Heise 2010

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Animal Reproduction Science 118 (2010) 48–53
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Animal Reproduction Science
journal homepage: www.elsevier.com/locate/anireprosci
Influence of seminal plasma on fertility of fresh and frozen-thawed
stallion epididymal spermatozoa
A. Heisea,∗, W. Kähnb, D.H. Volkmannc, P.N. Thompsona, D. Gerbera
a Department of Production Animal Studies, Faculty of Veterinary Science, 0110 Onderstepoort, South Africa
b Vetsuisse Fakultät Universität Zürich, Winterthurerstrasse 260, 8057 Zürich, Switzerland
c Missouri University, Veterinary Medical Teaching Hospital, A331 Clydesdale Hall, 379 Campus Drive, Columbia, MO 65211, USA
a r t i c l e i n f o
Article history:
Received 10 February 2009
Received in revised form 30 May 2009
Accepted 8 June 2009
Available online 18 June 2009
Keywords:
Stallion
Epididymal
Spermatozoa
Seminal plasma
Fertility
a b s t r a c t
The use of epididymal stallion spermatozoa for routine artificial insemination can secure
easy future use of valuable genetics after unforeseen death or injury of a valuable stallion.
The aims of this study were to (1) directly compare pregnancy rates for fresh and
frozen-thawed stallion epididymal and ejaculated spermatozoa after conventional artificial
insemination and (2) to investigate the effect of seminal plasma on the fertility of epididymal
spermatozoa after insemination.
Twenty-one mares were randomly assigned to three stallions. Mares were inseminated
at five consecutive oestrous periods using fresh ejaculated spermatozoa (Fr-E, n = 18), fresh
epididymal spermatozoa that had been exposed to seminal plasma (Fr-SP+, n = 12) or fresh
epididymal spermatozoa that had never been exposed to seminal plasma (Fr-SP−, n = 9),
frozen-thawed ejaculated spermatozoa (Cr-E, n = 18), frozen-thawed epididymal sperma-
tozoa that had been exposed to seminal plasma prior to freezing (Cr-SP+, n = 18) and
frozen-thawed epididymal spermatozoa that had never been exposed to seminal plasma
(Cr-SP−, n = 15). Pregnancy examinations were performed 14 days after each ovulation.
Pregnancy rates were 55.6% (Fr-E, 10/18), 75% (Fr-SP+, 9/12), 22.2% (Fr-SP−, 2/9), 38.9% (Cr-
E, 7/18), 27.8% (Cr-SP+, 5/18) and 6.7% (Cr-SP−, 1/15). Overall pregnancy rates for fresh and
frozen-thawed epididymal spermatozoa that had been exposed to seminal plasma were sig-
nificantly better than for epididymal spermatozoa that had never been exposed to seminal
plasma (Ph intervals for ovulation. Cycles during which
frozen-thawed spermatozoa were used and during which
ovulation had not occurred within 12 h after insemination
were excluded from the trial. Fourteen days after ovula-
tion mares were examined for pregnancy and treated with
250 �g Cloprostenol (Estrumate®, Schering-Plough Animal
Health) by i.m. injection to induce luteolysis and the next
oestrus.
Stallions used in this study were selected on fresh and
frozen-thawed (≥30%) progressive spermatozoal motility,
spermatozoal concentration and total spermatozoal num-
bers.
Semen was collected, frozen and stored every second to
third day prior to the first inseminations with fresh semen.
2.2. Semen freezing
Immediately after collection the sperm rich fraction
was separated from the gel fraction using a sterile gauze
filter. Gel-free semen was evaluated for volume, concentra-
tion and percentage of progressively motile spermatozoa.
