Optimization of a Filter-Lysis Protocol to Purify Rat Testicular
Homogenates for Automated Spermatid Counting
SARA E. PACHECO, LINNEA M. ANDERSON, AND KIM BOEKELHEIDE
From the Department of Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island.
spermatid heads (HRSH) is a powerful indicator of spermatogen-
esis. These counts have traditionally been performed manually
using a hemocytometer, but this method can be time consuming
and biased. We aimed to develop a protocol to reduce debris for the
application of automated counting, which would allow for efficient
and unbiased quantification of rat HRSH. We developed a filter-lysis
protocol that effectively removes debris from rat testicular homog-
enates. After filtering and lysing the homogenates, we found no
statistical differences between manual (classic and filter-lysis) and
automated (filter-lysis) counts using 1-way analysis of variance with
Bonferroni’s multiple comparison test. In addition, Pearson’s
correlation coefficients were calculated to compare the counting
methods, and there was a strong correlation between the classic
Quantifying testicular homogenization-resistant
manual counts and the filter-lysis manual (r 5 0.85, P 5 .002) and
the filter-lysis automated (r 5 0.89, P 5 .0005) counts. We also
tested the utility of the automated method in a low-dose exposure
model known to decrease HRSH. Adult Fischer 344 rats exposed to
0.33% 2,5-hexanedione in the drinking water for 12 weeks
demonstrated decreased body (P 5 .02) and testes (P 5 .002)
weights. In addition, there was a significant reduction in the number
of HRSH per testis (P 5 .002) when compared to controls. A filter-
lysis protocol was optimized to purify rat testicular homogenates for
automated HRSH counts. Automated counting systems yield
unbiased data and can be applied to detect changes in the testis
after low-dose toxicant exposure.
Key words:Methods, spermatogenesis, toxicology.
J Androl 2012;33:811–816
estimate daily sperm production rates and is a com-
monly used method in studies of toxicant-induced
testicular injury or dysfunction (Blazak et al, 1993;
Ashby et al, 1997; Omura et al, 2000; Wade et al, 2006;
Assinder et al, 2007). The use of a hemocytometer in this
method necessitates that multiple counts be recorded as
a result of the high levels of variation and error inherent
in the technique. It has been previously reported
(Freund and Carol, 1964; Zrimsek, 2011) that mean
differences of greater than 20% are common in manual
sperm counts, even when the counts are performed by
the same individual, highlighting the need for a reliable
Previous work describes the use of the Coulter
Counter for automated semen analysis, but cellular
contaminants within the semen tend to inflate the counts
(Evenson et al, 1993). In addition, the computer-assisted
uantification of testicular homogenization-resis-
tant spermatid heads (HRSH) can be used to
sperm analyzer (CASA) technology has been applied to
enumerate rodent testicular spermatids; however, the
CellSoft system also overestimates spermatid number
by misidentifying testicular debris as spermatid heads
(Working and Hurtt, 1987). Through the addition of
filtration and somatic cell lysis steps and the use of an
automated counter that can identify trypan blue–stained
cells, the classic protocol can be modified for automatic
quantification of testicular spermatid heads.
Here we describe a novel update to the classic
protocol for counting testicular HRSH to eliminate
cellular debris and purify spermatid heads. Using the
pure lysates in an automated counting system produces
efficient, reliable, and unbiased results that can be
applied to detect low-dose toxicant-induced testicular
Materials and Methods
The 2,5-hexanedione (HD; CAS 110-13-4) used in the applica-
tion study was purchased from Sigma Aldrich (St Louis,
Adult male Fischer 344 rats weighing 175–225 g (Charles River
Laboratories, Wilmington, Massachusetts) were maintained
The research was funded by Superfund Research Program (NIH/
NIEHS) grant P42ES013660 and T32ES007272-17 for ‘‘Training in
Environmental Pathology’’ (S.P.).
Correspondence to: Dr Kim Boekelheide, Department of Pathology
and Laboratory Medicine, Brown University, Box G-E5, Providence,
RI 02912 (e-mail: email@example.com).
Received for publication August 2, 2011; accepted for publication
January 11, 2012.
Journal of Andrology, Vol. 33, No. 5, September/October 2012
CopyrightEAmerican Society of Andrology
in a temperature- and humidity-controlled vivarium with a
12-hour alternating light-dark cycle. All rats were housed in
community cages with free access to water and Purina Rodent
Chow 5001 (Farmer’s Exchange, Framingham, Massachu-
setts). The Brown University Institutional Animal Care and
Use Committee approved all experimental animal protocols in
compliance with National Institutes of Health guidelines.
