Effect of long-term storage in Safe Cell+ extender on boar sperm DNA integrity and other key sperm parameters
Bielas et al. Acta Vet Scand
Effect of long-term storage in Safe Cell extender on boar sperm DNA integrity + and other key sperm parameters
Wiesław Bielas 0
Wojciech Niżański 0
Agnieszka Partyka 0
Anna Rząsa 2
Ryszard Mordak 1
0 Department of Reproduction and Clinic of Farm Animals, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences , pl. Grunwaldzki 49, 50-366 Wrocław , Poland
1 Department of Internal Medicine and Clinic of Diseases of Horses, Dogs and Cats, Faculty of Veterinary Medicine
2 Department of Immunology, Pathophysiology and Veterinary Prevention Medicine, Faculty of Veterinary Medicine, Wrocław University of Environmental and Life Sciences , ul. C. K. Norwida 31, 50-375 Wrocław , Poland
Background: There is some controversy about the extent of changes in different sperm cell features in stored boar semen, especially regarding the potential role of the DNA fragmentation assay for assessment of sperm fertilizing ability. The aim of this study was to assess the effect of time of storage and the dynamic changes in sperm cell characteristics in normospermic boar semen stored in long-term extender, in order to determine the susceptibility to damage of particular structures of spermatozoa during cooling and storage at 17 °C for 240 h post collection. The study included five ejaculates from each of seven boars of the Polish Large White breed (n = 35 ejaculates). The sperm characteristics were assessed using a flow cytometer and a computer assisted sperm analyzer on samples at 0, 48, 96, 168 and 240 h post collection. Results: The sperm chromatin structure assay (SCSA) showed a significant abrupt increase (P < 0.01) in the DNA fragmentation index (%DFI) after 48 h of semen storage with only subtle changes thereafter, not exceeding 5% on average after 240 h of storage. The use of a combination of SYBR-14/PI stains did not reveal any significant changes in the percentage of live sperm cells up to 168 h of semen storage. A significant (P < 0.01) decrease in the percentage of live spermatozoa with intact acrosomes was observed after prolonged semen storage (168 h). A significant and progressive decrease in sperm motility was recorded during the whole period of semen storage. Conclusions: Storage of boar semen extended in long-term diluent at 17 °C for 48 h initially induced a decrease in the integrity of sperm DNA. This suggests that the structure of boar sperm DNA is susceptible to damage, especially during semen extension and at the beginning of sperm storage. These findings support the opinion that the SCSA test has only a low potential for routine assessment of boar semen preserved in the liquid state and for assessment of sperm quality changes during 10 days of semen preservation. Remarkably, the integrity of acrosomes and plasma membranes remained nearly unchanged for 7 days.
Boar; Spermatozoa; DNA fragmentation; Flow cytometry; Acridine orange; SCSA; CASA
In the pig industry, the vast majority of sows are still
subjected to artificial insemination (AI) with extended liquid
semen, so that preservation of the fertilizing capacity of
boar spermatozoa for several days remains an important
target for the industry [
]. Up until now, there has been
no breakthrough in the use of frozen boar semen [
mainly due to the high sensitivity of boar spermatozoa to
cooling, freezing and thawing [
]. Commonly, boar
spermatozoa are stored in liquid at 15–17 °C for routine use
in artificial insemination, but extenders for boar semen
for storage even at lower temperatures have also been
available for a number of years [
]. Therefore, there
is a growing interest in the development of new
extenders and determination of optimal storage conditions for
diluted boar spermatozoa. To preserve the quality of
spermatozoa in diluted boar semen during long-term
storage, the choice of long-term extender is critically
]. Long-term extenders have certain
advantages: they allow for better organization in collection
centers, support long-distance transport and provide the
ability to conduct research on the semen before use [
