Absorption of Calcium and Phosphorus Along the

Gastrointestinal Tract of the Laying Fowl as

Influenced by Dietary Calcium and
Egg Shell Formation '
S. HURWITZ AND A. BAR
The Volcani Institute of Agricultural Research, The National and
University Institute of Agriculture, Rehovot, Israel

ABSTRACT The apparent absorption of calcium, phosphorus and total dry matter
along the gastrointestinal tract of the laying hen, was followed using yttrium-91 as a
non-absorbed tracer. Percentage calcium and phosphorus absorption appeared to be
greater in the proximal parts of the intestine than in the distal parts. This differ
ence was smaller for total dry matter. Percentage calcium absorption was not signifi
cantly influenced by its dietary level, nor did the latter influence the absorption of dry
matter. Percentage absorption of phosphorus was, however, depressed by the higher
dietary calcium level. Egg shell deposition was associated with increased calcium
and to a smaller degree, phosphorus absorption. It did not influence dry matter ab
sorption. A heavy endogenous phosphorus excretion was observed in the duodenum.

The identification of the sites of mineral available carbohydrate absorption along

absorption is important for understanding
the mechanism involved in the mineral
absorption. This problem was studied
mainly by in vitro and in situ experimenta
tion. The in vitro studies such as those of
Schachter and associates (1, 2) provide
information on the capacity of various
intestinal segments to absorb calcium.
However, the rate and percentage of cal
cium absorption may also depend on the
rate of passage of food, which is quite
variable in the various intestinal segments
(3, 4). Therefore, in vitro methods can
not measure the relative contribution of
each segment to total calcium absorption
in vivo. A different method for studying
calcium absorption is the use of oral doses
of 45Caor its injection into different parts
of the intestine (5, 6). This method, how
ever, measures mainly the outflux of cal
cium from the intestine and not the net
absorption which is the difference between
the outflux and the influx.
Chandler and Cragle (7) used I44Ceas
a non-absorbed tracer to study calcium and
phosphorus absorption in calves. The ratio
of calcium and phosphorus to 144Ceat any
point in the intestine measures the cumu
lative percentage absorption up to this
point. Similarly, Bolton (8) used cellulose
as a tracer for estimation of protein and
the tract of chickens. Marcus and Lenge-
mann (3) reported that 81Ywas not ab
sorbed in the rat.
Preliminary trials in this laboratory
showed that this isotope was not absorbed
by colostomized chickens. Following an
oral dose of 91Ymixed in some feed, 93%
of the dose was detected in the feces voided
during the first 24-hour period, and 3% in
the following 24-hour period. No activity
could be detected in the urine of these
animals.
Therefore, 91Ywas used in the present
trial to follow the absorption of calcium
and phosphorus along the intestinal tract
of laying hens. The possibility that dietary
calcium and the presence of a calcifying
shell in the uterus may modify the pattern
of calcium and phosphorus absorption was
also investigated.
EXPERIMENTAL
The experimental diets are shown in
table 1. These diets were all-vegetable, of
the practical type, and were prepared as
follows: the calcium carbonate and the
dicalcium phosphate portions of each ra-
Received for publication March 3, 1965.
i Contribution from the National and University
Institute of Agriculture, Rehovot, Israel, 1965, Series
no. 840-E. Supported by a grant-in-aid from the U.S.
Department of Agriculture, project no. A10-AH-20.

