MOLYBDENUM IN THE NUTRITION
OF THE RABBIT AND RAT
LEWIS ROBERTS ARRINGTON
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
UNIVERSITY OF FLORIDA
3 12112 08552 27811 i
3 1262 08552 2786
In the planning of this research, in conducting the
experiments and in preparation of this dissertation, acknowledgement
is made of the help and counsel of many individuals. The author
wishes to express sincere appreciation to all those who have con-
tributed to the completion of this dissertation.
Dr. George K. Davis has made available necessary facilities
for this research and as chairman of the supervisory committee has
guided the research program.
Dr. R. B. Becker, Dr. T. i. Stearns, Dr. Ray L. Shirley,
Dr. IL D. ;allace, Dr. A. F. Novak and Dr. R. L. Enerson served as
members of the special supervisory committee.
Mr. Sam L. Hansard has given material aid and advice in
the experiment involving the use of radioactive isotopes.
Mr. J. D. Burke made blood volume determinations on the
Dr. John P. Feaster has given personal assistance and co-
Mr. John T. lIcCall and other members of the staff of the
Nutrition Laboratory have aided in the laboratory analyses.
Dr. Max A. Jeter aided in the planning of some of the experi-
ments and other Praduate students have assisted in other phases of
vr. Juan 3!la, Mr. Richard Sparrow and !.r. Max Garhart have
contributed by attending the experimental animals.
TABLE OF CONTENTS
INTRDUCTI ON. .......................... 1
Molybdenum in Plant and Animal Nutrition . . . . 2
Molybdeamm and Copper Interrelationships in
the Animal Organism. . . . . . a $
Molybdenum and Phosphorus Ietabolis* . . .. . 7
EXPERIMENT I. Molybdenum Taxicity in the Rabbit . . . . 10
Experimental Procedure . . . . .. . . . . 10
Results and Discussion . . . . . . . 11
SXPERfIE!T II. The Effects of Varying Levels of Molybdenum
upon the Growth of Rabbits ... . .. . ...... 21
Experimental Procedure . . . . . . . . . 21
Results and Discussion... .... . . ... 22
EXPERIMENT II. The Effects of Varying Levels of Molybdenim
upon the Hemoglobin, Red Hlood Cell Count
and Blood Volume of Rabbits .... . . 25
Experimental Procedure . . . . . . . . . 25
Results and Discussion . ........ .. ... ... 26
EXPERIEIT IV. The Role of Copper in Relation to Molybdenu
Toxicity . . a ......... . .. . .a 32
Experimental Procedure . . . . . . . . . 32
Results and Discussion . . .. M . .. .. 33
EXPERIElIT V. The Effects of Low Intakes of Dietary Molybdeau
and of Vitamins A and E in these Diets upon the Grorth
and Reproduction of Rats . ... . ..... 37
Experimental Procedure . . . ... 37
RBsults and Discussion .. . . . . . 39
EXPER EINT VI. Phosphorus Metabolism and Distribution of
Phosphorus32 in the Rabbit and Rat .. . . .. 46
Experimental Procedure .. . . . . . . b6
Results and Discussion . . . . . ... LB
GENERALSUMARY.. ..... *-..........a*@ e s so 58
SmAuary wnd Col .usions . . . . . . 58
Recommendations for Future Research . . . . .. 60
BIBLIOGRAPHY . .......... ...... 63
List of Tables
1. Sumary of Molybdenum Toxicity S3l toms in :abbita . . 12
2. Mean Weekly Weights of Experimental Rabbits . . . . 23
3. Mean Hemoglobin Values for qkperlental Rabbits . . 27
4. Hemoglobin of Some Individual Rabbits RBceiving
Dietary Molybdenum . . . . a 28
5. Hemoglobin and Red Blood Cell Counts of Individual Rabbita 29
6. Blood Volume, Hemoglobin and Red Blood Cells of Normal
Rabbits and Rabbits Anemic with Molybdeanu Toxicity . . 31
7. Effects of Copper Therapy upon eight, Henmorlobin Level,
and Red Cell Count of Rabbits Affected with !.'olybden
Toxicity. . . . . . . . . 35
8. Accumulation of Copper in the Livers of Experimental
Rabbits on Different lHolybdemnu and Copper Intakes . 36
9. Gamposition of Rat Rations . . . . . . 39
10. Mean weekly y Weights and Total Gain of Male Rats . ... 0O
11. Mean Weekly ?eights and Total Gain of Female Rats . 41
12. Fertility of Male and Female Rats *eceivinr Dietary
Molybdemn and Copper . . . *. * .. L3
13. Effects of Dietary Uolybdenum nnd Methods of Adcministratin
upon the EBcretion of Phonphorus32 and of Total Phosphorus
by the Rabbit . . . . . . . . . . . 49
31. Rffects of Dietary Molybdenum, Age and Tim after Dosing
upon the Excretion of Orally Administered Phosphorau32
bytheRat. ....**. ... ... ... .... $51
15. Effects of Molybdenum upon the Deposition of Phoephoru32
in Selected Tissues of the Rabbit . . . . .. 52
16. Effects of Dietary Volybdenmm upon the Deposition of
Phosphorus32 and Total Fhosphorus in Selected Tissues
of Mature Rats . .. .............. ... 53
17. Deposition of Phosphorus32 and Amount of Total Phosphorus
in Selected Tissues of Young Rats . . .. . 5
18. Total Phosphorus Content of Femur Bones of Rabbits Fed
Varying Levels of Molybdenum and Copper . . . . . S6
List of Illustrations
1. Rabbits Receiving Dietary Molybdenum and
Exhibiting Typical Alopecia . . . ... . 1
2. Young Rabbits Exhibiting Front Leg Abnormality . . 16
3. Bones from Young Rabbit Showing Front Leg Abnormality . 19
4. Forelegs of Young Rabbit Showing Shifted Carpus Joint . 19
5. Male Rats on Molybdenum Diets Bdhibiting alopecia
over the Back ... ... .. ...... ... 45
Although molybdenum is one of the scarcer mineral elements, its
compounds are widely distributed in nature. It occurs in subterranean
deposits and in small but varying amounts in the surface layers of
soil where it is available to plants. It is present in plant and
animal tissues. Its wide occurrence in plants and animals has stimu-
lated much interest in evaluating its function in these forms of life.
Thus it is established that molybdenum is essential to the growth of
certain microorganisms and many higher plants. It is not known to be
a dietary requirements for any animal species; rather, its presence is
harmful under certain conditions.
Animal species vary in their tolerance to molybdenum in the
diet, ruminants being less tolerant than are non-ruminants. The
action of molybdenum in the animal body whereby the toxicity is pro-
duced is not known.
There are areas of the world where the presence of molybdenum
in the soil and in the plants makes grazing hazardous or impossible.
'While these areas are rather restricted, a potential problem exists
which cannot be limited to areas now affected. Since many plants
respond to the presence of molybdenum and reflect this to some extent
in their tissues, the practice of molybdenum fertilization creates a
problem which may be extended to other areas.
This study has been designed to extend observations of the
effects of dietary molybdenm to the rabbit and expand studies with
the rat. The herbivorous dietary habit of the rabbit and the size of
the animal make it possible to study a herbivorous species under
laboratory conditions. The difference in the response of ruminant
and non-ruminant animals toward molybdenum makes it desirable to
establish the relative position of this herbivorous, but non-ruminant
species, with regard to its tolerance for dietary molybdenum.
Molbdenum in Plant and Animal Nutrition
The wide distribution of molybdenum in the plant and animal
kingdom has been observed by several investigators. In 1931 ter Meulen
(59, 60) reported the presence of molybdenum in microorganisms, soil,
wood, peat and in the blood, liver and spleen of domestic animals and
man. Since that time it has been found in hens eggs (32, 56) and in
human and c oWs milk (4, 33, 34).
The role of molybdenum in the nutrition of certain micro-
organisms was recognized before its significance in higher plants was
established (10). In 1931, Bortels (11) reported that molybdenum is
essential to biological nitrogen fixation. Since that time there have
been many reports of investigations which have established the role of
molybdenum as a stimulant to growth and nitrogen fixation in certain
microorganisms (13, 19, 31, 47, 72, 73).
Some higher plants have shown favorable response to the presence
of molybdenum in the soil. Arnon and Stout (5) in 1939 demonstrated
the need for molybdenum in the nutrients of to&mto seedlings and later
its value for lettuce waa established (16). Molybdenum is required by
many plants (29, 46, 64, 77). Leguminous plants exhibit a greater
response to molybdenum than do non-legumes. In these plants it has been
observed to increase growth or yield, to improve the color and stimulate
nitrogen fixation (1, 2, 12, 48, 78).
