Vol.1, No.2, 33-40 (2011) Open Journal of Animal Sciences
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
Characterization of proteins in cryopreserved and
non-cryopreserved seminal plasma of dairy bulls of
differing fertility
——Seminal plasma proteomics
J. F. Odhiambo1,2, R. A. Dailey1*
1Division of Animal and Nutritional Sciences, West Virginia University, Morgantown, USA; *Corresponding Author: rdailey@wvu.edu
2Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Canada;
Received 6 May 2011, revised 25 May 2011, accepted 10 June 2011.
Seminal plasma is composed of secretions from
accessory sex glands, which are mixed with
sperm at ejaculation and contribute the majority
of semen volume. Seminal plasma is considered
a transport and support medium for sperm in
the female reproductive tract. Because seminal
plasma is not required for fertilization, the im-
portance of its constituents to the establishment
of normal pregnancy has been overlooked. Four
seminal plasma proteins, Osteopontin, Sper-
madhesin Z13, BSP 30 kDa and Phospholipase
A2, have been identified as markers of fertility in
dairy bulls [1-3]. The objective of the present
study was to characterize the expression pat-
terns of these proteins and other proteins found
to be of interest in seminal plasma of cryopre-
served and non-cryopreserved bull semen.
Seminal plasma samples were obtained from 16
mature Holstein- Friesian bulls at Select Sires
Inc. Samples were divided into two groups
based on assigned fertility score expressed as
the percentage point deviation (PD) of the bull’s
non-return rate (NRR) from the average NRR of
all bulls in the Select Sires Inc. reproductive
management program. Group 1 (high fertility
bulls, n = 8) 1.9% PD 2.7%, and group 2 (low
fertility bulls, n = 8) - 6.5% PD 1.8%. Addi-
tionally, the samples were categorized as proc-
essed (cryopreserved) or unprocessed (non-
cryopreserved) for protein analysis. Protein ex-
pression was analyzed by 2 - D fluorescence
difference gel electrophoresis (2D-DIGETM). Pro-
tein spots were picked from a reference gel,
analyzed by mass spectrometry and, subse-
quently identified by MS/MS ion searches per-
formed on the SwissProt database. Protein ex-
pression did not differ (P > 0.05) with fertility
grouping but displayed two distinct pa- tterns
among the processing groups: majority of the
functional proteins were highly expressed in
seminal plasma of non-cryopreserved semen
while the cryopreserved semen contained mainly
structural/ extender derived proteins. Functional
proteins identified included Spermadhesin Z13,
BSP A1/2, BSP 30 kDa, Nucleobindin-1 and
metalloproteinase inhibitor 2. Some of these
proteins have been identified as anti-fertility or
fertility enhancing agents in males. Whether this
alteration in protein expression after processing
might affect semen fertility is worthy of further
Keywords: Seminal Plasma; Proteomics;
Bull Fertility
Secretions from the accessory sex glands are mixed
with sperm at ejaculation and contribute to the majority
of semen volume and components. However, during
cryopreservation, most seminal plasma is replaced with
semen extenders, mainly egg yolk or milk proteins. Se-
minal fluid contains signaling agents that influence fe-
male reproductive physiology to improve chances of
conception and pregnancy success. These factors include
cytokines, sex hormones, and prostaglandins [4]. Ex-
periments in rodents and pigs showed that seminal
plasma is the second most vital component of the ejacu-
late, absence of which at mating reduced fertilization
and increased fetal loss after implantation [5]. Artificial
inseminations with adjunctive seminal plasma consis-
tently tended to have little or no effect on pregnancy
outcome [6]. The precise nature of active constituents
J. F. Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
that might influence pregnancy and their relative
amounts remain relatively unknown.
High concentrations of TGF-β cytokines were de-
tected in boar seminal fluid [7], and their characteristic
immunosuppressive activity was associated with protein
fractions of appropriate size in boar seminal fluid [4].
Four proteins (osteopontin, spermadhesin Z13, bovine
seminal plasma protein (BSP) 30 kDa, and phospholi-
pase A2) were identified as markers of fertility in dairy
bulls [1-3]. In those studies, proteins were visualized
with Coomassie brilliant blue staining after 2-D gel
separation, a process that might not reveal some of the
low abundance proteins in seminal plasma that might be
of biologic importance.
Therefore, a more sensitive protein detection tech-
nique that would give a broader approach was used in
the present study to identify seminal plasma proteins.
