494
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Asian-Aust. J. Anim. Sci.
Vol. 21, No. 4 : 494 - 498
April 2008
www.ajas.info
Evaluation of Boar Sperm Viability by MTT Reduction Assay in
Beltsville Thawing Solution Extender*
J. W. Byuna, S. H. Choo1, a, H. H. Kim, Y. J. Kim, Y. J. Hwang and D. Y. Kim**
Division of Biological Science, Gachon University of Medicine and Science, Incheon, 406-799, Korea
ABSTRACT : MTT (3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) reduction assay is a method that validates the
viability of an active cell. Dehydrogenase in mitochondria converts yellow colored insoluble tetrazolium salt to purple colored watersoluble formazan. Sperm also have mitochondria in the midpiece, therefore sperm viability could be evaluated by MTT reduction assay.
Several studies have already demonstrated the capability of application of the MTT reduction assay to sperm of several species in
Hepes-BSA buffer. Because most liquid semen was diluted in extender like BTS (Beltsville Thawing Solution), Modena or Androhep
when it is used or transferred, semen needed another dilution in Hepes-BSA buffer to assess sperm viability. In this study, we evaluated
boar sperm viability especially in BTS extended semen and compared the efficiency of this test with eosin-nigrosin staining. We used the
fresh BTS extended semen from a local A.I center. Semen sample was diluted to 3.0×107 sperms/ml in BTS. The rates of formazan
production were measured in 96-well microtiter plates immediately and 1h after incubation at 17°C using a spectrophotometer at wave
length 560 nm. Simultaneously, split samples of the same semen were tested, using eosin-nigrosin staining to compare the efficiency of
the MTT assay of sperm viability in BTS. The correlation between the results of these tests was calculated using Student-t test and
ANOVA. The results revealed a strong correlation between the results of MTT reduction rate and the results that were simultaneously
determined by eosin-nigrosin staining at 1 h. In conclusion, the MTT reduction test was an effective and simple method to validate
sperm viability and it could be used as a simple tool to evaluate sperm viability in the local A.I center and laboratory. (Key Words :
MTT Reduction Assay, Sperm Viability, Eosin-nigrosin Staining, BTS Extender)
INTRODUCTION
There are several methods for evaluating sperm viability.
Visual estimation of viable sperm using microscope is
commonly used for it. Eosin-nigrosin method and HOST
(Hypo-Osmotic Swelling Test) are simple and inexpensive;
however the result can be influenced by experience of
analyst (McNiven et al., 1992; Nasr-Esfahani et al., 2002;
Jang et al., 2006). By contrast, CASA (Computer Assisted
Sperm Analysis) and flow cytometry are the more objective
method. CASA measure the speed of sperm and
abnormality of shape. It uses the computer and camera to
measure these characters of sperm (Arman et al., 2006).
* This work was supported by a grant (No.20070301-034-040007-03-00) from BioGreen 21 Program, Rural Development
Administration, Korea.
** Corresponding Author: D. Y. Kim. Tel: +82-32-820-4544,
Fax: +82-32-821-2734, E-mail: [email protected]
1
Graduate School of Medicine, Gachon University of Medicine
and Science, Incheon, 406-799, Korea.
a
These two authors contribute equally to this work.
Received August 25, 2007; Accepted November 23, 2007
Flow cytometry is a technology that allows a single cell to
be measured for a variety of characteristics. It is different
technical applications takes many advantages for the
analysis of sperm quality. Because flow cytometry evaluates
individual cell with rapid speed, it is objective and accurate
method (Cordelli et al., 2005).
MTT (3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide) is a yellow water-soluble tetrazolium
salt. Succinate dehydrogenase system of active
mitochondria converts this dye to water-insoluble purple
formazan on the reductive cleavage of its tetrazolium ring
(Slater et al., 1963). Thus, the amount of formazan formed
can be determined spectrophotometrically and serves as an
estimate of the number of mitochondria and hence the
number of living cells in the sample (Denizot and Lang,
1986; Song et al., 2007). In many studies, MTT assay was
used which were related to viability of different cells
(Mosmann, 1983; Levitz and Diamone, 1985; Carmichael et
al., 1987; Campling et al., 1988; Freimoser et al., 1999).