Preparation of Testes and Homogenization Procedures
Body weights were recorded at the time of necropsy and the
testes were removed and weighed. The right testis was
detunicated, and one-third of the parenchyma was weighed,
flash-frozen, and stored at 280uC for later evaluation. At the
time of processing, each sample was thawed on ice and
homogenized using a Brinkmann Kinematica Homogenizer
Polytron PT 10/35 (Brinkmann Instruments, Westbury, New
York) in saline-merthiolate-triton (SMT) buffer, following a
previously published protocol (Blazak et al, 1993). Briefly,
testis samples were homogenized in 25 mL SMT at maximum
speed (27 000 rpm) for 2 minutes and used immediately for
Additional Filter and Lysis Protocol
After the homogenization procedure, the testis homogenates
were filtered through 10-mm nylon mesh (Dynamic Aqua-
Supply Ltd, Surrey, Canada). The filtered homogenates were
then combined in a 1:1 ratio with an optimized somatic cell lysis
buffer (0.3% sodium dodecyl sulfate and 1% Triton-X 100)
derived from a protocol used for lysing somatic cell contami-
nation in human semen (Goodrich et al, 2007). Each sample
containing the homogenate and lysis buffer mixture was
incubated on wet ice for 5 minutes prior to counting. The lysis
of debris was confirmed using phase contrast microscopy, and
photographs of trypan blue (Invitrogen, Eugene, Oregon)–
stained homogenates and lysates were taken using the Nikon
Diaphot microscope (640; Melville, New York) and a Nikon
D40 digital camera.
Manual Testicular Spermatid Head Counts
Testis homogenates or lysates were combined with 0.2%
trypan blue in a 1:1 ratio and 10 mL was loaded into both
chambers of 2 hemocytometers, resulting in 4 counts per
sample that were averaged together to obtain 1 value. The
hemocytometers were placed in a humidified chamber for
5 minutes prior to counting, and the samples were counted
according to previously published methods (Blazak et al,
Automated Testicular Spermatid Head Counts
Testis homogenates or lysates were combined with 0.2%
trypan blue in a 1:1 ratio and 10 mL was loaded onto both sides
of a cell-counting chamber slide (Invitrogen), resulting in 2
counts per sample that were averaged together to obtain 1
value. HRSH were counted using the Countess Automated
Cell Counter (Invitrogen), following manufacturer guidelines.
The gating parameters ‘‘sensitivity,’’ ‘‘minimum size,’’ ‘‘max-
imum size,’’ and ‘‘circularity’’ were optimized for rat testicular
spermatid heads and were determined to be 5, 5 mm, 10 mm,
and 30%, respectively.
Control testis samples (n 5 10) were homogenized. Each
homogenate was divided and one-half was left ‘‘as is,’’ while
the other half was subjected to the additional filter-lysis steps
described earlier. Both preparations of each sample were
counted manually by 2 individuals (S.P. and L.A.) and
automatically using the automated counter. Coefficients of
variation were calculated for each approach ([standard
deviation (SD)/mean] 6100), and the manual counts of S.P.
and L.A. were averaged to obtain a single value to reduce
variability. This resulted in 4 counts for each sample: 1) classic
manual, 2) classic automated, 3) filter-lysis manual, and 4)
filter-lysis automated. One-way analysis of variance (ANOVA)
with the Bonferroni’s multiple comparison test was applied to
determine if there was a statistical difference in the average
number of HRSH per testis among the 4 counting approaches.
Correlation between the groups was determined using Pear-
son’s correlation coefficients. Results from both tests were
considered significant if P was less than .05. A scatter diagram
of the number of HRSH obtained using the classic manual
method vs the filter-lysis automated method was fitted with a
Deming regression line. In addition, a Bland-Altman plot was
generated to determine the agreement between the classic
manual and filter-lysis automated approaches (Zrimsek, 2011).
To determine if the adapted protocol was capable of detecting
changes in the testes after low-dose toxicant exposure, rats
were exposed to either water (control, n 5 8) or 0.33% HD in
the drinking water (n 5 10) for 12 weeks. We chose this dose of
HD because it induces minimal but detectable testicular injury
in Fischer 344 rats (Moffit et al, 2007) and because pilot
experiments in our laboratory indicated that HD decreased
the number of HRSH in the testis when counted manually
(Pacheco and Boekelheide, unpublished). The testes were
homogenized and the homogenates were filtered, lysed, and
counted automatically as described earlier. The total numbers
of HRSH in the testis of the control and HD rats were
calculated and compared using a 2-tailed Student’s t test, and
the results were considered significant if P was less than .05.