Under certain soil and climatic conditions, plants accumulate
excessive quantities of molybdenum which may become critical in
relation to their use as pasture or hay crops. Some of these factors
which may alter the accumulation of molybdenum have been established.
A species difference has been established from the observations that
leguminous plants concentrate more of the soil molybdenum than do non-
legumes (8, 40, 52). An alkaline reaction of the soil favors an
increase of the element in plant tissues whereas very little molybdenum
is taken up under acid soil conditions (3, 8, 53). Young growing
plants contain more molybdenum than mature plants on a dry matter basis
(8, 37). The amount of the element in the soil is also a factor although
its effect upon plant tissue accumulation is often masked by the form
in which it is found and other soil conditions (53). Whore those con-
ditions exist, serious abnormalities in grazing cattle or sheep have
been common. Such abnormalities have been reported from areas in
California and Southern Florida in the United States and in England,
Australia and New Zealand. whilee the conditions are not the same in
all of these areas, the forage from these areas contains a relatively
high amount of molybdenum.
Beath and associates (9) in 1935 observed certain abnormalities
in cattle which were grazing pastures containing 89 ppm molybdenum.
The first specific description of pathological conditions in cattle
which were attributed to excess molybdernm was made by Ferguson in
1938 (39). Since that time his observations have been confirmed and
extended in the other areas of the world where cattle and sheep are
The external appearance of the plants gives no evidence of
abnormality and only by analysis is the excess molybdenum detected.
When plants from an affected area become mature or are made into hay,
the toxicity is much reduced. It is possible that the green forage
contains a form of molybdenum which is more toxic or that it contains
another substance which renders the molybdenum more effective. When
molybdate salts are fed to animals receiving dry hay and to those
receiving green succulent feed, the toxicity is less severe or even
absent in those receiving dry hay (37, 38, 39).
The toxic symptoms which have been observed in cattle and
sheep are characterized by diarrhea, loss in weight, change in the
color of the hair coat, anemia and eventual death unless removed from
the affected area or treated (8, 17, 25). Horses grazing on the same
areas which cause trouble in ruminant animals are not affected (17,
Investigations concerning the effects of dietary molybdenum
have not been limited to farm animals. Studies have been undertaken
using laboratory animals, but these have been principally confined to
experiments using rats (21, 36, U1, 42, 49, 63, 75). The results are
in general agreement that rats are more tolerant of dietary molybdenum
than ruminant animals; nevertheless, rats are subject to some toxic
effects of excess molybdenum. The manifestations of the toxic symptoms
observed in rats do not parallel those in cattle and sheep. Severe
diarrhea and anemia which are common symptoms in ruminants have not been
consistently produced in rats.
Molybdenum as molybdenum trioxide, calcium molybdate, and
amoninm molybdate supplying 1200 to 6000 milligrams of molybdenum
per kilogram was fatal to guinea pigs and rats (36). An equivalent
amount of molybdenite was not fatal.
Other abnormalities which have been more recently attributed
to molybdenum are interference with normal reproduction. Jeter (49)
in 1951 found that a high percentage of male rats which were fed 80
and 140 ppm molybdenum in the diet were sterile. In female rats fed
the same rations there was an irregular estrus cycle. Thomas and
Moss (76) have observed infertility and a lack of libido in young
dairy bulls. In both of these species, the male infertility was
accompanied by damage to the germinal epithelium and interstitial
cells of the testes.
olydenum and Copper Interrelationships
in the Ani Organism
One of the most significant discoveries with regard to molyb-
denum toxicity is the therapeutic action of copper. When adequate
amounts of copper are present in the dietary (the quantity necessary
depends upon the molybdenum present) the symptoms of molybdenum toxicity
seldom develop. Furthermore, toxic symptoms which may be present can be
corrected with copper supplementation. This action of copper has been
demonstrated in practically all species for which a toxicity has been
reported (25, 37, 38, 63). When there is an excess of molybdenum and
a deficiency of copper in the soil which is normally reflected in the
plant, the adverse effects upon animals grazing these plants can be
corrected by copper fertilization (25).
The abnormalities caused by copper deficiency and molybdenum
excess are such that it is frequently difficult to distinguish between
the two (28, 74). Because of the therapeutic effect of copper, it
might be expected that excess molybdenum should deplete the body of
its stores of copper and create a copper deficiency. Such a decrease
has not been consistently observed. Dick and Bull (30) reported a
decrease in the copper content of the liver of ewes which were fed
copper and molybdenum, but others (51, 58) have observed an increase
in liver copper under similar conditions. 'When the copper intake is
below that necessary to insure positive copper balance, molybdenum
will reduce the copper stores more rapidly. However, 'hen adequate
copper is provided, molybdenum causes an increase in the copper store
but at the same time may precipitate the symptoms of copper deficiency.
Molybdenum apparently tends either to fix the copper in a form which is
not available to the tissues or to antagonize by hindrance the copper
containing enzymes (58).
The physiological action of molybdenum and the mechanism through
which copper exerts its therapeutic effect are unknown. Based upon
some evidence, several different theories have been proposed which are
designed to explain these functions. McGowan and associates (55)
believe that the activity of bacteria in the gastrointestinal tract
is controlled by catechols and that molybdenum renders the catechols
ineffective by formation of complexes. The uncontrolled activity of
the bacteria thus causes the severe diarrhea. The therapeutic effect
of copper is thought to be due to its toxic action upon bacteria.
This theory is not generally accepted in light of present knowledge.
Wisconsin workers (63) have suggested that the action of molybdenum
may be due to the formation of a copper molybdate complex which is
poorly absorbed. The effect of molybdates upon certain enzymes in
vitro and the interaction of copper and molybdenum in vivo have
suggested to others that molybdenum interferes with some enzyme
sy8tems, particularly the copper enzymes (7, 50). None of these
theories has yet been supported by adequate evidence to explain the
action of molybdenum.
Molybdenm and Phosphorus Metabolism
Among the symptoms of molybdenum toxicity which have been
observed in cattle has been an abnormal skeletal development. This
has been accompanied by other evidences of abnormal phosphorus meta-
bolism. In areas of Florida where there is a deficiency of copper,
and molybdenum is present, abnormal bone formation has resulted (26,
27). Specific symptoms have been stiffness, broken bones, enlarged
joints, erosion of the joints and a condition resembling rickets in
young animals and osteomalatia in older cattle. Thomas and w oss (76)
have observed stiffness and bone involvement in young dairy bulls fed
molybdenum. The plasma inorganic phosphorus and serum phosphatase were
normal, but there was erosion of same joints and union and fusion in
one joint. The ash content of rib bones was normal. The stiffness
and brittleness of bones which have been observed by others (17, 25,
26) also reflect abnormal bone development, possibly related in some
manner to the metabolism of molybdenum.
Sane of the effects of molybdenum upon absorption and excretion
of phosphorus have been studied using radioactive phosphorus3. N;hen
phosphorus and molybdenum were administered simultaneously to rats,
the phosphorus uptake was increased. When molybdenum was fed previously
there was the reverse effect upon phosphorus (21). The normal excre-
tion of phosphorus by cattle where most of the phosphorus is excreted
in the urine has been observed to change to a pathway where more is
excreted in feces when the animals were given copper and molybdenum
supplements (68). No similar effect could be observed in rats,
although there was a change in the rate of excretion (69). These
results further indicate some abnormality in phosphorus metabolism.
Other possible relationships between molybdenum and phosphorus
metabolism have been shown through the action of molybdate ions upon
phosphatases. Bossard (14, 15, 22) has reported an inhibitory effect
of the molybdate ion upon certain phosphatases including a slight
inhibitory effect upon calf bone phosphatase. Also observed was an
increase in serum alkaline phosphatase and blood inorganic phosphorus
in copper deficient cattle (26).
The literature concerning molybdenum and its relation to other
elements in the animal organism has been reviewed by several authors.
The relation of certain minerals to animal health has been reviewed
by Russell (65) and the relation of mineral elements to health of
ruminants in Great Britain has been sumnarizod by Green (43). The
role of molybdenum and other minerals in the nutrition of animals and
plants has been more recently reviewed by Marston (57).
MOLYBDENUM TOXICITY IN THE RABBIT
Some differences in species with relation to their tolerance
for molybdenum have been observed. Limited studies with horses and
swine have indicated a relatively high tolerance for these species
and the numerous investigations with cattle and sheep show that these
animals have a low tolerance for molybdenum. Because of the different
effects of molybdenum upon ruminant and non-ruminant animals, it
appeared desirable to determine whether or not the element was toxic
to rabbits and to establish its relative toxicity in this species.
