BIOLOGY OF REPRODUCTION 58, 379-384 (1998)
Sperm Mobility: A Quantitative Trait of the Domestic Fowl (Gallus domesticus)
David P. Froman 2 and Allen J. Feltmann
Department of Animal Sciences, Oregon State University, Corvallis, Oregon 97331
ABSTRACT
Sperm from each rooster within a base population (n = 271)
were evaluated with a mobility assay validated in previous work.
Frequency analysis confirmed a normal distribution for the vari-
able of sperm mobility. Repeated-measure analysis of males cat-
egorized by phenotype demonstrated that average and high
sperm mobility phenotypes were distinct and independent of
time. Sperm morphology, fertilizing ability, and ATP content
were compared between phenotypes. Fertility and sperm ATP
content differed (p < 0.001) between phenotypes, whereas
sperm morphology did not (p > 0.05). Experimentation with
washed sperm demonstrated that phenotype was fully expressed
when mitochondrial respiration was the only source of ATP.
Sperm mobility increased (p < 0.001) when sperm from average
males were exposed to calyculin A, a protein phosphatase in-
hibitor. Correlation analyses were performed with data from a
subpopulation (n = 46) whose range, mean, and variance were
equivalent to those of the base population. Neither body weight
nor the combined weight of the testes was correlated with
sperm mobility (r = -0.02 and 0.01, respectively). In contrast,
sperm ATP content was correlated with sperm mobility (r =
0.80). We attribute phenotypic differences in sperm mobility to
differential rates of mitochondrial ATP synthesis.
INTRODUCTION
As reviewed by Froman and McLean [1], spectropho-
tometry has been one of four principal methods used to
objectively measure the motility of poultry sperm. Whereas
prior applications were based upon the rheotactic properties
of sperm [2-4], Froman and McLean [1] used spectropho-
tometry to quantify the net movement of fowl sperm into
6% (w:v) Accudenz. Thus, sperm penetration of an Accu-
denz solution affords a means of measuring sperm mobility.
Accudenz is a nonionic, biologically inert compound typi-
cally used as a medium for density gradient centrifugation.
While Accudenz can be used to wash fowl sperm [5], it
has been proven useful for other purposes. For example,
sperm penetration into Accudenz was used as a diagnostic
tool in an investigation of heritable subfertility in the roost-
er [6]. Likewise, selection of semen donors based upon
sperm penetration of Accudenz increased the number of
progeny obtained from hens over the course of a long-term
fertility trial [7].
We hypothesized that the sperm penetration test could
be used as an investigative tool because sperm mobility
appeared to be a normally distributed variable [1]. Like-
wise, the assay detected extreme variability among normal,
fertile males in preliminary experimentation. For example,
coefficients of variation for sperm viability, sperm mor-
Accepted September 16, 1997.
Received August 5, 1997.
'Oregon State University Technical Paper Number 11,212. This re-
search was supported by the Oregon Agricultural Experiment Station pro-
ject entitled "Factors Affecting the Reproductive Efficiency of Male Poul-
try."
2
Correspondence: D.P. Froman, Department of Animal Sciences, 112
Withycombe Hall, Oregon State University, Corvallis, OR 97331-6702.
FAX: (541) 737-4174; e-mail: fromand@ccmail.orst.edu
379
phology, percentage of motile cells, straight-line velocity,
and sperm penetration of Accudenz were 5%, 6%, 27%,
31%, and 65%, respectively (n = 27 roosters). Therefore,
the objectives of the present research were as follows: 1)
to confirm that sperm mobility is a normally distributed
variable using a larger base population than that used in
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initial research, 2) to conduct a time-course study with in-
dividual males to determine whether the trait was indepen-
dent of time, and 3) provided that the trait was independent
of time, to identify a cellular attribute that could account
for the trait.