All statistical analyses were performed using the Prism 5
software (GraphPad Software, La Jolla, California).
The Additional Filter-Lysis Protocol Removes Testicular
Debris—A comparison of the samples prepared using
the classic protocol (Figure 1A and B) and those
prepared following an additional filter-lysis (Figure 1C
812Journal of Andrology
and D) clearly demonstrated the efficacy of the updated
method to remove the majority of cellular contaminants
that would inflate automated counting methods.
Manual Counting Has High Interobserver Variability
Compared to Automated Counts—Correlation coeffi-
cients were calculated among the 4 sets of manual
counts and there was poor correlation between the 2
individuals doing the counting (Table 1). The correla-
tion coefficient calculated comparing ‘‘Classic Manual
#1’’ vs ‘‘Filter-Lysis Manual #2’’ was low (0.425).
Likewise, the correlation coefficient calculated compar-
ing ‘‘Filter-Lysis Manual #2’’ and ‘‘Filter-Lysis Manual
#1’’ was also low (0.438). These findings indicate that
the variability in the counts was mainly due to the
variation between the 2 individuals counting and not to
the methods used to prepare the samples. The
coefficients of variation (CV) for the mean manual
counts (‘‘Classic Manual #1,’’ ‘‘Classic Manual #2,’’
‘‘Filter-Lysis Manual #1,’’ and ‘‘Filter-Lysis Manual
#2’’) for all 10 samples were 32.2%, 28.6%, 26.4%, and
33.7%, respectively. However, the ‘‘Filter-Lysis Auto-
mated’’ had less variation (CV 5 25.5%). To reduce
variability we averaged the 2 individual counts for the
classic manual and filter-lysis manual approaches, and
the CVs were reduced to 28.72% and
There Is High Correlation Between the Classic Manual
and the Filter-Lysis Automated Approaches to Counting
Testicular Spermatid Heads—One-way ANOVA deter-
mined that there were no significant differences between
the observer-averaged manual (both classic and filter-lysis)
using the classic automated method compared to the other
Figure 1. Filter-lysis optimization. Testis homogenates were prepared using each of the 2 methods described (A, B, classic; C, D, filter-lysis)
and stained with trypan blue for visualization. White arrowheads indicate rat spermatid heads, while white arrows indicate cellular debris.
Photomicrographs were taken at 640, and the scale bar 5 20 mm.
Table 1. Correlation coefficients and P-values calculated for manual counts
Classic Manual #1Classic Manual #2
Filter-Lysis Manual #1
Filter-Lysis Manual #2
Classic Manual #1
Classic Manual #2
Filter-Lysis Manual #1
Filter-Lysis Manual #2
bP-values. Data were generated comparing the average number of homogenization-resistant spermatids per testis.
Pacheco et al
Purification of Testicular Spermatids813
additional filter-lysis steps did not alter overall testicular
HRSH numbers and that the automated cell counter
produced accurate HRSH counts. Furthermore, Pearson’s
correlation coefficients were calculated to compare the
similarity among the 4 preparation techniques. The classic
manual protocol was highly correlated with the filter-lysis
manual (r 5 0.85, P 5 .002) and automated protocols (r 5
0.89, P 5 .0005) but not with the classic automated counts
(r 5 0.65, P 5 .04; Table 2). The strongest correlation
occurred when the filter-lysis manual and automated
counts were compared (r 5 0.90, P 5 .0004; Table 2).
Scatter plots with a fitted Deming regression line
(Figure 2B) and Bland-Altman plots (Figure 2C) were
used to get the best overview of comparative data
generated using the classic manual and filter-lysis auto-
the relationship between the 2 methods that were both
generated with error, taking into account the analytical
imprecision of each method. This regression analysis
yielded a best-fit line with a slope and Y-intercept (when
X 5 0) of 0.84 6 0.15 (95% confidence interval [CI], 0.50–
1.18) and 0.20 6 0.25 (95% CI, 20.37–0.78), respectively
(P 5 .005). The Bland-Altman plot calculated a bias of
0.06 when comparing the classic manual and the filter-lysis
automated approaches (Figure 2C, dashed line) with a SD
of 0.21. The SD of the differences between the 2 assay
methods was used to calculate the limits of agreement
according to the followingformula: bias 6 1.966SD. Our
95% limits of agreement are between 20.35 and 0.47
(Figure 2C, solid lines). As expected, the 2 methods give
very similar results on average, and the level of agreement
among the samples is good, with 90% of the data falling
within the limits.
