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The composition of the anal glands of Dasypus novemcinctus.

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The Composition of the Anal Glands of
Dasypus novemcinctus'
Department of Biology, Rice University, Houston, Texas
The nine-banded armadillo ( D a s y p u s
novemcinctus) is monestrus (Hamlett, '32)
and apparently ovulates spontaneously
(Talmage and Buchanan, '54). The vagina
is separated from the exterior by an extensive urogenital sinus and cornification does
not occur at any stage of the reproductive
cycle (Enders and Buchanan, '59). The
inaccessibility of the vagina and lack of
prominent cycle make the use of vaginal
smears in this animal of limited value.
Furthermore, no changes in the external
genitalia of sufficient magnitude to serve
as an index of the reproductive cycle have
been observed. It has long been known
that mammalian cutaneous glands serve
definite functions in marking territory and
locating trails, and appear to play a part in
the reproductive cycles of many mammals
(Bourliere, '54). Recently, Bruce ('60)
has shown that in mice the scent of a
strange male can disrupt an early pregnancy, and Parkes ('60) has emphasized
that sense of smell may be more important
than it had previously been considered to
The armadillo possesses a pair of highly
developed anal glands (Owen, 1866) which
produce a characteristic scent. Although
the development and histology of the integument of the nine-banded armadillo have
been examined (Wilson, '14) and a historical study has also been given (Cooper,
'30), the integumentary derivatives which
form the anal gland complex have not been
studied. It was the twofold purpose of this
investigation to study the structure of this
complex and to determine whether there
is evidence of variation in secretion in relation to the reproductive cycle.
abrupt vertebral fracture and their anal
glands removed, fixed, dehydrated, double
imbedded in celloidin and paraffin, and
sectioned. The histology of the glands was
studied using 10-cl serial sections stained
with hematoxylin and eosin, and representative sections from other glands
stained either with Ehrlich's hematoxylin,
chromotrope 2R, orange G , and fast green,
or with Weigert's hematoxylin and fast
green. Whole mounts of several glands
were prepared by staining the intact gland
with paracarmine, dehydrating and clearing in cedar oil and then dissecting away
the outer connective tissue capusle. Glycogen was demonstrated by the application
of the periodic acid-Schiff test to sections
from glands fixed in Carnoy's or Rossman's
fluids and controlled by digestion in a 1%
malt diastase solution at pH 6. Cytoplasmic basophilia was observed using Zenker's
fixed material stained with eosin methylene blue at pH 5.2. For the demonstration
of lipids pieces of the glands were fixed
in buffered formalin and sections were
colored with Sudan black B; phospholipids
were revealed by the Elftman method
(Elftman, '57).
To induce ovulation pregnant mares
serum (Equinex) was administered to 5
animals for a period of three days in doses
ranging from 25 to 500 I.U. Subsequently,
a single injection of 250 I.U. of chorionic
gonadotropin ( Antuitrin-S) was administered. The animals were killed after an
interval of at least 24 hours. Examination
of the ovaries revealed that multiple ovulation had taken place in all cases.
The anal glands are a pair of peanutshaped sacs, ranging from 1-2.5 cm in
A total of 20 armadillos from all stages
of the reproductive cycle were killed by
1 Supported in part by grant G-9113 from the
United States National Science Foundation.
length, situated at either side of the anus
(fig. 1). These structures are distinctly separated from the surrounding tissue by a
heavy collagenous capsule. When this overlying fascia is dissected away, the gland is
frequently found to be bilobed with the
lobes twined about each other. The eversion
of the top one-quarter of the sac may be
brought about by the contraction of a large
mass of muscle inserting on the connective
tissue surrounding the orifice of the gland.
On closer examination the gland is seen
to consist of an epidermal sac surrounded
by connective tissue, in which are situated
both apocrine and sebaceous glands (figs.
2 and 3 ) .
The sac is lined with a highly keratinized epidermis from which extensive
sheets of cells are sloughed. This epidermis, together with a thin layer of connective tissue, forms several large folds which
separate the lumen into sacular compartments. Embedded in the connective tissue
surrounding the sac are from 8 to 12
branched tubular apocrine glands. When
one of these is teased apart it is found to
be composed of a series of flat epithelial
plates. Each of these plates is formed from
a branched anastomosing tubule, which
is recoiled within a single plane rather
than being coiled to form a lobular mass
as are the tubules of apocrine sweat
The secretory cells lining the tubules of
the apocrine glands vary from tall columnar to low cuboidal. Interspersed between
a thick PAS positive basement membrane
and the apocrine cells are a number of
myoepithelial cells. The following description pertains primarily to the tall columnar
cells which appear to be the active secretory stage. The apical portions of many
of the glandular cells protrude into the
lumen. The presence of discontinuous portions of cytoplasm within the lumen suggests dehissence. All of the cells are distinctly basophilic. Discreet regions of basophilia are present apically and frequently
in a juxtanuclear position (fig. 9 ) . These
regions may be separated by an area in
which fine eosinophilic granules are found.