MATERIALS AND METHODS
Evaluation of Base Population
Individually caged 25-wk-old New Hampshire roosters
(n = 271) were assigned randomly to be ejaculated on one
of 6 consecutive days. Each ejaculate was evaluated with
the sperm penetration assay [1] modified as follows. Sperm
concentration was determined by diluting 10 l of semen
with 2 ml of 3% (w:v) NaCl in a disposable cuvet, mea-
suring absorbance at 550 nm with a Turner Model 340
spectrophotometer (VWR Scientific, Seattle, WA), and then
calculating the unknown concentration from a standard
curve (r = 0.999). Thereafter, a 50-pl volume of semen
was diluted to 5 x 108 sperm per milliliter with 50 mM N-
tris[hydroxymethyl]methyl-2-amino-ethanesulfonic acid
(TES), pH 7.4, containing 120 mM NaCl, 10 mM glucose,
and 2 mM CaC1 2 (Sigma Chemical Co., St. Louis, MO). A
60-iil volume of this sperm suspension was overlaid upon
600 1Al of prewarmed 6% (w:v) Accudenz (Accurate Chem-
ical & Scientific Corporation, Westbury, NY) in a semi-
micro polystyrene cuvet and incubated at 41°C for 5 min,
after which the absorbance of the Accudenz layer was mea-
sured at 550 nm.
Data were analyzed by single-classification analysis of
variance (ANOVA) [8] in order to test for a time effect.
Pooled data were assigned to frequencies on the basis of
increments of 0.090 absorbance units. The Kolmogorov-
Smirnov test for goodness of fit [9] was used to determine
whether observed frequencies approximated a normal dis-
tribution.
Selection of Experimental Animals
Estimates of the mean and standard deviation were used
to calculate the normal probability density function [10],
which was used for selection as follows. Roosters (n = 271)
were ranked by initial estimates of sperm mobility. Those
males whose values were at either extreme of the distri-
bution or in proximity to the mean were retained for sub-
sequent experimentation (n = 49). At 28 wk of age, each
male was ejaculated 3 times on an every-other-day basis.
Order of ejaculation was randomized daily. Sperm mobility
was measured with the sperm penetration assay described
above. Data were analyzed by two-way ANOVA [11]. Av-
erage values were used to assign a subset of roosters to one
of two phenotypes. Any male whose average value was >
380 FROMAN AND FELTMANN
1.5 standard deviations above the population mean was cat-
egorized as having high sperm mobility. Likewise, any
male whose average value was between 1 standard devia-
tion below the mean and the mean was categorized as hav-
ing average sperm mobility.
Evaluation of Sperm Mobility Phenotypes
Males categorized by phenotype were evaluated with the
sperm penetration assay described above at 45, 53, 58, and
62 wk of age (n = 10 males per phenotype). These data
were combined with data obtained when roosters were 28
wk of age. Data were analyzed with a split-plot design [12].
In addition, males categorized by phenotype were used for
the following analyses: sperm morphology, fertility, sperm
metabolism, ATP content, and the effect of protein phos-
phatase inhibition on sperm mobility. Morphology of sperm
from average and high sperm mobility phenotypes (n = 10
males per phenotype) was evaluated as follows. A 50-pl
volume of semen was diluted to 5 X 108 sperm/ml with 50
mM TES, pH 7.4, containing 136 mM NaCl (TES-buffered
saline). A 50-x1 volume of this sperm suspension was di-
luted to 2 x 10 7 sperm/ml with TES-buffered saline in a
1.5-ml microcentrifuge tube. Sperm were immobilized by
heat denaturation at 56C by incubating tubes in an Eppen-
dorf Model 5320 Constant Temperature Heater (Brinkman
Instruments Co., Westbury, NY) for 10 min. Thereafter,
sperm were viewed at x400 by phase contrast microscopy,
and percentages of abnormal sperm were estimated from
samples of 500 sperm per male. Arcsine transformations of
percentages were analyzed by single-classification ANOVA
[8].
Fertility of average and high sperm mobility phenotypes
(n = 10 males per phenotype) was determined as follows.
Phenotype was confirmed before insemination with the
sperm penetration assay described above. Thereafter, each
ejaculate was diluted to 1 10 9 sperm/ml with TES-buf-
fered saline containing 10 mM glucose and 2 mM CaCI 2,
i.e., the same buffer as used for the assay. Then, each of
12 Single Comb White Leghorn hens was inseminated with
5 x 10 7 sperm. Egg collection commenced on the second
day after insemination and continued for 7 consecutive
days. Eggs identified by hen and date were stored daily at
18°C. Eggs were set en masse, and fertility was determined
by examining the contents of each egg after 4 days of in-
cubation. Percentages of fertilized eggs were calculated on
the basis of the total number of eggs laid per group of hens
during the 7-day egg collection interval. Percentages were
transformed to logits, and logits were analyzed with a log
odds model [13].
