Adenosine Triphosphatase in the Uterus and Duodenum of Chicken Hens During Eggshell Formation

Abstract

Plasma calcium concentration and uterine and duodenal adenosine triphosphatase (ATPase) activities were determined during shell formation for high (H) and low (L) shell strength lines of hens selected from the last of four consecutive generations. The H and L lines were divided into three groups according to shell formation at 0, 15, and 22 h following oviposition. Plasma total calcium was determined from blood samples collected from the common carotid artery. Activity of ATPase was determined in uterine and duodenal mucosa.
Shell strength, shell weight, percentage of shell per egg and shell thickness of the H line hens significantly exceeded those of the L line. During shell formation, no significant fluctuation in plasma calcium levels was observed within a line, but overall mean plasma calcium concentrations were higher in the H line than L line. Uterine ATPase activity increased with time after oviposition in both lines, with that of the H line being greater. Duodenal ATPase activity of H line hens remained fairly constant throughout the period, but this value showed fluctuations in the L line hens. It thus appears that laying hens with high and low shell strength may vary in their ability to use calcium for shell formation.

Full text

Adenosine Triphosphatase in the Uterus and Duodenum of
Chicken Hens During Eggshell Formation
EMIKO WATANABE, SHIOEKI KOBAYASHI, YOSHIAKI TERASHIMA, and HIROSHI ITOH
Faculty of Animal Science, Kitasato University, Towada-shi, Aomori 034, Japan
(Received for publication June 30, 1987)
ABSTRACT Plasma calcium concentration and uterine and duodenal adenosine triphosphatase (ATPase)
activities were determined during shell formation for high (H) and low (L) shell strength lines of hens
selected from the last of four consecutive generations. The H and L lines were divided into three groups
according to shell formation at 0, IS, and 22 h following oviposition. Plasma total calcium was
determined from blood samples collected from the common carotid artery. Activity of ATPase was
determined in uterine and duodenal mucosa.
Shell strength, shell weight, percentage of shell per egg and shell thickness of the H line hens
significantly exceeded those of the L line. During shell formation, no significant fluctuation in plasma
calcium levels was observed within a line, but overall mean plasma calcium concentrations were higher in
the H line than L line. Uterine ATPase activity increased with time after oviposition in both lines, with
that of the H line being greater. Duodenal ATPase activity of H line hens remained fairly constant
throughout the period, but this value showed fluctuations in the L line hens. It thus appears that laying
hens with high and low shell strength may vary in their ability to use calcium for shell formation.
(.Key words: adenosine triphosphate, uterine Ca2+-adenosine triphosphatase, duodenal Ca2+-adenosine
triphosphatase, plasma calcium, eggshell)
1989 Poultry Science 68:564-568
INTRODUCTION
During shell formation large quantities of
calcium are secreted into the avian uterus to be
deposited as calcium carbonate on the egg
shell (Mueller, 1976). Calcium secretion in the
shell gland at shell calcification occurs mainly
by active transport (Ehrenspeck et al., 1967;
Eastin and Spanziani, 1978). The data of
recent reports indicated Ca2+-dependent or
activated adenosine triphosphatase (Ca2+-
ATPase) may be involved in active transport
of calcium across uterine tissue and duodenal
mucosa (Pike and Alvarado, 1975; Coty and
McConkey, 1982; Grunder and Tsang, 1984).
Pike and Alvarado (1975) showed that uterine
Ca2+-ATPase activity at shell formation is
higher than with no shell calcification. From
this,
it is reasonable to consider that activity
may differ according to the stage of shell
formation.
Various studies with high and low shell
strength lines showed that the low lines
produced thin and light egg shells compared
with the high lines (Hamilton et al., 1981;
Saito et al., 1985). Therefore differences may
exist between the lines with respect to calcium
movement across the uterine wall during shell
formation. The present experiment was carried
out to determine variations in uterine and
duodenal Ca2+-activated ATPase (Ca2+-
ATPase) activities and plasma calcium concen-
tration during shell formation in laying hens
selected for either high or low egg shell
strength.
MATERIALS AND METHODS
Birds. This study used two lines of Single
Comb White Leghorn laying hens at 180 days
of age selected on the basis of high (H) and
low (L) shell strength from a fourth generation
produced by half-sib mating in Shaver strain
birds at Aomori Poultry Experimental Station
in Japan (Saito et al., 1985). Birds were
housed individually in laying cages (18 x 35
cm),
fed a commercial type layer diet (17%
CP,
2,750 kcal ME/kg, 3.0% calcium, .63%
total phosphorus) and had free access to feed
and water. They were exposed to a 16-h
photoperiod from 0400 to 2000 h throughout
the experiment.
