted with 99 μl of 3% NaCl solution
containing 0.02% eosin and sperm cell
concentration was estimated using an improved
Neubauer hemocytometer chamber.
Sperm motility was assessed in stored semen after
reactivating in a water bath at 35◦C for 30 min
before examination. Sperm motility was evaluated
by keeping a drop of semen on a pre-warmed slide
and estimating the percentage of sperm possessing
progressive motility at ×1000 magnification using
a phase contrast microscope (Leica DMLB 2000,
Germany) equipped with a heating stage (35◦C).
The motility was expressed as percentage of
progressively motile spermatozoa. The speed of
sperm motility was scored on a scale from 1 to 4
(slowest to fastest, respectively).
For sperm agglutination evaluation, a 10-μL
aliquot of each sample was placed onto a prewarmed glass slide, covered with a warm glass
cover slip, and observed at magnification x 125
under a phase-contrast microscope (Leica DMLB
2000, Germany) equipped with a warm stage at 37
°C. Approximately 50 motile sperm cells were
visually examined in each of four fields to
estimate the percentage of head-to-head
agglutinated sperm, which was defined as the
number of sperm agglutinated to at least one other
sperm per total motile sperm.
2. Materials and methods
Reagents
The
5,5Ά,
6,6Ά-tetrachloro-1,1΄,3,3΄
Αtetraethylbenzimidazolyl carbocyanine iodide (JC1) (218615) was obtained from Santa Cruz
Biotechnology Inc, USA. Eithidium homodimer
(EH-1) (46043) was obtained from Sigma-Aldrich
CO, (St. Louis, MO). Semen extender INRA 96
was obtained from IMV Technologies, France.
PorciPure® colloid was obtained from Nidacon
International AB, Göthenborg, Sweden.
Semen collection
Semen was collected from five mature fertile
Large White boars (2–3 years old) of proven
fertility from a pig farm in Pieria in North Greece.
Five ejaculates each from five boars were
Preparation of fluorescent probes
Preparation of 3-mM stock solution of JC-1
The 5-mg vial content (0.005 g) was dissolved in
2.55 ml of DMSO (1.96 mg dye/ml). Aliquots of
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Lymberopoulos A. G. et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
colloid column and centrifuge at 300 x g for 20
minutes at RT using a swing-out rotor. All the
fluid above sperm pellet was aspirated. Sperm
pellet was transferred to a new tube and was resuspended in 1 ml of INRA-96 medium (IMV
Technologies, France). After 48 hours post-semen
collection, stored semen was incubated at 37oC
and evaluated after 45 minutes for endpoints
described above.
0.1 ml stock solution were transferred in
eppendorf tubes and stored at -20oC until used.
Preparation of 1.167-mM stock solution of EH-1
The 1-mg vial content (0.001 g) was dissolved in
1 ml DMSO (1 mg dye/ml; 1μg dye/μl). Aliquots
of 0.1 ml stock solution were transferred in
eppendorf tubes and stored at -20oC until used.
Assessment of Mitochondrial Membrane Potential
All staining procedures were carried out at room
temperature (22-25oC). Semen samples were
extended
to
a
concentration
of
30100x106sperm/ml. A 1 μl of EH-1 stock solution
and 0.5 μl of the stock solution were transferred in
a light-protected tube containing 500 μl of sperm
suspension. The stains were mixed well with
sperm suspension to achieve final dye
concentrations of 1.96 μg JC-1/ml (2.99 μM) and
1.99 μg EH-1/ml (2.32 μM). Stained spermatozoa
were incubated at 37oC for 30 minutes before
analyses. A 10-μl sample of stained spermatozoa
suspension was placed on a slide, cover slipped
and evaluated immediately by fluorescent
microscope (LEICA DMLB, Germany) at 1000x·
magnification. Spermatozoa stained with JC-1
display either green fluorescence for mitochondria
with low to medium membrane potential, or
orange fluorescence for mitochondria with high
membrane potential in the tail [10]. An average of
200 cells was evaluated for each sample.
Statistical analysis
Data were presented as means ± SEM and
analyzed using independent t-test and one-way
ANOVA followed by Duncan’ multiple range test.
