IRON TOLERANCE IN YOUNG PIGS
AS INFLUENCEO BY DIETARY
VH'AMIN E AND SELENIUM

Thesis for the Degree at M S,
Mé‘CHiGAN STIR-“E UNIVERSITY;
NOAH. KiNG KAI
1974

ABSTRACT

IRON TOLERANCE IN YOUNG PIGS AS INFLUENCBD BY DIETARY
VITAMIN E AND SELENIUM

BY

Kuan King Kai

The work presented is an attempt to determine the
effects of iron, both oral supplementation and intramuscular
injection, in young pigs as influenced by dietary vitamin
E and selenium. Three experiments, using a total of 191
pigs, were conducted.

In Experiment 1, 127 pigs, from 17 litters, presumed
to be vitamin E - Se deficient, were employed. The animals
received their first iron injection at three days of age
amounting to 100 mg of iron from iron dextran intramuscu-
larly. They were weaned at 5 weeks of age to a low vitamin
E - Se starter diet. Two days after weaning, half of each
litter received an intramuscular injection of 34 1.0. of
vitamin B + 0.5 mg of Se. Two days after the E - Se injec-
tions, half of each of the resulting groups received a
second intramuscular injection of 100 mg of iron from iron

dextran. No sign of iron toxicity was observed.

Kuan King Kai

In the second experiment, pigs from sows fed a low
vitamin E - Se diet were weaned at 5 weeks of age to a
fortified grower corn - soy ration. The basal diet was low
in vitamin E and Se. Ferrous sulfate (FeSO4, 7HZO) and
vitamin E as d - a - tocopheryl acetate were added to the
basal ration in a 3 x 2 factorial design with 8 pigs per
treatment. The diets contained 345, 550 or 850 ppm of iron
and either no supplemental vitamin E or 22 I.U. vitamin
E/kg of feed. Weight gain, feed intake and feed efficiency
were recorded and calculated biweekly. There were no sig-
nificant differences between treatment groups, during the
entire study. There was a significant iron x vitamin E
interaction on gain during the growing phase of the trial
(P<.05).

Hematological analyses conducted at various intervals
of the trial revealed no significant treatment differences
in hemoglobin, hematocrit and serum iron levels during the
entire duration of the trial.

At the end of the study, the experimental animals were
slaughtered and samples of muscle, liver and pancreas were
collected for iron concentration determination. Here again,
it was found that no significant differences occurred
between treatment groups in the iron concentration of the
tissues taken.

In the third experiment, 16 pigs were used. Eight

were weaned from sows which had been fed a low vitamin

Kuan King Kai
E - Se gestation and lactation regime while the other 8 were
from sows whose feeds had been supplemented with vitamin E
at 22 I.U./kg of feed.

When the animals were about 9 weeks of age, one-half
of the presumed vitamin E deficient pigs as well as one-
half of the pigs from the vitamin E supplemented sows were
given a second iron injection, a massive dose of iron
(1000 mg) from iron dextran, intramuscularly. No signs of
toxicity were observed following the iron administration.

Blood was collected for hematological studies just
before the iron injection as well as at one day and again
at one week after treatment for comparative studies.

Serum iron levels had risen dramatically by one day
after the injection and were significantly greater than in
pigs not receiving the injection (P<.01). However, by one
week after the injection, serum iron levels had returned to
normal control levels leaving no significant treatment dif-
ferences in serum iron level. Hemoglobin and hematocrit
values of iron injected in pigs were not significantly
different from those of control animals. Again, there was

no evidence of iron toxicity in this study.

IRON TOLERANCE IN YOUNG PIGS AS INFLUENCED BY DIETARY
VITAMIN E AND SELENIUM

BY

Kuan King Kai

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Husbandry

1974

A

.535}
5;? ACKNOWLEDGEMENTS
X

(If ‘

Sincere gratitude is extended to the University of
Agriculture, Malaysia, for full financial support, in the
form of a scholarship, during the entire period of my
graduate studies.

I am deeply grateful to my major professor, Dr. E. R.
Miller, who has contributed so much to making my stay at
Michigan State University a very pleasant experience. As
a teacher, Dr. Miller has earned my heartfelt esteem
’through his patience, tremendous approachability and his
‘ever-willingness to allow me to share in his deep pool of
knowledge and vast personal experiences. I have indeed
profited greatly from his guidance and personal examples.
I am also indebted to Dr. C. K. Whitehair for his guidance
and continued personal interest in my graduate program.
Appreciation is also extended to Dr. D. E. Ullrey for
serving on my thesis committee.

I am grateful to Dr. Pao Kwen Ku, who has so patiently
taught and assisted me in the use of laboratory facilities
and in analytical procedures. The assistance of fellow
graduate students, laboratory staff and departmental

secretaries will always be remembered. I am also thankful

ii

to Miss Kim Miller and Miss Janice Fuller for their skillful

typing of this manuscript.
Above all, I wish to thank my wife, Rina, and daughter,

Valerie, whose love and encouragement have made this study

worthwhile.

iii

VITA
KUAN KING KAI
CANDIDATE FOR THE DEGREE OF
MASTER OF SCIENCE

DISSERTATION: IRON TOLERANCE IN GROWING SWINE AS
INFLUENCED BY DIETARY VITAMIN E AND
SELENIUM

OUTLINE OF STUDIES:

Main Area: Swine Management

Minor Area: Animal Nutrition

BIOGRAPHICAL ITEMS:
Born: April 18, 1939, Ipoh, Perak, Malaysia

Education: Agricultural University, East Pakistan
1962-1968. Veterinary Medicine (D.V.M.)
Michigan State University, 1972-1974
Animal Husbandry (M.S.)

Experience: 1959-61: Taught English after graduating
from high school

1961-62: Registration Officer

1968-72: Veterinarian for Merck Sharp
& Dohme (Aust.) Pty. Ltd.
Singapore Office

1974: Lecturer in Animal Science

Agricultural University of
Malaysia, Kuala Lumpur, Malaysia

iv

TABLE OF CONTENTS

LIST OF TABLES .

I.

II.

III.

INTRODUCTION .
'LITERATURE REVIEW.

Iron Metabolism.
Mechanism of Absorption of Iron Prepara-
tions Administered Intramuscularly . . . .
Iron Toxicity in Man and Laboratory Animals.
Reticuloendothelial Iron Overload.
Iron Toxicity in Farm Animals.
Iron Toxicity in Swine: Field Reports
Experimental Studies of Iron Toxicity
in Swine . .
Induced Rickets as a Result of High
Density Levels of Iron . .
Iron Toxicity in Swine Associated with
Low Levels of Vitamin E and High Levels
of Polyunsaturated Fatty Acids
Biochemical Studies in Piglets Made
Susceptible to the Toxic Effects
of Iron. . . . . . . . . . . . . .

EXPERIMENTAL PROCEDURE .

General Conduct of Experiments

1. Effect of a second iron injection on
vitamin E - Se deficient pigs
(Experiment 1) . .

2. Effect of dietary iron supplementation
to low vitamin E and vitamin E~
supplemented rations of young pigs
(Experiment 2) .

3. Effects of a large dose of iron given
intramuscularly to young swine
deficient in vitamin E and Se
(Experiment 3)

Page

vii

#AH

12
13
14

16
17

19

22

28

28

28

30

31

Page
B. Analytical Procedures. . . . . . . . . . . 32

l. Hemoglobin . . . . . . . . . . . . . . 32
2. Hematocrit . . . . . . . . . . . . . 32
3. Serum collection . . . . . . . . . . . 33
4. Serum iron . . . . 33
5. Determination of iron concentration

in feeds . . . . 33
6. Determination of tissue iron (liver,

muscle and pancreas) concentration . . 34

C. Statistical Analysis . . . . . . . . . . . 35

IV. RESULTS AND DISCUSSION . . . . . . . . . . . . 36
Experiment 1 . . . . . . . . . . . . . . . . 36
Experiment 2 . . . . . . . . . . . . . . . . 38
Experiment 3 . . . . . . . . . . . . . . . . 46

V. SUMMARY AND CONCLUSIONS. . . . . . . . . . . . 49
BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . 51

APPENDIX . . . . . . . . . . . . . . . . . . . . . . 58

vi

Table

A-8
A-9
A-lO

LIST OF TABLES

Composition of diets

Rectal temperatures, °F, after a second
IM iron injection. . . . .

Effect of oral iron and vitamin E on
performance. . . . . . . . . . . . . .

Effect of oral iron and vitamin E on
hematology . . . . . . . . . . . .

Tissue iron concentration in Experiment 2.

Effect of vitamin E and IM injection of
1000 mg Fe on hematology . . . . .

Vitamin-Trace Mineral premix .

Performance data, Experiment 2, low vitamin
E with 345 ppm iron.

Performance data, Experiment 2, high
vitamin E with 345 ppm iron. . . . . . . .

Performance data, Experiment 2, low vitamin
E with 550 ppm iron. . . . . . .

Performance data, Experiment 2, high
vitamin E with 550 ppm iron. . . . .

Performance data, Experiment 2, low vitamin
E with 850 ppm iron. .

Performance data, Experiment 2, high
vitamin E with 850 ppm iron. . . . .

Hematological data, Experiment 2 . . . . .
Tissue iron levels, Experiment 2

Hematological data, Experiment 3 .

vii

Page
29

37

4O

43
45

47
58

S9

60

61

62

63

64
65
66
67

I. INTRODUCTION

According to the usual classification of the mineral
content in an animal's body, iron is considered as one of
the trace elements since it is present in only minute amounts.
However, the essentiality of this mineral is beyond question.
The reactive part of the hemoproteins, myoglobin and hemo-
globin is the iron atom. These hemoproteins serve to store
and transport molecular oxygen. In addition, iron is an
essential part of various enzyme systems.

The subject of iron deficiency anemia in the pig has
been intensively studied and well documented. The baby pig
is born with a very low store of iron. Supplementation of
the sow's ration with various iron compounds or the paren-
teral administration of iron preparations during gestation
or following parturition does not significantly improve the
iron status of the baby pig. The low iron store at birth,
together with low iron concentration in the sow's milk plus
the rapid growth rate of the pig are factors which predis-
pose it to iron deficiency anemia. Different methods have
been proposed to provide a source of available iron to the
newborn pig. Allowing the piglets to forage in a trough
of soil mixed with iron or the swabbing of the sow's udder
with a concentrated iron solution are uncertain and

1

2

time-consuming practices which have no place in the present-
day swine farm. Oral iron administration is subjected to
a host of exogenous factors such as the size of the dose,
the state of iron repletion in the body, the presence of
phosphates and reducing substances. Numerous workers have
shown that intramuscular injection of iron preparations is
by far the superior method in terms of promoting maximum
hemoglobin synthesis and in increasing weight gain (Browlie,
1955; Gwatkin and L'Ecuyer, 1959; Hydberg et a1., 1959;
Wahlstrom et a1., 1960; Zimmerman et a1., 1959; Maner et
a1., 1959). Today, this method of iron administration to
the baby pig for anemia prophylaxis has become standard
practice.

In contrast to iron requirement, iron toxicity in the
pig is a subject that has not been well studied. In view
of the widely emphasized need to provide a supplemental
source of iron to the newborn pig, it is appropriate to
point out that the indiscriminate use of this mineral is
not without risk. There are numerous publications regarding
iron poisoning in man - especially children through the
accidental ingestion of iron tablets (Wallerstein and
Mettier, 1958; Reissmann et a1., 1955a,b; Bothwell and
Finch, 1962). Much work on the subject has also been done
in laboratory animals (Brown et a1., 1959; Goldberg et a1.,
1957; Taylor et a1., 1935). Field reports and actual experi-
mental work on iron toxicosis in farm animals are few and

far between. In recent years, however, there have been

3
sporadic field reports of iron toxicity in pigs following
either iron prophylaxis or therapy. Initial documented
reports were made by Scandinavian workers (Behrens, 1957;
Brag, 1958; Nilsson, 1960; Henriksson, 1962). Follow-up
studies and investigations by Scandinavian scientists
(Lannek et a1., 1962; Tollerz and Lannek, 1964; Arpi and
Tollerz, 1965) and British researchers (Patterson et a1.,
1967; Patterson et a1., 1969; Patterson et a1., 1971) have
implicated that vitamin E deficiency would render piglets
more susceptible to the toxic effect of iron. High levels
of polyunsaturated fatty acid in the gestating sow's diet
are believed to aggravate the problem. Studies by Tollerz
and Lannek (1964) have indicated that treatment with vitamin
E or synthetic antioxidants prior to the administration of
iron can afford protection.