Semen was diluted in a skim milk diluent to a concentration
of 100 × 106 spermatozoa/ml and centrifuged for 10 min at
600 × g. After removal of the supernatant, sperm pellets
were suspended in skim milk freezing extender to a final
concentration of 500 × 106 spermatozoa/ml. Semen was
packaged in 0.5 ml straws (0.5 cc semen straws, Minitüb,
Tiefenbach, Germany) and equilibrated for 60 min at 4 ◦C
(straws placed on a rack with a distance of 10 mm between
straws). Straws were then placed 3.5 cm above liquid nitro-
gen for 20 min and then plunged into liquid nitrogen.
Straws were subsequently thawed in a 37 ◦C water bath for
30 s.
2.3. Semen extender
Semen extenders used were skim milk based (modi-
fied INRA 82)(Palmer, 1984). The centrifugation medium
contained glucose (25 g), lactose (1.5 g), raffinose (1.5 g),
sodium citrate (dehydrate) (0.25 g), potassium citrate
(0.41 g), HEPES (4.76 g), UHT sterilised skim milk (500 ml),
deionised water (500 ml), penicillin G (50,000 IU), and
gentamicin sulfate (5 mg). The freezing medium con-
tained the same ingredients but also egg yolk (40 ml)
http://www.v-tech.co.za/
50 A. Heise et al. / Animal Reproduction Science 118 (2010) 48–53
Table 1
Experimental model of inseminations of one group consisting of seven mares and one allocated stallion. Within each group of mares spermatozoa of only
the allocated stallion were used for all inseminations during all five consecutive oestrus cycles. Example: all mares in group one were inseminated with
spermatozoa of stallion A. A cross-over design for inseminations with frozen-thawed semen (oestrous cycles 3–5) was chosen to compensate for possible
environmental influences.
Mares group 1 Consecutive oestrus cycles during which mares were inseminated
Oestrus cycle 1 Oestrus cycle 2 Oestrus cycle 3 Oestrus cycle 4 Oestrus cycle 5
Semen categories stallion A
Mare 1 aFr-E bFr-SP + dCr-E eCr-SP+ fCr-SP−
Mare 2 Fr-E Fr-SP+ Cr-E Cr-SP+ Cr-SP−
Mare 3 Fr-E Fr-SP+ Cr-SP+ Cr-SP− Cr-E
Mare 4 Fr-E Fr-SP+ Cr-SP+ Cr-SP− Cr-E
Mare 5 Fr-E cFr-SP− Cr-SP− Cr-E Cr-SP+
Mare 6 Fr-E Fr-SP− Cr-SP− Cr-E Cr-SP+
Mare 7 Fr-E Fr-SP− Cr-SP− Cr-E Cr-SP+
a Fr-E, fresh ejaculate.
b Fr-SP+, fresh epididymal spermatozoa that had been exposed to seminal plasma.
o semin
d to sem
exposed
c Fr-SP−, fresh epididymal spermatozoa that had never been exposed t
d Cr-E, frozen-thawed ejaculated spermatozoa.
e Cr-SP+, frozen-thawed epididymal spermatozoa that had been expose
f Cr-SP−, frozen-thawed epididymal spermatozoa that had never been
and glycerol (25 ml). For the preparation of egg yolk,
40 ml egg yolk were mixed with 40 ml deionised water
and centrifuged at 15,000 × g for 10 min. Forty millilitre
of the liquid component was harvested and used in the
extender.
2.4. Seminal plasma
To harvest seminal plasma from the stallions, semen
was collected and the sperm rich fraction was separated
from the gel fraction as described above and then cen-
trifuged at 600 × g for 15 min. The supernatant was placed
into a sterile 50 ml plastic tube and the centrifugation was
repeated twice more. Seminal plasma of all three stal-
lions was pooled and frozen at −18 ◦C in aliquots of 50 ml.
Aliquots of seminal plasma were thawed in a water bath at
37 ◦C before use.