Exposure to 0.33% HD Decreases Body and Testes
Weights—Rats were exposed to either water (control) or
0.33% HD in the drinking water for 12 weeks, and all rats
were weighed at the time of necropsy (Table 3). Rats
exposed to HD displayed significantly decreased body
weights when compared to control rats (P 5 .02). Testis
weights were also recorded during the necropsy, and rats
exposed to HD had significantly decreased testis weights
compared to controls (P 5 .002; Table 3).
Figure 2. Optimization experiment: Number of homogenization-
resistant spermatid heads (HRSH) per testis. (A) The average
number of HRSH per testis were calculated for 10 rats using each
protocol and values were graphed as follows: classic manual (black
circle); classic automated (open circle); filter-lysis manual (black
square); and filter-lysis automated (open square). The light gray
boxes represent the range of values and the dark gray boxes contain
the standard error of the mean, with the mean as the line in the
middle of the dark gray box. Statistical differences between the
average number of spermatids per testis among groups was
determined using 1-way analysis of variance with the Bonferroni’s
multiple comparisons test; *** P , .001 relative to all
other methods. (B) Scatter diagram of the number of HRSH obtained
using the classic manual method vs the filter-lysis automated
method, with Deming regression line fitted (solid line). (C) Bland-
Altman absolute bias plot of the number of HRSH obtained using the
classic manual method vs the filter-lysis automated method showing
the average bias (dashed line) and limits of agreement (solid lines).
814 Journal of Andrology
Exposure to HD Decreases the Average Number of
Homogenization-Resistant Spermatids in the Testis—
Following optimization of the technique, the utility of
the automated cell counter was tested in the context of
toxicant exposure that we had previously seen reduce
the number of HRSH in the testis when counted
manually (Pacheco and Boekelheide, unpublished).
Testes from HD and control rats were prepared with
the additional filter-lysis steps and counted using the
automated cell counter. The total number of HRSH per
testis was calculated for each sample, and consistent
with the significant decrease in testis weights, HD rats
displayed significantly decreased HRSH counts (P 5
.002) relative to control rats (Figure 3).
The most recent World Health Organization (2010)
laboratory manual for the examination and processing
of human semen emphasizes the necessity for automated
systems for analyzing sperm because they have the
potential for greater objectivity, precision, and repro-
ducibility than do manual systems. The CASA system
evolved because of the need to obtain objective and bias-
free data when analyzing sperm motility, but this system
has been incorporated into other aspects of the semen
analysis (Amann and Katz, 2004). Unfortunately, not
all laboratories with interests in male fertility have
access to a CASA system, and these laboratories have to
rely on manual methods to examine sperm parameters.
Here we present a novel update to the classic protocol
for quantification of rat testicular HRSH. This update
utilizes a filtration step followed by somatic cell lysis of
the homogenate in order to rid the sample of debris
while preserving spermatid heads. These additional steps
allow for the use of an automated cell counter, rather
than a hemocytometer, to quantify HRSH.
Optimization of the protocol was performed using
normal rat testes, and 4 sample preparation/counting
methods were compared to demonstrate the effective-
ness and reproducibility of the newly modified protocol.
We were able to show that automated counts of
classically prepared samples produced an artificially
high HRSH count due to inclusion of cellular debris, but
with the addition of filtration and lysis steps, the
automated counts were no different from those obtained
using a hemocytometer. In fact, we saw the strongest
correlation between the filter-lysis manual and auto-
mated protocols. These results indicate that the addi-
tional filter-lysis steps are beneficial for both manual
and automated counting, and this may be because the
lysates are cleaner, which reduces the misidentification
of debris as HRSH or prevents debris from masking the
Additionally, the automated method can be applied to
detect differences in testicular HRSH counts follow-
ing low-dose exposure to a known testicular toxicant.
Previous work in our lab has documented the deleteri-
ous effects of HD on the rat testis, including reductions
in testis weights and histological alterations to the
seminiferous tubules, including germ cell sloughing,
Sertoli cell vacuolization, and retained spermatid heads
(Moffit et al, 2007). These manifestations of testicular
injury can reduce the number of spermatids in the testis,
which we have observed in HD-exposed testes after
performing manual HRSH counts (Pacheco and Boe-
kelheide, unpublished). This is consistent with the
results presented here that show decreased body and
testis weights and decreased testicular HRSH with HD
exposure, thereby verifying the utility of the automated
protocol in animal studies of testicular injury.