The present research project was undertaken to further
investigate the toxic effects of iron in vitamin E
deficient young swine. An impression has also been gained
that selenium deficiency might be another factor which makes
the young pig more susceptible to the toxic effects of iron.
Selenium has been demonstrated to function as a biological
antioxidant. The element is an essential component of
glutathione peroxidase (Hoekstra et a1., 1973; Scott and
Noguchi, 1973). This enzyme is involved in enabling gluta-

thione to perform its role of a biological antioxidant.

II. LITERATURE REVIEW

Iron Metabolism:

In dealing with the subject of iron toxicity, a brief
review of iron metabolism seems relevant. Much more about
this topic is known in man and laboratory animals than in
other species. McCance and Widdowson (1938) were the first
to establish that "the amount of iron in the body is regu-
lated by controlled absorption.” Following this, the field
of iron metabolism and how the body achieves iron balance
was intensively studied. Hahn et a1. (1943) proposed the
"mucosal block" theory and this was later extended by
Granick (1954). According to this hypothesis, iron is
first reduced to its ferrous form; it is then transported
across the mucosal cells bound to ferritin; a second reduc-
tion occurs at the plasma membrane, and finally, there is
a re-oxidation to the ferric state when it becomes bound
to transferrin to be transported in the plasma. Moore
(1960) reviewed the mucosal block theory and found it to be
inconsistent with regard to the regulation and control of
iron transport in the mucosa.

Conrad et a1. (1964) put forward a modification of the
mucosal block theory. According to these workers, some
undefined fraction of the plasma iron serves as a

4

5
"messenger" to regulate the uptake of the dietary iron into
the mucosal cells where it is bound to protein. The rate
at which this iron subsequently enters the circulation is
regulated by a balance between the rate at which the epi-
thelial cells are exfoliated and the rate at which the
cellular iron enters the plasma.

Dowdle, Schachter and Schenker (1960) have put forward
another theory on iron absorption. Using isolated and
everted segments of the proximal small intestine of rats,
these workers reported that ferrous iron is actively trans-
ported; the mechanism is dependent upon oxidative metabolism
and the generation of phosphate-bond energy. Jacobs et al.
(1966) used a loop of gut with artificial circulation and
came up with similar findings. However, Brown and Justus
(1958) and Pearson and Reich (1965), also using everted gut
loops, were unable to fully confirm the theory that iron
is actively transported across the intestinal mucosa via
oxidative process. The findingsof Pearson and Reich (1965)
that soluble iron did not accumulate in the lumen of the
everted gut with increasing iron concentration led them to
suggest that iron enters the mucosal cell by diffusion.
These workers conceded that it is likely that iron absorp-
tion occurs through an active process when the iron concen-
trations are low, but at higher concentrations, passive
diffusion takes over. Charlton et a1. (1965) and Wheby
(1964) reported that the regulatory activity is lost when

iron intake exceeds physiological range. This probably

6

explains the linear relationships normally cited in that
there is always iron absorption at all levels of intake
(Bentler et al., 1963; Wallerstein and Mettier, 1958).

Yet another concept on iron absorption which has
gained much acceptance is that put forward by Saltman and
his associates (Saltman, 1965; Charley et al., 1960; Helbock
and Saltman, 1967; Pape et a1., 1968; Rubin and Princiotto,
1963). The basis for this hypothesis is directly related
to equilibrium binding and chelation phenomena. According
to Saltman (1965), Forth et al. (1965) and Ruliffson
(1966), both ferric and ferrous iron are available for
intestinal transport provided suitable chelates are present.
There is no direct dependence on metabolic energy for the
accumulation. The amount of iron available from the diet
is related to the relative concentrations of those agents
which prevent accumulation by forming insoluble or high
molecular weight complexes, such as phytic acid and proteins,
and low molecular weight substances able to solubilize the
iron and permit its absorption. Compounds that increase
iron uptake are ascorbic acid, sugars and polyols, amino
acids, gluconic acid, a-keto glutaric acid, citric acid,
synthetic chelates, alpha-tocopherol, diphenylparaphenylene-
diamine. Substances known to inhibit iron uptake are
endotoxins, alkalinizing agents, phosphates, oxalates,
synthetic chelates and malonic acid. The subject of phy-
tates and iron availability has been studied by various

workers. McCance et al. (1943) suggested that phytates

7
interfere with iron absorption by precipitating iron as
insoluble iron phytate, thus making the iron unavailable
for absorption. Indian workers (Apte and Venkatachalam,
1962; Sathe and Krishnamurthy, 1953) also demonstrated that
phytate considerably reduces the amount of iron available
for absorption. In contrast, Harrison and Mellanby (1942)
reported that the addition of sodium phytate to the diet
of anemic rats fed 0.3 mg iron/day did not appear to inhibit
hemoglobin regeneration when compared to control animals.
Again, Cowan et al. (1966) compared hemoglobin regeneration
in groups of anemic rats fed purified diets containing 10
or 20 ppm iron, in which 45% or 75% of the total phosphorus
was replaced with phytate phosphorus. Hemoglobin regenera-
tion was more rapid in the groups receiving 20 ppm iron
than in the groups receiving 10 ppm. However, the hemo-
globin values indicated that, even at the 10 ppm level, the
rate of hemoglobin regeneration was not affected by the
presence of either level of dietary phytate. Hence, it
was concluded that high levels of phytate have no effect
on iron absorption in the rat.

Davis, Luke and Deller (1966) reported the discovery
of a gastric inhibitory iron binding protein (gastroferrin),
secreted by the gastric mucosa of the stomach. In normal
animals and under normal circumstances, this protein com-
plexes with iron and makes the mineral less available to
the intestinal mucosa for absorption. This protein appears

to be absent in hemochromatosis and low in iron deficiency

8
anemia. Hence, it has been postulated that this substance
forms part of the physiological mechanism controlling iron
absorption. In contrast, Murray and Stein (1970a) have
studied the long—term effects of achylia gastrica in rats
on the absorption of dietary iron. Achylia gastrica was
produced in the animals by direct irradiation of the exposed
stomach. The treated animals were fed normal diets and it
was found that they had less liver iron stores, reduced
hemoglobin levels and body weights when compared to normal
control animals. The cause was attributed to a defective
assimilation of dietary iron as a result of the absence of
gastric secretion or acid which is thought to be essential
for optimal assimilation of dietary iron by rats.

In another study, Murray and Stein (1970b) confirmed
the findings of their previous study and further suggested
that defective iron absorption in the irradiated animals
can'be corrected by the addition of rat or human gastric
juice. It is significant to note that intrinsic factors
or gastric juice from patients with pernicious anemia did
not correct the abnormality. From this, the workers con-
cluded that there must be a factor in the gastric juice
necessary for optimal iron absorption in rats when an
increased demand for iron was needed.

Mechanism of Absorption of Iron Preparations
Administered Intramuscularly:
Beresford, Goldberg and Smith (1957) studied the mode

of absorption of a number of polysaccharide complexes

9
administered intramuscularly into rabbits. In all cases,
an acute inflammatory reaction with degenerative changes
developed at the injection site. Absorption was mediated
partly by this inflammatory reaction with enhancement of
lymphatic transport; this was evident from the fact that
iron was quickly picked up by the lymph nodes adjacent to
the site of injection. Most of the absorption occurred
within the first 72 hours; after this period, the iron
became rapidly fixed by tissue macrophages and this further
contributed to its unavailability. This study also sug-
gested that there is considerable variation in the absorba-
bility of different commercial iron preparations. Miller
et a1. (1965), in their study on iron retention and ham
discoloration in swine with five commercial parenteral iron
preparations, agree essentially with this observation.

In another study, Miller et al. (1967) also reported
iron injected intramuscularly was quickly picked up and
stored by adjacent lymph nodes. This store is then utilized
according to body needs for iron. This study concluded that
one intramuscular injection of 200 mg iron when piglets are
3 days of age is sufficient to satisfy the body needs for
this element and further iron administration later in life
could result in muscle discoloration.

Iron Toxicity in Man and
Laboratory Animals:

Cases of acute iron toxicity or simply an iron overload

in man are well recorded (Wallerstein and Mettier, 1958;

Bothwell and Finch, 1962).

10

Reissmann et al. (1955a) reported that 3-10 g of
ferrous sulfate usually prove to be fatal in younger
children. Most observers (Swift et a1., 1952; Smith et al.,
1950; Linquist et a1., 1952; Foucar, 1948) attributed death
to a local necrotizing action of the iron in the intestinal
canal resulting in shock from hemorrhages and fluid loss.
Reissmann and Coleman (1955b) disagree with this contention.
Working with dogs and rabbits these workers reported that
toxicity is due directly to the absorbed iron. The dosage
used was between 50 mg to 750 mg iron per kg body weight.
The route of administration was either by means of a stomach
tube, as an enema in 10% solution or intravenously. Irre-
spective of the route of administration, none of the animals
given 200 mg iron or more per kg body weight survived.
Survival time was roughly inversely proportional to the
dose administered. The cause of death was reported to be
due to a profound metabolic acidosis with blood pH values
as low as 6.7. This acidosis was reported to be due mainly
to a hydrolyzing effect of ferric iron and in part to an
increase in lactic and citric acids. It was suggested that
iron in toxic amounts inhibits the enzymes in the Kreb's
cycle. Earlier, Racker and Krimsky (1948) had reported that
iron shares the property of binding sulfhydryl groups and
thus may interfere with certain enzyme systems. These latter
workers also observed that glycolysis of tissue homogenates

was inhibited by ferrous sulfate.

ll

Bothwell and Finch (1962) reviewed the different types
of iron overload in man. Idiopathic hemochromatosis is
considered to be associated with excessive absorption of
iron as a result of some intrinsic metabolic defect with
alteration of the mucosal cell leading to an enhancement
of iron uptake. The condition is characterized by liver
enlargement with subsequent hepatic insufficiency, pigmenta-
tion of the skin and diabetes mellitus; heart failure is a
common complication. The condition is believed to be
inheritable.

Siderosis amongst South African Bantu is due to an
absolute increase in total body iron. This results from
the use of iron utensils for cooking and in the preparation
of beverages. The quantities present in an average daily
diet have been estimated to exceed 100 mg/day and, as the
amount of iron retained in the body increases with the size
of the dose, iron overload is a common occurrence in the
Bantu population. The condition first becomes evident in
late adolescence and reaches its peak between the ages of
40 and 50 years. The pathogenesis of the condition initially
manifests itself in the liver as parenchymal deposits.

With increasing absorption, splenic concentration of iron
also rises and often the level in this organ is higher than
that in the liver.

Oral iron overload in patients with refractory anemias
is due to an increased absorption from the gut. It is

associated with an increased, but usually ineffective,

12
erythropoietic activity and is especially common when
therapeutic amounts of iron have been given orally.
Parenchymal deposits are usually found and this appears
to be related to the fact that the plasma iron level is
often persistently raised and the unsaturated iron-binding
capacity reduced. The most prominent pathological features
are found in the liver; portal cirrhosis and portal tract

fibrosis are commonly encountered.

Reticuloendothelial Iron Overload:

Bothwell and Finch (1962) reported that parenteral
administration of iron preparations leads initially to an
accumulation in the reticuloendothelial system. This is
because the iron complexes consist of relatively large
molecules that are selectively removed from circulation by
the reticuloendothelial system (RES). The RES can tolerate
large amounts of iron because it requires very large doses
of iron injected into healthy individuals to elicit patho-
logic changes.

Nissim (1953) reported that subcutaneous injections of
"ferric hydroxide ferrous ascorbate" in total dosage of
l g per kilogram body weight caused hemorrhagic changes in
the lungs, patchy parenchymal damage in the liver and
adrenal hemorrhages in the guinea pig. Brown et al. (1959)
described the effects in dogs when iron was injected
parenterally in regular doses. Irrespective of the iron

preparations used (iron dextran or saccharated iron oxide)

13
or by the route of administration (intramuscular, intra-
peritoneal or intravenous) it was found that dosage of
between 2.5 to 3.3 g/kg body weight produced death within
5-10 months. The animals suffered from anorexia, apathy
and weight loss, and one dog developed anasarca, edema and
chronic leg ulcers. In another study, Brown et al. (1957)
reported that dogs which had received intravenously 0.5 to
1.0 g iron per kg body weight at regular intervals lived
for 4 to 7 years. Blindness was the only defect during
the observed period. After the animals were sacrificed,
histological studies revealed that almost no movement of
iron out of the reticuloendothelial cells had taken place.
Goldberg et al. (1957), using rats and rabbits also found
that these species are able to tolerate large doses of
iron given intramuscularly. Patterson et al. (1971), how-
ever, reported that 47 mg iron per kg body weight given
intraperitoneally was fatal to adult rabbits. The cause

of death was acute hepatic failure due to iron overload.

