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

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
20 40 60 ao ico significant interaction between dietary cal
Intestinol length, cm
cium and shell calcification on phosphorus
Fig. 3 Total dry matter/91Y ratio in gastro cumulative absorption in the colon.
intestinal segments of laying hens fed "Y-labeled Dry matter/91Y ratios in the various in
diets. Upper diagram, comparison between 1.90%
(•) and 3.56% (O) calcium diets. Lower dia testinal segments are shown in figure 3.
gram, comparison among periods of early egg The ratio was higher in the gizzard and
shell calcification (A), late shell calcification duodenum than in feed. It then decreased
(A), and of no shell formation (D). Standard at a progressively lower rate along the
errors were as follows: gizzard, 479; duodenum,
370; upper jejunum, 278; lower jejunum, 84; rest of the intestine. The calculated cumu
upper ileum, 62; lower ileum, 26; colon, 18. 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 PHOSPHORUS ABSORPTION 437

was about 73% , on the average. Although phosphorus and yttrium are assumed to
there is some tendency for dry matter to be move at an equal rate along the small
better absorbed by the low calcium birds, intestine. Preliminary results in this labo
this effect is only significant in one seg ratory appear to support this assumption.4
ment. There was also some, and at times Therefore, nutrient/91Y ratios appear to be
significant, diurnal variation in dry mat- suitable for calculation of cumulative ab
ter/9'Y ratio. However, this variation ap sorption along the intestine. However, more
pears small and inconsistent. evidence is necessary to clarify this point.
Nevertheless, the comparison of the pat
DISCUSSION terns of calcium and phosphorus absorp
On the basis of the known functions of tion as influenced by dietary and diurnal
the gizzard it appears unlikely that large variations, is apparently valid since it ap
amounts of calcium and phosphorus could pears unlikely that the movement of 91Y
be absorbed in this organ, as appears to be could be influenced either by dietary cal
suggested by the lower Ca and P/91Y ratios
cium or egg shell deposition. In this re
compared with those of the feed. It also spect it should be recalled that the late-
appears unlikely that very large quantities calcification hens and the no-calcification
of dry matter are secreted in this organ hens were killed at the same time of the
as suggested by the very high dry mat- day.
ter/91Y ratio. The examination of the giz
Those factors with respect to the rate
zard contents revealed the presence of of passage of the test nutrient relative to
relatively large feed particles, mostly the tracer, demonstrate clearly the difficul
grains, with few smaller ones. Apparently, ties in the use of such tracer.
therefore, the gizzard retains the larger The pattern of dry matter/91Y indicates
feed particles longer than the smaller absorption of dry matter along the entire
ones and the solutes. Calcium and phos intestine with a somewhat reduced rate at
phorus which are mainly in solution in the posterior segments. The high dry-
the gizzard (12) might be emptied more matter/9'Y ratio in the duodenum is prob
rapidly than the larger grain particles. On ably due to secretion of digestive sub
the other hand, 91Ytends to be partially
stances in this organ. The pattern in
adsorbed on solid particles (3) even at a general is similar to that observed by
low pH such as that in the gizzard. This Bolton (8) in chicks, for the absorption of
isotope will therefore be slower than cal protein and available carbohydrate.
cium and phosphorus to leave the gizzard, Assuming the same passage time for
but more rapid than the solids. This inter calcium, phosphorus and yttrium along the
pretation is also supported by a prelimi intestine, the present results appear to in
nary study in this laboratory with a single dicate that the major portion of calcium
dose of 45Ca and 91Yand may explain the
low Ca and P/"Y ratios and the high dry and phosphorus absorption is located at
matter/91Y ratio in the gizzard.3 In the the anterior parts of the intestine. It can
be easily shown that calcium concentration
steady state of feed passage from the giz in the total dry matter is actually increas
zard it may be assumed that the latter ing along the intestine and therefore could
passes the various feed components at not be a factor in the reduced absorption
constant rate. This assumption is sup in the posterior parts. The latter may be
ported by the dry-matter/91Y ratios which
due either to the greater capacity of the
show only slight variations that appear anterior segments to absorb calcium (1,2,
to be more of diurnal nature rather than 5, 14, 15) or to the reduced solubility of
related to shell deposition. There was calcium due to increase in pH with the dis
also no significant difference in the nutri- tance from the gizzard (12, 16).
ent/91Y ratios in the duodenum, suggesting
Dietary calcium, at the levels used, did
uniform emptying of yttrium, calcium, not influence the relative absorption of cal
phosphorus and total solids from the cium, in agreement with previous balance
gizzard. studies (9). The absence of a calcifying
In accordance with the observations of 3 Unpublished data, S. Hurwitz and A. Bar, 1965.
Marcus and Lengemann (13), calcium, 4 See footnote 3.