The relationship between sperm mobility and ATP syn-
thesis was evaluated as follows. Sperm mobility was mea-
sured as described above. Then sperm were washed by cen-
trifugation through 12% (w:v) Accudenz prepared with
TES-buffered saline according to McLean et al. [5].
Washed sperm were resuspended to a concentration of 5 x
108 sperm/ml in each one of the following media: TES-
buffered saline, TES-buffered saline containing 10 mM glu-
cose, TES-buffered saline containing 10 mM glucose and
1 mM KCN, and TES-buffered saline containing 1 mM
KCN. Each of the 4 media contained 2 mM CaC12. Each
suspension was overlaid, in random order, upon 6% (w:v)
Accudenz prepared with the same respective buffer, and the
sperm penetration assay was performed as described above.
A paired comparison (n = 20) was performed [11] using
pre- and post-wash data obtained with TES-buffered saline
containing glucose and Ca 2+ as the suspending medium.
Single-classification ANOVA [8] was used to test for phe-
notypic differences in sperm mobility when ATP synthesis
was limited to either oxidative phosphorylation or glycol-
ysis.
Sperm ATP content was determined immediately after
ejaculation with a bioluminescence assay kit and lumino-
meter (FireZyme Diagnostic Technologies, Ltd., Halifax,
Nova Scotia, Canada). In each case, measurements were
based upon a sample of 12.5 106 sperm cells. Single-
classification ANOVA [8] was used to test for phenotypic
differences in sperm ATP content. The effect of protein
phosphatase inhibition on sperm mobility was determined
as follows. A paired comparison [11] was performed with
sperm from the average phenotype. Sperm were diluted to
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5 x 108 sperm/ml either with TES-buffered saline contain-
ing 2 mM CaC12 or the same buffer containing 500 nM
calyculin A (Sigma Chemical Co.). Each suspension was
overlaid, in random order, upon 6% (w:v) Accudenz pre-
pared with the same buffer, and the sperm penetration assay
was performed as described above.
Correlations
At 65 wk of age, sperm mobility and sperm ATP content
were measured from subsamples of a single ejaculate from
each surviving rooster selected from the base population (n
= 46). Thereafter, each rooster was weighed and killed by
cervical dislocation. Testes were excised and weighed.
Sperm mobility was correlated to sperm ATP content, body
weight, and total testicular mass [14].
RESULTS
Evaluation of Base Population
Sperm mobility data gathered from 271 roosters over the
course of six consecutive days did not differ among days
(p > 0.05). Therefore, data were pooled, and values were
assigned to frequencies (Fig. 1). The hypothesis that ob-
served frequencies approximated a normal distribution was
not rejected (p > 0.05).
60 -
45
Ir
o
30 -
0
U)
o
0
HH
15-
0
n · · · L L--
--
- -
- -- L- - -
-- --
SPERM MOBILITY CATEGORIES
S
-
- f
· · ·· _
FIG. 1. Frequency distribution derived from categorization of sperm mo-
bility data obtained from a single measurement from each of 271 New
Hampshire roosters. Sperm mobility was quantified as described in Ma-
terials and Methods. The category with the maximal number of roosters
represented those males whose values ranged from 0.361 to 0.450 ab-
sorbance units.
 SPERM MOBILITY 381

1.2

2.19- E a
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O, 0.8
A A
1.69-
A A

m o o
0.4n
1.19 - 0 8o_ 8 8Q 0 _
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AVERAGE: \HIGH 0 §
0.69-
30 40 50 60
AGE (wk)
0.19I

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0.0 0.2 0.4 0.6 0.8
ABSORBANCE UNITS
FIG. 2. Normal probability density function based upon estimates of
0.401 and 0.1819 for the mean and standard deviation, respectively. The
function depicts the distribution of fowl sperm mobility as measured by
sperm penetration into 6% (w:v) Accudenz at body temperature using a
base population of 271 roosters. Absorbance indicates the extent to which
sperm penetrated the Accudenz layer after a 5-min incubation. Areas un-
der the curve denoted by dashed lines represent two sperm mobility phe-
notypes used for a long-term study of individual roosters.
