Journal of Proteomics 162 (2017) 52–61

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Journal of Proteomics

journal homepage: www.elsevier.com/locate/jprot

Proteomic identification of seminal plasma proteins related to the

freezability of carp semen
Mariola A. Dietrich a,⁎, Ilgiz Irnazarow b, Andrzej Ciereszko a
a
Department of Gametes and Embryo Biology, Institute of Animal Reproduction and Food Research, Polish Academy of Sciences, Tuwima 10, 10-748 Olsztyn, Poland
b
Institute of Ichthyobiology and Aquaculture of Polish Academy of Science Gołysz, 43-520 Chybie, Poland

a r t i c l e i n f o a b s t r a c t

Article history: The variation in sperm freezability among individuals within a fish species is a major factor justifying the identi-
Received 12 December 2016 fication of useful predictive indicators of cryopreservation success. It is unknown at present whether the protein
Received in revised form 4 April 2017 composition of fish seminal plasma affects sperm freezability. Therefore, the aims of this study were to compare
Accepted 14 April 2017
the proteome of carp seminal plasma from semen rated as good (GF) and poor (PF) freezability by two-dimen-
Available online 24 April 2017
sional difference gel electrophoresis followed by MALDI-TOF/TOF mass spectrometry. The semen was classified
Keywords:
as GF and PF based on sperm motility assessment after freeze/thawing. Five spots representing three proteins
Cryoresilience were more abundant in GF, while ten spots representing seven proteins were more abundant in PF seminal plas-
Cryopreservation ma. The majority of proteins present in higher abundance in PF seminal plasma were associated with the innate
Proteome immune response. On the other hand, higher freezability was associated with proteins involved in the mainte-
Seminal plasma nance of sperm membrane integrity and antioxidative protection. These results indicate that carp semen
Fish freezability levels may be related to different seminal plasma protein profiles. Lower usefulness of spermatozoa
in cryopreservation may be related to previous infection or stress leading to sublethal changes to sperm structure.
Significance: Sperm quality parameters such as motility, viability and sperm concentration have been used as pre-
dictive tools of sperm cryopreservation potential in fish species However, the usefulness of initial motility param-
eters as indicators of freezability varies among fish species and between individuals within a species. Recent
studies in mammals revealed that male-to-male variability in cryoresistance can be attributed to differences in
seminal plasma protein composition. To the best of our knowledge, no proteomic studies linking the protein
composition of fish seminal plasma and freezing resilience have been performed in fish. Our results indicate
for the first time that factors regulating how carp semen tolerate cryopreservation may be related to the different
protein profiles of carp seminal plasma. The obtained results provide new insight into understanding the molec-
ular mechanisms underlying cryoresistance of carp semen and provide a tool for the improvement of a long-term
sperm preservation procedure.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction from some individuals of carp, and cryoinjuries are characterized by

Cryopreservation of fish sperm is an important tool for the conserva-
tion of biological diversity in fish populations (fish sperm cryobanks)
and endangered species, efficient and selective breeding and the syn-
chronization of artificial reproduction [1–4]. Carp breeding programs in-
volve the use of a variety of strains, and cryopreservation can be an
effective strategy to secure the sperm of desired strains and to protect
their genetic variability. However, each cryopreservation procedure
has deleterious effects on the sperm compartments and their functions
[5,6,7]. Several effective cryopreservation protocols have been devel-
oped for carp semen [8,9,10,11]. It is noteworthy that these protocols
do not produce satisfactory results for the cryopreservation of semen
⁎ Corresponding author.
E-mail address: m.dietrich@pan.olsztyn.pl (M.A. Dietrich).
inter-male variability [12]. The mechanism of such a phenomenon re-
mains unclear. Therefore, it is of paramount importance to define the
variables that can accurately predict cryoresilience and to use these in-
dices to improve cryopreservation procedures by precise selection of
samples prior to freezing.
Recent research in fish species has identified a number of semen
quality indices of successful cryopreservation (i.e., motility, viability,
sperm concentration, osmolality, pH, enzymatic activity, ATP concentra-
tion, membrane lipid composition and antioxidant activity) [4,12,13–
17]. However, the usefulness of such bioindicators varies among fish
species and between individuals within a species [18]. Recent studies
in mammals revealed that male-to-male variability in cryoresistance
can be attributed to differences in seminal plasma protein composition.
A number of proteins were found to be more or less abundant in high
and low freezability groups in bull, buffalo, boar, stallion and ram