Iron Toxicity in Farm Animals:

Nutritional siderosis in beef cattle was reported by
Hartley et al. (1959) following the grazing of these animals
on pastures containing high levels of iron. The condition
was only discovered when the animals were slaughtered; the
livers and hepatic and pancreatic lymph nodes were dis-
colored. Bones were osteoporotic and there was hyperplasia
of the bone marrow. Biochemical studies of the livers of

affected animals revealed very high iron levels.

14

Effects of high oral iron on dairy cattle were reported
by Coup and Campbell (1964). Scouring, loss of weight,
reduced milk and butterfat yield were observed when animals
were either dosed orally with 30 to 60 g iron per day or-
when allowed to graze on pastures which were irrigated with
water containing high levels of iron.

Lawlor et a1. (1965) observed that lambs scoured and
performed less well when they were fed rations containing
more than 200 ppm supplemental iron. These workers, however,
did not discount the possibility of a mineral imbalance in

their study.

Iron Toxicity_in Swine: Field Reports

Behrefis (1957) administered 100 mg iron dextran intra-
muscularly to 5 one-week-old pigs. Within a few days, all
the treated animals exhibited clinical signs such as
anorexia, weakness and loss of weight. They died between
8 and 45 days after treatment. Other pigs in the same
litter which were not given the iron treatment remained
healthy. Postmortem findings in the dead animals included
emaciation, edema of the muscles and blackish discoloration
of the livers and spleens. Microscopic examination of the
livers showed variable amounts of hemosiderin. It was sug-
gested that in the absence of anemia, there is insufficient
apoferritin to combine with the iron, which becomes precipi-
tated as hemosiderin.

Brag (1958) described iron toxicity in piglings follow-

ing a conventional method of anemia prophylaxis. The method

15
involved the spraying of soil with ferrous sulfate and
allowing the piglings to forage in it. Animals reared on
concrete were usually 2 weeks old when they were first
exposed to this treatment. Signs of illness were observed
within a couple of hours after the animals had access to
the iron treated soil. As a rule, the whole litter became
111; if there were exceptions, they were usually the smallest
and weakest pigs which had been unable to reach the iron
soil mixture. Clinical signs included paleness, unsteady
gait, convulsion with paddling of the front limbs and diffi-
cult breathing; body temperature was usually subnormal and
heart rate was accelerated. Within 24 hours the whole
litter might be dead. Postmortem findings included pale-
ness of the carcass, waxlike skeletal and cardiac muscles,
coagulated milk in the stomach mixed with soil, gastroenteri-
tis and sometimes ulceration of the stomach and intestine.
The author reviewed some of the cases of iron poisoning in
children and pointed out the similarities in symptoms in
both man and pig.

Nilsson (1960) reported the death of 10 piglets 12
hours after receiving 1 to 2 ml of a high molecular weight
iron carbohydrate complex intramuscularly. Upon autopsy,
the myocardium had a striking tiger stripe pattern with
alternating pale and hyperemic parts. Hydropericardium and
hydrothorax were also encountered. Microscopically, the

myocardial lesion was found to be a focal hydropic

l6
degeneration. Large amounts of glycogen were present in
the degenerated muscles.

Blandford and Lodge (1966) reported the case of a 15-
week-old pig which died following an injection of an iron
dextran preparation. The pig was given 200 mg of the iron
preparation at 3 days of age. When it appeared anemic at
about 15 weeks of age, a further 300 mg of the same iron
preparation was given by subcutaneous route behind the ear.
Three days later it was found dead. At autopsy the carcass
appeared severely jaundiced and the liver was enlarged.
Sections of liver stained with Prussian blue contained only
moderate amounts of iron in the Kupffer cells; however, the
nuclei of a few small groups of parenchymal cells exhibited
either a diffuse Prussian blue reaction or contained discrete
Prussian blue positive bodies.

Ueberschfir (1966) reported sudden deaths in suckling
piglets following administration of iron dextran prepara-
tions. At necropsy, severe inflammatory edema and focal
muscular necrosis and hemorrhages at the site of injection
were found. He concluded that toxic properties of the iron
preparation were the cause of these changes.

Experimental Studies of Iron
Toxicity in Swine:

Campbell (1961) determined the toxic dose of iron
(ferrous sulfate) in piglets 3 to 10 days of age. The iron
was administered either by means of a stomach tube or

directly into the stomach or duodenum after these organs

17
were exposed following surgical procedure. Absorption was
reported to be rapid both from the stomach and duodenum.
It was suggested that when high level of iron is adminis-
tered directly into the gastrointestinal tract, the mechanism
of absorption is by diffusion. The toxic dose was found to
be 0.6 g iron per kilogram body weight. Clinical signs and
symptoms of toxicity were observed 1 to 3 hours after the
toxic dose was given; these included incoordination, marked
shivering, hyperpnea and, on handling most of the pigs,
tetanic convulsion. Some developed posterior paralysis and,
in all cases, there was a profuse diarrhea. Death usually
occurred in about 6 hours following the appearance of
symptoms. Histopathological studies of animals that had
survived up to 24 hours or more had extensive edema along
the entire stomach wall. The mucosa showed extensive
necrosis and hyperemia. The necrotic tissue contained
deposits of yellowish brown to black pigments which gave a
positive test for iron with Perl's stain.

It was postulated that the mechanism of toxicity was
either due to damage to the gastrointestinal mucosa which-
led to shock, or directly as a result of high level of
plasma iron.

Induced Rickets as a Result of High
Density Levels of Iron:

Various researchers working with different species of

animals have reported that high levels of dietary iron

interfere with phosphorus utilization, especially when the

18
latter mineral content in the diet is low or just on the
borderline of normal requirement (Cox et al., 1931; Deobald
and Elvehjem, 1934; Brock and Diamond, 1934). Prolonged
excess of dietary iron without at the same time increasing
the phosphorus intake can eventually lead to gross phos-
phorus deficiency with subsequent development of rickets.
The condition is believed to be brought about through the
precipitation of phosphorus as the insoluble phosphate.

O'Donovan et al. (1963), in their studies with early
weaned pigs, reported that pigs fed 3000 ppm iron or less
performed well. Pigs fed 4000 ppm iron had slightly
depressed serum inorganic phosphorus and phosphorus
deficiency rickets was first observed after 6 weeks of the
experiment. Pigs which received 5000 ppm iron in their
diet had significantly lower serum inorganic phosphorus and
femur ash content; rickets with its characteristic symptoms
was evident on the 4th week. Symptoms were particularly
severe in animals where the levels of serum inorganic
phosphorus were lower than 4 mg/100 ml.

This study also demonstrated that the phosphorus level
in the diet is important when high levels of iron are present
in the diet. Pigs receiving 0.60 or 1.20% phosphorus per—
formed well even though the diet contained up to 5000 ppm
iron. However, when animals were fed 0.35% phosphorus and
2500 or 5000 ppm iron, rickets developed.

A separate experiment within the same study indicated

that the source of phosphorus was related to the toxic level

19
of iron in the diet. When the diet contained 5000 ppm iron
and the source of phosphorus was either defluorinated rock
phosphate, Curacao Island phosphate or commercial dicalcium
phosphate, there was a significant reduction in serum
inorganic phosphorus values; when monosodium phosphate or
dicalcium phosphate (U.S.P.) were used to supply the same
level of phosphorus in the diet (0.6%), serum inorganic
phosphorus levels were normal.

The work of these investigators are in agreement with
those of Hegsted et al. (1949), who reported that rats fed
a low phosphorus ration absorbed and consequently stored
more iron in the liver than animals fed a similar diet sup-
plemented with phosphorus. Phosphorus (0.8%) was required
to prevent marked accumulation of liver iron when the diet
contained 0.3% iron as ferric citrate.

Iron Toxicity in Swine Associated with Low
Levels of Vitamin E and High Levels of
Polyunsaturated Fatty Acids:

Lannek et al. (1962) fed a specially prepared diet,
high in polyunsaturated fatty acids (PFA) and low in vitamin
E to 3 sows 6 to 27 days before parturition and throughout
the lactation period. The sows fed the experimental diet
produced milk with about twice the amount of PFA as compared
to sows fed a commercial sow ration. The average vitamin
E content in the milk fat of treated sows was only about
25% of that in the control sows (average of 11.7 ug/g in

treated animals compared to 47.2 ug/g milk fat in control

20
sows). Seventeen out of a total of 22 piglets from the 3
treated sows were given 150 mg of an organic iron complex
intramuscularly when they were between 10-27 days of age.
After a few hours or a day following treatment, the animals
developed signs of apathy, dyspnea and incoordination. Nine
of the 17 treated animals died within 2% days following
treatment. Gross postmortem findings were similar to those
described by Brag (1958) and Nilsson (1960): hydropericardium
and waxy degeneration of body musculature. There was a
significant elevation of plasma glutamic-oxalacetic trans-
aminase (GOT) in affected piglets. In surviving piglets
plasma GOT level returned to normal within a week. Two pig-
lets with high plasma GOT on the first day of iron treatment
were given a-tocopheryl acetate at 6 mg/kg body weight intra-
muscularly for 4 consecutive days; on the 5th day, the
organic iron complex was again administered. No deaths and
no elevation of plasma GOT occurred.

The workers believe that vitamin E exerts an antioxi-
dant effect within the cells. The introduction of iron
into tissues containing high PFA and low in vitamin E is
thought to speed up the oxidation of PFA and enhance the
reactivity of the oxidation products.

In another study, Tollerz and Lannek (1964) obtained
vitamin E deficient piglets by the same method as in the
previous study. The objective was to demonstrate that
vitamin E or synthetic antioxidants can increase the resis-

tance of vitamin E deficient piglets to iron administration.

21
Each litter was divided into two groups; one group was given
only the iron preparation whereas the other group received
in addition the substance that was under test. Treatment
began when the piglets were 3-8 days old. The iron prepa-
ration used was an organic iron complex. It was clear that
vitamin B when given 24 hours before the administration of
iron protected piglets from death. However, when both
vitamin E and iron were given simultaneously, there was no
protection. Ethoxyquin (l,2-dihydro-6-ethoxy-2,2,4-
trimethquuinoline) when given together with iron was
effective in preventing mortality. Selenium, as sodium
selenite, did not give any significant protective action.
The results of this study support the hypothesis that at
relative over-dosing of iron, vitamin E is active by means
of its antioxidant properties.

Arpi and Tollerz (1965) described in detail the post-
mortem changes in experimental and spontaneous cases of
iron-poisoning in piglets. The experimental piglets were
made hypersensitive to both oral and parenteral iron admin-
istration by feeding sows a diet deficient in vitamin E.

The spontaneous cases were from a farm where deaths occurred
following standard prophylactic measures against piglet
anemia (100-150 mg of iron from iron dextran intramuscularly).

Postmortem findings in piglets that died following
intramuscular injection of iron dextran, iron dextrin or
the oral administration of ferrous fumarate were very

similar. The skin and muscles were very pale. The muscles

22
and subcutis around the sites of injection were edematous.
The colon, rectum and, to some extent, the small intestine
had varying degrees of grayish black discoloration.. In
some cases there was mild to acute catarrhal enteritis.
Slight hydrothorax was reported only in some of the spon-
taneous cases.

The most striking microscopic finding was a severe
waxy muscle degeneration. The pathological condition was
characterized by swelling and loss of striation of the
muscle fibers and, in some cases, by the total destruction
of large areas of muscle fibers. In contrast to the study
by Nilsson (1960), who observed a striking tiger stripe
pattern of the myocardium, the present workers reported
only slight cardiac muscle degeneration in only one of a
total of 78 fatalities. Fatty degeneration of muscle fibers
and some dystrophic calcification were also reported.

Necropsy findings for piglets poisoned by ferrous
sulfate were different from that of the above in two
respects. Marked to moderate skeletal muscle degeneration
was found only in 3 out of a total of 7 deaths. In all 7
cases, there was a severe acute catarrhal to necrotizing
gastroenteritis with necrosis and hemorrhages in the mucous
membrane.

