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Bull Vet Inst Pulawy 50, 503-507, 2006
INFLUENCE OF BULL SEMINAL PLASMA ON POST-THAW
RAM SEMEN PARAMETERS AND FERTILITY
ULGEN GUNAY, IBRAHIM DOGAN, ZEKARIYA NUR, IVAN MANOLOV1,
HAKAN SAGIRKAYA, MUSTAFA KEMAL SOYLU, CUNEYT KAPTAN2 AND LATIF AKPINAR2
Department of Reproduction and Artificial Insemination, Faculty of Veterinary Medicine,
Uludag University, 16059, Gorukle, Bursa, Turkey
1
Department of Animal Reproduction, Agricultural Faculty,
Thracian University, 6000, Stara Zagora, Bulgaria
2
Bandirma Research Institute, 10200, Balikesir, Turkey
[email protected]
Received for publication March 24, 2006.
Abstract
The aim of the study was to evaluate the effect of the
addition of 20% bull seminal plasma to semen extenders on
post-thaw sperm motility, morphology, and fertility. Two
Merino rams at 3-5 years of age, 43 ewes, and one Holstein
bull were used. Fourteen ejaculates were collected by the use
of an artificial vagina, twice a day, at 60 min interval. Six
semen ejaculates were extended with Tris-egg yolk extender
containing 20% bull seminal plasma (TEY-BSP group), and 8
ejaculates were extended with Tris-egg yolk (TEY group), at a
rate to obtain 150x106 spermatozoa/straw. The ewes in
oestrous were divided at random into two groups. Twenty-two
ewes were inseminated with TEY-BSP and 21 ewes were
inseminated with TEY. The mean percentage of motility,
defected acrosome, and total morphological defect for the
equilibrated and post-thaw semen parameters of the TEY, were
69.4%, 8.6%, and 17.0% and 42.8%, 49.9%, and 51.2%,
respectively. The mean percentage of non-return rate (NRR)
(n=43) was 30.2% and for the semen frozen with TEY-BSP
(n=22) and TEY (n=21) - 31.8%, and 28.6%, respectively.
There were no significant differences between TEY-BSP and
TEY for NRR. In conclusion, the addition of the bull seminal
plasma to freezing extender has a supportive effect on
extended, equilibrated, and thawed semen. Moreover, the
addition of bull seminal plasma to ram semen extender
improved fertility.
Key words: ram, frozen semen, bull seminal
plasma, fertility.
Cervical insemination with frozen-thawed
semen has not been widely applied for commercial
artificial insemination (AI) of sheep (32). The
cryopreservation process results in reduced motility and
fertilizing capacity compared with fresh semen (1, 8,
34). Unlike other farm animals, ram spermatozoa are
more susceptible to cryopreservation (1, 8, 19), and the
cervix of ewes restricts the transcervical passage of
insemination instruments, and consequently AI (17, 18,
20, 29, 30). The success of AI with frozen ram semen
depends on the physiology of sperm transport (21, 24,
30, 31, 33), and on the survival of spermatozoa in the
female reproductive tract (21). Also, cooling and
freezing rates (8, 26), type of cryoprotective agents and
their concentration, extender composition, dilution rates,
temperature at which glycerol is added to the semen,
equilibration time, and thawing rate interact with the
success of AI with frozen semen (1-4, 25-27, 32).
The extenders composition assists in stabilizing
the cells during the freezing and thawing process (4, 6,
10, 13, 14). Several studies (1, 2, 6-9, 13, 15, 34) have
been conducted to determine the optimal extender to
enhance post-thaw motility, morphology, and fertility of
ram semen, including addition of egg yolk, bull seminal
plasma, milk, and sodium sulphate (6, 19, 21-23, 25,
28). In various studies (4, 6) the effects of seminal
plasma on the post-thaw motility, morphology, and
fertility abnormalities were examined. Some researchers
(7, 28, 32) agreed that dialysed egg yolk, bull seminal
plasma, milk, and sodium sulphate could prevent
spermatozoa from the post-thaw side effect of lipid
peroxidation
The aim of the present study was to evaluate
effect of addition of bull seminal plasma semen
extenders on post-thaw sperm motility, morphology, and
fertility.
Material and Methods
Animals. Two Merino rams at 3-5 years of age,
43 Merino ewes housed at Bandirma Research Institute,
and one Holstein bull housed at Veterinary Faculty Farm
of Uludag University were used in the study.
Semen collection and freezing. Bull semen
was collected by an artificial vagina and centrifuged at
5 000 rpm for 10 min to separate its seminal plasma.
Fourteen ejaculates from rams (2x7) were collected by
504
the use of an artificial vagina, in the presence on
oestrous ewe, twice a day, at 60 min intervals. Semen
samples were placed in a water bath at 37°C. Soon after
the collection, volume, wave activity, motility,
concentration, and percentage of spermatozoa,
exhibiting morphologic abnormalities were evaluated
according to standard procedures (16). Only ejaculates
with a volume higher than 0.5 ml, and wave motility
higher than (+++) were used. The volume was recorded
from the collection tube, which was graduated in 0.1 ml
divisions. Sperm concentration was assessed using
haematocytometer. To assess the wave activity, a drop
of semen deposited on a glass slide and placed on a
warm stage (40°C) was examined under the light
microscope (x100). The semen sample was scored using
a scale ranging from 0 (no wave movement), to 5
(extreme wave movement).