http://dx.doi.org/10.1016/j.jprot.2017.04.015
1874-3919/© 2017 Elsevier B.V. All rights reserved.
 M.A. Dietrich et al. / Journal of Proteomics 162 (2017) 52–61
specimens [19–24]. However, to the best of our knowledge, no proteo-
mic studies linking the protein composition of fish seminal plasma
and freezing resilience have been performed.
The seminal plasma of fish is designed to provide an optimal envi-
ronment for the storage of spermatozoa before spawning [25]. Our re-
cent proteomic analyses of the protein composition of carp seminal
plasma [26] revealed potential functions of seminal plasma proteins
(transferrin, apolipoproteins, Wap65 and alpha1-antiproteinase) in
the protection of sperm motility and viability against proteolytic and ox-
idative attack as well as heavy metals [26,27,28]. Moreover, proteomic
studies suggest that lipid and cholesterol binding is an important func-
tion of seminal plasma proteins. This function seems to be performed by
apolipoproteins (ApoA and ApoE) and may be related to the mainte-
nance of sperm membrane integrity and the energy requirements of
sperm. Such data suggest that carp seminal plasma proteins exert an im-
portant effect on sperm function and structure, which may influence
inter-male variation in the freezing resilience of carp semen. Therefore,
the identification of proteins responsible for such effects on sperm func-
tion and freezability are justified.
The post-thaw motility is commonly used as indicator of sperm
quality both in mammals and fish. This parameter has been used in
the dairy industry for evaluation of cryopreservation success and as a
discriminating factor in proteomic studies concerning freezability of
mammalian semen [22,24,29,30]. Recently Horokhorvatskyi et al. [12]
used post-thaw sperm motility to separate carp semen according to its
cryoresistance. In fish the importance of sperm motility of frozen-
thawed milt was confirmed in several studies indicating the correlation
of post-thaw motility and fertilization capacity [14]. Moreover the Com-
puter-assisted sperm motility (CASA) assessment is of significant value
in predicting the ability of sperm to achieve fertilization since the per-
centage of motile sperm and the sperm progressive velocity are the
sole predictors of fertility in fish, including carp [14,31,32].
The aims of the present study were (1) to characterize variation in
the resilience of carp spermatozoa to cryopreservation using post-
thaw motility measured by CASA system, (2) to compare the proteome
of carp seminal plasma of poor and good freezability semen using the
2D difference gel electrophoresis (2D–DIGE) technique and (3) to iden-
tify differentially abundant proteins among these fluids.
2. Material and methods
2.1. Semen collection and preparation of seminal plasma
The milt of the common carp was obtained from fish maintained at
the Institute of Ichthyobiology and Aquaculture of the Polish Academy
of Sciences in Gołysz, Poland. Healthy looking males were randomly se-
lected out of a larger population of spawners on the basis of external
maturity signs during the natural spawning period. The group of 5 to
7 years old males (n = 20) was placed in the tank of 2.5 m3 in volume
in aerated water at 19–20 °C and kept for two-day adaptation period.
Twenty-four hours before the collection of carp semen, the males
were injected intradorsally with Ovopel (one pellet containing 18–20
μg of a GnRH analogue and 8–10 mg of metoclopramide per 1 kg of
fish body weight; Interfish Ltd., Hungary). The milt was obtained during
the middle of the spawning season (July 2nd) by gentle abdominal mas-
sage, taking care not to pollute them with blood, faeces or urine. Ap-
proval from the Animal Experiments Committee in Olsztyn, Poland
(no. 93/2011) was obtained prior to experimentation.
Briefly, the quality of the fresh carp semen was first evaluated to ver-
ify if the minimum quality standard for sperm cryopreservation was ful-
filled (80% sperm motility, sperm concentration at least 10 × 109/ml).
Each semen sample was divided into two aliquots after collection. The
first aliquot was used for seminal plasma separation from spermatozoa
through centrifugation at 700 ×g for 30 min (4 °C), followed by centri-
fugation of the supernatants for 10 min at 12,000 ×g and subsequent
53
storage at −80 °C until analysis was carried out. The second semen ali-
quot was subjected to cryopreservation.
2.2. Cryopreservation of semen
The freezing protocol recommended for cryopreservation of carp
semen described by Kopeika [33] as modified by Li et al. [34] was ap-
plied. This protocol was also used by Dzyuba et al. [35] and
Horokhovatskyi et al. [12] and has been routinely applied for three con-
secutive years by our team for cryopreservation of semen from selected
carp strains maintained in Gołysz. The usefulness of this protocol was
confirmed by high values of post-thaw motility ranging from 40 to
70% (Dietrich et al., unpublished data).
Semen samples were diluted (1:9) in an extender composed of
59 mM NaCl, 6.3 mM KCl, 0.68 mM CaCl2, 2.1 mM Mg2SO4, 27 mM
NaHCO3, 3.4 mM sucrose, 69 mM D-mannitol, 118 mM Tris-HCl,
pH 8.1 and 16% ethylene glycol. After dilution with the extender, the
samples were immediately loaded into 0.5-ml straws and kept in liquid
nitrogen vapour 3 cm over the surface of the liquid nitrogen for 5 min.
Following freezing, the straws were placed directly into liquid nitrogen
for storage. The straws were subsequently thawed by immersing them
in a water bath (40 °C) for 13 s. The thawing procedure was performed
in duplicate. Thawed sperm samples were immediately used for the
analysis of sperm motility parameters.
2.3. Analysis of fresh and cryopreserved semen
The motility parameters of fresh and cryopreserved spermatozoa
were determined with Computer Assisted Sperm Analysis (CASA) as de-
scribed by Wojtczak et al. [36]. The two-step method for motility mea-
surement described by Rurangwa et al. [37] was used. As a first step,
carp semen were diluted 100-fold in an immobilizing buffer (94 mM
NaCl, 27 mM KCl, 50 mM glycine, 15 mM Tris–HCl, pH 7.5) in a 1.5-ml
polyethylene tube. Sperm motility was then activated by mixing 1 μL
of this solution with 19 μL of distilled water containing 0.5% albumin
(final sperm dilution 1:2000). Immediately after sperm activation, 0.7
μL of sperm subsamples was placed into a well of a 12-well multi-test
glass slide and covered with a coverslip. Sperm motility parameters
were analysed with a 15–30 s post-activation time using a Hobson
Sperm Tracker (Hobson Vision Ltd., Baslow UK) with the setting as de-
scribed by Wojtczak et al. [36]. Each sample was analysed in duplicate.
The sperm tracker simultaneously assessed fifteen sperm motility pa-
rameters, but, for brevity, only five representatives were chosen for fur-
ther analysis: straight line velocity (VSL), curvilinear velocity (VCL),
amplitude of lateral head displacement (ALH) and % of motile sperm
(MOT). The sperm concentration was measured by spectrophotometry
[38]. Protein concentration in seminal plasma was determined using the
Pierce 660 nm Protein Assay (Pierce/Thermo Scientific, Rockford).
2.4. Sample classification into good and poor freezability group
To distinguish seminal plasma samples between two groups of good
(GF) and poor (PF) freezability, carp semen was cryopreserved and
thawed and sperm quality assessments were carried out using the com-
puter assisted sperm analysis system (CASA). Carp with good
freezability were characterized by reduced sperm motility of no N25%
compared to fresh semen, while semen classified as poor freezability
showed reductions of N 40% in sperm motility.
2.5. Preparation of seminal plasma for proteomic analysis
Prior to proteomic analysis, the seminal plasma of GF and PF males
was concentrated using Amicon ultra centrifuge filter with cut off
3 kDa (Millipore). Aliquots containing approximately 800 μg of seminal
plasma proteins were processed using a Clean-Up Kit (GE Healthcare,
Uppsala, Sweden) according to the manufacturer's protocol. Samples
54 M.A. Dietrich et al. / Journal of Proteomics 162 (2017) 52–61
