No.269
No.269
IN
IN VITRO
VITRO FERTILITY
FERTILITY OF
OF BOAR
BOAR SPERMATOZOA
SPERMATOZOA PRESERVED
PRESERVED AT
AT 10
10ooC
C FOR
FOR 22
22 DAYS
DAYS
H. Funahashi
Graduate School of Natural Science & Technology, Okayama University,
University, 700700-8530 Japan
Materials
Materials and
and Methods
Methods
Fertility of boar spermatozoa after artificial
insemination can be maintained during liquid
preservation at 10-15oC for several days, although
prolonged liquid preservation reduces the pregnancy
rate rapidly. However, it is not clear if spermatozoa
can penetrate into oocytes in an IVF system even after a
prolonged liquid preservation. Oxidative stress could
be one of possible detrimental factors in liquid
preservation of spermatozoa. In the present study, the
fertility of liquid-preserved spermatozoa was examined.
It was also determined if cysteine can improve the
fertility. Spermatozoa (from four Berkshires) was
resuspended at 1 x 108 cells/ml in Modena solution
containing 20% (v/v) boar seminal plasma and 0 or 5
mM cysteine after washing. Sperm suspensions (1 ml)
were then preserved at 10oC for 22 days after cooling
down with a program (to 15oC for 4 h, keeping at 15oC
for 12 h and then to 10oC for 6 h). At 1, 8, 15, 22, 29
and 36 days after the start of preservation, spermatozoa
were co-cultured with IVM oocytes in an IVM/IVF
system (Funahashi et al., BOR 57;49-53). Viability and
functional status of spermatozoa were also examined at
Days 8 and 15 of preservation by using LIVE/DEAD
sperm viability kit and CTC fluorescence assay. Data
(mean±SEM) from 4-6 replicates were analyzed by
ANOVA and Fisher’s protected LSD test. Penetration
rates with spermatozoa preserved without cycteine
(Cys-) were not different (P>0.05) from those with
cysteine (Cys+) at Day 8 of preservation (91.4±3.4%
in Cys- and 99.3±0.7% in Cys+), but lower (P<0.02) at
Days 15 and 22 (72.6±13.6% and 33.8±8.4% in Cys-;
94.8±2.1% and 71.1±10.8% in Cys+, respectively).
Both viability and proportion of uncapacitated live cells
were higher (P<0.05) in Cys+ than Cys- at Days 8 and
15. These results demonstrate that boar spermatozoa
can penetrate into oocytes in vitro even after a liquid
preservation at 10oC for 22 days and that cysteine can
improve the viability and penetrability in vitro of
spermatozoa during liquid preservation. Supported by
the Ito Foundation.
Spermatozoa (from four Berkshires) was resuspended at 1 x
108 cells/ml in modified Modena solution* containing 20% (v/v)
boar seminal plasma and 0 or 5 mM cysteine after washing.
Sperm suspensions (1 ml) were then preserved at 10oC for
57 days after cooling down with a program (to 15oC for 4 h,
keeping at 15oC for 12 h and then to 10oC for 6 h).
At 1, 8, 15, 22, 29 and 36 days after the start of preservation,
spermatozoa were co-cultured with IVM oocytes in an IVM/IVF
system (Funahashi et al., 1997).
Viability and functional status of spermatozoa were also
examined at Days 8 and 15 of preservation by using
LIVE/DEAD sperm viability kit and CTC fluorescence assay.
Data (mean±SEM) from 4-6 replicates were analyzed by
ANOVA and Fisher’s protected LSD test.
Percentage of live cells
100
*
*
P*<0.05
*
*
*
*
20
0
0
8 15 22 29 36 43 50 57
Days after the start of preservation
80
60
a
40
20
0
1
8
15
22
29
36
Days after the start of preservation
Fig. 1 Effect of cysteine on the viability of
liquid-preserved boar spermatozoa
Fig. 3 Effect of cysteine on the penetrability in vitro
of liquid-preserved boar spermatozoa
Day-15
Day-8
b
152.60
23.46
11.90
6.31
46.66
15.10
(0.025g/L)
80
b
a
100
Control
Cysteine
a
60
Pab<0.05
%
40
a
a
20
Basic solution for liquid preservation
modified Modena + 20% (v/v) Seminal plasma
Control
: without Cysteine
Exp. group : with 5 mM Cysteine
b
b
0
Introduction
Introduction
Percentage of cells
100
80
F
B AR
CTC-patterns
F pattern
B pattern
AR pattern
60
40
20
0
F
B AR
CTC-patterns
Fig. 2 Effect of cysteine on the CTC-pattern
of liquid-preserved boar spermatozoa
Fertility of boar spermatozoa after artificial
insemination can be maintained during liquid
preservation at 10-15oC for several days (Johnson,
2000). However, prolonged preservation period
gradually reduces the penetrability of the cells,
probably due to ageing occurred during storage in
vitro (Johnson, 2000). It is not clear if
spermatozoa can penetrate into oocytes in an IVF
system even after a prolonged liquid preservation
and how the cells changes during liquid
preservation. Lipid peroxidation due to oxidative
stress during preservation could be one of
possible detrimental factors of sperm.
