/ . Embryol. exp. Morph., Vol. 17,1, pp. 131-138, February 1967
With 1 plate
Printed in Great Britain
131
On the constancy
of the number of villi in the duodenum of the
post-embryonic domestic fowl
By RUFUS CLARKE 1
From the Physiological Laboratory, University of Cambridge
INTRODUCTION
Since the classical paper of Leblond & Stevens (1948), much information on
cell renewal in the intestinal epithelium has become available. The introduction
of tritiated thymidine autoradiography has diverted attention from conspicuous
lacunae which still exist in our knowledge of intestinal structure and growth.
This paper attempts to fill two of these elementary gaps. The first is well defined
by Fry, Lesher, Kisieleski & Sacher (1963): 'we still remain ignorant even of the
complete three-dimensional structure of the mucosa in any one region'. It
seems incredible that such a problem was not attacked by the great German
microanatomists of the last century, but no accurate account of the threedimensional structure of the intestinal mucosa is known to me; the accounts in
present-day textbooks of histology in the English language are superficial and
even inaccurate (see, for example, Bloom & Fawcett, 1962; Ham, 1965). The
second point of ignorance concerns the growth of the intestinal mucosa from the
embryonic to the adult form, and the elucidation of this is the primary objective
of this paper.
The opportunity to shed light on these problems arose accidentally. Work
intended to provide an histological baseline for studies of chicken intestine in
vitro resulted in the rediscovery of the observation (Clara, 1927) that the rigid
zigzag pattern of villi, so evident in the chick embryo (Romanoff, 1960), persists
in the adult fowl. It was difficult to reconcile this persistence of an organized
regular pattern with the notion that the post-embryonic growth of the intestinal
mucosa takes place by the addition of new villi, and the only obvious alternative
is that the number of villi in the small intestine remains constant during postembryonic growth.
Berry (1900) counted the number of villi per square millimetre of mucosal
surface in the developing pig embryo, and found that the number per unit area
increased with the age of the embryo. Such a method is open to error from
stretching or shrinkage.
Direct confirmation of the hypothesis could theoretically be obtained by
1
Author's address: The Physiological Laboratory, Cambridge, England.
9-2
132
R. CLARKE
counting the number of villi in an individual at intervals as it grew. This is
impossible, since to see the villi the intestine must be turned inside-out, or slit
open, and neither manoeuvre is compatible with its continued development.
Therefore different individuals must be used, but an attempt to achieve uniformity may be made by examining material from individuals of similar genetic
constitution. To count the number of villi in the entire small intestine would be a
Herculean task, the more so since opening the gut must damage substantial
numbers of villi. The numbers involved can be reduced to manageable proportions by considering only a portion of the intestine lying between fixed
embryological landmarks. The extent to which the constancy of these landmarks may be assumed will be discussed later.
In mammals, where there is no obvious ordered pattern in the arrangement
of villi, the prospect of counting individually the large number of villi still involved is daunting. In the chicken, however, the villi are arranged in longitudinal
rows, each of which zigzags transversely in a regular manner, each zig (or zag)
being one villus, and each lateral projection of the zigzag fitting snugly into the
corresponding hollow of its neighbour. In the chicken, therefore, it is not
necessary to count each villus individually, since the rigid pattern of villi
demands that any increase in the number of villi be reflected in an increase in
the number of longitudinal rows, or zigzags, or both. The estimate of the number
of longitudinal rows must necessarily be rendered inaccurate by the longitudinal
slitting of the intestine, but the error introduced is small, relatively constant, and
unimportant if the absolute number of villi is not considered.
MATERIALS AND METHODS
The landmarks between which the number of zigzags was counted were the
pylorus and the most cranial of the group of bile and pancreatic ducts, usually
the duct of the left ventral pancreatic anlage. The number of longitudinal rows
of villi was counted, in embryo and adult, at three sites: near the pylorus, at the
apex of the duodenal loop, and just cranial to the pancreatic duct.