3. Results
The effect of colloidal purification of boar semen
on sperm kinetic activity and incidence of sperm
agglutination are shown in table 1. Before and
after purification of boar semen no significant
difference on sperm total motility was noticed.
However, a significant difference on sperm
progressive motility (P<0.035), velocity (P<0.012)
and agglutination (P<0,006) was observed.
Table 1. Effect of colloidal purification of boar semen
on sperm kinetic activity and incidence of sperm
agglutination
Sperm
Semen
Pvalue
Before
After
purification purification
Total motility 72.0±3.8
86.0±5.6
< 0.06
(%)
Progressive 63.0±3.4
80.0±5.5
<0.035 *
motility (%)
Velocity
1.4±0.2
2.6±0.3
<0.012*
grade(0-4)
Agglutination 1.9±0.4
0.0±0.0
<0.006*
grade (0-5)
*Significant effect for semen purification on mean (±
SEM) values of five ejaculates collected from five
different boars. Each ejaculate was purified after 4-10
hours of storage at 18oC.
Experimental design
Ejaculates (n= 5) were collected from five boars
(1ejaculate/boar) and incubated at 37oC until
initial assessment of fresh semen. Aliquots of
fresh semen were split, diluted in one step (3040x106sperm/ml) with commercial extenders and
incubated at the same temperature for 5-10
minutes. Semen was cooled to 18oC (–
0.10oC/minute) and packaged-stored in 15-ml
sterile propylene tubes. The air-filled spaces above
semen were less than 4.5% of the tube’s capacity.
Semen tubes were placed horizontally in a cooling
box and rotated at least twice a day throughout the
whole storage period.
After 4-10 hours post-semen collection, stored
semen was purified by PorciPure® (Nidacon
International AB, Göthenborg, Sweden) colloidal
centrifugation. Four ml of the colloid were
transferred to a sterile conical centrifuge tube. One
ml of diluted semen was layered on the top of the
In figure 1 four groups of sperm population were
observed after using JC-1 assay: (i) sperm cells
with intact plasma membrane and high ΔΨm
(IPM+HMP), (ii) cells with intact plasma
membrane and low ΔΨm (IPM+LMP), (iii) cells
with intact plasma membrane and high/low ΔΨm
(IPM+HLP) and (ιv) cells with damaged plasma
membrane and low ΔΨm (IPM+LMP). After
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Lymberopoulos A. G. et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
Table 2. Influence of storage of boar semen at 18oC on
sperm kinetic activity and incidence of sperm
agglutination
Sperm
Semen storage
Pvalue*
duration
4-10 hours 48 hours
Total motility
72.0±3.7
69.0±3.7
< 0.6
(%)
Progressive motility 63.0±3.4
57.0±4.1
< 0.3
(%)
Velocity grade
1.4±0.2
1.7±0.3
< 0.5
(0-4)
Agglutination grade 1.9±0.4
2.0±0.3
< 0.9
(0-5)
purification there was a significant decrease in the
sperm cells with high and low ΔΨm, while a
significant increase (99.8%) in the sperm cells
with high/low ΔΨm was noticed.
Storage of boar semen at 18oC for 48 hours had no
influence on sperm kinetic activity and incidence
of sperm agglutination compared to the storage for
4-10 hours (Table 2).
Boar semen ‐ Sperm mitochondrial membrane potential (ΔΨm)
Before purification
After purification
f
99.8
c
38.2
a 19.6
e
22.6
b
d
0
0
IPM+HMP
IPM+LMP
c
19.6
d
0.2
IPM+HLMP
DPM+LMP
Figure 1. Effect of colloidal purification of boar semen on incidences (%) of spermatozoa with intact plasma membrane
and high ΔΨm (IPM+HMP), intact plasma membrane and low ΔΨm (IPM+LMP), intact plasma membrane and high/low
ΔΨm (IPM+HLP) and damaged plasma membrane and low ΔΨm (IPM+LMP). Means with dissimilar letters are
significantly different at: P < 0.01a-b, < 0.001e-f and < 0.0001c-d. Five ejaculates were used from five different boars. Each
ejaculate was purified after 4-10 hours of storage at 18oC.
