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ABSTRACT

INFLUENCE or CALCITONIN IN PREVENTING SOFT TISSUE
CALCIFICATION BY DIHYDROTACHYSTEROL IN RATS

BY
Jorge A. Villar

Three experiments, using a total of 60 rats, were
conducted to determine the influence of calcitonin administra-
tion in preventing or diminishing the clinical signs and tissue
calcification associated with dihydrotachysteroi (DHT), a vitamin
derivative.

Daily oral adninistration of SOpg. of DHT in corn oil
produced clinical signs of progressive emaciation, muscular
weakness and death. Kyphosis and enteric disturbances were
evident. They were gross lesions of mineralization on the
surface of the myocardium and kidneys. Microscopically there
was extensive calcification in the aorta, heart, kidneys,
stomach and to a lesser extent in the small intestine and
lungs. Rats given the same amount of DHT and 20 MRC units/kg.
of calcitonin had the same clinical signs and lesions as those
given only the DHT. The degree of soft tissue Calcification
was dose-related. Decreasing the interval of calcitonin
administration had no influence in decreasing the soft tissue
calcification. Calcification of soft tissue by DHT was not

prevented by calcitonin.

INFLUENCE OF CALCITONIN IN PREVENTING SOFT TISSUE
CALCIFICATION BY DIHYDROTACHYSTEROL IN RATS

By

lb 2"”

Jorge A? Villar

A THESIS

Submitted to
Michi an State University
in partial fulgillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Pathology
l970

lamp,

TABLE

INTRODUCTION . . . . .
LITERATURE REVIEW. . .
Calcitonin. . . .
Existence. . .

Source . . . .

Mode of action

Protection against

Protection against

OF CONTENTS

hypercalcemia . . . . . .

soft-tissue calcification

Vitamin D intoxication. . .

MATERIALS AND METHODS. . . . . . . . .

Source and maintenance of animals

Chemicals . . . . . . . . . . .

Experimental procedure. . . . .

Experiment l . . . . . . . .

Necropsy procedures . . .

Histologic techniques . .

Hematologic

Experiment 2 .
Experiment 3 .
RESULTS. . . . . . . .
Experiment I. . .
Body.weight. .

Clinical signs

determinations.

Page

N-PUJNNN

IO
l2
I2
I2
I3
I3
I“
Ih
lh
IS
IS
I7
I7
l7
I7

Weight of organs . . . . .
Gross lesions. . . . . . .
Microscopic lesions. . . .

Statistical interpretation
le5ions. O O O O O O O O O

Hematologic determinations
Experiment 2. . . . . . . . .
Body weight. . . . . . . .
Microsc0pic lesions. . . .
Experiment 3. . . . . . . . .
Body weight. . . . . . . .
Clinical signs . . . . . .
Gross lesions. . . . . . .
Microscopic lesions. . . .

Statistical interpretation
leS‘ons. O O O O O O O O O

Hematologic determinations

DISCUSSION . . . . . . . . . . . .

Dosage of calcitonin. . . . .

of microscopic

of microscopic

Depletion of endogenous calcitonin. . . . . .

Compensatory parathormone secretion . . . . .

DHT action in adult rats. . .

Calcitonin action in adult rats . . . . . . .

"Escape phenomenon" . . . . .

Page

I7
20
20

3|
36
36
38
38
38
38
hi
hi
hi

In

an
us
as
1.6
1+7
#8
as
1.9

Page

Diet. . . . . . . . . . . . . . . . . . . . . . . . 49
SUMMARY 5|
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . . 52
VITA 51+

iv

LIST OF TABLES

Table Page

i Treatments, average initial and terminal
weights and selected organ weights in
Experiment I . . . . . . . . . . . . . . . . . I9

2 Grading of microscopic soft tissue calcifi-
cation in selected tissues from rats in
Experiment l . . . . . . . . . . . . . . . . . 21

3 Terminal hematologic determinations from rats
‘n EXPerImnt I. O O O O O O O O O O O O O O O 37

h Changes in body weight of rats treated with
different doses of HT in Experiment 2 . . . . 39

5 Microscopic assessment of degree of calcifica-
tion and percentage of changes in body weight
inrats in Experiment 2. . . . . . . . . . . . #0

6 Grading of microscopic soft tissue calcifica-
tion in selected tissues from rats in
Exper 'ment 3 O O O O O O O O O O O O O O O O o 42

7 Terminal hematologic determinations from rats
in Experiment 3. . ... . . . . . . . .w. . . . 4h

Figure

 

LIST OF FIGURES

Page
Extreme loss of weight, emaciation, dry and
inelastic skin and kyphosis in rat treated
with DHT. Similar signs were seen in rats
treated with DHT plus calcitonin. Rat 8,
Group 2, Experiment I. . . . . . . . . . . . . l9

Small arteries in the vicinity of the thyroid

gland with calcification in the media.

Rat l6, Group 3, Experiment I - H 8 E stain

x 560. O O O O O O O O O O O O O O O O O O O O 22

A cross-section of the aorta showing uniform
impregnation with calcium salts in the media.

Note absence of inflammatory reaction. Rat I0,
Group 2, Experiment I - H 8 E stain x 560. . . 23

involvement of coronary arteries with calci-
fication of the wall and heavy calcium salt

deposits between the myocardial fibers.

Rat l0, Group 2, Experiment l - H 8 E stain

x 560. O O O O O O O O O O I O O O. O O O O O 0 21*

Myocardial calcification. The deposition of
calcium was frequently found around coronary
arteries or in the subendocardial re ion.

Rat l3, Group 3, Experiment I - H 5 stain

x 560. O O O O O O C O O O O O O O O O O O O O 26

Heavy deposition of calcium salts in coronary
arterial branches of different dimensions.

Rat l7, Group 3, Experiment I - H 8 E stain

x 5620 O O O 0 O O O O 0 O O O O O O O O O O O 27

Disruption of the architecture of the renal

tissue with deposition of large masses of

calcium salts in the cortical area. Rat 9,

Group 2, Experiment I - H 8 E stain x 560. . . 28

Branch of the renal artery in hiIus of kidney

with mineralization in the media and disrup-

tion of the muscles'fibers. Rat l0, Group 2,
Experiment I - H 8 E stain x 560 . . . . . . . 29

vi

Figure

IO

l2

l3

lh

Page

Tunica muscularis, acid-secreting mucosa and

lamina propria of stomach with severe

cellular destruction and massive deposition

of calcium salts. Rat l6, Group 3, Experi-

ment l - H 8 E stain x 560 . . . . . . . . . . . 30

Wall of small artery of the duodenum with
mineralization of the media and disruption of
muscle fibers. Rat l2, Group 2, Experiment
I‘H‘ESta'nXSGOaaeaaaaaaaaaaa32

Lung. Notice calcification in the alveolar
and peribronchial tissue. Rat l6, Group 3.
Experiment l - H 8 E stain x 560 . . . . . . . . 33

Tibia. Increased density of trabecular bone.

