Acridine Orange Staining Techniques
The use of Acridine Orange to evaluate damage in stallion and llama
spermatozoa
Drs. Joyce Hofman
Tutors:
Dr. Edita Sostaric
Dr. Deborah Neild
Introduction
Routine evaluation of equine and llama sperm quality includes sperm concentration,
total and progressive motility and morphology (Erenpreiss et al., 2006). However in
the last decade there have been developments in research that enable us to detect other
factors in sperm cells that influence fertility. One of these factors is sperm chromatin
structure and integrity. While the conventional parameters influence the capability of
the spermatozoa to fertilize the oocyte, chromatin integrity is essential for the further
development of the embryo. It has been postulated that poor chromatin packaging
and/or damaged DNA may contribute to failure of sperm decondensation and,
consequently, in fertilization failure (Sakkas et al., 1996; Samocha-Bone et al., 1998).
Assisted reproductive techniques (ART), such as Intracytoplasmatic Sperm Injection
(ICSI) bypass the normal and physiological sperm selection barriers. As a result
sperm with damaged DNA may fertilize. Damaged DNA might be thereby be a factor
of increasing importance in stallion subfertility (Silva and Gadella, 2005).
To avoid fertilisation by spermatozoa with damaged DNA, it is necessary to evaluate
the DNA quality of a sperm sample. At the moment there are several tests available to
evaluate this, measuring DNA damage at different levels. For example the Single Cell
Gel Electrophoresis assay (COMET) and Terminal Deoxynucleotidyl Transferase
assay (TUNEL) evaluate DNA fragmentation. Toluidine Blue (TB) staining detects
DNA decondenation and Acridine Orange (AO) staining and the related Sperm
Chromatin Structure Assay (SCSA) evaluate the susceptibility of DNA to
denaturation. These different forms of damage though seem to be possibly correlated
in a certain way ( Erenpreiss et al., 2004 ). This might mean that the outcome of one
of these tests could can be used to predict the outcome of another.
The TUNEL, COMET and SCSA are reliable but expensive tests, therefore they are
not useful in daily practice (Beletti et al., 2004),being related mostly to researche.
Instead, nuclear dyes like TB and AO provide a simple, and inexpensive and sensitive
alternative. The toluidine blue (TB) stain is now the most widely used nucleic stain,
in human as well as in other species (Erenpreiss et al., 2006). The percentage of
abnormal cells seen after TB staining has been shown to correlate to the percentage of
abnormal cells shown by AO staining and by SCSA. An possible explanation for this
could be that the susceptibility to denaturation evaluated by AO and SCSA may
perhaps be related to the condensation status of the nucleus (Kosower et al.,1992)
which is measured with TB staining.
Most of the research with these tests has been done on fresh semen. Nevertheless,
with the increasing number of artificial inseminations carried out in domestic animals
today, cooled and frozen stored semen is becoming increasingly important. Cooling
and especially freeze/thawing are known to decrease fertility. (Blottner et al.,2001).
These processes influence different components of the cell in a negative way. The
effect of these processes seems to differ a lot between animals. SCSA is one of the
tests,which have been used in different researches to assess the damage that occurs to
the DNA during cooling and/or freezing and thawing. In fertile stallions cooling (46
h, 5° C) doesn’t change SCSA values, although in subfertile stallions it does (Love et
al., 2004). Cryopreservation seems to have the most significance influence on the
morphological and functional integrity of the cell, nevertheless SCSA values which
indirectly measure DNA quality, only show a very slight increase (Blottner et al.,
2001). This small increase in DNA danage after freeze/thawing was also observed
using the TB stain in stallion spermatozoa ( Sardoy et al, 2008 ).
While DNA evaluation is often carried out by SCSA, it is unlikely that most semen
collection, processing and cryopreservation facilities would possess a flow cytometer.
Nevertheless the importance of carrying out a multiple sperm functions test when
working with cooled and cryopreserved semen is has been emphasized.
(Colenbrander et al,. 2003). As mentioned before, as one of these test SCSA could
perhaps be replaced by TB staining.