Supplementation with antioxidants has been
demonstrated to improving the viability and motility
of liquid- and cyro-preserved mammalian
spermatozoa (Maxwell & Stojanov, 1996 ;
Bilodeau et al., 2001; Cerolini et al., 2001; Foote et
al., 2002; Pena et al., 2003). In the present study,
the fertility of liquid-preserved spermatozoa in the
presence of cysteine was examined.
Pab<0.05
b
a
*
Control
5 mM Cysteine
40
Control
5mM Cysteine
b
100
60
modified
modifiedModena
Modenasolution*
solution*
Conc. (mM)
Glucose
Sodium citrate•2H2O
NaHCO3
EDTA-2Na
TRIS
Citric acid
Gentamicin
*
80
Sperm penetration (%)
Abstract
Abstract
8
15
22
29
36
43
50
57
Days after the start of preservation
Fig. 4 Change of CTC-patterns of boar spermatozoa
liquid-preserved with 5 mM cysteine
Results
Results (Summary)
(Summary)
Cooled down the
semen sample by
using a program
freezer (EYELA
MPF-40)
℃
Viability of spermatozoa was higher in the presence of cysteine throughout the preservation period (Fig 1).
Proportion of uncapacitated live cells was higher after liquid-preservation in the presence of 5 mM cysteine
for 8 and 15 days (Fig 2).
Penetration rates with spermatozoa preserved without cycteine were not different (P>0.05) from those with 5
mM cysteine at Day 8 of preservation, but lower at Days 15 and 22 (Fig 3).
Proportion of uncapacitated live cells decreased gradually as the preservation duration was prolonged even
in the presence of 5 mM cysteine (Fig 4).
Kept the samples in a
dry thermo bath
（IWAKI CHT-101） at
10 ± 0.1°C.
25
15
8~57days
10
4h
12h
6h
Liquid
Liquidpreservation
preservation of
ofBoar
Boarspermatozoa
spermatozoa
Samples were
warmed up at
room temperature
for 15-20 min and
then mixed with
TL-HEPES-PVA for
washing.
Conclusion
Conclusion
These results demonstrate that boar
spermatozoa can penetrate into oocytes in
vitro even after a liquid preservation at 10oC
for 22 days and that cysteine can improve the
viability and penetrability in vitro of
spermatozoa during liquid preservation.
This research was supported by the Ito
Foundation.
References
Bilodeau JF, Blanchette S, Gagnon C & Sirard MA (2001) Thiols prevent
H2O2-mediated loss of sperm motility in cryopreserved bull semen.
Theriogenology 56 275-286
Cerolini S, Maldjian A, Pizzi F & Gliozzi TM (2001) Changes in sperm
quality and lipid composition during cryopreservation of boar semen.
Reproduction. 121 395-401
Foote RH, Brockett CC & Kaproth MT (2002) Motility and fertility of bull
sperm in whole milk extender containing antioxidants. Anim Reprod Sci.
71 13-23
Funahashi H, Cantley TC & Day BN (1997) Synchronization of meiosis in
porcine oocytes by exposure to dibutyryl cyclic adenosine
monophosphate improves developmental competence following in vitro
fertilization. Biol Reprod 57 49-53
Johnson LA, Weitze KF, Fiser P & Maxwell WMC (2000) Storage of boar
semen. Anim Reprod Sci 62 143-172
Maxwell WM & Stojanov T (1996) Liquid storage of ram semen in the
absence or presence of some antioxidants. Reprod Fertil Dev 8 10131020
Pena FJ, Johannisson A, Wallgren M & Rodriguez Martinez H (2003)
Antioxidant supplementation in vitro improves boar sperm motility and
mitochondrial membrane potential after cryopreservation of different
fractions of the ejaculate. Anim Reprod Sci. 78 85-98