Biochemical Studies in Piglets Made
Susceptible to the Toxic Effects
of Iron:
Patterson et a1. (l967a,b), following similar experi-

mental procedures used by Lannek et al. (1962), obtained

23
litters of pigs susceptible to the toxic effects of iron.
Sows on the last 4 weeks of pregnancy were fed a diet
deficient in natural antioxidants. Nine piglets from one
litter were given 47 mg iron dextran per kg body weight
intraperitoneally. Five died within 4 hours of treatment
and the others were killed for comparative studies.
Chemical analysis and enzymatic assays were determined for
muscle, serum and plasma samples. There was a significant
increase in the peroxide content of the muscle which was
believed to be due to peroxidation of muscle lipid. Raised
plasma levels of acid phosphatase are thought to result
from lysosomal damage; there was a significant leakage of
muscle enzymes such as aspartate aminotransferase into
the plasma; more important was the finding that muscle
potassium also leaked into the plasma. Death was thought
to have resulted from cardiac arrest as a result of
hyperkalemia.

In a more thorough study, Patterson et al. (1969)
examined in greater detail the biochemical aspects of iron
toxicity in susceptible piglets. Two— to three-day-old
susceptible piglets were given 47 mg iron dextrose per kg
body weight intraperitoneally. Deaths occurred within 5
hours following the iron injection. Pigs that were alive
after this period were killed by the intrathecal injection
of xylocaine. Blood and muscle were collected for bio-

chemical and histological studies.

24

The gross and histopathological findings were in
general agreement with the description given by Arpi and
Tollerz (1965). Coagulative necrosis, loss of striations
and, in more severe cases, total destruction of muscle
fibers were reported. However, fatty degeneration and
dystrophic calcification, which were features described by
the latter workers, were absent. An increased amount of
stainable iron was detected in the reticuloendothelial
cells of the liver. Histochemical studies revealed distri-
bution of succinic dehydrogenase which suggested mito-
chondrial clumping and probably the disruption of the
centers of activity of the mitochondria.

Analysis of muscle and serum iron concentrations
showed an absence of a relationship between these criteria
and mortality, i.e., there was no critical muscle or serum
iron concentration above which the outcome was fatal. There
was no significant alteration in muscle dry matter, pro-
tein, lipid and nucleic acid contents even in proven cases
of myodegeneration. However, in severe cases there was a
loss of muscle potassium with corresponding increases in
sodium and chloride contents. The raised potassium in the
circulation was associated with a significant electrocardio-
graphic response. Fatal cases occurred when the plasma
potassium values exceeded about 10 mEq/liter.

Increased iron concentration in the muscle following
iron injection brought about an enhanced rate of peroxi—

dation - giving rise to an increased level of tissue

llll I, ll. III

25

peroxides. This is thought to be responsible for the damage
to the lipoprotein envelope of muscle fibers with resultant
leakage of cell constituents - potassium and enzymes; the
plasma level of aspartate aminotransferase, a muscle enzyme,
was raised 2% to 5 hours following iron injection. There
was also an increase in the level of plasma alkaline
phosphatase which indicated liver damage. Increased values
of acid phosphatase and beta-glucuronidase were thought to
be due to early and specific damage to lysosomes. Release
of these lysosomal enzymes was thought to initiate the
disintegration of the myofibrils. Thus, the underlying
mechanism for muscle damage was attributed to an iron-
catalyzed peroxidation reaction.

The toxic effect of iron was also manifested by a
rise in plasma concentration of glucose. Plasma levels of
up to about 500 mg glucose/100 ml were recorded in severe
cases. Normal values for three-day-old piglets varied
between 115 to 160 mg/100 ml. This increase in plasma
potassium is believed to exert an effect on the adrenal
medulla and stimulates an adrenalin-sensitive phosphorylase.

The rate of iron absorption following the injection
of iron was greater in susceptible piglets than in normal
piglets. In normal piglets, maximum muscle iron concentra-
tions were reached 2-3 hours after injection; in susceptible
piglets, very much higher maximum muscle values were

attained after about one hour. From this it was concluded

26
that the skeletal muscles of susceptible piglets have an
increased affinity for iron.

Analysis of muscle and serum iron concentration prior
to iron injection indicated that susceptible piglets had
significantly higher values than normal piglets. This
coupled with an increased affinity for iron is believed to
predispose the muscle to overload following an iron
injection.

In yet another study, Patterson et al. (1971) compared
the toxic effects of iron in susceptible piglets at 2 and
8 days of age. Clinical manifestations of iron toxicity
were found only in the younger age group; 9 out of 16 iron
treated animals in this group showed clinical signs such
as vomiting and diarrhea, although only 2 of the affected
piglets died. Hyperkalemia, though severe in the 2 fatal
cases, was not significant when the whole group was taken
into consideration. Similarly, plasma aspartate amino-
transferase activity was raised in individual piglets but
also without significantly affecting the mean value.

The mean muscle iron concentration of the clinically
affected group was significantly higher than for the con-
trols; some affected animals which did not have a signifi-
cant increase in muscle iron concentration were observed
to excrete iron stained urine. They believed that, as
long as the kidneys were not too severely damaged, and
especially in the older piglets, excretion of iron and

potassium were protective mechanisms.

27
Muscle peroxide concentrations were significantly
raised only in the 2-day-old piglets that were clinically
affected. This finding confirms the hypothesis in an
earlier study (Patterson et al., 1969) that peroxidation
of muscle lipids is an early step in the acute degenera-

tion of muscle induced by iron dextrose.

III. EXPERIMENTAL PROCEDURE

Animals used in thestudieswere from either purebred
Hampshire or Yorkshire or from crossbred Yorkshire-
Hampshire sows which had been fed a low vitamin E - Se or
a vitamin E supplemented diet throughout gestation and
lactation. The piglets produced by these sows were used
in three experiments to examine the tolerance of iron as
influenced by the vitamin E and Se status of the animals.
A. General Conduct of Experiments

1. Effect of a second iron injection on vitamin

E - Se deficient pigs (Experiment 1)

In this experiment, piglets were obtained from
sows which had been fed a low vitamin E - Se ration through-
out gestation and lactation. The gestation and lactation
diets are shown in Table l. A total of 127 piglets from
17 litters were used. At three days of age the piglets
were given their first intramuscular injection of 100 mg
of iron from iron dextran. At 5 weeks of age, these pigs
were weaned to a low vitamin E - Se starter ration (Table
1). Feed was provided on an ad libitum basis. Two days
after weaning each litter was divided at random into two

groups. Pigs in one group received an intramuscular

28

29

Table 1. Composition of diets

 

Gestation Lactation Starter Grower

 

 

 

 

Ingredient % % % %
Ground shelled corn 85.0 77.5 51.45 78.75
Soybean meal (49%) 11.5 19.0 20.0 18.0
Rolled dehulled oats - - 10.0 -
Dried skim milk - - 10.0 -
Sucrose - - 5.0 -
Ground limestone 1.0 1.0 1 0 1.0
Dicalcium phosphate 1.5 1.5 0.8 1.25
Salt 0.5 0.5 0 5 0.5
MSU VTM premixa 0.5 0.5 1.0 0.5
Antibiotic premixb - - .25 -
100.0 100.0 100.0 100.0
Analyses:
Crude protein, % 13.2 16.1 19.5 16.0
Selenium, ppm 0.04 0.05 0.09 0.05
d-a-tocopherol, mg/kg 7.6 7.4 -C 7.5

 

3See Appendix A.

bSupplied to the ration 110 ppm chlortetracycline,
110 ppm sulfamethazine and 55 ppm penicillin.

CNot analyzed.

3O
injection of 34 I.U. of vitamin E plus 0.5 mg of Se, the
other group served as a control. Two days after the vitamin
E - Se injection, each group of animals was further divided
into two sub-groups. One group of vitamin E - Se injected
pigs and one group of non-injected animals were given a
second intramuscular injection of 100 mg of iron from iron
dextran.

Rectal temperature was taken at 0, l, 4 and 6 hours
after the second iron injection, to determine whether or
not there was anaphylactic reaction.

2. Effect of dietary iron supplementation to low

vitamin E and vitamin E-supplemented rations

of young pigs (Experiment 2)

In this second experiment, 48 pigs from sows on
a low vitamin E - Se diet were used. The pigs were weaned
at 5 weeks of age. They were allotted randomly by litter
and weight into 6 groups of 8 pigs per group. Initially
the pigs were kept on aluminum slatted pens until they
were approximately 2% months old, when they were moved to
cement slatted pens.

The feed provided following weaning and throughout
the experimental period was the grower ration (Table 1).
This was a corn-soy diet which contained about 0.05 ppm
Se. The basal diet contained 345 ppm Fe. Additions of
iron as ferrous sulfate (FeSO4 7H20) and vitamin E as
d - a - tocopheryl acetate to the basal diet were used in
a 3x2 factorial design. The diets contained 345, 550 and

850 ppm iron and contained either a low level of vitamin

31
E or were supplemented with 22 I.U. of vitamin E/kg of
feed. Feed was provided on an ad libitum basis and animals
were allowed free access to fresh water at all times.

The animals were weighed individually at the beginning
of the trial, then weekly or at fortnightly intervals.
Following each weighing, feed remaining in the feeders was
measured. Feed consumed for the period was recorded and
average feed consumption and feed/gain ratios were calculated.

Blood for hematological studies and serum Fe analysis
was collected at various intervals during the course of the
experiment. Half the number of animals from each group
were bled and bleeding was done on the same animals at
each subsequent bleeding. Blood was taken from the anterior
vena.cava by means of a sterilized 18 gauge needle and 20
ml syringe. Samples for hemoglobin (Hb) and hematocrit
(Hct) determination were put into acid washed heparinized
vials, while samples for serum Fe analysis were placed into
acid washed centrifuge tubes.

The length of the experiment was 126 days, after which
the pigs were slaughtered. Samples of liver, pancreas and
flank muscle were collected from each animal for Fe analysis.
Samples were placed in separate polyethylene bags and frozen
in the freezer until required for subsequent iron analysis.

3. Effects of a large dose of iron given intramuscu-

larly to young swine deficient in vitamin E and
Se (Experiment 3)
Sixteen pigs were used in this study. Eight were

weaned from sows fed a low vitamin E - Se gestation and

32
lactation ration, while the other 8 were from sows whose
diet had been supplemented with 22 I.U. of vitamin E/kg
of diet. All 16 animals received their first iron injec-
tion at three days of age as 100 mg of iron from iron
dextran intramuscularly.

At 9 weeks of age, each group of animals was again
divided into two sub-groups. One sub-group from each of
the two main groups received a second iron injection
intramuscularly at a dose of 1000 mg Fe from Fe dextran.
The animals were observed for signs of toxicity following
the administration of the massive dose of iron.

Blood was collected prior to the administration of
the massive dose of iron and then at 1 day and again at 1
week after the iron treatment for hemoglobin, hematocrit,

and serum iron determination.
B. Analytical Procedures

1. Hemoglobin
Hemoglobin was determined by the cyanmethemoglobin
method of Crosby et al. (1954). A Coleman Junior 11
spectrophotometer was used for optical density

determinations.

2. Hematocrit
Hematocrit was determined by the micro-capillary
tube method (McGovern et al., 1955). Blood samples were
centrifuged for 5 minutes at 1000 rpm in an International

"Hemacrit" centrifuge.

33
3. Serum collection
Blood samples were collected in acid washed centri-
fuge test tubes and allowed to coagulate. Following the
removal of the clot, samples were centrifuged at 550 g for
15 minutes. The serum was then pipetted into acid washed

vials and stored at 5° C.

4. Serum iron

Serum samples were thawed at room temperature and
then precipitated with 20% trichloroacetic acid. The
mixture was heated in a water bath at 90° C for 15 minutes.
It was then removed and kept at room temperature. Upon
cooling, the mixture was centrifuged for 5 minutes at 550
g. The supernatant was decanted into acid washed test
tubes. Iron concentration was determined by atomic absorp-
tion spectrophotometry, using a Jarrell-Ash model 82-516
spectrophotometer. Samples were aspirated into an air-
hydrogen flame. An absorption wavelength of 2480.5 A was
used. Samples from pigs which had received the 1000 mg
Fe injection were too concentrated and were diluted 1:5

with deionized distilled water prior to iron determination.

5. Determination of iron concentration in feeds
Iron levels in the feeds in Experiment 2 were
determined. A wet ash method was used. Depending on the
level of iron supplemented, 0.4 to 1.5 g of feed samples
from each treatment group were weighed out and placed into

separate 250 m1 tared Phillips beakers. Sixty milliliters

34

of concentrated nitric acid were added and digestion was
carried out on a hot plate to near dryness and allowed to
cool. Seven milliliters of concentrated perchloric acid
were added and digestion was allowed to continue again to
near dryness. Following cooling, samples were diluted to
constant weight with deionized distilled water. Standards
were prepared in a similar manner.