The aim of this research was to compare the Toluidine Blue stain with the Sperm
Chromatin Structure Assay when used in fresh, cooled and frozen equine. If the
Toluidine Blue Stain proves to be a reliable test this could provide clinicians and
cryopreservation facilities with an extra affordable extra parameter to evaluate the
quality of semen samples after they have been processed for preservation.
Materials and methods
Semen collection and routine evaluation of sperm quality
All semen samples were collected between October and December 2008 in Buenos
Aires, Argentina. 14 samples from 3 different stallions Due to changes made in the
research plan during the period materials and methods differed.
All stallion samples were obtained with a Missouri model artificial vagina. After
filtering the Gel free volume was determined and Sperm progressive motility was
estimated visually under a light microscope (400x). Sperm concentration was
determined using a Neubauer hemocytometer.
For these samples volume, motility (which is physiologically about 0%) and
concentration were estimated in the same way.
Toluidine blue staining protocol
The TB staining protocol was based on previous research at the faculty (Sardoy et al.,
2008). Dried smears were fixed with 96%ethanol for 2 minutes. After that they were
stained with 10 % TB for 5 minutes. The stock TB solution consisted of 0,4 gr
toluidine blue in 200 ml distilled water and was kept at 4º C for no longer than 1 year.
The working solution was made up freshly several times a week with one part of the
stock solution in 9 parts of buffer (potassium ftalatum acid sodium hydroxide, pH 4.0)
and kept at 4º C. Slides were rinsed with distilled water after staining and left to air
dry before evaluation.
A minimum of 200 cells per slide were evaluated at 1000x magnification using a
Leica light microscope.
.
SCSA protocol
The SCSA protocol was based on Erenpreiss et al., 2004 (human semen) and Love et
al., 1998 (stallion semen). An aliquot of unprocessed semen was diluted to a
concentration of 1–2 x 106 sperm/ml with TNE buffer (0.01 mol/l Tris–HCl, 0.15
mol/l NaCl and 1 mmol/l EDTA, pH 7.4). This cell suspension was treated with an
acid detergent solution (pH 1.2) containing 0.1% Triton X-100, 0.15 mol/l NaCl and
0.08 mol/l HCl for 30 s, and then stained with first 4 g/ml purified AO (stock
solution of AO 1 mg/ml in distilled water) in a phosphate-citrate buffer, pH 6.0. The
samples were kept in the dark by wrapping in aluminium foil and analysed using a
Facs Calibur Becton Dickinson. Laser strength 15.05 nW; wave length 488 nm.
Flow cytometer at a flow rate of 100-200cells/sec.
.
Sample processing
For both species the conventional parameters (volume, concentration and motility)
were assessed immediately after ejaculation. Three direct smears were made of the
fresh ejaculate; also two aliquots of the ejaculate were added to a solution of DDT
(1% in distilled water) for 3 min before making a smear. DTT has the ability to reduce
covalent disulfide bonds, necessary for the stability of the chromatin. In this way it
increases the staining with TB and these smears can thus be used as a positive control
for the staining method. All smears were stained with Toluidine Blue. TB binds with
phosphate residues in loosely packaged (decondensed) DNA. The ability of TB to
bind determinates the colour of the cell, which is seen when evaluated with a light
microscope.
In this way the colour of the cell characterizes the DNA conformation.
For evaluation with the Flow Cytometer different samples were prepared for both
species. The first sample consisted of only semen and buffer and was used to
eliminate the background fluorescence.
The second sample consisted of semen + buffer + detergent + Acridine Orange ( the
detergent induces DNA denaturation). The susceptibility of sperm nuclear DNA to
denaturation differs and depends on the thiol-disulfide status of DNA-associated
protamines (Kosower et al., 1992). Denaturation of the DNA means that the strands
are unwound, assuming a single stranded form. Acridine Orange is a metachromatic
dye which binds in a different way to single and double stranded DNA: single
stranded DNA fluoresces red, while normal, double stranded DNA fluoresces green.
In this way the percentage of cells that fluoresce red shows the subset of sperm which
are susceptible to acidic treatment (Love et al., 2004) , the advantage being that the
flow cytometer is able to analyze a large number of cells in a short time.