J. NUTRITION, 86: '65 433

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 434 S. HURWITZ AND A. BAR

TABLE 1 oviposition. The time of killing ranged

Composition of the experimental diets between 7 and 11:30 A.M. Since the egg
Diet 1 Diet 2 is laid shortly after the end of shell calci
fication, results obtained with this group
Yellow
cornSoybean represent absorption during the period of
protein)Soybean
oil meal (45% late calcification, (c) Five birds of each
refinedVitamin
oil, lot were killed between 9 and 11 A.M.
'Mineral mixture
*DL-MethionineAlfalfa
mixture Those birds had not laid any eggs during
the morning, but uncalcified egg was found
dehydratedCalcium
meal, either in the magnum or in the isthmus
carbonateDicalcium at the time of killing. Results from this
phosphateMiloCalcium
group indicate the absorption of calcium
and phosphorus when no shell is formed.
%Phosphorus
content, assayed, The gastrointestinal tract of each hen
content, assayed, %30.0024.004.000.250.300.102.003.501.7034.151.900.6330.0024.004.000.250.300.102.007.501.7030.153.560.63
(excluding the esophagus and crop) was
1Commercial premix, supplied per kg of diet: vita removed and separated into gizzard, duo
min A, 10,000 IU; vitamin D3, 1400 ICU; riboflavin,
3 mg; Ca pantothenate, 3 mg; niacin, 10 mg; choline
chloride, 400 mg; vitamin Biz, 8 /ig; butylated hydroxy-
denum, upper jejunum, lower jejunum,
toluene, 125 mg. upper ileum, lower ileum and colon. The
2Feed grade salt supplied in mg per kg of diet : cecums were not taken for analysis.
Mn, 60; zinc, 50; iodine, 1.2; cobalt, 0.2; iron, 25; Meckel's diverticulum was arbitrarily taken
copper, 2.
as the point of demarcation between the
tion were mixed well. A solution contain
ing 91Ywas added to the mixture. After jejunum and ileum, which were each di
vided into 2 parts of equal length. The
drying in an oven, the latter was passed
3 times through a thin-mesh wire screen. into contents of each segment were emptied
a crucible, oven-dried and ashed at
Count rates of samples were taken to as 700°. The ash was then dissolved in 2 N
sure proper mixing. The Ca-"'Y mixture
hydrochloric acid. For 9'Y assay, an aliquot
was then added to the other pre-mixed die
tary ingredients, all ground in a hammer was dried on a glass planchet and counted
in a gas-flow detector. Within the range of
mill. This was followed by thorough mix
ing. Concentration of "Y was approxi the weights of samples measured, there
mately 25 nc/kg and 55 uc/kg in diets 1 Calcium was no variation due to self absorption.
was determined by EDTA titra-
and 2, which contained 1.90% (low Ca)
and 3.56% (high Ca) calcium, respec tion as described previously (9) except
that murexide was replaced by hydroxy-
tively. naphthol blue.2 Phosphorus was deter
Forty White Leghorn laying hens, 16
months old, were selected for the experi mined by the method of Gomori (10).
ment on the basis of previous egg produc Results are expressed as ratios of cal
tion records. For a 1-week preliminary cium, phosphorus or dry matter to counts
per minute (cpm) X 10~4 of 91Yobserved
period they were fed a control diet identi
cal to diet 2, but containing no radioactive in the respective segment. This ratio de
supplement. At the end of this period they creases with progressive absorption of the
were divided into 2 lots receiving the re test nutrient. The cumulative percentage
spective labeled low calcium and high cal absorption in any segment is given by
( _ nutrient/9IY in intestine \
cium diets. After 3 days of feeding the ex 100 X ( 11 - •nutrient/slY in feed / '
perimental diets, the birds were killed by Because of the differences in the 91Ycon
dislocation of the neck in the following
order, (a) Four birds of each lot were killed tent of the 2 diets, the appropriate correc
at 8 P.M. after the presence of a shelled tions were made to bring the results to the
egg in their uteri had been established. At same scale.
this time calcification had already pro Analyses of variance of calcium, phos
phorus and dry matter to 91Yratios in each
ceeded for 3 to 5 hours. Results from this
group represent the absorption during the intestinal segment were run according to
period of early calcification, (b) Four birds Dixon and Massey (11). Pooled standard
of each lot were killed immediately after 2Mallinkrodt.