In an attempt to reduce any variation due to genetic factors, three 1-year-old
White Leghorn hens were isolated from cockerels for a fortnight, then mated
with a single White Leghorn cockerel, and kept in separate cages thereafter.
The fertile eggs produced were incubated at 39 °C. After 16-19 days of incubation, the embryos were dissected and the duodenum and ventriculus removed.
The organs were dissected in warm 0-9 % sodium chloride, the pancreas removed, and the duodenum freed so that it lay straight. It was then placed on
moist blotting paper, slit longitudinally and opened out flat, displaying the
mucosa. Fixation for 24 h in 10 % formalin was followed by dehydration,
staining in 1 % eosin in 98 % alcohol, clearing in xylol, and mounting in
DePeX. The number of longitudinal rows of villi, and of zigzags, was counted
under the microscope at a magnification of x 40 on a mechanical stage.
Number of duodenal villi
133
In the autumn, after the hens had ceased to lay, they were killed by cervical
dislocation. The cockerel was killed in the following spring. The duodenum of
each bird was freed as before, and fixed entire in 10 % formalin, after it had been
pinned out straight on a sheet of cork. After washing in tap water, it was treated
for 4 h with 98 % alcohol to harden it, slit longitudinally, and again pinned out,
flat, mucosa upwards on a sheet of cork. Unfolding from its cylindrical form,
after moderate hardening, caused the mucosa to tend to crack apart, and the
zigzag pattern became evident. The number of zigzags and longitudinal rows
was counted by reflected light under a dissecting microscope on a rackwork
arm, at a magnification of x 10. The specimen was held in position, and the
microscope racked across it.
Sixteen embryos and four adults were examined.
Each bird was weighed after its ventriculus and duodenum had been excised.
The embryos were sexed by macroscopic inspection of the gonads.
SOURCES OF ERROR
There are two main sources of error, which cannot easily be separated.
(1) Observer error in counting. The zigzags are moderately easy to count,
especially in the embryonic specimens.
(2) Non-rigidity of pattern. Variations occur in both dimensions; a longitudinal row may die out, or fuse with one of its neighbours. Alternatively a
length of intestine which houses, perhaps, five zigzags in one row may only
house four zigzags two or three rows away transversely (Plate 1, fig. A). Thus
counts made between the same fixed points on different rows of villi may contain different numbers of zigzags, but this will tend to even out over a long
distance. The counts were all performed on the whole duodenum, but with a
different starting-point on the circumference of the pylorus, or at the pancreatic-duct level, for each count in any one direction, so that a better estimate
of the mean number of zigzags might be obtained. Single villi not conforming
to the pattern were not found, and it is concluded that any non-rigidity of
pattern which may exist does not affect the conclusions drawn.
The small size of the standard deviations of the counts suggests that these
sources of error are not important.
RESULTS
It is reasonable to assume that carcase weight, rather than length of incubation, is the better guide to the stage of development of the embryo. If carcase
weight is plotted against the number of longitudinal rows of villi (Text-fig. 1) it is
clear that the number of rows about doubles as the weight doubles, from 12 to
24 g. This is due to the appearance of the fifth and last rank of longitudinal folds,
as suggested by Coulombre & Coulombre (1958), and not to division of existing
villi from the apex downwards (Pap, 1933). The sixtyfold increase in weight which
134
R. CLARKE
occurs during growth to the adult size is accompanied, if anything, by a slight
decline in the number of longitudinal rows.
The result of the counting of zigzags is less striking. There is variation
between individual embryonic specimens, with the suggestion that some increase in the number of zigzags does take place up to a carcase weight of 25 g;
possibly also the constancy of the position of the pylorus and/or pancreatic
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Text-fig. 1. Carcase weight plotted against: (a) number of longitudinal rows (each
point represents one observation); (b) number of zigzags (each point represents
mean ± S.D. of six observations for each embryo, or of three observations for each
adult). O, • , x : values for each of three hens, and their offspring; c, cockerel.
duct is less than had been assumed. There is no a priori reason why the distance
between these two landmarks should be accurately fixed. The mean ± standard
error of the mean for the number of zigzags in the sixteen embryonic specimens
is 361 ± 10; that for the four adults 376 ± 8; the difference is not significant at the
5 % level. There is no obvious effect of sex or parentage on the embryonic
counts.