2.5. Harvesting of epididymal spermatozoa
Castrations were performed 3–4 weeks after the last
semen collection and each stallion had both testes recov-
ered via open castration under general anaesthesia. The
cauda epididymis and the vas deferens were separated from
each testis. Connective tissue was removed from the cauda
epididymis. The epididymal duct was cut at the site in the
tail of the epididymis where the tubular diameter became
distinctly narrower. A blunted 18G four cm hypodermic
needle connected to a 20 ml syringe filled with flushing
medium was inserted into the open end of the vas deferens.
Spermatozoa were then flushed in a retrograde direction
from the vas deferens through the cauda epididymis into a
sterile 50 ml plastic tube.
For each stallion one epididymal tail was flushed with
20 ml seminal plasma and the other with 20 ml freezing
medium. Left and right epididymides were randomly allo-
cated to the different treatment groups.
Each epididymal semen sample was divided into two
parts. One part was used for the AI with fresh epididy-
mal spermatozoa (3–4 insemination doses of 200 × 106
al plasma.
inal plasma prior to freezing.
to seminal plasma.
progressively motile spermatozoa). The other part was cry-
opreserved. The aliquot of epididymal spermatozoa flushed
with seminal plasma and destined for cryopreservation
was diluted with centrifugation medium to a concentra-
tion of 100 × 106 spermatozoa/ml and further treated like
ejaculated semen (see semen freezing). Epididymal semen
flushed with freezing medium was further diluted with
freezing medium to a final concentration of 500 × 106 sper-
matozoa/ml and then cryopreserved as described above
without centrifugation.
2.6. Statistical analysis
Comparison of pregnancy rates between all SP+ groups
(Fr-E, Fr-SP+, Cr-E and Cr-SP+) and all SP− groups (Fr-SP−
and Cr-SP−). Univariable comparisons of pregnancy rates
between the six groups were initially done using Fisher’s
exact test and relative risks with 95% confidence intervals
calculated. Multilevel (mixed effects) logistic regression
models (Dohoo et al., 2003) were then used to estimate
the effect of flushing medium and freezing on pregnancy
rates. The models accounted for the hierarchical structure
of the data where pregnancy rates (level 1) were clustered
within mares (level 2) and mares within stallions (level
3). Such clustering violates the assumption of indepen-
dence inherent in most regression models and failure to
account for it may result in invalid estimates of effects, in
particular their standard errors. Fixed effects were flush-
ing medium (skim milk extender or seminal plasma) and
semen treatment (fresh or frozen). Random effects were
mares and stallions. Three models were constructed: a
combined model using all the data and separate models
for inseminations using fresh and frozen-thawed sperma-
tozoa. In addition, the effect of each individual stallion
on pregnancy rate was investigated by including stallion
as a fixed effect rather than a random effect in the com-
bined model. Data were analysed in Stata 8.2 (StataCorp,
College Station, TX, USA) using the “gllamm” procedure
(Rabe-Hesketh and Skrondal, 2005).
A. Heise et al. / Animal Reproduction Science 118 (2010) 48–53 51
Table 2
Effects of flushing medium and freezing on pregnancy rate in mares: results of multilevel logistic regression models.
Model Variable b SE (b) Odds ratio (OR) 95% CI P
Fresh and frozen-thawed combined SP+ vs. SP− 2.05 0.81 7.81 1.59, 38.31 0.014
E vs. SP− 1.93 0.78 6.92 1.49, 32.13 0.011
Frozen-thawed vs. fresh −1.43 0.54 0.24 0.08, 0.70 0.009
Fresh SP+ vs. SP− 2.63 1.14 13.83 1.47, 130.0 0.022
E vs. SP− 1.65 1.00 5.21 0.73, 37.16 0.100
Frozen-thawed SP+ vs. SP− 1.64 1.21 5.13 0.48, 55.32 0.178
2.2
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E vs. SP−
P+, epididymal spermatozoa that had been exposed to seminal plasma.
P−, epididymal spermatozoa that had never been exposed to seminal pl
, ejaculated spermatozoa.