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 CALCIUM AND PHOSPHORUS ABSORPTION 435

errors (n = 4) were calculated from the

error term of the analysis of variance.
RESULTS
Since the trial was designed as a fac
torial of 2 calcium levels and 3 physiologi
cal states with respect to egg shell forma
tion, the results are grouped according to
the factorial variables.
With only one exception, there was no
significant effect of any of the experimen
tal variables on the dry-matter contents of
the various intestinal segments. These
were in milligrams as follows (average
±SE): gizzard, 3018 ±254; duodenum,
671 ±151; upper jejunum, 785 ±85;
lower jejunum, 1266 ±333; upper ileum,
1075 ±279; lower ileum, 845 ±169; and
colon, 268 ±74.
The calcium, phosphorus and dry matter
to 91Yratios in the various intestinal seg
ments are shown in figures 1, 2, and 3 re
spectively. The probability levels for the
effect of each factorial variable in any 20 40 60 80
Intestinal length, cm
intestinal segment are shown in table 2.
Following an increase from the gizzard to Fig. 2 P/91Yratio in gastrointestinal segments
the duodenum, there was a marked decline of laying hens fed 91Y-labeleddiets. Upper dia
gram, comparison between 1.90% calcium (•)
and 3.56% calcium diets (O). Lower diagram,
15 comparison among periods of early egg shell
calcification (A), late shell calcification (A)
10
and of no shell formation (G)- Standard errors
e
are as follows: gizzard, 0.40; duodenum, 6.14;
upper jejunum, 2.67; lower jejunum, 1.03; upper
6 - ileum, 1.27; lower ileum, 0.41; colon, 0.32.
6* in the Ca/"Y ratio down to the lower
&
jejunum with a smaller change in the
I
following segments.
The average cumulative percentage ab
sorption of calcium was 50.3% at the lower
jejunum and 53.2% at the colon. The
Ca/91Y ratio in the duodenum was lower
than that of feed, suggesting considerable
absorption of calcium in this segment. In
the high calcium hens with no shell pres
ent, the upper ileum showed an increase
O 20 40 60 80
Intestinal length, cm
100 120 in the ratio, indicating net endogenous ex
cretion of calcium. There was no signifi
Fig. 1 Ca/"Y ratio in gastrointestinal seg cant effect of dietary calcium level on the
ments of laying hens fed 91Y-labeleddiets. Upper percentage absorption of this mineral.
diagram, comparison between 1.90% calcium (•) Hence, the total amount of calcium ab
and 3.56% calcium (O) diets. Lower diagram,
comparison among periods of early egg shell cal sorbed was higher in the high calcium
cification (A), late shell calcification (A) and birds. Percentage calcium absorption was
with no shell formation (D)- Standard errors significantly greater during the 2 periods
were as follows: gizzard, 0.90; duodenum, 1.24; of shell calcification than when no forming
upper jejunum, 0.85; lower jejunum, 1.09; upper
ileum, 1.16; lower ileum, 0.81; colon, 1.09. shell was present. However, there was no