Sperm motility was assessed subjectively using
a phase-contrast microscope (x400) with a warm slide
(40°C). Defected acrosome (DA) and total
morphological defect (TMD) were evaluated using
Giemsa staining procedure (16). A total of 200
spermatozoa were examined under a light microscope
(x1000), to determine the percentages of spermatozoa
with DA and TMB. Semen evaluation (motility, DA and
TMD) was carried out at 3 stages (after dilution,
equilibration, and thawing).
Six ejaculates were extended with Tris-egg yolk
extender containing 20% bull seminal plasma (TEYBSP group), and 8 ejaculates were extended with Trisegg yolk (TEY group), at a rate to obtain 150x106
spermatozoa/straw. Extended semen was cooled to 5°C
for 120 min. Cooled semen was glycerolized in 5 steps,
at 5 min intervals with Tris-egg yolk extender
containing 10% glycerol. Final concentration of glycerol
was 5%. After glycerolisation, it was subjected to
equilibration for 2 h. Semen in 0.25 ml straws was
frozen horizontally on a rack, about 7 cm above liquid
nitrogen vapour for 10 min, and stored in liquid nitrogen
containers at -196°C. At least 3 straws from each
ejaculate were thawed at 50˚C for 15 s, in a water bath
to evaluate post-thaw semen motility and morphology
(14x3=42 straws).
Artificial
insemination.
Intra-cervical
insemination was performed on ewes during the
transition period from non-breeding to natural breeding
season in the region, under natural lighting. Oestrous
was detected with a teaser ram every day. The ewes in
oestrous were divided at random into two groups.
Twenty-two ewes were inseminated with TEY-BSP and
21 ewes were inseminated with TEY.
Fertility. To detect ewes returning to oestrous,
all the ewes were checked from days 12-30 after
insemination using teaser rams. Ewes which did not
return to oestrous were considered pregnant and
recorded as non-return rate 30 d (NRR-30) (28).
The mean semen parameters of motility, DA,
and TMD calculated for the 14 ejaculates obtained from
the rams, were subjected to analysis of variance (oneway NOVA), and the differences among means were
tested for significance by the Fisher’s PLSD. Pregnancy
rates were analysed using the chi-square test. The SPSS
10.0 software was used for all statistical analyses (Instat,
1990–1993).
Results
The results of the study are summarised in
Table 1. The mean of volume, wave activity, and
concentration for the diluted semen of the TEY-BSP
group were 0.7 ml, 3.5, and 2.8X109 /ml, and in the TEY
group they were 1.0 ml, 3.6, and 3.2X109 /ml,
respectively. The mean percentage of motility, DA, and
TMD for the equilibrated and post-thaw semen
parameters of the TEY-BSP was 68.3%, 9.6%, and
12.5% and 48.3%, 47.3%, and 48.3%, respectively. The
mean percentage of motility, DA, and TMD for the
equilibrated and post-thaw semen parameters of the
TEY was 69.4%, 8.6%, and 17.0% and 42.8%, 49.9%,
and 51.2%, respectively (Table 1). No significant
differences in all the studied parameters for diluted,
equilibrated, and post-thaw semen were found between
rams (P>0.05).
The NRR-30 according to extenders and rams
was summarised in Table 2. The mean percentage of
NRR (n=43) was 30.2% and for the frozen semen with
TEY-BSP (n=22) and TEY (n=21) was 31.8% and
28.6%, respectively (Table 2). These differences were
not statistically significant (P>0.05). After the use of
both extenders, similar results were obtained.
Discussion
The cryopreservation cycle for semen samples,
includes the entire process from sperm collection,
preparation, and dilution through to post-thawing
maintenance of fertilizing ability. Many extenders have
been used for freezing ram semen (1, 2, 4, 6, 12). The
present study was aimed at investigating effects of bull
seminal plasma (BSP) on post-thaw ram sperm motility,
morphology and fertilizing ability. At the mean volume,
wave activity, and concentration for the diluted semen of
the TEY-BSP and TEY groups were in accordance with
previous reports (15, 16). For the extended and
equilibrated semen, the addition of bull seminal plasma
to freezing extender had supportive effect on motility,
acrosome, and morphological integrity. The mean
percentage of motility, DA, and TMD for the diluted and
equilibrated semen parameters of the TEY-BSP and
TEY groups, were in agreement with previous studies
(6, 15).
Furthermore, the addition of bull seminal
plasma to freezing ram semen extender protects
spermatozoa from the damaging effect of low
temperatures, and can also support post-thaw motility
(48.3% vs. 42.8%), DA (47.3% vs. 49.9%), and TMD
(48.3% vs. 51.2%). TEY-BSP provided higher post-thaw
motility, acrosomal, and morphological integrity than
TEY extender, there were no significant differences
between
post-thaw
motility,
acrosomal,
and
morphological integrity in the two groups (P>0.05).