Unfortunately, even if extenders and lower temperatures
can prolong the lifespan of spermatozoa,
physiological senescence of sperm cells still cannot be completely
avoided. Aging-related changes may occur, consisting
of non-regulated, capacitation-like modifications [
structural and functional changes [
], oxidative processes
in cell membranes [
] and damage to DNA integrity
]. These changes can only be partially delayed by using
different extenders [
Among the different indicators of sperm quality
during storage, motility and integrity of the sperm plasma
membrane have been the most evaluated characteristics
in boars. Motility is assessed by means of
computerassisted semen analysis (CASA) [
], and sperm plasma
membrane integrity through flow cytometry [
These methods are good tools for sensitive assessment of
storage effects on sperm quality as well as for evaluation
of new extenders and preservation methods . Both
sperm evaluation systems have been shown to be
accurate, precise and repeatable and have greatly improved
the accuracy, objectivity and reproducibility of sperm
]. However, assessment of motility
and sperm membrane integrity during storage only
partially addresses the lowering of sperm fertilizing
potential caused primarily by aging due to free radicals. Many
factors including storage length, extender type, male
effect, boar age and breed affect boar sperm quality. With
respect to this, assessment of the capacity of a specific
extender to maintain the quality of stored boar
spermatozoa should also include DNA integrity [
], mitochondrial activity , bacterial
contamination, pH determination [
] and apoptotic changes [
One of the key features related to sperm fertility is
the integrity of the nuclear DNA, whose stability largely
depends on the integrity of the chromatin. Therefore,
some authors recommend assessment of chromatin
integrity as a good, complementary and independent
indicator of sperm quality [
]. Sperm DNA
fragmentation tests, such as the DNA fragmentation index (%DFI),
may provide a reliable guide to identify individuals that
are at risk of failing to initiate a healthy pregnancy [
There are several methods to assess sperm DNA
fragmentation, which have been used in the assessment of
boar spermatozoa. These methods are the TUNEL assay
], the sperm chromatin structure assay (SCSA) [
], the Comet assay [
] and the sperm chromatin
dispersion test (SCD) [
A number of studies have indicated the potential of
the SCSA for assessment of boar sperm quality [
] and fertility [
]. The negative, damaging effect
of semen handling and storage on boar sperm DNA has
previously been described, both with respect to liquid
12, 16, 28, 34, 35
] and frozen storage [
Dilution conditions [
28, 35, 39
], time of storage of
liquid semen [
] and age of boars [
], as well as variation
between ejaculates within boars [
12, 32, 41
], may be to
some extent implicated and responsible for the
damage to boar sperm DNA integrity. Thus, damaged DNA
is considered to be one element responsible for reduced
capability of sperm cells to bind to the oviductal
], as well as to underdevelopment of embryos
], and can lead to early embryonic or fetal death or
have a dramatic impact on health of the offspring . In
recent studies, which investigated the relationship of flow
cytometric sperm integrity assessments with boar
fertility, none of the individual membrane integrity variables
was significantly related to fertility except the amount of
DNA damage. These studies have shown that only sperm
chromatin stability had a relationship with fertility from 7
to 10 days and again from 14 to 15 days after ejaculation,
dilution and long-term storage of semen [
Contradictory results were obtained in some recent experiments
showing that the level of DNA fragmentation in liquid
stored boar semen is very low for a long time [
The aim of this study was to investigate changes in
the %DFI determined by SCSA along with changes in
key sperm parameters in boars to elucidate effects of
boar and time of storage on sperm cell quality. During
the study, semen was diluted and stored in Safe Cell+, a
long-term extender for 240 h at 17 °C.