Bone marrow spaces are narrow and hematogenous
marrow is almost absent. Rat l2, Group 2,
Experiment 1 - H 8 E stain x 560 . . . . . . . . 3h

Tibia. Notice irregularity of epiphyseal

late, few marrow spaces and absence of

ematogenous marrow. Rat l3, Group 3.
Experiment l - H S E stain x $60 . . . . . . . . 35

Muscular layer of stomach. Observe necrosis

and fragmentation of the muscle cells. Rat

l3, Group l3, Experiment 3.- H 5 E stain

x 560. O O O O O O O O O O O O O 0 O O O O O O O “3

vii

ACKNOWLEDGMENTS

I wish to express my gratitude to Dr. S. D. Sleight, my
major professor. His guidance, experience and patience made
this research possible. I also wish to extend my sincere
appreciation to Dr. C. C. Morrill, Chairman of the Department
of Pathology, Dr. C. K. Whitehair, Dr. R. F. Langham,

Dr. Vance L. Sanger and Dr. G. L. Waxler who made me aware of
the unlimited possibilities that the field of animal pathology
has.

The author is very thankful to Mrs. D. Fenner for the
hematologic determinations; Mr. M. K. Sunderlin, Mrs. N. L.
Miller and Mrs. F. M. Whipple for preparing the histOpathologic
sections.

Final appreciation is extended to the Agency for Inter-
national Devel0pment (AID) whose generosity made it possible

for me to carry out this work.

To my wife, Olga
and
my children Ana and Martin

INTRODUCTION

Calcitonin and hypervitaminosis D have Opposite effects
in the animal body. Calcitonin inhibits the removal of mineral
from bone, reduces the level of calcium and phosphate in the
blood and apparently promotes the deposition of new bone. On
the other hand, vitamin D has a hypercalcemic effect by facili-
tating the absorption of calcium from the gastrointestinal tract.
Massive doses of vitamin D promote a syndrome characterized by
metastatic calcification of the soft tissues.

As long as the end product of vitamin D intoxication, the
calcification of soft tisSues, is common to several diseases of
known or unknown etiology, it was of interest to investigate
if calcitonin were able to prevent or diminish the clinical
signs and tissue structural changes associated with hyper-
vitaminosis D.

The objective of this research was to determine whether
calcitonin has any prophylactic effect against the signs and

lesions caused by chronic massive doses of dihydrotachysterol

in adult rats.

LITERATURE REVIEW

Calcitonin

Existence. it was believed until recently that regulation
of plasma calcium was achieved by apprOpriate alteration in the
secretion rate of the parathyroid hormone. The stimulus for
the production of the hormone was a decrease in plasma calcium
(McLean and Urist, I955), and this promoted osteolysis and
mobilization of calcium from bones. The control of hyper-
.calcemia was attributed to the suppression of parathyroid
hormone production. Nevertheless, by using dogs devoid of
parathyroid glands,-SandersOn.é£f gl,'(l960) found that the
control of induced hypercalcemia was still inefficient. Copp
gt 21. (l962) carried perfusion experiments in the dog upon
the parathyroid-thyroid gland mass as a unit. Generalized
depression of blood calcium resulted when the glands were
perfused with blood high in calcium; but when therparathyroidec-
tomy was performed, the blood calcium increased beyond normal
limits, thus providing evidence that some subStance produced
by the glands had a hypocaicemic effect. They postulated the
existence of a new calcium lowering hormone which they called
"calcitonin" and deduced erroneously that it was produced by
the parathyroid gland. The existence of calcitonin was soon

confirmed by further perfusion experiments using a different

technique (Kumar'gt‘gl., l963; Macintyre g£_gl., I965) but
without distinguishing between the parathyroid source claimed
by Copp 2; 31. and an origin from the thyroid gland.

Source. Foster §£_§l, (l96h) carried out further per-
fusion experiments in the goat, since in this species the
thyroid and parathyroid glands could be perfused Separately.
They reported that calcitonin is of thyroid origin and is
secreted by the thyroid when this gland is perfused with high-
calcium blood. Hirsch gg'gl. (I963) reported that a factor
liberated from the rat thyroid gland was effective in causing
a reduction in blood calcium. In their experiments, the para-
thyroid tissue was separated from the thyroid tissue and the
thyroid gland was identified as the source of the hypocalcemic
factor. They named the hormone "thyrocalcitonin". Although
doubts were expressed at first (Copp, I969), it now seems
generally agreed that calcitonin and thyrocalcitonin are
synonymous.

The cells in the mammalian thyroid which secrete calci-
tonin have been Called C cells by Pearse (I966). The cells are
parafoilicular in the dog but in other species, Such as the
pig, they occupy epifollicular and follicular positions.
Bussolati and Pearse (I967) identified calcitonin in the C
cells in the dog and the pig by immunofluorescence techniques.

The thyroid gland of such animals as the chicken does not produce

calcitonin (Kraintz and PuiI, I967). The hormone in that
species is produced by the ultimobranchial bodies, which are
distinct organs in birds, fish, amphibians and reptiles (COpp
.25‘g1., I967). In mammals, the ultimobranchial tissue normally
fuses with the thyroid gland.

Mode of action. The effects of calcitonin and parathyroid
hormone appear to be antagonistic. Whereas parathyroid hormone
promotes resorption of bone and thus hypercalcemia, calcitonin
inhibits bone resorption (Aliapoulios g£_§1,, l966) and causes
a decrease in blood calcium levels (Gudmundsson gt gl,, l966).
Calcitonin can cause hypocalcemia in the absence of the para-
thyroid gland or parathormone (Hirsch gt 31,, l963) and can
act independently of the pituitary gland (Milhaud and Moukhtar,
I965), the kidney and the gastrointestinal tract (Munson gt‘gl.,
l968). Both calcitonin and parathormone decrease serum phos-'
phate and their effects are additive (Milhaud and Moukhtar, l966b).
Both cause phosphaturia (Robinson 3; 21., I966) but their
effects on blood calcium levels tend to cancel out each other.