In electron micrographs these basophilic
regions show parallel arrays of endoplasmic reticulum with associated ribonucleoprotein (RNP) particles (fig. 10). Lipid
droplets are present throughout the apocrine glands. The more readily discernible
droplets are situated in the basal portions
of the cells and may be demonstrated by
either Sudan black B in frozen preparations or by osmification. The lipid present
in frozen sections can be removed by acetone extraction and no phospholipids are
demonstrable by Elftman’s method.
The cells of the apocrine glands of animals examined during either pregnancy
or prior to the development of mature follicles were devoid of demonstrable glycogen
(fig. 6). The extreme apical portions of
the cells are PAS positive and diastase resistant. The contents of the lumina are
intensely PAS positive and cannot be extracted by digestion with diastase. In 4 of
the 5 animals artifically ovulated using
PMS and PU the apocrine tubules showed
accumulations of glycogen (fig. 7). However, even during these periods the amounts
present were not extensive. No other
changes were observed during the cycle.
The cytoplasmic basophilia remained constant and there was no increase in the
amount of lipid present. The anal glands
of the two males examined were identical
to those of the females. The anal glands
from a single spayed female were histologically similar to those of normal females.
In addition to the apocrine glands there
are numerous large sebaceous glands opening into the sac (fig. 5). These glands may
share a duct with a group of apocrine tubules, but more frequently the openings to
the lumen of the sac are separate. These
glands are directly under the epidermis
and are superficial to the apocrine glands.
The peripheral cells in the individual
lobules of the sebaceous gland show appreciable diffuse cytoplasmic basophilia which
is less intense in cells towards the center
of the lobule. The distribution of glycogen
is similar to the distribution of cytoplasmic
basophilia, except that glycogen is especially heavy in the cells of the duct region
(fig. 8). The amount of glycogen diminishes as the sudanophilia of the gland increases. Montagna (’56) suggested that
the similar decrease in glycogen in the
human sebaceous gland was the result of
the conversion of carbohydrates into lipids.
The ensheathing connective tissue is
well vascularized with capillary beds frequently surrounding the apocrine glands.
This connective tissue contains patches of
lymphatic tissue which are rich in plasma
cells. Massive numbers of granular leucocytes (especially eosinophils) are often
found in the apocrine tubules (fig. 4).
Such areas of invasion are localized and
occur only when the surrounding connective tissue is rich in granular leucocytes.
The content of the anal sac is sloughed
cornified epidermal cells impregnated with
the products of apocrine and sebaceous
glands. Both types of glands are present
in sufficient numbers to be significant in
contributing to the odor of the final product.
The outstanding feature of the apocrine
glands is cytoplasmic basophilia in the
form of discrete bodies. In electron micrographs these bodies are seen to consist of
membranes of the endoplasmic reticulum
with associated RNP particles. This type
of structure is associated with the production of a specific protein product, whereas
free RNP is associated with the production
of new cytoplasm (see Slautterback and
Fawcett, '59). Consequently the eosinophilic granules seen in the tall columnar
cells probably represent an accumulation
of protein secretion. The presence of PAS
positive diastase resistant substance in the
distal fringe of the apocrine gland cells
and the presence within the lumina of
such substance is indicative of the inclusion of mucopolysaccharides or mucoproteins in the secretion product.
The organization of the armadillo anal
gland complex is very similar to that of
the dog as described by Montagna and
Parks ('48). However, not all anal glands
have the same components. For example,
examination of mink and nutria anal
glands during the course of this investigation revealed that the former contains
neither sebaceous glands nor a large epidermal sac while the latter has an anal or
perineal gland composed almost entirely
of sebaceous glands.
A number of functions have been attributed to anal and other cutaneous
glands. It has been speculated that in the
dog these glands aid in defecation. This is
an improbable function in the armadillo
since the openings of the glands are excluded from the anus during the act of
defecation. The anal sacs of the armadillo
contain secretory products and sloughed
epidermis at all times of the year and at all
stages of the reproductive cycle. A large
number of active secretory cells are also
present throughout the year. However, not
all the apocrine glands appear active at
any one time. Usually some glands are
composed of cuboidal cells which lack both
eosinophilic granules and the secretion
droplets seen in electron micrographs of
active cells. The constant activity of the
gland throughout the year indicates that
i t may serve a routine function such as
marking trails. The use of scent as a
means of orientation by the armadillo is
highly probable as they lack good eye sight
but possess a very well developed olfactory
sense (Clark, '51). When an armadillo is
excited, it will commonly evert the anal
sac. This may mean that the anal gland
can serve as some sort of repulsive mechanism. However, Kalmbach ('43) noted in
studying stomach contents of various animals that dogs and mountain lions killed
considerable numbers of armadillos. He
failed to find evidence of predation by
There stiIl remains the possibility that
the character of the secretion might change
in the female animal during the preovulation and ovulation periods. Our experimental evidence suggests that a change in
the characteristics of the secretion is possible. At any rate, the injection of PMS results in an increase in glycogen content of
the apocrine glands, suggesting that the
physiology of these structures is altered at
this time. It would be necessary to obtain
the glands from spontaneously ovulating
animals to determine whether a change in
glycogen content similar to that observed
in experimental animals occurs in a natural population.