Animals and semen collection
Seven mature boars of the Polish Large White breed
were used, ranging from 18 months to 3 years of age and
selected according to the normal semen quality criteria,
i.e., >50 × 108 total sperm cells per ejaculate, initial
motility >70%, and containing >70% morphologically normal
spermatozoa. The boars in this study were routinely used
in our AI center as semen donors. Boars were randomly
selected among all the AI males. Thirty-five ejaculates were
used in this experiment. Five ejaculates were collected from
each male. Semen was collected once a week in one
Polish boar station, for 5 consecutive weeks. The sperm-rich
fraction of the ejaculate was collected using the gloved
hand technique. Immediately after collection, the following
procedures were done: initial assessment of motility with a
phase-contrast microscope at 200× magnification;
measurement of sperm concentration with a SpermaCue
photometer Porcine (Minitüb GmbH, Tiefenbach, Germany);
and preparation of smears for subsequent staining with
Giemsa stain [
] and routine sperm morphology
assessment (1250× magnification). The sperm-rich fraction was
diluted with Safe Cell+ (IMV Technologies, l’Aigle, France)
long-term extender to a final concentration of 30 × 106
spermatozoa/mL to prepare conventional AI doses for
fresh semen. The extended semen doses of 100 mL
containing approximately 3 × 109 sperm were packaged in plastic
bags. They were slowly cooled down to 17 °C and
subsequently transported to the Laboratory of Andrology at the
Department of Reproduction, Faculty of Veterinary
Medicine in Wrocław within 5 h after collection. In the
laboratory, the semen doses were stored at 17 °C in a boar semen
incubator (Minitüb). Samples for computer assisted sperm
analysis and assessment in a flow cytometer were taken
immediately after arrival at the laboratory (0 h) and again
after 48, 96, 168 and 240 h of storage at 17 °C.
Assessment of sperm cell characteristics
Sperm motion characteristics in extended semen were
evaluated using CASA (Hamilton-Thorne Sperm
Analyser IVOS version 12.2l, Hamilton Thorne Biosciences,
MA, USA), under 1.89 × 10 magnification. A 3 µL
aliquot of semen was placed in a Leja4 analysis chamber
(Leja, Nieuw-Vannep, Netherlands) at 35 °C and
evaluated. Settings of the IVOS were the following: frame
acquired 45, frame rate 60 Hz, minimum cell contrast
46, minimum cell size 7, straightness threshold 45%, path
velocity threshold 45 µ/s, path velocity cut off 20 µ/s,
straight line velocity cutoff 5 µ/s, head size non-motile 7,
head intensity non-motile 50, static head size 0.65–4.90,
static head intensity 0.50–2.50, static elongation 0–87.
Six fields randomly selected by a computer were analyzed
for each semen sample. The motility parameters obtained
by the IVOS analyzer were: VAP (average path velocity,
µm/s), VSL (straight line velocity, µm/s), VCL
(curvilinear line velocity, µm/s), ALH (amplitude of lateral head
displacement, µm), BCF (beat cross frequency, Hz), LIN
(linearity, %), MOT (total motility, %), PMOT
(progressive motility, %), subpopulation of RAPID cells
(velocity > mean velocity of sperm population, %). CASA was
set for analysis (5 microscopic views), more than 200
spermatozoa per sample were examined.
Sperm membrane integrity
Sperm membrane integrity was assessed using dual
fluorescent probes, SYBR-14 and propidium iodide (PI)
(Live/Dead Sperm Viability Kit, Life Technologies Ltd.,
Carlsbad, CA, USA). Samples with a concentration of
30 × 106 spermatozoa/mL were taken for the analysis.
Portions (300 µL) of the samples were pipetted into
cytometric tubes and 5 µL of SYBR-14 working solution was
added. The working solution was obtained by diluting
SYBR-14 in DMSO at a ratio of 1:49. Samples were mixed
and incubated at room temperature for 10 min and then
the cells were counterstained with 5 µL PI (2.4 mM
working solution) for 5 min before analysis [
Acrosomal damage was assessed using PNA Alexa Fluor
488 (Lectin from Arachis hypogaea, Merck Biosciences,
Darmstadt, Germany). Ten microliter PNA Alexa Fluor
488 working solution (1 µg/mL) was added to 500 µL of
sperm sample (30 × 106 spermatozoa/mL) and incubated
for 5 min at room temperature in the dark. Following
incubation, the supernatant was removed by
centrifugation (500×g for 3 min) and the sperm pellets were
re-suspended in 500 µL of Safe Cell+. Then, 5 µL of PI (2.4 mM
working solution, AO; Life Technologies Ltd.) was added
to samples before cytometric analysis [
Assessment of chromatin status
Sperm samples were diluted in Safe Cell+ diluent to a
final concentration of 1 × 106 spermatozoa/mL. The
suspension (200 µL) was subjected to brief acid
denaturation by mixing with 400 µL of lysis solution [Triton
X-100 0.1% (v/v), NaCl 0.15 M, HCl 0.08 M, pH 1.4], held
for 30 s and mixed with 1.2 mL acridine orange solution
(AO; Life Technologies Ltd.) (6 µg AO/mL in a buffer:
citric acid 0.1 M, Na2HPO4 0.2 M, EDTA 1 mM, NaCl
0.15 M, pH 6). After 3 min samples were aspirated into a
flow cytometer [
Assessment of mitochondrial activity
The percentage of spermatozoa with functional
mitochondria was estimated by combining fluorescent stains:
Rhodamine 123 (R123; Life Technologies Ltd.) and PI.