Soliman 2; 21. (I967) pointed out that removal of the kidney
did not alter the plasma calcium-lowering response to calcitonin
injection and therefore, that effect is not mediated by.a renal
mechanism. Furthermore, they stated that the plasma calcium
lowering effect was not accompanied by an increase in soft tissue
calcium. They concluded that bone-was the most likely site of

action of calcitonin.

Reports conflict as to whether calcitonin exerts an effect
upon intestinal calcium absorption. Krawitt (I967) found that
calcium absorption and lumen-to-plasma flux were slightly
decreased in calcitonin treated animals. Milhaud and Moukhtar
(l966a), on the other hand, reported that calcitonin increased
the amount of calcium absorbed during digestion and enhanced the
utilization of dietary calcium by the intestines.

That calcitonin inhibits osteolysis and thus calcium
resorption was first demonstrated by Aliapoulios g£_§l. (l966).
They utilized organ cultures of calvarian bones from young
mice and showed that calcitonin was effective in controlling
the osteolytic effect of added parathormone. Their findings
were confirmed by Friedman and Raisz (I965), Gaillard (I967)
and Reynolds (I967) who also utilized in XLELQ techniques.

l_n 1119 studies were conducted by Martin _e_g §_l_. (l966)
to provide further evidence of direct inhibition of bone
resorption by calcitonin. They found a significant reduction
in urinary hydroxyproline excretion after calcitonin adminis-
tration. Klein and Taimage (I968) also concluded that repeated
administration of calcitonin results in an inhibition of all
phases of bone resorption. This was reflected in the diminished
hydroxyproline levels in urine, measured as a reliable index
of collagen breakdown.

Giraud £3 £1, (I967) indicated that calcitonin increased
metaphyseal bone mineral and reduced the number of osteoclasts

in the affected area. The increase in fully mineralized

metaphyseal bone was associated with an increase in partially
mineralized osteoid. They explained the ability of the hormone
to inhibit bone resorption to an increase in the rate of bone
formation, a reduction of the rate of bone demineralization,
or to an interference with the resorption phase of unmineral-
ized collagen. Any of these mechanisms would result in a
decrease in the movement of calcium from bone to blood and a
lowered serum calcium concentration. That calcitonin acts
independently of parathyroid hormone in preventing bone
resorption was reported by Foster _£,_l, (l966b). They observed
reduced osteoclast counts and an accumulation of trabecular
bone in the tail bones of parathyroidectomized rats treated
with massive doses of calcitonin for 28 days.

More detailed isotOpic studies by Johnston and Deiss
(l966), Mazzuloli g£_§l, (l966) and Robinson 2; 21. (I967)
left little doubt that inhibition of bone resorption is an
adequate explanation of the acute effects of calcitonin.

in addition to inhibition of resorption, there is the possi-
bility that calcitonin may also increase accretion of bone.
Wase £5 £1. (l966a) found that when calcitonin was continuously
infused intravenously into rats along with “SCa for about

I hr., increased amounts of radioactivity were found in bone.

Wase g£.§1, (l966b) demonstrated that the subcutaneous

administration of calcitonin to young male rats for 2i days
resulted in significantly greater cortical thickness in the
tibia, femur and humerus, as measured from a tetracycline
marker, than in control rats. Kumar ggflgl. (I968) approached
the problem in the opposite manner, by using rats with chronic
calcitonin deficiency. Bone changes were compared in thyroid-
intact and thyroidectomized rats (with thyroxine replacement),
both groups having functional parathyroid transplants.
Significantly less bone formation occurred in the femurs of
the thyroidectomized rats than in those of thyroid-intact rats.
On the other hand, Milhaud and Moukhtar (l966a), using kinetic
analysis, found that administration of calcitonin resulted in
a decrease in bone anabolism as well as in bone catabolism in
both intact andthyroparathyroidectomized rats. Based on
histology and tetracycline labeling in thyroparathyroidectomized
rats, Baylink‘gg‘gl. (I969) also concluded that calcitonin
inhibited both bone resorption and bone formation. '
Protection against hypercalcemia. According to Hirsch
and Munson (I969), the same experiments involving hypercalcemic
perfusion of the thyroid gland that led to the discovery of
calcitonin, also provided evidence that the thyroid gland, by
releasing calcitonin, could protect against hypercalcemia. In
their opinion, other experiments in which hypercalcemia was
produced by injection of a calcium salt or by administration

of parathyroid hormone or vitamin D, further support that

possibility. The latter claim that the effect of the thyroid
gland in protecting against hypercalcemia is more prominent when
the hypercalcemia is produced by parathyroid hormone or large
doses of vitamin D, than when hypercalcemia is produced by in-
jection of calcium. Hypercalcemia induced by parathormone or
hypervitaminosis D apparently stimulates bone resorption,
whereas the injection of calcium decreases bone resorption by
inhibiting release of parathormone. Other reports also suggest
a marked effect of the thyroid gland in reducing the hyper-
calcemic effect of parathyroid hormone in rats (Hirsch and
Munson, I966; Gittes and Irvin, I965). Melancon and DeLuca
(l969) recently reported that rats which were hypercalcemic
due to large doses of vitamin D had an additional transient
hypercalcemia following thyroparathyroidectomy. Parathyroi-
dectomy alone did not duplicate that response. However,
thyroidectomy 8 hours after parathyroidectomy, also gave rise
to an additional hypercalcemia. The secondary serum calcium
elevation was attributed to the removal of the endogenous
calcitonin, since the injection of exogenous calcitonin
prevented hypercalcemia.

The ability of exogenous calcitonin to reduce or prevent
vitamin D-induced hypercalcemia has been established by
Mittleman g£_gl, (I967). They showed that in hypercalcemia

produced by the administration of a single dose of IO5 USP

units of vitamin D3, thyroparathyroidectomized rats had a
significant decrease in the plasma concentration of calcium
during calcitonin administration. A similar response was
achieved when the rats were given a IO-fold greater dose of
vitamin 03. in collateral studies by the same authors on

the release of 85Sr from the skeleton, thyroparathyroidectomy
decreased, and vitamin D3 administration markedly increased,
the ratio of urinary 85Sr to tibial 85Sr. On the other hand,
the administration of calcitonin produced decreases in the
excretion of 85Sr.