Iron concentration was determined by atomic absorption
spectrophotometry using a model IL453 Atomic Absorption
emission spectr0photometer.* An absorption wavelength of
2486 A was used.

6. Determination of tissue iron (liver, muscle and

pancreas) concentration

A homogenate of the tissue sample was first pre-
pared. Frozen samples of liver, muscle and pancreas were
separately cut into thin slices. About 5 g of each tissue
sample were used. Deionized distilled water was added to
each sample to make up twice the weight of the sample.
Homogenization was performed with a Brickmann polytron at
high speed for about 1 minute with liver and pancreas
samples and for about 2 minutes with muscle samples. In
the latter tissue, care was taken to avoid the inclusion
of excess fat. The homogenates were poured into polyethy-
lene bags and frozen in the freezer until ready for iron

determination.

M
r

t
Instrumentation Laboratory, Inc., 113 Hartwell
Avenue, Lexington, Mass. 02173.

35

Iron tissue concentrations were again performed by wet
ash procedure. The homogenates in the polyethylene bags
were thawed with warm water in a beaker. About 5 ml of
well mixed samples were pipetted out and placed in separate
tared 250 m1 Phillips beakers. The beakers containing the
samples were then weighed to determine the exact amount of
samples used. Digestion was performed as with the feed
samples.

Tissue iron concentration was determined by atomic
absorption spectrophotometry as mentioned previously for

serum-iron determination.

C. Statistical Analysis

The data for experiments 2 and 3 were analyzed for
statistical difference by the f-test of Snedecor (1950).
Individual treatment values were compared by Duncan's

(Bliss, 1967) multiple range test.

IV. RESULTS AND DISCUSSION

Experiment 1

The feeding of a vitamin E - Se deficient diet alone
to sows did not produce piglets with reduced tolerance to
iron dextran injection. When three days old, piglets from
these sows were given an intramuscular injection of 100 mg
iron from iron dextran; the animals did not exhibit any
signs or symptoms of iron toxicity. Growth was normal and
there was no mortality throughout the five week suckling
period.

Pigs given a second intramuscular injection of 100 mg
iron from the same iron dextran, four days after weaning
to a low vitamin E - Se starter diet, also did not develop
any reduced iron tolerance. One-half of the experimental
pigs had received a vitamin E - Se injection (34 I.U.
vitamin E + 0.5 mg Se) two days prior to the iron injection;
irrespective of whether the animals had been previously
treated with vitamin E - Se injection or not, there was no
evidence of iron toxicity.

Rectal temperatures were taken at 0, l, 4 and 6 hours
following the second iron injection, and results are pre-
sented in Table 2. Temperatures were all normal with no
evidence of anaphylaxis for any of the treatments. There

36

37

Table 2. Rectal temperatures, °F, after a second IM iron

 

 

injection

E - Se inj.a - - + +
Iron inj.b - + - +
Time

0 hour 102.2 102.3 102.3 102.4
1 hour 103.0 103.1 103.2 103.1
4 hours 103.1 103.4 103.1 102.9
6 hours 102.7 103.0 102.8 103.0

 

aIntramuscular injection of 34 I.U. vitamin E +
0.5 mg Se.

bIntramuscular injection of 100 mg iron from iron
dextran.

was a slight rise in temperatures during the first one to
four hours, but this was believed to be due to excitement
during the handling of the animals rather than to any other
cause. Six hours after the iron administration, temperatures
had all declined or remained constant.

The results of this study appear to be in contrast to
those of Lannek et al. (1962) and Patterson et al. (1969).
Lannek et al. (1962) reported obtaining piglets with lowered
resistance to injectable iron when sows were fed a diet
containing.10.4 mg vitamin E per kg of feed for 6 to 27 days
during gestation and throughout lactation. When 10- to 27-
day-old piglets from these sows were given 150 mg iron from

an organic iron complex intramuscularly, signs and symptoms

t

5444. I- 1
M
W—

ofiron
the inje
treated

Pa
were hy
gestatf
piglet

dextro

the L
the o
subje
of a'
furt
pres
t01
inc:
res
the
men

bee]

EXPQ

Vita

tria

and a

38
of iron toxicity were seen within hours or a day following
the injection. There was more than 50% mortality with the
treated animals.

Patterson et al. (1969) also obtained piglets that
were hypersensitive to the toxic effects of iron by feeding
gestating sows a low vitamin E diet. When 3 days old,
piglets from these sows were given 47 mg iron from iron
dextrose; toxicity and deaths were again reported.

It is important, however, to point out that in both
the Lannek et al. (1962) and Patterson et al. (1969) studies,
the oil and grain portions of the experimental diets were
subjected to oxidizing conditions by heating in a current
of air. This increased the peroxide value of the diet and
further aggravated the low vitamin E situation. In the
present study, no polyunsaturated fatty acids were added
to the experimental diets and the peroxide value was not
increased by heat treatment. The possibility that the
results of this study might have been similar to those of
the other workers, had the experimental diets been supple-
mented with polyunsaturated fats or had the peroxide value

been increased, is not ruled out.

Experiment 2:

One pig out of 8 fed the 850 ppm iron ration with no
vitamin E supplementation died in the third week of the
trial.

Gross postmortem lesions included pale skeletal muscle

and a mottled appearance of the cardiac muscles with

39
alternating areas of hyperemia and paleness. There was a
large ulcer at the cardia of the stomach and it was filled
with blood. Hydrothorax was also observed.

Histopathological examination revealed extensive
necrosis of the myocardium. There was infiltration of
mononuclear cells and loss of myocardial fibers. There
was some fibrosis in the liver - more than what is expected
from an 8-week-old pig.

The gross and microscopic findings are somewhat similar
to those described by Brag (1958) in pigs which died follow-
ing the ingestion of soil mixed with ferrous sulfate solu-
tion. The heart lesions correspond with those reported by
Nilsson (1960) following the intramuscular injection of iron
for anemia pr0phy1axis. However, in the present case, it
is impossible to say with any degree of certainty that the
death was the direct result of iron toxicity. Gross and
microscopic findings in pigs which died from vitamin E -

Se deficiency are also very similar to those described
above.

Pig performance data are shown in Table 3. Average
daily gains during the growing period tended to increase
with increasing levels of iron in pigs fedllow vitamin E
diets and decreased with increasing levels of iron on
high vitamin E diets. Treatment average daily gains did
not significantly differ during the finishing period or when

the-entire study was taken into consideration. Similarly

.mmo.vmv xHucmoHMAcmfim Hommfiw mumfigomueQSm pcmuemmfiw spa: 30h eewm on» a“ monam>nm

.ucmofimficmfim no: u .m.z
.cmoe ogu mo Rogue enmwcmum

 

40

 

H
mm.o mm.o mm.o mm.o mm.o mm.o eomm\efieo
w~.e HN.¢ mH.e em.e mfi.e mN.e BA .emmm AHARe .><

.m.z .m.z .m.z mo. mm.H ee.H oe.H om.H mm.H mm.H AH .efimm AHAem .><

Hampo>o
Hm.o om.o mm.o om.o wN.o mN.o e66m\:fleu
em.e mm.m mw.e mfl.m mm.e mo.m AH .emom AHAme .><

.m.z .m.z .m.z co. Nm.H mm.H H4.H em.fl Am.fi we.H AH .eAem Afifimm .><

woflnom wdflzmwafim
em.o mm.o mm.o mm.o Am.o mm.o e66m\eAmu

em.m mm.m AA.m Hw.m AA.m mA.m AH .eoom AHAme .><

 

 

 

 

 

mo.va .m.z .m.z oeo. om.H mm.H we.H oe.H mm.H NA.H AH .EAmm AHAee .><

m am a n on m wowuomw:HSOHo

mHmEfldm mo . 02

m x EOAH m .AA> :OAH I owe omm mam omw omm men sag .:6AH
mm+

ouEeUAmfiemAm H OH o nH\.=.H .m .pA>

 

oocmehomgom no m casewfi> was donfl Hugo mo uoewmm .m manmh

41
there were only small treatment differences in daily feed
consumption and gain/feed ratios.

The iron-vitamin E interaction in average daily gains
during the growing period was rather unexpected. The
dietary iron requirement for pigs at this age is far below
the levels that were used in this study. The optimum iron
requirements for the growing pig have been given at between
60 to 125 ppm (Matrone et a1., 1960; Ullrey et a1., 1960).
These values vary depending on the animal's iron store,
the type of ration fed, the phosphorus level in the diet,
and other factors. The National Research Council (1968)
has considered all these factors and has established 80
ppm as a level of iron required by the pig. Increasing the
level of dietary iron above optimal requirement does not
normally bring about better performance. The term "optimal"
is used here in a relative sense; optimal requirement for
one individual might not be so for another.

The decrease in daily gains during the growing period
with increasing levels of iron on the high vitamin E diet
was also unexpected. When the levels of iron are con-
sidered, the highest level used (850 ppm) is nowhere near
the level which has been found to bring about a depression
in growth rate. O'Donovan et al. (1963) reported that
young pigs receiving up to 3000 ppm iron performed well.

It was only when the diet contained 4000 ppm iron that
rate of gain became significantly reduced. A similar obser-

vation was made by Furugouri (1972). When 60-day-old pigs

42
were fed up to 3102 ppm iron, there was no significant
difference in weight gain and feed consumption. When
animals were given 5102 ppm dietary iron, both feed intake
and rate of gain were depressed. One possible explanation
of the results of the present study is that a high level
of vitamin E is somehow incompatible with a high iron
level. The exact mode of incompatibility (if it exists)
cannot be explained without further investigation.

There was a significant (P<.05) iron x vitamin E
interaction on gain during the growing period of the study.
Here again, the indication suggests that high vitamin E is
not compatible with high iron.

Hematological data for this experiment are presented
in Table 4. There were no significant treatment differences
in hemoglobin, hematocrit or serum iron. Hematological
values would all be considered normal and there was no
evidence of iron toxicity with increasing levels of iron.
These results suggest that once the animal's iron require-
ment is met, further availability of the element would not
contribute to greater hemoglobin synthesis or an increase
in the number or size of the erythrocytes. Again, as the
serum iron concentrations were not affected by increasing
the dietary iron levels, the excess iron must either be
excreted and/or stored. If the surplus iron or part of it
is excreted, there must be some active mechanism in the
normal gastrointestinal tract which brings about this

process.

 

XWOHOHNEOC CO 2 CHEMHH> DEN COMM HOMO NO HUONWW -V OHQME

43

.mmoe ogu mo gouge unaccepm

.ucmowmflamfim uo: u

.m.z

H

 

 

 

 

 

 

 

 

 

.m.z .m.z .m.z mm.vH m.uNH m.nma n.moa n.mva N.wNH m.va mxooz 5H
.m.z .m.z .m.z mm.u~ m.om~ e.wHN N.om~ v.mo~ o.mnm w.mmm mxeez ea
.m.z .m.z .m.z oo.ma m.moa o.omH H.on m.woa v.wo~ m.omH mxmoz oH
.m.z .m.z .m.z mm.m~ m.mmH H.HmH m.v- w.~o~ H.vma m.omH mxooz a
HE ooaww: New Enhom
.m.z .m.z .m.z om.a o.om o.mm m.mm e.em m.mm 0.0H mxooz 5H
.m.z .m.z .m.z H5.H o.vm n.0m v.5m N.mm v.mm N.mm mxemz ea
.m.z .m.z .m.z om.H H.5m 5.5m n.em m.mm m.wm m.om mxooz OH
.m.z .m.z .m.z mN.H H.0m v.mm m.¢m m.om m.om m.mm mMomz v
w .uwuuoumEm:
.m.z .m.z .m.z mm. oo.OH vu.oa ow.m ow.oa no.NH v¢.HH mxooz AH
.m.z .m.z .m.z no. Am.m No.oH Nv.oa we.HH Nn.oa mo.oa mxeoz ea
.m.z .m.z .m.z mm. «H.NH we.HH om.o~ mm.a~ mo.HH He.oa mxooz OH
.m.z .m.z .m.z mm. AH.OH oo.HH em.m mm.OH ow.m oN.HH mxooz e
As OOH\wt.:Anonosmm
v e e e e v maweficw mo .02
m x :opH m .pH> couH I omw omm mew omm omm mvm Ema .conH
mm+
muemufimAEMAm OH o AH\.=.H .m .DA>
Amoaoumfim: :o m cfiEmufi> paw cop“ Hugo mo uuommm .v manmh

44

The results of this experiment agree with those of
O'Donovan et al. (1963), Standish et a1. (1971) and Harmon
et al. (1970), but disagree with those of Koong et al.
(1970) and Furugouri (1972). Koong et al. (1970) reported
that when young calves were fed increasing levels of iron
(100 to 2000 ppm), blood hemoglobin and serum iron values
increased significantly. Furugouri (1972) also reported
that hemoglobin concentration increased linearly with
increasing levels of iron and suggested that absorption
of excess iron stimulates marrow activity.