Upon the initiation of this study there had been no reported investi-
gations of the effect of molybdenum upon rabbits. Smith and Ellis (70)
have described the symptoms of simple copper deficiency in the rabbit,
but there is no evidence that excess molybdenum was involved in the
The objective of this experiment was to describe the effects
of molybdenum excess upon rabbits.
Male and female Dutch rabbits of different ages were used for
this experiment. They were maintained in metal cages with metal wire
bottoms. The rabbitry was housed in an open barn with natural venti-
lation and without artificial heat.
The basal ration for all treatments was a commercial ration
in pellet form (Staf-O-Life Rabbit Pellets, manufactured by Royal
Staf-O-Life Mills, Memphis, Tennessee) and had promoted satisfactory
growth for rabbits in this laboratory. Feed was given ad libitum and
all unconsumed feed was removed every other day or more often if it
became dirty. Small quantities of fresh green feed were riven each
week. Fresh water was supplied from glass drinking bottles.
Molybdenum was added to the basal ration as sodium molybdate
(Na2Mo,02H2O) in amounts calculated to provide 0.014, 0.05, 0.1, 0.2
and 0.4 per cent molybdenum. The basal ration contained 2.7 ppm
molybdenum and 16.l ppm copper by analysis using the thiocyanate-
stannous chloride method (67) for molybdenum and the carbamate method
(66) for copper, Molybdate salt was added to the pellets by spraying
on an aqueous solution and allowing it to air dry. Analyses of
composite samples made from the rations indicated that it the higher
levels of molybdenum the amount incorporated was slightly less than the
Thirty-four weanling rabbits six to seven weeks of age w-ere
placed on the experimental rations and six mature rabbits were changed
from the basal ration to three of the treatment levels of molybdenum.
Observations were made daily for any development of toxic symptoms and
the order of appearance of any abnormalities was recorded.
Results and Discussion
Gross abnormalities which were attributed to excess molybdennm
developed in the rabbits fed 0.1 per cent or more molybdenum. Deaths
resulted from the higher levels of molybdenum. A summary of the results
is presented in Table 1. All of the young rabbits receiving 0.1 per
cent molybdenmn developed some symptoms of toxicity although the
condition became severe in only three of these rabbits. These three
were treated with copper when it became apparent that they would not
survive without treatment. No detrimental effects of 0.1 per cent
molybdenum were observed in the two mature rabbits over a period of
eight weeks. Five-hundredths per cent molybdenum in the ration
caused no gross abnormalities in any of the animals. There was some
evidence of retarded growth of the rabbits on this level of molyb-
denum in the early weeks after weaning. There was a slight loss of
hair which soon returned to normal, but other symptoms of toxicity
were absent. None of the rabbits receiving 0.01 per cent molybdenum
developed abnormalities which could be attributed to molybdenum in
SUI2ARY OF MOLYBDENUR TOXICITY SYMPTOMS IN RABBITS
No. of and Front leg
Treatment Rabbits Deaths Anemia Dermatosis Abnormality
0.4% Mo. 4 4 present O* 1
0.?% Mo. 5 4 present 4 2
'.1% Mo. 7 present 5 2
7.051 o. 5 0 absent 0 0
'0.01~, o. 12 0 absent 0 0
Basal 7 0 absent 0 0
* Deaths occurred early and no alopecia observed.
*4 Severe toxic symptoms developed in three rabbits with deaths
probable but copper therapy was initiated to prevent death.
The time of appearance of the abnormalities varied from about
four veeks in the -.reanling rabbits to several months in the mature
animals. Follo.ring the first appearances of the condition, the
syndrome became progressively worse and death usually occurred within
The syndrome which developed was characterized by a decrease
in feed intake, a decrease in weight gains or actual weight loss,
roughening of the hair coat with loss of hair beginning -rith complete
denudation over small areas which generally spread to practically the
entire body. The alopecia was accompanied by varying degrees of
dermatosis with rough,. dry, scaly skin. There was no evidence of
skin infection or inflammation. Anemia became severe and the animals
soon died unless treated. In some young rabbits an abnormality in the
front legs was present.
The anemia, alopecia and dermatosis make the condition appear
similar to the symptoms of copper deficiency in rabbits described by
Smith et al (70). In contrast to the simple copper deficiency symptoms,
no graying of the black hair in the rabbits on this experiment was
observed. In Figure 1 are pictures of rabbits exhibiting typical
Limited observations .'ere made of the sex interest of male
rabbits. Two males on high levels of molybdenum which were suffering
from moderate and severe symptoms of toxicity demonstrated normal sex
interest when placed with females. This observation is in contrast to
that made on young dairy bulls receiving molybdenum (76). The
Figure 1. Younp rabbits receiving dietary molybdenum and
exhibiting typical alopecia.
_ __ __
r - -- ------
IIL- -- ~ IIILIIICCIIII
fertility of rabbits in this experiment was not determined.
In each of the rabbits which developed the syndrome of toxicity,
with two exceptions, the condition became progressively worse until
the animal died or was given copper therapy. Two of the young rabbits
developed moderately severe symptoms of toxicity but gradually re-
covered spontaneously. The explanation for this is difficult, but
several facts should be observed. The symptoms did not develop in
the early wieks of the experiment as was common with the other weanling
rabbits, so the animals were older when the abnormalities were observed.
The level of molybdenum (0.1 per cent) was borderline for toxicity in
mature rabbits. The development of the toxicity was slow and the
recovery was slower than the recovery observed with copper treatment.
One of the more specific abnormalities not observed in other
species was a "break-down" in the front legs of some young rabbits
receiving high levels of molybdenum. This condition occurred in five
of the ten weanling rabbits which developed other toxic symptoms. It
did not appear in any of the mature rabbits nor any of the other
young rabbits after they had passed ten to eleven weeks of age.
In Figure 2 are pictures of typical rabbits which exhibit this
foreleg abnormality. The first evidence of this condition was an
apparent soreness and slight difficulty in walking. There was,
however, no evidence of soreness when the legs were examined. The
affected animals were observed to sit far back on their roar legs and
frequently rest the front legs in the feed cup in apparent effort to
shift the body weight from the front feet. They soon became unable
Young rabbits exhibiting front log abnormality.
to maintain their weight; the legs spread outward and the forepart of
the body rested on the floor. The ability to move the logs was not
lost and in efforts to walk the logs simply slid on the floor. Two
of the rabbits thus affected were treated -.ith copper sulfate when
the symptoms of toxicity became severe. There was only slight improve-
mont in this condition while there was complete recovery from all the
Examination of the bone as it was removed from a typical rabbit
after sacrificing revealed bending of the upper portion of the humerus
and a swollen joint at the junction of the humerus and scapula
(Figure 3). Apparently the twisting of the humerus permitted the
legs to spread outward. Not all of the cases showed gross bending of
the bone or swollen joints and apparently a slight twisting of the
bones permitted the legs to spread outward.
Hear the termination of this experiment somewhat different
abnormalities were observed in the front legs of two young rabbits
which died with coccidiosis. These two animals were receiving dietary
molybdenum but they have not been included in the results described
for the other experimental animals because of the presence of
coccidiosis. The infection was accompanied by anorexia, diarrhea and
other symptoms --hich may have complicated the effects of molybdenum,
In the case of one of these rabbits, the feet of both forelegs
bent inward at the carpus joint (Figure 2). An examination of the
bones as they were removed revealed that the carpus joint of both legs
had shifted out of place and the articular surface of the radius was
exposed (Figure i). The tendons of both joints had slipped out of the
normal position. The scapulo-humoral joint of the right leg was loose
but had not shifted out of place. There was no gross bending of the
bones in this rabbit. In the other young rabbit receiving molybdenum
which died with coccidiosis, significant bending of the lower portion
of the radius and ulna of one leg was observed. There was also slight
enlargement of the scapulo-humoral joint, but no slipped tendons.
Some of the abnormalities of the forelegs of rabbits in this
experiment appear to be similar to the crooked front legs described
by Smith et al (35, 71) and attributed to manganese deficiency. In
the manganese deficient rabbit, the crookedness was confined to the
radius and ulna. Gross bending or twisting of these bones has not
been a characteristic of the rabbits in this study, although these
bones in the leg of one young rabbit were thus affected. The results
of this experiment do not, however, eliminate the possibility that a
deficiency of manganese is involved and further studies should be made
to compare these conditions. Deformities in the legs of rabbits fed
a milk diet were observed as early as 1926 by Brouwer (18), but the
cause of the abnormality in these rabbits was not known.
In Experiment IV the results of copper therapy are presented.