Protection against soft-tissue calcification. Gabbiani
‘g£,gl. (I968), at the University of Montreal, Canada, found that
either calcitonin or thyroxine could partially prevent the
calcification of the kidney and heart and the appearance of
osteitis fibrosa produced by administration of parathyroid
extract to therparathyroidectomized rats. When adequate
doses of both calcitonin and thyroxine were administered,
development of the lesions was completely prevented. in a
similar study, Rasmussen and Tenenhouse (I967) observed
nephrocalcinosis in thyroparathyroidectomized rats infused for
l6 hours with parathyroid hormone. When calcitonin and para-
thyroid hormone were infused together over a period of 70

hours nephrocalcinosis did not develop.

IO

On the other hand, according to Hirsch and Munson (l969),
there is a possibility that endogenously secreted calcitonin
might aggravatesoft-tissue calcification. They cited Bajusz
gt 21. (l963), who reported that in rats with experimental
lesions of the coronary artery there was more myocardial
calcification in parathyroidectomized rats than in rats with
intact glands. This increased calcification of soft tissues
was not observed in thyroparathyroidectomized rats whether
they were untreated or given thyroxine. They postulated the
existence of a thyroid principle other than thyroxine that was
responsible for the greater severity of the lesions in para-
thyroidectomized rats. Because this study was conducted before
the discovery of calcitonin was published, the possible

association of calcitonin with the effect was not discussed.

Vitamin D Intoxication

Selye (I932) demonstrated that intoxication of young rats
with an impure vitamin D preparation (irradiated ergosterol)
produced osteitis fibrosa-like bone lesions with multiple
spontaneous fractures. in older rats, hypervitaminosis D tended
to produce calcification in various soft tissUes, especially
in the arteries, the heart and the kidneys. Numerous
additional investigations have since shown that purified vitamin

02,03 and AT-lO (Dihydrotachysterol, a vitamin derivative) can

ll

elicit essentially similar lesions. A suitable review of hyper-
vitaminosis D was made by Mulligan (l9h7). A complete description
of the syndrome and the lesions produced by dihydrotachysterol

was reported by Selye (I957) and Selye gt 21. (i963 and l965).

MATERIALS AND METHODS

Source agg_Maintenance‘gf Animals

Adult female Sprague-Dawley rats were used throughout,
except for the 6 immature rats used in Experiment 2. They were
housed in groups of 2 or 3 in galvanized steel cages with the
front and bottom of wire screen. Water was provided from glass
bottles with stainless steel tubes. Rats were fed commercial
feed*. During the second half of Experiment I the feed was

ground to enable the rats to be able to consume it.

Chemicals

The calcitonin was of porcine origin and provided through
the courtesy of Dr. J. P. Aldred of Armour Pharmaceutical
Company, Chicago, ill. The potency of the hormone was 9 MRC
Units/mg. in Experiment I and 60 MRC Units/mg. in Experiment 3.
One MRC Unit is equivalent to approximately A pg. of pure
porcine calcitonin (Capp, I969). Calcitonin solutions were
prepared immediately before use. The vehicle used was l6%
gelatin in Experiment I and 5% gelatin in Experiment 3. The
vehicle was maintained at 37 C before and after incorporation

of the calcitonin.

 

*Purina Laboratory Chow

12

l3

A solution of dihydrotachysterol* (DHT) in 50 ml. of corn

oil was prepared weekly and stored at h C.

Experimental Procedure

Experiment I. Twenty-four female, adult rats ranging in
weight from 2h5 to 265 g. were randomly divided into A groups
of 6 each. They were weighed daily and examined for the
presence of IeSions or other signs of abnormality. The experi-
ment lasted 30 days.

Each rat in Group I was given 0.5 ml. of cprn oil by
stomach tube and were given subcutaneous injections of 0.2 ml.
of l6% gelatin,once a day, during the experimental period.

Rats in Group 2 were treated daily with SO‘pg. of DHT in
0.5 ml. of corn oil by stomach tube. Dosage was lowered to
25 pg. after 2 weeks of treatment due to loss of weight.

Group 3 rats were treated daily with SO‘pg. of DHT in
0.5 ml. of corn oil by stomach tube. Calcitonin diluted to l6%
gelatin vehicle was injected daily at the dose of 20 MRC Units/kg.
of body weight subcutaneously. The dose of DHT was lowered to
25,pg. at the same time as Group 2.

Rats in Group A were given daily Injections Of calcitonin
in 16% gelatin vehicle at the rate of 20 MRC Units/kg. during

the experimental period.

 

*Mann Research Laboratories, Div. of Becton-Dickinson
5 Co., New York, N.Y.

lh

Necropsy procedures. At the end of the treatment period
the rats were anesthetized with ethyl ether and killed by
exsanguination., Blood samples were obtained by cardiac puncture
with 20 gauge, I inch needles. Heparinized 2 mi. vials were
used to collect blood for hemoglobin concentration and packed
cell volumes. Nonheparinized l0 ml. tubes were used to
collect blood for content of calcium, phosphorusand alkaline
phosphatase.

A gross necropsy examination was performed on each rat.
Weights of the liver, kidneys and heart plus lungs were recorded.

Histologic technigggg. Samples from the aorta, coronary
artery, myocardium, lungs, thyroid gland, stomach, duodenum,
liver and kidneys were collected and fixed in lQ% formalin
solution containing sodium acetate as a buffer. Portions of the
rib cage, tibia and tail were collected and decalcified before
embedding in paraffin. Sections were cut at 6,p and stained
routinely with hematoxylin and eosin. in selected cases, the

Von Kossa stain for calcium salts was applied.

Hematoiogic determinations. Hemoglobin concentrations were
determined by the cyanmethemoglobin method and packed cell
volumes by the microhematocrit method (Coles, I968). Serum
levels for calcium (Patton and Reeder, I956) and phosphorus

(Fiske and Subbarow, l929) were determined by use of a

l5

spectrophotometer*. Alkaline phosphatase concentrations were

obtained by using the Monitor method**.

Exppriment 2. This experiment was undertaken to provide
further information on the effect of DHT given at different
doses to immature and mature rats and to determine the most
appropriate dosage at which DHT could induce detectable lesions
without killing the rats.