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 438 S. HURWITZ AND A. BAR

shell was associated with a reduced rate 3. Marcus, C. S., and F. W. Lengemann 1962
of calcium absorption. This effect was Use of radioyttrium to study food movement
in the small intestine of the rat. J. Nutrition,
identical for the high calcium and low 76: 179.
calcium lots. The mechanism responsible 4. Cramer, C. F., and D. H. Copp 1959 Prog
for this variation is not clear, although it ress and rate of absorption of radiostrontium
may be explained in terms of the greater through intestinal tracts of rats. Proc. Soc.
Exp. Biol. Med., 102: 514.
need for calcium during periods of egg 5. Lengemann, F. W., R. H. Wasserman and
shell formation. It is accepted that absorp C. L. Comar 1959 Studies on the enhance
tion alone cannot supply all the calcium ment of radiocalcium and radiostrontium
needed during egg shell formation and that absorption by lactose in the rat. J. Nutrition,
68: 443.
bone reserves have to be utilized (17), 6. Lengemann, F. W., and C. L. Comar 1961
even when birds are fed sufficient calcium. Distribution of absorbed strontium-85 and
This conclusion is based on daily averages calcium-45 as influenced by lactose. Am. J.
of calcium retention not considering any Physiol., 200: 1051.
7. Chandler, P. T., and R. G. Cragle 1962
diurnal variations in calcium absorption. Gastrointestinal sites of absorption and en
The greater calcium absorption during dogenous secretion of calcium and phos
shell deposition, observed in this study, phorus in dairy calves. Proc. Soc. Exp. Biol.
could mean that the importance of bone Med., Ill: 431.
as a calcium reserve in birds fed sufficient 8. Bolton, W. 1961 The absorption of food
from the gut of the fowl. Proc. Nutrition
calcium, is not as great as previously be Soc., 20: xxvi.
lieved. 9. Hurwitz, S., and P. Griminger 1960 Ob
The absorption of phosphorus appears servations on the calcium balance of laying
to be closely related to that of calcium, and hens. J. Agr. Sci., 54: 373.
10. Gomori, G. 1942 A modification of the
is probably influenced by the latter. The colorimetrie phosphorus determination for
depressing effect of high levels of dietary use with photometric colorimeter. J. Lab.
calcium on phosphorus absorption is well Clin. Med., 27: 955.
known (18). The significant increase in 11. Dixon, W. J., and F. J. Massey, Jr. 1957
Introduction of Statistical Analysis. McGraw
absorption of phosphorus during shell for Hill Book Company, New York, p. 139.
mation is not as striking as with calcium 12. Tyler, C. L. 1946 Studies on the absorp
absorption, and appears to be secondary tion and excretion of certain minerals by
to the latter. poultry. II. J. Agr. Sci., 35: 275.
From the P/91Y ratios in the duodenum, 13. Marcus, C. S., and F. W. Lengemann 1962
Absorption of Ca45 and Sr85 from solid and
it is apparent that large quantities of en liquid food at various levels of the alimen
dogenous phosphorus are emptied into this tary tract of the rat. J. Nutrition, 77: 155.
segment. It may be calculated that the 14. Williams, G. A., E. N. Bowser, W. J. Hender
son and V. Uzgiries 1961 Effects of vita
ratio of endogenous to dietary phosphorus min D and cortisone on intestinal absorption
in this segment is at least 4:1. This ob of calcium in the rat. Proc. Soc. Exp. Biol.
servation is consistent with the heavy 3SP Med., 106: 664.
secretion into the duodenum, observed in 15. Harrison, H. E., and H. C. Harrison 1960
Transfer of Ca45 across intestinal wall in
the laying hen by Shirley et al. (19). vitro in relation to action of vitamin D and
cortisol. Am. J. Physiol., 199: 265.
ACKNOWLEDGMENT 16 Coates, M. E., and E. S. Holdsworth 1961
The authors wish to acknowledge the Vitamin Da and absorption of calcium in the
technical assistance of Mrs. M. Cotter. chick. Brit. J. Nutrition, 35: 131.
17. Simkiss, K. 1961 Calcium metabolism and
LITERATURE CITED avian reproduction. Biol. Rev., 36: 321.
18. deLange, D. J. 1961 Some factors which
1. Schachter, D., and S. M. Rosen 1959 influence the absorption and retention of
Active transport of Ca45 by the small intestine calcium and phosphorus in rats on a high
and its dependence on vitamin D. Am. J. cereal diet. Proc. Nutrition Soc. S. Africa, 2:
Physiol., 196: 357. 76.
2. Kimberg, D. V., D. Schachter and H. Schenker 19. Shirley, R. L., J. C. Daggers, J. T. McCall and
1961 Active transport of calcium by intes G. K. Davis 1952 Excretion of P32 and Ca45
tine: effects of dietary calcium. Am. J. into the various alimentary segments of hens.
Physiol., 200: 1256. Poultry Sci., 31: 316.

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