Feb 20, 1973 - john Company,. Kalamazoo,. MI 49001. 'Present address: Department ... Armour-Baldwin. Labs) given intravenously to induce ovulation. They.
BIOLOGY
OF
9,
REPRODUCTION
Progesterone,
116-124
(1973)
2OfLHydroxypregn4en3one,
Hormone
Levels
in Superovulated Postpuberal
C.
H.
SPILMAN,2 C. R.
Department
G. E. VUKMAN,
of Animal
and
Science,
Prepuberal
and
Cattle1
SEIDEL,
JR.,3 L. L. H. FOOTE
R.
AN])
Cornell
Accepted
Luteinizing
University,
February
20,
LARSON,4
Ithaca,
New
York
14850
1973
Pituitary and plasma luteinizing hormone (LH) and luteal and plasma progesterone were measured in 47 prepuberal Holstein calves and in eight postpuberal heifers for 30 days after induced ovulation or superovulation. Mean pituitary LH in six of the eight heifers sacrificed 30-31 days after superovulation averaged 1.1 mg LH/g of pituitary tissue. Plasma LH rose to 7.2 ng/ml the day after PMSG injection and gradually declined to normal levels of about 2 ng/ml. Plasma progesterone peaked at 63 ng/ml 10 days after LH injection to induce superovulation and declined to nearly undetectable levels in three of the heifers by Day 24. These three heifers ovulated spontaneously, plasma progesterone rose to 8.3 ng/ml by Day 28, and each had a new active corpus luteum at slaughter. The others had not ovulated when slaughtered on Day 30, and still had some old active luteal tissue. Mean pituitary LH in control calves was 5.0 mg/g of wet pituitary tissue and ranged from 0.7 to 1.6 mg/g during the 30 days after superovulation. Plasma LH in calves rose from 1.5 ng/ml before injection of pregnant mare serum gonadotropin (PM SC) to 5.5 ng/ml the day after PMSG and returned to the original level by 10 days after PMSG. Calf corpora lutea had a maximum average progesterone concentration of 43 g/g of luteal tissue 10-11 days after ovulatory LH. This declined to a minimum on Day 28, but some calves had as much as 36 g progesterone/g of luteal tissue on Day 30. Plasma progesterone rose to nearly 100 ng/ml 8-10 days after superovulation, and peak progesterone was correlated with the number of corpora lutea (r = 0.76, P .( 0.01). Plasma progesterone declined after Day 15, but detectable levels were present
throughout
the
experiment.
Plasma
progesterone
and
20p-hydroxypregn-4-en-3-one
rose and fell simultaneously in eight superovulated calves. The major difference among the prepuberal and postpuberal animals was the fact that plasma progesterone in the prepuberal animals never declined to essentially undetectable levels and none ovulated spontaneously. Whether this reflects a difference between uteri of immature and mature cattle in regulating corpus luteum function has not been investigated.
No
information
ture
was
concerning
1This
work
was
found
in
possible
cyclical
partially
supported
litera-
the
in
by
Grant
itary
from the National Institute of Child Human Development. ‘ address: Fertility Research, The Upjohn Company, Kalamazoo, MI 49001. ‘Present address: Department of Physiology and HD 03471 Health and
Biophysics, CO
Colorado
State
University,
“Present
address:
Department
of Nebraska,
of Lincoln,
#{174} 1973 by of reproduction
The
Society for in soy form
they
Ft. Collins, Animal SciNB 68503.
the Study reserved,
levels
of LH
of Reproduction.
can
(LH)
ovulation cattle, in dairy
(Desjardins
Calves normally at about 8-12
116 Copyright All rights
hormone
after induced in prepuberal
measured
80521.
ence, University
luteinizing
terone lation
changes
be
start months induced
or
calves and
Hafs,
to ovulate of age. to
proges-
or superovualthough pitu-
ovulate
ogenous
gonadotropin
treatment
month of and Hafez,
age (Marden, 1970; Seidel
1953;
have
been 1968).
and cycle However,
with
exat
Lineweaver et a!., 1971). Pro-
1
PROGESTERONE
AND
LII
IN
SuPEROvULATED
cor-
-15#{176}C for
pora lutea by Marden (1953). The in vitro steroidogenic capabilities of luteal tissue from superovulated calves have been deter-
determination.