1. The anal gland of the armadillo is
found to consist of an epithelial sac surrounded by apocrine glands and sebaceous
2. The cells of the apocrine glands have
a marked cytoplasmic basophilia and contain fine eosinophilic granules. Histological and cytological evidence indicates a
high rate of protein secretion.
3. At all times of the year the sac is
filled with sloughed epidermis and the
products of the apocrine and sebaceous
glands. The only variation seen is an increase in the amount of glycogen present
in the apocrine glands following induced
4. It is likely that the glands serve in
routine recognition functions. However, a
function in sexual attraction is possible.
Bourlisre, F. 1954 The Natural History of
Mammals. Alfred A. Knopf, New York.
Bruce, H. M. 1960 Further observations on
pregnancy block in mice caused by the proximity of strange males. J. Repro. Fertil., 1:
31 1-3 12.
Clark, W. T. 1951 Ecological life history of the
armadillo i n the Eastern Edwards Plateau region. Am. Midland Naturalist, 46: 337.
Cooper, Z. K. 1930 A historical study of the
integument of the armadillo. Am. J. Anat.,
45: 1-32.
Elftman, H. 1957 Osmichrome Fixation. Quart.
J. Micr. Sci., 98: 15-18.
Enders, A. C., and G. D. Buchanan 1959 The
reproductive tract of the female nine-banded
armadillo. Texas Rep. Biol. Med., 17: 323-340.
Hamlett, G. W. D. 1932 The reproductive cycle
i n the armadillo. Zeit. Wiss. Zool., 141: 143157.
Kalmback, E. R. 1943 The armadillo: its relation to agriculture and game. Game, Fish and
Oyster Commission, Austin, Texas.
Montagna, W. 1956 The Structure and Function of Skin. Academic Press, New York.
Montagna, W., and H. F. Parks 1948 A histochemical study of the glands of the anal sac
of the dog. Anat. Rec., 100: 297-318.
Owen, R. 1866 On the Anatomy of Vertebrates.
Longmans, Green and Co., London.
Parkes, A. S. 1960 The role of odorous substances in mammalian reproduction. J. Repro.
Fertil., 1: 312-314.
Slautterback, D. B., and D. W. Fawcett 1959
The development of the cnidoblasts of Hydra.
J. Biophys. Biochem. Cytol., 5: 4 4 1 4 5 2 .
Talmage, R. V., and G. D. Buchanan 1954
The Armadillo ( D a s y p u s novemcinctus). The
Rice Institute Pamphlet XLI ( 2 ) .
Wilson, C. W. 1914 Development and histology
of the integument of the nine-banded armadillo. Bull. Univ. Texas, no. 308, Sci. ser. no.
36: 1-18.
The anal glands of a female armadillo protruding in response to irritation. In this position the anus is concealed between the two glands.
Longitudinal section of an entire anal gland. Note that the anal sac containing sloughed
epidermis is surrounded by glands. H. and E. X 65.
Section through the wall of the anal gland. Note that the sebaceous gland in the center of the picture lies between the epidermal sac and the apocrine gland (base of picture). Weigert’s iron hematoxylin and fast green. >: 110.
Apocrine glands in an area of leucocytic invasion. Note the presence of granular leucocytes beneath the basement membrane (small arrow) and within the lumen. Myoepithelial cell nuclei can be seen up against the basement membrane (large arrow).
Tetrachrome. X 435.
Julian F. Haynes and Allen C. Enders
Sebaceous gland. The dark (osmiophilic) material is lipid. Flemmings, tetrachrome.
x 110.
PAS-positive substances in a n anal gland of a non-experimental female. Note the absence of demonstrable glycogen and presence (especially in the upper left corner) of a
PAS-positive luminal fringe. Periodic acid-Schiff. x 435.
7 Glycogen in the apocrine gland of a n animal in which ovulation was induced. The position of the glycogen against the cell membranes is a diffusion artifact. Periodic acidSchiff. x 435.
Glycogen in sebacceous glands. Glycogen is abundant in a duct (left of picture). A
small amount is also present i n the peripheral cells of a lobule (lower right corner).
Periodic acid-Schiff. x 10.
Cytoplasmic basophilia i n columnar cells of the apocrine glands. Note that between
the juxtanuclear and apical regions there is interposed a non-basophilic region. Eosin
methylene blue. X 435.
10 A n electron micrograph through part of a region of basophilia of apocrine gland cells.
note the orderly arrangement of endoplasmic reticulum with associated RNP particles
in the cell at the upper left. G , Golgi zone; S , secretion droplet; M, cell membrane.
Buffered osmium tetroxide-sucrose. X about 15,000.
Julian F. Haynes and Allen C. Enders
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anal, dasypus, gland, composition, novemcincta
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