Six young female rats averaging lhs g. and 6 adult female
rats with a mean weight of 258 g. were used. One yoUng and one
adult rat were assigned to each of 6 pairs and all rats were
given DHT by stomach tube daily for lh days. Individual dosages
were: 5 us. for Pair i, l0 pg. for Pair 2, 20 pg. for Pair 3,
30 pg. for Pair 10, h0g9 for Pair 5 and 50 pg. for Pair 6.

Body weight was recorded daily and, at the end of the experiment,
rats were killed and the usual necropsy and histologic procedures

were perfonmed. Blood samples were not obtained.

Experimppt 3. Twenty-four female, adult rats ranging in
weight from 250 to 305 g. were randomly divided into A groups of

6 each, as in Experiment I. Experiment 3 differed from

 

*Coleman Jr., Coleman instruments Corp., Maywood, Ill.

**American Monitor Corporation, lndianapolis, ind.

l6

Experiment I in that the daily dosage of calcitonin (20 MRC/kg.)
was divided into thirds and given at 8 hour intervals. The
dose of DHT was ZSng. instead of 50 pg. and the experiment
lasted l5 days instead of 30 days.

Histologic techniques and hematologic determinations were
similar to those in Experiment l. Weights of organs were not

taken. Except for this, necrOpsy procedures were also similar.

RESULTS

Experiment I

figgx_ggjgh£, Differences in body weight among the groups
were recorded at the end of the experiment (Table l). Group I
gained II% with respect to initial weight. Group 2 and Group 3
decreased 35% and Group A gained 20%. Weight changes followed

a similar pattern in Groups I and h and in Groups 2 and 3.

Clinical pjgpg. There were no distinct clinical signs in
Groups I and h other than a steady increase in weight. In Groups
2 and 3 the outstanding signs were progressive emaciation and
muscular weakness. The skin became dry and inelastic, forming
numerous wrinkles. The bones and particularly the ribs were
visible through the skin and there was marked kyphosis in the
lower thoracic and upper lumbar region (Fig. I). Five rats
died, I in Group 2 and h in Group 3. Extreme loss of weight
was a consistent feature in these rats. Persistent diarrhea
was also seen in these rats a few days before death but other-
wise scant and hard feces were common features in rats in

Groups 2 and 3.

Weight pf organs. Table I gives the weights of the organs
in the different groups and the percentages of body weights that
the organs represent. There were not significant differences in

the weightsof the liver, lungs and heart between Groups I and h

I7

l8

Table I. Treatments, Average initial and terminal weights and
selected organs weights of rats in Experiment I.

 

_-

 

 

 

 

 

Aver. Aver. Liver Kidneys Heart and
Group No. rats ini- wgt. Lungs
no. and at end tial at % % %
treatment of expt. wgt. necrop- Wt. -body Wt. bod Wt. bod
(g) sy wt. wt. wt.
(9) (g) (g) (g)
l Corn oil 6 257.6 290.8 Il.2 3.8h l.0 0.36 h.83 1.65
and gelatin
2 DHT* 5*** 26l.l l7l.0 7.u n.32 0.9 0.5u 3.06 I.78

 

3 DHT and
calcitonin** ‘ 2*** 250.0 l67.5 8.l h.82 l.0 0.6h 3.33 l.92

 

h Calcitonin 6 258.3 3l0.0 ll.8 3.79 I.0 0.3h h.50 l.h5

 

I*10HT was given daily by stomach tube to rats in Groups 2 and
3 at the rate of sogpg. for IS days and 25 pg. for the
remaining l5 days 0 the experiment.

** Calcitonin was given subcutaneously as a single daily
injection at the rate of 20 MRC Units/kg. to rats in
Groups 3 and 4.

*** One of 6 rats from Group 2, and A of 6 rats from Group 3,
died during the experiments.

 

 

Figure l

Extreme loss of weight, emaciation, dry and inelastic
skin and kyphosis in rat treated with DHT.’ Similar
signs were seen in rats treated with DHT plus calci-
tonin. Rat 8, Group 2, Experiment I.

20

and between Groups 2 and 3. Organs from Groups I and h were
generally heavier than the ones from Groups 2 and 3. The weights
of the kidneys did not vary appreciably; hence the ratio of
kidney weight to body weight was increased in Groups 2 and 3.

£393; lesions. At necropsy, rats in Groups 2 and 3 lacked
the normal amount of subcutaneous, perirenal and omental fat.
The muscles appeared atrophic and curvature of the spinal column
was pronounced. The skin appeared inelastic with atrophy of the
dermal tissue and of-the cutaneous musculature. There was
mineralization on the surfaces of the myocardium and kidneys.
Gross lesions were not observed in rats of Groups l and h.

Microscopic lesions. Lesions of hypervitaminosis D were
noticed in rats of Groups 2 and 3 (Table 2). Calcification was
most extensive in the aorta, heart and coronary vessels, kidney
and stomach and, to a lesser extent, in the small intestine and
lungs. A few arteries in the region of the thyroid and para-
thyroid gland were calcified but the glandular tissue appeared
to be spared (Fig. 2).

The media of the proximal aorta was almost totally calcified
in the majority of the rats in Groups 2 and 3 (Fig. 3). The
coronary arteries were involved in almost every rat, especially
the internal elastic membrane and the smooth muscle fibers of
the media (Fig. A). in the myocardium, the calcified muscle

cells were distributed irregularly but frequently around the

2I

Table 2. Grading of microscopic soft tissue calcification in
selected tissues from rats in Experiment I.

 

 

 

Gaoup :at Aorta Heart Kidney Stomach Duodenum Liver Lung
o. o. -

 

l l O 0 2 O 0 0 O
2 O O 2 O O O O

3 O O 2 O O 0 O

h 0 0 O O O O O

S O O 2 O O O O

6 O O O O 0 0 O

2 7* 3 3 3 3 3 0 3
8 3 3 3 3 3 O 0

9 3 3 3 2 2 0 0

l0 3 3 3 3 O O 0

ll 3 l 3 I 0 0 0

l2 3 3 3 3 3 0 O

3 l3 3 3 3 3 l 0 0
IA* '3 3 3 3 I 0 l

15* 3 3 3 3 3 0 2

l6 3 3 3 3 3 O 3

l7* 3 3 3 3 3 O 0

l8* 3 3 3 3 3 0 2

4 l9 0 O O O O O 0
20 O 0 0 O 0 0 0

2i 0‘ O l 0 O O 0

22 .0 O 2 O 0 O O

23 O 0 l O O O 0

2R 0 O 2 O 0 0 0

 

*Died during the course of the experiment.