were resuspended in 80 μL of DIGE labelling buffer consisting of 30 mM
Tris, 7 M urea, 2 M thiourea and 4% CHAPS to a protein concentration of
5–10 mg/mL. The protein concentration prior to and after the cleaning
procedure was measured by a Coomassie Plus Kit (Thermo Scientific,
Rockford, IL, USA) with bovine serum albumin as the standard.
2.6. Fluorescent labelling of seminal plasma of good and poor freezability
with CyDyes and 2D–DIGE analysis
Protein labelling with CyDye DIGE fluors (Cy2, Cy3, Cy5) and 2D
electrophoresis were performed under the same conditions as de-
scribed by Dietrich et al. [39]. Aliquots of 50 μg of protein from each
sample (seminal plasma of GF and PF, n = 6 for each group) were la-
belled with CyDye DIGE Fluor minimal dyes (GE Healthcare, Uppsala,
Sweden; reconstituted in fresh 99.8% anhydrous dimethylformamide)
at a concentration of 400 pmol dye/50 μg of protein. Cy2 was used to
label the internal standard consisting of 50 μg of protein aliquots from
each sample of all six biological replicates from each group, and Cy3
and Cy5 to label individual seminal plasma samples of GF and PF. A
dye swap (Cy3/Cy5) was performed between seminal plasma samples
of GF and PF males (Supplementary Table S1) to exclude dye bias. The
labelling reaction was performed in the dark on ice for 30 min.
Differentially labelled samples (50 μg each of Cy2-, Cy3-, and Cy5-la-
belled samples) were mixed together according to the scheme present-
ed in Supplementary Table S1, uploaded onto 24 cm IPG strips (pH 3–10
NL), and isoelectric focusing (IEF) was performed with an Ettan IPGphor
apparatus (GE Healthcare) as described by Dietrich et al. [39]. As a sec-
ond dimension, a 12.5% SDS-PAGE (gel size 25.5 × 19.6 cm, 1-mm thick-
ness, GE Healthcare) was run using the Ettan Dalt-Six apparatus (GE
Healthcare). Each gel contains seminal plasma of GF (Cy3 or Cy5), sem-
inal plasma of PF (Cy5 or Cy3) and an internal standard (Cy2). The
CyDye-labelled gels were analysed by post-run fluorescence imaging
with the use of Typhoon FLA 9400 (GE Healthcare) with parameters rec-
ommended for 2D DIGE experiments by the manufacturer. After images
were acquired, image analysis was performed using DeCyder Differen-
tial In Gel Analysis version 5.02 software (GE Healthcare). The number
of spots was estimated to 10,000, and a spot volume of 30,000 was
used as a cutoff filter. The DeCyder biological variation analysis module
was used to detect protein spots and simultaneously match all twelve
protein spot maps from six gels. Protein spots with a p-value b0.05 by
t-test analysis that showed at least a 1.5-fold increase or decrease in
their relative intensities in any comparison between seminal plasma
of GF and PF were considered as differentially expressed. False Discov-
ery Rate (FDR) correction was applied when the t-test was performed.
To properly pick and identify the selected spots, DIGE gels were
restained using Coomassie Brilliant Blue G-250 (Bio-Rad, Hercules,
CA). Spots presenting significant differences between seminal plasma
of GF and PF were manually excised, trypsin digested, and identified
with matrix-assisted laser desorption/ionization time-of-flight/time-
of-flight (MALDI-TOF/TOF) mass spectrometry (MS).
2.7. Mass spectrometry protein identification
Spots presenting significant differences between seminal plasma of
GF and PF semen were cut from gels, subjected to tryptic digestion
using modified sequencing grade trypsin (Promega, Madison, WI,
USA) and identified as described by Dietrich et al. [39]. After digestion,
the spots were concentrated and desalted using Zip-Tip C18 pipette
tips (Millipore) as described by Dietrich et al. [39]. Peptide samples
were analysed on a time-of-flight Autoflex-Tof/Tof mass spectrometer
(MALDI-TOF/TOF, Bruker Daltonics, Bremen, Germany). The MS and
MS/MS LIFT spectra of selected ions were collected and calibrated exter-
nally using monoisotpoic [M + H] + ion peptide calibration standards
(Bruker Daltonics) consisting of Angiotensin II (1046.54), Angiotensin
I (1296.68), Substance P (1347.73), Bombesin (1619.82), ACTH clip 1
(2093.086), ACTH clip 18 (2465.19) and Somatostatin 28 (3147.471).
MS peptide mass fingerprint (PMF) and fragment mass spectra (MS/
MS) from each individual spot were combined and used to search
against a NCBInr Bony Fishes database (database containing 3,005,095
proteins was generated on 2017.01.30) using MascotServer (Matrix Sci-
ences) with the following settings: cleavage enzyme: trypsin, max
missed cleavages: 2 and mass tolerance mono: 50 ppm, fragment ion
mass tolerance of 0.5 Da; parent ion mass tolerance of 200 ppm; alkyl-
ation of cysteine by carbamidomethylation as a fixed modification;
and oxidation of methionine as a variable modification. For the PMF
and MS/MS ion search, statistically significant (p ≤ 0.05) matches by
MASCOT were regarded as correct hits. Proteins identified as hypothet-
ical were searched in terms of protein sequence similarity using the
Basic Local Alignment Search Tool (BLAST).
2.8. Protein-protein interaction analysis
Protein–protein interactions were analysed using a Web-based
bioinformatic Search Tool for the Retrieval of Interacting Genes/
Proteins (STRING) software, version 10. STRING is a database of in-
teractions for known or predicted proteins, including physical and
functional associations from genomic contexts, high-throughput ex-
periments, coexpression and previous knowledge. The identified
proteins that were more abundant in seminal plasma of GF and PF
semen were entered into STRING for analysis of potential protein–
protein interactions. The interaction search was restricted to Homo
sapiens (Human, 9606) protein pairs. Each interaction has a com-
bined score (edge score), which represents the reliability of the in-
teraction between proteins.
2.9. Validation of 2D–DIGE results by Western blotting
To confirm the identification of ApoA-I as a protein differing in abun-
dance between GF and PF semen, antibodies against carp ApoA-I pro-
duced in our laboratory by immunization of rabbit with ApoA-I
isolated from carp seminal plasma were used [40]. Aliquots of seminal
plasma proteins of GF (n = 6) and PG (n = 6) semen (100 μg) in a
total volume of 125 μl of rehydration buffer were applied to 7-cm IPG
strips (pH range 4–7; GE Healthcare). Isoelectric focusing was per-
formed according to the manufacturer's protocol (GE Healthcare), and
then the IPG strips were equilibrated with 2% dithiothreitol and 2.5%
iodoacetamide. Each strip was laid onto a 15% SDS-PAGE gel (10 × 8 ×
0.1 cm) for second dimension electrophoresis. Western blots were per-
formed as described by Dietrich et al. [39]. Briefly, six samples of semi-
nal plasma of GF and PF semen were transferred to nitrocellulose
membranes after 2DE. The membranes were incubated overnight at 4
°C with anti-ApoA-I antibodies diluted 1:15,000 in TBS-T (0.05 M Tris-
HCl, 0.15 M NaCl, and 0.1% Tween 20, pH 7.6). After rinsing to remove
unbound primary antibodies, the membranes were incubated with alka-
line-phosphatase-(AP)-conjugated horse anti-rabbit antibodies (Sigma)
diluted 1:20,000 in TBS-T for 2 h at room temperature. Products were vi-
sualized by incubation with 5-bromo4-chloro-30-indolyphosphate and
nitro-blue tetrazolium (BCIP/NBT) reagent solvent in the dark for
10 min. Then the membranes were washed with 2 mM EDTA in order
to stop the colour reaction. Blots were then scanned with the VersaDoc
MP 4000 system (Bio-Rad). Using Quantity One Analysis Software, ver-
sion 4.6.9 (Bio-Rad), ApoA-I spots were quantified by Density/Area
(INT/mm2). The relative abundance of ApoA-I was calculated as a ratio
of spots 4 and 5 abundance to total abundance of ApoA-I (spots
4,5,a,b,c,d,e).
2.10. Statistical analysis
The data are presented as means ± SD. Prior to statistical analysis,
residuals were tested for normality (the D'Agostino and Pearson omni-
bus normality test) and homogeneity of variance (Bartlett test). For sta-
tistical procedures, percentage data of sperm motility were transformed
 M.A. Dietrich et al. / Journal of Proteomics 162 (2017) 52–61 55