gesterone
was
mined progestin
have
secretion
postpuberal
in
uteri
superovulate with
were
similarly
sexually of
In
were
and
heifers,
comwhich plasma
group
AND
METHODS
The superovulation regimen for calves has been described in detail (Seidel et al., 1971). Briefly, 47 Holstein calves, 2-5 months old, were given 1250-2000 IU of pregnant mare serum gonadotropin (PMSG) intramuscularly, followed in 5 days by 75 mg LH (PLH, Armour-Baldwin Labs) given intravenously to induce ovulation. They were laparotomized 3 days after the injection of LH in order to examine the ovaries and to recover ova. The calves were subsequently sacrificed at regular intervals from 5-30 days after LH administration. Eight cycling postpuberal heifers were injected intramuscularly on Day 15 or 16 of the estrous cycle with 3000 IU PMSG followed in 4 days by 2500 IU human chorionic gonadotropin (HCG) given intravenously. They were sacrificed at
either
30
or
31
days
after
HCC.
The
pituitaries
from all animals were quickly removed and frozen. The ovaries were removed and approximately 0.5 g of luteal tissue was taken from several corpora lutea of each animal and stored at -15#{176}C for later extraction. Ten to fifteen milliliters of blood were collected from the jugular vein of calves 5 days before the PMSG injection and from calves and heifers on alternate days from the day of PMSG injection to the day of LH or HCC injection. On the days that PMSG and LH or HCG were injected, blood was collected prior to the injection and then not again for at least 24 h. For the next 5 days, 200-250 ml of blood were collected from each animal on alternate days and thereafter every 4 days. More frequent bleeding was avoided to minimize stress and hemodilution. After centrifugation, the plasma was removed, frozen, and stored at
in
a
and
(1:2,
developed
systems of Armstrong ples were transferred developed in isopropyl
was
of
alka-
times
silicic a
by
with
3
were spotted on plates prepared acid
Chemical
(Silic
at
in
two-dimensional
thickness
et al. (1964). to a second ether-ethyl
AR in dis-
Works)
w/v) the
made
funnel
Samples (tic)
slurry
plasma
(1970)
three
separatory
LH
the
Hansel
extracted a
and
from
and Plasma
and
Mailinckrodt
7CF,
of
0.25
solvent Usually,
tic
plate
acetate
samand (5:2,
v/v). After
in one
modification.
spreading
mm
extracted
diethyl ether. chromatography
tilled water
or
of calves. MATERIALS
vol of thin-layer
extraction
of Kazama
shaking
with
ovulate
was
NaOH
cattle
addition,
determined
a slight
with
TLC
20/3-hydroxypregn-4-en-3-one
(20$-OH-P)
with
line
by
steroid
procedure
with
to
mature
treated.
the
cattle
et a!., 1971)
(Seidel
pared levels
induced
later
Progesterone
by
vigorous
to compare posLH and proges-
Prepuberal
were
calf
1972), but plasma not been reported.
prepuberal
cattle.
infantile
in
was initiated changes in
study
cyclical
terone
identified
et a!.,
(Spilman levels
This
sible
first
117
CArrLE
the
final tic
separation,
progesterone
and
20k-OH-P spots were eluted with diethyl ether or chloroform and quantitated by gas-liquid chromatography (glc). The glass columns were packed with either 2.5% SE-30 or 3% OV-225 on GasChrom Q. Sample progesterone and 20p-OH-P peaks were identified by comparing their respective gic retention times against the retention times for authentic compounds. Plasma progesterone values obtained by this method in the same laboratories for mature cycling cattle were normal (Hansel and Echternkamp, 1972). All samples were corrected for procedural losses. Tritiated 20p-OH-P was obtained by enzymatic reduction of [7-’HJ progesterone. Luteal tissue was minced and extracted by refluxing in ethanol (Seifart and Hansel, 1968). Progesterone was isolated by the same tic procedures used for plasma and quantitated by uv absorption at 240 nm. Corrections were made for background from 225-255 nm (Allen, 1950). Plasma LH levels were determined by radioimmunoassay (RIA) following the procedure given in detail by Harms et al. (1969). This method, in simultaneous studies on mature cycling cattle (Hansel and Echternkamp, 1972; Hobson and Hansel, 1972), measured about 1.5-2.0 ng LH/ml plasma during the estrous cycle. Three aliquots of 0.2 ml were used to determine plasma LH in each sample. Triplicate NIH-LH-B7 standards were run with each assay. To determine whether injected PMSG might affect the LH assay, levels of PMSG likely to be found in blood plasma after
an
intramuscular
injection
were
also
assayed.