R123 solution (10 µL) was added to 500 µL of diluted
sperm samples (50 × 106 spermatozoa/mL) and
incubated for 20 min at room temperature in the dark.
Samples were then centrifuged at 500×g for 3 min and the
sperm pellets were resuspended in 500 µL Safe Cell+
extender. Then PI (2.4 mM working solution) was added
as previously described [
Flow cytometry (FC)
Measurements were performed on a FACSCalibur
(Becton–Dickinson, San Jose, CA, USA) flow cytometer.
The fluorescent probes were excited by an Argon ion
488 nm laser. SYBR-14 fluorescence (cells with intact
plasma membranes), PNA Alexa Fluor 488 signal (cells
with damaged acrosomes), and Rhodamine 123
fluorescence (cells with active mitochondria) were detected on
detector FL2. PI fluorescence (cells with damaged plasma
membranes) was detected on detector FL1. Green
fluorescence of acridine orange (double-stranded DNA)
was detected on the FL1 detector and red fluorescence of
AO (single-stranded DNA) with detector FL3. This is the
standard protocol for flow cytometer analysis.
Gates were set according to forward and side scatters
to eliminate particles smaller than sperm in cell
aggregates. For SYBR-14/PI, PNA Alexa Fluor 488/PI and
Rhodamine 123/PI fluorochrome quadrants were set
on dot plots of the logs of green fluorescent events (live
spermatozoa, damaged acrosomes, active mitochondria),
and red fluorescent events (dead spermatozoa) and dual
The extent of DNA denaturation, expressed as the
DNA fragmentation index (%DFI), was calculated based
on the ratio of red/total (red + green) fluorescence for
each sperm cell in the sample [
]. For each sample, two
terms of DFI were evaluated: the percentage of
spermatozoa outside the main population with denatured
DNA (%DFI) and the percentage of spermatozoa with an
abnormally high DNA stainability (%HDS). The
percentage of HDS cells was calculated by setting the
appropriate gate above the upper border of the main cluster of the
sperm population with no detectable DNA denaturation,
mainly immature cells.
Acquisitions were performed using the CellQuest 3.3
software (Becton–Dickinson, San Jose, CA, USA). The
non-sperm events were gated out based on scatter
properties and excluded from analysis. A total of 40 × 103
events (spermatozoa) were analyzed for each sample.
The results obtained, presented as mean ± SD of
measurements on samples from 35 replicate determinations,
were analyzed by ANOVA considering the time of
storage and boars as the main variables. When ANOVA
revealed a significant effect, values were compared by
the least significant difference pairwise multiple
comparison post hoc test (Tukey’s test). Differences were
considered to be significant if the calculated probability
of their occurring by chance was <5% (P < 0.05). The
statistical model included the effect of time of storage and
the interaction between boar and time of storage. All
percentage data were arc sin transformed to normalize
The Spearman’s rank correlation coefficients were
calculated to measure the statistical dependence among all
variables, i.e., among all parameters assessed in the study
at 0, 48, 96, 168 and 240 h of sperm storage.