It should be observed at this point, however, that the only symptom of
molybdenm toxicity in these rabbits whichh was not cured by copper
therapy was abnormality of the forelegs. Two of the three rabbits
which were thus treated exhibited the typical front leg "break-down".
The condition in both rabbits improved slightly although all other
Bones from young rabbit showing front leg abnormality.
Forelegs of young rabbit showing shifted carpus joint,
manifestations were completely corrected. The strength of the legs
appeared to improve and in the case of one rabbit in which the left
leg was not severely affected, it returned to its normal position.
The lower photograph of rabbit number 385 in Figure 2 shows the partial
recovery following copper therapy. The bone injury appears to be
permanent. In attempting to relate this condition to that due to a
manganese deficiency, it would be desirable to know whether or not
the crookedness in manganese deficient rabbits can be corrected with
manganese therapy or whether the injury is permanent.
Two of the characteristic symptoms of molybdenum toxicity in
cattle, namely diarrhea and a change in color of the hair coat, have
been consistently absent in the syndrome observed in rabbits. In
view of the marked alopecia occurring in some animals it is possible
that achromotricia may have been masked. It should be observed,
however, that achromotricia was not present in those rabbits vhich
were receiving high levels of molybdeaum but which did not develop
THE EFFECTS OF VARYING LEVELS OF 'OLYBDiUM
UPON,THE GRO'ITH OF RABBITS
Comnon among the symptoms of molybdenum toxicity in species
which are subject to the toxic effects is retarded growth or a decrease
in body weight. Retarded growth has been observed in rats even though
other gross abnormalities were not present. The study of the action of
molybdenum upon the growth of young animals is desirable, therefore,
in evaluating the effects of sub-lethal quantities of this toxic element.
Much of the growth data reported relative to the effects of molybdenum
have not been accompanied by adequate feed consumption data so it is
difficult to determine whether or not molybdenum per se is the cause
of sub-normal growth. The recent experiments of Jeter (49), however,
indicate that the retarded growth of rats cannot be attributed to a
decreased food intake.
This experiment was designed to determine the relative rates
of growth of weanling rabbits fed varying levels of molybdenum.
Twenty-four weanling Dutch rabbits from eight litters were
randomized into five treatments consisting of a basal ration and four
levels of molybdenum added to the basal ration. The rabbits were six
to seven weeks of age and weighed 585 to 820 grams.
The basal ration was the same commercial pellet ration used in
Experiment I. The levels of molybdenum added were O.01h, 0.05, 0.1
and 0.2 per cent of the ration, added as sodium molybdate. Rabbits
were housed and managed under the same conditions as outlined in
Growth was determined from weekly weights made throughout
the fourteen week growth period. Feed intake measurements were made
periodically during the experimental period.
Results and Discussion
The mean weekly weights of the rabbits on the different levels
of molybdenum are shown in Table 2. Molybdenum at the level of 0.1
per cent and higher retarded growth. Below this level there was no
effect upon the rate of gain. A test of significance for the dif-
ference in the amount of gain at twelve weeks for the control group
and the group receiving 0.1 per cent molybdenum showed no statistical
significance (FPO5). At the higher levels of molybdenum, growth was
greatly restricted and most of the animals died before a reasonable
growth period was completed.
Limited observations show that the rabbits on the higher levels
of molybdenum consumed less feed. The intake was not materially
reduced, however, until symptoms of the toxicity appeared. There is
no evidence that the molybdenum rations were unacceptable, for during
the early weeks of the experiment, intakes were essentially normal.
After some weeks on the experiment, rabbits receiving the toxic levels
of molybdenum were observed to pick over the pellets and to scratch
wildly in the feed cups.
When the effects of molybdenum upon the growth of the rabbits
are compared with the action of molybdenum in causing anemia, alopecia
O4 '0O NO
P4 C 0D 0
.4 ?4 N I
NO .4 O
5 a 4
Vl rl 0 O4
44 Go '0 0 0
N 1lNo 0- m
I N \i
4 l 4 lr4 r4 .l
N% En 9 4 4
NO V O O
0 NO %n n 4I
M r 4 4 r-I
-C5 NM C
cl r *H
D N 0 t- N N
N\ No Go U rt
C M cm O- M 10
r .4 %1 % .4
t c c 9 1- m
r rr 9-4 fi
S ai .a *
1 oo ab cr
VU 1 L IA 1o No
0 *! i r4
A C03 CO C; CO CC
and other toxic symptoms, it can be observed that growth was retarded
only in those animals which developed the toxicity syndrome. iThen the
toxic symptoms were absent, even though the rabbits were receiving
molybdenum, growth was not retarded. This observation is in contrast
with the effect of molybdenum upon rats where growth has been retarded
in the absence of other gross abnormalities.
In evaluating the effects of molybdenum in this experiment, it
is necessary to consider the relatively high level of copper (16.l ppm)
in the basal ration. With this intake of copper a greater tolerance
for molybdenum would be expected. The action of molybdenum in retarding
growth is, however, demonstrated. Since the consumption of feed was
somewhat less in the groups in which growth was sub-normal, it cannot
be determined whether or not molybdenum per se retards growth or reduces
the feed intake.
THE EFFECTS OF VARYING LEVELS OF HOLYBDEUIU
UPON THE HEMOGLOBIN, RED BLOOD CELL COUNT
AND BLOOD VOLUME OF RABBITS
Anemia is common in the syndrome of molybdenum toxicity in
cattle and sheep. The investigations which have related anemia to
excess molybdenum have not included intensive studies relative to
other changes in the blood picture. Included in this experiment are
observations of the changes in hemoglobin, red blood cell count and
blood volume of normal rabbits and of rabbits suffering from moly-
Mood analyses were made on the rabbits in Experiments I and
II. The analyses were made concurrently with the development of the
toxic syndrome and with the observations upon the growth of the
rabbits. No alterations were made in the feeding or management pro-
Blood samples were taken from the marginal ear vein and hemo-
globin determined every two weeks on the experimental animals using
the acid hematin method (20). '.hen the first symptoms of toxicity
appeared, determinations were made more often in order to follow more
closely the changes in hemoglobin.
Red blood cell counts were made on the blood from representa-
tive control rabbits and those receiving high levels of molybdenum
before and after the hemoglobin bean to decrease. Cells were counted
in the standard red cell counting chamber after dilution in eyema's
The blood volume of two control rabbits and of six rabbits
receiving varying levels of dietary molybdenum was determined. The
determinations were made with phosphorus32 labelled cells (45) and
also by the dye dilution method (l4, 62). The rabbits were given
ether anaesthesia and both jugular veins were exposed. Injections
and withdrawals were made from opposite veins and the calculations
based upon samples of blood taken after five and ten minute intervals.
Results and Discussion
The mean hemoglobin values which were obtained from rabbits
on the control ration and the different levels of molybdenum are
presented in Table 3. These averages do not represent values from
all rabbits in each group, but are calculated from determinations
made on three to five rabbits in each treatment which were repre-
sentative of the group. The values obtained for the control rabbits
and those receiving the lower intakes of molybdenum (O.014 and 0.05
per cent) were within the normal range for this species (23). The
rabbits receiving 0.1 per cent or more molybdenum in the ration
developed anemia as evidenced by the low hemoglobin. Since the values
reported in Table 3 are averages, the extremely low hemoglobin which
was found in some animals is not evident. In Table 4 are shown the
hemoglobin values for some individual rabbits as anemia developed.