Determination of Shell Quality. One week
before birds were sacrificed, determination was
made of egg production of 12 birds/line and
shell strength and shell plus membrane weight.
Shell strength values were expressed as maxi-
mum compression force to fracture (Hamilton
564

UTERINE AND DUODENAL ADENOSINE TRIPHOSPHATASE 565
et al., 1979). The egg contents were removed,
and the shells with membranes attached were
washed with tap water, dried at 60 C
overnight, and weighed.
Blood,
Uterine, and Duodenal Samples. At
about 210 days of age, hens were observed
every 15 min in the morning between 0600 to
0900 h to determine time of oviposition. For
this purpose, 12 hens from each line were
used. Hens were selected that laid eggs on the
day of observation and the day before so as to
take into consideration the effect of production
status on plasma calcium level (Miller et al.,
1978).
Hens of both lines were divided into
three groups of four birds from each of which
samples were obtained at 8, 15, or 22 h
postoviposition. The presence of an egg in the
uterus was confirmed by palpation before
sampling. Birds were lightly anesthetized by
intravenous administration of pentobarbital
sodium in the amount of 35 to 40 mg/kg BW.
About 2 mL of blood were collected from the
common carotid artery. Blood plasma was
separated by centrifugation at 1,500 x g for 15
min. Plasma total calcium concentration was
measured by atomic spectrophotometry.
Enzyme Preparation. For enzyme prepara-
tions,
the uterus and upper duodenum were cut
open to expose the mucosa, which was rinsed
with homogenizing medium (250-mAf sucrose
and 1-mAf Na2EDTA) and then removed by
scraping with a microscope slide. All solutions
used in this procedure were maintained at 0 to
5 C. Each mucosa sample was placed in 4 vol
of homogenizing medium. Following homoge-
nization of the sample for 2 min with a teflon
pestle, the solution was centrifuged at 10,000 x
g for 1 h at 4 C to obtain the supernatant
fraction as the enzyme preparation for assay.
Determination of Ca2+-ATPase Activity.
Freshly prepared enzyme was used, as storage
for 24 h at 4 or -20 C has been found to lessen
activity by 10 to 30% (Pike and Alvarado,
1975).
The ATPase activity was determined
from inorganic phosphate (P;) released from
the added ATP by ATPase per unit weight of
supernatant protein in 30 min (Pike and
Alvarado, 1975). The final volume of the assay
mixture was 2.0 mL and consisted of 1.0 mL
of standard medium [100-mAf tris-(hydroxy-
methyl)-aminomethane (Tris)-Tris HC1, pH
7.4, 39 C, 10-mAf CaCl2, and 200- mM NaCl],
.2 mL of enzyme preparation, and .8 mL of
substrate solution (12.5-mM Na2ATP and 50-
mM Tris-Tris HC1, pH 7.4, 39 C). Before
adding the substrate solution, the assay mix-
ture was incubated in a water bath for 5 min at
39 C. The substrate solution was added, and
the sample was incubated for exactly 30 min.
The reaction was terminated by the addition of
1.0 mL of 10% trichloroacetic acid. A control
mixture was prepared without enzyme prepara-
tion which, after incubation, was centrifuged at
1,500 x g for 10 min at room temperature. The
supernatant was assayed for P; by the p-
methylaminophenol method and for protein by
the Lowry method (Lowry et al., 1951).
Finally, ATPase activity for each hen uterus
was expressed as micromoles P; per milligram
protein per 30 minutes, after correcting for P;
in the control mixture. For determination of
optimum condition in enzyme assay, effects of
substrate, Ca2+, and oubain on Ca2+- ATPase
activity in enzyme preparations were also
checked.
Statistical Analysis. Student's t test was
used for egg and shell traits to compare line
effects. Analysis of variance was carried out
according to a one-way design for determina-
tion of the optimum concentrations of Na2ATP
and Ca2+ and Ca2+-ATPase activities in the
assay mixture, and a two-way design for
plasma calcium and ATPase activities to test
line and time effects. The least significant
difference procedure was also used to test for
significant differences of time effects within
line on plasma calcium level and ATPase
activities.
RESULTS AND DISCUSSION
Egg Shell Quality. Shell strength, shell plus
membrane weight, percentage of shell (weight
of shell plus membrane/total egg weight x
100) and shell thickness of H line hens
significantly exceeded those of the L line
(Table 1). Egg weights were significantly
smaller for the H line than for the L line. The
H line hens deposited more shell per egg, thus
accumulating more calcium per egg than the L
line hens. These data indicate that the two lines
possibly differ in their ability to mobilize
calcium for eggshell formation.