The multiplexing capability of 2 - D fluorescence dif-
ference gel electrophoresis (2 - D DIGE) technology was
expected to broaden the number of markers included in
the assay and yield more robust predictions of bio-
markers for bull fertility. The objectives of this study
included: 1) large scale identification and differential
expression of seminal plasma proteins between high and
low fertility bulls, 2) correlation of expression of spe-
cific proteins to fertility phenotype, and 3) comparison of
the expression patterns pre- and post-cryopreservation.
Semen samples from 16 dairy bulls were obtained
from Select Sires Inc. (Plain City, Ohio). All samples
used (cryopreserved and non-cryopreserved) were ob-
tained from the same ejaculate of each bull and proc-
essed at Select Sires Inc. At each collection time, ejacu-
lates from the same bull were pooled and an aliquot was
obtained for the non-cryopreserved sample. The rest of
the semen was processed and extended for cryopreserva-
tion in 0.5 mL semen straws.
Semen was extended in egg-yolk-citrate (EYC) com-
posed of a 2.9% sodium citrate buffer supplemented
with 20% egg yolk (vol/vol) prepared with a glycer-
olated and non-glycerolated fractions containing 14 or
0% glycerol (vol/vol), respectively. Samples were di-
luted 1:3 (vol/vol) with non-glycerolated semen extender,
placed in a 200-mL water bath and allowed to equilibrate
to 5˚C over a 2-h period. Samples were extended to half
volume (80 × 106/ml) in non-glycerolated extender and
then an equal volume of glycerolated extender was
slowly titrated over a 10 min period. The final dilution
yielded a sperm concentration of 20 × 106 per 0.5-mL
French straw. Straws were then frozen in liquid nitrogen.
To obtain seminal plasma, semen was centrifuged at
3,000 g for 10 min and then aspirated into Eppendorf
tubes for volumes < 1 mL, or 15 mL centrifuge tubes for
larger volumes. Samples were then further clarified in
0.45 micron syringe filter and finally frozen in 0.5 mL
Samples were assigned to high or low fertility groups
based on fertility score expressed as the percentage point
deviation (PD) of the bull’s non-return rate (NRR) from
the average NRR of all bulls in the Select Sires Inc. re-
productive management program. These scores were
based on inseminations with frozen semen from the bulls
under this program. Range of scores for high fertility
bulls (n = 8) was 1.9% PD 2.7% and for low fertility
bulls (n = 8) was –6.5% PD 1.8%.
Following delivery, semen samples were thawed at
room temperature and centrifuged at 10,000 × g for 15
min at 4˚C to remove sperm and associated debris. Ali-
quots of 0.5 mL seminal samples were obtained from the
supernatant and stored frozen at –80˚C until further use.
2.1. Electrophoresis
Frozen samples of seminal plasma were thawed at
room temperature and centrifuged at 10,000 × g for 60
min at 4˚C. The supernatant was processed by 2-D
Clean-UP protocol (GE Healthcare, Piscataway, NJ) to
remove impurities such as nucleic acids, lipids and salts.
Samples were then assayed for protein content [8] using
BSA as standard, and aliquots were frozen at –800˚C.
Samples for electrophoresis were thawed at room tem-
perature, concentrated to 1 - 10 mg protein/mL and la-
beled with cyanine dye (CyDye) DIGE Fluor Cy3/5 (GE
Healthcare, Piscataway, NJ) at a ratio of 50 µg protein to
400 pmol fluor. A pooled internal standard was created
from equal aliquots of each sample and labeled with Cy2
dye. Samples were separated by isoelectric focusing on
an Ettan™ IPGphor™ apparatus (GE Healthcare, Pis-
cataway, NJ) using 24 cm Immobiline DryStrip gels (GE
Healthcare, Piscataway, NJ) containing a mixture of
ampholytes with pH ranging from 3 to 10.
Following isoelectric focusing, strip gels were trans-
ferred to 24 cm Tris-Tricine gradient gels (Bio-Rad La-
boratories, Hercules, CA) mounted on low-fluorescence
glass plates. Thereafter, proteins were separated by mo-
lecular mass in the second dimension using Ettan™ Dalt
II Electrophoresis System (GE Healthcare, Piscataway,
NJ). Dalt gels were scanned using a Typhoon 9400 Va-
riable Mode imaging densitometer (GE Healthcare, Pis-
cataway, NJ) at 100 µm resolution. A fully automated
image analysis software, Progenesis SameSpots™ (Non-
linear Dynamics, Durham, NC), was used to analyze the
protein expression data.