by arcsin square root transformation and velocity data by log10 trans- Table 1
formation to reach the assumption of normality and homoscedasticity. Quality parameters of good (n = 6) and poor (n = 6) freezability semen. Values are
expressed as the mean ± S.D.
The data of sperm motility and western blot signal intensity were sub-
jected to a Student's t-test for unpaired samples (P-value b0.05 was Parameters Good semen Poor semen
regarded as statistically significant). Correlations between ApoA-I pro- freezability freezability

tein abundance estimated by the DeCyder tool (standard abundance) Fresh semen
and relative abundance measured after Western blotting and between Sperm concentration × 109/ml 19.6 ± 0.7 19.3 ± 1.1
Motility (%) 92.5 ± 4.2 89.5 ± 9.9
carp age and sperm motility after cryopreservation were calculated
VCL (μm s−1) 125.4 ± 9.5 106.0 ± 9.7
using the Spearman correlation test (P-value b 0.05). VSL (μm s−1) 69.9 ± 7.4 54.1 ± 6.4
ALH (μm) 7.1 ± 1.2 6.4 ± 0.8
3. Results Osmolality 279.0 ± 2.4 261.5 ± 4.8*
Protein concentration of seminal plasma (mg/ml) 1.37 ± 0.09 1.19 ± 0.09

3.1. Variation in the resilience of carp spermatozoa to cryopreservation Cryopreserved semen

The variation in sperm motility parameters after cryopreservation
such as motility, VCL, VSL and ALH was observed among semen from
20 carp males (Supplementary Table S2). The highest range of variabil-
ity was found for the percentage of motile sperm and ALH while the
lowest was for sperm velocity (VSL and VCL). The decline in sperm mo-
tility between pre-freeze and post-thaw assessment ranged from 10% to
51% (Fig. 1). Within the experiment 30% of semen samples were charac-
terized by high freezability and 30% represented low freezability semen.
The reminder samples (40%) revealed medium resilience to cryopreser-
vation. There was no correlation between the fish ages and % of motile
sperm after cryopreservation (R = 0.003).
3.2. Sample classification into good and poor freezability groups and semen
quality parameters
A decrease in the percentage of sperm motility induced by cryopres-
ervation was used as a marker for semen freezability. The six semen
samples with the highest decrease of sperm motility (from 50 to 40%)
were arbitrary classified as poor freezability semen (PF), whereas six
semen samples with the lowest decrease (below 25%) were arbitrarily
classified as good freezability semen (GF) (Fig. 1). Good and poor
freezability carp semen differed in seminal plasma osmolality prior to
the cryopreservation procedure (i.e., fresh semen), while the other
sperm quality parameters such as VSL, VCL, ALH and sperm concentra-
tion as well as seminal plasma protein concentration did not differ be-
tween GF and PF (Table 1). After cryopreservation, the percentage of
motile sperm was significantly lower in PF semen than in GF semen.
Motility (%) 71.8 ± 7.4 46.2 ± 4.1***
VCL (μm s−1) 80.1 ± 3.1 72.7 ± 2.3
VSL (μm s−1) 38.2 ± 2.7 37.9 ± 2.4
ALH (μm) 6.0 ± 0.6 4.9 ± 0.7
Asterisks indicate significant differences between the seminal plasma of good and poor
freezability carp males (*p b 0.05, *** p b 0.001). VCL-curvilinear velocity, VSL-straight
line velocity, ALH-amplitude of lateral head displacement.
No significant differences between GF and PF were observed for VCL,
VSL and ALH after cryopreservation.
3.3. Comparison of fresh seminal plasma proteome between GF and PF
semen
The analysis of seminal plasma from good and poor freezability
semen using quantitative DIGE technology led to the detection of 840
matched spots, and 15 spots were found to significantly (p b 0.05) differ
between GF and PF (Fig. 2). Five spots representing three proteins were
enriched in GF and ten spots representing seven proteins were more
abundant in PF seminal plasma (Table 2, Supplementary Table S3).
3.4. Protein-protein interaction analysis
The protein-protein interactions network demonstrated medium
edge (score 0.4) confidence between all proteins present in higher
amounts in the seminal plasma of GF semen (Fig. 3A). On the other
hand, high interactions (score 0.7–0.9) were found between seminal
plasma proteins more abundant in PF semen (Fig. 3B). The highest

