Evaluation of Boar Sperm Viability by MTT Reduction Assay in Beltsville Thawing Solution Extender *

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 *

* This work was supported by a grant (No.20070301-034-040- 007-03-00) 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: davekim@gachon.ac.kr

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

J. W. Byuna,S. H. Choo1,a, H. H. Kim, Y. J. Kim, Y. J. Hwangand D. Y.Kim**

Division

of

Biological Science,GachonUniversity

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 water­

soluble 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 3.0x107 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

Thereare severalmethods

for

evaluatingsperm 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

SpermAnalysis)

and flow

cytometry arethe 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).

Flow cytometry

is a

technology

that

allows

a

single cellto be measured

for

a variety

of

characteristics. It

is

different

technical

applications

takes

many advantages

for

the analysis

of

spermquality.Becauseflowcytometryevaluates individual cell

with

rapid speed,

it

is objective

and

accurate method(Cordelliet

al.,

2005).

MTT

(3-(4,

5-dimethylthiazol-2-y1)-2, 5-diphenyl-

tetrazolium bromide)

is

a yellow

water-soluble

tetrazolium salt.

Succinate dehydrogenase system

of active

mitochondria converts this dye to water-insoluble purple formazanonthe 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;

Songet

al.,

2007).

In

many studies,

MTT

assay was used which were related to viability

of

different cells

(Mosmann, 1983;

Levitz

and

Diamone

, 1985;

Carmichaelet al.,

1987;

Campling et

al.,

1988;

Freimoser

et

al.,

1999).

Therefore,this

method

can

be

used

to

assess sperm

viability

and

MTTreductionassayalreadyhave successfullyapplied

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 (

卩

QuantTM Microplate Spectro­

photometer, 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

etal.,

2005;

Hu et al., 2006).

In

case

of boar

semen, no

research

hasbeen

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 (Joshiet

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-nigrosinstaining.

MATERIALS AND METHODS

Semensamples and analysis

Liquid

semen which was diluted in BTS (at 30x

106

sperm/ml) were

used in

this studyfrom

Local

A.Icenter. To evaluate sperm

viability, 1%

eosin-nigrosin

stain

method was

performed

according to BjOrndahl's method (2003).

The staining solution

for

the one-step eosin-nigrosin staining

contained

0.67

g eosin Y,

0.9gsodiumchlorideand 10g

of

nigrosin.

Aliquots of each

6 samples

(50

卩l) mixed

with

same

amount of

solution

in

microtube. The

suspension

was incubated

for 30 s at room

temperature. Then,

a

12 卩l droplet was

transferred

into slide where it was smearedby sliding

a cover slip. After smear

the

samples,

dried few second

and

examinedirectly 250 spermwere

assessed

three timesat

a

magnification400x

in each

samples.

MTTreductionassay andexperiments

The MTT

assay

was performed by routine methods (Mosmann, 1983;

Aziz

et al., 2005).

For

each sample, 6

wells

of

the 96-well

microplate

were

used.

One hundred microliters

of semen

sample

plus

10 卩l

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(卩 _QuantTM

Microplate

Spectrophotometer, Bio-Tek instruments, USA) at

a

wave

length of 560

nm.

MTT

reduction

rate

(optical density)

for each

sample was

calculated

byconcuringthe differencebetweenthe

first

and secondreading

of

the spectrophotometer.

The

freeze-killed

procedurewasused,

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 determinedimmediately

and each hour

upto4h

incubation

at17

°

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

MTTreductionweretaken 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

assessthe spermviability.

Statisticanalysis

Regression

analysiswasusedto

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

Initialresults

showed MTT reduction

rates

of

thesemen sample groups which contained different proportions of

viable

sperm inFigure

1.

The rate

of

MTT

reduction

assay

increased

gradually with

incubation

time,

in

all

groups.

Increasing the volume

of viable

sperm cells

in

semen samples resulted

in

a proportional

and

significant

increase

in

the rate

of

MTTreduction.