Characteristics of fresh semen
The mean volume of ejaculates collected from the boars
was 264.2 ± 47.4 mL. The percentage of progressively
motile spermatozoa assessed subjectively in fresh semen
and the concentration of spermatozoa per mL were
76.6 ± 6.1 and 516.5 × 106 ± 134.2 × 106, respectively.
The percentages of sperm cells with primary and
secondary defects of morphology were 12.9 ± 4.6 and 3.1 ± 1.7,
respectively (mean ± SD).
CASA and flow cytometric assessment of spermatozoa
A gradual decrease of MOT and PMOT of
spermatozoa was observed in samples stored in long-time
extender (Table 1). The decrease of MOT was
significant (P < 0.01) in all analysis periods, beginning from
48 h. The values for MOT of spermatozoa stored at 168
and 240 h were relatively low. Similarly, initial values
of PMOT were below 25%. There were no significant
differences between PMOT at 0, 48 and 96 h. A
significant drop of PMOT at 168 h was noted (P < 0.001).
The initial mean value of STR was 40.9% ± 4.7 and LIN
was 20.4% ± 2.7 (mean ± SD). STR and LIN increased
concomitantly with the decrease of ALH and PMOT.
A gradual decrease in the velocity of spermatozoa was
observed. A dramatic drop of VAP and the
subpopulation of RAPID cells at 96 and 168 h of semen storage was
observed. A significant effect on all sperm motion
characteristics was shown for time of storage (P < 0.0001),
and with the exception of MOT and BCF, for the
factors boar and interaction of boar and time (P < 0.05,
P < 0.0001) (Table 1).
The percentage of spermatozoa with an intact plasma
membrane (i.e., live spermatozoa) was relatively high and
nearly unchanged up to 168 h of sperm storage (Table 2).
At 240 h of storage a significant (P < 0.01) decrease in
live cells was observed. The percentage of dead cells
increased (P < 0.01) earlier, i.e., at 168 h of sperm
storage. The percentage of cells that exhibited a partly green
fluorescence and a partly red fluorescence (moribund,
dying cells) remained unchanged during the whole
The percentage of live cells with an intact acrosome
began to decrease significantly (P < 0.01) at 168 h of
storage (Table 2). However, even on the 10th day of storage
almost 80% of sperm cells possessed intact acrosomes.
The percentage of live spermatozoa with damaged
acrosomes remained constant during storage up to
240 h; a significant difference was detected only for the
factor boar (P < 0.0001). The percentage of dead
spermatozoa with intact acrosomes remained unchanged
for 168 h of sperm storage. The value of this parameter
increased significantly (P < 0.05) at 240 h of sperm
storage whereas the percentage of sperm cells with damaged
plasma membranes and damaged acrosomes remained
nearly constant during the whole period of sperm
The %DFI, describing the percentage of spermatozoa
outside the main population with denatured DNA, increased
significantly (P < 0.01) within a short time after semen
collection and dilution, and was already apparent at 48 h
of sperm storage (Table 2). At each time point of sperm
assessment, a significant increase in %DFI was observed.
A significant effect on sperm chromatin integrity was
shown for boar and time (P < 0.0001 and P < 0.05,
respectively) The percentage of spermatozoa with an
abnormally high DNA stainability (%HDS), i.e., immature
cells was similar at all times of sample analysis.
A gradual increase in the percentage of live
spermatozoa with inactive mitochondria was observed. There was
a significant difference (P < 0.05) between 0, 168 and
240 h of storage (Table 2). No significant differences were
noted in the percentage of live spermatozoa with active
mitochondria during storage (P > 0.05) between 0 and
240 h. There were no significant differences between the
percentages of dead sperm cells with active and inactive
mitochondria in consecutive measurements. However, a
significant effect on these subpopulations of dead sperm
cells was shown for boar and time of storage (P < 0.0001
and P < 0.05, respectively).
Relationships among boar sperm cell characteristics during storage
In Fig. 1, the relationship between the %DFI and
motility (MOT and PMOT) and the viability, acrosome and
plasma membrane integrity characteristics, is shown for
35 ejaculates, representing different patterns of changes
in these boar sperm cell parameters during storage.