Anemia was first observed at about the same time that other symptoms
of toxicity became evident and the hemoglobin continued to decrease
until death occurred or until copper therapy was initiated. Hemoglobin
as low as 2.0 grams per 100 ml blood has been observed before death
The number of erythrocytes in the blood of normal rabbits and
experimental rabbits before the toxic syndrome developed were within
the normal range for rabbits (23). .'hen anemia appeared and progressed,
there was a corresponding decrease in the red blood cell count. In
Table 5 are hemoglobin values and red blood cell counts of some individ-
ual rabbits. These data relate the hemoglobin measurements to the
number of erythrocytes and further relate the anemia to dietary molyb-
MEAN HEMOGLOBIN VALUiS FOR EXPERIMSMTAL RABBITS*
Per cent molybdnum in ration
Basal O.01O 0.05 0.1 0.2
Initial 12.5 12.1 12.1 11.0 1?.6
2 12.9 12.6 1.3 1?.5 13.2
4 13.0 11.3 12.8 %.5 9.6
6 12.8 11.3 12.9 9.1 7.6
8 13.8 ** 12.5 9.0 11.8
10 12.1 12.1 12.1 7.6 13.3
12 12.9 13.3 12.6 8.7 10.9
14 13. 13. 13.0 10.8 7.3
16 14., 1. 9 %.5 5.7
18 12.1 16.2 16.2 10.6
* Orans hemoglobin per 100 ml blood,
** Values not determined.
' -4 r4
% vr %
N C .4
V\ M l-
*n\ o r*-
a*^ fl (fl
,~t lbel %a
0 N- N %
N N N 4
o 0 0 0
A jN N
N .4 N
r-4 r-4 r-l
- En No
a^ r*' w"
o \ 0
HEMOGLOBIN AID RED BLOOD CELL COUNTS OF INDIVIDUAL RABBITS
7eek of Erythrocytesa
Rabbit No. Ration Expt. Hemoglobin* (millions)
331 Control 12 13,2 6.16
341 Control 17 15.0 6.42
301 Control 17 14.5 5.79
906 Control 17 12.0 5.08
304 0.014% Mo. 8 16.0 6.11
372 0.0O1% Mo. 23 13.1 6.29
332 0.05% Mo. 6 15.0 6.20
342 0.0o% Mo. 17 13.0 6.4o
333 0.1% Mo. 13 3.5 1.55
313 0.% 1- n. 17 10.7 5.54
353 0.1% !o. 7 6.3 3.39
382 0.1% 'o. 5 6.3 3.16
385 0.1% Mo. 3 8.0 3.hO
335 0.2% Mo. 4 5.5 1.89
344 0.2% Ho. 17 7.3 3.50
386 0.2% Mo. 13 3.2 1.20
909 0.2% Ho. 9 4.5 1.53
Crams per 100 ml Blood.
Cells per cubic millimeter.
Limited measurements of the red blood cell size indicated that
erythrocytes were smaller and more variable in size in anemic rabbits
than in normal rabbits.
The blood volume for the control rabbits and experimental
rabbits is recorded in Table 6. Other data relative to the blood
picture which were obtained at the same time are also presented.
The relationship of the plasma volume to the red cell count is also
reflected in the low hemoglobin and red cell count. The blood volume
of all of the rabbits is within the normal range for the age and
weight of the animal. It is significant that blood volume was main-
tained at a normal level even though severe anemia was present. The
results indicate, therefore, that the mechanism for maintaiining blood
volume is not affected in molybdenum toxicity.
NC Nc 0
N 0 0 '
* S S
r. O a N
CO ) r- 0
SO 00 CT^ S
% N In a0 I
O r4 0 N o 7
a 7' N 0 N N
p0 0 0
S0 0 0 C0 0
N (, r" N r\
- 1 H
THE ROLE OF COPPER IN RELATION TO MOL)YDEnUM TOXICITY
In considering the role of molybdenum in animal nutrition, it
is now evident that its effects must be related to the copper intake
and copper status of the animal. The therapeutic action of copper
upon the copper status of the animal has already been reviewed.
Included in this experiment are observations on the prevention of
molybdenum toxicity with dietary copper, the therapeutic action of
copper, and the relation of molybdenum to liver storage of copper in
Two weanling Dutch rabbits, six to seven weeks of age and
weighing 628 and 791 grams, were placed on a ration containing .02%
copper and 0.2% molybdenum. The basal ration was the commercial
rabbit pellets used in prior experiments. Feeding and management
procedures were the same as previously outlined. Daily observations
were made for appearance of any abnormalities and body weights made
weekly for growth estimation. Copper as CuS04.H20 and molybdenum
as NaMoO-S2H20 were sprayed onto the ration from aqueous solutions.
It was necessary to add each of the salts to separate portions of
the ration then mix after drying.
In order to further observe the therapeutic effect of copper,
three rabbits which were receiving high levels of molybdenmn and had
developed the typical toxicity syndrome wore treated with copper. The
clinical manifestations were allowed to progress until it appeared that
the animals would not longer survive without treatment. At this time
they were given a drench of 0.05 gn CuSO~-5H20 in 10 ml water and
thereafter they were supplied drinking water which contained 0.1 em
CuSOi1-H2O per 500 ml water. Clinical observations were continued in
order to follow the changes resulting from copper treatment.
At the termination of the experiment, rabbits were killed or
sacrificed for phosphorus balance and distribution studies. Livers
and femur bones were removed and copper storage in the liver determined
using the carbamate method (66).
Results and Discussion
Neither of the rabbits which was given supplementary copper
and molybderum in the ration developed any of the symptoms of molyb-
denam toxicity. The rate of gain was slightly less but not signifi-
cantly lower than the control rabbits (Table 2). The level of
molybdenum in this ration (0.2%) had proved to be toxic in the absence
of added copper so the protection afforded by the copper is demon-
From other observations made in this phase of the experiment,
an estimation of the ratio of copper required for protection may be
made, .'hen the copper sulfate solution was added to the ration, this
portion turned distinctly blue-rreen in color so that even when mixed
with the normal colored molyldenum treated portion, the copper pellets
were easily distinguishable. The rabbits picked over the ration,
leaving a large portion of the copper treated pellets. Because of the
difference in color, it could be observed that much less than the
calculated amount of added copper was actually consumed. Therefore,
the amount of copper required to protect against the 0.2 per cent
molybdenum was much less than 0.02 per cent added copper and more
than the 0.0016 per cent contained in the basal ration.
The three rabbits which had severe symptoms of toxicity and
were treated with copper responded rapidly to the copper therapy. In
Table 7 data are presented illustrating some of the changes which were
observed following the initiation of copper treatment.
Not shown in the table are the effects of the treatment upon
alopecia and dermatosis. Dermatosis disappeared and the hair began
to return to the denuded areas within about a week.
The return to normal weight gains, normal hemoglobin and full
hair coat was generally complete within one month. There was only
slight improvement in the foreleg abnormality after five months
treatment with copper. The failure of this condition to respond to
copper has been discussed in Experiment I.
The relative accumulation of copper in the livers of rabbits
on the different dietary levels of molybdenum is shown in Table 5.
The higher concentrations of copper in the livers from those rabbits
which received supplementary molybdenum show that molybdenum had a
tendency to cause an increase rather than a decrease in the copper
storage. The concentration of copper in the livers from those rabbits
receiving supplemental molybdenum and copper was considerably higher
than that found in any of the other groups. This intake of copper
EFFECTS OF COPPER TIERWY UFON 1EIJGHT, HLIOGLOBIN LEVEL, AND
R1D CZLL COUNT OF RABBITS AFFECTED 7TTH I.!OLY7DZNU TOXICITY
Rabbit Time after
No. treatment eightt Hemoglobin
333 Initial 1144 3.5
1 week 1137 7.0
2 weeks 1204 10.0
13 weeks 1362 10.8
5 weeks Ij48 12.6
382 Initial 853 5.4
1 week 915 9.0
3 weeks 1080 12.7
5 weeks 1276 13.0
385 Initial 907 7.5 3.75
1 day 8.0 4.15
3 days 871 7.5 3.80
1 week 94u 10.5 h.69
2 weeks 993 11.0
4 weeks 1296 10.3
-- I -- !
was not considered a high level but was estimated to be the amount
necessary to prevent the toxic action of molybdenum. The quantity
of copper found in the livers of the control rabbits is slightly
greater than the amount reported by Lorenzen and Smith ($5) and is
considerably higher than the quantity reported by Cunningham (24).
Although the liver copper was not greatly increased except in
the group receiving supplemental copper, it is significant that
molybdenum did not cause a decrease in the copper content of the liver.
This finding supports previous observations (51, 58) that molybdenum
tends to cause an accumulation of copper when the intake of copper is
ACCULULATIOH OF COPPER III LIVERS OF EXPERLE2ITAL RABBITS
ON DIFFEREI'T OLYBDENUM ANID COPPER INTAKES
Micrograms Cu per
No. of gram dry liver
:Iation Rabbits (average)
Control 5 29.7
C.01i% Mo. 5 30.2
0.05' Mo. 3 45.0
0.1% 1o. 5 35.7
0.2% Yo. 4 60.
THE EFFECTS OF LOT INTAKES OF DIETARY I'OLYBDENUK"
AND OF VITAMINS A A.D E InI THESE DIET3 UON
THE GR(ITH AND REPRODUCTION OF RATS
The results from several investigations of the role of
molybdenum in the rat show that the element is toxic, causing death
at high levels and retarded growth at sub-lethal levels. Previous
work in this laboratory has shown that 80 and 1hO ppm molybdenum
in a simplified ration significantly retards growth. Observed also
in this experiment was an interference with reproduction (49). From
these two observations it appeared that there mirht be a deficiency
of vitamin A or vitamin E as a result of the action of molybdenum.