Also, in figure 2 four groups of sperm population
were observed after using JC-1 assay: (i) sperm
cells with intact plasma membrane and high ΔΨm
(IPM+HMP), (ii) cells with intact plasma
membrane and low ΔΨm (IPM+LMP), (iii) cells
with intact plasma membrane and high/low ΔΨm
(IPM+HLP) and (ιv) cells with damaged plasma
membrane and low ΔΨm (IPM+LMP). After 48
hours storage of boar semen there was a
significant decrease in the sperm cells with high
and low ΔΨm, while a significant increase
(51.6%) in the sperm cells with high/low ΔΨm
was noticed.
4. Discussion
An important test for determining semen quality
may be the assessment of membrane
mitochondrial potential (ΔΨm) in intact
spermatozoa since mitochondrial function has
been related to sperm motility. JC-1 appears to be
the best probe for the detection of ΔΨm changes
in spermatozoa and to predict sperm fertility
potential.
The results of this study show a significant
correlation between the capacity of the ΔΨm and
the parameters of the routine semen analysis.
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Lymberopoulos A. G. et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
Sperm mitochondrial membrane potential (ΔΨm) of stored
boar semen at 18o C for:
4‐10 hours
48 hours
51.6
38.2
a 22.6
21.2
19.6
27.2
19.6
b
0
IPM+HMP
IPM+LMP
IPM+HLMP
DPM+LMP
Figure. 2: Effect of storage of boar semen at 18oC on incidences (%) of spermatozoa with intact plasma membrane
and high ΔΨm (IPM+HMP), intact plasma membrane and low ΔΨm (IPM+LMP), intact plasma membrane and
high/low ΔΨm (IPM+HLP) and damaged plasma membrane and low ΔΨm (IPM+LMP). Means with dissimilar
letters are significantly different at P < 0.01. Five ejaculates were used from five different boars.
documented [16- 19]. However, other studies have
not detected significant differences. This is
probably related to the high variability that could
be present in some boars, differences in the colloid
density procedures [20, 21] or differences in the
system of measurement. However, the differences
in the motility reported in this study showed that
the colloid is important to optimize recovery of a
higher percentage of spermatozoa with a high
velocity which could be related to the capacitation
process due to a significant increase of tyrosine
phosphorylation and calcium intake [22]. This
sperm subpopulation with high velocity could be
related to the fertilizing capacity [23, 24]. The
study reported here clearly demonstrates that boar
spermatozoa exhibit an enhanced function after a
combination
of
washing
and
colloid
centrifugation. These results support the extensive
data from human studies [25-29].
Among those showing the strong correlation of
ΔΨm with progressive motility and the velocity
which enhances previous results [11, 7, 8 and 12].
Our results showed an increase in capacity of the
mitochondrial membrane (ΔΨm) of spermatozoa
after purification of boar semen and its storage at
18°C for 48 hours. This increase indicates good
mitochondrial function, which leads to renewal of
ATP. The energy produced by increasing the
production of ATP may be responsible for the
improvement observed in the progressive motility
and velocity of sperm [13]. In contrast, a reduction
of capacity of the mitochondrial membrane
potential of spermatozoa was found in boar semen
preserved at 18 ° C for 0, 24, 48, 72 and 96 hours
without prior colloid centrifugation [14]. It has
been reported by the authors of the above findings
that this reduction may result in dysfunction of
mitochondria resulting in non-renewal of ATP.
Also, it has been found that the integrity of
mitochondria during programmed cell death may
be affected by the release of apoptotic agents
present in the inner and outer mitochondrial
membrane [15].
The effect of colloid centrifugation in selecting
porcine sperm with higher motility has been well
5. Conclusions
In conclusion, colloid purification improves the
quality of boar semen. Both purification and
storage had an influence on mitochondrial
membrane potential.
62
Lymberopoulos A. G. et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
for male infertility. Exp. Cell Res. 1998,
dx.doi.org.scopeesprx.elsevier.com/10.1006/excr.1998.