Plasma Calcium Concentration. The H line
hens had significantly higher plasma calcium
than those from the L line (P<.05, Table 2).
Plasma calcium levels did not differ with
respect to time after oviposition.
Plasma calcium level is affected by shell
formation (Miller et al., 1978), bone metabo-

566 WATANABE ET AL.
TABLE 1. Shell strength, egg weight, shell plus membrane weight, and shell thickness of high (H) and low (L)
shell strength lines (x ± SEM)
Line
H
L
n
12
12
Shell
strength
(kg/cm2)
4.0 ± .1
2.3 ±
.1**
Egg Shell + membrane
weight weight
53.9 ± .9 5.6 ± .1 10.4 ± .2
57.2 ± 1.1* 4.9 ±
.1**
8.7 ± .2**
Shell
thickness
(.01 mm)
36.8 ± .8
32.4 ± .4**
*P<.05.
**P<01.
lism (Ishibashi et ai, 1986), and dietary
calcium intake (Paul and Snetsinger, 1969) and
is regulated by hormonal control (Garlich and
Bryant, 1975). Wideman and Buss (1985)
showed that genetic selection for thick or thin
egg shell production resulted in higher plasma
calcium level in thick shell-producing hens
than in thin shell-producing hens; this is
confirmed by the results of the present study.
Hodges (1969) demonstrated that plasma
calcium level fell at 12 to 18 h postoviposition
and began to recover at 19 h for the next
oviposition. Parsons and Comb (1981) sug-
gested that plasma ionized calcium levels in
laying hens more clearly followed the stage of
egg shell formation than plasma total calcium
level, although the pattern of total calcium
level in their report was similar to that reported
by Hodges (1969). However, a similar pattern
in arterial plasma calcium with respect to stage
of shell formation was not seen in the present
experiment.
Effects of Substrate, Ca2*, and Oubain on
Ca2*-Adenosine Triphosphatase Activity in
Enzyme Preparations. As shown in Tables 3
and 4, optimum concentrations of Na2ATP
and Ca2+ for Ca2+-ATPase activity in the assay
mixture should be above 5 mAf and 5 mAf,
respectively. This finding is consistent with the
data of Pike and Alvarado (1975). The addition
of 1 mAf oubain to the assay mixture caused
slight inhibition of the enzyme activity (about
2%),
indicating Na+-K+-activated ATPase to be
present in the enzyme preparations from
organs not purified.
Uterine Ca2*-Adenosine Triphosphatase
Activity. The changes in uterine Ca2+-ATPase
activity during shell formation are shown in
Table 2. There were significant effects for time
after oviposition (P<.05) and line effect (P<
TABLE 2. Arterial plasma calcium concentration and uterine and duodenal Ca2*- adenosine triphosphatase
(ATPase) activity at 8, 15, and 22 h after oviposition (x ± SEM)
Line1
Time after
oviposition
Plasma Ca
concentration
Ca2+-ATPase activity
Uterus Duodenum
(h)
8
15
22
x
8
15
22
x
(mg/dL)
23.0 ± .5
22.0 ± 2.4
24.0 ±
23.1 ±
1.2
.7
23.3 ± 2.4
18.8 ±
19.4 ±
20.5 ±
.5
1.1
1.0*
.88 ± .17b
1.35 ± .23b
2.33 ± .16*
1.49 ± .21
.59 ± .11
.95 ± .18
1.27 ± .15*
.94 ± .12**
.67 ± .11
.71 ± .15
.75 ± .15
.71 ± .07
.39 ± .03"
.81 ± .07'
.74 ± .04'
.65 ± .07
'••"Within high and low egg shell strength lines, mean values in the same column with different superscripts differ
significantly (P<.05).
'H = High shell strength; L = low shell strength.
•Difference between lines was significant (P<.05).
••Difference between lines was highly significant (P<.01).

UTERINE AND DUODENAL ADENOSINE TRIPHOSPHATASE 567
TABLE 3. Effect of Na^ATP concentration on uterine
and duodenal Ca2*-ATPase activity1
Relative enzyme
Concentration ^1
of Na;ATP Uterus Duodenum
(mAf) — (% of 5-mAf NajATP value) —
0 .8e 4.0°
.5 14.6" 49.0bc
1
28.3C 67.3,b
3 76.8" 99.6,b
5 100* 100''
"Values in columns with no common superscripts differ
significantly (P<.05).