After placing a tube in the machine all the particles seen in the sample are presented
as dots in a scattergram. Based on granularity and cell size, the population of
spermatozoa is selected, and for each cell of this group the machine measures the
green and red fluorescence emitted creating a second scattergram. This scattergram
has the amount of red fluorescence at the X-axis and the amount of green
fluorescence at the Y-axis. In a healthy sample, most cells emit mainly green
fluorescence resulting in an ellipse of dots on the left side of the scattergram. Cells
with single stranded DNA appear more on the right, outside of this ellipse. These cells
make up what has been termed the COMP (Cells Outside Main Population). The
scattergram provides a value measured for each cell called -t., which is the ratio of
red/red + green. Thereby obtaining a value,which stands for each cell’s ‘redness’.
When the values of all of the counted cells are presented in a diagram the mean -t
and Standard Deviation of the whole population can be calculated, the mean -t
shows the extent of denaturation of the population and the SD stands for the
variability in between the cells of this population. In samples which have a high
percentage of sperm with single stranded DNA. the typical curve seen in ‘healthy’
samples shows an extra ‘shoulder’ on the left.
Results
The TB stained sperm heads were evaluated and classified into 3 categories: (1)
‘negative’ if they possessed a light blue head (condensed DNA); (2) ‘positive’ if they
possessed a dark blue or dark violet head (uncondensed DNA); and (3) ‘intermediate’
if they possessed a head only partly coloured dark blue or showed a colour of
blue/violet in between ‘positive’ and ‘negative’ TB staining was done on all semen
samples obtained of the four available stallions in October and November 2008. The
results were as followed:
Stallion n1 (Garfurio ) had an average of 41.2% negative, 55.2% intermediate and
3.6% positive cells ( 6 samples ).
Stallion n2 (Aiglefin) had an average of 32.75% negative, 61.5% intermediate and
5.75% positive cells ( 4 samples ).
Stallion n3 (Tuerto) had an average of 46.5% negative, 51.25% intermediate and
2.25% positive cells ( 4 samples ).
All 3 stallions used thus showed a large number of intermediate cells.
Due to Acridine Orange obstructing the tubing of Flow Cytometers, only one hospital
in Buenos Aires allowed us to use their machine for running the SCSA. For this an
appointment had to be made approximately one week in advance and in each
appointment we had about 1 hour to use the machine under the guidance of a certified
technician.
At the first appointment samples of both llama and stallion semen were used. The
samples were prepared at the veterinary faculty and transported in the dark (wrapped
in aluminium foil) to the hospital. Due to a miscalculation though, the stain was
diluted (40 g/ml instead of 4 g/ml). We tried to obtain a concentration of about 4
g/ml by diluting further at the hospital. Results showed all the cells in an
intermediate position, on the upper right side of the scattergram, emitting both green
and red fluorescence.
At the second appointment, the samples were split in two groups: one was stained
with AO at a concentration of 1 g/ml and the other 2 g/ml. Again all cells showed
an intermediate position, with the signal of the sample which had an AO
concentration of 2 g/ml being a bit more intense. By exposing the semen, before
processing, to low and high temperatures, we tried to achieve a positive control.
Aliquots of semen were put for 30 min. in an eppendorf and submerged in: (i) water at
100 C, (ii) liquid nitrogen (-196 C) or (iii) were subjected to 3 consecutive 5
minute cycles of water 100 C and liquid nitrogen. Results though were similar to that
of the unexposed samples. Another finding was that although set at a flow rate of 100200 cells/sec, the machine was counting at least 1000. The technician was not able to
get the rate down to the required speed.
At the third appointment, the flow rate was brought done to 600-700 cells/sec. Based
on the article of Kosower et al. (1992), one sample was mixed with DDT, in a
continued effort to obtain a positive control. DDT decondenses the chromatin and
thereby makes it more susceptible to denaturation when treated with an acid
detergent. The sperm population appeared in a different position on the scattergram;
more upright, meaning the cells were larger and more granulated. Nevertheless the
fluorescence of all the samples (with and without DDT) was similar to earlier results.