Grade 0 - no lesion
l u'minimal
2 - moderate
3 - maximal

22

 

 

Figure 2

Small arteries in the vicinity of the thyroid gland
with calcification in the media. Rat l6, Group 3,
Experiment I - H 8 E stain x 560.

23

 

 

Figure 3

A cross-section of the aorta showing uniform impreg-
nation with calcium salts in the media. Note a sence

of inflammatory reaction. Rat l0, Group 2, Experiment
I - H 5 E stain x 560.

 

2h

 

Figure A

Involvement of coronary arteries with calcification of
the wall and heavy calcium salt deposits between
the myocardial fibers._ Rat l0, Group 2, Experiment I -

H s E stain x 562.

25

coronary arteries or in the subendocardial region (Fig. 5).
In some instances, the deposition of calcium was so extensive
that it was detectable in coronary arterial branches of all
dimensions (Fig. 6). The endocardium, especially of the left
atrium, was occasionally severely calcified. Valves were
normal in all cases.

Kidneys were the only organs that presented some degree of
calcification in rats of all groups. However, the degree of
mineralization was much less in Groups I and A. There were
only isolated areas of mineralization situated in the vicinity
of the cortico-medullary junction. These were interpreted as
a common finding for adult rats and were not considered as
atypical calcification in the statistical treatment of the
different groups. In Groups 2 and 3, the kidneys were the
commonest site of severe calcification in all the rats. Calci-
fication usually involved extensive areas in the subcapsular
zone and localized areas in the medullary portion (Fig. 7).

The cells of the epithelium of the convoluted tubules were
frequently impregnated with calcium. Sometimes the basement
membranes were heavily calcfified, the same being true of branches
of the renal artery in the hilus of the kidneys (Fig. 8).

The stomach was also a common site of severe calcification
in the rats of Groups 2 and 3. Prominent changes were in the
wall of arteries, the tunica muscularis and muscularis mucosae

(Fig. 9). When mucosal changes occurred, they were restricted

 

26

 

Figure 5

Myocardial calcification.

The deposition of calcium was

frequently found around coronary arteries or in

the subendocardial region.
- H 8 E stain x 560.

Rat l3, Group 3, Experiment I

27

 

 

. Figure 6

Heavy deposition of calcium salts in coronary arterial
branches of different dimensions. Rat l7, Group 3,
Experiment I - H 5 E stain x 562.

 

 

 

Figure 7

Disruption of the architecture of the renal tissue
with deposition of large masses of calcium salts in
the cortical area. Rat 9, Group 2, Experiment I -
H s E stain x 560.

 

29

 

Figure 8

Branch of the renal artery in hilus of kidney with
mineralization in the media and disruption of the

muscle fibers. Rat l0, Group 2, Experiment I
- H 8 E stain x 560.

30

 

 

Figure 9

Tunica muscularis, acid-secreting mucosa and lamina
propria of stomach with severe cellular destruction
and massive deposition of calcium salts. Rat l6,
Group 3, Experiment I - H 8 E stain x 560.

3i

to the acid-secreting mucosa and lamina propria. Occasionally,
cells lining the glands were calcified and at times calcified
concretions were in the lumens.

The duodenal mucosa, tunica muscularis and muscularis mucosa
appeared normal, whereas calcium deposits often appeared in
the walls of small arteries and arterioles (Fig. l0).

Calcification of the lung tissue was not common. in some
rats of Groups 2 and 3 calcification occurred in the walls of the
alveolar ducts and alveoli. Sometimes the pulmonary elastic
tissue and the walls of interalveolar capillaries were calcified
(Fig. ll).

Bone changes were evident in Groups 2 and 3. increased
density of trabecular bone was the principal feature. This
was noticeable near the epiphyseal plate of the tibia (Fig. l2).
Narrowing of the marrow spaces, irregularity of the epiphyseal
plate and failure of osteoid to calcify properly were among the

more priminent changes (Fig. l3).

Statistical interpretation pfi_microscopic lesions. Samples
of aorta, heart, kidney, liver, stomach, intestine and lungs
were graded numerically as to the degree of mineralization and
the different groups were compared. The results are given in
Table 2. The 95% confidence limits for observations consisting
of 6 animals are: (l) for Groups I and h, in which no atypical

calcifications were noted, 0 to 39.3; (2) for Groups 2 and 3

 

 

Figure l0

Wall of small artery of the duodenum with mineralization
of the media and disruption of muscle fibers. Rat l2,
Group 2, Experiment I - H 8 E stain x 560. '

 

33

 

Figure ll

Lung. Notice calcification in the alveolar and
peribronchial tissue. Rat l6, Group 3, Experiment I
H 8 E stain x 560.

-__ ._

 

Figure l2

Tibia.~ Increased density of trabecular bone. Bone
marrow spaces are narrow and hematogenous marrow is
almost absent. Rat l2, Group 2, Experiment I - H 8 E
stain x 560.

35

 

 

Figure l3

Tibia. Notice irregularity of epiphyseal plate, few
marrow spaces and absence of hematogenous marrow.
Rat l3, Group63, Experiment I - H 8 E stain x 560.

36

in which all the subjects showed atypical calcifications, 6i
to l00 percent. Since there is no overlap in these confidence
limits, the probability of observing these effects by chance
is considerably less than 0.05 percent. Therefore, these
observations are statistically valid. The same conclusion

was drawn when the data were analyzed by the chi-square method

applying the Yates allowance (Lewis, l966).

Hematologic determinations. The results are given in
Table 3. Packed cell volumes and hemoglobin concentration
decreased significantly (P4:0.05) in Groups 2, 3 and h with
respect to the controls. ICaIcium values increased significantly
in Group 2 (P(0.05). Hemolysis occurred in some of the blood
samples and therefore the values given for inorganic phosphorus
are not sufficient to warrant any conclusions in Groups 3 and
h. For the same reason, the constant K that represents the
averages of the products of calcium and phosphorus is not
valid for Groups 3 and h. Hemolysis of the samples also
prevented the analysis for alkaline phOSphatase in many samples.

The few values obtained are not included in Table 3.

Experiment 2

Due to the limited objective of this experiment, the effects
of different doses of DHT when given to immature and mature rats,
only changes in body weight and microscopic lesions were used

~as bases for comparison.