As reported by Niswender et al. (1968), no cross reactivity was found between PMSG and the LH antibody used in the RIA. Pituitary LH was determined in the initial group of calves by the ovarian ascorbic acid depletion (OAAD) method (Parlow, 1981). Two levels of each pituitary, 0.4 and 1.6 mg wet weight, and
118 0.4, in
SPILMAN
1.6, 1.0
and
ml
6.4 g
were dissolved into rats (five
of NIH-LH-B5
of saline and injected which had been primed
per level), with PMSC and HCG. Statistical analysis of the assay data was done by the methods described by Finney (1952). Subsequently, additional calves were examined for a longer interval of time after superovulation. The pituitaries from these calves and from all heifers were analyzed for LH by RIA. They were homogenized in phosphate-buffered saline-1% egg white. Appropriate dilutions were made and the samples were assayed in triplicate. NIH-LH-B7 was used as the standard.
RESULTS
AL.
six
Pituitary
and
Mature
after of
to
the
heifers
was
nearly
be present
sacrificed
about 2 study, 30
or
averaged
pituitary.
content
Thus,
of adult
1.1
pituitary
tissue
superovulated
normal
after
animals
superovulation
Plasma (Fig. 1)
LH in sexually rose sharply after
and
declined
Day
5 the
at three
gradually
for
normally
had 15
et a!., 1972).
spontaneous
and
slaughter of
levels
cycling
1969; Henricks Echternkamp,
these
LH
although
26,
on
Days had
heifers injection
thereafter.
LH
plasma
mature PMSG
(Harms
1970; Five
Hansel of the
peaks it was
30 ovulated
or
After
similar
were cattle
31
between observed
that
only
recently.
S E 0, C C Ui
z 0 U)
Ui I-. U) Ui
A-
0 0 A.
U,
A.
DAYS
Fxc.
1. Blood
ovulated
with
AND
AFTER
OVULATORY
LII
progesterone concentrations in sexually mature heifers superThe number of samples assayed are noted at each point. After Day 22 the progesterone values for the three heifers that ovulated are shown separately (solid dots) from the rest (open circles). PMSG was injected on Day -4 and HCG on Day 0 after taking blood on those days. The standard errors for plasma LH were 0.2, 3.3, 0.1, 0.5, 0.2, 0.7, 0.3, 0.4, 0.3, 0.2, 0.3, 0.4, 0.4, 0.5, 0.1, 0.2, 0.2, and 0.3. The standard errors for plasma progesterone were 1.2, 5.7, 22.7, 20.3, 13.8, 24.4, 31.2, 8.5, 3.2, 3.2, and 1.0 ignoring the separation into two groups after Day 22. plasma
PMSG
LH
and
and
BEFORE
HCC.
31 mg
treatment.
Days
sexby
(1966) In
superovulation
LH
heifers
LH concentration of cattle has been reported
tissue.
postpuberal
days LH/g
and
LH
Plasma
The pituitary ually mature
Heifers
a!.