Selection of Experimental Animals
Sperm mobility differed among males (p < 0.0001), but
no time effect was observed (p > 0.05) when repeated mea-
surements from 49 males were analyzed by two-way ANO-
VA. The range, mean, and standard deviation of averaged
values (n = 3 observations per male) were 0.019 to 0.854,
0.415, and 0.2329 absorbance units, respectively. The
range, mean, and standard deviation of the base population
were 0.010 to 0.898, 0.401, and 0.1819. On the basis of the
similarity of these statistics, we deemed the sample of 49
males to be representative of the base population. Before
repeated-measure analysis, any male whose initial value
was less than a standard deviation below the population
mean or greater than a standard deviation above the mean
was deemed a low or high sperm mobility male, respec-
tively. Likewise, any male whose initial value was within
a standard deviation of the mean was deemed as having
average sperm mobility. Using these criteria, phenotypic
fidelity was 84% after repeated-measure analysis. All mis-
matched males were categorized as average. Phenotypes
selected for long-term study of sperm mobility are illus-
trated in Figure 2 relative to the predicted normal proba-
bility density function.
FIG. 3. Sperm mobility data from average (circles) and high (triangles)
sperm mobility phenotypes plotted as functions of time (n = 10 males
per phenotype). Sperm mobility was measured as described in Materials
and Methods. Solid lines represent the functions: y(x) = 0.729 +
0.0006(x) and y(x) = 0.296 - 0.0005(x). Slopes and y-intercepts were
estimated by the method of least squares. Neither slope differed from zero
(p > 0.05). Therefore, phenotypes were independent of time.
0.0001) after repeated sampling of individual males over
the course of 34 wk. In contrast, neither a time effect nor
a phenotype by time interaction were observed (p > 0.05).
These relationships are illustrated in Figure 3. Phenotypic
differences in sperm mobility were not explicable in terms
of varying sperm morphology: percentages of abnormally
shaped sperm averaged 3.5% and 3.6% for average and
high sperm mobility phenotypes, respectively. As shown in
Table 1, there was a difference in fertility (p < 0.0001)
between phenotypes.
Washing sperm by centrifugation through 12% (w:v) Ac-
cudenz did not affect sperm mobility (p > 0.05). Further-
more, resuspension of sperm in a glucose-free medium had
no effect (p > 0.05) upon the mobility of washed sperm
when the sperm penetration assay was performed under aer-
obic conditions. When washed sperm were forced to use an
unknown endogenous substrate and mitochondrial respira-
tion for ATP synthesis, the phenotypic difference in sperm
mobility was evident (Table 2). Mean values were equiva-
lent to the y-intercepts of the functions shown in Figure 3.
In contrast, washed sperm were immobilized in the pres-
ence of cyanide if glucose was not provided. Even though
glucose enabled sperm to be motile in the presence of cy-
anide, sperm mobility was reduced substantially for either
TABLE 2. Effect of mitochondrial respiration and anaerobic glycolysis
on sperm mobility.*

Evaluation of Sperm Mobility Phenotypes A Absorbance according to metabolic constraints

imposed upon washed sperm
Differences in sperm mobility were observed between
phenotypes and among males within a phenotype (p < ATP synthesis ATP synthesis
Sperm limited to Respiration limited to
mobility Males mitochondrial blocked by anaerobic
TABLE 1. Fertility of roosters categorized by sperm mobility phenotype. phenotype (n) respiration cyanide' glycolysis*

Hens Total eggs Average 10 0.380 0.0555A None 0.223 0.0474 a

Sires per sire laid* Fertility t High 10 0.743 0.0365B None 0.433 0.0 6 2 5 b
Phenotype (n) (n) (n) (%) * Measured by sperm penetration of 6% (w:v) Accudenz at 410 C from
Average 10 12 734 64 _ 6.8A an overlay of extended semen; sperm penetration induced a change in
High 10 12 734 94 + 1.0B absorbance at 550 nm.
t Sperm suspended in a glucose-free medium containing 1 mM KCN.