Statistically significant correlations between motility
parameters at consecutive hours of semen incubation
(Figs. 2, 3) were observed. The experiment also revealed
many statistically significant correlations among the
majority of the structural parameters of boar sperm cells
during incubation. Moreover, the %DFI and %HDS were
well correlated with structural parameters at 0 h of
incubation. After that only %DFI was strongly correlated with
sperm structural features while %HDS lost that feature.
At 240 h of incubation, neither parameter presented
significant correlations with structural characteristics of
sperm cells. Overall, there were no strong correlations
between motility parameters and structural parameters
of boar spermatozoa stored in the liquid state.
Artificial insemination in pigs is mostly done using boar
semen preserved in the liquid state at 16–17 °C.
Therefore, semen in the present study was diluted and stored
in extender at 17 °C. While extenders for boar semen for
storage at lower temperatures have been available for a
number of years [
], these are not sufficiently effective
for practical use in pig AI and further work is needed to
produce efficient low temperature extenders.
The decrease of MOT and PMOT is similar to values
obtained by others [
]. The percentages of motile
sperm stored for 5 days in long-term extenders X-cell,
Androstar and Mulberry III were 55.6, 49.9 and 80.5,
We found that the percentages of boar spermatozoa
with intact plasma membranes were usually more that
80% and did not change significantly during storage for
168 h. Similar results concerning sperm plasma
membrane integrity in stored boar spermatozoa were obtained
by others [
The study revealed a relative stability of sperm
acrosomal membranes during storage of boar semen in the
liquid state. It should be noted that there was a discrepancy
between apparent loss of sperm motility and unchanged
percentages of acrosome intact spermatozoa at
consecutive measurements up to 168 h of semen storage.
Based on a total of 35 ejaculates collected from seven
boars over a period of 5 weeks, 17.1% of the sperm
samples showed >3%DFI, 11.4% showed >5%DFI, and
5.7% showed >10% of %DFI. Thus, our findings confirm
0 h 48 h 96 h 168 h 240 h
Fig. 1 Changes in motility, progressive motility, acrosome integrity, plasma membrane integrity and DNA fragmentation in spermatozoa stored for
240 h at 17 °C (mean ± SD, n = 35). Characteristics assessed by computer assisted sperm analyzer: MOT-percentage of motile spermatozoa;
PMOTpercentage of progressively motile spermatozoa. Characteristics assessed by flow cytometer: PNA− PI− live spermatozoa with intact acrosome;
SYBR-14+/PI− live spermatozoa; %DFI the percentage of spermatozoa with DNA fragmentation
previous observations about relatively low levels of %DFI
in fresh boar semen [
16, 26, 31, 34
It has been proved that differences in sperm DNA
damage between ejaculates can result from external
factors such as collection procedure, handling, dilution or
internal factors, e.g., inherent chromatin packaging of the
spermatozoa, the composition of seminal plasma and of
accessory gland fluid including zinc ions, zinc-binding
proteins, low molecular weight antioxidants and proteins
with antiperoxidant properties [
]. There is a current
debate about whether these intrinsic or extrinsic factors
cause different reactions of the sperm chromatin to the
SCSA procedure. In our study, the influences of external
factors were minimized just as in earlier studies [
However, in the present study, sperm rich fractions were
used, which may mean that the antioxidant properties of
the entire seminal plasma were reduced [
In the present study, the average %DFI of spermatozoa
in fresh semen after extension (0 h) in 35 ejaculates from
seven boars was 3.55%. We found an initial significant,
abrupt rise of %DFI at 0 h and again at 48 h of
incubation in long-term extender. The %DFI obtained here
increased slightly but significantly during the 240 h of
storage after collection, up to an average of 4.71 ± 2.2%.
This increase was greatest (+34.48%) between day 0 and
day 2, and in most boars the percentages of spermatozoa
with fragmented DNA were almost always lower than 5%
up to 240 h of storage. Our data agree with the results
of Broekhuijse et al. [
] who reported that the %DFI at
day 0 was 3.15% and increased to 4.19% during 15 days
of liquid storage, and the greatest increase of %DFI was
observed between day 0 and day 1.