4064.
12.
Barroso G., Taylor S., Morshedi M., Manzur
F., Gavino F., Oehninger S. Mitochondrial membrane
potential integrity and plasma membrane translocation
of phosphatidylserine as early apoptotic markers: a
comparison of two different sperm subpopulations.
Fertil. Steril. 2006, doi:10.1016/j.fertnstert.2005.
06.046.
13.
Medeiros, C.M., Forell, F., Oliveira, A.T.,
Rodrigues, J.L.,. Current status of sperm
cryopreservation: why isn’t better? Theriogenology,
2002 dx.doi.org/10.1016/ S0093-691X(01) 00674 -4.
14.
Kumaresan A., Kadirvel G., Bujarbaruah
KM., Bardoloi RK., Anubrata Das., Satish Kumar.,
Naskar S Preservation of boar semen at 18 ◦C induces
lipid peroxidation and apoptosis like changes in
spermatozoa.
Anim
Reprod
Sci,
2009,
doi:10.1016/j.anireprosci.2008.01.006.
15.
Ravagnan, L., Roumier, T., Kroemer, G.
Mitochondria, the killer organelles and their weapons. J
Cell Physiol. 2002, DOI:10.1002 /jcp.10111.
16.
Berger, T., Parker, K. Modification of the
zona-free hamster ova bioassay of boar sperm fertility
and correlation with in vivo fertility. Gamete Res.
1989, DOI: 10.1002/mrd. 1120220405.
17.
Horan, R., Powell, R., McQuaid, S., Gannon,
F., Houghton, J.A. Association of foreign DNA with
porcine spermatozoa. Arch. Androl. 1991, doi:
10.3109/01485019108987631.
18.
Grant, S.A., Long, S.E., Parkinson, T.J.
Fertilizability and structural properties of boar
spermatozoa
prepared
by
Percoll
gradient
centrifugation.
J.
Reprod.
Fertil.
1994,
doi: 10.1530/jrf.0.1000477.
19.
Waberski, D., Magnus, F., Ardon, F.,
Petrunkina, A.M., Weitze, K.F., Topfer-Petersen, E.
Binding of boar spermatozoa to oviductal epithelium in
vitro in relation to sperm morphology and storage time.
Reproduction 2006, doi:10.1530/rep.1.00814.
20.
Berger, T., Horton, M.B. Evaluation of assay
conditions for the zona free hamster ova bioassay of
boar sperm fertility. Gamete Res. 1988, DOI:
10.1002/mrd.1120190110.
21.
Suzuki, K., Nagai, T. In vitro fertility and
motility characteristics of frozen–thawed boar
epididymal spermatozoa separated by Percoll.
Theriogenology
2003,
doi:10.1016/S0093691X(03)00115-8.
22.
Matás C., Vieira L., García-Vázquez F.A.,
Avilés-López K., López-Úbeda R., Carvajal J.A.,
Gadea J.. Effects of centrifugation through three
different discontinuous Percoll gradients on boar sperm
function.
Anim.
Reprod.
Sci.
2011,
doi:10.1016/j.anireprosci. 2011.06.009.
References
1.
Cerolini, S., Maldjian, A., Pizzi, F., Gliozzi,
T.M. Changes in sperm quality and lipid composition
during cryopreservation of boar semen. Reproduction
2001, doi:10.1530/ rep.0.1210395.
2.
Brezezinska-Slevbodzinska, E., Slebodzinski,
A.B., Prietas, B.,Wieezorek, G.. Antioxidant effect of
vitamin E and glutathione on lipid peroxidation in boar
seminal plasma. Biol. Trace Elem. Res. 1995, DOI
10.1007/ BF02790102.
3.
Chatterjee, S., Gagnon, C. Production of
reactive oxygen species by spermatozoa undergoing
cooling, freezing and thawing. Mol. Reprod. Dev.
2001, DOI: 10.1002/ mrd.1052.
4.
White, I.G. Lipids and calcium uptake of
sperm in relation to cold shock and preservation: a
review. Reprod. Fertil. Dev. 1993, doi:10.1071/RD
9930639.
5.