Fig. 1. Distribution of semen freezability among carp males. The percentage differences of motile sperm between fresh and cryopreserved samples were assessed for 20 males. The six carp
with the higher and the lower cryopreservation abilities were classified as good freezability (GF, blue fill) and poor freezability (PF, white fill) carps, respectively. The remaining carp males
(grey fill) were not selected.
56 M.A. Dietrich et al. / Journal of Proteomics 162 (2017) 52–61

Fig. 2. Representative pattern of spots in carp of good freezability (GF) semen (A) and poor freezability (PF) semen (B) after 2D-DIGE analysis of seminal plasma proteomes. (C)-overlay of
seminal plasma of GF and PF semen (Cy5, red - seminal plasma of PF; Cy3, green - seminal plasma of GF). Numbers 1–15 indicate spots that significantly differ between seminal plasma of
GF and PF (red colour indicates proteins more abundant in seminal plasma of PF, while green colour indicates proteins in higher abundance in seminal plasma of GF semen). Letters from a
to e indicate proteoforms of ApoA-I detected using anti-ApoA-I antibodies in seminal plasma of GF and PF via western blotting (see Fig. 4). (D) - the 3-D (representative images) and line
charts (data for six males) of the chosen spots (apolipoprotein A – ApoA-I and complement C3).

interaction (score 0.9) was determined between haptoglobin and
hemopexin like (warm-temperature-acclimation-associated 65-kDa
protein) and between haptoglobin and apolipoprotein E. Cofilin 2 and
intelectin were unconnected proteins. Such high interaction indicates
that the proteins are functionally linkage.
3.5. Western blot analysis of ApoA-I
The proteins identified through 2D–DIGE analysis were validated by
Western blot analysis. Polyclonal antibodies against carp seminal plas-
ma ApoA-I recognized seven spots of ApoA-I (marked as 4, 5, a, b, c, d,
e; Fig. 4A, B). Similar to the proteomic data a significant increase of
the relative abundance (defined as ratio of spot abundance to total
abundance of ApoA-I) of spot 4 and spot 5 of ApoA-I was observed in
the seminal plasma of GF compared to that of PF semen (Fig. 4C, D).
These ApoA-I proteoforms (spot 4 and 5) correspond to spot 4 and 5
identified as enriched in good freezability semen through 2D–DIGE
(Fig. 2). Spots from “a” to “e” and total abundance of ApoA-I did not dif-
fer in abundance between GF and PF semen. The densities (INT/mm2) of
all detected spots are presented in Supplementary Table S4. Fig. 4 shows
the representative protein spot pattern of ApoA-I from Western blot
analysis. The Western blots of all 6 biological replicates are present in
Supplementary Fig. S1. The abundance of spot 4 and 5 of ApoA-I esti-
mated by the DeCyder tool (standard abundance) correlated (R =
0.786 and R = 0.788 for spot 4 and 5, respectively) with relative abun-
dance measured after Western blotting analysis (Supplementary Fig.
S2).
4. Discussion
This is the first study that focused on the evaluation of the fish sem-
inal plasma protein profile with relation to sperm freezability. The appli-
cation of the 2D–DIGE approach revealed significant differences in the
seminal plasma proteome between carp males of good and poor
semen freezability and facilitated the identification of 15 protein spots
which differed in abundance between these groups. Given that both
analysed groups were characterized by similar initial sperm quality
 M.A. Dietrich et al. / Journal of Proteomics 162 (2017) 52–61 57

Table 2
Seminal plasma proteins differentially expressed between good and poor freezability semen of carp.

Spot Protein name Gene Organism Gi number Protein % Peptide Calc. Good/poor Poor/good P
no in name score sequence matched MW/pI ratio ratio value
Fig. 2 coverage (p b 0.05)

Proteins more abundant in seminal plasma of good freezability semen

1 Fetuin long form AHSG Cyprinus gi| 429 31 4 52.6/7.9 1.7 0.009
carpio 29501368
2 PREDICTED: hemopexin-like (82% identity with HPX Cyprinus gi| 382 20 5 51.1/7.1 1.5 0.028
warm-temperature acclimation- associated-protein carpio 1101518671
65 kDa protein Misgurnus mizolepis)
3 PREDICTED: hemopexin-like HPX Cyprinus gi| 203 10 3 51.1/7.1 1.7 0.018
carpio 1101518671
4 Apolipoprotein A-I APOA Cyprinus gi| 339 38 4 30.2/6.3 1.5 0.035
carpio 586636848
5 Apolipoprotein A-I APOA Cyprinus gi| 288 28 3 30.2/6.3 1.6 0.035
carpio 586636848