The number of villi in the post-embryonic duodenum is therefore in the region
of 24,000 (380 zigzags x 32 rows x 2 villi per zigzag), and remains reasonably
constant, although the internal surface area of the cylindrical duodenum increases approximately fiftyfold. The dimensions in Table 1 are approximate.
This may be compared with the approximate area covered by the base of a villus
(Table 2).
/. Embryo/, exp. Morph., Vol. 17, Part 1
PLATE 1
facing p. 135
Number of duodenal villi
135
At their first appearance on about the nineteenth day of incubation, the crypts
of Lieberkiihn are shallow pits growing from the lumen as outbulgings of the
epithelium in the gutters between the villi, and initially there are about six
crypts ranged along the gutter between two adjacent villi (Plate 1, fig. B).
During subsequent growth they increase enormously in number and in depth,
but are approximately the same diameter in the adult as those present just after
hatching. In the adult there are approximately forty crypts ranged along the
gutter between two adjacent villi (Plate 1, fig. D). The mechanism of this increase in number has not been investigated.
Table 1.
Duodenum
Length
Circumference
Area
19-day
embryo
Adult
Factor
40 mm
3 mm
120 mm2
300 mm
20 mm
6000 mm 2
x50
Table 2.
Villus base
i
Length
Breadth
Area
19-day
embryo
Adult
Factor
120 /*
30 /*
3600 fi2
1200/*
150/*
180000 /*2
x50
DISCUSSION
After the nineteenth day of incubation, the number of longitudinal rows and
zigzags appears to remain virtually constant, and, in conjunction with the retention of the relatively rigid pattern evident at the bases of the villi in the adult
PLATE 1
Fig. A. Whole mount of opened duodenum of embryo incubated 16 days. Stained with
eosin: x 64. Note geometrical pattern, and areas whose rigidity of pattern is broken.
Fig. B. Oblique horizontal section of opened duodenum of chick, 2 days after hatching.
Haemalum: x 64. Note increase in size of villi, and pattern and number of crypts.
Fig. C. Oblique horizontal section of opened duodenum of adult hen. Iron haematoxylin:
x 64. In this section at the level of the bases of the villi, note the persistence of the embryonic
pattern.
Fig. D. Oblique horizontal section of opened duodenum of adult hen. Iron haematoxylin:
x 64. This section passes through the crypts. They are less tightly packed than in the chick,
although of comparable diameter. The pattern is lost.
136
R. CLARKE
(Plate 1, fig. C), this suggests that there is little, if any, increase in the number of
villi in the duodenum during this time, especially as the difference between the
two groups is not greater than can be simply accounted for by the differences
between individuals.
This pattern of increase in the size of the villi, without increase in their
number, will be described as 'post-embryonic' to distinguish it from events
occurring before the nineteenth day, notwithstanding the fact that it dates from
about 2 days before hatching.
By contrast, the crypts undergo a sevenfold increase in numbers. This is less
than the fiftyfold increase in cylindrical surface area of the duodenum for two
reasons; first, because the surface area covered by a single villus has already
increased fourfold in the first three days of the 'post-embryonic' period; and
secondly, because the crypts are more widely spaced in the adult. There is a
relative increase in the amount of lamina propria between the crypts, and there
must also be space for the blood-vessels, lymphatics and nerves supplying the
villi.
The plasticity of the embryonic growth pattern (for detailed descriptions, see
Romanoff, 1960; Hinni & Watterson, 1963) reflects the greater freedom enjoyed
during this period of preparation for function. The onset of secretory, digestive
and absorptive function imposes a restriction on the growth possibilities
available to the 'post-embryonic' mucosa, since function must be maintained
while growth takes place. This restriction is most noticeable at the plane of the
crypt-villus junction (Plate 1, fig. C), where the embryonic architecture becomes
'frozen'. On the villus side of this plane, the growth potential of the lamina
propria is unknown, but the epithelium is known to consist of mature cells
migrating upwards to be sloughed from the tip of the villus. Thus the inherent
possibilities for change in structure appear small.