If the vitamins became deficient by being tied up in the alimentary
tract, or are otherwise rendered ineffective by molybdenum, the
deficiency should be corrected by therapeutic administration of the
vitamins. This experiment was designed to further study the effects
of low intakes of molybdenum upon growth and reproduction of rats
and to study the effects of vitamins A and E added to diets containing
Ninety weanling rats of the Sherman strain, 21 to 23 days old,
were randomized into three treatments of 30 rats each. These treat-
ments consisted of the basal ration and two levels of molybdenum
(O.014 and 0.02 per cent) added to the basal ration. These three
treatments were further subdivided into three sub-groups of 10 rats
each approximately equally distributed between males and females. One
of the sub-groups in each treatment was considered the basal treatment
for the addition of the vitamin A or vitamin E to each of the other
The rats were housed in metal wire cages maintained in an air-
conditioned roan at 800 F. Rations were fed ad libitum and pyrex-
distilled water was provided from glass watering bottles.
The composition of the basal ration is recorded in Table 9.
The whole milk powder was Powdered Klim, received in vacuum packed
containers. Rations were prepared fresh about each ten days. The
copper content of this ration was 5 ppm. Vitamins A and E were
administered directly to the rats with the use of a medicine dropper.
The vitamins were given twice weekly in amounts that supplied 5,000
units of vitamin A and 12 milligrams of alpha tocopherol in each dose.
Vitamin A was in the form of a natural ester concentrate and vitamin E
was alpha tocopherol acetate.
Growth was determined from weekly weights made throughout the
twelve week growth period. At 16 weeks of age females were bred to
males on the same ration and any rats which did not breed were later
exposed to other rats to check the cause of the breeding failure.
In order to observe the protective action of cop-er against
the effects of molybdenum, one ration was prepared which contained
0.02% molybdenum and 0.002% copper. The basal ration for the trial
was the same as previously used. The composition of both is shown in
Table 9. Five male and four female weanlinr rats were used in this
trial and procedures previously outlined were used for obtaining data.
These rats were not given vitamin.
GCC'OSITION OF RATIONS
Basal Basal Basal Basal +
S0.O14% Mo. 0.02% Mo J.302Z Ou
Powder (Klim) 3415.0 3415.0 3415.0 3415.0
Sucrose 3380.0 3380.0 3380.0 3380.0
NaC1 3h.3 3l.0 34.0 34.0
FeSO.-7 0 .O0 4.0 l.0
MnS. 20 2.52 ?.2 .52 2.52
Thianine hydrochloride 0.023 0.023 0.023 0.023
Pyridoxine hydrochloride 0.033 0.033 0.033 0.033
Ouso04.R20 0.10 0.10 0.10 0.528
NaaoO4. 2H20 (.0 2.38 3.43 3.43
Results and Discussion
The mean weekly weights of the rats throughout the 12 week
growth period are recorded in Tables 10 and 11. heights of the male
rats are found in Table 10 and females in Table 11. Also included in
the table are the average total gains for each group. The growth of
the male rats receiving molybdenum was significantly less than the
growth of those in the basal group. The difference in the amount of
gain for the group receiving 0.0l1 per cent molybdenmn and of the basal
group was highly significant (PGO1). For those receiving 0.02 per
cent molybdenun, the gain was significantly less (P
of A i i
O 0O 0.
X. .I x
0 0> 0>
-o r N
0 04 0 4
C; W C;W4
bb bb O
W4 g4 wM
O 00 0
+ n1* 1
* 4 14
The growth of the female rats receiving molybdenum appeared to
be retarded, but the differences in amount of gain were not statisti-
cally significant for either level of molybdenum. This sex difference
in response to molybdenum supports a previous observation (49) that
male rats are less tolerant to dietary molybdenum than are female rats.
The effects of the vitamins may also be observed in Tables 10
and 11. The addition of both vitamins A and E increased the growth
of female rats on all treatments including the basal ration. No
consistent differences in the growth of male rats could be attributed
to either vitamin. The objective of the vitamin supplementation was
to study its possible therapeutic effect upon molybdenum toxicity.
Although both vitamins appeared to increase the growth of the female
rats on both levels of molybdenum, they had a like effect upon the
females on the basal ration; therefore, the relation to molybdenum
could not be observed. In the male rats where the adverse effects of
molybdenum were more evident, it should be observed that neither
vitamin A nor vitamin E increased the growth. From this observation
it appears, therefore, that neither vitamin A or vitamin E is effective
in counteracting molybdenum toxicity.
The average weekly weights and total gain of the rats given
0.002 per cent copper and 0.02 per cent molybdenum are recorded in
Tables 10 and 11. This amount of copper was not considered a high
level of dietary copper but was the quantity estimated to be necessary
to counteract the effects of molybdenum. This ration, however, did not
support growth in the male rats equal to that of the controls. The
amount of gain was significantly less than the gains produced in control
male rats. There was no significant difference in the growth of these
female rats. The growth of these females which was approximately equal
to that of the control females cannot be attributed to the presence of
the additional copper since the female rats which received molybdenum
but without the additional cop-er made approximately equal gains.
The results of the breeding trials are summarized in Table 12.
FERTILITY OF 'ALE AND FI.LLE RATS RECEIVING
DIETARY !JOLYBDEITLU .AID COOPER
Ration nales Females
o. IHo. :Io. Ho. No. Io.
Ilatings Litters Sterile Hatings Litters Sterile
Basal 6 6 0 5 5 0
0.011% i o 6 6 0 4 4 0
0.025 Ho 5 .1 5 5 0
;.002 P Cu
.,2 o 5 5 4 4 0
FnT these results, no significant effect of the amount of
molybdenum fed can be observed. The proportion of fertile rats in the
groups receiving supplemental vitamins A and E was the same. Since
the molybdenum did not produce sterility in this experiment, the effects
of the vitamins upon reproduction cannot be determined. These results
are contrary to the results of fertility experiments with rats (:9).
wherein a large proportion of male rats on a similar dietary regime
were sterile. A higher level of molybdenum has been fed in the current
experiment without causing sterility. The basal rations and other
conditions of these two experiments were similar; however, the following
changes applied to the current experiment may account for the differences
in results: (1) Sherman (albino) rats were used in this experiment.
The Long-Evans strain was used in the former experiment. (2) The iron
and manganese content of the ration was increased. (3) Pyridoxine was
added to the ration. These changes apparently improved the ration, as
reflected in somewhat better growth of the animals, and with an improved
nutritional status of the animals, the greater tolerance to molybdenum
would be expected.
During the course of the experiment, a peculiar alopecia was
observed in some of the male rats receiving molybdenum and also in
those receiving the hiFher level of copper with molybdenum. It was
not present in all of the rats of any group nor was the loss of hair
permanent. This alopecia was much more caomon in the male rats and
was limited to a strip approximately an inch wide over the entire
length of the back. Figure 5 shows pictures of typical rats exhibiting
this alopecia. Alopecia appeared in some of the other rats, particu-
larly about the neck and shoulders, but it was not confined to the rats
receiving molybdenum, nor was it observed more in male rats than in
The alopecia which occurred over the back of the animals
appeared to be associated with molybdenum. Although less than one-third
of the male rats on the molybdenum diets exhibited this particular type
of alopecia, none of the rats of the basal group were so affected.
Alopecia has been observed in rats on similar diets, but it was more
characteristic of the female rats than of males (W9).
Figure 5. Male rats on molybdenum diets exhibiting alopecia over the
PHOSPHORUS METABOLISM AND DISTRIBUTION OF
PHOSPHORUS32 IN THE RABBIT AND RAT
Certain abnormalities in skeletal formation have been attributed
to excess molybdenum in the dietary. Some of the investigations which
have been undertaken to relate this abnormality to molybdenum have
demonstrated an abnormal phosphorus metabolism when molybdenum was in
excess. The observation made early in this experiment that the front
legs of some young rabbits develop abnormally when fed high levels of
molybdenum demonstrated that molybdenum interferes in some manner with
normal skeletal development in this species. In the absence of other
explanation, it appeared possible that there was an interference with
This experiment was undertaken to obtain data which would
afford an evaluation of phosphorus balance in normal rabbits and rats
and in rabbits and rats which were receiving dietary molybdenum. Also
included in the study are observations on the distribution of radio-
active phosphorus in these animals.
The animals used in this study were selected from those used in
previous experiments. They were continued on the same ration after cam-
pletion of the growth studies. Four young rabbits and 18 young rats
were introduced into the experiment at this time in order to obtain data
from your animals.
Phosphorus balance studies were made using radioactive phosphorus32
Followinr administration of the isotope, rabbits were confined for
72 hours to metabolism cages tfhich provided quantitative separation
of urine and feces. The 70 h'ur period was selected because pre-
liminary observations indicated that the greater portion of the isotope
was excreted within this time. Ten rabbits -ere riven oral adninis-
tration of the isotope and seven were riven intravenous injections
through the marginal ear vein. Administration to the rats was made
orally and collection periods were 48 and 72 hours.