. Results
.1. Pregnancy rates
Twenty-one mares were inseminated during consec-
tive oestrous cycles using fresh ejaculated spermatozoa
Fr-E, n = 18), fresh epididymal spermatozoa that had been
xposed to seminal plasma (Fr-SP+, n = 12) or fresh epididy-
al spermatozoa that had never been exposed to seminal
lasma (Fr-SP−, n = 9), frozen-thawed ejaculated spermato-
oa (Cr-E, n = 18), frozen-thawed epididymal spermatozoa
hat had been exposed to seminal plasma prior to freezing
Cr-SP+, n = 18) and frozen-thawed epididymal sperma-
ozoa that had never been exposed to seminal plasma
Cr-SP−, n = 15).
Pregnancy rates were 55.6% (Fr-E, 10/18), 75% (Fr-SP+,
/12), 22.2% (Fr-SP−, 2/9), 38.9% (Cr-E, 7/18), 27.8% (Cr-SP+,
/18) and 6.7% (Cr-SP−, 1/15).
Univariable comparisons of pregnancy rates showed
hat, for fresh and frozen-thawed semen combined, the
robability of pregnancy was greater for ejaculated sper-
atozoa (Fr-E and Cr-E) than for epididymal spermatozoa
hat had never been exposed to seminal plasma (Fr-SP− and
r-SP−) (relative risk (RR) = 3.8; 95% CI: 1.2, 11.5; P = 0.006).
able 3
nsemination results for individual stallions.
Insemination groups
aFr-E bFr-SP+
tallion A
Mares inseminated (n) 6 4
Mares pregnant (n) 3 3
Pregnancy rate (%) 50 75
tallion B
Mares inseminated (n) 6 4
Mares pregnant (n) 2 2
Pregnancy rate (%) 33 50
tallion C
Mares inseminated (n) 6 4
Mares pregnant (n) 5 4
Pregnancy rate (%) 83 100
a Fr-E, fresh ejaculate.
b Fr-SP+, fresh epididymal spermatozoa that had been exposed to seminal plasm
c Fr-SP−, fresh epididymal spermatozoa that had never been exposed to semin
d Cr-E, frozen-thawed ejaculated spermatozoa.
e Cr-SP+, frozen-thawed epididymal spermatozoa that had been exposed to sem
f Cr-SP−, frozen-thawed epididymal spermatozoa that had never been exposed
6 1.22 9.54 0.88, 103.8 0.064
The probability of pregnancy was 3.8 times greater for ejac-
ulated spermatozoa than for epididymal spermatozoa not
exposed to seminal plasma. Overall comparison between
fresh and frozen-thawed ejaculated spermatozoa with epi-
didymal spermatozoa that had been exposed to seminal
plasma showed that pregnancy rates were similar (RR = 1.0;
95% CI: 0.6, 1.7; P > 0.99). There was a significantly higher
pregnancy rate after AI with fresh and frozen-thawed epi-
didymal spermatozoa that had been exposed to seminal
plasma (Fr-SP+ and Cr-SP+) than after AI with epididy-
mal spermatozoa that had never been exposed to seminal
plasma (Fr-SP− and Cr-SP−) (RR = 3.7; 95% CI: 1.2, 11.5;
P = 0.009). Fr-SP+ achieved higher pregnancy rates than Fr-
SP− (RR = 3.4; 95% CI: 1.0, 12.0; P = 0.03). A similar trend
was seen for frozen-thawed epididymal spermatozoa (Cr-
SP+ compared to Cr-SP−), but the statistical significance
was lower (RR = 4.2; 95% CI: 0.5, 31.9; P = 0.19). Results of the
multilevel logistic regression models are shown in Table 2
and were very similar to the results obtained using the
Fisher exact test.
Insemination results for individual stallions are listed in
Table 3.