Proteins more abundant in seminal plasma of poor freezability semen

11 PREDICTED: haptoglobin-like isoform ×1 HP Cyprinus gi| 235 19 2 37.06/8.84 1.7 0.05
carpio 1101558456
9 Apolipoprotein E APOE Cyprinus gi| 310 43 3 31.7/5.15 1.9 0.007
carpio 372292218
6 Apolipoprotein E APOE Cyprinus gi| 84 17 2 31.7/5.00 6.5 0.015
carpio 385865224
8 Apolipoprotein E APOE Cyprinus gi| 374 47 4 31.7/5.15 4.2 0.05
carpio 372292218
13 PREDICTED: intelectin-like isoform ×2 ITLN1 Cyprinus gi| 800 29 6 35.3/6.35 1.5 0.015
carpio 1101614073
12 PREDICTED: intelectin-like isoform ×2 ITLN1 Cyprinus gi| 540 29 4 35.3/6.35 1.7 0.047
carpio 1101614073
7 Complement C3-H1 C3-H1 Cyprinus gi|4126589 530 27 5 70.1/6.81 6.3 0.006
carpio
14 hypothetical protein cypCar_00028289 MMP2 Cyprinus gi| 436 27 5 70.4/5.09 1.7 0.009
(98% identity with matrix metalloproteinase 2 carpio 966643297
Apostichopus japonicas)
10 PREDICTED: hemopexin-like HPX Cyprinus gi| 242 9 4 51.1/7.1 1.8 0.001
carpio 1101518671
15 PREDICTED: cofilin-2-like CFL2 Cyprinus gi| 221 33 3 18.9/6.21 1.5 0.05
carpio 1101613190

parameters, the variation in freezing resilience of carp spermatozoa may
be related to the protein composition of the seminal plasma.
Sperm quality parameters such as motility, viability and sperm con-
centration have been used as predictive tools of sperm cryopreservation
potential in fish species [4,2,10,14,18,41]. However, our results indicat-
ed that carp spermatozoa evaluated as being of high quality (motility
90% and sperm concentration 19 × 109/ml) prior to freezing differed
in their cryoresistance. Out of 20 semen samples selected for cryopres-
ervation procedure based on their high quality parameters, six semen
samples were characterized by low sperm motility after thawing (cryo-
preservation caused a 40% decrease of motility). The low usefulness of
initial motility parameters as indicators of freezability was previously

Fig. 3. Interaction analysis of seminal plasma proteins with higher abundance in good (A) and poor (B) freezability carp semen generated with the STRING v.10 database program. The
interaction search was restricted to Homo sapiens protein pairs. APOA1 - apolipoprotein A-I; HPX - hemopexin (warm-temperature-acclimation-associated 65-kDa protein); AHSG -
alpha-2-HS-glycoprotein; MMP2 - matrix metallopeptidase 2; C3 - complement component 3; APOE - apolipoprotein E; CFL2 - cofilin 2; ITLN1 - intelectin 1; HP – haptoglobin. The
line size indicates a high interaction score (tight lines indicate high score N 0.7; thin lines indicate a medium score N 0.5). Proteins identified in carp seminal plasma of GF and PF males
are shown in Table 3.
58 M.A. Dietrich et al. / Journal of Proteomics 162 (2017) 52–61

Fig. 4. Validation of 2D–DIGE results by Western blotting using anti-apolipoprotein A-I (ApoA-I) antibodies. Representative Western blot of seminal plasma of good freezability (GF) semen
(A) and poor freezability (PF) semen (B). Proteins were separated by 2DE, transferred to nitrocellulose membranes and incubated with anti-ApoA-I antibodies. Blots were scanned with the
VersaDoc MP 4000 system (Bio-Rad, Hercules, CA). Spots 4, 5 and a–e indicate proteoforms of ApoA-I detected using anti-ApoA-I antibodies in seminal plasma of GF and PF. Number 4 and
5 indicate ApoA-I isoforms differing in abundance between GF and PF semen and correspond to spot 4 and 5 in Fig. 2. Intensity/area (INT/mm2) of ApoA-I proteoforms in the seminal
plasma of GF semen (n = 6) and PF semen (n = 6) was quantified using Quantity One Analysis Software (Bio-Rad, Hercules, CA). The relative abundance of ApoA-I was calculated as a
ratio of spots 4 and 5 abundance to total abundance of ApoA-I (C, D). The data shown are means ± SEM. Asterisks indicate significant differences between the seminal plasma of GF
and PF carp semen (*p b 0.05).