Tissue iron concentrations are shown in Table 5. Liver
iron concentrations were generally higher than normal. This
was not unexpected as the lowest level of dietary iron fed
was 345 ppm - about 250% more than the level that is normally
found in the feed. Liver iron levels tended to increase
with increasing levels of iron in pigs fed the high vitamin
E diets. However, these increases are not statistically
significant. Furugouri (1972) reported that when higher
levels of dietary iron were used, there was a linear
increase in the total liver iron stores, which indicates
that the iron storage capacity of the liver is relatively
high. Furugouri (1972) believes that the liver iron storage
capacity alleviates iron toxicity from excess in dietary
iron. In man, it has been reported (Bothwell and Finch,
1962) that with excessive dietary iron over a prolonged
period of time there is an increased accumulation of the

element in the liver. However, it takes a period of many

4S

.uchAMACMAm uo: u .m.z

.cmoe esp mo nohum uhmwcmuma

 

0H.~ m.oa H.mH e.NH e.eH m.oH 4.4H echfl sea .meoAuema

 

 

 

 

.m.z .m.z .m.z «H.H m.eH o.HH m.HH m.HH e.HH N.~H eopA egg .maumsz

.m.z .m.z .m.z AH.©N w.Nwm N.AON m.AwH A.wNN m.ow~ e.emH :oAA sag .A6>Ag

o o e o o o mHaaAea mo .62

m x :oAH m .DA> eoAH I omm omm mam omw omm men and .eoAH
mm+

oueauAmfiemAm H OH o BH\.=.H .m .DA>

 

N ucoeflnomxm :fi :owumpuaoocoo coufl mammwh .m manme

46

years before clinical signs are manifested. In the pig,
this is not likely to constitute a practical problem, as
the animal's life is usually terminated before the iron
accumulated in the liver can give rise to clinical problems.
On the other hand, it must be remembered that excess die-
tary iron can interfere with phosphorus utilization and
bring about an induced rickets (O'Donovan, 1963).

Muscle and pancreas iron concentrations in pigs fed
high iron levels were not significantly different between

treatment groups .

Experiment 3:

Hematological data for Experiment 3 are presented in
Table 6. There were no significant treatment differences
in hemoglobin or hematocrit levels at 0, 1 day and 1 week
after the intramuscular injection of 1000 mg iron from
iron dextran. Serum iron levels, however, rose dramatically
and,the day following the injection, were significantly
(P<.01) greater than in pigs not receiving the injection.
Further, iron treated pigs from sows receiving the vitamin
E supplemented diet had significantly (P<.01) greater serum
iron concentrations one day after injection when compared
to pigs from sows fed the low vitamin E diets. However,
one week after the iron injection, serum iron levels of
treated pigs had returned to the baseline control levels,
leaving no treatment differences in serum iron level between

the control and treated animals. Observations on behavior,

47

HHo.vmv AHpnmuHchmHm ohm mumHHomHDQSm uaonmmme :uHs 30H eEmm exp qH moaHm>

.uaenemee
won
.xpsum

on“ mo x003 ape aw :muuxow :ouH Eopm :onH mo we OOOH mo :oHuuoncH pmHsomssmnchm

.ucmonchHm no: u .m.z

.cmee on» mo nouno unmeamum

 

 

 

 

 

A

.m.z .m.z .m.z EA.AH mo.AAH mm.oom mo.AAH mo.AmH He OOH\m: .mm esAom

.m.z .m.z .m.z om.H AA.AN AH.mm me.mm A~.em A .AAAUOAm26:

.m.z .m.z .m.z NA. om.o~ oo.HH Am.HH AA.HH Ha OOH\A .eAnon656m

Moo: H

.m.z .m.z Ho. m o~.oe~ mo.o~om AON.AONUAH.AAHA nmo.mmN Ha OOH\AE .mm eaAmm

.m.z .m.z .m.z H~.N mm.mm mo.om oo.mm AH.om A .DAAuooeaom

.m.z .m.z .m.z om. OH.OH om.m mo.m om.HH He OOH\A .eflnonoamm

me A

.m.z .m.z .m.z mm.AN oh.omH mm.~a~ Am.AON Ae.meH He OOH\m: .mm aseom

.m.z .m.z .m.z AH.H oo.em mm.mm OH.om om.Am A .DAAUOAAEom

.m.z .m.z .m.z me. AA.OH AE.OH mm.o OH.AH Ha OOH\A .eAponoaom

HmApAeH

v v v e mHmEHcm mo .02

m x nouH m :ouH I. + - + - «coHuooncH nouH
Hmm+

muemuAmfiemAm OH o AH\.=.H .m .uAs

 

HmOHoquo: no em we oooH Ho :oHuuomnH 2H wan m :HEmuH> Ho uuommm

.o OHQMH

48
performance and hematological data provided no evidence of
iron toxicity.

This study clearly suggests the tremendous tolerance
of iron by the young pig and demonstrates the innocuous
nature of iron dextran. This good tolerance was exhibited
both by the pigs from sows fed vitamin E supplemented
diets as well as by the presumed vitamin E deficient animals.
The fact that the serum iron levels in the pigs from sows
fed vitamin E supplemented diets were significantly higher
than those from sows with low E diets might be because
vitamin E enhances iron uptake when iron is given by the
intramuscular route. The high levels of iron in the serum
did not seem to be harmful to the animals in this short
term study. Iron is known for its peroxidation action.
This being so, one would expect an increase in the rate of
peroxidation to occur in the presumed vitamin E deficient
animals. Lipid peroxide formation could then bring about
hemolysis. However, normal hemoglobin and hematocrit
values in this study suggest that this has not occurred.
Again, the short term study conducted here does not permit
any real conclusion to be made as far as this area is

concerned.

V. SUMMARY AND CONCLUSIONS

Three experiments were conducted to study the iron
tolerance of young pigs as influenced by dietary vitamin
E and selenium. Intramuscular administration of up to
1000 mg of iron from iron dextran or oral administration
of up to 850 ppm of iron from ferrous sulfate in the diet
for 126 days warranted the following conclusions:

1. Sows fed low vitamin E - Se corn-soy fortified
diets during gestation and lactation did not produce pig-
lets with a reduced tolerance to the standard dose of 100
mg iron from iron dextran when given at three days of age.

2. Iron toxicosis was not produced when young pigs
weaned at five weeks from sows on a low vitamin E - Se diet
were given a second iron-dextran injection of 100 mg iron.

3. Pigs weaned from sows on low vitamin E - Se diets
and kept on a low E - Se corn-soy fortified diet up to
market weight did not exhibit any evidence of iron toxicity
when their feed contained up to 850 ppm iron.

4. There was an increase in liver iron storage with
excess dietary iron. However, this increase was not linear
and,'w'ith further additional dietary iron levels, there was

no significant proportional increase in liver storage.

49

50

S. Hemoglobin, hematocrit and serum iron levels were
not significantly affected by dietary iron levels above the
normal requirements.

6. An intramuscular injection of 1000 mg iron from
iron dextran given to nine-week-old pigs produced a tremen—
dous increase in serum iron levels; pigs on the vitamin E
supplemented diets had significantly higher serum iron
levels than those on the low vitamin E diet.

7. High serum iron levels did not produce any notice-
able ill effect on the animals.

8. Hemoglobin and hematocrit values were not affected
by a large dose of iron dextran given intramuscularly when
the iron requirement of the pig was already met.

9. Pigs can tolerate intramuscularly administered

iron dextran with a high degree of safety.

B I BLI OGRAPHY

BIBLIOGRAPHY

Apri, T. and G. Tollerz. 1965. Iron poisoning in piglets;
autopsy findings in spontaneous and experimental
cases. Acta Vet. Scand. 6: 360.

Apte, S. V. and P. S. Venkatachalam. 1962. Iron absorp-

tion in human volunteers using high phytate cereal
diets. Ind. J. Med. Res. 50: 516.

Behrens, H. 1957. Pigs poisoned by iron injection. J.
Am. Vet. Med. Assoc. 132: 169 (Abstract).

Bentler, E., V. F. Fairbanks and J. L. Fahey. 1963.
Clinical disorders of iron. Grune and Stratton,
New York.

Beresford, C. R., L. Goldberg and J. P. Smith. 1957.
Local effects and mechanism of absorption of iron

preparations administered intramuscularly. Brit.
J. Pharmacol. 12: 107.

Blandford, T. B. and G. A. Lodge. 1966. Acute hepatic
necrosis following an iron dextran injection. Vet.
Rec. 78: 117.

Bliss, C. I. 1967. Statistics in biology. Vol. 1.
McGraw-Hill Book Co., New York.

Bothwell, T. H. and C. A. Finch. 1962. Iron metabolism.
Little, Brown and Co., Boston.

Brag, S. 1958. Iron poisoning in piglets. Proc. VIIIth
Nordic Vet. Congress, Helsinki. 98: 102.

Brock, J. F. and L. K. Diamond. 1934. Rickets in rats by
iron feeding. J. Pediat. 4: 442.

Brown, E. B. and B. W. Justus. 1958. In vitro absorption
of radioiron by everted pouches of rat intestine.
Am. J. Physiol. 194: 319.

Brown, E. R. Jr., D. E. Smith, R. Dubach and C. V. Moore.
1959. Lethal iron overload in dogs. J. Lab. Clin.
Med. 53: 591.

51

52

Brown, E. R. Jr., R. Dubach, D. E. Smith, C. Reynafarje and
C. V. Moore. 1957. Studies in iron transportation

and metabolism. X. Long-term iron overload in dogs.
J. Lab. Clin. Med. 50: 862.

Brownlie, W. M. 1955. The treatment of pig anemia. Vet.
Rec. 67: 350.

Campbell, E. A. 1961. Iron poisoning in the young pig.
The Austral. Vet. J. 78: 83.

Charley, P., M. Rosenstein, E. Shore and P. Saltman. 1960.
The role of chelation and binding equilibria in
iron metabolism. Arch. Biochem. Biophys. 88: 222.

Charlton, R. W., P. Jacobs, J. D. Torrance and T. H. Bothwell.
1965. The role of intestinal mucosa in iron absorp-
tion. J. Clin. Invest. 44: 543.

Conrad, M. E., L. R. Weintraub, and W. H. Crosby. 1964.
The role of the intestine in iron kinetics. J.
Clin. Invest. 43: 963.

Cox, G. J., M. L. Dodds, H. B. Wigman and F. J. Murphy.
1931. The effect of high doses of aluminium and
iron on phosphorus metabolism. J. Biol. Chem. 92: xi.

Coup, M. R. and A. G. Campbell. 1964. The effect of exces-
sive iron intake upon the health and production of
dairy cows. N. Z. J. Agric. Res. 7: 624.

Cowan, J. W., M. Esfahani, J. P. Salji and S. A. Azzam.
1966. Effect of phytate on iron absorption. J.
Nutr. 90: 423.

Crosby, W. H., J. I. Munn and F. W. Furth. "1954. Stan-
dardizing a method for clinical hemoglobinometry.
U.S. Armed Forces Med. J. 5: 693.

Davis, P. S., C. G. Luke and D. J. Deller. 1966. Reduction
of gastric iron binding protein in hemochromatosis.
Lancet 1431.

Deobald, H. J. and C. A. Elvehjem. 1935. The effect of
feeding high amounts of soluble iron and aluminum
salts. Am. J. Physiol. 111: 118.

Dowdle, G. B., D. Schachter and H. Schenker. 1960. Active
transport of Fe59 by everted segments of rat duo-
denum. Am. J. Physiol. 198: 609.

53

Forth, W., W. Rummel and G. Seifen. 1965. Influence of
chelating agents on the resorption of iron-59 in
the isolated intestine of rats. Arch. Exp. Path.
Pharm. 252: 224.

Fourcar, F. H., B. S. Hordon and S. Kayes. 1948. Death
following ingestion of ferrous sulfate. Am. J.
Clin. Path. 18: 971.

Furugouri, K. 1972. Effect of elevated dietary levels of
iron on iron store in liver, some blood constituents
and phosphorus deficiency in young swine. J. An.
Sci. 34: 573.