2.2. Statistical Analysis
In 2D-DIGE experiments, the pooled internal stan-
J. F.Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
dards were essential for assessing biological and ex-
perimental (between gels) variations and increasing the
robustness of statistical analysis. Individual protein data
from sample groups (Cy3 or Cy5) were normalized
against the Cy2 labeled internal standard. Scanned im-
ages of the labeled proteins were sequentially analyzed
by differential in-gel analysis (DIA) that performed
Cy3/Cy5:Cy2 normalization, and then by biological var-
iation analysis (BVA) that performed inter-gel statistical
analysis to provide relative abundance in various groups.
Log abundance ratios were then compared between
sample groups using ANOVA and t-test in Progenesis
SameSpots. The analyzed spots were ranked by their
probability values and then grouped into experimental
groups for further evaluation. Principal component
analysis (PCA) was used to determine the presence of
outliers in the data and also to compare how well the
samples fitted to the experimental groups. The expres-
sion profiles of the selected proteins were then examined
by correlation analysis.
2.3. Protein Identification
A list of protein spots of interest (pick-list) was gener-
ated using the image analysis software and exported di-
rectly into a Spot Picking Ettan™ Spot Handling Work-
station (GE Healthcare, Piscataway, NJ) equipped to
automatically pick spots from the Dalt gels. Selected
protein spots were washed by 50 mM ammonium bicar-
bonate/50% (vol/vol) methanol in water, dried by vac-
uum centrifugation, and incubated overnight at 37 0C in
140 ng of sequencing grade trypsin [9]. Tryptic digests
were analyzed by capillary liquid chromatography-
nanoelectrospray ionization-tandem mass spectrometry
(CapLC-MS/MS, Thermo Finnigan, San Jose, CA). Pro-
teins were identified by MS/MS ion searches performed
on the processed spectra against the SwissProt and NCBI
protein databases using a Bioworks Browser 3.1
(Thermo Finnigan, San Jose, CA) search engine. The
identification of a protein was confirmed when the Bio-
works confidence interval was greater than 95%. The
protein mass and pI accuracy on the 2D gel was used as
a guide to confirm protein identification.
3.1. Distribution of Protein Spots in Seminal
Plasma of Dairy Bulls
Three patterns of protein expression were observed
consistently in seminal plasma samples of cryopreserved
semen (Figure 1). Spot volume analysis and peptide
identification indicated a higher expression of proteins
from semen extender at the molecular weight range be-
tween 30 to 60 kDa (Group 1) accounting for 55% of
protein spots by Coomassie staining. A “train” of spots
was visible within the 20 to 25 kDa range (Group 2)
accounting for about 35% of the spot volume, while the
remaining spots (10%) were expressed below the 20 kDa
range (Group 3).
There was a two-fold difference (P < 0.01) in total
protein content between extended and non-extended se-
minal plasma (41.6 ± 2.3 vs. 19.5 ± 2.1 ng/mL, respec-
tively). However, the expression patterns of proteins in
seminal plasma of high and low fertility bulls did not
differ (P > 0.05). Therefore, subsequent analyses were
done between cryopreserved (processed) and non-cryopre-
served (unprocessed) seminal plasma. A total of 54 spots
differed (P < 0.001) in expression pattern between proc-
essed and unprocessed seminal plasma (Figure 2(a)).
The spots were then examined by principal component
analysis and clustered into two groups: those 31 spots
(57.4%) that were highly expressed in processed seminal
plasma but not in unprocessed seminal plasma and those
23 spots (42.6%) that were highly expressed in unproc-
essed seminal plasma but not in processed seminal
plasma (Figure 2(b)).
3.2. Protein Identification
A reference image (Figure 3) was generated from the
expression data and used to pick spots for protein identi-
fication (Table 1). Extender derived proteins, mainly
chicken vitellogenin-2 (MW 20.5 kDa), fibrinogen β
chain (MW 52.70) and chicken albumin, were predomi-
nant proteins identified from spots above 20 kDa in se-
minal plasma of processed semen. In unprocessed semi-
nal plasma, which lacked extender proteins, several
spots above 20 kDa were identified. Notable among
these proteins were nucleobindin-1, clusterin, phosphol-
ipase A2 isoforms, seminal plasma protein BSP-30 kDa,
metalloproteinase inhibitor-2 and cathepsins (B and D).