Therefore, this method can be used to assess sperm viability
and MTT reduction assay already have successfully applied
Byun et al. (2008) Asian-Aust. J. Anim. Sci. 21(4):494-498
1.200
Absorbance at 560 nm
1.000
100
60
20
80
40
0
0.800
0.600
0.400
0.200
0.000
0
1
2
3
4
Incubation time (h)
Figure 1. MTT reduction rates of the semen samples containing
different portion of live and dead sperms. The optical density of
samples were measured immediately and after 1h of incubation at
17°C using a spectrophotometer (μQuantTM Microplate Spectrophotometer, Bio-Tek instruments, USA) at a wave length of 560
nm.
to check sperm viability in many species (Capkova et al.,
2000; Park et al., 2002, 2004; Aziz et al., 2005; Hu et al.,
2006). In case of boar semen, no research has been studied.
Fresh boar semen sample is diluted in BTS (Beltsville
Thawing Solution) extender. BTS extender is commonly
used to dilution the fresh boar semen for transfer and
storage (Joshi et al., 2006).
In this study, we evaluated the boar sperm viability
especially in BTS extender using simple and less costly
MTT reduction assay and compare the efficiency with
eosin-nigrosin staining.
MATERIALS AND METHODS
Semen samples and analysis
Liquid semen which was diluted in BTS (at 30×106
sperm/ml) were used in this study from Local A.I center. To
evaluate sperm viability, 1% eosin-nigrosin stain method
was performed according to Björndahl’s method (2003).
The staining solution for the one-step eosin-nigrosin
staining contained 0.67 g eosin Y, 0.9 g sodium chloride and
10 g of nigrosin. Aliquots of each 6 samples (50 μl) mixed
with same amount of solution in micro tube. The suspension
was incubated for 30 s at room temperature. Then, a 12 μl
droplet was transferred into slide where it was smeared by
sliding a cover slip. After smear the samples, dried few
second and examine directly 250 sperm were assessed three
times at a magnification 400× in each samples.
495
wells of the 96-well microplate were used. One hundred
microliters of semen sample plus 10 μl of MTT stock
solution (5 mg of MTT/ml of PBS) were placed in each
well. The optical density of samples were measured
immediately and after 1 h of incubation at 17°C using a
spectrophotometer (μ QuantTM Microplate Spectrophotometer,
Bio-Tek instruments, USA) at a wave length of 560 nm.
MTT reduction rate (optical density) for each sample was
calculated by concuring the difference between the first and
second reading of the spectrophotometer.
The freeze-killed procedure was used, in order to obtain
the standard curve and the relationship between the MTT
reduction rate and sperm viability. Liquid semen samples of
Duroc with good quality (about 80% viable sperms) were
used. After the dilution of Semen with BTS, 6 ml of the
diluted semen divided in two fractions; one fraction was
maintained at 17°C, while the sperms in the other fraction
were killed by two cycles of plunging into liquid nitrogen
and thawing at 37°C. Samples for analysis were made by
combining aliquots of viable and freeze-killed sperms at
ratios of 10:0, 8:2, 6:4, 4:6, 2:8 and 0:10 v/v, respectively.
The prepared samples were analyzed by (1) MTT and
spectrophotometer, and (2) Eosin-nigrosin stain. MTT
reduction rates were determined immediately and each hour
up to 4h incubation at 17°C.
The MTT test was applied to evaluate the sperm
viability in liquid semen of Duroc. After the dilution of
semen samples in BTS, samples and MTT stock solution
were distributed in the wells of the 96-wll microplate. The
rates of MTT reduction were taken immediately, 1 and 4 h
after incubation at 17°C. Simultaneously split samples of
the same semen were tested using eosin-nigrosin stain
method to evaluate the efficiency of MTT reduction assay
in assess the sperm viability.
Statistic analysis
Regression analysis was used to evaluate the efficiency
of the MTT test for the assessment of sperm viability of
boar semen. Data was analyzed using KCjunior (Bio-Tek
autoreader control Version 1.41.8), and p<0.05 was
considered as statistically significant.
RESULTS AND DISCUSSION
Analysis of the results
Initial results showed MTT reduction rates of the semen
sample groups which contained different proportions of
viable sperm in Figure 1. The rate of MTT reduction assay
increased gradually with incubation time, in all groups.