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37

38

fipgx,ggjgh£. The changes in body weight related to the
different doses of DHT are given in Table A. In young rats
there was no decrease in weight at any dose level. Gains in
weight were recorded in young rats treated daily with 5, IO,
20, 30 and ho pg. of DHT. No change in weight occurred in the
rate given the 50 pg. dose. In adult rats there was a slight
weight gain in rats treated with ID or 20 pg. of DHT. Rats
treated with either 30, no or 50 pg. steadily decreased in
body weight.

Microscopifi lesions. The microscopic lesions were evalua-
ted by using the same numerical scale as in Experiment l. The
results are given in Table 5. The relationship between micro-
scopic lesions and body weight changes were clear-cut in adult
rats. Lesions were more severe in adult rats, especially in

those given 30, #0 or 50 pg. of DHT.

Experiment;

§9g1_gglgh£. Differences in body weight between the groups
at the end of the experiment were not significant. Group I
gained h% with respect to initial weight. Group 2 lost 9%.
Group 3 lost 6% and Group A gained h%. The rate of gain or
loss followed a similar pattern in Groups I and h and in Groups

2 and 3.

39

Table h. Changes in body weight of rats treated with different
doses of DHT in Experiment 2.

 

 

 

Dosage . Age of Initial Final Difference Gain
of DHT rats weight weight or loss
(#9.) (9.) (9.) (9.) (%l
5 Young l50 l80 +30 +20
Adult 260 260 O O
10 Young I60 205 +h5 +28
Adult 275 285 +l0 +h
20 Young lhO I85 +h5 +32
Adult 250 255 +5 +2
30 Young l35 ’ I75 +h0 +30
Adult 2RD 225 -IS -6
#0 Young7 l55 I90 ~35 -22
Adult 250 225 -25 -IO
50 Young I30 I30 0

0
Adult 275 220 -55 -20

 

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Clinical pjgpg, There were no distinct clinical signs in
Groups I and h. In Groups 2 and 3 there were progressive
emaciation and muscular weakness but to a lesser degree than
in Experiment I. Loss of weight was not pronounced. Deaths
did not occur. Toward the end of the experimental period the
skin appeared somewhat dry and inelastic and kyphosis was

noticeable in the majority of the rats in Groups 2 and 3.

Gros§ lesionp. At necropsy, rats in Groups 2 and 3 had the
i
same type of lesions described in Experiment I. Nevertheless,
the intensity of the lesions was of a lesser degree. There

were no gross lesions in rats of Groups I and h.

Microscopjp lesions. Typical lesions of hypervitaminosis
D were noticed In rats in Groups 2 and 3. The numerical
assessment of the lesions is given in Table 6. The location
of lesions followed the same pattern as described in Experi-
ment l, but the intensity of the lesions was less. In some
rats the muscular layer of the stomach was fragmented (Fig. lh)
and the myocardium was somewhat hyalinized, but calcification

was not .a preminent feature.

Statistippl interpretation pf microsc0pic lesions. The
results of the analysis of data in Experiment 3 were similar to
the results obtained in Experiment I. Groups I (controls) and
h (calcitonin) differed significantly from Groups 2 (DHT) and
3 (DHT and calcitonin) with respect to the presence or absence

of typical lesions.

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Figure Ih

Muscular layer of stomach. Observe necrosis and
fragmentation of the muscle cells. Rat I3, Group I3,
Experiment 3, H 8,E Stain x 560.

Ah

Hematologic determinations. The results of the hematologic
determination on the terminal blood samples from treated and
control rats are given in Table 7. Packed cell volumes were
highest in Group 3. Hemoglobin concentration was similar in
all groups. Calcium levels were significantly higher in
Groups 2 and 3 (P<.0.05). Phosphorus levels in the blood were
not significantly altered In Groups 2 and 3. Values for inorganic
phosphorus in Group A are not necessarily representative of the
group, due to hemolysis of the majority of the samples. The
constant K value was significantly higher (P<.0.05) in Groups 2
and 3 as compared to the controls, even though the values for
inorganic phosphorus were essentially the same in Groups I, 2
and 3. Values for the constant K in Group h are not meaningful
due to the hemolysis mentioned previously. Alkaline phosphatase

values are not included for the same reason.

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DISCUSSION

These experiments indicate that calcitonin is unable to
Iarevent soft tissue calcification produced by DHT in adult rats.
Calcification was not prevented even by simultaneously reducing
the length of the experiment, lessening the dose of DHT and
reducing the intervals of administration of calcitonin. Hence,
under the conditions of these experiments, calcitonin adminis-
tration did not have a protective or prophylactic action._

There are many factors either singly or in combination that
could have affected the results already described. The following

might be the more likely:

Dosagéhpfficalcitonin. The dose level of calcitonin
utilized in the experiments described here was 20 MRC Units/kg.
and was similar to the dosage utilized by Farnell (l969). This
author was able to prevent calcification in magnesium-deficient
rats by using calcitonin. As a basis of comparison, Foster
25 pl. (l966) Utilized ho MRC Units/kg. four times daily or
80 MRC Units/kg. once a day for 28 days to produce measurable

effects on bone density in parathyroidectomized rats.

Depletion pfi_endogenous calcitonin. A depletion of calci-
tonin in the thyroid gland is a possibility pointed out by
certain workers. Gittes ppnpl. (l968) demonstrated that when

marked hypercalcemia was Induced by repeated calcium chloride

#6

“7

administration for 6 hours there was a 30 to 60 percent
reduction in the amount of calcitonin in the thyroid gland in
rats. Albrecht (I968) also reported depletion of endogenous
calcitonin after a treatment with daily injections of 5,000 I.U.
of Vitamin 03 for 7 days. Logically, in the present experiments
the release of endogenous calcitonin should have been slight
since the hypercalcemic factor was given for IS to 30 days.
Furthermore, if endogenous calcitonin was not being produced,
the exogenous calcitonin that was administered might have
served only to compensate for this lack of secretion. If, in
turn, the dose of exogenous calcitonin was too low, the
osteolytic effect of parathormone would not be neutralized. In
this hypothetical condition, then, 2 hypercalcemic mechanisms
could possibly be working together (hypervitaminosis D and

hyperparathyroidism) without an adequate compensatory mechanism.