* Eggs per group of 120 hens collected over the course of 7-days follow- *Sperm suspended in medium containing 10 mM glucose and 1 mM
ing a single intravaginal insemination of 5 x 107 sperm per hen. KCN.
t Each value is a mean SEM. AtBMeans within a column differ (p < 0.001).
A,B Means within a column differ (p < 0.0001). o,b Means within a column differ (p < 0.05).
382 FROMAN AND FELTMANN
TABLE 3. Sperm ATP content according to sperm mobility phenotype.
Males ATP*
Phenotype (n) (nmol/109 sperm)
Average 10 95 + 19.2A
High 10 220 t 22.2B
* Each value is a mean ± SEM; measurements were made immediately
after ejaculation.
A,B Means within a column differ (p < 0.001).
phenotype relative to observations made under aerobic con-
ditions (Table 2). The phenotypic difference in sperm mo-
bility observed under aerobic conditions was explicable in
terms of sperm ATP content (Table 3). In the case of av-
erage males, phenotypic expression was modulated by the
inhibition of protein phosphatase activity (Table 4) in ad-
dition to the ATP biosynthetic pathway (Table 3).
Correlations
The range, mean, and standard deviation for the sperm
mobility of 65-wk-old roosters (n = 46), as measured by
absorbance with the sperm penetration assay, was 0.017 to
1.007, 0.398, and 0.2696 absorbance units, respectively.
These statistics were comparable to those derived from the
same individuals on any day (n = 3), when sperm mobility
was measured at 28 wk of age. Mean ATP content was 154
nmol ATP/10 9 sperm. Mean body weight, right testis
weight, and left testis weight were 3.6 kg, 19.6 g, and 19.9
g, respectively. Estimates of product-moment correlation
coefficients for sperm mobility versus sperm ATP content,
body weight, and total testicular mass were 0.80, -0.02,
and 0.01, respectively. A plot of sperm mobility versus
sperm ATP content is shown in Figure 4.
DISCUSSION
The first objective of the present research was to confirm
an initial report [1] that fowl sperm mobility, as measured
by sperm penetration of 6% (w:v) Accudenz, is a normally
distributed variable. This objective was accomplished by
studying a base population of 271 roosters (Fig. 1). The
second objective was to study the sperm mobility of indi-
vidual males through time. We hypothesized that phenotype
1.000 o
C o o
0 o
0.800
o
o 00
0.600 o is located between the shell gland and vagina. Second,
0
z 0 0
0.400
0 are located distal to the vaginal sphincter and provide the
Z,
0.200
0.000
0 100 200 300
nmol ATP / 109 SPERM
FIG. 4. Correlation of sperm mobility to sperm ATP content. Each data
point (circles) represents data collected from a single ejaculate immedi-
ately after ejaculation (n = 46 roosters). Sperm mobility was measured
as described in Materials and Methods. Sperm ATP content was measured
with a bioluminescence assay kit and luminometer (FireZyme Diagnostic
Technologies, Ltd.). The solid line denotes the regression equation: ab-
sorbance = 0.1793 + (0.0025)(ATP). The product-moment correlation co-
efficient was 0.80.
TABLE 4. Effect of protein phosphatase inhibition on the mobility of
sperm from males categorized as having average sperm mobility.*
Males Absorbance
Treatment (n) units'
Control 10 0.226 ± 0.0301A
Calyculin A (500 nM) 10 0.456 + 0.03288
* Measured by sperm penetration of 6% (w:v) Accudenz at 41° C from
an overlay of extended semen; sperm penetration induced a change in
absorbance at 550 nm.
t Each value is a mean SEM.
AB Means within a column differ (p < 0.001).
would be independent of time. This hypothesis was based
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upon our observation that sperm mobility was independent
of time over 20 wk when measurements were based upon
ejaculates pooled by phenotype [7]. As shown in Figure 3,
sperm mobility was independent of time over the course of
34 wk. This interval represents the preponderance of time
during which the roosters under study were at their peak
reproductive performance. Our third objective was to iden-
tify a cellular attribute that could account for the trait, pro-
vided that the trait was independent of time. We attributed
the existence of sperm mobility phenotypes to variable rates
of sperm ATP synthesis (Table 3, Fig. 4). Furthermore, mi-
tochondrial function was identified as being fundamental to
the expression of phenotype (Table 2). We inferred from
protein phosphatase inhibition (Table 4) that axonemal
function was not the principal limitation on maximal sperm
mobility.
