Title: Molybdenum in the nutrition of the rabbit and rat
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Title: Molybdenum in the nutrition of the rabbit and rat
Physical Description: v, 69 leaves : ill. ; 28 cm.
Language: English
Creator: Arrington, Lewis Roberts, 1919-
Copyright Date: 1952
 Subjects
Subject: Molybdenum   ( lcsh )
Rabbits   ( lcsh )
Rats   ( lcsh )
Animal Science thesis Ph. D
Dissertations, Academic -- Animal Science -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Statement of Responsibility: by Lewis Roberts Arrington.
Thesis: Thesis (Ph. D.)--University of Florida, 1952.
Bibliography: Bibliography: leaves 63-69.
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
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Bibliographic ID: UF00098041
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000401971
oclc - 24859031
notis - ACE7829

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MOLYBDENUM IN THE NUTRITION

OF THE RABBIT AND RAT









By
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
June, 1952































UNIVERSITY OF FLORIDA
3 12112 08552 27811 i
3 1262 08552 2786











ACKNO"iLEDGE.5IENTS


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

experimental rabbits.

Dr. John P. Feaster has given personal assistance and co-

operation.

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

the research.

vr. Juan 3!la, Mr. Richard Sparrow and !.r. Max Garhart have

contributed by attending the experimental animals.

ii











TABLE OF CONTENTS


Page

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


iii











Page

GENERALSUMARY.. ..... *-..........a*@ e s so 58

SmAuary wnd Col .usions . . . . . . 58
Recommendations for Future Research . . . . .. 60

BIBLIOGRAPHY . .......... ...... 63



List of Tables

Table

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










Page
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

Figure

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










INTRODUCTION


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

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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
-2-










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

-3-











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

similarly affected.

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,

39).

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
-4i-











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

-5-










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)

-6-










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

-7-










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.

-0-










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).











EXPERI-MNT I

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

abnormality.

The objective of this experiment was to describe the effects

of molybdenum excess upon rabbits.


Experimental Procedure

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

-10-










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

calculated amount.

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

-11-











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

the ration.


TABLE 1

SUI2ARY OF MOLYBDENUR TOXICITY SYMPTOMS IN RABBITS


Alopecia
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.

-12-











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

two weeks.

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

alopecia.

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

-13-


































































































Figure 1. Younp rabbits receiving dietary molybdenum and

exhibiting typical alopecia.


-11-


_ __ __


r - -- ------


IIL- -- ~ IIILIIICCIIII


----------- --


,?H-\










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

-15-














)K


Young rabbits exhibiting front log abnormality.


-16-


Firure ?.










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

other symptoms.

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

-17-











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
-18-



































Bones from young rabbit showing front leg abnormality.



























Forelegs of young rabbit showing shifted carpus joint,


-10-


Figure 3.


Figure h.











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

alopecia.


-20-











EXPERIMENT II

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.


Experimental Procedure

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

-21-











were housed and managed under the same conditions as outlined in

Experiment I.

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

-22-













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-23-











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.











EXPERIMENT III

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-

denum toxicity.


Experimental Procedure

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-

cedure.

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

-25-










in the standard red cell counting chamber after dilution in eyema's

solution.

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

-26-










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

occurred.

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-

denum.


TABLE 3

MEAN HEMOGLOBIN VALUiS FOR EXPERIMSMTAL RABBITS*


Per cent molybdnum in ration
Week on
abperiment
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.


-27-



















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TABLE 5


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.


-29-











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.


-30-



















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-31-


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EXPERIaT IV

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

the rabbit.

Experimental Procedure

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
-32-










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-

strated.

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

-33-











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

-34-










TABLE 7


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


Erythrocytes
(millions)

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 -- !


-35-











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

adequate.


TABLE 8

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.
n.02% Cu.


-36-










EXPERMrfT V

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

molybdennm.


peerimcntal Procedure

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

-37-











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

two sub-groups.

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

-38-










trial and procedures previously outlined were used for obtaining data.

These rats were not given vitamin.

TABLE 9

GCC'OSITION OF RATIONS
(grams)


Basal Basal Basal Basal +
S0.O14% Mo. 0.02% Mo J.302Z Ou
0.02% Mo


holeoe Milk
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
-39-
















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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

-42-











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.

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
LMales Females


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).

-43-











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

females.

The alopecia which occurred over the back of the animals

appeared to be associated with molybdenum. Although less than one-third
S-,-










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
back.


-I5-











EXPEU.1~SET VI

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

phosphorus metabolism.

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.


Experimental Procedure

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

-46-











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

samples.

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

-L7-











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

-l18-
















0








14
o










1-1
Ra











6'
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gg


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em
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IS
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ee





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em





as
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I
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-69-


a
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e
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u
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4


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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

-50-










l8 hours. This is also reflected in the fecal excretion but to a

lesser degree.

TABLE 14

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
Control
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
High Mo.
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

-51-










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.


EFFECTS OF


TABLE 15

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

-52-












TABLE 16

EFFECTS OF DIETARY :IOLYEDE NUU UPON DEFOSTTION OF
PHOSPHORUS32 AID TOTAL PHOSPHORUS T
SELECTED TISSUES OF IEATURE RATSs


Control High Uo.

Tissueas
% 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.


-53-










TABLE 17


DEPOSITION


OF PHOSPHORUS32 ANlD AMOOU3T OF TOTAL
I SELECTED TISSMUS OF YOTUIG RATS*


PHOSPHORUS


lours Per Cent of Dose per Gram of Tissue
after
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

-55-










difference exists.

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.


TABLE 18

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
Tissue


Control 4 96.16
0.0112 Uo. 3 9P4. 0
0.1Z to. 6 87.o8
0,? Mlo. 0.02% Cu. I 89.r6


-56-











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.


-57-











GEcER.AI, SUI.'ARY

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

development.

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

rabbits.

5. Dietary copper was effective in preventing molybdenum toxicity

and in curing all symptoms of the toxicity except the front

leg abnormality.

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.
-5--












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

the ration.


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'

field follow.

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
-c6-











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

amount.

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

process.

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

therapy.

.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

-61-












in rabbits may not parallel the effects observed in other

species,

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.


-62-











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*t
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BIOGRAPHICAL ITEMS


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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