'NajATP
=
Sodium adenotriphosphate. Values represent
percentage of activity obtained with 5-mM NajATP. Mean
ATPase activity values (± SE) of uterine and duodenal
enzyme preparation in the presence of 5- mAf Ca2* were 1.
57 ± .02 and .75 ± .08 junolPj/mg protein/30 min, respec-
tively. Each preparation was run in duplicate.
.01) for uterine Ca2+-ATPase activity. There
were no significant differences in the interac-
tion of time after oviposition x line. From
these results, it follows that uterine Ca2+-
ATPase activity increases as shell formation
proceeds, and the activity is higher in H line
than L line birds.
Pike and Alvarado (1975) found the Ca2+-
ATPase activity in Japanese quail uterus to be
higher than in other tissues such as the heart,
skeletal muscle, intestine, kidney, and liver.
Enzyme-cytochemical studies on quail uterus
show higher Ca2+-ATPase activity in microvil-
lus tubular gland cells undergoing calcification
than in those cells prior to this process
(Yamamoto, 1984). Uterine Ca2+-ATPase thus
appears essential for active calcium transport
across uterine tissue during shell formation.
Following ovulation, an egg reaches the uterus
in approximately 4 h and during the next 3 h,
calcification occurs slowly; thereafter, the
calcification rate becomes more rapid but is
not constant (Talbot and Tyler, 1974; Farmer
et al., 1986). Uterine Ca2+-ATPase activity
was higher in H than L line hens at 22 h
postoviposition, indicating more active trans-
port of calcium. The difference between both
lines was greater at 22 h than at 8 h
postoviposition, suggesting more active trans-
port of calcium as shell formation proceeds.
This hypothesis does not agree with data from
previous work dealing with rate of shell
calcification (Talbot and Tyler, 1974; Farmer
TABLE 4. Effect of Co2* concentration on uterine and
duodenal C^-ATPase activity1
Relative enzyme
Concentration £££2
of Ca2* Uterus Duodenum
(mAf) (% of 5-mAf Ca2* value)
0 0d 26.0°
.5 35.3<=
72.8b
1 SS^ 64.0b
3 90.9b 73.6"
5 100" 100"
10
101.3'
98JS*
*_llValues in columns with different superscripts differ
significantly (P<.05).
'ATP = Adenosine triphosphate. Values represent per-
centage of activity obtained with 5-mAf
Ca2*.
Mean ATPase
activity values (± SE) of uterine and duodenal enzyme
preparations in the presence of 5 mAf NajATP, were 1.
27 ± .09 and .50 ± .12 ixmolP/mg protein/30
min,
respec-
tively. Each preparation was run in duplicate.
et al., 1986). Thus, differences between H and
L lines in shell-strength and the amount of
calcium deposited on a shell may be related in
some way to differences in the activity of
uterine Ca2+-ATPase.
Duodenal Ca2*Adenosine Triphosphatase
Activity. Changes in duodenal Ca2+-ATPase
activity during shell formation are shown in
Table 2. Duodenal Ca2+-ATPase activity in H
line hens was fairly constant during shell
formation. In L line hens, it was significantly
lower at 8 h and increased (P<.05) by 15 h
after oviposition. But at any of the three times,
the differences between the lines in levels of
activity were not significant. Calcium absorp-
tion in the small intestine occurs mainly by
active transport. Higher calcium absorption at
the level of the intestines has been observed
during shell formation than before this process
(Bar et al, 1976). The Ca2+-ATPase influences
absorption of calcium in the chick intestine
(Haussler et al., 1970). These reports indicate
duodenal Ca2+-ATPase activity may possibly
affect calcium absorption in the small intestine
during shell formation. In the present study, no
significant differences between measures of
duodenal Ca2+-ATPase activity of the two lines
were noted. Thus, both lines may possess the
same ability to absorb calcium from the
duodenum during shell formation. At present,
the low value of duodenal Ca2+-ATPase at 8 h
in L line hens cannot be explained, but may

568 WATANABE £T AL.
have some effect on calcium absorption in the
duodenum. The present data indicate that
uterine Ca2+-ATPase activity and arterial
plasma calcium concentration may be related
to the ability of hens to deposit calcium on egg
shells.
REFERENCES
Bar, A., D. Dubrov, U. Eisner, and S. Hurwitz, 1976.
Calcium- binding protein and calcium absorption in
the laying quail (Coturnix coturnix japonica). Poultry
Sci.