On the crypt side of the plane, the epithelium shows continuous and intense
mitotic activity, and part of this cell production is readily available for the postembryonic increase in the number of crypts of Lieberkiihn.
It is difficult to estimate the increase in absorptive area that occurs during
post-embryonic growth, but it is clear that the growth pattern described would
allow the increase to be made with minimal disturbance of function.
The exact numerical and spatial relationship of crypts and villi requires more
detailed study, but it is interesting to speculate on the effects that crypt geometry
may have on the upward migration of cells on to the villi, for which direct
experimental evidence was first provided by Leblond, Stevens & Bogoroch
(1948), using 32P. Streams of cells produced by the adjacent sides of two adjacent
crypts will, if they migrate directly up the crypts, meet each other head on where
the two crypt epithelia meet (* in Text-fig. 2); presumably these cells are
deflected to each side to climb the villi, but it is possible that there is an area of
stagnation in the centre (*), and it would be interesting to know whether such an
area of stagnation exists, and, if so, what sort of cells are to be found there. In
Number of duodenal villi
137
the cat duodenum, for example, where Paneth cells are not present at the bottom
of the crypt, these are commonly found cells, at the level of the junction of
crypts and villi and infrequently on the villi, containing large, acidophilic, faintly
PAS-reactive granules similar to those found in Paneth cells of other species
(unpublished personal observations). It is possible that in this site, as in the
depths of the crypt, cells may find a refuge from which they are only occasionally
swept upward on to the villi.
Villus
Crypts
Villus
Text-fig. 2. Diagrammatic, foreshortened view from above villi, looking into two
crypts. Arrows represent direction of movement of cells from crypts on to villi.
The results here described establish a baseline for quantitative studies of
growth and cell renewal in the chicken intestine, and indicate that the villus,
together with those crypts which supply cells to clothe it, may be regarded as a
stable anatomical and functional unit. The same may be true for mammals, but
further work is needed to establish this.
SUMMARY
1. Counts performed on whole mounts of late embryonic and of adult
chicken duodenum demonstrate that the number of villi present at these two
stages of development is approximately the same. Growth occurs by increase in
the size of the villi.
2. The crypts of Lieberkuhn increase greatly in number and depth, but change
little in diameter.
RESUME
Sur la Constance du nombre des villosites du duodenum,
aux stades post-embryonnaires chez la poule domestique
1. Sur des montages totaux de duodenum de poulet en fin de phase embryonnaire, et adulte, comptages du nombre des villosites presentes ont ete effectues.
138
R. CLARKE
Le nombre des villosites est approximativement le meme pour ces deux stades.
La croissance se traduit par l'augmentation de la taille des villosites.
2. Les cryptes des glandes de Lieberkiihn croissent considerablement en
nombre et en profondeur. Leur diametre change peu.
This work forms part of a dissertation to be submitted for the degree of Ph.D. in the
University of Cambridge. Part of this work was performed during the tenure of a Medical
Research Council Scholarship. I wish to express my gratitude to the Medical Research
Council. I thank Professor E. N. Willmer and Dr F. W. Campbell for help in the preparation
of the manuscript, and Dr T. Vickers for criticism, encouragement and advice.
REFERENCES
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CLARA, M. (1927). Beitrage zur Kenntnis des Vogeldarmes. VIII. Das Problem des Rumpfdarmschleimhautreliefs. Z. mikrosk.-anat. Forsch. 9, 1-48.
COULOMBRE, A. J. & COULOMBRE, J. L. (1958). Intestinal development I. Morphogenesis of
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LEBLOND, C. P., STEVENS, C. E. & BOGOROCH, R. (1948). Histological localisation of newlyformed desoxyribonucleic acid. Science, N.Y. 108, 531-3.
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BERRY,
(Manuscript received 25 July 1966)