Below 20 kDa range, greater amounts of spermadhesin
(SPAD1 and Z13) isoforms were expressed than the ma-
jor bovine seminal plasma proteins (PDC - 109 and
BSP-A3) in seminal plasma of processed semen. How-
ever, in unprocessed seminal plasma, PDC - 109 and
BSP-A3 predominated over the spermadhesins below the
20 kDa range.
The main finding of this study was that expression
pattern of seminal plasma proteins differed between,
cryopreserved and non-cryopreserved dairy bull semen.
Indeed, during cryopreservation, major seminal plasma
proteins are replaced with extender proteins. Most artifi-
cial inseminations in cattle involve use of processed
(cryopreserved) semen, and as a consequence, most fer-
tility data have been derived from inseminations with
J. F. Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
Table 1. Proteins identified from corresponding spots in Figure 3.
Gel ID Fold increase* Protein ID Accession number Coverage % Molecular
weight pI
Membrane stabilizing proteins
1 95.4 Spermadhesin Z13 P82292 44.0 15.2 6.3
19 30.2 Spermadhesin Z13 P82292 43.3 15.2 6.3
3 54.4 Spermadhesin 1 P29392 78.4 15 5.1
20 30.1
Epididymal secretory pro-
tein E1 P79345 55.0 16.6 8.4
ECM interacting proteins
6 38.8 Cathepsin B P07688 16.1 36.7 --
26 27.3 Cathepsin D P80209 5.1 42.5 --
43 20.4 Clusterin P17697 31.4 51.1 6.0
12 33.9
Metalloproteinase inhibitor
2 P16368 42.7 24.3 7.8
Capacitation/acrosome reaction proteins
9 37.3 BSP-A1/A2 P02784 44.8 15.5 4.9
19 30.2 BSP A1/A2 P02784 44.8 15.5 4.9
34 24.6 BSP-30kDa P81019 27.9 21.3 5.9
51 17.1 Phospholipase A2 IPI00760435.1 47.1 50.1 6.5
90 7.9
Platelet-activating factor
acetylhydrolase Q28017 53.2 50.1 6.5
100 5.5 Nucleobindin-1 Q0P569 63.1 54.9 5.2
Ubiquitination proteins
14 33.3 Kelch-like protein 9 Q2T9Z7 24.5 153.6 8.8
Motility associated proteins
30 26.4
Fast myosin heavy chain
extraocular IPI00829549.2 30.6 186.1 5.7
*Spot volumes differed in relative amounts compared to internal standard
Protein was identified at this spot only in unprocessed semen.
Identification at this
spot was enhanced by semen processing.
processed semen. Therefore, it was prudent to utilize
cryopreserved semen in this study to examine changes in
its protein profile between low and high fertile bulls, and
also between processed and unprocessed semen.
Abundant low molecular weight bovine seminal plasma
proteins were replaced during cryopreservation by high
molecular weight extender-derived chicken structural
proteins. Major bovine seminal plasma proteins
PDC-109 (BSP-A1/A2), BSP-A3 and BSP-30 kDa play
important roles in fertility by maintaining sperm in an
appropriate state in the female tract until the oocyte
reaches the site of fertilization [10-13]. It is unclear
whether the effect of processing that decreased the ab-
undance of these BSP proteins by more than half would
render them ineffective in preventing premature capaci-
tation and acrosome reaction of sperm from normal fer-
tility bulls in the female tract. Spot volume data indicated
that removal of the major seminal plasma proteins ex-
posed less abundant low molecular weight proteins like
spermadhesins, especially spermadhesin Z 13 which has
been identified as an antifertility factor [2,14] in bovine
semi-nal plasma.
Killian et al. [14] suggested that four bovine seminal
plasma proteins were associated with fertility. These pro-
teins were later characterized as osteopontin and BSP-30
kDa in high fertility bulls and spermadhesin Z
J. F.Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
Figure 1. Reference 2D gel depicting distribution of protein spots in seminal plasma of dairy bulls. Protein spots were
characterized as: 1) probable extender-derived proteins, 2) probable medium and high molecular weight seminal
plasma proteins, and 3) major bovine seminal plasma proteins.
J. F. Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
Figure 2. Cluster analysis of protein expression in seminal plasma of dairy bulls. Reference 2D gel (a) depicting
protein spots that differed (P < 0.001) and their standard expression profiles (b) in processed (Red outlines) and
unprocessed (Green outlines) semen.