Our experimental outcomes have three implications.
First, in vitro measurements of fowl sperm mobility are
relevant to reproductive efficiency. The data in Table 1
corroborate previous research [7] in which a 10% differ-
ence in hatchability was realized by selecting semen do-
nors based upon sperm mobility. In this case, semen was
collected weekly for 14 wk, pooled by phenotype, and
then used to artificially inseminate hens. The difference
in progeny was attributable to a difference in fertility.
For many years sperm evaluation tests have been sought
that use only a few semen samples per male to identify
males with the potential to be highly fertile. Amann and
Hammerstedt [15] predicted that achieving this goal was
highly unlikely. To date, this seems to be true for mam-
mals. However, this is no longer true for the domestic
fowl. We attribute the predictive value of the sperm pen-
etration assay to the following facts. First, immotile tes-
ticular sperm can fertilize oocytes if placed in the hen's
oviduct above the vaginal sphincter [16]. This sphincter
fowl sperm must be motile in order to ascend the vagina
and enter the sperm storage tubules [17]. These tubules
oviduct with a resident population of sperm when the
oviduct becomes patent at oviposition. Third, fowl sperm
do not undergo capacitation before fertilization as mam-
400 malian sperm do [18]. Therefore, the sperm penetration
assay simulates a critical step for internal fertilization in
the hen: the net movement of a population of motile
sperm cells, presumably against a resistance.
The second implication of our experimental results
pertains to the inheritance of sperm mobility. As evi-
denced by frequency analysis (Fig. 1) and subsequent
performance of individuals (Table 1) selected by pheno-
type (Fig. 2), sperm mobility may be a polygenic trait
that affects fitness. Repeatability was estimated to be
 SPERM MOBILITY
0.84 on the basis of repeated-measure analysis of 49 in-
dividuals. This statistic is an upper estimate of heritabil-
ity [19]. Therefore, the trait may be highly heritable. In
this regard, it is noteworthy that there is a paucity of
information about quantitative reproductive traits in male
poultry [20]. To date, breeders have viewed the motile
nature of sperm as an essential but uncontrollable vari-
able. However, genetic progress may be possible through
selection.
The third implication of our experimental results per-
tains to the study of fowl sperm motility itself, which has
focused on the axoneme [21-30] and the role of second
messengers, extracellular Ca2 + in particular [31-36]. To
date, biochemical data, including that shown in Table 3,
reflect averaged values. Such data obscure differences
among males within a population as well as differences
within a sperm population from any given male. Before
ejaculation, sperm within the rooster's deferent duct are
essentially immotile at body temperature [37]. Conse-
quently, there is no variation in sperm cell motion among
males at this time. In contrast, a profound difference ex-
ists among males after ejaculation (Figs. 1-3). Sperm cell
motion is a function of the cell's physical environment
in addition to temperature, chemical environment, met-
abolic capacity, and structural integrity [38]. Therefore,
phenotypic differences in sperm mobility may be expli-
cable in terms of how sperm react to the interface be-
tween the sperm suspension overlay and the Accudenz
solution. In any event, we anticipate that biochemical and
biophysical analyses of sperm cell populations following
auto-segregation via sperm penetration of Accudenz will
reveal molecular mechanisms underlying phenotypic dif-
ferences in fowl sperm mobility.
ACKNOWLEDGMENTS
Accudenz was provided as a gift from Rudi Rosenberg Sr. of Ac-
curate Chemical & Scientific Corporation. Derek McLean assisted with
the evaluation of sperm cell morphology. ATP measurements were
made with reagents and equipment provided by Paul Wegener of
FireZyme Diagnostic Technologies, Ltd. The senior author thanks Paul
Bernier, Oregon State University (OSU) Emeritus Professor of Genet-
ics, for his mentoring over the course of a decade and Walther Ott,
OSU alumnus, for his endowment promoting research within OSU's
Department of Animal Sciences.
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