55:622-628.
Coty, W. A., and C. L. McConkey, Jr., 1982. A high-affinity
calcium-stimulated ATPase activity in the hen oviduct
shell gland. Arch. Biochem. Biophys. 219:443-453.
Eastin, Jr., W. C, and
E.
Spanziani, 1978. On the mechanism
of calcium secretion in the avian shell gland (uterus).
Biol. Reprod. 19:505-508.
Ehrenspeck, G., H. Schraer, and R. Schraer, 1967. Some
metabolic aspects of calcium movement across the
isolated avian shell gland. Proc. Soc. Exp. Biol. Med.
126:392-395.
Farmer, M., D. A. Roland, Sr., and A. J. Clark, 1986.
Influence of dietary calcium on bone calcium utiliza-
tion. Poultry Sci. 65:337-344.
Garlich, J. D., and D. M. Bryant, 1975. Relationships
between dietary and plasma concentrations of calcium
and phosphorus in intact and ultimobranchialecto-
mized chickens. Poultry Sci. 54:388-395.
Grunder, A. A., and C.P.W. Tsang, 1984. Effects of vitamin
D3
deficiency on adenosine triphosphatase activity of
jejunums from White Leghorn hens. Poultry Sci.
63:1073-1075.
Hamilton, R.M.G., A. A. Grunder, B. K. Thompson, and K.
G. Hollands, 1981. Relationship between blood ion-
ized calcium levels and shell strength of eggs laid by
White Leghorn hens. Poultry Sci. 60:2380-2384.
Hamilton, R.M.G., B. K. Thompson, and P. W. Voisey,
1979.
The effect of age and strain on the relationships
between destructive and non-destructive measure-
ments of eggshell strength for White Leghorn hens.
Poultry Sci. 58:1125-1132.
Haussler, M. R., L. A. Nagode, and H. Rasmussen, 1970.
Induction of intestinal brush border alkaline phospha-
tase by vitamin D and identity with Ca-ATPase. Nature
228:1199-1201.
Hodges, R. D.,
1969.
pH and mineral ion levels in the blood
of the laying hen (Gallus domesticus) in relation to egg
shell formation. Comp. Biochem. Physiol.
28:1243-1257.
Ishibashi, T., T. Sunahara, and M. Yamazaki, 1986. Time
course of distribution of single dose oral or intravenous
calcium-45 in laying hens. Jpn. Poult. Sci. 23:75-82.
Lowry,
O.
H.,
N.
J. Rosebrough,
A.
L. Fair, and
R.
J. Randal,
1951.
Protein measurement with the Follin phenol
reagent. J. Biol. Chem. 193:265-275.
Miller, E. R., H. R. Wilson, and R. H. Harms, 1978. The
relationship of production status to serum calcium and
phosphorus in hens. Poultry Sci. 57:242-245.
Mueller, W. J., 1976. Reproduction in the female and egg
production. Egg shell and skeletal metabolism. Pages
312-323 in: Avian Physiology. P. D. Sturkie, ed. 3rd
ed. Springer-Verlag New York Inc., New York, NY.
Parsons, A. H., and G. F. Combs, Jr., 1981. Blood ionized
calcium cycles in the chicken. Poultry Sci.
60:1520-1524.
Paul, H. S., and D. C. Snetsinger, 1969. Dietary calcium and
variations in plasma alkaline phosphatase activity in
relationships to physical characteristic of egg shells.
Poultry Sci. 48:241-250.
Pike,
J. W., and R. H. Alvarado, 1975. Ca2+-Mg2+-activated
ATPase in the shell gland of Japanese quail (Coturnix
coturnix japonica). Comp. Biochem. Physiol.
51B:119-125.
Saito,
K., H. Ohkubo, S. Yoshida, and T. Ogawa, 1985.
Two-way selection for egg shell breaking strength.
Bull. Aomori Prefectural Poult. Exp. Stat. 21:12-
21.
Talbot, C. J., and C. Tyler, 1974. A study of the progressive
deposition of shell in the shell gland of the domestic
hens.
Br. Poult. Sci. 15:217-224.
Wideman, R. F., and E. G. Buss, 1985. Percent shell and
plasma mineral concentrations in three strains of do-
mestic fowl selected for thick or thin egg shell produc-
tion. Poultry Sci. 64:388-395.
Yamamoto, T., 1984. Ultrastructural and enzyme-cyto-
chcmical studies on the shell gland in egg-laying
Japanese quail. With special reference to calcium
secreting cells. Niigata. Dent. J. 14:13-35.