Compensatory parathormone secretion. Due to the experi-
mental design it is not known if the hypercalcemia noted in the
terminal analysis of blood was a feature throughout the
experiments. if hypercalcemia were constant during the entire
experiment, parathormone secretion would not be expected. But
if this were not the case the action of calcitonin could have
resulted in a compensatory secretion of parathormone. Foster

t I. (l966) was unable to produce changes in bone in intact

rats treated with calcitonin and concluded that parathormone

AB

secretion was responsible since parathyroidectomized rats

responded to calcitonin treatment by marked changes in bone.

Qfll action jp.pppl£.;p£g, Another factor to consider is
that the action of DHT on adult rats is extremely severe.
Selye (I957) reported the different sensitivity of adult and
young rats. Young rats had lesions less severe than older
rats and usually the lesions occurred only in the cardio-
vascular system. In Experiment 2 none of the younger rats lost
weight at any dose level of DHT, and the lesions that occurred

were not severe. Adult rats, on the other hand, were more

susceptible to weight loss and lesions were more prominent.

Calcitonin action 1p adult rats. 0n the contrary, adult

 

rats have been found less responsive to calcitonin. The
evidence for this has been provided by several authors.

Milhaud _£_pl, (I967) reported that calcitonin was most
effective in the young, rapidly growing rat. The rate of
hypercalcemia was also found to be slower in the old rats than
in the young rats (Orimo, I967). This suggests a less vigorous
endogenous calcitonin response with advancing age. Similar
results have been obtained by C00per p£_pl. (l967) in rats

and by Care and Duncan (l967) in sheep and lambs. Rasmussen
and Tenenhouse (l967) pointed out that a change from a l25-g.

rate to a ZSO-g. rat usually leads to l- to 2-fold decrease in

A9

sensitivity to parathyroid hormone, but at least an 8- to
IO-fold decrease in responsiveness to calcitonin. Pechet

.2; pl. (l967) anticipated that calcitonin ”will be found to
play a more prominent role in younger animals with high rates
of bone growth than in older animals, since the greater the
rate of bone turnover, the greater the need for the regulation
of bone resorption promoted by calcitonin".

The above considerations regarding the action of both DHT
and calcitonin on adult rats led to the conclusion that the age
of the rats utilized in these experiments is probably the most
important single factor in explaining the failure of calcitonin

to prevent soft tissue calcification.

"gppppg‘phenomenon". Possibly what Raisz p£_§l, (l967)
described as an "escape phenomenon" could have occurred. This
is mainly a reduced effect of calcitonin after repeated injections.
Bone in culture failed to respond to further additions of calci-

tonin after #8 hours. In an jp_vivo experiment, the hypocalcemic

 

effect of calcitonin was less in rats that had been previously
treated with calcitonin than in control rats with no prior

treatment with calcitonin.

Diet. Workers in several laboratories have mentioned that
dietary calcium and phosphate has an influence on the response

to exogenous calcitonin. Hirsch and Munson (l969) pohwted out

50

that the dietary intake of calcium and phosphate and, especially,
the calciumzphosphorus ratio influence calcium absorption and
excretion, the level of serum calcium and hence the secretion
of parathyroid hormone, the rate of bone resorption and, most
likely, the secretion of calcitonin. The hypocalcemia produced
by calcitonin in rats fed a diet containing 0.05% calcium and
0.3% phosphorus (standard Harvard low-calcium diet) was much
greater than that produced in rats fed a commercial diet which
was low in phosphate and calcium. This probably indicates that
the response to calcitonin administration could have been
considerably increased by adding phosphate to the diet used in

our experiment.

SUMMARY

Three experiments, using a total of 60 rats, were conducted
to determine the influence of calcitonin administration in
preventing or diminishing the clinical signs and tissue calcifi-
cation associated with administration of dihydrotachysterol
(DHT), a vitamin derivative.

Daily oral administration of 50 pg. of DHT in corn oil
produced clinical signs of progressive emaciation, muscular
weakness and death. Kyphosis and enteric disturbances were
evident. There were gross evidences of mineralization on the
surface of the myocardium and kidneys. Microscopically there
was extensive calcification in the aorta, heart, kidneys, stomach,
and, to a lesser extent, the small intestine and lungs. Rats
given the same amount of DHT and 20 MRC Units/kg. of calcitonin
had the same clinical signs and lesions as those given only the
DHT. The degree of soft tissue calcification was dose-related.
Decreasing the interval of calcitonin administration had no
influence in decreasing the soft tissue calcification. Calci-

fication of soft tissue by DHT was not prevented by calcitonin.

SI

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388 -389 a

VITA

. The author, Jorge Antonio Villar, was boen on September 2#,
I928 in Buenos Aires, Argentina. His father, Antonio Villar was
a businessman and his mother, Maria isabel Rubinchik is a
housewife.

The author received his elementary and high school education
in the Colegio San Jose, Buenos Aires, Argentina. In l9#7 he
entered the Veterinary School, University of Buenos Aires.

After being drafted for I# months, he received the degree of
D.V.M. in I953. In l95#, the author was hired by the Animal
Health Division of the Department of Agriculture and Livestock,
Argentina. In I958, he joined the National institute of
Agricultural Technology (INTA) and was Head of the Artifical
Insemination Center and Breeding Diseases in the Pergamino
Experiment Station, Buenos Aires Province, Argentina. In
September l96l he received a U.S./A.i.0. scholarship and came
to the United States of America. He obtained a Master of
Science degree in the Department of Surgery and Medicine, Kansas
State University, Manhattan, Kansas in I962.

Upon returning to his country, he worked on Project No. 53
of the United Nations doing research in the area of fertility
in ruminants. He also taught in the School of Agronomy,

University of Mar del Plata, Balcarce, Argentina.

60

6l

In September I968, again with a scholarship provided by
U.S./A.I.D. he entered the Department of Pathology, Michigan
State'University to train himself in a new area of interest.
Upon returning to Argentina he will be assigned to a Diagnostic
Laboratory Unit that is being deveIOped in the Balcarce
Experiment Station, Balcarce, Buenos Aires Province.

The author is a member of a number of professional and
honorary organizations in his country. He has published about
l0 journal articles describing his research in the area of
animal reproduction and artificial insemination.

The author married Olga Beatriz Bonaldo of Gualeguay,
Entre Rios Province, Argentina in I959. He and his wife have
two children: Ana Valeria, age 6 years and Martin Jorge, age

3 years.

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