It was also previously reported that extended boar
spermatozoa showed an increase in DNA instability from
day 0 to day 4 in some extenders [
]. Contrary to
this, De Ambrogi et al.  suggested that the customary
storage of boar semen for 96 h at 17 °C was too short an
interval to cause loss of integrity in nuclear DNA. Similar
results were obtained by Waberski et al. [
] in the first
part of their study. However, in the second part of their
study, they obtained a slight but significant increase in
mean %DFI results from 2.2% initially up to 2.7% at 120 h
of semen storage.
In our study, only two out of 35 ejaculates showed an
increase of %DFI above 10% (12.6; 10.8) during 240 h
of storage. This is in accordance with findings of other
authors who showed a significant increase of DFI during
168 h of storage in only three out of 42 ejaculates [
The results support the concept of relatively low
sensitivity of boar sperm DNA to defragmentation during
storage of liquid semen [
12, 16, 19, 34
]. The differences
in DNA fragmentation on different days of storage were
generally rather low compared to changes in motility or
sperm viability. It can be assumed that these slight
differences may have no biological significance. This
assumption supports the conclusion presented by Hernandez
et al.  who stated that the low overall DNA damage
observed in frozen-thawed spermatozoa seemed to have
little biological importance. Waberski et al. [
demonstrated that evaluation of sperm chromatin
structural integrity by the SCSA has only limited value for
identifying deficiencies in normospermic fresh or stored
The values of %HDS in boars of the Polish White Large
breed were similar to values obtained by others [
Landrace, Danish Large White and Hampshire boars.
We did not observe any trend of decrease or increase of
%HDS during sperm storage. The similarity of HDS
values during the whole period of semen storage is probably
due to the fact that the %HDS value is mainly determined
by the initial integrity of DNA structure resulting from
the quality of spermatogenesis. In humans, the
population of %HDS is supposedly composed of immature cells
that lack chromatin condensation [
] and may also
represent doublets or triplets of spermatozoa. This is
consistent with our observation that storage of boar semen
did not increase the %HDS population. Thus, it seems
that %HDS is not a useful marker of changes in the
quality of liquid stored boar spermatozoa.
Significant differences in chromatin structure of
stored spermatozoa between individual boars were
also detected. The study included only seven boars, but
there were differences among males with respect to
the quality of their sperm characteristics during
storage. We discovered a significant influence of boar on
chromatin integrity (Table 2) in spermatozoa stored
for 240 h at 17 °C, and also on the sperm motion
characteristics (with the exception of BCF) (Table 1), on
plasma membrane integrity (Table 2), on acrosome
integrity (Table 2) and on mitochondrial activity (with
the exception of dead sperm cells with active
mitochondria) (Table 2). These study reveal that there is
individual variation among boars concerning preservation of
DNA integrity during storage, which is in accordance
with Fraser and Strzeżek [
] and Sutkeviciene et al.
]. This indicates that the effect of individual boar is
of great importance concerning sperm quality during
longtime storage and must always be taken into account
in the assessment both of DNA fragmentation and other
sperm variables. No previous studies have investigated
sperm DNA integrity using the SCSA parameters in the
semen of normospermic, healthy boars of the Polish
Large White breed. However, our preliminary results
need to be investigated further in a larger study to
evaluate and understand the precise mechanism
maintaining sperm DNA integrity.
Sperm characteristics, especially all motility and
structural parameters, were also significantly affected by the
storage time. This means that the time points when these
characteristics were assessed during storage had a
significant impact on these properties of the stored boar sperm
The interaction of boar and time of incubation as a
source of variability only affected motility parameters
(without MOT) assessed by CASA during storage, which
indicates that the boar factor as well as time of semen
storage play important roles in assessment of these
motility traits during long-term liquid storage of boar semen.