At this point discussions were carried out whether or not to continue working with the
flow cytometer. Several doctors, working at the hospital were the machine was
situated, expressed their doubts about the reliability of the SCSA when carried out on
human semen samples, having decided not to use SCSA as an assay when evaluating
their samples.
The problems we encountered could be brought under in three categories: (i) machine
related (flow rate), (ii) assay related (no positive control) and technique related
(appearance of only intermediate cells, i.e. stained both red and green). Doubts about
the technique led us to decide to try other AO staining techniques which could be
evaluated visually at the veterinary faculty. This might provide us with information
about the activity of the test before using it at a large numbers of cells, as in the flow
cytometer.
Materials and methods
AO staining protocols
Due to problems with the flow cytometer, changes were made in the research plan.
These changes involved the use of 2 Acridine Orange staining techniques: Tejada
(Tejada et al., 1984) and RAOO (Erenpreiss et al., 2001)
RAOO protocol: Briefly, air-dried smears were fixed with ethanol 96%-acetone (1:1)
at 4º C for 30 minutes-24 hours. After that they were rehydrated at ambient
temperature in a cycle of ethanol 96% for 5 min, ethanol 70% for 5 min. and ethanol
30% for 3 min. The samples were then incubated in PBS for 5 min. followed by
treatment with 1 N HCl for 1 min. at 60º C. After this they were rinsed 3 times for 2
min. in distilled water and once for 5 min. in McIlvain citric phosphate buffer (Mc
Ilvain; 0.1 M citric acid, 0.2 M disodium hydrogen phosphate (pH4)).
The smears were stained in the dark for 15 min. with AO (0.038 mg/ml; 10 -4M) in
McIlivain buffer (pH 4.0), which was prepared weekly out of a stock of 7.6 mg AO in
1 ml of distilled water. After removing the stain by softly rinsing the slides with
distilled water they were stained again. This time for 5 min. with AO 10 -6M ( in Mc
Ilvain) the same buffer. Subsequently they were rinsed again. This staining-rinsing
was repeated another 2 times.
After staining, the slides were rinsed with distilled water and, when still wet, covered
with a cover glass. The cover was firmly pressed on the slide with a paper towel to
remove underlying air. The borders were then sealed with nail polish to keep the
sample hydrated. All samples were kept in the dark until evaluation, which took place
in the dark at 1000x magnification using a Leica light microscope with the
corresponding fluorescence filter. Due to fading of the slides, the evaluation had to
take place within 48 hours.
Tejada protocol (TAO method); Briefly 1 ml of semen was centrifuged at 1300g at
least twice in 3 ml Tyrodes solution for 5 min.. The pellet was resuspended each time
until a final concentration of 50 million/ml. Smears were made and they were fixated
in Carnoys solution (3 parts of methanol and 1 part of glacial acid) for 2 hours or
overnight.
The smears were stained with a solution of 10 ml of stock AO (1 mg in 1000 ml
distilled water) in 40 ml of 0.1M citric acid and 2.5 ml of 0.3M Na2HPO4, 7H2O. The
Final working solution had a pH of 2.5 and a concentration of 0.19 mg/ml. and was
prepared weekly. All the solutions were kept at 4º C.
For staining the slides were covered for 5 min. with 2-3 ml of working solution. They
were then rinsed with distilled water and covered with a cover slip as described for
the RAOO method. All slides were always kept in the dark. Due to fading of the
slides, evaluation with the microscope had to take place as soon as possible and was
always done within 4 hours.
Sample processing
Samples from 3 stallions and 3 llamas were collected. All samples were split, with
half being used for RAOO and the other half for the Tejada staining technique. For
both tests fresh samples were used, a part of the sample being directly used while the
other part was used to try to obtain a positive control. This was done by exposing
aliquots of the sample to high temperature (30 min. in boiling water), to a cycles of
low (liquid nitrogen, -196 C) and high (boiling water) temperature (5 times for 5
min. each ), exposure to acid ( HCl 1 N, 4.5 N and 12.5 N for 30 min ), DTT (3 min.)
and UV light (45 min.). The samples which were exposed to acid and DDT were
mixed with these substances for the required period and then centrifuged, after which
the supernatant was removed.