. Jones, R., Mann, T., Sherins, R.J.
Peroxidative breakdown of phospholipids in human
spermatozoa: spermicidal effects of fatty acid peroxides
and protective action of seminal plasma. Fertil. Steril.
1979, 1:531–537.
6.
Cummins, J.M., Jequier, A.M., Kan, R., 1994.
Molecular biology of the human male infertility: links
with ageing, mitochondrial genetics and oxidative
stress.
Mol.
Reprod.
Dev.
1994,
DOI:
10.1002/mrd.1080370314.
7.
Kasai T., Ogawa K., Mizuno K., Nagai S.,
Uchida Y., Ohta S., Fujie M., Suzuki K., Hirata S.,
Hoshi K: Relationship between sperm mitochondrial
membrane potential, sperm motility and fertility
potential. Asian J. Androl. 2002, 4, 97–103
8.
Marchetti C., Jouy N, Leroy-Martin B.,
Defossez A., Formstecher P., Marchetti P. Comparison
of four fluorochromes for the detection of the inner
mitochondrial membrane potential in human
spermatozoa and their correlation with sperm motility.
Hum. Reprod. 2004, 19, 2267–2276.
9.
Ericsson SA., Gamer DL, Thomas CA,
Downing TW, Marshall CE. Interrelationships among
fluorometric analyses of spermatozoa1 function,
classical semen quality parameters and the fertility of
frozen-thawed bovine spermatozoa. Theriogenology
1993,
dx.
doi.org.scopeesprx.elsevier.com/10.1016/0093691X(93
)90002-M.
10.
Garner DL, Thomas CA, Joerg HW,
DeJamette JM, Marshall CE. Assessment of
mitochondrial function and viability in cryopreserved
bovine
sperm.
Biol.
Reprod.
1997,
doi:
10.1095/biolreprod57.6.1401.
11.
Troiano L., Granata AR., Cossarizza A.,
Kalashnikova G., Bianchi R., Pini G. Mitochondrial
membra ne potential and DNA stainability in human
sperm cells: a flow cytometry analysis with implication
63
Lymberopoulos A. G. et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1)
Nycodenz and Percoli density gradients for the
selection of motile spermatozoa Fertil and Steril 1988,
49 334—341.
27.
Guerin JF, Mathieu C, Lornage J, Pinatel MC
and Boulieu, D. Improvement of survival and fertilising
capacity of human spermatozoa tested in an in vitro
fertilisation program, by selection on discontinuous
Percoli gradients Hum. Reprod. 1989, 4(7):798-804.
28.
McCIure RD, Nunes L and Tom R. Semen
manipulation: improved sperm recovery and function
with a two-layer Percoli gradient Fertil Steril 1989, 51
874-877.
29.
Englert Y, Van den Bergh M, Rodesch C,
Bertrand E, Biramane J and Legreve A. Comparative
auto-controlled study between swim-up and Percoli
preparation of fresh semen samples for in vitro
fertilisation Hum. Reprod. 1992, 7(3):399-402.
23.
Quintero-Moreno, A., Rigau, T., RodríguezGil, J. Regression analyses and motile sperm
subpopulation structure study as improving tools in
boar semen quality analysis. Theriogenology 2004,
doi:10.1016/S0093-691X(03)00248-6.
24.
Jedrzejczak, P., Rzepczynska, I., TaszarekHauke, G., Pawelczyk, L., Kotwicka, M., Warchot, W.
Effect of sperm subpopulation’s kinetics on human
fertilization in vitro. Syst. Biol. Reprod. Med. 2005,
doi: 10.1080/014850190898791.
25.
Akerlof E, Fredricson B, Custafsson O,
Lundin A, Lunell NO, Nylund I, Rosenberg L and
Pousette A. Comparison between a swim-up and a
Percoli gradient for the separation of human
spermatozoa.
Int.
J.
Androl.
1987,
DOI:
10.1111/j.1365-2605.1987.tb00367.x.
26.
Gellert-Mortimer ST, Clarke GN, Gordon
Baker HW, Hyne RV and Johnston WIH. Evaluation of
30.
64