At

each time

of

measurement, the MTT reduction rate correlated (p<0.001, r>0.915)

Figure 2. MTT purple green formazan dye formation in sperm midpiece (400x).

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.

positively

with proportion of freeze-killed

deadsperms.

The

color

changes

of

MTT

from

yellow to

purple was

very clear

after 1 h of

the incubationtime

and

up to 4

h,

especially

in

midpiece are shown Figure

2. Two

standard curves (at 1

h,

y = 1.7545x-0.1684

and

4 h, y = 0.8268x- 0.0804)

for

therelationship between theMTT

reduction rate and

thepercentage

of viable

spermswere acquired

(Figures

3

and 4). These

two standard

curves

were

applied

laterto acquire the percentage

of

viable spermcells

in each sample

inaccordancewiththe

MTT

reduction

rate.

The results

of

MTT reduction rate

and

that

of

eosin- nigrosin

stain

are

shown in

Table

1.

The results showed

a high

correlation (p<0.001) betweenthe results

of

MTT

test

at 1

and 4

h

of incubation

time

and

the results

of

eosin- nigrosin

stain

which evaluated the sperm

viability. The

MTT

reduction

rate was significantly (p<0.001) correlated

with

the

results

that simultaneously determined by eosin- nigrosin staining, yielding

correlation

coefficients

of

r =

0.9493 for

1

h and

r

=

0.9564

for 4

h.

Discussionand further study

The MTT

assay

was used

in

many studies to evaluate the viability

of

different

cells.

This test depends on the ability

of viable

cells to convert the

MTT

to purple formazan. In this study, the diagnostic

value of

the MTT reduction assay to evaluate the boar sperm viability

was

Figure 4. Linear regression between the MTT reduction rate at 4 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.

investigated by

comparison of

other method which is already

believed

reliable, that

is, eosin-nigrosin

staining especially

in

BTSextended

semen

(Zhouet

al.,

2004).

In contrast to the procedure that was published previously, the

MTT

reduction rate was

taken

successfully

after

1 h

of incubation

time. This is expected

because

spermatozoa are veryactivecells

and

rich

in

mitochondria;

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

Live:dead

MTT reduction assay Eosin-nigrosin staining

MTT absorbance rate at 560 nm % of viable sperm No. of sperm

Viability (%)

1 h 4 h 1 h 4 h Liv 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

than other

cells.

A

similar

observation

wasreported

in

other previous

study in

equine

and

bovine(Azizetal., 2005;

Aziz,

2006). Because other studies have already

revealed that

sperm

viability is

positively related to sperm quality parameters like acrosome integrity, mitochondrial activity

and

even

these

parameters also correlate positively with fertility (Garner et

al.,

1997),

these

findings

is useful

in evaluating

liquid

boar semen quality. Furthermore, we observed

similar results

with other experiments

in

BTS extended semen,

so

liquid semen

wouldn't

need any other

treat

whichcan

affect

spermviability.

The

advantages of

the

MTT test

are that it

is

feasible

and

reproducible (Ciapettietal., 1992).Additionally, results from this

study

suggest

other

advantages of this

test

in evaluating thebovine

semen.

Firstly, this test

is

fast (1 h);

secondly,

many samples

(up

to 10)

canbe examinedat the

same

time;

these

test

wouldn't need

any

other

treat. When we would apply this method

to

assess sperm viability in laboratoryorlocalA.Icenter,

some

problems seems

to

have to be solved. Firstly, we have to plate same number of sperm

in testing well; secondly,

identify

whether

the

formazan affect

sperm

viability

or DNA.

The

MTT reduction testwas effective

and

simple methodtovalidate spermviability

and

itwould

be used

simpletool

to

evaluate sperm viability

in

local A.I

center and laboratory.

ACKNOWLEDGMENTS

We would like to acknowledge the

other

members of

our

laboratory

for

helpful discussion.

The

study

was

supported by grants

from

the Rural Development Administration(20070301-034-040-007-03-00).

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