The inclusion of stallion as a fixed effect rather than
a random effect resulted in no material changes to the
cFr-SP− dCr-E eCr-SP+ fCr-SP−
3 6 6 6
1 2 1 1
33 33 17 17
3 6 6 6
0 2 0 0
0 33 0 0
3 6 6 3
1 3 4 0
33 50 67 0
a.
al plasma.
inal plasma prior to freezing.
to seminal plasma.
oduction
52 A. Heise et al. / Animal Repr
estimates or significance of the coefficients for the other
predictors. There were increased odds of pregnancy using
stallion C (Welsh Cross) over stallion B (Boerperd) (odds
ratio (OR) = 8.3; 95% CI: 2.2, 31.3; P = 0.002). There was also
some evidence for increased odds for pregnancy using stal-
lion C (Welsh Cross) over stallion A (Warmblood) (OR = 3.0;
95% CI: 0.9, 10.0; P = 0.07) and using stallion A (Warm-
blood) over stallion B (Boerperd) (OR = 2.7; 95% CI: 0.8, 9.9;
P = 0.12).
4. Discussion
The most important finding of the present study, that
included the results of a total of 90 inseminations, is
that exposing epididymal spermatozoa to seminal plasma
improved pregnancy rate after routine AI into the uterine
body. A significantly higher pregnancy rate was achieved
after AI with fresh and frozen-thawed epididymal sperma-
tozoa that had been exposed to seminal plasma (Fr-SP+
and Cr-SP+) than after AI with epididymal spermatozoa
that had never been exposed to seminal plasma (Fr-SP−
and Cr-SP−) (P = 0.009). Furthermore the overall pregnancy
rate obtained with fresh and frozen-thawed epididymal
spermatozoa that had been exposed to seminal plasma
(Fr-SP+ and Cr-SP+) was similar to that after use of fresh
and frozen-thawed ejaculated spermatozoa (Fr-E and Cr-
E).
Our results indicate a definite advantage of using semi-
nal plasma to increase fertility of epididymal spermatozoa,
stand in contrast to previously published data (Morris et
al., 2002) that showed that seminal plasma did not have
an effect on fertility, regardless of the method of insem-
ination (hysteroscopic or routine AI) with frozen-thawed
epididymal spermatozoa. However, the study by Morris
et al. (2002) was based on a small number of insemi-
nations and methods used were less well defined with
regard to the exposure of epididymal spermatozoa to sem-
inal plasma and insemination dose. The report by Morris
et al. (2002) also did not state whether or not seminal
plasma was removed prior to freezing. Differences in the
methods of processing of the epidiymal spermatozoa may
thus have contributed to the contradictory results between
the two studies. Our results demonstrated that the expo-
sure of epididymal spermatozoa to seminal plasma during
harvesting from the tail of the epididymis increased the
fertilising ability of epididymal spermatozoa. But, even
though the exposure of epididymal spermatozoa to sem-
inal plasma increased the odds for conception, it was
not absolutely necessary to achieve conception, a find-
ing that is well supported (Melo et al., 2008; Morris et
al., 2002; Papa et al., 2008). Looking at pregnancy rates
after AI with fresh and frozen-thawed epididymal sperma-
tozoa separately, Fr-SP+ achieved higher pregnancy rates
than Fr-SP− (P = 0.03). A similar trend, though not statis-
tically significant (P = 0.19), was seen for frozen-thawed
epididymal spermatozoa (Cr-SP+ compared to Cr-SP−).
This difference was only very obvious for spermatozoa
of stallion C. A number of different explanations can be
offered for the positive effect of seminal plasma on the
fertility of stallion epididymal spermatozoa. It is well
documented that seminal plasma components influence
Science 118 (2010) 48–53
sperm capacitation which is necessary for the acrosome
reaction to occur. Seminal plasma contains decapacita-
tion factors or coating factors which can reversibly inhibit,
or even reverse, capacitation (Oliphant et al., 1985). This
may be a reason for prevention of premature capacitation
in Fr-SP+. Therefore, fresh epididymal spermatozoa that
have never been exposed to seminal plasma may undergo
spontaneous acrosome reactions, thus preventing fertilisa-
tion.