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 436 S. HURWITZ AND A. BAR

significant difference in calcium absorp

20 40 60 ao
Intestinol length, cm
Fig. 3 Total dry matter/91Y ratio in gastro
intestinal segments of laying hens fed "Y-labeled
diets. Upper diagram, comparison between 1.90%
(•) and 3.56% (O) calcium diets. Lower dia
gram, comparison among periods of early egg
shell calcification (A), late shell calcification
(A), and of no shell formation (D). Standard
errors were as follows: gizzard, 479; duodenum,
370; upper jejunum, 278; lower jejunum, 84;
upper ileum, 62; lower ileum, 26; colon, 18.
tion between periods of early and late
calcification. The former difference was
observed along the entire intestine but did
not reach significance in the duodenum.
The calculated cumulative percentage of
calcium absorption in the colon was 67
and 39%, during periods of calcification
and no calcification, respectively.
Changes in the P/91Y ratios (fig. 2) were
similar to those observed for calcium, indi
cating that the main sites of phosphorus
absorption are at the anterior intestine,
i.e., jejunum. The P/91Y ratio in the duo
denum is much higher than that of feed,
suggesting a large endogenous phosphorus
excretion in this segment. Unlike calcium,
the relative absorption of phosphorus was
influenced by dietary calcium level; the
high calcium level increased the P/"Y
ratio in the 3 lower portions of the intes
tine. During the 2 periods of egg shell
calcification, there was a significant in
crease in phosphorus absorption in the
lower portions of the intestine, although
the same tendency was observed in the
upper segments as well. There was also a
ico significant interaction between dietary cal
cium and shell calcification on phosphorus
cumulative absorption in the colon.
Dry matter/91Y ratios in the various in
testinal segments are shown in figure 3.
The ratio was higher in the gizzard and
duodenum than in feed. It then decreased
at a progressively lower rate along the
rest of the intestine. The calculated cumu
lative dry-matter absorption in the colon

TABLE 2
Levels of probability for the effects of the several factorial components for the various feed
ingredients and intestinal segments 1

Factorial variation
calciumCa/YnsnsnsnsnsnsnsP/Ynsnsnsns0.01ns0.01Dm/Y
variableParameterGizzardDuodenumUpper formation)Ca/Ynsns0.010.010.010.010.01P/Yn
(shell
2nsnsnsns0.05nsnsDiurnal
snsnsns0.010.050.01Dm/Y0.01nsnsns0.05ns0.01InteractionCa/Y0.01nsnsnsnsnsnsP

jejunumLower
jejunumUpper
ileumLower
ileumColonDietary

1Probability levels calculated on the basis of 21 analyses of variance, each for one feed ingredient and
intestinal segment.
2Dm = dry matter.