At the end of the metabolism period, the animals were sacri-
ficed and selected tissues taken for distribution studies. Urine and
feces were collected and the radioactivity determined on representative
The radioactive phosphorus which was received in the f?rm of
phosphoric acid was neutralized with sodium carbonate and made isotonic
with sodium chloride prior to administration. The dosare administered
to rabbits ranged from 1.5 to 5 microcuries and the rats w-ere riven
1 to 2 microcuries.
Radioactivity measurements were made in the usual manner using
a Geirer-Lullcr counter fitted -ith a dipping tube. Urine and blood
samples were counted directly without ashinr. Before detormininr the
activity of the rabbit urine, it was necessary tn acidify the urine
with hydrochloric acid in order to dissolve the solid portion of the
urine. This portion was observed to contain a larpg proportion of
the phosphorus and unless it was dissolved or thoroughly suspended,
the measurements were inaccurate. Feces and tissue samples were met
digested with nitric acid and heat prior to activity measurements.
Calculations were made of the per cent of total dose excreted
in the urine and feces during the balance period. The per cent of
total dose per gram of tissue was determined for the liver, kidney,
femur, muscle and blood and the per cent of dose was corrected to the
average body weight of the animals.
The total phosphorus content of femur bones and certain other
tissues was determined using a modification of the volumetric method
outlined in Methods of Analysis of the Association of Official Agri-
cultural Chemists (6).
Results and Discussion
Balance Studies The data representing the balance studies
with rabbits are summarized in Table 13. The values reported are
mean values for the per cent of total dose which was excreted in 72
hours by way of the urine and the feces. Results are given for both
oral and intravenous administration of the isotope. The results show
that a greater proportion of the orally administered phosphorus32 was
excreted by the rabbits which were receiving dietary molybdenum. There
was a significant increase in both urinary and fecal excretion of the
labeled phosphorus. Following intravenous administration, there was
no observed difference in the total amount excreted nor in the pathway
of excretion. This indicates that in the rabbit molybdenum did not
alter the pathway of excretion as has been observed in steers (68).
On the high nolybdenum rations, tho urinary excretion of total phos-
phorus increased almost fourfold while fecal excretion of total
phosphorus was not significantly affected. The increased excretion
of both labeled and total phosphorus by way of the urine in animals
on high molybdenum diets indicates that molybdenum may decrease the
absorption of phosphorus and at the same time increase the excretion
of that which has been absorbed. Since there was no significant
alteration in the excretion of labeled phosphorus following intra-
venous administration, it appears that the primary effect of molyb-
denum is in the gastro-intestinal tract.
The excretion data for the rats on control and high molybdenum
diets are presented in Table 14. These values represent only oral
administration of the isotope. Results from both mature and young
rats and of two intervals of time after administration are recorded.
The amount of total phosphorus in the urine could not be accurately
determined because of contamination from feed. The total excretion
of phosphorus32 was not increased by molybdenum feeding in the case
of the rats. There was an increase in urinary excretion of the labeled
phosphorus, but this was not accompanied by an increased excretion
through the feces. The effect of molybdenum upon phosphorus32 excre-
tion in the urine parallels the results observed ith oral administra-
tion to rabbits. Both total phosphorus and labeled phosphorus vere
less in the feces from the rats receiving molybdenum. The urinary
excretions at L8 and 72 hours indicated that molybdenum in the young
rat caused more rapid excretion of the absorbed labeled phosphorus.
There was no increase at 72 hours over that excreted at 48 hours,
";ith mature rats, the excretion at 72 hours was almost double that at
l8 hours. This is also reflected in the fecal excretion but to a
EFFECTS OF DIETARY MOLYBDENUM, AGE A31D TI E AFTER DOTTIG UPON THE
EXCRETION OF ORALLY ADMINISTERED PHOSPHORUS32 BY THE RAT
Hours Urine Feces
Ration after Age No.
Dqse (weeks) Rats % Dose % Dose Ug total P
25 4 8.60 27.97 47.95
48 8 a 6.98 16.06 5 5-.I5
25 2 15.72 33.59 66.63
72 2 18.73 1.97 33.87
25 4 12.19 15.65 36.55
48 8 L 12.07 6.01 3P.11
25 2 22.57 25.25 55.78
72 8 3 13.49 12.70 38. 7
Tissue Distribution Data representing the deposition of
labeled phosphorus in the tissues under the different conditions are
summarized in Tables 15, 16 and 17. The values reported have been
corrected to the average weight of the animals within the groups for
which the comparisons are made.
The deposition of phosphorus32 was very similar for comparable
tissues of the animals on different rations and there were only slight
differences which may be attributed to the effects of molybdenum. The
per cent of dose of the labeled phosphorus was slightly higher in the
liver and kidney of both the rats and rabbits which were on the high
molybdenum diets. The femur bone of the rabbits receiving molybdenum
accumulated a greater percentage of the administered dose than did the
control rabbits, but no similar increase was observed in the rats.
IZOOLYBD4SIU UPON THE DEPOSITION QF PHOSPHORUS32
IN SELECTED TISSUES OF THE RABBIT*
Per Cent of Dose per Gram of Tissue
Tissue Oral Administration Intravenous Administration
(3 rabbits) (2 rabbits)
Control High Mo. Control High Mo,
Blood 0.016 0.012 0.018 0.014
Liver o.105 0.125 lJ8 0.172
Kidney 0.085 0.087 0.109 0.126
Muscle 0.026 0.019 0.04j4 0.070
Femur shaft 0.149 o.154 r.136 0.271
Femur epiphysis 0.255 0.315 n.?72 0. 88
* 72 hours after administration.
The effects of molybdenum and of two intervals of time after
administration upon accumulation of phosphorus32 in the tissues of
young rats are shown in Table 17. Also shown is the amount of total
phosphorus in the fenurs of these rats. The per cent of the total dose
EFFECTS OF DIETARY :IOLYEDE NUU UPON DEFOSTTION OF
PHOSPHORUS32 AID TOTAL PHOSPHORUS T
SELECTED TISSUES OF IEATURE RATSs
Control High Uo.
% Dose % Dose mg. % Dose Z Dose mg.
Per im. Fer Total Per gm. Per Total
Tissue age P. F. Tissue mg, P. F.
Blood 0.019 :-*, 0.030
Liver 0o.55 0.35 2.37 0.638 0.34 2.69
Kidney n.116 0.066 2.61 0.118 0.064 1.67
muscle 0. 00 0.05?7 2.34 0.037 0.030 2.29
Fenur 117 0.020 85.87 1.167 0.019 85.89
* 48 hours after administration.
-o Tissues represent pooled samples
c-* Total phosphorus not determined.
of h rats.
OF PHOSPHORUS32 ANlD AMOOU3T OF TOTAL
I SELECTED TISSMUS OF YOTUIG RATS*
lours Per Cent of Dose per Gram of Tissue
Tissue Dose Control High Mo.
L48 0.026 0.029
ood 72 o0.oU 0.0o-3
L r 48 0o.56 'n 507
Liver 72 0. 375 -. 363
48 0.211 ?0.226
Kidney 72 0.375 0.3858
48 0.089 o.-65
Muscle 72 0.203 0. 17
/ Dose Z Dose mg. % Dose / Dose mg.
Per total Per total
mg. P. F. mg. P. P.
L8 2.81 0.0o6 60.33 2.96 0.058 56.96
Femur 72 3.49 0.057 60.90 3.86 0.080 47.75
* Tissues represent pooled samples
at 72 hours.
of h rats at 48 hours and 3 rats
of labeled phosphorus found in the tissues at 72 hours was greater than
that found at 48 hours in all of the tissues except the liver. These
increases were cannon to the rats on both rations; therefore, no
specific effect of molybdenum upon the rate of accumulation can be
observed in the young rats. The increase in the labeled phosphorus
of the kidney at 72 hours is also reflected in the urinary excretion
at 72 hours.
The decrease in the amount of total phosphorus and the
increased specific activity of the bone of young rats receiving molyb-
denum (Table 17) indicate a greater rate of exchange of phosphorus and
more interference with normal phosphorus deposition in the young rat
than in the mature rat.
;hen the concentration of labeled phosphorus per unit of total
phosphorus was calculated there was little difference in the specific
activity of the tissues of rats on the different rations (Table 16).
The difference in the per cent dose per milligram of total phosphorus
in the bone of old and young rats reflects the larger amount of total
phosphorus present in the bone of mature rats.