reported by Butts et al. [18] whose inferred that cod spermatozoa of
high quality (i.e., have the fastest swimming sperm) prior to freezing
were not necessarily those most suited to endure the freezing and
thawing process. Nevertheless our results clearly demonstrated varia-
tion in the resilience of carp semen to cryopreservation indicating that
only 30% of semen specimens have excellent usefulness for cryopreser-
vation. The variation of sperm cryopreservation ability has also been re-
ported previously for mammals [29,30] and fish species such as
northern pike [42], turbot [43], rainbow trout [44] and whitefish [45].
The mechanism underlying variation in freezing resilience of fish sper-
matozoa has not been elucidated, and future studies addressing this as-
pect, especially at molecular level, are justified.
The only useful index for predicting the cryopreservation ability of
carp spermatozoa was osmolality. This parameter was also selected as
a predictor of cryopreservation success in rainbow trout and cod
semen [18,42]. The slightly lower osmolality value recorded in PF
semen may result from disturbances in the mineral content of the
semen caused by maturation state and/or stress and contamination
with urine [14,46]. Since osmolality is important for sperm motility
and activation in fish [47], our results suggest that even a minor differ-
ence in the environment of spermatozoa can affect the freezing ability of
carp semen.
The majority of proteins present in higher abundance in seminal plas-
ma of poor freezing resilient carp were found to be involved in the innate
immune response. Apolipoprotein E was identified as the most discrim-
inating protein between GF and PF semen showing a 6-fold increase in
PF. ApoE is a polymorphic, multifunctional protein that in addition to
its well-established role in lipid transportation, bears immunomodulato-
ry properties by modulating the functions of macrophages and suppress-
ing the proliferation of T cells. Moreover ApoE can play an anti-
inflammatory role or stimulated production of the pro-inflammatory cy-
tokine interleukin-1β in an isoform-dependent manner [48,49]. Many
other proteins are involved in the immune response including: comple-
ment C3 and MMP-2- regulator of inflammation. Other such example
include haptoglobin which has been reported to be involved in modulat-
ing the immune response, autoimmune diseases and major inflammato-
ry disorders [50] and intelectin-an infection induced antimicrobial
protein [51]. The latter specifically recognize pathogens and bacterial
components containing galactofuranosyl residues in mammals [51],
and its higher level in human seminal plasma is characteristic of genital
tract infection [52]. The elevated level of proteins involved in immune re-
sponse suggest the presence of reproductive tract infections or inflam-
mations in carp of poor semen freezability.
We believe that infection or inflammation of the carp male repro-
ductive tract might be caused by the transport of fish and their acclima-
tion to the new housing conditions, which is a routine procedure for the
controlled carp spawning. It has been demonstrated that such proce-
dures are stressful (hypoxia, crowding, mechanical injuries) and can
have a negative effect on fish health, lowering the immune system re-
sponse and thus leading to an increase in disease or bacterial infection
[53,54]. Subsequent studies are intended to focus on a detailed evalua-
tion of immunological response factors within the reproductive tract re-
garding health status before sampling, handling conditions during
transport and acclimation conditions.
The mechanism affecting the freezing ability of spermatozoa involv-
ing the immune response proteins is unknown. We believe that the
higher level of immune response proteins in PF semen did not directly
affect the decrease in semen cryoresistance but reflected the processes
occurring within the testes, such as infection or stress. Stress and im-
mune response activation are the main causes of semen oxidative stress
[55]. It is well known that ROS can negatively affect spermatozoa includ-
ing DNA fragmentation, lipid peroxidation and protein oxidation. There-
fore, the generated ROS can cause sublethal damage to sperm structure,
which could result in lower freezability of sperm resulted in the de-
crease in sperm motility. Such sublethal damage would have a minor in-
fluence on the quality of fresh spermatozoa but would compromise the
cryoresistance of the sperm. Our hypothesis supports the results
concerning mammalian sperm. Evangelista et al. [56] showed that
 M.A. Dietrich et al. / Journal of Proteomics 162 (2017) 52–61
infection of dog semen compromised the total motility of cryopreserved
but not fresh semen. In summary, our results suggest that lower useful-
ness of spermatozoa to cryopreservation may be related to previous in-
fection or stress leading to sublethal changes to sperm structure.
We interpret the presence of cofilin at higher levels in carp seminal
plasma of PFS as indirect evidence of sublethal changes to sperm struc-
ture. The cofilin present in carp seminal plasma has been found to orig-
inate from spermatozoa [57,58], and its release from carp spermatozoa
into the extracellular medium due to cryopreservation has been dem-
onstrated [57,34]. Cofilin localizes to mammalian sperm tails and is an
ubiquitous actin-binding protein stimulating the severance and depoly-
merization of F-actin and correlates with sperm motility [59,60] there-
fore, its increased release from carp sperm of PF strongly suggests that
spermatozoa from such semen were subjected to damage of the flagel-
lum structure. Consequently, the leakage of cofilin from sperm may be a
part of the mechanism responsible for the decrease in motile sperm in
the PF group after cryopreservation.
The ability of carp spermatozoa to survive cryopreservation was as-
sociated with warm-temperature acclimation-related 65 kDa protein
(Wap65), which is present in multiple proteoforms in carp seminal
plasma [26]. In this study, distinct Wap65 proteoforms were associated
with differentially affected carp semen freezability. Poor freezability
semen was characterized by the presence of a Wap65 proteoform
with lower molecular mass, and a higher pI may have resulted from pro-
teolysis occurring within the PF due to inflammation or infection as
mentioned above. On the other hand, two proteoforms of Wap65
were more abundant in GF semen. Wap65 is a homolog of mammalian
hemopexins that functions as a scavenger of free heme and acts as an
antioxidant in mammalian seminal plasma [61]. Its beneficial role in
GF can be postulated in the protection of spermatozoa from oxidative
stress induced by cryopreservation. In conclusion, proteoforms of
Wap65 may be critical for either the positive or negative relationship
with cryopreservation success. This should be taken into account in fu-
ture studies.
ApoA-1 is a part of high-density lipoprotein (HDL) and is involved in
reverse cholesterol transport from the sperm membrane to HDL in
mammals [62]. The composition of the phospholipids (PL) and choles-
terol (CHO) determine plasma membrane fluidity and CHO regulates
the lipid chain order and molecular organization of membranes [62,
63]. Therefore, ApoA-I in carp seminal plasma may be involved in regu-
lating membrane lipid components of spermatozoa, which is consider-
ably modified through changes in CHO and PL composition during
spermatogenesis. In addition to its role in reverse cholesterol transport,
ApoA-I in fish is recognized as a potential immune regulator or antimi-
crobial protein in blood and skin, playing an important role in the innate
immune system and acute phase response [64–67]. Our recent studies
revealed antibacterial properties of ApoA-I from carp and rainbow