Goldberg, L., J. P. Smith and L. E. Martin. 1957. The
effects of intensive and prolonged administration

of iron parenterally in animals. Brit. J. Exp.
Path. 38: 297.

Granick, S. 1954. Iron metabolism. Bull. N.Y. Acad.
Med. 30: 81.

Gwatkin, R. and C. L'Ecuyer. 1959. Test of some injectable
iron products in pigs. Canad. J. Comp. Med. 23: 29.

Hahn, P. F., W. F. Bale, J. F. Ross, W. M. Balfour and G.
H. Whipple. 1943. Radioactive iron absorption by
gastrointestinal tract. Influence of anemia, anoxia
and antecedent feeding. Distribution in growing
dogs. J. Exp. Med. 78: 169.

Harmon, B. G., D. E. Becker, A. H. Jensen and H. W. Norton.
1968. Effect of NazEDTA in diets of different iron
levels on utilization of calcium, phosphorus and
iron by rats. J. An. Sci. 27: 418.

Harrison, D. C. and E. Mellanby. 1942. Phytic acid and
iron absorption. Lancet 242: 595.

Hartley, W. J., J. Mullins and B. M. Lawson. 1959. Nutri-
tional siderosis in the bovine. N. 2. Vet. J. 7: 99.

Hegsted, D. M., C. A. Finch and T. D. Kinney. 1949. The
influence of diet on iron absorption. II. Inter-

relationship of iron and phosphorus. J. Exp. Med.
90: 147.

Helbock, H. J. and P. Saltman. 1967. The transport of iron

by the rat intestine. Biochem. Biophys. Acta 135:
979.

Henriksson, K. 1962. Poisoning of piglets during iron
therapy. Finsk Veterinfirtidskr. 68: 293.

54

Hoekstra, W. G., D. Hafeman, S. H. Oh, R. A. Sunde and
H. E. Ganther. 1973. Effect of dietary selenium
on liver and erythrocyte glutathione peroxidase in
the rat. Fed. Proc. 32: 885 (Abstract).

Hopping, J. M. and W. S. Ruliffson. 1966. Roles of citric
and ascorbic acids in enteric iron absorption in
rats. Am. J. Physiol. 210: 1316.

Jacobs, P., T. H. Bothwell and R. W. Charlton. 1966.
Intestinal iron transport: Studies using a loop
of gut with an artificial circulation. Am. J.
Physiol. 210: 694.

Koong, Ling-Jung, M. B. Wise and E. R. Barrick. 1970.
Effect of elevated dietary levels of iron on the
performance and blood constituents of calves. J.
An. Sci. 31: 422.

Lannek, N., P. Lindberg and.G. Tollerz. 1962. Lowered
resistance to iron in vitamin E deficient piglets
and mice. Nature 195: 1006.

Lawlor, M. J., W. H. Smith and W. M. Beeson. 1965. Iron
requirement of the growing lamb. J. An. Sci. 24:
742.

Lindquist, N. 1949. Acute iron poisoning. Acta Paediat.
38: 447.

Maner, J. H., W. G. Pond and R. S. Lowrey. 1959. Effect
of method and level of iron administration on growth,
hemoglobin and hematocrit of suckling pigs. J.

An. Sci. 18: 1373.

Matrone, G., E. L- Thomason Jr. and C. R. Bunn. 1960.
Requirement and utilization of iron by the baby pig.
J. Nutr. 72: 459.

McCance, R. A., C. N. Edgecome and E. M. Widdowson. 1943.
Phytic acid and iron absorption. Lancet 245: 126.

McCance, R. A. and E. M. Widdowson. 1938. The absorption
and excretion of iron following oral and intravenous
administration. J. Physiol. 94: 148.

McGovern, J. J., A. R. Jones and A. G. Steinberg. 1955.
The hematocrit of capillary blood. New Eng. J. Med.
253: 308.

55

Miller! E. R., D. E. Ullrey, B. E. Brent, R. A. Merkel,

' ' V. A. Laidlaw and J. A. Hoefer. 1965. Iron reten-
tion and ham discoloration: A comparison of five
injectable iron preparations. J. Am. Vet. Med.
Assoc. 146: 331.

 

Miller, E. R., D. E. Ullrey, B. E. Brent, R. A. Merkel,
B. L. Bradley and J. A. Hoefer. 1967. Effects of
age and form of parenteral iron upon tissue iron
concentration and ham discoloration at slaughter.
J. Am. Vet. Med. Assoc. 150: 735.

Moore, C. V. 1960. Iron metabolism and nutrition. Harvey
Lectures 55: 67.

National Research Council Publication No. 1137. Revised
1968.

Nilsson, P. O. Nagra fall av akut jfirnfargiftning med.
myokardskada hos smfignisar. Nord. Vet. Med. 12:
113.

Nissim, J. A. 1953. Experimental siderosis: A study of
the distribution, delayed effects and metabolism
of massive amounts of various iron preparations.
J. Path. Bact. 66: 185.

O'Donovan, P. B., R. A. Pickett, M. P. Plumlee and W. M.
Beeson. 1963. Iron toxicity in the young pig.
J. An. Sci. 22: 1075.

Pape, L., J. S. Multani, C. Stitt and P. Saltman. 1968.
The mobilization of iron from ferritin by chelating
agents. Biochemistry 7: 613.

Pearson W. N. and M. Reich. 1965. In vitro studies of
§9Fe absorption by everted intestinal sacs of the
rat. J. Nutr. 87: 117.

Patterson, D. S. P., W. M. Allen, D. C. Thurley and J. T.
Done. 1967. The role of tissue peroxidation in

iron induced myodegeneration of piglets. Biochem.
J. 104: 28.

Patterson, D. S. P., W. M. Allen, D. C. Thurley, and J. T.
Done. 1967. The toxicity of iron dextran in pig-
lets. Vet. Rec. 80: 333.

Patterson, D. S. P., W. M. Allen, S. Berrett, D. Sweasey,
D. C. Thurley and.J. T. Done. 1969. A biochemical
study of the pathogenesis of iron induced myodegenera-
tion of piglets. Zentralblat fur Vetemarmedizin.
16: 199.

56

Patterson, D. S. P., W. M. Allen, S. Berrett, D. Sweasey
and J. T. Done. 1971. The toxicity of parenteral
iron preparations in the rabbit and pig with a com-
parison of the clinical and biochemical responses
to iron-dextorse in two day old and eight day old
piglets. Zentralblat fur Vetemarmedizin. 18: 453.

Reissmann, K. R., T. J. Coleman, B. S. Budai and L. R.
Moriarty. 1955a. Acute intestinal iron intoxica-
tion. 1. Iron absorption, serum iron and autopsy
findings. Blood 10: 35.

Reissmann, K. R. and T. J. Coleman. 1955b. Acute intestinal
iron intoxication. II. Metabolic, respiratory and
circulatory effects of absorbed iron salts. Blood
10: 46.

Rubin, M. and J. V. Priciotto. 1963. Chelation as a basic
biological mechanism. J. Agri. Food Chem. 11: 98.

Rydberg, M. E., H. L. Self, T. Kowalczyk, and R. H. Grummer.
. 1959. The.effectiveness of three different methods
of iron administration to young pigs. J. An. Sci.
18: 410.

Sathe, V. and K. Krishnamurthy. 1953. Phytic acid and
absorption of iron. Ind. J. Med. Res. 41: 453.

Saltman, P. 1965. The role of chelation in iron metabolism.
J. Chem. Educ. 42: 682.

Scott, M. L. and T. Noguchi. 1973. .Metabolic function of
selenium in prevention of exudative diathesis in
chicks. Fed. Proc. 32: 885 (Abstract).

Smith, R. P., C..W. Jones and W. E. Cochran. 1950. Ferrous
sulfate toxicity: Report of a fatal case. New Eng.
J. Med. 243: 641.

Snedecor, G. W. 1950. Statistical methods. The Iowa State
College Press, Ames.

Standish, J. P., C. B. Ammerman, A. Z. Palmer and C. F.
Simpson. 1971. .Influence of dietary iron and
phosphorus on performance, tissue mineral composi-
tion and mineral absorption in steers. J. An.
Sci. 33: 171.

Swift, S. C., V. Cefalu and E. B. Rubell. 1952. Ferrous
sulfate poisoning. J. Pediat. 40: 6.

Taylor, J., D. Stiven and E. W. Reid. 1935. Experimental and
idiopathic siderosis in cats. J. Path. Bact. 41: 397.

57

Tollerz, G. and N. Lannek. 1964. Protection against iron
toxicity in vitamin E deficient piglets and mice
by vitamin E and synthetic antioxidants. Nature
201: 846.

Ueberschfir, S. 1966. Todesfalle bei Sangferkeln nach
Applikation von.Eisen-dextran Prfiparaten (Fatalities
among piglets given iron dextran preparations).
Dtsch. tierfirztl. Wschr. 73: 145.

Ullrey, D. E., E. R. Miller, D. A. Thompson, 1. M. Ackermann,
D. A. Schmidt, J. A. Hoefer and R. W. Luecke. The
requirement of the baby pig for orally administered
iron. J. Nutr. 70: 187.

Wahlstrom, R. C. and E. W. Juhl. 1960. A comparison of
different methods of iron administration on rate of
gain and hemoglobin level of the baby pig. J. An.
Sci. 19: 183.

Wallerstein, R. O. and S. R. Mettier. 1958. Iron in
clinical medicine. Univ. of California Press,
Berkeley.

Wheby, M. S., L. G. Jones and W. H. Crosby. 1964. Studies
on iron absorption.. Intestinal regulatory mechan-
isms. J. Clin. Invest. 43: 1433.

Zimmerman, D. R., V. C. Speer, V. W. Hays and D. V. Catron.
1959. Injectable iron dextran and several iron
treatments for the prevention of iron deficiency
anemia of baby pigs. J. An. Sci. 19: 1409.

APPENDIX

APPENDIX A

Table A-1. Vitamin-Trace Mineral premix

 

 

Nutrient Amount in 10 lbs of premix
Vitamin A, million 3.0 I.U.
Vitamin D2, million 0.6 I.U.
Riboflavin 3.0 g
Nicotinic acid 16.0 g
d-pantothenic acid 12.0 g
Choline chloride 100.0 g
Vitamin B12 18.0 mg
Zinc 68.0 g
Manganese 34.0 g
Iodine 2.5 g
Copper 9.0 3
Iron 54.0 g
Antioxidanta 45.0 g
Carrier (ground yellow corn) to bring to 10 lbs

 

aButylated hydroxyanisole (BHA) and/or butylated
hydroxytoluene (BHT).

58

59

 

 

 

 

uo.m mm.~ um.~ om.m mm.~ on.~ eo.~ mv.m mN.N ow.H :Hmm\vmom
m~.e AH.v mm.m mw.m mn.m om.m ~m.m om.~ Hw.~ mm.~ pH .voem NAHHmH. .><
“mam mmom whom mHoN omHN woOH omNH NHm mhe ONH pH .oumw on .mcoo ween
Am.H oe.H mm.H em.H NA.H om.H AN.H 0H.H N~.H AN.H AH .eAem AHAme .><
emH owH ocH oeH NNH mOH mm no me mm mm n-0oH
mmH omH ooH AQH mNH NHH mm mm mm mm om o-H>
Hem NMN mmH OAH evH OHH om mo mm em 5N ~-mo~
eom NmH NAH AmH va OHH mm no om um um H-N:
mnH NAH omH NeH mNH HHH mm on mm om mm m-oHH
NmH whH mmH me mHH mm mm we om mm mm m-~>
ANN -~ emH HnH oeH ONH om Hm mm mm mm N-HHH
- - - - - - - co am He Hm n-mpH
Ana .uemfimz .6: Am

x3 H x3 H #3 H x3 H H3 HH H3 a VH3 A H3 m H3 m x3 H HmHuHaH voted
:oAH Ema mew :qu m :HsmuH> 30H .N pcoeHummxm .mumw ounmfiuemuom .~-< oHamH

60

 

 

 

AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AAAAAAAAA
AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA .AAAA AAAAA .><
AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAA AAA AAA AA .AAAA AA .Aeoo AAAA
AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA .eAAA AAAAA .><
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AAA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA IANWHH
AAA .AAAAAA .6: AA

AAA AAA AAA AAA AA: AA A: AAA AAA AAA AAAAAAA AAAAAA

 

GOAH Sam men :qu m :HEAAH> AMH: .N udoaHAomxm .mumv ooamshomhom .m-< oHan

61

 

 

 

AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A :AAAAAAAA
AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA .AAAA AAAAA .><
AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAA AAA AAA AA .AAAA AA .Aeou AAAA
AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA .AAAA AAAAA .><
AAA AAA AAA AAA AAA AAA AA AA AA AA AA AA-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-A»
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-A:
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA AA-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA H-mAH
AAA .AAAAAA .6: AA

AAA AAA: VAAAA AAA AAA AA A: AA AAA AAA AAAAAAAA AoAAAA

 

:OAH Ema omm nqu m :HEwuH> 30H .N unoEHhemxm .muww ouamEAoonm .AL< oHan

62

 

 

 

AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AAAAAAAAA
AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA .AAAA AAAAA .><
AAAA AAAA AAAA AAAA AAAA AAAA AAAA AAA AAA AAA AA .AAAA AA .Aeou AAAA
AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA .AAAA AAAAA .><
AAA AAA AAA AAA AAA AA AA AA AA AA AA AA-AAA
AAA AAA AAA AAA AAA AA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA AAA AAA AA AA AA AA AA-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-AAA
AAA AAA AAA AAA AAA -AA AA AA AA AA AA AA-AAA
AAA AAA AAA AAA AAA AAA AA AA AA AA AA A-mAA
AAA .AAAAAA .6: AA

VEAA V3AA AAA AAA 63: AAA DEA AAA AAA AzA AAAAAAA AoASA

 

couH Egg omm :AHS m :HEAAH> AMHA .N ucoeHAeaxm .mumw ouawanompom .m-< oHan

63

 

 

 

 

HO.N OA.N AA.N OO.N NO.N HO.N OO.N OA.N AA.N AO.H :Hmm\womm
mm.A AN.A AH.A NO.A Nw.m mo.m Hm.m HH.m mO.m ON.H pH .Oomm AAHHAO .><
Aowm HOmm Hmom HOmN «NON mmOH AmNH AAA AAA OHH 9H .eumw op .mcoo Omen
AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA.A AA .AAAA AAAAA .><
mNH HHN NOH HAH mAH OHH AO HA mm on ON H-HNH
AOH NNH OAH AmH AmH OHH mm mo AA mm mN H-NOH
NON OOH NAH OmH NNH OOH mm . NO we om mN m-AON
AmN ANN mON NAH mmH mNH OO AA mm om ON m-NON
AHN AON OAH mmH NNH mOH mm OO me ON HN O-m>
OOH mON NAH OmH OmH OHH OO ON OO NA Hm OH-OHH
NAN AAN NHN mOH emH H mmH OHH V cm no HA HA A-N>
AooH: - Avm NH m weH: Hm ON N-wbn

AAA .AAAAAA .6: AA

H3 OH x3 AH #3 mH #3 H x3 HH x3 O H3 A H3 m x3 m x3 H HmHuHAAH voHAom
:OAH flan 0mm AHA: m :HawuH> 30H .N uaoaHuonHm .auwv oucmlhomuom .c-< oHan

A ____ ._. _ __ ..._

 

64

mo.m No.m Am.N ow.N mm.N HA.N Ao.N Am.N mm.N 0A.H chM\wmom

 

AN.A NN.A AA.A AA.A AN.N NN.N NA.N NN.N NN.N oo.N NN .Aomm ANNAA .><
NANA NANA NNAN NNNN NNNN NNNN NANN NAN NNA ANN NN .AAAA 0N .Ncou ANNA
NN.N NN.N NN.N NA.N NN.N NN.N NN.N AN.N AN.N NN.N NN .aNAN ANNAA .><
NNN NNN NAN NAN NNN NON AN AN NN NN NN N-NNN
NNN NNN NAN NAN NNN ANN NN AN AN NN NN NN-NoN
ANN ANN NAN NNN NNN NNN NN No NN NA NN A-NA
NON ANN NAN AAN NNN NON AN No NA NN NN N-N:
NON ANN NNN AAN NNN ANN NN No NA NN AN N-ANN
ANN NNN NAN ANN NNN ANN AN No NN NN NN N-oNN
NNN ANN NNN AAN AAN NNN NNN NN NA NA NN N-oNN
NNN NNN NNN NAN NAN NNN NON NN NA NA NN -
NNN «uanoz .o: WAN

 

NzNN NzAN NzNN VENN VENN N: NzA NzN sz NzN NANANaN N528

 

:OAN Ema omm HHH: m :NEmuH> nmwn .N unoefinomxm .mpmu munmaAOWNom .A-< mHan

6S

 

 

 

 

O.NNN N.NAN N.AAN N.NNN A.AN N.NN N.AN A.NN ON.NN NN.O NO.NN ON.O NN-NON
N.OO N.NNN A.NAN O.NNN N.NN N.AN N.NA N.ON NN.ON AN.ON OO.NN AO.O N-Nm
O.NNN A.ONN N.OO N.NAN N.AN N.AN N.ON N.NN NN.N NN.ON NO.NN ON.O N-ANN AN ENN ONN
N.NAN O.NNN N.NNN N.NNN N.NN N.AN N.NA N.ON AA.NN OA.ON NA.NN AN NN N-ONN m .NNN .O.N ON
N.OON A.NNN A.AON N.ANN A.AN N.NN N.AN O.NN OO.NN NN.NN NA.NN OA.ON NNNON
A.NNN N.NNN N.ONN N.NAN N.AN N.NN A.NN N.AN NN.NN OO.NN NN.NN NN.NN O-NA
A.NNN N.ONN N.NNN O.ONN N.NN A.NA N.OA N.ON OO.NN NN.NN NA.NN NN.ON ON-ONN 0N ENN ONN
N.NNN N.NNN A.NAN N.NNN A.NN O.NA N.OA A.NN OO.O ON.O NN.NN ON.ON A-NA m .NN3 .:.N O
O NAN N.AON O.NNN O.ANN N.OA N.AN A.AN O.Nm ON.ON NN.O NN.ON OA.ON A-NON
N.ANN N.ONN N.AAN O.NON A.NN N.AN O.AN N.AN NA.ON ON.O ON.NN ON.NN NN-ONN mN ENN ONN
N.NAN N.NNN N.AAN N.OON A.NN N.NA N.NA N.AN ON.NN NA.NN NA.NN AN.NN N-NON N .NN3 .:.N ON
N.NAN N.AAN A.NNN N.OON N.NN N.NN N.ON N.NN AN ON ON.NN OO.NN NN.NN A-ONN
N.ANN N.NAN N.AAN N.AA O.NA N.OA O.NA N.ON NA.NN AN.ON NO.NN ON.O A-NN
N.OA N.ANN 0.0NN O.NNN N.ON O.NN N.AN O.NN NA.NN OA.ON ON.N NN.N N-NON 0N eNN ONN
N.OAN N.NNN 0.0NN O.NNN 0.0A O.NN A.AN O.NN NN.NN NN.ON ON.N AO.N O-NA N .NN3 .=.N O
N.NAN N.ONN A.AON N.AAN N.NN N.AN N.AN N.AN NA.NN NA.ON NN.NN AO.N N-NN
N.NNN N.ANN N.ONN N.ANN A.NN N.NN N.NN N.NN AN.NN NN.O NA.ON NN.N N-AON
N.NA O.NNN A.NON N.NAN A.NN N.AN A.NN N.NN AN.NN ON.NN NN.NN ON.ON N-NON AN aNN NAN
N.NN A.AON N.NNN O.NNN N.ON N.NN N.ON N.NN ON.NN OA.O AN.O AO.O N-NNN N .NN3 .:.N ON
N.NNN N.NAN N.NNN N.NNN N.AN N.ON O.NN O.NN NA.NN ON.NN NN.O NN.NN O-NA
O.NNN O.ONN N.AON N.OON N.ON N.AN N.NN N.NN AN.NN NO.N NA.O NN.ON N-NON
O.NAN N.NAN A.NNN N.NNN N.ON A.AN N.ON N.NN AA.NN NO.NN OO.NN NA.NN N-NN 0N aNN NAN
A.ANN N.ANN O.NNN N.NAN N.AN N.AN N.NN N.NN ON.ON NA.ON NO.NN NN.NN N-NNN m .NN3 .:.N O
N3 AN N3 AN N3 ON .NNN N3AN N3AN N3ON .NNN N3 AN N3 AN N3 ON .NNN .o: NNN AamsumeN
Ne OONANO N Na OONAN
:ONN Sapom awhuoumEmm :NQOHono:
N unmaHNomxm .wumw HNUNMOHONNSm: .w-< mHnmA

66

 

 

 

Table A-9. Tissue iron levels, Experiment 2
PPm
Treatment Pig No. Liver ‘Muscle Pancreas
0 I.U. vit. E 111-2 155.9 8.7 12.8
345 ppm Fe 205-2 160.4 9.7 9.2
- 110-3 247.1 17.5 20.2
106-7 266.9 12.0 16.2
H2-1 141.9 13.3 13.8
Y1-6 194.4 12.2 14.4
10 I.U. vit. E 205-1 103.4 15.6 8.5
345 ppm Fe 207-6 201.6 7.4 15.0
Y1-2 268.3 11.8 14.3
121-3 125.2 9.3 11.5
106-6 281.1 11.1 12.9
205-8 144.2 14.0 11.9
0 I.U. vit. E 205-5 194.3 10.8 11.3
550 ppm Fe H3-4 159.0 11.3 11.4
Yl-l 223.4 8.8 13.0
108-1 145.2 11.3 37.4
102—10 209.8 14.6 9.4
Y2-6 186.3 11.4 16.5
10 I.U. vit. E 203-1 127.6 10.0 11.1
550 ppm Fe 202-4 147.1 7.7 11.9
108-12 202.6 10.7 18.3
106-12 299.2 12.0 15.1
102-8 258.5 14.4 18.9
110-11 207.2 11.0 15.1
0 I.U. vit. E 110-10 253.5 11.3 22.9
850 ppm Fe 102-1 258.4 14.3 10.1
121-1 206.6 8.9 16.4
207-5 268.9 11.9 11.8
202-3 155.9 10.9 10.9
Y3-9 228.7 11.5 14.4
10 I.U. vit. E 110-8 274.9 20.0 13.3
850 ppm Fe 234-9 138.2 8.7 16.4
Hl-l 135.4 10.5 15.0
102-12 303.3 15.2 19.5
121-6 221.3 14.6 16.0
110-1 382.8 16.5 17.7

 

67

 

 

 

 

 

A.NON O.NmoA O.NN o.oN o.om N.mm H.HH o.OH o.m H-HHH
o.oo~ o.moov m.mmN m.Nm m.Am m.mm o.OH m.oH A.NH m-00H cowpommcH
m.on H.NHmm m.AOH o.mm N.mm A.Nm m.m o.oH A.m O-NOH mm we OOOH
m.mAH m.Hmom w.wOH w.mm o.mm m.om o.HH m.m m.HH w-OOH m .uH> .D.H OH
o.AHN O.NNN o.mAH A.om o.om N.Am N.HH N.OH H.NH A-NOH
m.AAH H.omH N.NOH A.om w.mm N.AN m.QH A.m o.m A-HNH :ONuuoncN
N.HAN N.NHN c.AwH. H.Nm N.NH m.om A.OH o.OH w.m N-OOH :ONN oz
m.mmH w.HAN o.AAN m.Am m.Am m.mm H.HH o.m N.HH w-HNH m .uH> .D.H OH
H.moH H.Nomm o.mON o.wm A.cm H.om N.HH m.m m.m A-HHH
m.AmN m.owom N.NNN H.mm H.mm A.Am A.HH A.w N.m m-H> :oNuuonaN
N.AAH A.©AwN N.ANH w.om o.mN N.Nm o.HH m.w H.OH A-©OH om we oooH
w.OHN ~.mmwm w.AwH O.Nm N.mm A.mm o.HH N.HH m.m 0HuooH m .HH> .D.H o
m.AAH o.AOH o.mmH A.mm m.om O.Nm o.HH H.NH O.NH o-HHH NONuuoncw
N.AmH O.NNN o.mAN m.om H.mm H.Nm N.OH A.NH o.NH m-oOH NONN oz
o.AoH H.NAN N.omH m.mm o.mm m.Am m.m N.m o.HH A-¢HH m .NH3 .=.H o
#3 H Amw H .HNH #3 H .Amw H nmnH x: H Amw H .NaH .0: mam ucoaumohe
HE oOH\m: ANONN Sahmm N AuwpuoumEo: HE OOmewwnflnonoam:

m Newswnomxm .mumw kuwmoHOHNEm: .oH-< mHnmA

HICHIGQN STRTE UNIV. LIBRARIES

Hill!
3

 

l llfllll IIIIIHI llli ‘| "I IN“ lllfll

31293102043 0