Results
With RAOO, all samples showed only cells with green fluorescence (double stranded
DNA).
With Tejada, one llama sample showed 1% red, 10 % yellow-orange and 89% green
cells. All other samples showed only cells with green fluorescence.
At this moment a colleague commented that exposure to high temperature should
denaturate DNA, but that our temperature (100 C) might not be high enough to do
this. Besides which she pointed out that DNA has the ability to recombine when
brought back to ambient temperature.
We decided to try once again, this time making the smears while the slide was in a
pan over a high flame. The slides were left on the pan for 30-40 min. After this they
were directly put into fixative (Carnoy’s solution) and further treated according to the
Tejada protocol. When evaluated with the microscope all cells showed dark yellow to
red fluorescence.
After obtaining these results, the original research plan was again modfied.
The aim of the new research plan was to compare different ‘cooking’ times to
denaturate the DNA of spermatozoa and to compare different ways to maintain the
DNA single strand after heat treatment. The scope for this was to find a useful
positive control to use for both Tejada and the SCSA. Due to time pressure and
availability of animals it was decided to work only with samples of llama semen.
Materials and methods
Semen of 5 llama males was obtained by electro-ejaculation. From one llama two
samples were obtained. According to the technique described by Director et al.
(2007). Briefly, general anesthesia was performed with xylazine (0.2 mg/kg
Rompun, Bayer ) and 1.5 mg/kg ketamine hydrochloride ( Ketamina  Holliday,
Argentina). An electroejaculator ( P-T Electronics model 304, Oregon, USA ) and a
4 probe were used. Electrical stimulation was conducted for 6-12 min. Semen was
collected in a glass tube surrounded by water 37 C which maintained the temperature
constant throughout the process.
Standard parameters were evaluated (volume, concentration). All samples were split
in two fractions. The first was divided into four eppendorfs. Two eppendorfs were
exposed to continuously boiling water for 30 min, the other two for 40 min. After this
the samples were kept on ice for 2 hours before a smear was made. Directly after
making the smear the samples were fixed in Carnoy’s solution.
An aliquot of the second fraction was mixed with collagenase (1:1). In general llama
semen has a low concentration and a high viscosity. The low concentration can make
evaluation difficult. To increase this concentration centrifugation can be used but only
with samples treated previously with collagenase.
The smears from the second fraction were exposed to high temperature by either
putting them in a pan with the heating on maximum or by placing them on an
electrical heater. Slides were exposed for 15 or 30 min.
After the ‘heat treatment’ the slides were: (i) put directly into the fixative (Carnoy’s
solution), (ii) kept on ice for 2 hours, (iii) kept in a freezer for 2 hours or (iv) kept at
ambient temperature for either 10 min or 60 min. After, they were all put into fixative
for at least 2 hours. As a negative control, two smears were made which were not
exposed to high temperature, but directly put into the fixative.
Figure 1 shows an overview of the processing of the samples.
Figure 1. Processing of the ejaculate
Fresh smear
Semen in eppendorf
Boiled for 30 min.
2 hrs on ice
fresh ejaculate
2 hrs in freezer
evaluation of volume and
concentration,
Boiled for 30 min.
2 hrs on ice
2 hrs in freezer
Smear made in pan
Boiled for 30 min.
Boiled for 30 min.