trout seminal plasma [68]. These antimicrobial properties of fish semi-
nal plasma ApoA-I coincides with the similarity and/or identity of
ApoA-I proteoforms from seminal and blood plasma. In summary
ApoA in carp seminal plasma could be involved in the maintenance of
testis homeostasis. This could be manifested through lipid transport to
developing germ cells or through protection of reproductive tissue
and sperm against bacteria.
ApoA-I is present in multiple proteoforms in carp seminal plasma
[40], and only two of them were found to be of higher intensity in GF.
The origin of this high variation of carp ApoA-I is unknown at present.
It is known that in mammals ApoA-1 is processed to three different
lengths, including a pro-form (amino acids 19–276), a mature form
(25–267), and a truncated form (25–266), and can be modified by in
vivo oxidation as well as by N-linked glycosylation or glycation [69].
In mammals these PTMs may affect function of ApoA-I in the anti-in-
flammatory actions as well as the efficiency of HDL in the reverse cho-
lesterol transport pathway and were found to be diagnostic markers
in atherosclerotic disease [70]. The variety of ApoA-I proteoforms in
carp seminal plasma can potentially result from alternative splicing
59
[71] and post translational modifications (PTMs) such as removing of
propeptide, phosphorylation, tyrosine nitration, O-glycosylation or
truncation determined based on sequence analysis [40]. It should be
underlined that proteoforms of ApoA-I detected in our study as poten-
tial markers of GF were previously identified in our laboratory as specif-
ic for carp reproductive tract [26]. These proteoforms had lower
molecular mass and pI than proteoforms common for blood and semi-
nal plasma and can be specifically related to the maintenance of
sperm membrane structure and motility. This suggestion coincides
with the recent study of Horokhovatskyi et al. [12] who demonstrated
the difference in carp sperm PL, CHO and free fatty acid concentration
among males of differ cryoresistance and suggested that these differ-
ences could have an influence on plasma membrane fluidity, lipid
phase transition and permeability to water and other molecules. Our re-
sults strongly suggest that better freezability of semen is related to par-
ticular proteoforms/PTMs of ApoA-I and their functional significance is
related to maintenance of sperm membrane structure and consequently
to enhance motility of cryopreserved semen.
However it should be emphasized that in contrast to ApoA, some
proteoforms of another apolipoprotein – ApoE, which plays a central
role in lipid transport, were negatively related to cryoresistance (see
above). Recently Setarehbadi et al. [72] revealed the difference in distri-
bution of ApoE genotypes in fertile and infertile men and suggested that
expressed ApoE alleles differed in their efficiency in promoting sperm
maturation during epididymal transport. In our opinion, additional
studies are necessary to determine whether carp ApoE is also a poly-
morphic protein. If carp ApoE is indeed polymorphic, it would be a
basis for a selective program aimed to increase the freezability of carp
semen.
One proteoform of fetuin long form (fetuin-A-type), which belongs
to the cystatin superfamily of cysteine protease inhibitors, was found
to be positively associated with carp semen cryopreservation. Our re-
cent studies revealed its presence in multiple proteoforms in both rain-
bow trout and carp seminal plasma [26,73]. It is known that fetuin is
subjected to posttranslational modifications (PTMs) such as proteolytic
processing, complex glycosylation, phosphorylation (Ser and Thr) and
sulfation, which may regulate protein expression levels, stability, and
biological activity. Therefore, it is possible that such PTMs can be re-
sponsible for the creation of the multiple proteoforms of carp fetuin ob-
served in our study. Fetuin-A is a pleiotrophic glycoprotein with both
the pro- and anti-inflammatory attributes. As a positive acute phase
protein (APP), fetuin-A was reported to induce the expression of inflam-
matory cytokines in adipocytes and macrophages and was regarded as
an inflammatory marker. On the other hand, as a negative APP, was
found to play a protective or anti-inflammatory role in various disease
conditions such as infection, sepsis, endotoxemia, autoimmune disor-
ders [74]. Moreover Sarıözkan et al. [75] revealed that fetuin had a pro-
tective role in maintaining glutathione peroxidase antioxidant activity,
sperm motility and plasma membrane integrity of frozen/thawed rabbit
semen. Perhaps these functions are related to better cryoresistance of
carp spermatozoa; however, it should be stressed that such protection
strongly depends on the specific proteoforms rather than general activ-
ity of fetuin.
Almost all proteins related to freezability were previously identified
and characterized in carp seminal plasma [26,76]. Apolipoproteins A-1
and E, complement C3 and fetuin all belong to major proteins as they
were found among the ten most abundant carp seminal plasma pro-
teins. They also have important roles in acute phase response signalling
and LXR/RXR and FXR/RXR activation, which are predicted as top ca-
nonical pathways [28]. These two latter pathways are involved in lipid
metabolism and inflammation. Moreover MMP2, cofilin, particular
proteoforms of fetuin, ApoE and ApoA-I (see above) were found as pro-
teins more abundant in carp seminal plasma compared to blood plasma.
This confirms their specific functions in the male reproductive tract of
carp. We extended the list of carp seminal plasma proteome with
intelectin, formerly not yet identified in fish seminal plasma. Its
60 M.A. Dietrich et al. / Journal of Proteomics 162 (2017) 52–61
presence supports the importance of carp seminal plasma proteins in
sperm protection against microbes.
In summary, the results of the present study revealed variation in the
ability of carp sperm to sustain cryogenic freezing and thawing. The fac-
tors regulating how carp semen tolerate cryopreservation may be relat-
ed to the different protein profiles of carp seminal plasma. A low
usefulness of sperm to cryopreservation is related to higher concentra-
tions of proteins reflecting infection or inflammation in the reproductive
tract. On the other hand, higher freezability seems to be related to the
presence of proteins involved in maintenance of sperm membrane in-
tegrity and antioxidative protection. Particular proteoforms of these
proteins may be critical for either the positive or negative relationship
with cryopreservation success. The identified proteins may serve as po-
tential bioindicators of freezing resilience to screen carp males and pre-
empt freezing success leading to improved artificial fertilization and
preservation techniques in carp. This suggestion should be tested in
the future studies with the use of several semen samples obtained
from the same male.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jprot.2017.04.015.
Conflict of interest
The authors have declared no conflict of interest.
Acknowledgements
We thank Sylwia Judycka and Grzegorz Dietrich for the analysis of
sperm motility parameters and Halina Karol and Ewa Liszewska for
their excellent technical assistance. The authors would like to thank dr
Anders Aufderhorst-Robertsthe for English-editing assistance and two
anonymous reviewers for their valuable comments and suggestions.
This work was supported by Project 2011/01/D/NZ9/00628 from the
National Science Centre, Poland (Identification and characterization of
specific carp seminal plasma proteins – proteomics and a classical ap-
proach) and funds appropriated to the Institute of Animal Reproduction
and Food Research.
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