Seminal plasma also contains factors that enhance
capacitation. Several bull seminal plasma proteins (BSP
proteins) facilitate cholesterol efflux from the plasma
membrane (Therien et al., 1998) which decreases mem-
brane stability and could induce or enhance a reorgani-
sation of the sperm plasma membrane. Incubation of bull
epididymal spermatozoa with BSP proteins enhanced their
ability to undergo the acrosome reaction (Therien et al.,
1997). The capacitation enhancing factors contained in
seminal plasma are therefore another possible explanation
for the higher pregnancy rate with Fr-SP+. This has also been
shown for boar epididymal spermatozoa in vitro, where a
time-dependent improvement of zona pellucida binding
was achieved after exposure of epididymal spermatozoa to
boar seminal plasma (Harkemaet al., 2004).
A third explanation could be the fact that seminal
plasma suppresses complement activation, polymor-
phonuclear neutrophil (PMN) chemotaxis and phagocyto-
sis in vitro (Dahms and Troedsson, 2002; Troedsson et al.,
2002; Troedsson et al., 2000) therefore playing a regu-
latory role in breeding-induced endometritis. A transient
uterine inflammation, caused by chemotaxis of sperma-
tozoa through activation of complement, is physiological
and necessary in order to clear excess spermatozoa and
seminal plasma from the uterus (Troedsson et al., 2001).
An inflammatory uterine environment has however been
shown to be detrimental to spermatozoa motion charac-
teristics in vitro. PMNs in uterine secretions form slow
moving spermatozoa/PMN aggregates (Alghamdi et al.,
2001) which may prevent a large number of fertile sper-
matozoa from being transported to the oviduct, thus
reducing the number of spermatozoa available for fertili-
sation.
Transportation of spermatozoa from the equine uterus
to the oviduct is complete within 4 h of breeding (Brinsko
et al., 1991) but PMNs have been observed in the uterine
lumen within 30 min after AI (Katila, 1995). Even though
seminal plasma did not improve spermatozoa motion
characteristics, it significantly reduced the formation of
spermatozoa/PMN aggregates (Troedsson et al., 2001). This
could enable more epididymal spermatozoa that had been
exposed to seminal plasma to reach the oviduct. Although
most of the seminal plasma is removed prior to cryop-
reservation a small amount remains with the spermatozoa,
possibly exerting a protective effect in utero on frozen-
thawed epididymal spermatozoa. Similar results have been
reported in dogs (Hori et al., 2005). Exposure to seminal
plasma had a significant positive effect on the motility and
viability of canine epididymal spermatozoa, as well as preg-
nancy rates after AI. Hori et al. (2005) hypothesised that this
effect could be due to a longer life span of spermatozoa that
had been exposed to prostatic fluid.
oduction
5
p
t
s
b
t
A
S
m
R
A
B
B
B
B
C
D
D
H
H
J
J
K
A. Heise et al. / Animal Repr
. Conclusions
Exposure of epididymal spermatozoa to seminal plasma
rior to any other dilution or extension increases the fer-
ility of both, fresh and frozen-thawed, epididymal stallion
permatozoa. Due to the fact that only three stallions have
een used for this trial it can be seen as a pilot study guiding
he way to a future large scale study.
cknowledgements
The authors would like to thank P. Botha, Geyerspan
tud, North West Province, South Africa for providing the
ajority of the mares used in this study.
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	Influence of seminal plasma on fertility of fresh and frozen-thawed stallion epididymal spermatozoa
	Introduction
	Materials and methods
	Animals and experimental design
	Semen freezing
	Semen extender
	Seminal plasma
	Harvesting of epididymal spermatozoa
	Statistical analysis
	Results
	Pregnancy rates
	Discussion
	Conclusions
	Acknowledgements
	References