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 CALCIUM AND
was about 73% , on the average. Although
there is some tendency for dry matter to be
better absorbed by the low calcium birds,
this effect is only significant in one seg
ment. There was also some, and at times
significant, diurnal variation in dry mat-
ter/9'Y ratio. However, this variation ap
pears small and inconsistent.
DISCUSSION
On the basis of the known functions of
the gizzard it appears unlikely that large
amounts of calcium and phosphorus could
be absorbed in this organ, as appears to be
suggested by the lower Ca and P/91Y ratios
compared with those of the feed. It also
appears unlikely that very large quantities
of dry matter are secreted in this organ
as suggested by the very high dry mat-
ter/91Y ratio. The examination of the giz
zard contents revealed the presence of
relatively large feed particles, mostly
grains, with few smaller ones. Apparently,
therefore, the gizzard retains the larger
feed particles longer than the smaller
ones and the solutes. Calcium and phos
phorus which are mainly in solution in
the gizzard (12) might be emptied more
rapidly than the larger grain particles. On
the other hand, 91Ytends to be partially
adsorbed on solid particles (3) even at a
low pH such as that in the gizzard. This
isotope will therefore be slower than cal
cium and phosphorus to leave the gizzard,
but more rapid than the solids. This inter
pretation is also supported by a prelimi
nary study in this laboratory with a single
dose of 45Ca and 91Yand may explain the
low Ca and P/"Y ratios and the high dry
matter/91Y ratio in the gizzard.3 In the
steady state of feed passage from the giz
zard it may be assumed that the latter
passes the various feed components at
constant rate. This assumption is sup
ported by the dry-matter/91Y ratios which
show only slight variations that appear
to be more of diurnal nature rather than
related to shell deposition. There was
also no significant difference in the nutri-
ent/91Y ratios in the duodenum, suggesting
uniform emptying of yttrium, calcium,
phosphorus and total solids from the
gizzard.
In accordance with the observations of
Marcus and Lengemann (13), calcium,
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PHOSPHORUS ABSORPTION 437
phosphorus and yttrium are assumed to
move at an equal rate along the small
intestine. Preliminary results in this labo
ratory appear to support this assumption.4
Therefore, nutrient/91Y ratios appear to be
suitable for calculation of cumulative ab
sorption along the intestine. However, more
evidence is necessary to clarify this point.
Nevertheless, the comparison of the pat
terns of calcium and phosphorus absorp
tion as influenced by dietary and diurnal
variations, is apparently valid since it ap
pears unlikely that the movement of 91Y
could be influenced either by dietary cal
cium or egg shell deposition. In this re
spect it should be recalled that the late-
calcification hens and the no-calcification
hens were killed at the same time of the
day.
Those factors with respect to the rate
of passage of the test nutrient relative to
the tracer, demonstrate clearly the difficul
ties in the use of such tracer.
The pattern of dry matter/91Y indicates
absorption of dry matter along the entire
intestine with a somewhat reduced rate at
the posterior segments. The high dry-
matter/9'Y ratio in the duodenum is prob
ably due to secretion of digestive sub
stances in this organ. The pattern in
general is similar to that observed by
Bolton (8) in chicks, for the absorption of
protein and available carbohydrate.
Assuming the same passage time for
calcium, phosphorus and yttrium along the
intestine, the present results appear to in
dicate that the major portion of calcium
and phosphorus absorption is located at
the anterior parts of the intestine. It can
be easily shown that calcium concentration
in the total dry matter is actually increas
ing along the intestine and therefore could
not be a factor in the reduced absorption
in the posterior parts. The latter may be
due either to the greater capacity of the
anterior segments to absorb calcium (1,2,
5, 14, 15) or to the reduced solubility of
calcium due to increase in pH with the dis
tance from the gizzard (12, 16).
Dietary calcium, at the levels used, did
not influence the relative absorption of cal
cium, in agreement with previous balance
studies (9). The absence of a calcifying
3 Unpublished data, S. Hurwitz and A. Bar, 1965.
4 See footnote 3.
 438 S. HURWITZ
shell was associated with a reduced rate
of calcium absorption. This effect was
identical for the high calcium and low
calcium lots. The mechanism responsible
for this variation is not clear, although it
may be explained in terms of the greater
need for calcium during periods of egg
shell formation. It is accepted that absorp
tion alone cannot supply all the calcium
needed during egg shell formation and that
bone reserves have to be utilized (17),
even when birds are fed sufficient calcium.
This conclusion is based on daily averages
of calcium retention not considering any
diurnal variations in calcium absorption.
The greater calcium absorption during
shell deposition, observed in this study,
could mean that the importance of bone
as a calcium reserve in birds fed sufficient
calcium, is not as great as previously be
lieved.
The absorption of phosphorus appears
to be closely related to that of calcium, and
is probably influenced by the latter. The
depressing effect of high levels of dietary
calcium on phosphorus absorption is well
known (18). The significant increase in
absorption of phosphorus during shell for
mation is not as striking as with calcium
absorption, and appears to be secondary
to the latter.
From the P/91Y ratios in the duodenum,
it is apparent that large quantities of en
dogenous phosphorus are emptied into this
segment. It may be calculated that the
ratio of endogenous to dietary phosphorus
in this segment is at least 4:1. This ob
servation is consistent with the heavy 3SP
secretion into the duodenum, observed in
the laying hen by Shirley et al. (19).
ACKNOWLEDGMENT
The authors wish to acknowledge the
technical assistance of Mrs. M. Cotter.
LITERATURE CITED
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Active transport of Ca45 by the small intestine
and its dependence on vitamin D. Am. J.
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2. Kimberg, D. V., D. Schachter and H. Schenker
1961 Active transport of calcium by intes
tine: effects of dietary calcium. Am. J.
Physiol., 200: 1256.
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AND A. BAR
3. Marcus, C. S., and F. W. Lengemann 1962
Use of radioyttrium to study food movement
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4. Cramer, C. F., and D. H. Copp 1959 Prog
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16 Coates, M. E., and E. S. Holdsworth 1961
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