Meanwhile, the non-significant interactions between
boar and time points in the remaining assays of sperm
characteristics indicate that influences of boar and time
of semen storage were homogeneously distributed among
the sperm parameters studied. This may mean that these
characteristics of spermatozoa provide an additive value
in assessing the quality of long-term liquid-stored boar
Significant negative correlations among SCSA variables
and most classical sperm quality parameters in fresh and
cryopreserved semen were shown in rams [
], bulls [
and humans [
]. The significant correlations between
SCSA and classical sperm quality parameters suggest
that, taken together, both types of assays are better
predictors of sperm quality and male fertility than each one
separately. The negative correlation between %DFI and
sperm viability was also detected in stored boar
]. In another study [
] when ejaculates from
only four boars were included, the increase of %DFI was
accompanied by increased deterioration of sperm plasma
membrane integrity during storage. Other researchers
] did not find a significant correlation between the
%DFI and the standard boar sperm variables during
longterm storage. We found significant correlations between
%DFI and %HDS and structural sperm parameters at
0 h of storage after which the only significant
correlations were observed for %DFI. It may be concluded that
%DFI changes are associated with the disruption of other
sperm structures during storage. On the other hand,
%HDS is the parameter associated with abnormalities
of spermatogenesis and is only partly independent from
storage time. Correlations between both parameters and
structural features were lost at 240 h of storage. This
may indicate that assessing DNA integrity has an
additive value for standard sperm assessment only in cases of
extremely long storage times.
It is noteworthy that detailed analysis of correlations
between values obtained in the present study revealed
high, significant correlations among the majority of
motility parameters. Therefore, it may be suggested that
routine analysis on 1–2 motility parameters is adequate
for proper evaluation of motion properties of stored
boar spermatozoa. It should be borne in mind that we
revealed almost no correlation between motility
features and structural sperm parameters. The high
correlation between all motility parameters and the lack of
correlation between motility and structural features was
characteristic during the whole time of sperm storage.
Therefore, motility and structure may be treated as
separate, partly independent features that always have to be
We did not observe abrupt changes of mitochondrial
activity over time in the populations of live and dead
spermatozoa. It is obvious, that in the population of
dead spermatozoa the proportion of cells with inactive
mitochondria remained nearly unchanged at
consecutive assessment points. However, it is more difficult to
understand why changes in mitochondrial potential were
so subtle in the group of live cells in spite of the rapid
decrease in progressively motile spermatozoa. It appears
that the dynamics of the increase in percentages of live
cells with inactive mitochondria were similar to the
dynamics of other tests performed on the flow cytometer
rather than the dynamics of motility changes recorded on
The most sensitive method for assessing changes in
sperm cell features during storage at 17 °C are those
describing populations of motile cells and parameters
related to speed of motility. Plasma and acrosome
membrane integrity and mitochondrial function
characteristics are relatively resistant to storage in long-term
semen extender and change to a lesser degree. Although
increased DNA fragmentation was revealed, the extent
of these changes was relatively low and it appears that
extenders efficiently protect DNA structure. These
findings support the opinion that the SCSA test has
relatively little value for routine evaluation of changes in boar
sperm characteristics during semen storage in long-term
WB participated in the study design and was involved in manuscript
preparation; WN contributed to the study design, interpretation of the results,
coordinated clinical work and was responsible for revision of the manuscript; AP
performed semen analysis, and participated in statistical analysis; AR participated
in collection of samples and coordinated clinical work; RM participated in the
collection of samples. All authors read and approved the final manuscript.
Wrocław University of Environmental and Life Sciences, pl. Grunwaldzki 47,
50-366 Wrocław, Poland.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from
the corresponding author on reasonable request.
Consent for publication
The experimental protocol was previously approved by the Local Ethical
Committee for Experimentation with Animals, Wrocław Poland.
This study was supported by a Polish Government grant provided by the
Ministry of Science and Higher Education/National Science Center in Poland-N308
008 31/3067. Project supported by Wrocław Centre of Biotechnology,
programme The Leading National Research Centre (KNOW) for years 2014–2018.
Springer Nature remains neutral with regard to jurisdictional claims in
published maps and institutional affiliations.
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