Increasing the volume of viable sperm cells in semen
MTT reduction assay and experiments
samples resulted in a proportional and significant increase
The MTT assay was performed by routine methods in the rate of MTT reduction. At each time of measurement,
(Mosmann, 1983; Aziz et al., 2005). For each sample, 6 the MTT reduction rate correlated (p<0.001, r>0.915)
496
Byun et al. (2008) Asian-Aust. J. Anim. Sci. 21(4):494-498
80
% of viable sperm
70
60
50
40
y = 1.7545x-0.1684
R2 = 0.9011
30
20
10
0
0
0.1
0.2
0.3
0.4
0.5
0.6
MTT reduction rate
Figure 2. MTT purple green formazan dye formation in sperm
midpiece (400×).
Figure 3. Linear regression between the MTT reduction rate at 1 h
of incubation time and the percentage of viable sperm, which was
evaluated by eosin-nigrosin staining. Data was analyzed using
KCjunior (Bio-Tek autoreader control Version 1.41.8), and p<
0.05 was considered as statistically significant.
% of viable sperm
positively with proportion of freeze-killed dead sperms.
80
The color changes of MTT from yellow to purple was
70
very clear after 1 h of the incubation time and up to 4 h,
60
especially in midpiece are shown Figure 2. Two standard
50
curves (at 1 h, y = 1.7545x-0.1684 and 4 h, y = 0.8268x40
0.0804) for the relationship between the MTT reduction rate
y = 0.8268x-0.0804
and the percentage of viable sperms were acquired (Figures
30
R2 = 0.9147
3 and 4). These two standard curves were applied later to
20
acquire the percentage of viable sperm cells in each sample
10
in accordance with the MTT reduction rate.
0
0
0.2
0.4
0.6
0.8
1
1.2
The results of MTT reduction rate and that of eosinnigrosin stain are shown in Table 1. The results showed a
MTT reduction rate
high correlation (p<0.001) between the results of MTT test
at 1 and 4 h of incubation time and the results of eosin- Figure 4. Linear regression between the MTT reduction rate at 4 h
nigrosin stain which evaluated the sperm viability. The of incubation time and the percentage of viable sperm, which was
MTT reduction rate was significantly (p<0.001) correlated evaluated by eosin-nigrosin staining. Data was analyzed using
with the results that simultaneously determined by eosin- KCjunior (Bio-Tek autoreader control Version 1.41.8), and p<
0.05 was considered as statistically significant.
nigrosin staining, yielding correlation coefficients of r =
0.9493 for 1 h and r = 0.9564 for 4 h.
investigated by comparison of other method which is
already believed reliable, that is, eosin-nigrosin staining
Discussion and further study
especially in BTS extended semen (Zhou et al., 2004).
The MTT assay was used in many studies to evaluate
In contrast to the procedure that was published
the viability of different cells. This test depends on the previously, the MTT reduction rate was taken successfully
ability of viable cells to convert the MTT to purple after 1 h of incubation time. This is expected because
formazan. In this study, the diagnostic value of the MTT spermatozoa are very active cells and rich in mitochondria;
reduction assay to evaluate the boar sperm viability was therefore, the reduction of MTT by spermatozoa is faster
Table 1. Analysis of the liquid semen samples using MTT test and eosin-nigrosin staining for evaluating viability
MTT reduction assay
Eosin-nigrosin staining
Live:dead
MTT absorbance rate at 560 nm
% of viable sperm
No. of sperm
Viability (%)
1h
4h
1h
4h
Live
Dead
10:0
0.528
1.014
75.80
75.80
175
81
68
8:2
0.440
0.834
60.36
60.92
181
114
61
6:4
0.367
0.671
47.55
47.44
138
93
59
4:6
0.319
0.545
39.13
37.02
90
131
41
2:8
0.220
0.416
21.76
26.35
62
183
25
0:10
0.178
0.236
14.39
11.47
12
226
5
Byun et al. (2008) Asian-Aust. J. Anim. Sci. 21(4):494-498
497
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ACKNOWLEDGMENTS
Joshi, A., A. K. Mathur, S. M. K. Naqvi and J. P. Mittal. 2006.
Influence of osmolality of complete semen extender on motion
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sperm viability in local A.I center and laboratory.
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