505
Table 1
Mean parameters of diluted, equilibrated, and thawed sperm for the individual ram, according to extenders
Diluted
Extender
Equilibrated
Post-thaw
Ram
No
Thawed
straw
(n)
Volume
(ml)
Wave
activity
(1-5)
Concentration
(X109/ml)
Motility
(%)
DA
(%)
TMB
(%)
Motility
(%)
DA
(%)
TMB
(%)
Motility
(%)
DA
(%)
TMB
(%)
60
9
0.6±0.1
3.7±0.3
2.7±0.6
83.3±1.6
4.8±0.4
5.5±0.8
71.7±1.7
9.2±1.2
10.8±1.3
51.1±2.0
43.7±2.1
44.8±2.1
570
9
0.9±0.1
3.3±0.3
2.7±0.2
81.7±1.7
4.2±0.8
5.5±1.0
65.0±2.9
10.0±3.5
14.2±4.8
45.6±1.0
50.9±1.4
51.8±1.4
18
0.7±0.1
3.5±0.2
2.8±0.9
82.5±1.1
4.5±0.4
5.5±0.6
68.3±2.1
9.6±1.7
12.5±2.3
48.3±1.3
47.3±1.5
48.3±1.5
60
12
1.0±0.2
3.5±0.3
2.9±0.7
80±2.9
6.1±1.2
10.0±0.7
67.5±1.4
9.0±0.8
17.9±0.6
41.1±6.3
52.7±5.4
54.1±5.6
570
12
1.0±0.2
4.0±0.0
3.7±0.3
78.8±2.4
5.4±1.1
14.9±1.3
71.3±1.3
8.1±0.9
16.1±1.4
44.4±2.9
47.2±1.2
48.2±1.3
24
1.0±0.1
3.6±0.2
3.2±0.2
79.4±1.8
5.6±0.8
15.5±0.7
69.4±1.1
8.6±0.6
17.0±0.8
42.8±3.2
49.9±2.8
51.2±2.9
TEY-BSP
General mean
TEY
General mean
± SD, DA-defected acrosome; TMD-total morphological defects; TEY-BSP-Tris-egg yolk-bull seminal plasma; TEY-Tris-egg yolk
Table 2
Fertility according to TEY-BSP and TEY extenders
Extender
Ram No
Number of inseminated ewes
(n)
NRR-30
(%)
60
11
3/8 (27.3)
570
11
4/7 (36.4)
mean
22
7/15 (31.8)
60
11
4/7 (36.4)
TEY
570
10
2/8 (20.0)
mean
21
6/15 (28.6)
60
22
7/15 (31,8)
570
21
6/15 (28.6)
General mean
mean
43
13/30 (30.2)
NRR-30-non return rate 30 days; TEY-BSP-Tris-egg yolk-bull seminal plasma; TEY-Tris-egg yolk
TEY-BSP
505
506
The post-thaw motility observed in the present study for
the semen extended with TEY, is in accordance with the
results of researchers (1, 13, 15) who used different
glycerol and egg yolks rates, in various extenders. The
post-thaw motility of the TEY-BSP was higher than that
reported by Baran et al. (6), who used bull seminal
plasma in different rates (7.5% and 15%). The reason for
this difference is thought to be that semen samples were
centrifuged by the last authors.
(6). Moreover, it might be associated with beneficial
effect of different rates of bull seminal plasma for ram
semen. DA and TMB for the two groups were similar to
findings (40-60%) demonstrated by some researchers
(13, 15). However, our results were not similar to those
reported by Baran et al. (6).
The physiological status of the ewe, lambingAI interval, its age and breed (3, 11, 12), insemination
technique (9, 11, 27), inseminator (27), and farming
conditions, have an effect on fertility rates. Other
important factors affecting fertility are, post-thaw sperm
motility and morphology (3, 8, 21, 28, 29, 34). Fertility
was slightly higher in the TEY-BSP than TEY group for
NRR-30 (31.8% vs. 28.6%), although these differences
were not statistically significant. The NRR-30 in the
present study, though lower than results reported by
Anel et al. (3) and Paulenz et al. (28), was in agreement
with results reported by Danovan et al. (11), King et al.
(20), and Ataman and Coyan (5) who used different
NRR, lambing rate, extender, and insemination
technique.
Maxwell et al. (21) observed that the
improvement in the post-thaw motility reflected in
fertilizing capacity. Although the ram fertility results,
including both extenders, were higher for ram 60 than
570, these differences were not significant (P>0.05).
This result was not surprising, because post-thaw
spermatological findings of ram 60 were better than of
ram 570 for TEY-BSP and TEY, generally.
In conclusion, the present study indicates that
the addition of the bull seminal plasma to freezing
extender has a supportive effect on extended,
equilibrated, and thawed semen. In addition, freezing
ram semen with bull seminal plasma, improved fertility
after artificial insemination. In the future, research on
different various rates of bull seminal plasma; on postthaw ram semen quality and fertility, can also be
profited from.
Acknowledgments: This work was funded
by the Research Fund of Uludag University, BAPB
(2001/20). The authors wish to thank the Bandırma
Research Institute for providing the animals.
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