of fresh
et a!., Superovulated
et
Desjardins
mg/g
to those
DISCUSSION
AND
ET
PROGESTERONE
LH
AND
IN
SUPEROVULATED
TABLE LH
CONTENT
OF
ANTERIOR
1
PITUITARIES
LH
119
CATTLE
FROM
SUPEROVULATED
concentration,
CALVES
Total
LH
No. Treatment Untreated Days
_________________
calves
Mean
4
5.0
3.7-7.0
2.3
1 .6-3.3
controls after
of
content,
m g’
mg/gb
Range
Mean
flange
LH
5
3
1.2
0.7-1.5
0.6
0.4-0.7
10
4
1.6
0.9-2.5
0.9
0.5-1.2
15
4
1.3
0.9-1.6
0.7
0.4-0.9
20
5
0.9
0.5-1.3
0.5
0.3-0.9
22
6
1.0
0.2-2.2
0.3
0.1-0.5#{176}
24
6
0.9
0.2-1.7
0.6
0.1-1.2
26
6
0.7
0.3-1.0
0.5
0.3-0.7
28
5
1.5
0.6-2.4
1.0
0.4-1.6
30
4
1.6
0.8-2.2
0.8
0.3-1.0
Samples from control calves and from treated calves taken for 20 days after LH were assayed by the OAAD method. Subsequent samples were assayed by RIA. There were no differences (P > 0.10) among the LH-treated groups, but the untreated controls were different from all other groups (P < 0.005). ‘The
mean
lost from
total LII
the other
Luteal
and
At
Plasma
slaughter,
two
for this group
three
corpora
heifers,
again,
lutea
which
averaged
g progesterone! the progesterone lutea in some
is based
0.8,
from
had
9.5,
not
and
21.4
g of corpus luteum. Thus, concentration of corpora animals was maintained
longer than in normal cycling heifers (Hafs and Armstrong, 1968) in which luteal progesterone declined sharply after Day 18. Three
other
heifers
each
had
corpus Days
luteum formed before 30 or 31. The progesterone
tion
was
measured
was 52.1 Plasma by Days (P
in
two
between
Days
days
sampled.
Lamond
(1972)
study heifers. reported
found
values
similar
in
the
present
higher
than
normally
cycling
or
(Henricks
kamp,
single
slaughter on concentraof
these
10-22
than
and to the
those pregnant
are
reported dairy
et a!., and
The lutea
calves,
three when
since
some
pituitary
heifers that slaughtered
undergoing
a nearly
progesterone nearly
in
tissue
increased
Day
23.
was
that
reported
animals. into
by
during Three
(1972)
estrus
Hansel
and
midcycle heifers
in that
ovulate
levels
Day of
slightly
on Day
slaughtered
ng/ml
Plasma
declined
on
is
and
plasma
tectable
6.3
value
corpora were
cycle.
animals levels
to
This
had new apparently
normal
these
undetectable
then
to
24
and
plasma higher
by than
Echternkamp mature did not
dairy come
maintained
de-
of progesterone
until
30 or 31.
Superovulated
Pituitary The
on of
Caddy present
study
et a!., 1970; Plotka et a!., 1968; Hansel 1972).
three
and
luteum. had risen greater
on Day 15 in superovulated beef The levels of plasma progesterone
nificantly
Shemesh
a new
of corpus (Fig. 1) significantly
and 84.3 zg/g progesterone 3 to 5 and was
< 0.01)
other
on
at slaughter.
Progesterone
postpuberal
ovulated
content
calves
sig-
for cows
1967; Echtern-
and
calves ously (Desjardins ovulation,
LH
Plasma
mean
pituitary
LH tissue
(Table published
1)
Calves
concentration for
of 5.0
untreated
agrees with for calves
mg/g
prepuberal
values previof this age
and Hafs, 1968). After superpituitary LH levels decreased
markedly (P < 0.005), approximating those found in mature animals (Desjardins et al., 1966). Levels remained low,
al-
though
it appears
in-
creasing
slightly
they
may
by 30 days.
have
been
120
SPILMAN
ET
AL.
12 .
5.5 5.0
7-\
!16
100
/
4.5 4.0
14
/
E
/
01 C
18
.
:
3.0
-J
Ill
\
/
9o
\
0)
\
Ui
z
\
/
2.0
:
28
U, Ui
0
50
0
:
:
..
25
A.
9
40
‘C
..
:
26
....#{149}.. 39
12
Ui
I-.
25
13
60
.
29
18
0
:
-i
0.
C
:
Progesterone
-
2.5 .1l
S -_
80
: U)
110
26
16. ...I#{149}
18
18
30
U,
:
A.
‘C
0
#{149}k#{149} II
0
III
III
-10
5
DAYS
Fic. 2. and LH. -5 and were 0.1, and 2.7. 26.1, 9.3,
Plasma
values
postpuberal Henricks
ment
a!.,
the
1970;
and
AND
the
declined
et
(Harms Hansel
gradually
(P < 0.01).
levels
control
6 and 26. The observed due calves sions
rise
injection solely
(Fig.
to LH
creases tration.
LH
apart
in plasma Levels
of
plasma after not detectable
LH
assay Niswender
used.
in
plasma
1)
is believed
30
LH
on
two
LH after PMSG
PMSG likely
intramuscular when checked agrees
This
et
a!.