Placed directly in
fixative
2 hrs in freezer
10 min in AT
60 min in AT
Placed directly in
fixative
2 hrs in freezer
10 min in AT
60 min in AT
Results
Fresh smear:
Animal
Cola Marron
Green cells
0
Yellow cells
0
Orange cells
0
Red cells
0
Cutini
99.8
0
0.2
0
Sisson
99
0.75
0.25
0
Carozo
Carozo
95.85
92.5
3.95
2.1
0
3
0.2
2.5
Gonzalito
95.35
4.65
0
0
Processed semen:
1= 15 min, fixation
2= 30 min, fixation
3= 15 min, 2 hrs ice
4= 30 min, 2 hrs ice
5= 15 min, 10 min AT
6= 30 min, 10 min AT
7= 15 min, 60 min AT
8= 30 min, 60 min AT
9= 15 min in eppendorf , 2 hrs ice
10= 30 min in eppendorf , 2 hrs ice
11= 40 min in eppendorf , 2 hrs ice
results sisson
100%
90%
80%
70%
red cells
60%
orange
cells
yellow
cells
green cells
50%
40%
30%
20%
10%
0%
1
2
3
4
5
6
7
8
9
10 11
red
results cuttini
100%
90%
80%
70%
60%
red cells
orange cells
50%
yellow cells
40%
green cells
30%
20%
10%
0%
1
2
3
4
5
6
7
8
9 10 11
results cola marron
100%
90%
80%
70%
60%
red cells
orange cells
50%
yellow cells
40%
green cells
30%
20%
10%
0%
1
2
3
4
5
6
7
8
9 10 11
Results Gonzalito
100%
90%
80%
70%
60%
red cells
orange cells
50%
yellow cells
40%
green cells
30%
20%
10%
0%
1
2
3
4
5
6
7
8
9 10 11
results carozo (1)
100%
90%
80%
70%
60%
red cells
orange cells
50%
yellow cells
40%
green cells
30%
20%
10%
0%
1
2
3
4
5
6
7
8
9 10 11
results Carozo (2)
100%
90%
80%
70%
60%
red cells
orange cells
50%
yellow cells
40%
green cells
30%
20%
10%
0%
1
2
3
4
5
6
7
8
9 10 11
Discussion
Too few results were obtained in the present study to formulate statistically based
conclusions. Further research is necessary before suggestions can be made about how
to receive and maintain a positive control for the Acridine Orange staining (Tejada
protocol) and the Sperm Chromatin Structure Assay.
The present results demonstrate that it is possible to create a positive control for the
Tejada Acridine Orange staining technique by treating the sample with high
temperature. Very high temperature seems to be essential to denaturate the DNA.
Samples exposed to boiling water show less denaturation than the samples which are
placed on a very hot pan. Due to the technique though, the used temperature is liable
to variations, which might influence the results. In further research, use of an
electrical heater with a fixed temperature, should make results more reproducible.
Results also indicated that the susceptibility of sperm DNA to heat treatment differs a
lot between animals. For some animals the DNA can be denaturated after 15 min. of
heat treatment, while for others 30 min. is necessary before denaturation starts.
Different samples from the same animal show variation as well.
Many problems were experienced while using the flow cytometer. Only one Flow
Cytomter in Buenos Aires would accept to run samples stained with Acridine Orange.
Nevertheless, the results obtained for both animal and human samples, are not
consistent with the results published in different articles about the assay. In spite of
the effort the technician expended, he was unable to correct settings to those
necessary to run the assay. Technical problems thus seem to restrict further research
using the SCSA so far. It is debatable whether more effort should be put into further
developing this test or whether more useful alternatives should be further explored.
The aim to create a sample which might be used as a positive control for SCSA has
not been reached. Exposing an aliquot to boiling water does not seem to denaturate
the DNA. Longer exposure to a higher temperature might cause more damage,
although the practical implementation of this may be difficult.
The DNA does not seem to recombine after 2 hours kept on ice (max.time we tried) or
1 hour at ambient temperature. This would allow the transportation of a treated
sample to the flow cytometer.
The llamas used in this study have no known history of fertility problems. For the
untreated, raw samples most animals show only cells with intact (double-stranded)
DNA. Positive (single-stranded DNA) cells were found for only one llama (Carozo).
This does not coincide with findings in other species, which indicate that about 014% (Kosower et al.,1992) or 6-37% ( Erenpreiss et al., 2004 ) of spermatozoa in
human samples and 4-19% in stallion samples (Morrell et al.,2008) would have DNA
susceptible to denaturation.
For further research with the Tejada technique it would be interesting to analyse
samples of animals with known fertility problems or find a way of mixing different
proportions of damaged cells with the raw samples. This would give more of an idea
about the reliability of the test and it possible use in veterinary practice.
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