There appears to be a species difference in the tissue accumu-
lation of labeled phosphorus. The soft tissues and the femur bones of
the rats accumulated a considerably greater proportion of the dose of
the isotope per unit of tissue than did the rabbit tissues. A portion
of this difference may be attributed to the difference in time after
administration of the isotope. rEperiments involving a large number of
animals should be undertaken in order to establish whether such a
The total phosphorus content of the femur bones from 17 rabbits
receiving different dietary levels of molybdenum has been determined.
The results which represent mean values for the number of bones
examined are suimarized in Table 18. There were wide variations in
individual rabbits, but the averages indicate a slight decrease in the
phosphorus content of the bones of rabbits which were receiving molyb-
denum. Two of the rabbits in the rroup receiving copper and molybdenum
had previously received molybdenum without copper supplementation. They
had developed typical symptoms of toxicity, one of which exhibited the
foreleg trouble. Both rabbits were treated with copper and the copper
supplementation was continued until they were sacrificed and the total
phosphorus of the bones determined.
TOTAL PHOSPHORUS CONTNT OF FEMUR BODIES OF RABBITS
FED VARjYIG LEVELS OF IOLYBDEI~ M AID COPPER
(mg per Cram fresh tissue)
Ho. of me P.
Ration Rabbits per gram
Control 4 96.16
0.0112 Uo. 3 9P4. 0
0.1Z to. 6 87.o8
0,? Mlo. 0.02% Cu. I 89.r6
It appears that the effects of molybdenum upon the metabolism
of phosphorus under the conditions of this experiment were limited to
alteration in the excretion of phosphorus. while e there were slight
changes in the deposition of labeled phosphorus in some of the tissues,
these differences were not significant and not as pronounced as the
changes in excretion which have been described. It should be noted
that none of the animals exhibited symptoms of molybdenum toxicity at
the time when these phosphorus metabolism studies were undertaken. The
quantity of molybdenum (0.1%) fed to the rabbits in this experiment had
produced toxicity in other young rabbits and had produced moderate
symptoms in some of the rabbits which were later used for those phos-
phorus studies, but none exhibited any toxic symptoms at the time of
this experiment. If the phosphorus studies had been made when evi-
dence of molybdenum toxicity first appeared, it is possible that the
effects of molybdenum may have been exhibited in a different manner.
Summary and Conclusions
The role of molybdenum in the nutrition of the rabbit has been
studied through Abservations of the effect of suprlenontary molybdenum
in the diet. A molybdenum toxicity was produced and the symptoms have
been described. Anemia was characteristic of the toxic syndrome and
was further studied -rith respect to hemoglobin, red blood cell counts
and blood volume. The therapeutic action of copper in curing the
toxicity was studied by following the changes resulting from copper
therapy. Growth experiments were conducted with weanling rabbits and
rats receiving, varying levels of molybdenum. Some effects of molyb-
denum upon the metabolism of phosphorus were studied through balance
experiments and tissue distribution of radioactive phosphorus32.
Some of the results have been of a confirmatory nature while
other findings have demonstrated actions of molybdenum which appear
to be specific for the rabbit. These observ-tions have been related
to results of experiments -ith other species and have been discussed
with respect to their possible im-lications. Conclusions which may be
made from these experiments are summarized in the following statements:
1, Molybdenum is toxic to rabbits. Under conditions of this
experiment, 0.1 per cent added to a natural ration was toxic
to young rabbits and was borderline in toxicity for mature
rabbits. Two-tenths and 0.4 per cent nolybdenum was toxic
to all acres of rabbits.
2. Molybdenum toxicity in the rabbit was characterized by loss
of weight, anemia, alopecia and dernatosis and abnormal bone
3. The abnormal bone development was observed in your rabbits
receiving 0.1 per cent or more molybdenum. The front legs
of affected rabbits bec-me weakened so that they could not be
maintained in a normal position and could not support the
body. Observed in the abnormality were swollen joints, bent
and twisted bones, slipped joints and slipped tendons.
4. -Ixcess molybdenum caused anemia with low hemoglobin and low
red blood cell counts, but did not affect the blood volume of
5. Dietary copper was effective in preventing molybdenum toxicity
and in curing all symptoms of the toxicity except the front
6. Molybdenum tended to increase the accumulation of copnor in
the liver of experimental rabbits.
7. Molybdenum fed at the level of 0.014 and 0.02 per cent in a
simplified diet retarded the growth of male rats, but did not
significantly restrict the gro-rth of female rats.
I. Vitacin A anre vitamin 2 did not improve the rrowth of male rats
fed -.01 and 0.02 per cent molybdenum
9. Molybdenum did not affect the reproduction of rats under con-
ditions of this experiront.
10. Alopecia 4.*hich was confined to thinning of the hair over the
back was observed in rsome mnle rats reccivinr dietary molybdenuiw.
11. There :as 'an increase in urinary and fecal excretion of orally
administered radioactive phosphorus32 in rabbits receiving
0.1% molybdenum in the ration.
1?. Molybdenum fed at a level of 0.01O1 and 0.0? per cent in the
diet of rats increased the urinary excretion of phosphorus32
but did not increase fecil excretion.
13. The distribution of phosphorus32 and the amount of total
phosphorus in the tissues of rabbits and rats was not
significantly affected by the presence of molybdenum in
Recommendations for Future Research
The results of this research havc been presented and discussed
with relation to results of similar research -.-ith other species. In
addition to the results which have been recorded, other observations
have been made which imply still other actions of nolybdenun. These
proposals, if submitted to properly designed research, should provide
additional approaches to the study of the role of molybdenum in the
animal organism. Some recommendations for specific research in this'
1. Differences in species tolerance and differences in the mani-
festation of molybdenum toxicity have been established for
several species. These different responses to molybdenum
should be more specifically related to the physiology of the
animal. In example of the variations in species response is
the occurrence of severe diarrhea in sono species and absence of
such a symptom in others when molybdenum is in excess.
Further studies with rabbits should employ simplified or purified
diets so that the intake of copper may be limited to a smaller
3. The abnormal development of front legs in young rabbits sug-
gested that molybdenum may create a manganese deficiency or
may interfere *"ith the metabolism of manganese. This bone
abnormality should be studied with relation to manganese or
to other physiological functions concerned with bone formation.
h. A comparison of the growth of rats in this study rwith previous
experiments indic-ted that an improved nutritional status of
the animal increases the tolerance to molybdenum. The effects
of nolybdenun should be further related to nutritional status
of the animal.
5. The more adverse effects of molybdenum upon younr animals
indicated an action of molybdenum associated with the growth
The anemia with Ir.v red blood cell counts in rabbits surrested
that cobalt or vitamin B12 might be deficient. It should be
determined whether the anemia will respond to vitamin 12
.7. The type of anemia which develops in the different species
should be more specifically identified.
. The apparent normal libido in male rabbits suffering from
molybdenrm toxicity suggested that the effect ur- n reproduction
in rabbits may not parallel the effects observed in other
C. Factors responsible for the greater toxicity of molybdenum
when fed .ith zreen succulent feed could be studied on a
laboratory scale usinr the rabbit as a laboratory animal.
In. Phosphorus metabolism studies indicated that molybdenum
interfered with absorption of phosphorus. Wdditinnal balance
studies made at a tine -rhen animals show symptoms of molyb-
denui toxicity should establish more clearly the effect of
molybdenum upon phosphorus absorption and excretion.
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Lewis Roberts Arrington was born May 1, 1919 at Kirksey,
Greernood County, South Carolina. He was graduated from high school
at Ninety-Six, South Carolina in May 1936. He entered Clemson Collee
in 1936 and received the Bachelor of Science degree from that insti-
tution in June 1940. Following graduation from Clemson College, he
entered Virginia Polytechnic Institute in September 1940 and received
the Master of Science degree in June 1941.
1While in residence at Virginia Polytechnic Institute he held
a teaching fellowship. During World WIar II he served four years as a
commissioned officer in the United States Army. In September 19h6
he was employed as Assistant Professor in the Department of Dairy
Science at the University of Florida. He held this position until
entering graduate school at the University of Florida in February
1950 to begin studies leading toward the Doctor of Philosophy degree.
While in residence for this degree he held a grant-in-aid fellowship
from the Nutrition Foundation, Inc.
He is a member of Alpha Zeta honorary fraternity, Sigma Xi
Society and Phi Sipma Biological Society.
This dissertation was prepared under the direction of the
Chairman of the candidate's Supervisory Committee and has been
approved by all members of the Committee. It was submitted to the
Graduate Council and was approved as partial fulfilment of the re-
quirements for the degree of Doctor of Philosophy.
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