Figure 3. Pick list for protein spots on a reference gel of a pooled internal standard of seminal plasma from processed
and unprocessed dairy bull semen. Thirty spots from this list were picked and digested with trypsin for protein identi-
fication by CapLC-MS/MS mass spectrometry.
J. F.Odhiambo et al. / Open Journal of Animal Sciences 1 (2011) 33-40
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/OJAS/
13 in low fertility bulls [2]. In the present study, seminal
plasma protein expression did not differ between high
and low fertility dairy bulls. A difference in protein ex-
pression between fertility groups had been demonstrated
in a previous study [2]. It was anticipated that by utiliz-
ing the multiplexing capability of the 2-DIGE technol-
ogy, experimental errors would be minimized, and a
more robust analysis would be achieved as opposed to
the densitometric analysis utilized in the former study.
The discrepancy in outcome between the two studies can
be attributed to sample type utilized or fertility grouping,
or both. Samples in the present study were from proc-
essed insemination straws and unprocessed seminal
plasma from the same ejaculates as opposed to fresh
seminal plasma samples utilized in the previous study
that avoided secretions from epididymis and vas diferens.
Therefore, differential expression of proteins in samples
examined in this study might have been affected by
sample type as well as semen processing as evidenced
by the comparison of processed and unprocessed semi-
nal plasma protein profile. The narrow range of fertility
differences in the bulls used in this trial also impeded the
classification of semen samples into distinct high and
low fertile groups as was done in the previous study [2].
Bulls used in the present study had percentage point de-
viations (PDs) from the average of +2.7% to –6.5%. In
contrast, the previous study had PDs from +7.7% to
18.1%. Consequently, low and high fertility groups in
the present study corresponded to the intermediate fertil-
ity groups in that study. Because the previous authors
reported no differences in expression levels of osteopon-
tin, spermadhesin Z13, phospholipase A2 and BSP
30kDa among these two groups, it was not surprising
that the groups did not differ in their seminal plasma
protein profile in the present study. This is a major lim-
iting factor in fertility studies in farm animals because it
is rare to find sires at the lower extremes of fertility due
selection pressure.
Seminal plasma proteins have been characterized by
other investigators and their association with male fertil-
ity continues to be explored [2,3,15-17]. Functions of
sperm that may be affected by seminal plasma proteins
include capacitation, acrosome reaction, motility, DNA
integrity and interaction with the oocyte [3,18].
Major BSP proteins (BSP-A1/A2, A3 and BSP-30
kDa) are known to influence capacitation by their ability
to modulate membrane cholesterol [18]. Phospholipase
A2 (PLA2) and osteopontin are involved in acrosome
reaction and sperm-oocyte interaction and possibly early
embryonic development [3]. Proteins that might be asso-
ciated with interaction and modulation of extracellular
matrix (ECM) components are TIMP-2, clusterin and
cathepsins. These functions may be important during
fertilization when the sperm is required to interact with
and cross barriers established by the cumulus cells, zona
pellucida and oocyte membrane. Albumin, aSFP and
clusterin are involved either directly or indirectly in
mechanisms aimed at preventing damage to sperm
membrane, oxidative stress, and immune attack. Proteins
associated with sperm motility in the female reproduc-
tive tract include BSP A1/A2, aSFP, PLA2 and ecto
5'-nucleotidase (5'-NT). Spermadhesin Z13 might also
be included in the motility associated group because it
shares 50% homology with aSFP. However, expression
of spermadhesin Z13 in seminal plasma of dairy bulls
was inversely related to fertility [2].
Although expression pattern of seminal proteins was
not associated with fertility ranking in this study, altera-
tion of protein expression might affect fertility in bulls
that show more divergence in fertility ranking. In addi-
tion, expression patterns of seminal plasma proteins
might be altered during cryopreservation as has been
demonstrated in this study. Whether this alteration might
affect fertility remains to be explored.
The authors acknowledge the support of Select Sires Inc for provi-
sion of fertility data and semen samples. We greatly acknowledge the
support of Ms. Linda Corrum, Mr. Steve Wolf and Dr. Peter Perotta of
the Core Proteomics facility at West Virginia University. This work is
published with the approval of the director of the WV Agriculture and
Forestry Experiment Station as a scientific paper from the Division of
Animal and Nutritional Sciences and was supported by Hatch Project
421, NE1007 and Select Sires grant.
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