This applicability of the pure testicular lysates to an
automated basic cell culture counting platform repre-
sents a significant improvement to the established
manual protocol for counting testicular HRSH, a
Table 2. Optimization experiment: Correlation coefficients and P-values
Classic Manual Classic Automated Filter-Lysis Manual Filter-Lysis Automated
bP-values. Data were generated comparing the average number of homogenization-resistant spermatids per testis.
Table 3. Application experiment: Average body and testis weights after toxicant exposurea
TreatmentBody Weights, g Testis Weights, g
Control (n 5 8)
2,5-hexanedione (n 5 10)
315.8 6 6.1
286.9 6 8.9*
1.58 6 0.02
1.48 6 0.02**
aTestis weight is the mean of the average left and right testis weights. Values are presented as mean 6 standard error of the mean. * P 5 .02
and ** P 5 .002 relative to the control.
Pacheco et al
Purification of Testicular Spermatids 815
procedure that provides an important measure of
spermatogenic capacity in animal models. Manual
counts using the hemocytometer are variable, time
consuming, and susceptible to bias. We have developed
a newly modified protocol that is effective and sensitive,
allowing testicular HRSH counts to be obtained more
efficiently and reliably.
Amann RP, Katz DF. Reflections on CASA after 25 years. J Androl.
Ashby J, Tinwell H, Lefevre PA, Odum J, Paton D, Millward SW,
Tittensor S, Brooks AN. Normal sexual development of rats
exposed to butyl benzyl phthalate from conception to weaning.
Regul Toxicol Pharmacol. 1997;26:102–118.
Assinder S, Davis R, Fenwick M, Glover A. Adult-only exposure
of male rats to a diet of high phytoestrogen content increases
apoptosis of meiotic and post-meiotic germ cells. Reproduction.
Blazak WF, Treinen KA, Juniewicz PE. Application of testicular
sperm head counts in the assessment of male reproductive toxicity.
In: Chapin RE, Heindel, JJ, eds. Methods in Toxicology,
Volume 3A: Male Reproductive Toxicology. Waltham, MA:
Academic Press; 1993:86–94.
Evenson DP, Parks JE, Kaproth MT, Jost LK. Rapid determination
of sperm cell concentration in bovine semen by flow cytometry.
J Dairy Sci. 1993;76:86–94.
Freund M, Carol B. Factors affecting hemacytometer counts of sperm
concentration in human semen. J Reprod Fertil. 1964;8:149–155.
Goodrich R, Johnson G, Krawetz SA. The preparation of human
spermatozoal RNA for clinical analysis. Arch Androl. 2007;
Moffit JS, Bryant BH, Hall SJ, Boekelheide K. Dose-dependent effects
of Sertoli cell toxicants 2,5-hexanedione, carbendazim, and mono-
(2-ethylhexyl) phthalate in adult rat testis. Toxicol Pathol.
Omura M, Masuda Y, Hirata M, Tanaka A, Makita Y, Ogata R,
Inoue N. Onset of spermatogenesis is accelerated by gestational
administration of 1,2,3,4,6,7-hexachlorinated naphthalene in male
rat offspring. Environ Health Perspect. 2000;108:539–544.
Wade MG, Poon R, Li N, Lee A, McMahon A, Chu I. Testicular
toxicity of candidate fuel additive 1,6-dimethoxyhexane: compar-
ison with several similar aliphatic ethers. Toxicol Sci. 2006;
Working PK, Hurtt ME. Computerized videomicrographic analysis of
rat sperm motility. J Androl. 1987;8:330–337.
World Health Organization. WHO Laboratory Manual for the Ex-
amination and Processing of Human Semen. Geneva, Switzerland:
WHO Library Cataloguing-in-Publication Data; 2010.
Zrimsek P. Evaluation of a New Method and Diagnostic Test in Semen
Analysis. InTech Web site. http://www.intechopen.com/books/
and-diagnostic-test-in-semen-analysis. Accessed May 23, 2012.
Figure 3. Application experiment: Number of homogenization-
resistant spermatid heads (HRSH) per testis after toxicant exposure.
Testicular spermatid head counts were obtained from 2,5-hexane-
dione (HD; n 5 10; black square) and control rats (n 5 8; black circle)
using the filter-lysis automated technique and graphed as the total
number of HRSH per testis. The light gray boxes represent the range
of values and the dark gray boxes contain the standard error of the
mean, with the mean as the line in the middle of the dark gray box.
** P 5 .002 relative to control.
816 Journal of Andrology