(1968),
with
of
LH
1971).
Estrogen
after
in-
release
of
1969;
PMSG
estrogen
and has
follicles.
was on
the
injection by the
this
injection
cattle
(Hobson
from
may
the
previous on
injection
be
3 h,
than
Since
plasma
injection, of the
on
the
day
because
The small LH due to residual of 75 mg of
be
day.
a!.,
phenomenon in superovu-
administered first.
1
24 h after in the blood less
et
and
collected Day
the
(Coding
A similar to occur
was
to a!.,
et causes
sheep
(Midgley et a!., 1968). peaks were observed LH
es-
calves
administration
in
et a!., 1971)
lated rats No LH blood
these (Seidel
by
Hansel, 1972). been reported
peak
secretion Sufficient
estrus
LH
Bolt
LH
found
following
behavioral
hormone
work
between
produced
exogenous
occa-
reactivity
PMSG-stimulated
administo be in
the who
release
was
to be
similar
cross
the antibody used in our RIA. The in LH presumably reflects endogenous
trogen induce
additional
showed
little
and rise
had in-
in one
LH
Four
PMSG
with
days
the were of
OVULATORY
by
to pretreat-
Plasma
secretion.
injected 32-33
AFTER
25
20
and
calf that did not ovulate after LH injection varied from 0.6 to 1.2 ng/ml between Days
PMSG
1111111
15
very
al.,
These values day after PMSG
1972).
considerably
jection
BEFORE
(Fig. 2) are for normally
cattle
et
Echternkamp,
risen
during
before superovulation to values reported
cycling 1969;
liii
JO
Blood plasma LH and progesterone concentrations in calves superovulated with PMSC The number of samples assayed are noted at each point. PMSG was injected on Day LH on Day 0 after taking blood on those days. The standard errors for plasma LH 0.1, 0.4, 0.6, 0.4, 0.3, 0.2, 0.3, 0.2, 0.1, 0.2, 0.1, 0.1, 0.2, 0.1, 0.3, 0.1, 0.1, 0.2, 0.5, The standard errors for plasma progesterone were 0.7, 0.4, 1.5, 9.9, 7.8, 14.7, 33.0, 13.3, 12.0, 8.2, 2.3, and 2.7.
LH
period similar
111111
.1111111
0
LH
the was
effect
of
minimal
the half-life of calf would appear
LH to
PROGESTERONE
Five 5.2,
to
had
calves
4.8,
and
20.
4.8
Owing
pling,
undetected
cured
in
contrast heifers
LH
peaks
calves.
had The
of
5.4, 15
blood
may
up
to
Day
in one
the these
of the
were
and
Plasma
Superovulation sive production tissue.
sg/g
rise in an LH
calves
days
lutea
slaughtered
after
LH
luteal
tissue
3,
10,
15,
respectively. lutea per
often
exceeded
PMSG
0.9,
Also,
With animal
an the
corpora maintenance. Plasma
Average
progesterone
concentration
luteal (Table
tissue from superovulated 2) was similar to that
cycling
cows
through
Armstrong,
1968).
Luteal
began Day 11
to and
tained tle
in which
than
of
progesterone decline reached
in most a mini-
cycling
levels
or about Day 18 (Armstrong 1966; Hafs and Armstrong,
decline and 1968).
on
CONCENTRATION
OF
CALF
Day Item
Number
5-8
10-11
and lutea
Hafez,
blood
the
1970;
flow
greatly
to
different
the
plasma
levels
indicating
not
a calf
that
4
and Peak
correlated 0.76, low at
=
to have lower titers just prior to slaugh-
was
from
of were
of CL (r Calves having concentrations
progesterone
Progesterone
were
high mass
Days times.
samples
calf lutea
were large
between at other
tended
prorest.
corpora levels
of
progesterone
Mar-
enlarged
affected
tissue
also
after
1971), thereby older than the
significantly higher 26 (P < 0.01) than
TABLE PROGESTERONE
days
Levels
ter.
Black, They
20
present.
slaughter of plasma
cat-
calf
et a!.,
a result
luteal
the
ovulaby the
have
as
calves,
progesterone/g
result from a few being induced
with the number P 0.05).
122
SPILMAN
not
cause
progesterone
absence
of luteal
Plasma Since
20/3-OH-P of
with
may
superovulated
clear
that
was
much peak
higher.
The to
(Hafs
(Fig.
3).
a
same as of pro-
data
indicate was
it was
reported
6-18%
and
It
et a!.,
in 20-OH-P have
The lation
Levels
from
that
of
progesterone
levels Woody
been
shown
that
after
plasma
tissue
heifers
1968;
1968)
recattle.
and in
there levels
of pro-
Discussion
level of indicates
pituitary
LH
that
after a rate
is
superovuof release to
similar
1.
130
E 110 C
A.
-Progesterone
I
20f1.OH.P
0 0
03
z Lii
z 0 :. U., I‘I,
\
.:
U.,
0 0 A.
‘C
‘C A.
..
1-2
3-5
10-11
5-7
DAYS Fic. OH-P) The
3.
Relationship
in blood standard
between plasma
errors
plasma
of eight
35.0, 23.7, and 9.4. Those 9.2, 12.1, 0.1, and 3.2.
for 20p-OH-P
8
were
after
20
22
OVULATORY
progesterone
calves
for progesterone
14-15
AFTER
and inducing
were
24
26
28
LH 20p-hydroxypregn-4-en-3-one
superovulation
0.1, 1.0, 0, 16.2,
the
is a sharp
fallen.
plasma of calves the
been
luteal
postpuberal
has
General
being
not
in
Armstrong, 1968).
gesterone
have
or postpuberal
20/3-OH-P
about
rise
It
the value
20/3-OH-P.
20/3-OH-P prepuberal
in
Ginther, rat (Wiest
followed
apparently
reduced
were
cattle,
progesterone
also
the
progesterone
continually
of
very
but
major
in
compared
calves
pattern
plasma
first
it were
20/3-OH-P
progesterone,
gesterone
the
concentrations
eight
of
however, levels of
20/3-OH-P
be of
is
that
and
progesterone
in
curvilinear
the
tissue.
concentrations plasma
in
AL.
ported
Progesterone
metabolite plasma
production
ET
4.4, 47.6,
3.2, 3.1, 3.2, 10.8,
(20p-
with
PMSG
and
56.5,
31.0,
20.0,
0.9, 21.9,
17.0,
47.3,
LH. 39.1, 7.7,
that
PROGESTERONE
in
normally
the 1)
calf was
cycling
AND
mature
pituitary content not replenished
LH
cattle.
of after
IN
Since
LH (Table superovula-
SUPEROVULATED
ovulations. Consequently, no “cycles” apparently were induced in these calves. It would be of interest to determine whether
tion, a reduced rate of LH synthesis is indicated. This is evidenced by the fact that the plasma levels of LH were very similar
the
before
waska
superovulation the
whereas
significantly superovulation. feedback of tion
of
Either progesterone
LH
in
some duction
higher would
itary
LH.
level, release
and
on
pituitary content higher before than
the
a
Days
5-25,
of LH it was
was after
direct on the
anterior
pituitary
In
spite
of
this
LH low
low
calves
32
or
33
days
after
on
for into
original
A plasma
LH peak from six each
by a terone.
sharp These
ovulation
calf,
was calves the
measured on Days
LH
decrease changes
precocious However,
unable
to
cycling if so, the
respond
since
The high plasma these superovulated
tissue,
culating the high
plasma
precipitously
mature
heifers,
terfered
with
gression. lay, three
PMSG
kindly
was
(Lukasze-
LH-B5
and
tion, C.
Inc.,
by
We E.
LH
concerning
tance
Mrs.
Cole,
Mr.
Trout NIH-
Study
grateful the
Sec-
to
LH
Dr.
antiserum
Jr. for supplying
Dr.
puri-
Hansel for suggestions The technical assisMiss J. Wiebold, Mr.
W.
manuscript.
Pierro,
R.
Lindsay
Dr. the
Endocrinology
Reichert,
L.
Dr. by
and
supplying
and
the
of
PLH
especially
for
L.
to Dr.
the
are
Niswender
fied bovine
by
the
Laboratories,
B7
NIH. D.
supplied
Laboratories,
and
Stehn,
P.
Mr.
Ball
is
typical cyclical of progesterone a subsequent
might
no
pituitary seemed
of calves
progesterone primarily not of
normal
progesterone and heifers production by the mass
elevated
levels
required plasma
of
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J. Gun.
ARMSTRONG,
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